Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Structural-functional relationships in the human thrombin A-chain Carter, Isis Sarah Rosemary 2011

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
24-ubc_2011_fall_carter_isis.pdf [ 4.23MB ]
Metadata
JSON: 24-1.0072069.json
JSON-LD: 24-1.0072069-ld.json
RDF/XML (Pretty): 24-1.0072069-rdf.xml
RDF/JSON: 24-1.0072069-rdf.json
Turtle: 24-1.0072069-turtle.txt
N-Triples: 24-1.0072069-rdf-ntriples.txt
Original Record: 24-1.0072069-source.json
Full Text
24-1.0072069-fulltext.txt
Citation
24-1.0072069.ris

Full Text

STRUCTURAL-FUNCTIONAL RELATIONSHIPS IN THE HUMAN THROMBIN A-CHAIN by Isis Sarah Rosemary Carter B.Sc., The University of Canterbury, 2000 PGDipSci, The University of Canterbury, 2001 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Genetics) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) August 2011 © Isis Sarah Rosemary Carter, 2011  ABSTRACT Thrombin is the terminal protease in the coagulation cascade and plays a pivotal role in haemostasis, affecting both amplification and down-regulation of coagulation. Although prothrombin is one of the most widely studied enzymes in biology, the role of the thrombin A-chain region has been neglected in comparison to the other domains. While originally considered to be simply an activation remnant with little physiological function, mutations in the prothrombin A-chain region lead to bleeding disorders. There is evidence that the thrombin A-chain may play a role as an allosteric effector in enzymatic reactions and may also represent a structural scaffold to stabilize the protease domain; however, the exact role(s) of the A-chain remain to be elucidated. In this thesis, the roles of the A-chain region in prothrombin folding and activation, thrombin Ca2+ binding, enzyme stability and function were investigated. The results from this study suggest that the A-chain region is not required for prothrombin folding and secretion out of the cells; however, the A-chain is required for prothrombin activation. In an independent study using x-ray crystallographic techniques, NMR and activity assays, no evidence of a Ca2+ binding site was found in the thrombin A-chain or elsewhere in the thrombin molecule. During prothrombin activation, nascent thrombin undergoes autolysis of a 13residue N-terminal peptide of the A-chain to produce α-thrombin. Nascent thrombin and α-thrombin were compared to assess the effects of the A13 peptide. Contrary to expectation, autolysis of the A13 peptide at the N-terminus of the thrombin A-chain was very slow, with a half life of 46 minutes. Investigation of whether retention of this  ii  peptide affected thrombin structure and activity revealed that nascent thrombin was significantly different than α-thrombin in terms of 1) chromogenic activity and fibrinogen clotting activity, 2) thermal stability, 3) heparin binding and 4) inhibition by antithrombin. These studies further our knowledge of the roles the A-chain plays in the zymogen prothrombin and protease thrombin, and demonstrate that the A-chain A13 peptide of nascent thrombin may be a procoagulant stabilizer of thrombin in coagulation.  iii  PREFACE Sections of the Introduction relating to the prothrombin A-chain region have been published (Carter ISR, Vanden Hoek AL, Pryzdial ELG, MacGillivray RTA. Thrombin A-chain: activation remnant or allosteric effector? Thrombosis. 2010; vol. 2010: 9 pages, [1]). I was responsible for designing the review article, creating the figures and writing much of the article. The work outlined in Chapter 3 was performed in collaboration with Dr. James Huntington at Cambridge University, UK. I was responsible for designing and carrying out the experiments along with data analysis. The protein structures were solved by Dr. Huntington; in addition, members of the Huntington laboratory provided technical assistance for the NMR experiment described in Chapter 3. The work outlined in Chapter 4 is currently being prepared for publication. I was responsible for conceiving the work, and for generating the data for all experiments except for the MALDI TOF analysis. Amanda Starr was responsible for running the MALDI TOF MS and generating Figure 16. Dr. James Huntington provided intellectual assistance with experimental design of the heparin experiments. Dr. Ross MacGillivray was the principal investigator for the work. I created all of the text, figures and tables in this thesis apart from Figures 16 and 20.  iv  TABLE OF CONTENTS ABSTRACT ....................................................................................................................... ii PREFACE ......................................................................................................................... iv TABLE OF CONTENTS ................................................................................................. v LIST OF TABLES ............................................................................................................ x LIST OF FIGURES ......................................................................................................... xi LIST OF SYMBOLS AND ABBREVIATIONS ......................................................... xiii ACKNOWLEDGEMENTS .......................................................................................... xix 1. INTRODUCTION......................................................................................................... 1 1.1 Haemostasis ............................................................................................................. 1 1.1.1 Primary haemostasis: vasoconstriction and the platelet plug ............................ 1 1.1.2 Secondary haemostasis: the coagulation cascade .............................................. 2 1.1.3 Anticoagulant mechanisms ................................................................................ 4 1.1.4 Fibrinolysis ........................................................................................................ 6 1.2 Plasma prothrombin/thrombin.............................................................................. 6 1.2.1 Prothrombin structure and biosynthesis ............................................................. 7 1.2.2 Prothrombin domain organization ..................................................................... 8 1.2.3 Conversion to thrombin ..................................................................................... 8 1.2.4 Proteolytic activity of thrombin ....................................................................... 10 1.2.5 Modulation of activity...................................................................................... 14  v  1.3 Thrombin A-chain ................................................................................................ 16 1.3.1 Evolution .......................................................................................................... 16 1.3.2 Naturally occurring mutations in the prothrombin A-chain region ................. 20 1.3.3 Thrombin A-chain biochemistry ...................................................................... 23 1.3.4 Mutagenesis studies ......................................................................................... 25 1.3.5 Signalling and disease ...................................................................................... 28 1.4 Thrombin cation binding ..................................................................................... 30 1.4.1 Monovalent cation binding .............................................................................. 30 1.4.2 Divalent cation binding .................................................................................... 31 1.5 Project outline and rationale................................................................................ 32 1.6 Hypotheses ............................................................................................................. 35 2. MATERIALS AND METHODS ............................................................................... 37 2.1 Materials ................................................................................................................ 37 2.2 Proteins .................................................................................................................. 37 2.3 Bioinformatic analysis of A-chain rigidity .......................................................... 38 2.4 Recombinant protein expression and characterization ..................................... 38 2.4.1 PT and PT A constructs .................................................................................. 38 2.4.2 Recombinant human prethrombin-2 (rhpreII) ................................................. 44 2.4.3 Enzyme quantification ..................................................................................... 46 2.4.5 MALDI-TOF molecular weight determination ............................................... 47 2.4.6 A13 autolysis time course ................................................................................ 47 vi  2.5 Assessment of procoagulant function .................................................................. 47 2.5.1 Activation of prothrombin zymogens by TSV................................................. 48 2.5.2 Activation of prethrombin-2 zymogens by ECV ............................................. 48 2.5.3 Thrombin chromogenic activity ....................................................................... 48 2.5.4 Thrombin clotting activity ............................................................................... 49 2.6 Assessment of anticoagulant function ................................................................. 49 2.6.1 PC activation .................................................................................................... 49 2.7 Assessment of inhibition ....................................................................................... 50 2.7.1 Heparin inhibition of thrombin amidolytic activity ......................................... 50 2.7.2 Kinetics of thrombin inhibition by antithrombin (AT) .................................... 51 2.7.3 Stoichiometries of inhibition............................................................................ 51 2.8 Structural determination...................................................................................... 52 2.8.1 Circular dichroism (CD) .................................................................................. 52 2.8.2 Crystallization of thrombin with Ca2+.............................................................. 53 2.8.3 Thrombin NMR ............................................................................................... 55 2.9 Effect of Ca2+ on thrombin amidolytic activity .................................................. 56 3. ROLE OF THE A-CHAIN REGION IN PROTHROMBIN FOLDING AND ACTIVATION................................................................................................................. 57 3.1 Rationale and overview ........................................................................................ 57 3.2 Results .................................................................................................................... 58 3.2.1 Mutagenesis and expression of rhPT A .......................................................... 58  vii  3.2.2 Characterization of rhPT A ............................................................................ 59 3.3 Discussion............................................................................................................... 65 3.4 Conclusions ............................................................................................................ 67 4. ROLE OF D318 IN THROMBIN CALCIUM ION BINDING ................................ 68 4.1 Rationale and overview ........................................................................................ 68 4.2 Results .................................................................................................................... 69 4.2.1 Crystallization of pl.-thrombin and soaking in of Ca2+.................................... 69 4.2.2 Crystallization of thrombin in the presence of inhibitors and calcium ............ 70 4.2.3 Thrombin calcium NMR .................................................................................. 75 4.2.4 Effect of calcium on thrombin chromogenic activity ...................................... 75 4.3 Discussion............................................................................................................... 76 4.4 Conclusions ............................................................................................................ 80 5. FUNCTIONAL DIFFERENCES BETWEEN NASCENT THROMBIN AND THROMBIN: ROLE OF THE A13 PEPTIDE ............................................................ 81 5.1 Rationale and overview ........................................................................................ 81 5.2 Results .................................................................................................................... 82 5.2.1 Bioinformatic analysis of the human thrombin A-chain.................................. 82 5.2.2 Mutagenesis of recombinant prethrombin-2 R284Q ....................................... 87 5.2.3 Preliminary characterization of R284Q-thrombin ........................................... 87 5.2.4 A13 generation time ......................................................................................... 92  viii  5.2.5 Substrate hydrolysis is faster for R284Q-thrombin ......................................... 92 5.2.6 Assessment of anticoagulant function ............................................................. 99 5.2.7 Inhibition ........................................................................................................ 101 5.4 Discussion............................................................................................................. 105 5.5 Conclusions .......................................................................................................... 109 6. SUMMARY AND GENERAL DISCUSSION ....................................................... 110 6.1 Role of the A-chain region in prothrombin folding and activation ................ 111 6.2 Role of D318 in thrombin calcium coordination .............................................. 112 6.3 Role of A13 peptide in nascent thrombin ......................................................... 112 6.4 Significance of the work ..................................................................................... 113 6.5 Future studies ...................................................................................................... 116 BIBLIOGRAPHY ......................................................................................................... 120  ix  LIST OF TABLES  Table 1. Oligonucleotides used in the prethrombin-2 studies........................................... 40 Table 2. Prothrombin A-domain sequence alignment across 12 vertebrate species. ........ 83 Table 3. Conservation of the thrombin A13 peptide in vertebrates. ................................. 84 Table 4. S-2238 hydrolysis in the presence and absence of Na+. ..................................... 97 Table 5. Procoagulant activity of recombinant thrombins. ............................................... 98 Table 6. Effect of the A13 peptide on AT inhibition of thrombins in presence and absence of heparin................................................................................................... 106  x  LIST OF FIGURES Figure 1. The coagulation cascade of secondary hemostasis. ............................................. 5 Figure 2. Activation of thrombin by the prothrombinase complex. ................................. 12 Figure 3. Thrombin topography. ....................................................................................... 15 Figure 4. Pymol generated figure of nascent thrombin using S195A thrombin (PDB accession 3GIS) [39]. ................................................................................................ 17 Figure 5. Naturally occurring prothrombin mutations. ..................................................... 21 Figure 6. The thrombin A-chain. ...................................................................................... 29 Figure 7. Thrombin-PN1 crystal structure. ...................................................................... 33 Figure 8. Thrombin bound to Ni2+. .................................................................................. 34 Figure 9. Recombinant human prothrombin expression constructs.................................. 60 Figure 10. Selection of prothrombin clones. ..................................................................... 62 Figure 11. TSV-catalysed cleavage of rhPT and PT A monitored by SDS-PAGE. ........ 63 Figure 12. Near and far-UV CD spectrum for purified rhPT and PT A. ......................... 64 Figure 13. PPACK-thrombin with Ca2+ soak.................................................................... 71 Figure 14. PPACK-rh-thrombin/FVIII peptide1 crystallization. ...................................... 72 Figure 15. PPACK-thrombin/hirudin54-65. ....................................................................... 74 Figure 16.  15  N 1H NMR of S195A-thrombin. .................................................................. 77  Figure 17. Effect of [CaCl2] on thrombin chromogenic activity in the presence and absence of solubilizing additives. ............................................................................. 78 Figure 18. Thrombin A-chain structural flexibility. ......................................................... 86 Figure 19. Coomassie Blue stained SDS PAGE of purified recombinant thrombin species. ................................................................................................................................... 89 xi  Figure 20. MALDI-TOF molecular weight determination. .............................................. 90 Figure 21. Far and near-UV CD spectrum for rh -thrombin and R284Q-thrombin. ....... 91 Figure 22. Effect of the A13 peptide on the thermal denaturation of thrombin. .............. 93 Figure 23. A13 peptide autolysis from thrombin. ............................................................. 94 Figure 24. S-2238 hydrolysis in the presence and absence of Na+. .................................. 96 Figure 25. Activation of PC by thrombin/TM. ............................................................... 100 Figure 26. Elution of thrombins from heparin-Sepharose. ............................................. 102 Figure 27. Heparin inhibition of thrombin S-2238 activity. ........................................... 103 Figure 28. Heparin inhibition of thrombin chromogenic activity toward S-2238. ......... 104 Figure 29. Proposed model for the influence of the A13 peptide on nascent thrombin. 114  xii  LIST OF SYMBOLS AND ABBREVIATIONS  -IIa-HRP  anti-human thrombin heavy chain monoclonal primary antibody conjugated to horseradish peroxidase  IIa  thrombin  IIa-B  thrombin B-chain  IIa_PN1  thrombin protease nexin-1 complex  APC  activated protein C  aPL  anionic phospholipids  ATIII  antithrombin III  BCA  bicinchoninic acid  BHK  baby hamster kidney  BSA  bovine serum albumin  dH20  Distilled water  DMEM-F12  Dulbecco‟s modified Eagle‟s medium/F- 12  DTT  dithiothrieitol  ECV  Echis carinatus venom, a FXa homolog  xiii  EDTA  ethylene diamine tetra acetic acid  F1  fragment 1 of prothrombin  F1.2  fragment 1.2 of prothrombin  FII  Prothrombin gene  FLH  Full length heparin (15kD)  FPR  H-D-Phe-Pro-Arg-p-nitroanilide  FV  factor V  FVa  Factor Va  FVII  Factor VII  FVIIa  Factor VIIa  FXIII  Factor XIII  FXIIIa  Factor XIIIa  GAG  glycosaminoglycan  Gla  γ-carboxyglutamic acid  Gp 1b  Glycoprotein 1b  HBS  HEPES buffered saline  HEPES  4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid  xiv  ITS  insulin transferrin selenium  kcat  first-order rate constant, also called the turnover number  Kd  dissociation constant  k2  second order rate constant  K  app d  apparent dissociation constant  Km  Michaelis constant  Kmapp  apparent michaelis constant  mIIa  meizothrombin  MALDI TOF MS  Matrix assisted laser desorption/ionization-time of flight mass spectroscopy  MES  2-(N-morpholino)ethanesulfonic acid  Mr  molecular mass which is the mass of one molecule of that substance relative to the unified atomic mass unit u (equal to 1/12 the mass of one atom of 1C2)  MRW  mean residue molecular weight  MTX  methotrexate  PARs  protease activated receptors  PBST  Phosphate buffered saline with Tween  xv  PC  protein C  PCR  polymerase chain reaction  PEG  polyethylene glycol  PPACK  d-Phe-Pro-Arg-chloromethylketone  Pl.-IIa  plasma derived human thrombin  PN1  protease nexin1, a serpin  pNA  p-nitroaniline  PreI  prethrombin-1  PreII  prethrombin-2  PS  phosphatidylserine  PT  prothrombin  PT∆a  recombinant prothrombin expressed without the A-chain  PVDF  polyvinylidine difluoride  rh -IIa  recombinant human alpha thrombin  rhPT  recombinant human prothrombin expressed in BHK cells  S-2238  H-D-phenylalanyl-L-pipecolyl-L-arginine-P-nitroanilide  SDS-PAGE  sodium dodecyl sulphate - polyacrylamide gel electrophoresis  xvi  Serpin  serine protease inhibitor  SVTLEs  snake venom thrombin like enzymes  sTM  soluble thrombomodulin  TAFI  thrombin activatable fibrinolysis inhibitor  TAME  tosyl- Arg-methyl-ester  TAT  thrombin antithrombin inhibition complex  TBS  tris buffered saline  TBST  Tris buffered saline plus 0.5% Tween-20  TEA.Cl  triethanolamine hydrochloride  TF  tissue factor  TFA  trifluoroacetic acid  TFPI  Tissue factor pathway inhibitor  TM  thrombomodulin  t-PA  tissue type plasminogen activator  TRIS  tris (hydroxymethyl) aminomethane  TROSY NMR  transverse relaxation optimized spectroscopy nuclear magnetic resonance  TSV  Taipan snake venom, a prothrombinase complex homolog xvii  VKOR  vitamin K epoxide reductase  WT  wild type (the native form of the enzyme)  vWF  von Willebrand factor  Xase  tenase complex  xviii  ACKNOWLEDGEMENTS  I would like to thank a number of people who have been instrumental in my PhD experience at UBC. I greatly appreciated the guidance from my committee, Drs. Ed Pryzdial, Grant Mauk and LeAnn Howe. Thanks also to Drs. Jim Huntington, Ed Conway, Fred Rosell, Dana Devine and Cedric Carter for their encouraging and enthusiastic attitude to my project. A special thanks to Ed Pryzdial and Jim for providing a varied and holistic perspective on coagulation. I am grateful for the numerous and enlightening conversations I have had with members of the MacGillivray, Pryzdial, Conway, Strynadka and Mauk Labs, and for their support and camaraderie. Special thanks to Amanda, Scott, Kimmi, Michael, Mike Krisinger, Jeff, Val, Ann, Gerd, Frank and Barbara. You all made coming to work so much fun! Special thanks to Brad, my family and friends for embarking on, and supporting me in this endeavour, your encouragement was invaluable. Lastly, thank you to my supervisor, Ross MacGillivray, for taking me on and for your kindness, generosity and patience. Thank you for giving me so many wonderful opportunities, such as writing reviews and grants, attending conferences and visiting the UK– the learning experiences I have received are irreplaceable and everlasting.  xix  1. INTRODUCTION 1.1 Haemostasis Haemostasis is the process by which blood flow and volume are maintained in response to damage, by changing blood from a fluid state to solid and arresting bleeding. Haemostasis is comprised of four interconnected and highly regulated processes: primary haemostasis, secondary haemostasis or the coagulation system, negative regulation of coagulation through anticoagulant mechanisms and finally fibrinolysis (for a review see [2]). 1.1.1 Primary haemostasis: vasoconstriction and the platelet plug Upon damage to a vessel wall, smooth muscle contractions cause blood vessel constriction, slowing the blood flow. In the high shear environment of arterial flow, this reduction in flow rate allows platelets to „roll‟ along the endothelial wall and adhere to the site of injury where exposure of subendothelial structures (including collagen and collagen-bound von Willebrand factor (vWF)) provide platelet binding sites through platelet-bound collagen receptors and the platelet complex GP1b-IX-V [3]. The adhesion of platelets at the wound site creates a temporary platelet plug causing weak activation of the platelets to undergo signalling and cytoskeletal changes to stop rolling and allow spreading out at the wound site [4, 5]. The weak activation of platelets causes Ca2+ release from intracellular stores into the cytoplasm initiating a G-protein coupled receptor cascade that causes platelets to degranulate and activate. The Ca2+ dependent signalling events also cause rearrangement of the phospholipids in the platelet membrane bilayer, allowing exposure of negatively charged phospholipids at the platelet membrane surface, 1  and on microparticles shed from activated platelets [6-9]. Anionic phospholipids (aPL) can complex Ca2+ to allow binding of the vitamin K-dependent coagulation factors, causing colocalization of the coagulation factors on the activated platelet surface and subsequent synergistic amplification of platelet recruitment to the wound site [7, 10]. 1.1.2 Secondary haemostasis: the coagulation cascade Activation of the coagulation cascade involves the sequential activation of a series of inactive enzyme precursors (zymogens) and their non-enzymatic cofactors (procofactors) culminating in the formation of fibrin. Fibrin acts to stabilize and reinforce the platelet plug at the site of injury (see Figure 1 for a schematic of the coagulation pathway). Tissue factor (TF) is the vascular trigger required for coagulation initiation in vivo, and TF association with aPL is required for significant procoagulant activity [11, 12]. In healthy, intact blood vessels, TF is located in the subendothelial matrix between endothelial cells – as a result, it is „extrinsic‟ to blood until injury exposes it to the vasculature [13]. Endothelial damage exposes both membrane bound TF and subendothelial aPL to platelets and coagulation proteins circulating in blood to initiate clot formation through this extrinsic pathway. TF binds the serine protease factor VIIa (FVIIa) and the TF-FVIIa complex (also known as the extrinsic tenase complex) then activates the circulating zymogen factor X (FX) to the serine protease factor Xa (FXa) [13]. Approximately 10-15% of FVII circulates in an activated form (FVIIa) [14]. However FVIIa is zymogen-like until it is bound by TF, and stabilized in the proteinase form, capable of cleaving FX [14]. FVII can also be activated to FVIIa once bound to TF by FVII activating protease or FXa [15, 16]. Newly generated FXa binds its 2  nonenzymatic cofactor FVa in the presence of Ca2+ and aPL, forming the prothrombinase complex. FXa in the prothrombinase complex proteolytically activates the zymogen prothrombin to its active form, thrombin [17-19]. Thrombin is the terminal protease in the coagulation cascade and is responsible for converting circulating soluble fibrinogen monomers to insoluble fibrin [20]. Fibrin monomers spontaneously polymerize to form a fibrin clot, enmeshing the platelet plug and red and white blood cells caught at the site of injury. The clot is then further stabilized by the cross-linking enzyme factor FXIIIa, which is also activated by thrombin [21]. Once sufficient FXa is generated by the extrinsic tenase complex to initiate coagulation, serine protease inhibitors (serpins) such as antithrombin and heparin cofactor II, and the kunitz type inhibitor; tissue factor pathway inhibitor (TFPI) terminate the FXa generation reaction (reviewed in [22-24]). Sustained thrombin generation is then maintained by positive feedback of coagulation through thrombin-induced activation of factors V, VIII and XI. FX can be activated by another activating complex comprised of factors IXa (FIXa) and VIIIa (FVIIIa) (referred to as part of the intrinsic coagulation pathway as they are „intrinsic to blood). The zymogen FIX can be activated either by the TF-FVIIa complex or by factor XIa (FXIa) which is activated by the initial burst of thrombin generated through the extrinsic pathway [25]. Sustained thrombin generation via amplification of FX activation is critical for the formation of a stable fibrin blood clot as evidenced by the prevalence of haemophilia A (FVIII deficiency) and B (FIX deficiency) which affect approximately 1 in 5,000 males combined worldwide [26, 27].  3  1.1.3 Anticoagulant mechanisms Clot dissemination into intact vasculature is prevented by several mechanisms. For example, the coagulation propagation and inactivation steps are localized on different cell surfaces. Resting endothelial cells express membrane-bound antithrombotic proteins to prevent migration of the clot away from the damaged vasculature and plasma protease inhibitors localize coagulation to membrane surfaces by inhibiting any wayward proteases from diffusing away from the clot site. While TF-bearing cells and activated platelets are strongly procoagulant, resting endothelial cells have anti-coagulant features. Thrombomodulin (TM) is a receptor protein that is expressed at high levels on intact vessel endothelium and is able to bind excess thrombin [28]. TM possesses both direct and indirect anticoagulant properties. The direct anticoagulant function of TM is to alter thrombin specificity upon TM binding, preventing the cleavage of fibrinogen or activation of platelets [29]. TM-bound thrombin is also rapidly endocytosed and degraded. Indirectly, when bound to TM, thrombin becomes effective at activating the endothelium-bound protein C (PC) to activated protein C (APC) [30]. APC forms a complex with its non-enzymatic cofactor, protein S to proteolytically degrade FVa and FVIIIa to shut down thrombin generation [31]. As a result, formation of the prothrombinase and tenase complexes is inhibited thus preventing uncontrolled thrombus growth [32, 33] (Figure 1). The surface of endothelial cells is decorated by negatively-charged glycosaminoglycans (GAGs) which serve as a scaffold to carry out the reaction between excess thrombin with the plasma borne serpins AT and heparin cofactor II (HCII) thereby enhancing thrombin inhibition [34, 35]. Heparan sulphate is a surface-exposed 4  Figure 1. The coagulation cascade of secondary hemostasis. During initiation, a small amount of FX is activated by the FVIIa-TF complex on TFbearing cells. Upon adhesion at the site of injury, platelets are partially activated, promoting the release of partially activated FV (FVa). FVa complexes with FXa on TFbearing cells and is activated by FXa or other proteases. The FVa-FXa prothrombinase complex converts a small amount of prothrombin (II) to thrombin (IIa), which plays a major role in amplification. Thrombin completes platelet activation, releases activated FVIIIa from vWF to platelet surfaces, and activates FV and FXI on the platelet surface. Clot formation is propagated through a series of events on activated platelet surfaces: FIX activated by FVIIa-TF complex or FXIa complexes with FVIIIa to form the tenase complex; tenase activates FX to FXa, which joins FVa to form the prothrombinase complex and activates FII to IIa; IIa converts fibrinogen to fibrin and activates FXIII to FXIIIa to cross-link the fibrin clot (Reviewed in [24]).  5  endothelial GAG that acts as a scaffold to enhance AT inhibition of thrombin by approximately 1000 fold by bridging AT and thrombin [36]. TFPI is the major FXa inhibitor in plasma, while AT is the major thrombininactivating protein (Figure 1) [22, 23]. Protease nexin-1 (PN-1) is also a serpin protein that inhibits thrombin, factor Xa and other serine proteases. PN-1 has been located on the surface of vascular endothelial cells, fibroblasts and platelets (reviewed in [22, 24]). Together, these mechanisms prevent the uncontrolled dissemination of a clot into intact vasculature. 1.1.4 Fibrinolysis The fourth phase of haemostasis commences once vascular repair is underway. Fibrinolysis degrades the fibrin clot to restore normal blood flow to the area. The degradation of the thrombus is controlled by tissue type plasminogen activator (t-PA) which converts plasminogen to the active clot lysis enzyme, plasmin [37, 38]. Plasmin degrades insoluble fibrin to soluble fibrin degradation products, thus clearing the clot [39].  1.2 Plasma prothrombin/thrombin Thrombin is the central enzyme in haemostasis, interacting with a multitude of substrates, cofactors and inhibitors to participate in both procoagulant and anticoagulant pathways. Its zymogen, prothrombin is a single chain glycoprotein of Mr 72,000 that circulates in plasma at a concentration of 100-200 g/mL [40].  6  1.2.1 Prothrombin structure and biosynthesis The cDNA and amino acid sequences of human prothrombin have been determined [41-43]. The amino acid sequence provides information about the structure and domain organization of the protein. A schematic model of prothrombin protein organization is shown in Figure 2. Prothrombin is synthesized in the liver as a precursor containing an N-terminal prepro-peptide of 43 amino acids [44]. The pre-sequence, or signal peptide, functions in the co-translational transfer of the protein across the endoplasmic reticulum membrane and is removed by signal peptidase. Prothrombin undergoes several posttranslational modifications prior to secretion. These include cleavage of the prepro-peptide of prothrombin, vitamin K-dependent γ-carboxylation and glycosylation [45]. The pro-peptide comprises in part the recognition sequence for a vitamin K-dependent carboxylase that converts the 10 glutamic acid residues in the Gla domain (at the N-terminal region of prothrombin) to -carboxyglutamic acid, or Gla [46]. These modified residues bind calcium (Ca2+) leading to a conformational change that is essential for assembly of the substrate, prothrombin, with prothrombinase on a procoagulant phospholipid membrane for subsequent activation to thrombin [47-49]. Following γ-carboxylation the pro-peptide is removed by enzymatic cleavage Cterminal to an Arg-Arg sequence generating the new N-terminus of the mature zymogen found in plasma [43, 45]. Prior to secretion into the bloodstream, the final posttranslational modification to prothrombin is addition of the three N-linked carbohydrate moieties at residues N78, N100 (both located in the kringle domain 1) and N373 (present in the serine protease domain) [45, 50].  7  1.2.2 Prothrombin domain organization As shown in Figure 2, plasma prothrombin consists of four structural domains: the Gla domain, a region containing 10 γ-carboxylated glutamic acid residues which mediate prothrombin binding to procoagulant phospholipid surfaces; two kringle domains, which are thought to be involved in protein-protein interactions [51, 52]; and lastly the trypsinlike serine protease domain, which contains the enzyme active. Disulfide bonds are also shown in Figure 2. 1.2.3 Conversion to thrombin Prothrombin and homologous serine proteases in the coagulation cascade are produced and circulate in plasma as zymogens to allow for rapid activation by limited proteolysis when required. Irreversible peptide cleavage activation of zymogens provides rapid temporal and spatial regulation of the hemostatic response. Coagulation initiation is doubly regulated by the requirement for activation of both cofactors and proteinases in coagulation enzyme complexes, in addition to the formation of the complexes on membrane surfaces exposed upon injury [53]. When coagulation is initiated at a site of vascular injury, prothrombin is converted to thrombin by the prothrombinase complex; consisting of the protease FXa and its cofactor FVa on the surface of anionic phospholipid in the presence of Ca2+ (reviewed in [24]). Prothrombin cleavage is modulated by the presence of FVa in two ways; FVa interacts with prothrombin via kringle 2 and the heavy chain of FVa and additionally causes FXa to undergo a conformational change to expose an exosite for prothrombin binding. While the exact contact points for FXa and FVa in the prothrombinase complex are currently not known, the FVa contact points on FXa include all of the heparin binding 8  residues of FXa in addition to D185-D189 and V231-R245 (reviewed in [54]). Factor Xa in the prothrombinase complex cleaves two peptide bonds in prothrombin, initially cleaving at the R320-I321 bond to generate meizothrombin (Figure 2). Meizothrombin then spontaneously changes to a different conformation (ratchets) to present the R271-T272 bond for cleavage to release fragment 1.2, consisting of the Gla domain and the two kringle domains [55, 56]. The two prothrombin bonds are located approximately 36 angstroms apart on the surface of prothrombin and sequential cleavage occurs by prothrombinprothrombinase exosite interactions that tether each substrate (prothrombin and meizothrombin) to the enzyme and facilitate presentation of the scissile bond to the active site of the catalyst [55]. The cleavage of the two scissile bonds in prothrombin activation is driven by sequential presentation of substrate bound either in the zymogen or proteinase conformations. Prothrombinase ordered cleavage of prothrombin is accomplished by ratcheting of the substrate from the zymogen to the proteinase-like states [55]. Kim and Nesheim have further proposed that there are two functional forms of prothrombinase, each selective for one of the two bond cleavages [57]. Their model suggests a classical “ping-pong” catalytic mechanism whereby the two forms spontaneously interconvert whilst channelling the substrate [57]. In the absence of FVa, cleavage of the prothrombin R271-T272 bond occurs slowly to generate prethrombin2/fragment 1.2 which then is cleaved at R320 to generate thrombin [58]. Wood et al. have also shown that on the surface of activated platelets, prothrombin actvation occurs predominantly through a prethrombin-2 intermediate with cleavage of R271 occuring first [59]. This was suggested to be a function of a different and unique form of platelet derived FVa [59].  9  Through either activation pathway, the proteolytically-active thrombin molecule is comprised of a 49-amino acid light chain (A-chain) linked by a single disulphide bond to the 259-residue heavy chain (B-chain), which contains three intrachain disulphide bonds. The serine protease domain of thrombin is located in the B-chain. Nascent human thrombin undergoes autoproteolysis at R284-T285 of the A-chain, releasing a 13-residue Nterminal peptide and yielding a 36-residue A-chain to form -thrombin [60-63] (Figure 2). It is not currently known whether full A-chain nascent thrombin, truncated A-chain αthrombin or both forms of thrombin are present when coagulation is initiated in vivo or in plasma [45]. 1.2.4 Proteolytic activity of thrombin The primary haemostatic role of thrombin is the generation of fibrin. At the site of vascular injury, a series of proenzymes and procofactors are activated to facilitate the thrombin-mediated proteolytic conversion of fibrinogen into fibrin which ultimately results in the formation of a stabilized fibrin blood clot. The initial small quantities of thrombin generated through the extrinsic pathway participate in a positive feedback mechanism to amplify the coagulation response by activating coagulation factors V (FV), FVIII, FXI, and FXIII [64-66]. Thrombin also contributes to the generation of a stable clot by interacting with a variety of platelet receptors including protease-activated receptors (PARs) and glycoprotein Ibα to induce platelet activation and aggregation [67]. 1.2.4.1 Physiological substrates Thrombin is characterized by high substrate flexibility that allows it to interact with upwards of 12 physiological substrates in procoagulant, anticoagulant and signalling pathways [68, 69]. 10  1.2.4.1.1 Procoagulant The cleavage of soluble fibrinogen to insoluble fibrin monomers and the activation of platelets through PARs 1 and 4 are the main procoagulant roles of thrombin, catalyzing the production of the fibrin mesh that forms the mechanical clot and creation of the procoagulant surface upon which the upstream coagulation factors can coalesce [68, 70]. Thrombin positively regulates its own production through the activation of factor XI and the pro-cofactors FV and FVIII [68]. Thrombin also activates factor XIII (when bound to fibrin monomer as a cofactor) resulting in the formation of factor XIIIa which transglutaminates fibrin monomers to give a stable cross linked network [21]. 1.2.4.1.2 Anticoagulant When in the presence of thrombomodulin (TM), thrombin substrate specificity is changed towards the activation of the anticoagulant pathway zymogen protein C, thus down-regulating pro-coagulant thrombin production [71]. TM cofactor binding also allows thrombin to activate the enzyme thrombin activatable fibrinolysis inhibitor (TAFI), which breaks up the stable fibrin network to allow clot dissolution [72]. Activated protein C achieves downregulation of coagulation by inactivating the cofactors FVa and FVIIIa for the prothrombinase and tenase complexes respectively [73]. 1.2.4.1.3 Signalling Thrombin activates platelets through the binding of a platelet glycoprotein GP 1b , which then allows binding to PAR-1 for platelet activation [74].  11  Figure 2. Activation of thrombin by the prothrombinase complex. Prothrombin is colored by domain in this schematic, highlighting the A-chain (pink) and B-chain (yellow) (A). The gamma carboxylated Gla residues are noted by (Y) at the Nterminus of prothrombin, the carbohydrate attachment sites in kringle 1 (K1) and the B domain are noted by the shaded star, and the disulfide bridges are shown. The arrows show how sequential cleavage intermediates are formed from prothrombin in the generation of thrombin. Experimental constructs used in biochemical studies of the thrombin A-chain include prethrombin-1 (B) and prethrombin-2 (C). Factor Xa initially cleaves Prothrombin at R320 to produce meizothrombin (D), followed by cleavage at R271 to release fragment 1.2 from nascent thrombin (E). Thrombin then undergoes intermolecular autolysis to cleave the R284/T285 bond (F), liberating the A13 peptide (pale pink) to generate α-thrombin.  12  1.2.4.2 Thrombin specificity While thrombin acts on many substrates in coagulation, thrombin specificity is comparatively more restrictive than other trypsin-like proteases in the chymotrypsinogen family [75], requiring an arginine residue in the primary binding pocket and is restricted to a small hydrophobic residue (such as valine or proline) at P2 due to the hydrophobic lid imposed by 60-insertion loop [76]. Hydrophobic residues are required at P3 and P4 [77, 78]. Thrombin will cleave after an arginine residue within the substrate sequence A-BPro-Arg-X-Y, where A and B are hydrophobic residues and X,Y are nonacidic residues. Alternatively, a glycine residue at both P2 and P1‟ is also cleavable by thrombin [79]. Thus, synthetic substrates with the thrombin recognition sequence, such as S-2238 (H - D - Phe - Pip - Arg - pNA) and TAME (tosyl-Arg-methyl ester) allow cleavage after the arginine residue and release either a chromogenic or fluorogenic product. 1.2.4.3 Inhibitors To prevent widespread thrombosis caused by the wayward migration of thrombin away from the wound site, there are plasma serpins (such as AT and HCII) that bind and inactivate thrombin [22]. Serpin inhibition of thrombin is accelerated approximately 1000 fold by thrombin binding to endothelial cell-exposed glycosaminoglycan chains, such as heparin sulphate, chondroitin sulphate and dermatan sulphate [80]. These long chain negatively-charged polysaccharides form a scaffold between thrombin and the serpin to bridge them together in two dimensional space. Serpins inhibit their substrates by the formation of a covalent bond between the serpin and the protease, causing a  13  conformational change that drastically alters the protein structure denaturing the enzyme‟s active site [81]. 1.2.5 Modulation of activity Thrombin activity is modulated though the binding of nonenzymatic cofactors such as TM, GP Ib , glycosaminoglycans, fibrin and also by sodium ions (Na+). These cofactors alter thrombin substrate specificity by binding to surface exposed patches on the thrombin molecule including exosites I or II and the Na+ binding loop (Figure 3). Cofactors and substrates compete for access to the exosites both temporally and spatially during coagulation to steer thrombin towards different activities [82]. While the exosites and Na+ binding sites are far removed from the active site of thrombin, their influence is exerted through long range allosteric effects [83-86]. Exosite 1 is a positively-charged patch comprised of several arginine and lysine residues centred on K70 in thrombin, and is homologous to the Ca2+ binding loop in trypsin and chymotrypsin (Figure 3). This patch of basic residues is flanked by a hydrophobic patch that spans the exosite. The intense positive charge of exosite I provides electrostatic steering and preorientation for fibrinogen, PAR-1, TM and the leech derived thrombin inhibitor hirudin [69]. Exosite II is located on the opposite side of the thrombin active site in relation to exosite I and is comprised of numerous positively charged residues that serve to bind the negatively charged GAGs, GP -1b and the fibrinogen γ peptide (Figure 3). Na+ binding between the 220 and 180 loops of thrombin promotes a prothrombotic state in thrombin, capable of cleaving fibrinogen and PAR1 [87-89]. When Na+ is absent, thrombin  14  Figure 3. Thrombin topography. Pymol generated figure of nascent thrombin using S195A thrombin (PDB accession 3GIS) [33]. A) Thrombin structure showing active site residues in red and location of the A-chain on the opposite face of the molecule in green. Exosite 1 residues are colored purple, exosite II residues are colored yellow and the Na+ binding loop residues are marked in orange. (B). Back view of thrombin showing the 36 amino acid residue Achain colored in green, and the covalently bound A13 peptide segment marked in pink.  15  activity toward these substrates is reduced; however, it retains the same PC activation anticoagulant activity [69].  1.3 Thrombin A-chain As shown in Figure 4, the A-chain of thrombin extends along the surface of the Bchain in a shallow curved groove, arranged in a boomerang-like shape opposite the active site [90]. The covalent interaction between the A and B-chains is a disulfide bridge through C293(1)-C439(122) (prothrombin numbering is indicated, with chymotrypsinogen numbering provided in parentheses where applicable). NMR structures of human thrombin show that the A-chain interacts with the two six-stranded beta barrels of the Bchain [91]. The A-chain contacts the B-chain through a network of buried salt bridges and ionic interactions, and ten interchain H-bonds that stabilize the orientation of the Bdomain barrels (Figure 4). The A-chain is further stabilized through several intramolecular ionic and hydrophobic interactions [92]. 1.3.1 Evolution Genetic analysis of the clotting factor genes demonstrates that the clotting proteases of the chymotrypsinogen superfamily have evolved as a result of several gene duplications, exon shuffling and intron sliding events [93]. Gene organization studies reveal that factors VII, IX, and X are closely related and have evolved separately from the homologous genes for factor XII, tissue-type plasminogen activator and urokinase [94, 95]. Prothrombin has a unique exon organization and is thought to be the ancestral gene in this clotting factor family [96].  16  Figure 4. Pymol generated figure of nascent thrombin using S195A thrombin (PDB accession 3GIS) [97]. A) Thrombin structure showing active site residues in red and location of the A-chain on the opposite face of the molecule. The covalent disulfide bridge between the A and B chains is through C293(1)-C439(122), as shown in orange (A). Residue numbering is based on prothrombin, with chymotrypsin numbering provided in parentheses). The 36 amino acid residue A-chain is colored in green, and the covalently bound A13 peptide segment is colored magenta. B) Interactions between the A-chain and the B-chain of thrombin are shown with dotted lines. C) A13 peptide hydrogen bonding with the A and B chains of αthrombin. D) Hydrophobic interactions between the A13 peptide and the α-thrombin Achain.  17  1.3.1.1 Prothrombin evolution Analysis of prothrombin genes across seven vertebrate species has established that the regions of highest conservation in prothrombin are the propeptide region, Gla domain, and the thrombin B-chain. The least conserved regions are the A-chain region and the interconnecting regions between the Gla and kringle domains [96]. There is 54% amino acid identity for the B-chains of thrombin encoding the protease domain while only 40% amino acid identity for the A-chain residues [98]. When conservative substitutions were accounted for, the thrombin B-chains were found to have 75% identity across nine vertebrate species while the A-chain was found to have 42% sequence identity [99]. 1.3.1.2 Serine Protease Clan PA subfamily S1 The subfamily S1A of clan PA encompasses serine proteases bearing the chymotrypsinogen fold and includes proteases with diverse extracellular functions from digestion (e.g. chymotrypsin, trypsin and proelastase) to complement-mediated immunity (factor B, factor D, factor I, C1r/s and C2), fibrinolysis (urokinase, tissue type plasminogen activator, plasminogen, kallikreins) to coagulation (factors VII, IX, X, XI, prothrombin, PC, and PS), apoptosis (granzymes) to bone remodelling (osteocalcin) [100]. A detailed review of the serine peptidase family and clan classification system is provided in [101]. While some homologous proteases such as trypsin lack an A-chain altogether, other homologous clotting proteins that remain tethered to phospholipid membranes, such as factor Xa, IXa, APC, and VIIa retain the remainder of the zymogen through a covalent disulfide bridge with C438(122) on the B domain [101]. While it is not currently known 18  whether associated zymogen protein domains influence the protease domain in any S1 peptidase, it is considered a plausible scenario [101]. When present in other serine proteases, including urokinase and tissue plasminogen activator, a non-catalytic peptide (that is disulphide bonded to the B-chain) appears to have an allosteric effect on the enzymatic activity [102]. The non-catalytic chains of plasmin and factor XI also contain binding sites for physiological substrates [103, 104]. Six residues of the prothrombin A-chain region are homologous to the propeptide of chymotrypsinogen. However, the chymotrypsinogen propeptide is not involved in substrate or inhibitor binding [90]. A recent NMR study has also revealed that in the absence of stabilizing ligands, the protease domain regions of highest stability are flanked by the light chain of thrombin. The authors suggest that the A-chain may play a ligand-like role to stabilize and maintain the integrity of the protease domain in the absence of other ligands [91]. 1.3.1.3 Snake venom thrombin-like enzymes (SVTLEs) The venoms of many snake genera contain serine proteases that share approximately 26-33% sequence identity with thrombin [105]. One class of these venom proteases is recognized as thrombin-like due to their ability to cleave fibrinogen to release fibrinopeptide A and/or B [106]. Other thrombin-like activities are found in snakederived proteases such as cerastobin, batroxobin, ancrod, crotalase, Russell‟s Viper Venom-V and thrombocytin, which activate platelets, fibrinogen, factor V, factor XIII, and PC [106]. SVTLEs and thrombin share a similar catalytic mechanism and have a conserved structure that may have evolved from a common ancestral protease [107]. As compared to thrombin‟s three intrachain and one interchain (A-B) disulfide bridges, 19  SVTLEs contain twelve cysteine residues. Ten of these cysteine residues form disulfide bridges in the same arrangement as trypsin [108]. The remaining two cysteines form a unique and highly conserved disulfide bond in the C-terminal tail of SVTLEs. SVTLEs are either one- or two-chain proteins synthesized as zymogens with proposed activation peptides of six amino acid residues (Q-K-S-S-E-L) [109]. However, none appear to have a light chain that bears homology to thrombin [105]. Additionally, the conserved C122 (chymotrypsin numbering) which forms the A-B interchain disulfide of thrombin has been identified as a serine residue in all known SVTLEs [105]. Previous studies have suggested that the amino acid sequences of venom gland serine proteases have diversified in an accelerated manner and that the SVTLE subclass belongs to the most primordial phylogenetic lineage of serine proteases [105]. It is possible that these enzymes diverged from the chymotrypsinogen family prior to the emergence of the A-chain. 1.3.2 Naturally occurring mutations in the prothrombin A-chain region Of the 50 inherited prothrombin mutations reported in the literature, seven involve mutations in the A-chain resulting in either hypoprothrombinemia or dysprothrombinemia (reviewed in [26, 110]) (Figure 5). Documented missense mutations of human thrombin include in-frame deletions of three nucleotides in exon 9 at two positions (7484/7489 del. GAA), resulting in omission of one of two lysine residues (K301 or K302) [111, 112]. The deletion of either K301 or K302 leads to the removal of a salt bridge interaction between K301 and D292, which is thought to stabilize the centre of the A-chain. The authors suggested that hypoprothrombinemia is caused by the incomplete folding of the A-chain, which is then unable to stabilize the B-chain structure. Additionally, activation of recombinant K301 prothrombin was observed to be  20  Figure 5. Naturally occurring prothrombin mutations. Reviewed in [26]. The prothrombin gene is found on chromosome 11 p11-q12 and consists of 14 exons spanning 20kB. A schematic of the prothrombin gene is mapped in grey with exons corresponding to the prothrombin protein domains shown with black lines. The prothrombin protein domains are abbreviated; SP (signal peptide), PP (prepropeptide), Gla (gamma carboxyglutamic domain), K1 (kringle 1), K2 (kringle 2), A (thrombin A-chain region), B(thrombin B-chain region). Mutations in the prothrombin gene are shown above the exons. There are 41 naturally occurring missense/nonsense mutations noted with an asterisk (*), one small insertion (+), three small deletions ( ) and one gross deletion of the entire exon 11. Three splicing mutations are noted with a downward arrow ( ).  21  significantly slower than WT prothrombin, (two hours as compared to 20 minutes in vitro with the prothrombinase homolog TSV) [111, 112]. Two recent studies on the K301 deletion prothrombin mutant have suggested that A-chain structure affects the conformation and catalytic properties of thrombin through long range allosteric effects on the active site and insertion loops [112, 113]. Another patient was identified as a compound heterozygote for two prothrombin A-chain missense mutations also located in exon 9 (E300K and E309K) [114]. These mutations were designated prothrombin Denver I and II, respectively, and both of these glutamic acid residues are conserved throughout vertebrate species from fish to humans (Table 2). The authors hypothesized that these mutations interfere with the autocatalytic cleavage of the A-chain or activation by factor Xa; however, no confirmatory activity studies have yet been reported. Prothrombin San Antonio is an A7543G missense mutation resulting in an arginine to histidine substitution at residue 320. The R320-I321 bond is one of two prothrombinase-mediated cleavage sites that generates thrombin. Replacement of arginine with histidine at this site prevents cleavage by factor Xa, forming a dysfunctional molecule [115]. Similarly, Akhavan et al.,[116] discovered an A-chain missense mutation resulting from a G to A transition at nucleotide 7312, producing an R271H substitution. In plasma prothrombin, the R271-T272 bond is cleaved by factor Xa to form thrombin. The alternative R271 replacement by H prevents cleavage by factor Xa at this site and results in the formation of meizothrombin, which has amidolytic activity with the chromogenic substrate S-2238 but little fibrinogen clotting activity compared  22  with normal human α-thrombin [117]. This observation supports a role for the fully formed A-chain in substrate recognition. Akhavan et al.,[118] also discovered prothrombin Segovia, a G-A mutation at nucleotide 7539 of exon 9 of the prothrombin gene. The G7539A mutation resulted in a G319R substitution. The authors proposed that the substitution, which occurs near the site of factor Xa-mediated cleavage of prothrombin (R320-I321), altered the conformation of the protein making the cleavage site inaccessible to factor Xa. Taken together, mutational analysis of these naturally occurring A-chain variants suggests that the A-chain of thrombin plays a functional role or roles in vivo. 1.3.3 Thrombin A-chain biochemistry Initial modeling and structural studies of thrombin suggested that the A-chain may contribute to the determination of substrate specificity based largely on its proximity to the active site [119-121]. Early work on plasma-derived bovine thrombin, however, suggested that while the A-chain is strongly associated with the B-chain through an array of non-covalent interactions and a disulphide bond, it does not appear to play a substantial role in the catalytic activity of thrombin [122]. Disulphide reduction, removal of the A-chain, and subsequent refolding of the B-chain demonstrated that the A-chain is not required for proper B-chain folding and that intrachain interactions of the B-chain are sufficient to yield an active enzyme upon refolding [122]. The refolded B-chain cleaved fibrinogen, tosyl-L-arginine methyl ester and other small peptide substrates at similar rates as refolded two-chain thrombin. These results lead the authors to conclude that the A-chain does not significantly contribute to either the catalytic activity or substrate specificity of thrombin. A similar study by Pirkle et al. [123] confirmed that isolated 23  bovine thrombin B-chain cleaves fibrinogen and is also able to activate factor XIII as shown by the generation of cross-linked fibrin polymers during clotting assays. These studies concluded that prothrombin activation may require the A-chain; however, once mature thrombin is generated the A-chain is superfluous. These results suggest the Achain is an activation peptide only. Removal of the A-chain of human thrombin by disulphide bond reduction has also been attempted [124]. Reductive unfolding resulted in sequential generation of two partially reduced intermediates prior to conversion to the fully reduced form. Partial loss of B-chain disulphide bonds prior to reduction of the interchain disulphide link was observed, leading to denaturation of the B-chain structure and loss of activity before the A-chain was released. From this study, the authors concluded that the presence of a covalently bound A-chain may be required for structural stability of the thrombin protease domain, and through noncovalent interactions with the B-chain, the A-chain is required for normal functioning of the active site of thrombin. The authors also conducted disulphide scrambling experiments, demonstrating that thrombin is one of the few proteins that contain disulphide bonds formed between cysteines that occur consecutively in the primary structure of the protein. The thrombin disulfide arrangement more typically resembles extensively scrambled protein isomers and was found to result in weak conformational stability as the native protein is easily converted to disulphide scrambled isomers at low denaturant concentrations [124]. The authors proposed that this unique disulphide arrangement and consequent conformational flexibility may be crucial for the multiple bioregulatory functions of thrombin.  24  A number of linkage studies have investigated the effects of salt, pH and temperature on thrombin amidase activity, highlighting not only the importance of both cation and anion binding sites, but also the allosteric communication between these sites and the active site of thrombin [125-132]. In particular, substrate binding to the catalytic pocket was found to be controlled by two ionizable groups while the catalytic activity of thrombin was controlled by a more complex linkage scheme involving three ionizable groups [132]. The authors postulated that the two ionizable groups that affect both kcat and km were the active site histidine and the B-chain amino terminus, as these residues play similar roles in other serine proteases. The third ionizable group, which affects catalytic activity but not substrate binding, was suggested to be part of the anion binding exosite of thrombin [130, 132]. While these early studies did not specifically identify the A-chain as an allosteric effector of thrombin activity, they did emphasize the biological importance of allostery in thrombin function and served as the foundation for future mutagenesis studies addressing potential long-range linkages between thrombin's active site and the A-chain. 1.3.4 Mutagenesis studies Prothrombin and prethrombin-2 mutagenesis and recombinant expression studies of A-chain residues have revealed that at least some A-chain residues impart functional effects on thrombin activity (Figure 6). In an extensive mutagenesis study, Tsiang et al., [133] functionally mapped surface exposed residues of thrombin that were capable of participating in H-bonds and electrostatic interactions (Figure 6). Charged and polar residues were chosen for mutation as these were considered most likely to participate in the binding of charged ligands. Prothrombin A-chain residues S288, E290, D292, K301, K302,  25  S303, K307, R310, E311, E314, and D318 were individually mutated to alanine, expressed in COS7 cells, and the unpurified mutant proteins were functionally analyzed for amidolytic activity with S-2238, fibrinogen clotting, PC activation, and inhibition by thrombin aptamer. A triple mutant (S288A/E290A/D292A) had significantly enhanced S-2238 amidolytic and fibrinogen clotting activity while a double mutant (E314A/ D318A) had reduced fibrinogen clotting activity, but slightly enhanced PC activation. Conversely, the K307A mutant possessed significantly reduced amidolytic and clotting activity, as well as reduced PC activation. These results demonstrate that charged A-chain residues contribute in thrombin activity toward both pro and anticoagulant substrates. More recently, a site-directed mutagenesis study was performed on charged Achain residues known to be involved in inter- and intra-molecular interactions [134] (Figure 6). Prethrombin-1 A-chain mutants were expressed in BHK cells and purified prior to functional analysis. Of the analyzed mutants, E300, D306, E309, and R296 were each found to be functionally compromised in terms of hydrolysis of the chromogenic substrate H-D-Phe-Pro-Arg-p-nitroanilide (FPR), release of fibrinopeptides A and B, activation of PC, and PAR1 cleavage. In particular, the R296A mutant was most severely compromised with a 65-fold reduction in FPR hydrolysis. The concomitant reduction in cleavage of fibrinogen, PAR1 and PC confirmed the molecular origin of active site perturbation more than 20 angstroms from the active site. The R296 side chain is stabilized by strong ionic contacts in the ion quartet R296-E300-D306-E309 and also interacts with W334(29) and W539(207) in the B-chain via van der Waals forces. Alanine substitution of R296 abrogates the ionic interaction, weakening the A-chain ion quartet and disrupting hydrophobic interactions with W334(29) and W539(207). Perturbation of the A-chain stability  26  through R296A substitution is thought to propagate long-range to the Na+ binding site via W334(29) and W539(207), abrogating Na+ binding and resulting in reduced catalytic activity [134]. Papaconstantinou et al. [134] also used alanine scanning mutagenesis to investigate the functional effects of the naturally occurring mutations prothrombins Denver I and II (E300K and E309K), Segovia (G319R) and San Antonio (R320H). Alanine substitution at all four of these positions produced poorly activatable prothrombins when incubated over two days with the prothrombinase complex [134]. R320H and G319R affect the factor Xa P1 and P2 recognition sites respectively, and these mutations have previously been attributed to perturbation of the zymogen activation process. The bleeding diatheses associated with E300K and E309K may result from their interference with the autocatalytic cleavage of the A-chain or factor Xa-mediated activation [114]. However, considering that these two mutations occur in the central region of the A-chain, it seems plausible that these mutations disrupt the structural stabilization of the A- and B-chains after thrombin activation. Alanine substitution of E300 and E309 was found to result in a reduction in fibrinogen cleavage and PAR1 activation. It is expected that the charge reversal of the naturally occurring E300K and E309K mutations is substantially more disruptive than the alanine substitution conducted. The naturally occurring prothrombin K301 deletion has been the most extensively studied of all A-chain mutations. K301 deletion mutation resulted in a prothrombin that was activated to thrombin with the prothrombinase homolog Taipan snake venom (TSV). The K301 deletion thrombin had reduced substrate turnover for Phe-Pip-Arg-pnitroaniline, reduced antithrombin and PC interaction, less robust platelet activation, and 27  reduced sodium ion sensitivity. By contrast, thrombin-thrombomodulin and thrombinplatelet glycoprotein Ibα interactions were unaffected [112]. Further structural characterization revealed decreased stability of the K301 deletion mutant and a weakening in the A-B interchain interactions, resulting in faster dissociation of the A-chain upon disulfide scrambling [113]. Molecular dynamics simulation of the effects of the K301 deletion revealed geometric distortion of the catalytic triad and alteration to the aryl-binding site within the active site, resulting in substrate restriction to the active site cleft. In particular, there were changes to the S2 subsite (W370(60d) loop) and transition of W547(215) (S3 subsite). These changes collectively resulted in reduced catalytic activity of the K301 deletion thrombin, confirming that alterations in A-chain residues have the potential to transmit long-range allosteric effects to the active site [113]. 1.3.5 Signalling and disease Thrombin plays a role in many processes linked to cancer including thrombosis, inflammation, and tissue repair and remodeling (reviewed in [135]). Recent work by Ebert et al., [136] has identified the thrombin A-chain as a potential diagnostic tool for gastric cancer. Patients with dyspeptic symptoms could be distinguished from those with gastric cancer by comparing levels of circulating liberated A-chain with both specificity and sensitivity of this marker found to be 80%. The authors speculated that the decreased circulating A-chain levels observed in serum samples of cancer patients correspond to higher concentrations of intact active thrombin in the tumor microenvironment, possibly due to reduced local proteolytic degradation of thrombin. While this does not imply a specific role for the thrombin A-chain in the tumor environment, it does suggest that 28  Figure 6. The thrombin A-chain. Prothrombin numbering provided. The A13 peptide is removed by autolysis in human thrombin (A), The mature A-chain of thrombin (B). Residues found to functionally compromise thrombin activity upon alanine substitution are shown in red text [134] or blue text [133]. Residues with no functional effects on thrombin activity upon alanine substitution are indicated with a pink outline [133]. C293 forming the interaction disulfide is indicated with a black outline, and A-chain interactions are shown with dashed lines between residues [92].  29  additional proteolytic events occur to liberate the A-chain from the localized thrombin molecule. Given that activation peptides released from prothrombin and other coagulation proteins have signalling effects [137-139], it is possible that released A-chain peptides may have a similar function. A recent study investigated the ability of various thrombin fragments to act as host defence peptides [140]. While no antimicrobial or anti-inflammatory capabilities were detected for A-chain peptides examined under physiological conditions in this study, future research involving (pro)thrombin fragmentation by a wider array of host, bacterial and viral proteases may identify A-chain peptides that function within the immune system, further expanding the biological roles of thrombin.  1.4 Thrombin cation binding The effect of cations on thrombin activity was first described by Orthner and Kosow [127]. They showed that Na+, K+, Rb+, Ca2+, Mn2+ but not NH4+ or Li+ increased the rate of cleavage of various chromogenic substrates. Subsequent studies have demonstrated that small cations as well as anions may play an important role in controlling thrombin catalytic activity, and thrombin is capable of discriminating between different cations [128, 129]. 1.4.1 Monovalent cation binding The most effective modulator of human thrombin activity in solution is Na+, which triggers the enzyme transition from an anticoagulant (slow) form to a procoagulant (fast) form [141]. Concentrations of Na+ in the 100mM range were found to increase thrombin activity by up to 349% for the chromogenic substrate S-2238 [127]. The  30  binding affinity for Na+ was determined both kinetically and spectroscopically to be represented by a Kd of ~20mM at 30 C [127] although other values have been reported in the literature [88]. The Na+ bound fast form is considered procoagulant as it cleaves fibrinogen and the PARs more specifically, whereas the slow form is considered anticoagulant because it displays normal PC activation (through interaction with TM), but is slower at cleaving procoagulant substrates. Physiologically, the importance of Na+ binding is highlighted by the existence of naturally occurring prothrombin mutations resulting in compromised Na+ binding and bleeding phenotypes [142]. K+ has been shown to have a much lower affinity for thrombin than Na+ [126, 128]. In contrast to Na+, high concentrations of Li+ have been shown not to result in major conformational changes in the thrombin active site [128]. 1.4.2 Divalent cation binding To date, very few studies have investigated the biochemical effects of bivalent cation binding on thrombin activity [127-129]. One study indicated that both calcium and manganese affect thrombin amidolytic activity, and at under saturating substrate concentrations up to 100mM, Ca2+ titration increases thrombin activity by 41% [127]. Landis et al. found that increasing concentrations of CaCl2 markedly decreased the thermal stability of thrombin suggesting that Ca2+ selectively interacts with the enzyme. By contrast 2M Mg2+ only slightly enhanced the thermal stability of thrombin at high concentrations [128]. In many thrombin crystal structures, the A-chain N- and C-terminal residues are characterized by very low electron density, suggesting lability on the surface of the molecule [90]. A recent, unpublished crystal structure of recombinant human thrombin 31  (S195A) in complex with protease nexin 1 (PN1) indicated the presence of a calcium ion held coordinated between the thrombin light chain residue D318(14l) and the backbone of G453(133) on the heavy chain (Figure 7). Coordination of Ca2+ increased the rigidity of the thrombin A-chain C-terminus and generated interest as to whether it was of functional importance to thrombin activity and stability. Another recent crystal structure of thrombin complexed with sulfo-hirudin found that K301 participates in a divalent metal binding site between the A- and B-chains of thrombin (Figure 8) [143]. The authors hypothesized that this cation binding site contributes to the stabilization of the A-chain conformation and may modulate thrombin activity, offering an alternative biochemical explanation for the hemorrhagic diathesis seen in patients with the K301 deletion prothrombin mutation [143].  1.5 Project outline and rationale Thrombin is considered a model enzyme for studies of structure and function of homologous coagulation proteases in the chymotrysinogen family. Although thrombin is one of the most widely studied enzymes in biology, the role of the thrombin A-chain has been neglected in comparison to the B-chain or the other domains of prothrombin [90]. The function of the coagulation serine proteases A-chains within the chymotrypsinogen family is still unclear [101]; however as mentioned earlier, homologous snake venom thrombin-like enzymes lack an A-chain altogether [105]. While originally considered to be simply an activation remnant with little physiologic function, this peptide in thrombin is postulated to play a role as an allosteric  32  Figure 7. Thrombin-PN1 crystal structure. Pymol image showing calcium ion coordination between D318(14l) in the thrombin A-chain and G453(133) of the B-chain. (Huntington laboratory, unpublished 2009).  33  Figure 8. Thrombin bound to Ni2+. Pdb reference 2PW8. Ni2+ is coordinated between D292 and K301 of the thrombin A-chain and H407 of the B-chain [143]. Figure produced using Pymol [144].  34  effector in enzymatic reactions [112, 113, 133, 134]. The A-chain may provide a structural scaffold for protease domain function as the naturally occurring thrombin Achain mutations would suggest [91, 112-115, 145], or may act allosterically to alter thrombin function. To explore these alternatives, this project aimed to examine the thrombin A-chain using site directed mutagenesis to probe the role of A-chain residues in the overall structure and function of thrombin. This was achieved using recombinant prothrombin and prethrombin-2 (preII) mutated at A-chain residues including the entire A-chain region in prothrombin and the A13 peptide cleavage site (R284). I also conducted a protein crystallography study to explore whether D318 is a physiological Ca2+ binding residue. These studies investigated previously unexplored parts of the thrombin A-chain and help to elucidate the role of the thrombin A-chain, including the entire mature Achain, the impact of A13 peptide autolysis, and the D318 putative Ca2+ binding site respectively.  1.6 Hypotheses The hypotheses in this dissertation were threefold: 1.  Based on the conservation of at least a partial thrombin A-chain throughout  vertebrate species, the A-chain is essential for proper thrombin folding and/or stability. 2.  Thrombin is a Ca2+ binding enzyme, and the Ca2+ ion binding site is located  between D318(14l) and G453(133).  35  3.  Nascent thrombin has an additional 13-residue peptide at the N-terminus of the A-  chain which makes nascent thrombin enzymatically and structurally distinct from thrombin.  36  2. MATERIALS AND METHODS 2.1 Materials Heparin octa-saccharide was purchased from Iduron (www.iduron.com, Manchester, UK). D-Phe-Pro-Arg-chloromethylketone (PPACK), puromycin and heparin sodium salt were obtained from Sigma Co. (Oakville, Ontario). Restriction enzymes, Taq polymerase and Antarctic Phosphatase used for cloning were from New England Biolabs (Pickering, Ontario). FuGENE 6 Transfection Reagent was purchased from Roche Applied Science (Laval, Quebec). DMEM-F12 and Newborn Calf Serum (NCS) were purchased from Invitrogen (Burlington, Ontario). Methotrexate was from Mayne Pharma Canada Inc (Kirkland, Quebec) and vitamin K was from Sandoz Canada Inc (Boucherville, Quebec).  2.2 Proteins Human plasma-derived proteins including protein C (PC), thrombin (IIa) and antithrombin (AT) were purchased from Haematologic Technologies Inc. (Essex Junction, Vermont). Echis carinatus venom (ECV), Taipan snake venom (TSV), hirudin54-65 and human fibrinogen were obtained from Sigma Co. (Oakville, Ontario). Prothrombin deficient human plasma was from George King Bio-medical, Inc (Overland Park, Kansas) and S-2238 was purchased from Diapharma Group Inc (West Chester, Ohio). Recombinant human soluble thrombomodulin (sTM) was a generous gift from Dr. Edward Conway, University of British Columbia. Sheep anti-human thrombin-HRP was purchased from US Biological (Swampscott, Massachusetts).  37  2.3 Bioinformatic analysis of A-chain rigidity To ascertain the A-chain flexibility and interchain communication for human thrombin with a variety of interaction partners, 26 different human thrombin crystal structures of S195A (1DM4, 1JMO, 1JOU, 1OA5, 1TB6, 1UUA, 2B5T, 2PV9, 3B9F, 3GIS, 3K65, 3LU9) and -thrombin (1OOK, 1PPB, 1P8V, 1W7G, 1WAY, 1WBG, 1VZQ, 2AFQ, 2BVR, 2BXT, 2C8W, 2C9O, 2HWL, 2V3H) were taken from the Protein Data Bank. The B-chains were aligned to S195A thrombin (2BVR) using BLOSUM62weighted dynamic programming sequence alignment with refinement in Pymol and the A-chain intra and interchain interactions were determined using Pymol [144].  2.4 Recombinant protein expression and characterization This study utilized two established expression systems for recombinant protein production. 2.4.1 PT and PT A constructs This study utilized the established Baby Hamster Kidney (BHK) cell expression system for prothrombin, which produces stably transfected cells using the pNUT plasmid [146]. Expression of cloned cDNAs is controlled by the mouse metallothionein I promoter and human growth hormone transcription termination signals. pNUT contains a mutated dihydrofolate reductase cDNA under transcriptional control of the SV40 promoter and hepatitis B virus termination signals, allowing selection of high copy numbers of pNUT in wild type cell lines by high levels of methotrexate [147]. The 5‟ and 3‟ ends of the human cDNA encoded by the plasmid pcHX11501 were modified using the polymerase chain reaction (PCR) to eliminate untranslated regions  38  and add Not1 restriction sites for cloning into the pNUT plasmid. At the 3‟ end additional modifications were performed to add an ECV recognition sequence followed by a His6 tag. The newly modified FII cDNA was then digested with Not1 at both the 5‟ and 3‟ ends and ligated into Not1 cleaved pNUT vector that had been treated with Antarctic Phosphatase (New England Biolabs, Pickering, Ontario) to prevent religation. Because pNUT does not allow for colour selection, 24 colonies from each ligation were analyzed by PCR. An oligonucleotide within the 5‟ pNUT vector was used in conjunction with an internal reverse hFII oligonucleotide to confirm both the presence of the FII cDNA and the orientation of the gene. The correct orientation of the gene was confirmed by DNA sequence analysis. To delete the region encoding the prothrombin A-chain (PT A), internal primers were designed flanking the A chain with an overlapping Xba1 site. The 5‟ signal peptide through to kringle two encoded region of the cDNA were subjected to PCR using the oligonucleotides hIIPTSPF and hIIPTSPR/B. The protease domain of prothrombin was amplified using the oligonucleotides hIIPTBF and hIIPTNot1EcR. (Table 1). The two fragments were digested with Xba1 and ligated together before PCR reamplification using hIIPTSPF and hIIPTNot1EcR. The new full construct fragment was then digested with Not1 and ligated into the pNUT vector. The Xba1 restriction site was then deleted by Quickchange reaction using the oligonucleotides hIIPTBQCXbaF and hIIPTBQCXbaR, and the full construct was sequenced to ensure the absence of errors, and confirm the orientation of the gene fragment in the vector for expression in tissue culture. Quikchange Mutation of the B-chain cysteine residue to alanine was performed with hIIPTBC439AF and R primers to create PT AC439A.  39  Primer name  Oligonucleotide sequence 5'-3'  hIIPTSPF  ATCGCGGCCG CATGGCGCAC GTCCGAGGCT TGCAG  hIIPTK2R(Xba)  GAT TCT AGA ACG CCC TTC GAT GGC CCT GTC TGA G  hIIPTBF(Xba)  ACT TCT AGA ATT GTG GAG GGC TCG GAT GCA G ACT GCG GCC GCT CAT CAG TGG TGG TGG TGG TGG TGC CGG  hIIPTNot1EcR  CCA TCG ATC TCT CCA AAC TGA TCA ATG ACC  hIIPTBQCXbaF  GAC AGG GCC ATC GAA GGG AGA ACC TTT GG  hIIPTBQCXbaR  CCA AAG GTT CTC CCT TCG ATG GCC CTG TC  hIIPTBC439AF  GT GAC TAC ATT CAC CCT GTG GCT CTG CCC GAT AGG G  hIIPTBC439AR  CCC TAT CGG GCA GAG CCA CAG GGT GAA TGT AGT CAC  R284QF  CAG ACT TTC TTC AAT CCG CAG ACC TTT GGC TC  R284QR  GAG CCA AAG GTC TGC GGA TTG AAG AAA GTC TG  Table 1. Oligonucleotides used in the prethrombin-2 studies.  40  2.4.1.1 Stable expression of recombinant prothrombin pNUT-hPT plasmids were transfected into BHK cells using Fugene according to the manufacturer‟s protocol. Briefly, plasmid and the transfection reagent were combined in serum free DMEM-F12 medium and incubated for ~20 min. at room temperature before being used to transfect BHK cells (at ~85 % confluency) in 6-well plates. Lipoplexes were added directly to the cell medium that was bathing the cells in a dropwise fashion. After 12 hours incubation at 37 C and 5 % CO2, the medium was replaced with DMEM-F12 supplemented with 5 % NCS, 1 % L-glutamine, 1 % penicillin/streptomycin, supplemented with 6 ug/mL vitamin K and 0.22 mM methotrexate (MTX) to begin selection (selection media). MTX resistant colonies containing the pNUT plasmid were evident 10 days following selection. After approximately 10-14 days after selection, MTX-resistant colonies were isolated by trypsin treatment at the tip of a pipette and selected for expansion into T150 flasks (Corning) which were allowed to reach ~90 % confluence prior to removal of small samples of conditioned medium to be assayed for prothrombin production by Western blot analysis. For the estimation of prothrombin antigen secretion from BHK clones, 2-3 samples from each clone were analysed by prothrombin Western blots to determine how much prothrombin antigen was expressed by each clone and the number of days that conditioned media yielded significant prothrombin levels. Samples were diluted in 3x SDS PAGE buffer (NEB, Pickering Ontario) and heated at 95 C for 5 min. They were then subjected to SDS-PAGE on 10 % acrylamide gels and transferred to polyvinylidine difluoride (PVDF) membranes prior to blocking in 5 % skim milk in 50 mM sodium  41  phosphate buffer (pH 7.4), 150 mM NaCl, 0.05 % Tween-20 (PBST) at room temperature for 60 min or overnight. Blocked PVDF membranes were incubated with 1:5000 horseradish preoxidase (HRP) conjugated sheep anti-human thrombin heavy chain (α-IIaHRP) polyclonal antibody (US Biological) in 5 % skim milk (in PBST) for 60 min. at room temperature. Membranes were then washed in PBST prior to detection with Amersham ECL-Plus chemiluminescent substrate (GE Healthcare, Piscataway, New Jersey) using the ChemiGenius imaging equipment (PerkinElmer). To increase expression levels of functional recombinant prothrombin, the vitamin K epoxide reductase (VKOR) gene-containing plasmid VKOR-pIRES was stably transfected into recombinant prothrombin-expressing BHK cells as described above and previously [148]. Selection media for these doubly transfected cells was supplemented with both methotrexate and 1.75 µg/mL puromycin, the selection reagent for the VKORpIRES plasmid. At least 2-3 vials of cells from each clone were frozen in selection media containing 5 % DMSO and stored in liquid nitrogen for large-scale growth after clone selection (see below). For the highest expressing and secreting clones, ~4x106 cells were seeded into triple flasks or roller bottles cells and after reaching ~80-90 % confluence, selection media was replaced with expression media (DMEM-F12 supplemented with 1x insulintransferrin-selenium (ITS), 1 % L-glutamine, 1 % penicillin/streptomycin, and 10 ug/mL vitamin K1). Conditioned media was collected every 1-2 days and stored at -80 C in the presence of 10 mM benzamidine. Small samples of uninhibited conditioned media were also stored at -80 C for use in Western blots.  42  Constructs which did not demonstrate any secretion (PT AC493A) were further analysed to detect possible intracellular expression. Briefly, the cells (~106) were treated with trypsin, resuspended in media and recovered by centrifugation. The cells were resuspended in 1 mL dH20 and boiled for 5 minutes before centrifuging again and analyzing the supernatant fluid fraction by SDS PAGE immunoblot. 2.4.1.2 Purification of recombinant prothrombin Conditioned media (5-10 L) was thawed at 37 C then immediately stored at 4 C or on ice for the duration of the purification process (except when flowed over columns at room temperature). The conditioned medium was centrifuged at 15,000 g for 30 min. to remove cell debris. Medium was concentrated 10x using a Sartorius Vivaflow 10kD MWCO filter and then dialyzed overnight against 2x 5L of loading buffer (10mM TrisHCl pH 7.4, 500mM NaCl, 10mM imidazole). The dialyzed protein was then loaded on a GE HisTrap column equilibrated in the same buffer. The column was washed with 5 column volumes of equilibration buffer containing 40mM imidazole before the protein was eluted over 10 column volumes with a gradient of imidazole (40mM - 250mM). Fractions were assayed for prothrombin activity by chromogenic assay. Small samples of each fraction were incubated for 20 min. at room temperature with TSV (125 nM) and CaCl2 (2 mM) in a 96-well microplate to generate thrombin. Prothrombin activation was quenched with 20 mM EDTA in Hepes-Buffered Saline (HBS). The chromogenic substrate S-2238 (150 L, 200 M) was added, and thrombin activity was monitored kinetically at 405 nm using a Spectramax190 microplate reader (Molecular Devices). Prothrombin-containing fractions were pooled and concentrated at 13,000 g in Microcon centrifugal filter devices (Amicon) with a 10 kDa molecular weight cut off. Buffer  43  exchange to HBS was also carried out in these microtubes. Purified recombinant prothrombin proteins were stored in 50 % glycerol at -20 C. 2.4.2 Recombinant human prethrombin-2 (rhpreII) An E.coli expression system was used for recombinant human prethrombin-2 [149]. The wild type human prethrombin-2 cDNA subcloned into the pET23(+) vector (Novagen) was provided by Dr. James Huntington (Cambridge, UK). The prethrombin-2 construct has DNA coding for the following amino acid sequence: MAIEGRTATSEYQTFFNPRTFGSGEADCGLRPLFEKKSLEDKTERELLESYIDGRI VEGSDAEIGMSPWQVMLFRKSPQELLCGASLISDRWVLTAAHCLLYPPWDKNFT ENDLLVRIGKHSRTRYERNIEKISMLEKIYIHPRYNWRENLDRDIALMKLKKPVA FSDYIHPVCLPDRETAASLLQAGYKGRVTGWGNLKETWTANVGKGQPSVLQVV NLPIVERPVCKDSTRIRITDNMFCAGYKPDEGKRGDACEGDSGGPFVMKSPFNNR WYQMGIVSWGEGCDRDGKYGFYTHVFRLKKWIQKVIDQFGE. MAIEGR is an expression insert and provides an initiator methionine followed by a factor Xa cleavage site. This insert should be removed upon thrombin activation. The inactive mutant S195A-prethrombin-2 plasmid was also provided by Dr. Huntington and has the active site serine mutated to an alanine residue, as underlined on the sequence. 2.4.2.1 Site directed mutagenesis of preII R284Q Substitution of R284 to glutamine was performed as in the previous section using the R284Q Quikchange primers listed in Table 1. PCR mutation was carried out according to the manufacturers protocol (Stratagene). The mutation was confirmed by  44  DNA sequence analysis on both DNA strands before transformation into the E. coli expression strain BL21*(DE3)pLysS (Invitrogen). 2.4.2.2 Expression and refolding of prethrombin-2 Recombinant human prethrombin-2 was expressed and refolded with some modifications to the previously described method [149]. Once the plasmid was transformed into E. coli strain BL21(DE3)pLysS, the cells were cultured at 37 C in 2 x TY broth containing 50µg/mL ampicillin and 34µg/mL chloramphenicol to an OD600 of 0.8, followed by induction with 1mM IPTG for 4.5 hours. Harvested cells were resuspended in 20 mLs of 20mM Tris-HCl, 1% (v/v) Triton X-100, 20mM EDTA, and 20mM DTT, pH 7.4 and stored at -80 C. Thawed cells were sonicated on ice for 5 minutes in 10 second bursts followed by a 10 second recovery. After centrifugation at 18,000x g for 10 minutes, the pellets (containing inclusion bodies) were washed with 20mLs of 20mM Tris-HCl, 100mM NaCl, 20mM EDTA, and 20mM DTT, pH 7.4, centrifuged again and then washed with 20mM Tris-HCl, 20mM EDTA, pH 7.4. The pellet was then resuspended in 0.1% TFA and 7.0M guanidinium chloride before resuspension into 50mLs of 6M guanidinium chloride, 20mM Tris-HCl, 2mM L-cysteine and 0.5mM EDTA, pH 8.0 before incubation at room temperature for 3 hours. Refolding was initiated by dropwise dilution with 2L 50mM Tris-HCl, 0.6M arginine, 20mM CaCl2, 10% (v/v) glycerol, 0.2% (w/v) Brij-58, pH 8.5 at room temperature for 24 hours. The 2L of refolded protein was concentrated (Sartorius Vivaflow 10Kd MWCO filter) to 100mLs and then dialysed against 5L of 25mM Tris-HCl 2mM EDTA, 0.1% (w/v) PEG 6000, pH 7.4 at room temperature overnight. The precipitate was removed by centrifugation and filtration before purification of folded prethrombin-2 on a 5mL heparin-Sepharose  45  column (GE Healthcare, Piscataway, NJ) eluting with a linear gradient of NaCl (150mM to 2M). The prethrombin-2 was activated to thrombin overnight at room temperature with 1:50 molar ratio of ECV (Sigma, Oakville, Ontario), before dilution 5-fold in 50mM TrisHCl, pH 7.4 and repurification by heparin-Sepharose chromatography. Correct refolding of the protein was confirmed by fibrinogen clotting and S-2238 amidolytic activities compared to WT -thrombin and human plasma derived thrombin [150]. 2.4.3 Enzyme quantification Prothrombin protein concentrations were determined by BCA assay (Thermo Scientific Pierce, Rockford, Illinois) using BSA standards. Protein concentrations were confirmed by ultraviolet (UV) absorbance at 280 nm using an extinction coefficient for prothrombin of 1.38 mL mg-1 cm-1 [151]. Recombinant thrombins were quantified in three ways: 1) BCA assay using the molecular weights 35.3kD and 33.8kD for nascent and α-thrombin, as determined by matrix-assisted laser desorption/ionization time of flight mass spectroscopy (MALDI TOF MS) analysis, 2) measuring the absorbance at A280 nm using an extinction coefficient for thrombin of 1.83 mL mg-1 cm-1 [152] and 3) the concentration was confirmed by the formation of a 1:1 stoichiometric complex with antithrombin [153]. 2.4.4 Confirmation of rh-thrombin identities N-terminal sequence analysis was performed (Advanced Protein Technology Centre, The Hospital for Sick Children, Toronto) to determine the A-chain sequences of both rhα- and R284Q-thrombin.  46  2.4.5 MALDI-TOF molecular weight determination Matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF MS) analysis of mass was used to determine the molecular weights of rhαand R284Q thrombin (Dr Christopher Overall laboratory, UBC, Vancouver). 2.4.6 A13 autolysis time course To determine the time required for liberation of the A13 peptide after thrombin activation, S195A-thrombin (1.38μM) was incubated with plasma-derived thrombin (pl.IIa) (0.34μM) at 37oC in 20mM Tris-HCl, 150mM NaCl, 0.1% PEG 8000, pH 7.4. The reaction (250 μL) was started by the addition of pl.-IIa and allowed to proceed for three hours. Samples (23 μL) were removed at 30 minute intervals and added to non-reducing SDS PAGE buffer prior to boiling for 5 min. Samples were electrophoresed on a 12% polyacrylamide-SDS gel followed by staining with Coomassie blue. Scanning densitometry was performed using a Li-Cor Odyssey infrared imaging system (Dr. Edward Conway laboratory, UBC, Vancouver) and data were analyzed using Origin version 8.1 software (Northampton, Massachusetts). The half-life of A13 peptide liberation was determined by analysis of the exponential decay of nascent S195Athrombin and the concomitant generation of α-thrombin.  2.5 Assessment of procoagulant function Prothrombin and prethrombin-2 zymogens may be activated to thrombin by either FXa or snake venom FXa homologs, such as Taipan Snake Venom (TSV) or Echis carinatis venom (ECV). After activation, procoagulant activity was assessed either by fibrinogen clotting, prothrombin deficient plasma clotting or hydrolysis of S-2238 [150].  47  2.5.1 Activation of prothrombin zymogens by TSV Plasma-derived and purified recombinant prothrombin (10 µM) were activated at room temperature with the prothrombinase homolog TSV (125 nM) and CaCl2 (2 mM) for up to 60 min. At the indicated times, samples were removed from the main reaction and quenched with Laemmli sample buffer. Samples were then heated at 95 C for 5 min. and subjected to SDS-PAGE (10 % acrylamide) and Coomassie staining. 2.5.2 Activation of prethrombin-2 zymogens by ECV Recombinant prethrombin-2 and the R284Q mutant were activated at room temperature overnight with a 1:50 molar ratio of ECV before 5-fold dilution in 50mM Tris-HCl, pH 7.4 and repurification by heparin-Sepharose chromatography. 2.5.3 Thrombin chromogenic activity The ability of plasma-derived and recombinant thrombins (rh and R284Q) to cleave small substrates was determined by chromogenic assay. The proteases were diluted in 20mM sodium phosphate buffer containing 100mM NaCl, 0.1% PEG 8000, 0.1% BSA, pH 7.4 in a 96-well microplate. Experiments were also performed in the absence of sodium where potassium phosphate buffer was used and the ionic strength was kept constant by substituting 100mM Choline Chloride (ChCl) in the buffer instead of NaCl [112, 154] or KCl [128]. S-2238 was added to 1-100µM final concentration and thrombin activity was monitored kinetically using a Spectramax190 microplate reader (Molecular Devices) at 23 C. The initial velocity of substrate hydrolysis yielding pnitroaniline (pNA) was determined by the change in absorbance at 405nm over 150 seconds. The absorbance change was converted to micromoles of product formed using ε405= 9.92x103 M-1 cm-1 for pNA [152]. Data were fit to the Michaelis-Menton equation 48  and values for Km and kcat were determined from analysis of progress curves using the program Origin, version 8.1 (Northampton, Massachusetts). 2.5.4 Thrombin clotting activity To confirm that rh-thrombin displays normal procoagulant function, plasmaderived and recombinant thrombin (rhα- and R284Q) were assayed for thrombin activity by using single stage clotting assays. Samples were diluted in 20mM Tris-HCl buffer, pH 7.4 containing 0.1% BSA, 0.1% PEG 8000, 100mM NaCl (20 L) and incubated for 1 minute at 37 C. Fibrinogen (2mg/mL) or prothrombin deficient plasma was added (100 L) to initiate clotting, and clot formation was monitored electro-mechanically at 37 C using an ST4 coagulation analyzer (Diagnostica Stago). Thrombin activity was obtained by comparing the clotting times to a standard curve generated using plasmaderived commercial thrombin, and data were expressed as a percentage of plasma derived thrombin activity [133]. Assays were conducted in triplicate for each experiment and results were averaged over three experiments.  2.6 Assessment of anticoagulant function Through allosteric modulation, thrombin acts on both the pro and anticoagulant pathways of coagulation. In the presence of thrombomodulin, thrombin‟s activity is directed toward activation of PC. PC in the presence of the non-enzymatic cofactor protein S is anticoagulant, inactivating factor Va and factor VIIIa. 2.6.1 PC activation Human Protein C (hPC) (200nM) was incubated with 200nM soluble thrombomodulin (sTM) in 700 L of 10mM HEPES, 20mM Tris-HCl, 150mM NaCl,  49  1mM CaCl2, 10mg/mL BSA, pH 8.3 at 37 C [155]. The reaction was initiated by the addition of 5nM thrombin. Activated protein C (APC) generation was determined by quenching thrombin activity with a molar excess of hirudin (6 units) and following the rate of S-2238 hydrolysis at 405nm as a function of time. Protein C activation in the absence of sTM was determined by incubating 50nM thrombin with 200nM hPC under the same conditions.  2.7 Assessment of inhibition Thrombin is irreversibly inhibited by serpins such as antithrombin to prevent coagulation beyond damaged vasculature. Negatively charged sugar chains cover the surfaces of intact endothelial cells to bridge thrombin and circulating serpins, enhancing the inhibition reaction. 2.7.1 Heparin inhibition of thrombin amidolytic activity Thrombin (5nM) was added to 20mM phosphate buffer, 100mM NaCl, 0.1% PEG 8000, 0.1% BSA, pH 7.4 and various concentrations of heparin (0-40nM) in a total volume of 200μl in a 96-well plate. The reaction was initiated by the addition of 100µM S-2238, and thrombin activity was monitored at 23°C. The rate of substrate hydrolysis to yield pNA was followed by the change in absorbance at 405nm over 150 seconds to determine the initial velocity. The dissociation constant for heparin was determined as previously described with some modifications [156]. Briefly, heparin (0-3nM) was incubated with 1nM thrombin in the buffer described above. S-2238 (1-60uM final concentration) was added, and thrombin activity was monitored at 23 C. The rate of substrate hydrolysis to yield pNA was followed by the change in absorbance at 405nm  50  over 60 seconds to determine the initial velocity. The Kd was determined as described previously [156]. 2.7.2 Kinetics of thrombin inhibition by antithrombin (AT) Assay solution (20μL) containing 0.5μM AT, 20mM phosphate buffer, 100mM NaCl, 0.1% PEG 8000, 0.1% BSA, pH 7.4 and various concentrations of heparin (0-2nM) was incubated in a 96-well plate. The reaction was initiated by the addition of thrombin (50nM) and quenched by dilution at various time points by the addition of 200μM S-2238 (180μL). Residual thrombin activity was monitored at 23 C. The rate of substrate hydrolysis yielding pNA was followed by the change in absorbance at 405nm over 60 seconds to determine the initial velocity for the reaction. Pseudo first-order rate constants (kobs) were obtained from the slopes of the plots of the natural logarithm of residual protease activity versus time of incubation. Second-order rate constants for the uncatalyzed reactions were obtained by dividing kobs by the inhibitor concentration. Second-order rate constants, kcat/Km, for the heparin-catalyzed reactions were taken as the slope of kobs versus the total heparin concentration [36]. 2.7.3 Stoichiometries of inhibition The stoichiometry of inhibition (SI) by AT was measured in the presence and absence of heparin as previously described by Mushunje et al. [36]. Briefly, 0.5μM rh thrombin or R284Q-thrombin were incubated for 2 hours with plasma derived AT at concentrations ranging from 0 to 0.6μM in 20mM sodium phosphate, pH 7.4, 150mM NaCl, 1mM EDTA, 1mg/mL BSA, 0.1% PEG 8000. All reactions were carried out in 100μL volumes at room temperature (23 C). The residual protease activity was determined by diluting 10μL into 100μL of 200mM S-2238 and monitoring the 51  absorbance (405nm) over 60 seconds with a Spectramax190 microplate reader at 23 C. The residual protease activity was plotted against the molar ratio of AT to thrombin, and the stoichiometry was taken as the x-intercept from the linear regression [36].  2.8 Structural determination Circular dichroism, x-ray crystallography and NMR techniques were utilized to probe the putative calcium binding proterties of thrombin and to assess the contribution of the thrombin A-chain A13 peptide to thrombin structure and stability. 2.8.1 Circular dichroism (CD) 2.8.1.1 Far and near -UV CD spectroscopy Solutions of both prothrombin and thrombin were dialysed into 20mM Tris-HCl pH 7.4, 150mM NaCl and concentrated to 0.113-0.194 mg/mL. Far and near-UV CD spectra were recorded with a Jasco J-810 spectropolarimeter (1 nm slit width and a four second averaging time) (University of British Columbia Laboratory for Molecular Biophysics, www.lmb.ubc.ca). The cell path length was 0.2cm, and the spectrum was scanned from190-300nm wavelength. An average of 10 scans was acquired for each sample. The molar ellipticity was calculated as: [ ] = where  MRW/lc is the measured ellipticity (in millidegrees) at a wavelength , MRW is the  mean residue molecular weight calculated from the protein sequence [157] (for PT the MRW is 124.4, PB A the MRW is 107, -thrombin the MRW is 114.6, R284Q-thrombin the MRW is 114.5), l is the cell path length (in millimetres), and c is the protein  52  concentration (in mg/mL). The temperature was maintained at 23 C with a Pelletier device. 2.8.1.2 Circular dichroism (CD) determination of thermal denaturation (Tm) Thrombin proteins were dialysed into 20mM Tris-HCl, 150mM NaCl, pH 7.4 and concentrated to 5µM. Thermal denaturation was carried out under computer control with a Jasco spectropolarimeter (model J-810) equipped with a Pelletier device. Thermal denaturation was determined from 10oC to 82oC with a temperature gradient of 1oC per minute. Thermal denaturation curves were calculated as previously described [158]. 2.8.2 Crystallization of thrombin with Ca2+ Human plasma-derived and rhprethrombin 2 were activated with ECV (1/50 w/w) overnight and purified by heparin affinity chromatography into 20mM Tris-HCl pH 7.4, 0.6M NaCl. The proteins were concentrated to 4-5mg/mL at 10,000xg in Microcon centrifugal filter devices (Amicon) with a 10 kDa molecular weight cut off. (Crystals were imaged and diffraction data was processed by members of the Huntington Laboratory, Cambridge, UK). 2.8.2.1 PPACK inhibition To prevent autolysis of plasma-derived and rh-thrombin, proteins were incubated for two hours at room temperature with a 10 fold molar excess of PPACK. Complete inhibition was assessed by no residual thrombin activity, as monitored by S-2238 amidolytic assay as before with a Spectramax190 microplate reader (Molecular Devices) at 23 C. Samples of PPACK inhibited proteins (10 L) were added to 190 L 300 M S2238 and monitored for 90 seconds at 405 nm. Proteins were then dialyzed to remove  53  excess PPACK using a 10 kDa MWCO filter (Slide-A-Lyzer, Thermoscientific, Canada) and concentrated to approximately 4-6 mg/mL. 2.8.2.2 Plasma-derived thrombin-Ca2+ soaking crystallization The inactivated, purified thrombin was dialysed overnight into 20mM Tris-HCl, pH 7.4, 100mM calcium acetate (Ca(OAc)2) and concentrated to 5.65 mg/mL. Drops of concentrated proteins (200nL) were subjected to a broad screen in sitting drops at 22oC using PEG/ion crystallization screens (Hampton Research, Aliso Viejo, CA) to produce thrombin crystals for soaking into Ca(OAc)2 solutions after crystallization. 2.8.2.3 PPACK rh-thrombin/ FVIII peptide 1 rh-thrombin was inactivated with PPACK and dialysed overnight into 50mM Tris-HCl pH 7.4, 150mM NaCl, before concentration to 4.0 mg/mL. FVIII peptide 1 was added on a 10/1 molar ratio of peptide to protein to help stabilize the thrombin protein and aid crystallization. The sequence of FVIII P1: H-N-N-E-E-A-E-D-W-D-D-L-OH. 2.8.2.4 rh-thrombin/FVIII peptide-Ca2+ Crystallization Crystals were grown by the hanging drop method at 22oC in (50mM MES pH 7.0, 10-100mM Ca(OAc)2 , 21-26% isopropanol conditions). 2.8.2.5 PPACK pl.-thrombin/hirugen The protein data bank was searched for high resolution thrombin structures of high symmetry to find conditions for addition of Ca(OAc)2 during the crystallization process, in the hope of allowing the calcium coordination with G453(133)/D318(14l) prior to crystal packing. The crystallization conditions for PDB 2UUF using thrombin in complex with the C-terminal peptide of hirudin (residues 54-65; hirugen) was chosen as the  54  crystals were found to be in a high symmetry space group, C121 and many of the structures using these conditions were published to diffract to less than 1.4 Å (PDB 2UUF, 2JH6, 2CF8). rh- thrombin was inactivated with PPACK and dialysed overnight into 50mM Tris-HCl pH 7.4, 150mM NaCl, before concentration to 4.0 mg/mL. Hirugen was added on a 10/1 molar ratio of peptide to protein. Crystals were grown by the hanging drop method at 22oC in 50mM HEPES pH 7.0, 500mM NaCl, 24-36% PEG 3350/ 0-720mM Ca(OAc)2. 2.8.3 Thrombin NMR 15  N labelled S195A rhprethrombin-2 was expressed in E. coli, refolded and  activated overnight at 22oC by 1/50 w/w addition of ECV followed by heparin affinity chromatography purification into 50mM Tris-HCl pH 7.4, 0.6M NaCl. The protein was then dialysed over four steps into 50mM Tris-HCl pH 7.4, 100mM LiCl, 50mM LiOAc, 5% D2O and the protein was concentrated to 31.8µM. To investigate the effect of calcium on thrombin, 100mM Ca(OAc)2 was added to the protein after the first NMR spectrum was determined. NMR spectra were acquired with a Bruker Avance 2+ 700 MHz spectrometer (Cambridge University, UK) equipped with a cryoprobe and single-axis gradient. Samples were prepared with a volume of 350μL in microtubes (Shigemi), degassed, and stored under argon. A 2D 1H 15N NMR spectrum was taken with 16 scans and a 14.8µs pulse time. The S195A thrombin spectra were recorded at 298°K (25°C), due to reduced sample stability. (This experiment was carried out with the assistance of Bernard Lechtenburg, Huntington laboratory, Cambridge, England.)  55  The resulting spectra were processed with Topspin 2.1 (Bruker, Karlsruhe) and analyzed with Sparky (Goddard and Kneller, SPARKY 3, University of California, San Francisco). Backbone resonance assignments were obtained from TROSY tripleresonance experiments [91] with S195A thrombin in the presence of Na+ based on the software MAPPER [159]. Weighted chemical shifts were calculated as described in [91].  2.9 Effect of Ca2+ on thrombin amidolytic activity The formation of p-nitroaniline was monitored at 405nm following the addition of 10nM pl.-thrombin to a 200µL reaction volume of 50mM TEA.Cl pH 7.5, 250µM S-2238 chromogenic substrate and varying CaCl2 concentration from 0-100mM. Addition of 0.1% PEG 8000, 0.1% BSA, and 0-200mM LiCl were tested to ensure that thrombin activity increases in the presence of calcium were not attributed to increasing enzyme solubility. LiCl was used as 3M Li+ does not alter thrombin structure unlike other monovalent cations such as Na [128].  56  3. ROLE OF THE A-CHAIN REGION IN PROTHROMBIN FOLDING AND ACTIVATION 3.1 Rationale and overview Although prothrombin is one of the most widely studied enzymes in biology, the role of the thrombin A-chain region has been neglected in comparison to the other domains. Recently there has been some interest in the A-chain; however, there are conflicting data on the functional importance of this peptide [123, 124, 133, 134]. The function of the A-chains of the coagulation serine proteases of the chymotrypsinogen family is still not known. Homologous snake venom thrombin-like enzymes lack an Achain altogether. Based on the conservation of at least a partial thrombin A-chain throughout vertebrate species, we hypothesize that the A-chain is essential for proper thrombin folding and/or stability. In this chapter, I describe a study to ascertain the role of the prothrombin A-chain region in protein structure and function. A deletion mutant of prothrombin lacking the Achain region (PT A) was expressed in mammalian cells and was characterized. The PT A protein was secreted from cells if the B-chain cysteine residue remained intact. However, PT A was non-activatable by factor Xa homologs and had significantly different secondary structure to recombinant WT prothrombin, as determined by CD.  57  3.2 Results 3.2.1 Mutagenesis and expression of rhPT A Prothrombin contains two factor Xa cleavage sites and one site susceptible to thrombin. On longer exposure to thrombin, a second site within the A-chain of thrombin is also cleaved [63]. Three cDNAs were constructed by PCR mutagenesis, restriction digestion and ligation (Figure 9). 3.2.1.1 Confirmation of constructs Upon sequence analysis of the hPT cDNA, two polymorphisms were identified. N121 which is encoded by AAC in the published prothrombin cDNA sequence [43] is substituted to threonine (ACC) and T122 (ACG) is substituted to methionine (ATG). Both changes are conservative and are encoded by exon 6 of the gene. Both polymorphisms have also been reported previously [43, 160]. All of the prothrombin cDNAs were ligated into the pNUT expression vector and the construct sequence confirmed by DNA sequence analysis on both strands using the computer program Chromas 2.33. (Technelysium Pty Ltd, Tewantin, Australia) and the basic local alignment search tool (BLAST) from the national Center for Biotechnology Information (NCBI). Nucleotide numbering was assigned according to NCBI accession number NM_000506.3. 3.2.1.2 Selection of clones For each construct, 6-20 colonies were cloned and cultured for analysis of expression/secretion levels. A first screen was performed by Western blot analysis after which a high-secreting clone was selected for large-scale expression. The criteria used for  58  determining the best cell line were the secretion level, the doubling time and the general cell phenotype as an indicator of health. Because integration events are random and may disrupt critical genes leading to slow growth times and unusual phenotypes, healthy looking cells with normal doubling times were preferentially chosen. 3.2.1.2.1 Secretion levels Variations in secretion levels of 5-10 fold were observed between clones derived from the transfection of a single construct. Figure 10A and B illustrate these differences by Western blot analysis of various clones of rhPT and PT A. No secretion was observed for PT AC439A, where expression was determined by boiling the cells and analyzing the supernatant fluid fraction by Western blotting. The cell lines chosen for large scale expression in triple flasks were: rhPT1 and PT A3. Secretion levels of approximately 10mg/L were detected for rhPT. BHK expression of the PT A construct with no mutation of the B-chain interchain disulfide cysteine yielded low concentrations of secreted PT A protein (209ug/L of culture media) as determined by BCA assay and absorbance at 280 nm after purification. 3.2.2 Characterization of rhPT A SDS PAGE of the rhPT preparation showed the presence of a band the expected molecular weight of meizothrombin after purification and before activation (Figure 11A, T0) as purification with Q Sepharose did not allow for separation of prothrombin alone. PT A purification revealed the presence of a high molecular weight species in addition to the expected 59.6kD PT A protein upon non-reducing SDS PAGE (Figure 11C, T0). Upon reducing SDS PAGE the high molecular weight species was lost, however a  59  Figure 9. Recombinant human prothrombin expression constructs. cDNAs were cloned into pNUT under the control of the constitutive MMP promoter. rhPT encodes the wild type prothrombin. PT A encodes the prothrombin deletion mutant for the entire A-chain region, retaining a factor Xa site between kringle 2 and the B-chain region of prothrombin. PT A retains the native B-domain cysteine residues involved in the A-B interchain disulfide bridge. PT AC439A is the same prothrombin A-chain region deletion construct with the B-domain interchain cysteine mutated to an alanine residue.  60  new band appeared at approximately 68kD (Figure 11D). SDS PAGE analysis and Coomassie brilliant blue staining of the TSV activation time course revealed that rhPT was fully converted to thrombin within 5 minutes while there was negligible PT A conversion to thrombin within 10 minutes (Figure 11). At 10 minutes of TSV activation the PT A band was lost while no lower molecular weight bands appeared in the range expected for thrombin B-chain. Further activation of PT A overnight also failed to yield any S-2238 chromogenic activity. Removal of the A-chain was expected to reduce amidolytic activity based on previously conducted experiments [124]; however, an intact factor Xa cut site is present but must not be accessible in this construct once folded. The lack of cleavage activation of the PT A protein suggests the A-chain removal may alter the tertiary structure of the zymogen or binding of FXa. Near UV CD spectroscopy did not show appreciable differences however with the protein concentrations available (Figure 12). Far-UV CD spectroscopy showed that PT A has a very different spectra than rhPT and significantly more beta sheet than rhPT (Figure 12). The CD spectrum of rhPT had a minimum at 203nm and a broad sholder at 215-230nm, in accordance with previously published studies [161, 162]. The spectra for PT A showed a minimum at 218nm and no shoulder at 215-230nm, suggesting an altered secondary structure. In contrast to the secreted PT A with a native B-chain cysteine I found that alanine mutation of the B-chain cysteine involved in the A-B interchain disulfide caused the rhPT AC439A protein to be retained within the cells, allowing no observable secretion. Because of the problems encountered with the non-secreting rhPT AC439A mutant and with the inability to activate the PT A protein, I proceeded to work with prethrombin-2 constructs instead.  61  Figure 10. Selection of prothrombin clones. Following SDS PAGE (10% acrylamide) under reducing conditions, the proteins were subjected to Western blot analysis using a sheep anti-human thrombin polyclonal antibody conjugated to HRP. A) Prothrombin proteins: Lane 1, prestained PAGE Ruler protein standard; lane 2, medium from BHK cells transfected with empty pNUT plasmid; lanes 3-7, medium from cloned rhPT cell lines cultured in flasks; Lane 8, human plasma derived thrombin positive control. B) PTB A proteins: lane 1, prestained PAGE Ruler Plus protein standard; lane 2, medium from BHK cells transfected with empty pNUT plasmid, lanes 3-10 PTB A clone culture medium; lanes 11-12, human plasma derived prothrombin positive control. In all cases, the medium was DMEM-F12/ ITS/ 6ug/mL vitamin K.  62  Figure 11. TSV-catalysed cleavage of rhPT and PT A monitored by SDS-PAGE. The reaction was initiated by the addition of TSV (125nM) and samples were withdrawn from ongoing activation and subjected to SDS-PAGE under non-reducing (A and C) and reducing (B and D) conditions. Sampling with purified rhPT (A and B) and PT A (C and D) from left to right were 0, 30, 60, 120, 150, 300, 600 seconds after addition of TSV. The abbreviations used are: PT: recombinant human prothrombin; mIIa, meizothrombin; 1.2, fragment 1.2; IIa, thrombin; IIa-B, thrombin B chain.  63  Figure 12. Near and far-UV CD spectrum for purified rhPT and PT A. Both spectra were recorded at 23 C, in 20mM Tris-HCl buffer, pH 7.4 in the presence of 150mM NaCl. CD spectra of rhPT is shown with a dashed line, PT A is shown with a continuous line. Ellipticity values were calculated as described in the methods section.  64  3.3 Discussion In this study, I investigated the contribution of the prothrombin A-chain region to prothrombin folding and activation. Mutagenesis and splicing of the prothrombin cDNA were performed to delete the A-chain region followed by expression of the recombinant constructs in BHK cells. The PT A mutant construct was secreted from the BHK cells, as was the WT prothrombin protein suggesting that the endogenous cellular misfolded protein response (ERAD) and unfolded protein response (UPR) pathways were not activated by the mutant protein construct [163]. By contrast, removal of the free thiol from the PT AC439A mutant resulted in sequestration of the recombinant protein within the cell. This intracellular retainment of the protein suggests that it may be preferable for prothrombin to have a free cysteine residue present at this position in the B-chain rather than an alanine substitution in the absence of the A-chain. The secretion of PT A suggests that the A-chain region is not required for folding of prothrombin; as the endogenous misfolded protein response in the BHK cells did not cause intracellular protein degradation. While the PT A mutant protein has an intact native factor Xa cleavage site between kringle 2 and the B-domain, activation of the purified PT A protein with TSV did not produce any thrombin, as determined by Coomassie blue-stained SDS-PAGE. ECV was also used to test whether PT A could be activated; however, no activation was observed. These results suggest that in the absence of the A-chain region, the factor Xa recognition sequence is not in a conformation accessible for cleavage by prothrombinase or FXa homologs (TSV and ECV respectively), indicating the A-chain region has a role as a spacer peptide in prothrombin. 65  To ascertain whether the A-chain region influences prothrombin structure, I investigated the secondary structure using far UV CD spectroscopy. I found that the spectra for rhPT and PT A were extremely different, and the secondary structure of PT A had significantly more predicted beta sheet than rhPT. The A-chain region predominantly forms -helical structure on the surface of thrombin so it was expected that removal of this region would affect the -helical content of the recombinant protein [90]. However, given that the A-chain region of thrombin is surface exposed and maintains contact with the B-chain through the central region only, it was not expected that the secondary structure would be altered so much compared to WT prothrombin. The recent crystallization of prethrombin-1 shows that the A-chain N-terminal region covalently bound to Fragment 2 has the same conformation as in thrombin. However, the C-terminal region bound to the B-chain forms a loop that is in close contact with the 186loop (K586 and G319 coordinate a water molecule) [164]. Fragment 2 was missing 17 residues on the C-terminus connecting to the A-chain region in this structure; thus, it is not possible to extrapolate the effect of the A-chain deletion on the orientation of the kringle 2 C-terminus in relation to the B-chain N-terminus. As there are currently no NMR or crystal structures of prothrombin it is not known how the A-chain region interacts with the rest of the prothrombin molecule. However, the differences in the far UV spectra for PT A may be due to altered disulfide bonding caused by the lack of the A-chain. Previous studies have shown that human plasma derived prothrombin has a minimum at 203nm of -8000 ± 300 and a broad shoulder between 215-230nm [162]. rhPT used in this study showed a similar CD spectrum to the previously published results [161, 162], while PT A showed a minimum at 218nm with a wide trough between 208-  66  230nm. The effect of pH on prothrombin has previously been studied and at pH 3 a deepening of the minimum at 208nm through to 222nm was observed corresponding to acid-induced structural transition to a partially unfolded state [165]. The authors suggest that at pH 3-4 hydrophobic patches on the interior of prothrombin are exposed corresponding to a decrease in the magnitude of the mean residue ellipticity at 222nm, and indicating a reduced level of secondary structure [165]. The increase in the depths of the minimum around 208-230nm of the PT A CD spectrum suggests similar loss of secondary structure upon removal of the A-chain.  3.4 Conclusions In this study, I expressed recombinant prothrombin deleted in the A-chain region. The PT A protein was secreted from cells, but was not activated by a prothrombinase homolog (TSV) or factor Xa homolog (ECV). The observation of inactivatibility by proteolysis validates the hypothesis that the prothrombin A-chain region is required for prothrombin activation. CD analysis indicated significant differences in the secondary structure of PT A compared to WT prothrombin suggesting that the A-chain region influences prothrombin conformation. However, in the absence of a prothrombin crystal structure it is not known how the A-chain region interacts with the remainder of the prothrombin molecule.  67  4. ROLE OF D318 IN THROMBIN CALCIUM ION BINDING 4.1 Rationale and overview An interaction between thrombin and calcium has long been proposed based on the observations that increasing Ca2+ concentrations affect thrombin amidolytic activity. Ca2+ titration to 100mM increased thrombin activity by 41% [127]. Landis et al. (1981) found that increasing concentrations of CaCl2 markedly decreased the thermal stability of thrombin. However, in the presence of CaCl2, thrombin autolysis was retarded and enzyme activity was maintained suggesting that Ca2+ selectively interacts with thrombin [128]. While the plasma Ca2+ concentration is in the range of 2-5mM, activation of platelets at a wound site causes release of Ca2+ from dense granules into the cytoplasm, and efflux into the extracellular medium [166]. It is possible that Ca2+ may play a role in stabilization of thrombin near the forming clot, enhancing activity and preventing clearance. Recently the Huntington laboratory (Cambridge, UK) solved a crystal structure of recombinant human thrombin (S195A-thrombin) in complex with protease nexin 1 (PN1) (IIa_PN1) which indicated the presence of a calcium ion held coordinated between the thrombin light chain residue D318(14l) and the backbone of G453(133) on the heavy chain (Figure 7, unpublished). Ca2+ coordination resulted in a more rigid structure of the thrombin A-chain C-terminus compared to other thrombin structures deposited in the  68  PDB and generated interest as to whether it was of functional importance to thrombin activity and stability. To address whether the binding of a Ca2+ at D318 was of functional and structural importance, I crystallized plasma derived and recombinant human thrombin in the presence of calcium to ascertain whether Ca2+ coordination could be observed. I also studied the effects of calcium addition to unliganded thrombin in solution by NMR to ascertain the effect on thrombin crystal packing by PPACK and inhibitory peptides such as FVIII p1 and Hirudin54-65 especially in terms of Ca2+ binding during crystallization. Finally, I investigated the amidolytic activity of thrombin in the presence and absence of Ca2+ to determine the basis for the Ca2+ effect reported previously [127].  4.2 Results 4.2.1 Crystallization of pl.-thrombin and soaking in of Ca2+ Crystallization by vapour diffusion in hanging drops consisting of 1µL PPACK inhibited human plasma derived thrombin (PPACK-Thrombin) with 1µL crystallization mother liquor produced diffraction quality crystals within a week (Figure 13A). Crystals of PPACK-Thrombin were screened by briefly soaking crystals in cryogenic solutions containing 20% glycerol, 34% PEG 4000, 0.3M NaOAc, 100mM Ca(OAc)2 before data collection at the CIMR using a MAR dtb Xray set using a crystal to detector distance of 250 mm. On one crystal 180° of data was collected from 117-297 with an oscillation of 1° and 5 minute image time. The crystal (space group P21) diffracted to a resolution of 3.0 Å. The data were processed using the Mosflm package [167] and programs from the CCP4 Suite [168]. The structure was solved by molecular replacement using the program  69  Phaser with the PPACK-bound thrombin structure (PDB accession code 1PPB) as the search model, and two molecules were placed in the asymmetric unit. To identify Ca2+ coordination between D318/G453, electron density mapping was performed with Coot [169]. The G453 backbone oxygen expected to coordinate the Ca2+ had it been present, was found to orientate away from the A-chain C-terminus in this structure (Figure 13B). No density for the D318 residue and no calcium ion density at D318/G453 was observed. 4.2.2 Crystallization of thrombin in the presence of inhibitors and calcium Thrombin was crystallized in the presence of the ligands FVIII peptide 1 and hirugen in order to improve crystallization conditions and obtain high resolution crystal structures to view calcium binding. 4.2.2.1 PPACK-rh-thrombin/ FVIII peptide 1 E. coli expressed and refolded recombinant human thrombin was inactivated with PPACK (PPACK-rh-thrombin) and dialysed overnight into 50mM Tris-HCl pH 7.4, 150mM NaCl, before concentration to 4.0 mg/mL. FVIII peptide 1 was added on a 10/1 molar ratio of peptide to protein. 1:1 and 1:2 ratios of protein to crystallization mother liquor were set up in 24 well plates. Crystals were grown by hanging drop method at 22oC in (50mM MES pH 7.0, 10-100mM Ca(OAc)2, 21-26% isopropanol) and plates grew within one week (Figure 14A). Crystals were soaked with 100mM Ca(OAc)2 in the cryogenic protection solution (25% isopropanol, 25% MPD, 50mM MES pH 6.0, 10mM MgCl2) before data collection over 720 (117-297 ) produced diffraction to 2.4 Å. Analysis of the diffraction reflections indicated the crystals were of space group P21. Molecular replacement and analysis was performed using Phaser [170]. Density mapping by Coot revealed the R390(75) side chain in an orientation that precluded Ca2+ ion 70  A  B  Figure 13. PPACK-thrombin with Ca2+ soak. A) PPACK-thrombin crystals soaked with 100mM Ca(OAc)2. B) Electron density map of Ca(OAc)2 soaked PPACK-thrombin crystal indicating no density for a Ca2+ ion between D318(14l) and G453(133). The orientation of the backbone oxygen of G453(133) in this structure would prevent coordination of Ca2+ and no density was observed for D318.  71  A  B  Figure 14. PPACK-rh-thrombin/FVIII peptide1 crystallization. A) Crystals of PPACK-rh-thrombin/FVIII peptide 1 grown in 50mM MES pH 7.0, 10100mM Ca(OAc)2 , 21-26% isopropanol conditions). B) Coot electron density image of PPACKrhIIa/FVIII peptide 1. No calcium coordination was observed between D318(14l) and G453(133). The orientation of the backbone oxygen of G453(133) is altered in this structure to prevent coordination of a metal atom. Additionally R390(75) is now occupying the hole previously accommodating the Ca2+ in the IIa_PN1 structure.  72  coordination as it occupied the space between D318 and G453. No calcium ion density at D318/G453 was observed (Figure 14B). 4.2.2.2 PPACK-rh-thrombin/hirudin54-65 A PDB search for high resolution thrombin structures of high symmetry was performed to find conditions for addition of Ca(OAc)2 during the crystallization process. Ca(OAc)2 addition during the crystallization process was attempted to allow the calcium coordination with G453(133)/D318(14l) prior to crystal packing. The crystallization conditions for PDB accession 2UUF was chosen as the crystals were found to be in a high symmetry space group (C121) and many of the structures determined under these conditions resulted from crystals that diffracted to <1.4 Å. Crystals were grown by the hanging drop method at 22oC in 50mM HEPES buffer pH 7.0, 500mM NaCl, 24-36% PEG 3350/ 0-720mM Ca(OAc)2. Phase separation occurred immediately, and rhomboid crystals grew within a week for all conditions (Figure 15A). Increasing Ca(OAc)2 concentration resulted in a shift to a new crystal form, needles. The needles grew into long plates and were found exclusively in 520mM Ca(OAc)2 and above. Analysis of the diffraction pattern of the rhomboid crystal form indicated that they were high symmetry space group P43212, and collection of data with 20-minute images produced diffraction to 2.4 Å. However, molecular replacement analysis and density mapping for an anomalous calcium signal indicated that there was no Ca2+ coordination to thrombin in the structure (Figure 15B). The orientation of the backbone oxygen of G453(133) was away from the Achain again, and the C-terminal region of the A-chain was observed to bend down onto itself. A water channel appeared to occupy the space filled by Ca2+ in the IIa_PN1 structure.  73  A  B  Figure 15. PPACK-thrombin/hirudin54-65. A) Effect of [Ca(OAc)2] on thrombin crystallization. B) Coot electron density image of PPACK-thrombin/hirudin54-65. No calcium coordination between D318(14l) and G453(133) was observed.  74  4.2.3 Thrombin calcium NMR To determine whether Ca2+ binding was inhibited in the liganded thrombin crystals (PPACK, hirudin54-65, FVIII p1) an inactive thrombin mutant (S195A) was studied by NMR in the presence and absence of Ca2+. 15N labelled S195A rhprethrombin-2 was expressed in E. coli, refolded and activated overnight at 22oC by 1/50 w/w addition of ECV followed by heparin affinity chromatography purification into 50mM Tris-HCl pH 7.4, 0.6M NaCl. The protein was then dialysed over four steps into 50mM Tris-HCl pH 7.4, 100mM LiCl, 50mM LiOAc, 5% D2O. The final protein concentration was 31.8µM. To investigate the effect of calcium on thrombin, 100mM Ca(OAc)2 was added to the protein after the first NMR spectrum was determined. The 2D spectra of thrombin with and without Ca(OAc)2 were indistinguishable (Figure 16) indicating no change to the environments of the residues when calcium was added. When the chemical shifts of individual residues were mapped for both spectra, no residues underwent significant chemical shifts within the margin of error (0.01 weighted chemical shift perturbation in ppm) [91]. Thus, calcium did not cause significant conformational changes to any residue in the thrombin structure. 4.2.4 Effect of calcium on thrombin chromogenic activity The formation of p-nitroaniline was monitored at 405nm following the addition of plasma derived thrombin (10nM) to S-2238 and varying CaCl2 concentration from 0100mM. Addition of 0.1% PEG 8000, 0.1% BSA, and 200mM LiCl were tested to control for thrombin solubility. LiCl was used to substitute for the physiological monovalent cation Na+ as the large sodium effect has previously been shown to overwhelm the effects of other ligands [127]. It has also been demonstrated that high  75  concentrations of lithium cations do not induce conformational changes in the thrombin active site [128]. Thrombin-dependent hydrolysis of S-2238 was found to be dependent on CaCl2 concentration in the absence of the buffer additive, 1g/L BSA (0.1%). BSA addition provided maximum protein solubility as determined by thrombin activity in the absence of CaCl2 (Figure 17). The presence of 0.1% PEG 8000 and 200mM LiCl increased thrombin solubility as determined by an increase in thrombin chromogenic activity compared to thrombin activity in buffer alone. The addition of CaCl2 with these additives further increased the S-2238 chromogenic activity. However, when protein solubility was controlled for by the presence of BSA, calcium did not act as a cation activator of thrombin activity (Figure 17).  4.3 Discussion Early spectroscopic and biochemical studies have demonstrated that thrombin activity, thermal stability and autolysis are modulated by the presence of mono and divalent cations [127-129]. The effects of Na+ on thrombin structure and activity are well established; however, other cations such as Ca2+, which have been shown to increase thrombin amidolytic activity and retard autolysis, have not been investigated since the initial studies were published in 1991. A recent crystal structure of thrombin in complex with PN1 suggested a putative Ca2+ binding site between the A and B-chains of thrombin located at D318(14l) and G453(133). The purpose of my study was to confirm Ca2+ binding by using both crystallography and NMR. Three different crystallization conditions were tested to account for possible packing effects caused by stabilizing ligands. Thrombin crystals were formed under all conditions tested; however, no Ca2+ coordination was seen in any 76  Figure 16.  15  N 1H NMR of S195A-thrombin.  2D TROSY NMR for S195A-thrombin in the absence (green) and presence (red) of 100mM Ca(OAc)2 indicates that there are no significant shifts in the electron resonances for any thrombin residues, demonstrating calcium does not bind.  77  Figure 17. Effect of [CaCl2] on thrombin chromogenic activity in the presence and absence of solubilizing additives. Thrombin activity was determined by S-2238 hydrolysis in 50mM TEA.Cl, pH 7.5 at 23 C (black), with 0.2M LiCl (red), 0.2M LiCl and 0.1% PEG 8000 (dark blue), 0.1% PEG 8000 (teal), 0.1% BSA (purple), 0.2M LiCl and 0.1% BSA (green), 0.2M LiCl, 0.1% PEG 8000, 0.1% BSA (magenta). For each condition CaCl2 was varied from 0100mM. All data were collected in triplicate, and error bars represent 1 S.D.  78  condition. Additional analysis for any calcium binding sites in thrombin failed to reveal calcium even with addition of 750mM Ca2+ in the crystallization conditions. As PPACK was used for all three of these crystal structures, it is possible that PPACKinhibited thrombin may be too rigid to allow G453 movement for a crystal contact to form with a calcium ion after crystal packing. Catalytically inactive S195A-thrombin was used for the II_PN1 crystal with Ca2+ coordination. To test whether Ca2+ can bind to uninhibited thrombin, I tested the effect of Ca2+ addition to S195A-thrombin by NMR. 2D TROSY NMR of thrombin in the presence and absence of 100mM Ca2+ showed no significant changes to the environments of any residues, indicating calcium does not bind. These results suggest that the original IIa-PN1 crystal showing calcium ion coordination may have been a crystal artefact, or may indicate that Ca2+ is uniquely coordinated with thrombin in the presence of PN1. Repetition of the previously published chromogenic substrate activity assays reporting that increasing concentrations of Ca2+ increases thrombin activity toward S2238 showed that when protein solubility was maintained, Ca2+ did not increase thrombin activity. The control activity assays in this study were performed in the presence of thrombin solubilizing agents BSA and PEG 8000 to ensure protein solubility was not limiting activity. BSA is the most abundant plasma protein (3.4-5.5 g/dL) and a strong Ca2+ ion binder [171]. While this study did not directly measure thrombin solubility, the calcium concentration used exceeds the calcium binding capacity of the BSA used [172]. This study used 1g/L BSA, and 0-100mM CaCl2. As the Ca2+ concentrations used in this study far exceed the physiological 2-5mM plasma Ca2+ concentration and the BSA was  79  one third the physiological concentration the results from this study do not support the hypothesis that Ca2+ affects thrombin activity.  4.4 Conclusions In this study, I investigated Ca2+ binding to thrombin both functionally and structurally. Contrary to previous reports, no evidence was found to support the hypothesis that thrombin is a Ca2+ binding enzyme. The Ca2+ effect on thrombin chromogenic activity was found to be due to protein solubility increasing with Ca2+ concentration in the absence of stabilizing agents. Taken together with the thrombin crystallography results and NMR, I conclude that thrombin is not a calcium-binding enzyme under the experimental conditions tested. It is possible that thrombin can bind divalent cations weakly, and may require additional ligands such as PN1 to stabilize the interaction, however it is not likely to be physiologically relevant.  80  5. FUNCTIONAL DIFFERENCES BETWEEN NASCENT THROMBIN AND -THROMBIN: ROLE OF THE A13 PEPTIDE 5.1 Rationale and overview The nascent thrombin enzyme is comprised of a 49-amino acid light chain (Achain) linked by a single disulphide bond to the 259-residue heavy chain (B-chain). Nascent human thrombin undergoes additional proteolysis at R284-T285 of the A-chain releasing a 13-residue N-terminal peptide to yield a 36-residue A-chain in -thrombin (Figure 2) [60, 63, 90]. It is not currently known whether 1) full A-chain nascent thrombin, truncated A-chain α-thrombin or both forms of thrombin are present in plasma [45], 2) if α-thrombin is present in the thrombin-antithrombin (TAT) complex (a marker of coagulopathy), or 3) if α-thrombin is merely an artifact of thrombin activation in vitro. In contrast to other vertebrate species such as cattle and trout, it is also unclear why human thrombin is capable of complete autolysis of the A13 peptide [173, 174] (Table 3). Given that only human thrombin is known to autolyse the A13 peptide while bovine and murine thrombin are known to retain this peptide, I considered whether the truncated α-thrombin could be a physiologically relevant form of thrombin in plasma, or whether truncated α-thrombin is a degradation product formed later in the haemostatic response. Since human thrombin is known to undergo autolysis of the A13 peptide, here I have investigated whether the immediate precursor to human α-thrombin (nascent thrombin) has altered structural and functional properties. In this study I aimed to 1) identify the approximate autolysis time for the A13 peptide at the N-terminus of the thrombin light chain; and 2) determine if retention of this peptide has any functional 81  consequences for human thrombin structure and activity. A recombinant human (rh)prothrombin-2 mutant R284Q was generated that cannot autoproteolyse the A-chain N-terminal 13-residue peptide. Both R284Q and α-thrombin were compared in biochemical assays to determine if retaining this peptide alters thrombin activity with respect to both procoagulant and anticoagulant functions such as cleaving fibrinogen, binding thrombomodulin and activating protein C.  5.2 Results 5.2.1 Bioinformatic analysis of the human thrombin A-chain Sequence alignment of the prothrombin A-chain region across 12 vertebrate species is shown in Table 2. Of the ten thrombin A-chain residues that interact with the B-chain, five residues are completely conserved, three are partially conserved between the various species, and S288 and T308 are not conserved (Table 2). To provide insight into the possible importance of the thrombin A-chain A-13 peptide I performed a multiple alignment across 12 vertebrate species (Table 3) from fish through to humans. T272, F280 and F281 are completely conserved in the sequences of the 12 vertebrate species analyzed and six other residues have positive weighted frequencies, indicating conservation (E276, Y277, Q278, N282, P283, R284). S275 is not conserved, but contributes to hydrogen bonding with N537(204b) and R538(206) in the B-chain of thrombin in some S195A thrombin structures. Y277, F280, F281 and N282 contribute to H-bonding to other residues in the A-chain. Thrombin substrate recognition usually requires a basic residue at P1 and is restricted to a small hydrophobic residue at P2 [76]. Residue 284 corresponding to the P1 82  Table 2. Prothrombin A-domain sequence alignment across 12 vertebrate species. NCBI protein family Conserved Domain Database Pfam09396 [174]. Residues with complete identity in all species are shown in black, conserved residues are shown in red, and non-conserved residues are marked in blue. Twelve human prothrombin A-chain residues interact with the B-chain, and are shown underlined. Human prothrombin numbering is provided underneath the sequences. Multiple alignment and residue conservation was determined by a position specific scoring matrix used in protein BLAST searches with a model specific score threshold (2.0).  83  Table 3. Conservation of the thrombin A13 peptide in vertebrates. Prothrombin A13 sequence alignment across twelve vertebrate species. NCBI Conserved Domain Database Pfam09396 [174]. Conserved residues are shown in red, and nonconserved residues are marked in blue. Six of the A13 peptide residues are partially conserved and three residues completely conserved. The residue at P1 is marked in bold, and the substrate subsite positions are provided with an arrow indicating the scissile bond. Mouse and cow sequences are indicated in bold as these species do not cleave off the A13 peptide. Multiple alignment and residue conservation was determined by a position specific scoring matrix used in protein BLAST searches with a model specific score threshold (2.0).  84  subsite for A13 peptide autolysis is an arginine or lysine in all vertebrate species examined, except for rainbow trout, with a glutamate at P1 (Table 3). However, bovine thrombin does not release this peptide [173, 175] and mouse thrombin also has a lysine at P1 and appears not to release the A13 peptide as this peptide is retained in crystal structures [154, 176]. Both species have a glutamic acid residue at P2, and this may be preventing autolysis of the A13 peptide. It is not known if other vertebrate species also retain this peptide; however, across six of the twelve species, the P2 residue is large and acidic, suggesting unfavourable interactions with the thrombin S2 subsite. 5.2.1.1 Human thrombin A-chain rigidity When expressed as a recombinant molecule in E. coli, mutation of the catalytic triad serine residue (S195) to alanine results in a thrombin species that cannot autoproteolyse the A13 peptide from the N-terminus of the A-chain. Pymol was used to align 26 different human thrombin structures deposited in the Protein Data Bank. The sequences aligned with a global root mean square deviation (RMSD) of 0.332 across 250 C atoms. Superposition reveals that the central portion of the A-chain is most rigid between residues A291(A0b)- S315(S14i) due to the presence of strong conserved salt bridge interactions while both termini of the A-chain are disordered (Figure 18). The flexibility and disorder of both termini suggests that these sequences do not interact strongly with other parts of the thrombin molecule and may not contribute significantly to the overall structural stability of the enzyme. However, there have been no studies published previously to exclude other biological functions.  85  Figure 18. Thrombin A-chain structural flexibility. Alignment of 26 different human S195A and -thrombin structures deposited in the PDB (see methods for PDB codes). A) S195A thrombin showing the 49 amino acid A-chain in dark grey and the B-chain in light grey. B) Superposition of the human thrombin Achains demonstrates the central portion of the A-chain is most rigid while the termini have increased flexibility. The nascent thrombin A-chain N terminus is T272 and thrombin A-chain N terminus is T285. The covalent disulfide bridge between the A- and B-chains is through C293(C1)-C439(C122).  86  5.2.1.2 Human thrombin A-chain residue interactions Comparison of S195A thrombin to wild type α-thrombin reveals additional hydrogen bonding between the S195A thrombin 49 residue A-chain and the B-chain. In these structures, the A-chain is further stabilized by the R284(R1i)-E579(E247) and S288(S1e)D355(D49) ion pairs. The ionic interactions include S275(S1r)-N536(N204b)-R538(R206)A291(A1b) or E276(E1q)-N536(N204b)-F535(F204a), Y277(Y1p)-E290(E1c)-F280(F1m)-F281(F1l), R284(R1I)-R356(R50)-G246, T285(T1h)-W357(W51) and F286(F1g)-S354(S48) or L440(L123). There are also hydrophobic stacking interactions between F280(F1m)-F281(F1l)-F286(F1g) in the first 13 residues of the A-chain in addition to the Y316(Y14j)-P534(P204) interchain interaction. These polar and hydrophobic interactions involving the A13 peptide may have long range electrostatic or steric effects on the overall thrombin structure. In αthrombin, it is likely that once cleaved autolytically, the A13 peptide rapidly dissociates from α-thrombin; however, it is not clear whether thrombin structure, stability and/or function are affected prior to cleavage. 5.2.2 Mutagenesis of recombinant prethrombin-2 R284Q Prethrombin-2 is the immediate precursor to thrombin and is comprised of the prothrombin cDNA catalytic domain (A-chain and B-chain regions). R284Q mutation was carried out by QuickChange reaction and the mutation confirmed by DNA sequence analysis. 5.2.3 Preliminary characterization of R284Q-thrombin Recombinant prethrombin-2 proteins were expressed in E. coli, activated with ECV, and purified to homogeneity. Recombinant thrombins had slightly faster electrophoretic mobility than plasma-derived thrombin, due to the absence of 87  glycosylation. No degraded forms of thrombin were detected upon gel electrophoresis before or after reduction (Figure 19). Both rhα and R284Q-thrombin were capable of removing the expression insert (MAIEGR), generating the true α and nascent thrombin A-chain N-termini; this was determined by N-terminal sequence analysis and the correlation of the calculated molecular weight (35.38kD) and the experimental molecular weight analyzed by MALDI TOF MS (35.36kD) (Figure 20). To ensure accuracy, concentrations of recombinant thrombin species were quantified in three ways: (1) BCA assay using the molecular weights 35.3kD and 33.8kD for nascent and α-thrombin, as determined by MALDI TOF MS analysis (Figure 20), (2) measuring the absorbance at A280 nm using an extinction coefficient of 1.83 mL mg-1 [152] and (3) formation of a 1 to 1 stoichiometric complex with AT [36].There was found to be excellent agreement between both the BCA and absorbance concentration measurements (rhα-thrombin: BCA: 5.7 M, A280(1) 5.65 M, (2) 5.67 M; R284Q-thrombin: BCA: 6.57 M, A280(1) 6.57 M, (2) 6.3 M). The concentration used was the average of the two A280 meansurements. 5.2.3.1 Near and Far UV CD spectrum CD analyses demonstrated no appreciable differences in the overall conformation of the enzymes, as determined by the near UV spectra for R284Q and rhα-thrombin within the margin of error (1 mDeg). However a deepening of the 225nm trough was seen for the mutant (Figure 21). Previously, shifts in this region have been attributed to increased -helix with either partial neutralization of charged groups or shielding of electrostatic repulsions due to the formation of salt shells around ionized groups [177].  88  Figure 19. Coomassie Blue stained SDS PAGE of purified recombinant thrombin species. Prethrombin-2 (lanes 1 and 5), WT rh -thrombin (lanes 2 and 6) and R284Q-thrombin (lanes 3 and 7). A molecular marker was run for both non-reducing and reducing conditions (PageRuler, Fermentas) and molecular weights are shown in kD.  89  Figure 20. MALDI-TOF molecular weight determination. WT) rh -thrombin, Q) R284Q-thrombin, K) R284K-thrombin. No degradation peptides were seen in this analysis. R284K-thrombin was found to autoproteolyse to α-thrombin.  90  Figure 21. Far and near-UV CD spectrum for rh -thrombin and R284Q-thrombin. Both spectra were recorded at 23 C, in 20mM Tris-HCl buffer, pH 7.4 in the presence of 150mM NaCl. CD spectra of rh -thrombin is shown with a dashed line, R284Q-thrombin is shown with a continuous line. Ellipticity values were calculated as described in the methods section.  91  5.2.3.2 CD determination of Tm CD thermal denaturation profiles for rhα- and R284Q thrombin indicated αthrombin has a thermal denaturation temperature of 51.5 C while nascent thrombin has an increased thermal denaturation temperature at 55.3 C, suggesting increased stability of the mutant protein that retains the A13 peptide (Figure 22). Based on the absorbance change and near complete loss of ellipticity at 222nm it appears that protein aggregation and precipitation accompany the thrombin denaturation process. 5.2.4 A13 generation time As the A-chain is located on the opposite face of thrombin than the active site, the A13 peptide cleavage must occur through intermolecular cleavage by a second thrombin protein. Scanning densitometry of A13 peptide cleavage from the catalytically inactive nascent thrombin S195A-thrombin was performed to determine the approximate lifetime of nascent thrombin in vitro. The rate constant for A13 peptide autolysis from S195Athrombin was 0.015 min-1 and the half-life was 46 minutes at 37 C under physiological conditions of ionic strength, pH and temperature in the presence of 0.34µM plasma derived-thrombin. This suggests that the activation of prothrombin to α-thrombin occurs through a relatively long-lived nascent thrombin intermediate, even at thrombin concentrations far above that required to form a fibrin clot (Figure 23). 5.2.5 Substrate hydrolysis is faster for R284Q-thrombin The rh-thrombins were characterized enzymatically for chromogenic activity against S-2238 and physiological substrate activity toward fibrinogen in a purified system and in a plasma clotting assay.  92  Figure 22. Effect of the A13 peptide on the thermal denaturation of thrombin. CD thermal denaturation profiles for rhα-thrombin (black) and R284Q thrombin (grey). rh -thrombin had a thermal denaturation temperature of 51.5 C while R284Q thrombin had an increased thermal unfolding temperature at 55.3 C.  93  Figure 23. A13 peptide autolysis from thrombin. Progress curve for consumption of S195A-thrombin following autolysis of the A13 peptide were obtained by quantitative scanning densitometry. Pixel intensity (PI) for protein bands were graphed against time of incubation. The line for the consumption of S195A-thrombin was drawn according to the equation for one-phase exponential decay (R2= 0.996).  94  5.2.5.1 Thrombin chromogenic activity In the presence of 100mM NaCl, the values for Km and kcat for S-2238 hydrolysis for the rhα-thrombin were similar to plasma-derived thrombin, whereas R284Q-thrombin had a lower Km and higher kcat, indicating a faster enzyme for S-2238 hydrolysis (Table 4). No significant differences were seen for the best fit Km and kcat values between rhαthrombin and R284Q-thrombin in the absence of sodium (Figure 24 and Table 4). Plasma derived thrombin was used as a positive control to allow comparison to established studies, however rhα-thrombin was found to have lower Km values for S-2238 hydrolysis. The reason for the difference is not immediately apparent but might be attributable to the different preparations of the two enzymes. Previously similar results have been seen between E. coli-derived recombinant human thrombin and plasmaderived thrombin [178]. Na+ has been well established to enhance thrombin activity toward S-2238 and an increase in activity was expected upon Na+ addition [141]. However, the additional increase in activity for R284Q-thrombin was not anticipated as the Na+ binding site is located near the active site, far from the A-chain. These results suggest that the presence of the A13 peptide improves Na+ binding for nascent thrombin or enhances the sodium activation of thrombin through allosteric effects. 5.2.5.2 Thrombin clotting activity R284Q-thrombin was found to clot fibrinogen 12% faster than rhα-thrombin (Table 5), suggesting that the A13 peptide may alter the tertiary structure of thrombin in some way since the interaction of fibrinogen with thrombin involves exosite I, the S1 specificity pocket and the catalytic site [179]. Similarly, R284Q thrombin clotted plasma 95  Figure 24. S-2238 hydrolysis in the presence and absence of Na+. Measurements were made at 23 C, in 100mM ionic strength, 20mM phosphate buffer, 0.1% PEG 8000, 0.1% BSA, pH 7.4. Sodium free: plasma derived thrombin (Pl.-IIa; open circles), Recombinant -thrombin (WT-IIa; open squares), R284Q-thrombin (R284Q-IIa; open triangles). Sodium added : Pl.-IIa (closed circles), WT-IIa (closed squares), R284QIIa (closed triangles). Data are expressed as the mean of three or more determinations ± SD.  96  Vmax Enzyme  Km  Kcat  Kcat/Km  (μM PNA/min)  (μM)  -1  (s )  (M s )  7.41 ± 0.15  3.98 ± 0.67  123.66 ± 2.44  3.11 x 107  0.97  (5.99 ± 0.37)  (3.82 ± 0.66)  (99.97 ± 6.30)  (2.62 x 107)  0.93  7.45 ± 0.06  2.78 ± 0.34  124.24 ± 1.09  4.47 x 107  0.97  (5.51 ± 0.17)  (2.64 ± 0.22)  (91.90 ± 2.86)  (3.48 x 107)  0.98  8.97 ± 0.09  2.12 ± 0.14  149.61 ± 1.43  7.06 x 107  0.98  (5.17 ± 0.17)  (2.41 ± 0.33)  (86.27 ± 2.94)  (3.58 x 107)  0.98  -1 -1  R2  Pl.-IIa  rhα-IIa  R284Q-IIa  Table 4. S-2238 hydrolysis in the presence and absence of Na+. For hydrolysis of S-2238, values for Km and Vmax were determined from analysis of progress curves using Origin 8.1. Kinetic parameters determined in the presence of Na+ are listed and the Na+ free parameters provided in parentheses. Measurements were made at 23 C in 100mM ionic strength, 20mM phosphate buffer, 0.1% PEG 8000, 0.1% BSA, pH 7.4. Experiments were conducted on three occasions and in triplicate.  97  Enzyme  Fibrinogen (%)  Plasma (%)  Pl.-IIa  100 ± 1  100 ± 1  rhα-IIa  112 ± 1  95 ± 1  R284Q-IIa  124 ± 2  111 ± 1  Table 5. Procoagulant activity of recombinant thrombins. Clotting times were converted into equivalent concentrations of plasma-derived thrombin (Pl.-IIa ) using a pl.-IIa standard clotting curve. Protein concentrations were determined previously by BCA assay and A280 as described in the methods. The fibrinogen and plasma clotting activity of rh -thrombin and R284Q-thrombin were then expressed as a percent of Pl.-IIa activity. Clotting times were determined electro-mechanically at 37 C. Measurements were made in 100mM NaCl, 20mM phosphate buffer, 0.1% PEG 8000, 0.1% BSA, 2mg/mL fibrinogen, pH 7.4. Experiments were conducted in triplicate.  98  16% faster than rhα-thrombin. There were differences seen between rh -thrombin and plasma-derived thrombin in both clotting assays. E.coli-derived rhα-thrombin does not have a native glycosylation at N373 , however previous studies on deglycosylated human thrombin have shown that glycosylation should not interfere with fibrinogen clotting [180]. However, as with the S-2238 activity assays differences have been previously identified for fibrinogen cleavage between E. coli-derived rhα-thrombin and plasmaderived thrombin [178]. The plasma derived thrombin was commercially produced and stored in 50% glycerol, and thus was not as fresh at the recombinant thrombin used. It is possible that the storage of the plasma-derived control may have resulted in degradation to  and thrombins, which cannot cleave fibrinogen [181].  5.2.6 Assessment of anticoagulant function To determine whether the A13 peptide in nascent thrombin affects the anticoagulant function of thrombin, the activation of hPC in the presence and absence of sTM was investigated [182]. 5.2.6.1 Activation of human protein C Plasma-derived thrombin, rhα-thrombin and R284Q-thrombin all activated hPC at approximately the same rate, indicating that the A13 peptide does not confer any alteration to PC activation and thus has no significant effect on the anticoagulant function of thrombin (Figure 25). This experiment was performed in both the presence and absence of thrombomodulin, with no difference seen between the three enzyme species within each experiment. This result taken with the enhanced clotting results suggest that nascent thrombin may be a procoagulant precursor to α-thrombin.  99  Figure 25. Activation of PC by thrombin/TM. Activation of hPC by plasma derived thrombin (black), recombinant α-thrombin (grey), R284Q-thrombin (white) in the presence of soluble thrombomodulin (sTM). At the indicated time intervals the activity of thrombin was inhibited by a molar excess of hirudin and the rate of APC generation was measured by S-2238 hydrolysis as described in Methods. Data are expressed as the mean of three determinations ± SD.  100  5.2.7 Inhibition Due to spatial proximity of the A13 peptide to exosite II in published crystal structures [97, 183, 184], affinity for heparin was assessed by a NaCl gradient elution chromatography using heparin Sepharose [179] (Figure 26). Prethrombin-2 eluted at 764mM NaCl, Rhα-thrombin eluted at 787mM NaCl while R284Q thrombin eluted at 805mM NaCl. While this is a minor difference in elution profile, this method has been used previously to map the heparin binding residues on thrombin exosite 2 with similar differences seen between mutants and WT thrombin Sheehan and Sadler [185]. We speculated that the proximity of the A13 peptide in R284Q thrombin would enhance heparin affinity for exosite II residues. To address this hypothesis, we studied the effect of heparin on S-2238 hydrolysis, in the presence and absence of AT as in [36, 179, 186]. 5.2.7.1 Heparin binding affinity In the absence of AT, 15kDa heparin was found to be 5% more effective at inhibiting S-2238 hydrolysis by R284Q-thrombin than by plasma-derived or rhαthrombin (Figure 27). To assess the magnitude of the increased heparin affinity the relative affinities for heparin were determined for rhα- and R284Q-thrombin by S-2238 hydrolysis. As with some other synthetic substrates, heparin decreased the Kmapp for S-2238, but had little effect on V [156]. The dissociation constant (Kd) for heparin was found to be 40nM ± 1.5nM and 9nM ± 1.7nM for rhα-thrombin and R284Q-thrombin respectively using the method described in [156] (Figure 28). The rhα-thrombin value is consistent with the apparent dissociation constant determined in previous studies [187], while the R284Q-thrombin value is considerably lower. The lower dissociation constant for  101  Figure 26. Elution of thrombins from heparin-Sepharose. Affinity for heparin was assessed by a NaCl gradient elution (green) from heparin Sepharose (GE). Prethrombin-2 eluted at 764mM (blue), rh -thrombin eluted at 787mM NaCl (orange) while R284Q-thrombin eluted at 805mM NaCl (grey).  102  Figure 27. Heparin inhibition of thrombin S-2238 activity. Thrombin S-2238 amidolytic substrate activity inhibition by binding heparin (15-17kD). Inhibition rates in the presence of heparin were normalized to the enzyme activity in the absence of heparin for each protein and converted to percentage inhibition. Plasma derived (black) and rhα-thrombin (grey) S-2238 amidolytic activities were both inhibited approximately 5% by heparin. R284Q-thrombin (white) hydrolysis of S-2238 was inhibited 9% by comparison. Data are expressed as the mean of three or more determinations ± SD.  103  Figure 28. Heparin inhibition of thrombin chromogenic activity toward S-2238. Lineweaver-Burk plots showing the effect of heparin on thrombin S-2238 hydrolysis. No heparin (squares), 1nM heparin (diamonds), 3nM heparin (circles). rh -thrombin (A) shows very little inhibition of S-2238 hydrolysis by heparin whereas heparin inhibited R284Q-thrombin substrate hydrolysis in a competitive manner (B). Data are expressed as the mean of three determinations.  104  R284Q-thrombin suggests that heparin binding to nascent thrombin is tighter than to αthrombin. 5.2.7.2 Inhibition by AT While rhα- and R284Q-thrombin were found to have similar AT inhibition constants in the absence of heparin (Table 6), R284Q-thrombin was found to have reduced AT inhibition in the presence of heparin. A reduction in heparin-dependent AT inhibition for R284Q-thrombin was a surprising result given the enhanced heparin affinity of the mutant. In order to determine whether the apparent differences in rates of inhibition were caused by a change in the fraction of R284Q-thrombin binding to AT, the stoichiometries of inhibition (SI) were determined (Table 6). The AT SI was found to be 1:1 for both rhα-thrombin and R284Q-thrombin in the absence of heparin. However, in the presence of heparin the SI for R284Q-thrombin increased to 1.38, while rhα-thrombin and the plasma derived thrombin control were 1.23 and 1.14 respectively. It has previously been established that heparin increases the AT SI by increasing the stability of thrombin [36]. These results demonstrate a heparin-dependent perturbation of the AT inhibition of R284Q-thrombin, which may be due to increased thrombin stabilization by the presence of the A13 peptide.  5.4 Discussion This study has investigated the effects of the A13 peptide on human thrombin to assess the functional differences between nascent thrombin and α-thrombin. We chose to study nascent thrombin as in other species such as mouse and cow, thrombin do not undergo autolysis at R284 to cleave off the A13 peptide. To address the contribution of the A13 peptide in nascent human thrombin we have compared R284Q-thrombin with α105  Rate constant (k2) of inhibition M-1 min-1  AT k2  SI (AT)  heparin AT k2 No heparin  0.6µM heparin  5.96 ± 0.00  n.d  1.14 ± 0.01  5.60 ± 0.04  5.78 ± 0.02  1.01 ± 0.05  1.23 ± 0.01  5.40 ± 0.24  4.82 ± 0.06  1.01 ± 0.02  1.38 ± 0.01  Enzyme  (x 10 )  (x 10 )  Pl.-IIa  4.48 ± 0.24  rhα-IIa  R284Q-IIa  5  8  Table 6. Effect of the A13 peptide on AT inhibition of thrombins in presence and absence of heparin. Measurements were made at 25 C in 100mM NaCl, 20mM phosphate buffer, 0.1% PEG 8000, 0.1% BSA, 50nM thrombin, 0.5μM AT, pH 7.4, 0-2nM heparin. Data are expressed as the mean of three or more determinations ± SD.  106  thrombin. The R284Q mutation was chosen as glutamine is similar to arginine in terms of residue size. Lysine was also substituted to account for residue charge, however lysine was found to be autolytically cleaved in position 284 for the human thrombin sequence. It is possible that the R284Q mutation may be responsible for the differences between nascent and -thrombin, however this conservative mutation is often used by researchers in the field instead of the more commonly accepted alanine mutation [19]. To test the effects of the R284 mutation, repetition of the experiments with R284A nascent thrombin could be considered. The results described in the current work indicate that prior to the autolytic release of the A13 peptide, nascent thrombin is a procoagulant form of the protease without compromising TM binding for APC generation and anticoagulant function. Two other studies have identified thrombin mutations that increase fibrinogen cleavage while decreasing antithrombin inhibition. Prothrombin K154E was found to increase fibrinogen cleavage by 18%, and decrease inhibition by AT by 4 times [179]. The same study also identified K252E to increase fibrinogen cleavage by 8% whilst reducing AT inhibition 10 times. Both of these mutations map to exosite 2 in thrombin. Another mutagenesis study by Tsiang et al. identified several A-chain mutations that altered thrombin activity. A triple mutation (S288A/E290A/D292A) in the thrombin A-chain that increased S-2238 amidolytic activity 282% and fibrinogen clotting 175% compared to WT thrombin [133]. K307A modestly increased S-2238 amidolytic activity 112% and fibrinogen cleavage 115%, while E311A and the double mutant E314A/D318A reduced fibrinogen clotting and S-2238 activity. Similarly the naturally occurring K301/K302 deletions decrease  107  fibrinogen clotting and Na+ binding [112, 113]. This study has identified that the A13 peptide increases thrombin activity toward fibrinogen and S-2238 in the presence of Na+. To our knowledge there are no published mutations in thrombin that increase Na+ binding or S-2238 hydrolysis in the presence of Na+. The double mutation of W215A/E217A is well known to decrease fibrinogen cleavage without compromising protein C activation by blocking Na+ binding [188-190]. Thus the A13 peptide may also allosterically affect Na+ binding to ensure nascent thrombin is maintained in the 'fast' form [191]. Similar results have been seen with EGF5 and 6 of thrombomodulin and hirugen, which are exosite 1 binding ligands that impart long range allosteric effects on the thrombin active site to alter thrombin activity toward peptidyl substrates, increasing activity toward some and decreasing activity toward others [192]. I propose that the A13 peptide may stabilize the enzyme, as shown by thermal denaturation profiling and AT inhibition stoichiometry for α- and nascent thrombin. No other studies have identified mutations that increase thrombin stability. Additionally, the half-life for A13 peptide autolysis was found to be 46 minutes under physiological conditions in vitro, which is far in excess of time required for clot formation during coagulation. The long autolysis half-life raises the possibility that nascent thrombin is the physiological form of human thrombin at the site of a clot and truncated α-thrombin may be a degradation product formed later in the haemostatic response when less fibrinogen clotting activity is required. Nascent thrombin was also shown in this study to bind heparin more tightly than -thrombin. The dissociation constant (Kd) for heparin was found to be 40nM ± 1.5nM and 9nM ± 1.7nM for rhα-thrombin and R284Q-thrombin respectively. A previous study  108  by Cunningham and Nelsestuen [193] compared the heparin catalyzed AT interaction for human and bovine thrombin, which retains the A13 peptide. The authors found that there were significant species differences in the way the thrombins interacted with heparin and concluded that the bovine protein would have a substantially tighter interaction with heparin/AT, as determined by a lower Km and kcat.  5.5 Conclusions I propose that in the initial burst of thrombin production during coagulation, nascent thrombin plays a role in tipping the balance of thrombin activity towards procoagulation, thus ensuring adequate clot formation. The A13 peptide may serve to stabilize nascent thrombin, preventing the inherent flexibility required by α-thrombin for interaction with diverse cofactors and substrates in the multitude of roles thrombin plays. The increased heparin binding affinity of nascent thrombin may provide the physiological balance required to ensure wayward thrombin migrating away from the wound site is quickly sequestered on the surface of intact endothelium, and neutralized to prevent pathological clotting. Subsequent autolytic cleavage of the A13 peptide may then permit thrombin to assume the flexible and less stable structure we have come to regard as αthrombin, allowing the equilibrium between procoagulant and anticoagulant pathways to be assumed.  109  6. SUMMARY AND GENERAL DISCUSSION Thirty years since human thrombin was first cloned and recombinantly expressed, the roles of the A-chain region of prothrombin and the A-chain in thrombin remain poorly understood [43, 150, 178, 194]. The paucity of literature on the thrombin A-chain may be attributed to the fact that the thrombin A-chain is homologous to the activation peptide of chymotrypsinogen, which has been demonstrated to have no involvement in chymotrypsin activity [90]. However, there are instances in which mutations in the Achain region of prothrombin have caused bleeding phenotypes due to a lack of circulating prothrombin (hypoprothrombinemia) or dysfunctional prothrombin (dysprothrombinemia) suggesting the A-chain region of prothrombin is involved in prothrombin folding, secretion and/or activation [111-118]. Recently there has been some interest in the A-chain; however, there is conflicting data on the functional importance of this peptide [123, 124, 133, 134]. The function of the A-chains of the coagulation serine proteases of the chymotrypsinogen family is still not known [101]. Homologous snake venom thrombin-like enzymes lack an A-chain altogether [105]. Based on the conservation of at least a partial thrombin A-chain throughout vertebrate species, we hypothesized that the A-chain is essential for proper thrombin function and/or stability. An approach to study the necessity of the thrombin Achain region is described in this thesis through investigation of a potential Ca2+ binding site in the thrombin A-chain, and investigation of the autolytic A13 peptide.  110  6.1 Role of the A-chain region in prothrombin folding and activation Recombinant WT prothrombin and mutants lacking the entire A-chain region (PT A and PT AC482A) were produced to investigate the contribution of the prothrombin A-chain region to prothrombin folding and activation. PT A mutant protein was secreted from the BHK cells, suggesting that the endogenous cellular misfolded protein response (ERAD) and unfolded protein response (UPR) pathways were not activated by the mutant protein construct [163]. The secretion of PT A suggests that the A-chain region is not required for folding of prothrombin; as the endogenous misfolded protein response in the BHK cells did not cause intracellular protein degradation. The prothrombin deletion mutant (PT A) was found to be non-activatable by the FXa and prothrombinase homologs ECV and TSV even though an intact native FXa cleavage site is formed between kringle 2 and the B-domain. This result suggests that the A-chain region of prothrombin is required to orientate the FXa recognition sequence for cleavage. It is also possible that the A-chain region contains additional FXa or FVa interaction sites although this remains unknown in the absence of a prothrombinase complex crystal structure. CD analysis indicated significant differences in the secondary structure of PT A compared to WT prothrombin suggesting that the A-chain region influences prothrombin conformation. However, in the absence of a prothrombin crystal structure it is not known how the A-chain region interacts with the remainder of the prothrombin molecule. Due to poor protein yields from purification and the likelihood of nonactivatable proteins from recombinant prothrombin protein studies, I pursued investigation of the A-chain with prethrombin-2 constructs to test the contribution of the A-chain to thrombin activity.  111  6.2 Role of D318 in thrombin calcium coordination The role of Na+ in thrombin activity has been well established [127, 195]; however, the influence of other monovalent and divalent cations on thrombin activity, stability and autolysis have not been thoroughly investigated [127-129]. While there was some recent crystallographic evidence that Ca2+ interacts with thrombin through the Achain residue D318 and the B-chain residue G453, it remained unclear whether this was a unique example and whether Ca2+ confers function to thrombin. The results from my study showed that crystallization of thrombin under several different conditions and in the presence of up to 700mM Ca(OAc)2 did not produce evidence of Ca2+ binding, and there was no observed magnetic resonance shifts in any thrombin residues upon 100mM Ca2+ addition in an NMR study of unliganded thrombin. These results alone suggest that thrombin does not have a calcium binding site anywhere, let alone between D318/G453. The results from this study also indicate that the basis for the increase in thrombin activity upon Ca2+ addition previously reported could be attributed to enzyme solubilization with increasing salt concentration. Other studies have shown that thrombin activity, stability and autolysis may be affected by Mn2+, Co2+ and K+, but full characterization of the effect of these cations remains to be elucidated [128].  6.3 Role of A13 peptide in nascent thrombin This research suggests that the A13 peptide is autolytically cleaved so slowly from nascent human thrombin that it is unlikely for -thrombin to be the physiological form of thrombin acting during propagation of coagulation. My findings suggest that the A13 peptide alters substrate specificity and inhibition such that the A-chain A13 peptide plays an allosteric regulatory role to control thrombin activity, steering thrombin activity 112  toward procoagulation and imparting stability as evidenced by a higher thermal denaturation temperature and AT inhibition resistance. The A13 peptide is located on the back side of the thrombin protease domain (far from the active site). Therefore its effects must be transmitted over a long range to the active site, to account for the observation of enhanced amidolytic and fibrinogen clotting activities of nascent thrombin. A theoretical model of the A13 peptide influence on thrombin activity is proposed in Figure 29. Through the methods commonly used for zymogen activation, -thrombin is produced in vitro [196, 197]. As such, -thrombin has been used in laboratory experiments throughout the coagulation research field as thrombin. The results described in this thesis suggest that -thrombin may be a degradation product of nascent thrombin on the cleavage pathway prior to the production of  and -thrombin [181]. However, in  vivo sampling of thrombin during clot formation, circulation in plasma and inhibition would be required to confirm this is the case physiologically.  6.4 Significance of the work Hemostasis is a highly controlled process consisting of both temporally and physically regulated interactions between components to prevent uncontrolled bleeding or widespread thrombosis. The multifunctional thrombin enzyme is the final protease in the coagulation cascade and plays a pivotal role to both enhance and down-regulate coagulation. In addition, thrombin participates in cell signalling, wound regeneration and the complement immune response. Modulation of thrombin activity is achieved through allosteric regulation and cofactor interactions that alter thrombin substrate specificity. Through mutagenesis and biochemical studies the thrombin A-chain is now gaining  113  Figure 29. Proposed model for the influence of the A13 peptide on nascent thrombin. Prothrombin activation produces nascent thrombin (nascent-IIa) with the A13 peptide covalently attached (as shown in magenta). Nascent thrombin has increased clotting activity, enhanced thermal stability, and increased heparin binding, but decreased antithrombin inhibition in the presence of heparin. The presence of the A13 peptide may impart long range effects to the thrombin active site and may order other parts of the thrombin B-chain (blue), or affect the exosites such as anion binding exosite II (ABEII) making nascent thrombin more stable. Upon autolytic cleavage of the A13 peptide, thrombin ( -IIa) is generated and activity is reduced, as depicted by a smaller active site (shown in red).  114  recognition as an allosteric regulator in addition to its role as a structural stabilizer of the protease domain [91, 134] . This research demonstrates the potential for therapeutic drug development for coagulation, as thrombin is used extensively in surgical procedures and for treatment of burn victims [198-201]. Elucidation of the thrombin A-chain contribution may allow the simpler production of recombinant human thrombin for fibrin glues (fibrinogen, thrombin and factor XIIIa) to replace the current plasma-derived and recombinant human thrombin in these preparations [202-204]. Additionally, novel thrombin proteins with distinctive function and potential therapeutic value may be discovered, such as that of the R284Qthrombin described within this thesis. Using nascent thrombin as procoagulant enzyme scaffold, additional procoagulant mutations could be added, such as S288A/E290A/D292A [133] or stability and longevity could be increased by removing the  and thrombin cleavage sites at R390 and K474 [181, 205]. Therefore, these studies  have the potential to have an impact in surgical and pre-hospital settings to reduce dependency on blood products. Using thrombin as a model enzyme for these studies, we expect to gain a better understanding of the A-chain function of homologous coagulation proteases in the chymotrysinogen family. Six residues of the A-chain are equivalent to the propeptide of chymotrypsinogen, which is not involved in substrate and inhibitor binding [90]. Homologous proteases, such as trypsin, lack the A-chain, and other homologous clotting proteins, such as factor Xa, IXa, APC and VIIa, retain the remainder of the zymogen through the conserved A-B interchain disulfide bridge. Features and functions gleaned from investigation of the thrombin A-chain thus present the opportunity to identify 115  potential homologous roles in the rest of the chymotrypsinogen family, and may allow for hypothesis development for future studies on other proteases.  6.5 Future studies Although prothrombin is one of the most widely studied enzymes in biology the role of the thrombin A-chain has been neglected in comparison to the other domains. Of the few studies investigating the function of the A-chain, conflicting reports have prevented clarity [123, 124, 133, 134]. There has been no comprehensive study of all Achain residues to date. While originally considered to be simply an activation remnant with little physiologic function [122, 123], the A-chain in thrombin is now thought to play a role as an allosteric effector in enzymatic reactions [112, 113, 133, 134]. The A-chain may provide a structural scaffold [91] for protease domain function as the naturally occurring thrombin A-chain mutations would suggest [112-115, 145], or may provide or mask recognition motifs for other plasma proteins. To explore these alternatives, a full biochemical examination of the thrombin A-chain using site directed mutagenesis and recombinant protein expression would probe the role of individual residues in the overall structure and function of thrombin. It is expected that mutation of highly conserved residues would impact thrombin stabilization or functional activity. Naturally occurring mutations in the A-chain causing dysprothrombinemia, such as F299V, E309K and E311K require characterization as the mutations do not interfere with prothrombin activation and are likely to structurally destabilize the thrombin enzyme, or affect the activity through long range allosteric effects, as seen with the K301 and K302 deletions [112, 113]. 116  The results from this study indicate that nascent thrombin is a procoagulant thrombin, and potentially more stable than α-thrombin. This stabilization may serve to rigidify nascent thrombin, preventing the inherent flexibility required by α-thrombin for interaction with diverse cofactors and substrates in the multitude of roles thrombin plays [68]. To test the effect of the A13 peptide in nascent thrombin on thrombin conformation, CD and intrinsic fluorescence provide information on the tertiary structure of nascent thrombin in the presence of ligands [206, 207]. This study has identified that the A13 peptide increases the fibrinogen clotting activity of nascent thrombin, however the effect of the A13 peptide on other exosite binding procoagulant substrates, such as FV, FVIII, FXIII and platelet activation remain to be explored [150, 208, 209]. The effect of the A13 peptide on Na+ binding should also be elucidated [210]. In addition, thrombin is involved in wound regeneration and complement activation [211, 212], the presence of the A13 peptide may alter thrombin activity in these pathways also. The nascent thrombin studies described in this thesis have utilized purified proteins expressed from E. coli. In order to accurately measure conversion of nascent thrombin to α-thrombin in blood it would be ideal to use ELISA with an antibody targeted to the A13 peptide. However, at this time there are no commercially available antibodies for the A13 peptide of human thrombin. As the A13 peptide on nascent thrombin confers enhanced fibrin clotting and antithrombin resistance in the presence of heparin, it would be valuable to produce an A13 antibody to test on idiopathic prothrombotic patients [213]. It is plausible that some patients with a prothrombotic tendency may be explained by mutations disrupting the A13 thrombin cleavage motif, resulting in nascent thrombin that cannot autoproteolyse to α-thrombin. However, the  117  prothrombotic phenotype is expected to be mild in the absence of other environmental and genetic risk factors such as G20210A and FV Leiden [214-217]. To identify the physiological differences between nascent thrombin and thrombin a murine model could be employed [218]. While prothrombin deletion is incompatible with survival in mice [219], liver specific transgene expression allows rescue of the pheontype [220]. As murine thrombin does not undergo autolytic cleavage of the A13 peptide, and is also not Na+ activated, human prothrombin transgene expression would be used [176]. Transgenic recombinant human prothrombin constructs could be tested in a mouse model to assess the effects of the A13 peptide on thrombin activity in normal coagulation by mechanical or laser-induced injury [221]. One could also test for the effects of nascent thrombin in a thrombosis model by administering ferric chloride as a thrombogenic agent [221]. Ideally the transgene constructs would include a WT prothrombin positive control, which would autolyse to -thrombin after activation in vivo; a R284Q-prothrombin mutant would test the effect of retaining the A13 peptide after thrombin activation. An A13 deletion mutant could be used to test for the effects of the A13 peptide on prothrombin folding and activation. I would expect that this study would show no differences between the WT prothrombin control and the R284Q mutant for activity during the clotting response as the autolysis rate has been established to be very slow in vitro. However, the effect of nascent thrombin on later stages of coagulation after the burst of thrombin has been generated has not been ascertained. This research has also identified that the A13 peptide enhances heparin binding to thrombin. There has been only one other report of thrombin mutations increasing heparin affinity, and map to exosite 2 of thrombin [179]. We would like to confirm the molecular 118  basis for the enhanced heparin binding and reduced AT inhibition by crystallizing R284Q thrombin/ heparin and R284Q thrombin/ heparin/ AT [184].We hope to determine whether the A13 peptide produces additional or altered heparin binding contacts to explain the increased heparin binding affinity and the reduced heparin-dependent AT inhibition seen for R284Q thrombin.  119  BIBLIOGRAPHY 1.  Carter ISR, Vanden Hoek AL, Pryzdial ELG, MacGillivray RTA. Thrombin Achain: activation remnant or allosteric effector? Thrombosis. 2010; vol. 2010: 9 pages.  2.  Hoffman M, Monroe DM. Coagulation 2006: a modern view of hemostasis. Hematol Oncol Clin North Am. 2007; 21: 1-11.  3.  Stassen JM, Arnout J, Deckmyn H. The hemostatic system. Curr Med Chem. 2004; 11: 2245-60.  4.  Davie EW. Biochemical and molecular aspects of the coagulation cascade. Thromb Haemost. 1995; 74: 1-6.  5.  Kahn ML, Nakanishi-Matsui M, Shapiro MJ, Ishihara H, Coughlin SR. Proteaseactivated receptors 1 and 4 mediate activation of human platelets by thrombin. J Clin Invest. 1999; 103: 879-87.  6.  Wolfs JL, Comfurius P, Rasmussen JT, Keuren JF, Lindhout T, Zwaal RF, et al. Activated scramblase and inhibited aminophospholipid translocase cause phosphatidylserine exposure in a distinct platelet fraction. Cell Mol Life Sci. 2005; 62: 1514-25.  7.  Zwaal RF, Comfurius P, Bevers EM. Surface exposure of phosphatidylserine in pathological cells. Cell Mol Life Sci. 2005; 62: 971-88.  8.  Daleke DL, Lyles JV. Identification and purification of aminophospholipid flippases. Biochim Biophys Acta. 2000; 1486: 108-27.  9.  Engelmann B. Initiation of coagulation by tissue factor carriers in blood. Blood Cells Mol Dis. 2006; 36: 188-90.  10.  Heemskerk JW, Bevers EM, Lindhout T. Platelet activation and blood coagulation. Thromb Haemost. 2002; 88: 186-93.  11.  Manly DA, Boles J, Mackman N. Role of tissue factor in venous thrombosis. Annu Rev Physiol. 73: 515-25.  12.  Owens AP, 3rd, Mackman N. Tissue factor and thrombosis: The clot starts here. Thromb Haemost. 104: 432-9.  13.  Mackman N. The role of tissue factor and factor VIIa in hemostasis. Anesth Analg. 2009; 108: 1447-52.  120  14.  van't Veer C, Mann KG. The regulation of the factor VII-dependent coagulation pathway: rationale for the effectiveness of recombinant factor VIIa in refractory bleeding disorders. Semin Thromb Hemost. 2000; 26: 367-72.  15.  Rao LV, Rapaport SI, Bajaj SP. Activation of human factor VII in the initiation of tissue factor-dependent coagulation. Blood. 1986; 68: 685-91.  16.  Kanse SM, Etscheid M. Factor VII activating protease. Single nucleotide polymorphisms light the way. Hamostaseologie. 2011; 31(3):174-8.  17.  Kamath P, Krishnaswamy S. Fate of membrane-bound reactants and products during the activation of human prothrombin by prothrombinase. J Biol Chem. 2008; 283: 30164-73.  18.  Bukys MA, Orban T, Kim PY, Nesheim ME, Kalafatis M. The interaction of fragment 1 of prothrombin with the membrane surface is a prerequisite for optimum expression of factor Va cofactor activity within prothrombinase. Thromb Haemost. 2008; 99: 511-22.  19.  Kroh HK, Panizzi P, Tchaikovski S, Baird TR, Wei N, Krishnaswamy S, et al. Active Site-labeled Prothrombin Inhibits Prothrombinase in Vitro and Thrombosis in Vivo. J Biol Chem. 2011; 286: 23345-56.  20.  Farrell DH, Siebenlist KR. Fibrinogen containing gamma' chains. Blood. 2006; 107: 3011-2; author reply 2.  21.  Lorand L. Factor XIII: structure, activation, and interactions with fibrinogen and fibrin. Ann N Y Acad Sci. 2001; 936: 291-311.  22.  Rau JC, Beaulieu LM, Huntington JA, Church FC. Serpins in thrombosis, hemostasis and fibrinolysis. J Thromb Haemost. 2007; 5 Suppl 1: 102-15.  23.  DelGiudice LA, White GA. The role of tissue factor and tissue factor pathway inhibitor in health and disease states. J Vet Emerg Crit Care (San Antonio). 2009; 19: 23-9.  24.  Monroe DM, Hoffman M. What does it take to make the perfect clot? Arteriosclerosis, thrombosis, and vascular biology. 2006; 26: 41.  25.  Smith SB, Gailani D. Update on the physiology and pathology of factor IX activation by factor XIa. Expert Rev Hematol. 2008; 1: 87-98.  26.  Carter I, Wong, AY, Bleakley, MR, Vashchenko, G, Fox, HD, MacGillivray, RT. Molecular basis of human coagulopathies. In: Matsumoto A, Nakano, M., editor. The human genome: features, variations and genetic disorders. New York: Nova Science Publishers Inc.; 2009. p. 101-24.  121  27.  Peyvandi F, Jayandharan G, Chandy M, Srivastava A, Nakaya SM, Johnson MJ, et al. Genetic diagnosis of haemophilia and other inherited bleeding disorders. Haemophilia. 2006; 12 Suppl 3: 82-9.  28.  Esmon CT, Owen WG. The discovery of thrombomodulin. J Thromb Haemost. 2004; 2: 209-13.  29.  Esmon CT, Esmon NL, Harris KW. Complex formation between thrombin and thrombomodulin inhibits both thrombin-catalyzed fibrin formation and factor V activation. J Biol Chem. 1982; 257: 7944-7.  30.  Esmon NL, Carroll RC, Esmon CT. Thrombomodulin blocks the ability of thrombin to activate platelets. J Biol Chem. 1983; 258: 12238-42.  31.  Esmon CT. The roles of protein C and thrombomodulin in the regulation of blood coagulation. J Biol Chem. 1989; 264: 4743-6.  32.  Walker FJ. Regulation of activated protein C by protein S. The role of phospholipid in factor Va inactivation. J Biol Chem. 1981; 256: 11128-31.  33.  Dahlback B. Protein S and C4b-binding protein: components involved in the regulation of the protein C anticoagulant system. Thromb Haemost. 1991; 66: 4961.  34.  van 't Veer C, Mann KG. Regulation of tissue factor initiated thrombin generation by the stoichiometric inhibitors tissue factor pathway inhibitor, antithrombin-III, and heparin cofactor-II. J Biol Chem. 1997; 272: 4367-77.  35.  Baglin TP, Carrell RW, Church FC, Esmon CT, Huntington JA. Crystal structures of native and thrombin-complexed heparin cofactor II reveal a multistep allosteric mechanism. Proc Natl Acad Sci U S A. 2002; 99: 11079-84.  36.  Mushunje A, Zhou A, Carrell RW, Huntington JA. Heparin-induced substrate behavior of antithrombin Cambridge II. Blood. 2003; 102: 4028-34.  37.  Huber K. Plasminogen activator inhibitor type-1 (part one): basic mechanisms, regulation, and role for thromboembolic disease. J Thromb Thrombolysis. 2001; 11: 183-93.  38.  Brownstein C, Falcone DJ, Jacovina A, Hajjar KA. A mediator of cell surfacespecific plasmin generation. Ann N Y Acad Sci. 2001; 947: 143-55; discussion 556.  39.  Lord ST. Molecular mechanisms affecting fibrin structure and stability. Arterioscler Thromb Vasc Biol. 2011; 31: 494-9.  122  40.  Lundblad RL, Kingdon HS, Mann KG. Thrombin. Methods Enzymol. 1976; 45: 156-76.  41.  Degen SJ, Davie EW. Nucleotide sequence of the gene for human prothrombin. Biochemistry. 1987; 26: 6165-77.  42.  Hewett-Emmett D, Czelusniak J, Goodman M. The evolutionary relationship of the enzymes involved in blood coagulation and hemostasis. Ann N Y Acad Sci. 1981; 370: 511-27.  43.  Degen SJF, MacGillivray RTA, Davie EW. Characterization of the complementary deoxyribonucleic acid and gene coding for human prothrombin. Biochemistry. 1983; 22: 2087-97.  44.  von Heijne G. Signal sequences. The limits of variation. J Mol Biol. 1985; 184: 99-105.  45.  Davie EW, Kulman JD. An overview of the structure and function of thrombin. Semin Thromb Hemost. 2006; 32 Suppl 1: 3-15.  46.  Suttie JW, Hoskins JA, Engelke J, Hopfgartner A, Ehrlich H, Bang NU, et al. Vitamin K-dependent carboxylase: possible role of the substrate "propeptide" as an intracellular recognition site. Proc Natl Acad Sci USA. 1987; 84: 634-7.  47.  Houston DF, Timson DJ. Interaction of prothrombin with a phospholipid surface: evidence for a membrane-induced conformational change. Mol Cell Biochem. 2011; 348: 109-15.  48.  Soriano-Garcia M, Padmanabhan K, de Vos AM, Tulinsky A. The Ca2+ ion and membrane binding structure of the Gla domain of Ca-prothrombin fragment 1. Biochemistry. 1992; 31: 2554-66.  49.  Soriano-Garcia M, Park CH, Tulinsky A, Ravichandran KG, Skrzypczak-Jankun E. Structure of Ca2+ prothrombin fragment 1 including the conformation of the Gla domain. Biochemistry. 1989; 28: 6805-10.  50.  Wu W, Suttie JW. N-glycosylation contributes to the intracellular stability of prothrombin precursors in the endoplasmic reticulum. Thromb Res. 1999; 96: 918.  51.  Kotkow KJ, Deitcher SR, Furie B, Furie BC. The second kringle domain of prothrombin promotes factor Va-mediated prothrombin activation by prothrombinase. J Biol Chem. 1995; 270: 4551-7.  123  52.  Deguchi H, Takeya H, Gabazza EC, Nishioka J, Suzuki K. Prothrombin kringle 1 domain interacts with factor Va during the assembly of prothrombinase complex. Biochem J. 1997; 321: 729-35.  53.  Kalafatis M, Egan JO, van 't Veer C, Cawthern KM, Mann KG. The regulation of clotting factors. Crit Rev Eukaryot Gene Expr. 1997; 7: 241-80.  54.  Bock PE, Panizzi P, Verhamme IM. Exosites in the substrate specificity of blood coagulation reactions. J Thromb Haemost. 2007; 5 Suppl 1: 81-94.  55.  Bianchini EP, Orcutt SJ, Panizzi P, Bock PE, Krishnaswamy S. Ratcheting of the substrate from the zymogen to proteinase conformations directs the sequential cleavage of prothrombin by prothrombinase. Proc Natl Acad Sci U S A. 2005; 102: 10099-104.  56.  Krishnaswamy S, Mann KG, Nesheim ME. The prothrombinase-catalyzed activation of prothrombin proceeds through the intermediate meizothrombin in an ordered, sequential reaction. J Biol Chem. 1986; 261: 8977-84.  57.  Kim PY, Nesheim ME. Further evidence for two functional forms of prothrombinase each specific for either of the two prothrombin activation cleavages. J Biol Chem. 2007; 282: 32568-81.  58.  Doyle MF, Mann KG. Multiple active forms of thrombin. IV. Relative activities of meizothrombins. J Biol Chem. 1990; 265: 10693-701.  59.  Wood JP, Silveira JR, Maille NM, Haynes LM, Tracy PB. Prothrombin activation on the activated platelet surface optimizes expression of procoagulant activity. Blood. 2011; 117: 1710-8.  60.  Downing MR, Butkowski RJ, Clark MM, Mann KG. Human prothrombin activation. J Biol Chem. 1975; 250: 8897-906.  61.  Lanchantin GF, Friedmann JA, Hart DW. Two forms of human thrombin. Isolation and characterization. J Biol Chem. 1973; 248: 5956-66.  62.  Bishop PD, Lewis KB, Schultz J, Walker KM. Comparison of recombinant human thrombin and plasma-derived human alpha-thrombin. Semin Thromb Hemost. 2006; 32 Suppl 1: 86-97.  63.  Petrovan RJ, Govers-Riemslag JW, Nowak G, Hemker HC, Tans G, Rosing J. Autocatalytic peptide bond cleavages in prothrombin and meizothrombin. Biochemistry. 1998; 37: 1185-91.  64.  Camire RM, Bos MH. The molecular basis of factor V and VIII procofactor activation. J Thromb Haemost. 2009; 7: 1951-61.  124  65.  von dem Borne PA, Meijers JC, Bouma BN. Feedback activation of factor XI by thrombin in plasma results in additional formation of thrombin that protects fibrin clots from fibrinolysis. Blood. 1995; 86: 3035-42.  66.  Muszbek L, Bereczky Z, Bagoly Z, Komaromi I, Katona E. Factor XIII: A Coagulation Factor With Multiple Plasmatic and Cellular Functions. Physiol Rev. 2011; 91: 931-72.  67.  Coughlin SR. Protease-activated receptors in hemostasis, thrombosis and vascular biology. J Thromb Haemost. 2005; 3: 1800-14.  68.  Adams TE, Huntington JA. Thrombin-cofactor interactions: structural insights into regulatory mechanisms. Arterioscler Thromb Vasc Biol. 2006; 26: 1738-45.  69.  Di Cera E, Cantwell AM. Determinants of thrombin specificity. Ann N Y Acad Sci. 2001; 936: 133-46.  70.  Coughlin SR. Thrombin signalling and protease-activated receptors. Nature. 2000; 407: 258-64.  71.  Esmon NL, Owen WG, Esmon CT. Isolation of a membrane-bound cofactor for thrombin-catalyzed activation of protein C. J Biol Chem. 1982; 257: 859-64.  72.  Bajzar L, Morser J, Nesheim M. TAFI, or plasma procarboxypeptidase B, couples the coagulation and fibrinolytic cascades through the thrombin-thrombomodulin complex. J Biol Chem. 1996; 271: 16603-8.  73.  Walker FJ, Fay PJ. Regulation of blood coagulation by the protein C system. Faseb J. 1992; 6: 2561-7.  74.  De Candia E, Hall SW, Rutella S, Landolfi R, Andrews RK, De Cristofaro R. Binding of thrombin to glycoprotein Ib accelerates the hydrolysis of Par-1 on intact platelets. J Biol Chem. 2001; 276: 4692-8.  75.  Krishnaswamy S. Exosite-driven substrate specificity and function in coagulation. J Thromb Haemost. 2005; 3: 54-67.  76.  Stubbs MT, Bode W. The clot thickens: clues provided by thrombin structure. Trends Biochem Sci. 1995; 20: 23-8.  77.  Chang JY. Thrombin specificity. Requirement for apolar amino acids adjacent to the thrombin cleavage site of polypeptide substrate. Eur J Biochem. 1985; 151: 217-24.  125  78.  Schechter I, Berger A. On the size of the active site in proteases. I. Papain. Biochem Biophys Res Commun. 1967; 27: 157-62.  79.  Le Bonniec BF, Myles T, Johnson T, Knight CG, Tapparelli C, Stone SR. Characterization of the P2' and P3' specificities of thrombin using fluorescencequenched substrates and mapping of the subsites by mutagenesis. Biochemistry. 1996; 35: 7114-22.  80.  Izaguirre G, Swanson R, Raja SM, Rezaie AR, Olson ST. Mechanism by which exosites promote the inhibition of blood coagulation proteases by heparinactivated antithrombin. J Biol Chem. 2007; 282: 33609-22.  81.  Huntington JA, Read RJ, Carrell RW. Structure of a serpin-protease complex shows inhibition by deformation. Nature. 2000; 407: 923-6.  82.  Lane DA, Philippou H, Huntington JA. Directing thrombin. Blood. 2005; 106: 2605-12.  83.  Ng NM, Quinsey NS, Matthews AY, Kaiserman D, Wijeyewickrema LC, Bird PI, et al. The effects of exosite occupancy on the substrate specificity of thrombin. Arch Biochem Biophys. 2009; 489: 48-54.  84.  Qureshi SH, Yang L, Manithody C, Iakhiaev AV, Rezaie AR. Mutagenesis studies toward understanding allostery in thrombin. Biochemistry. 2009; 48: 8261-70.  85.  Di Cera E. Thrombin. Mol Aspects Med. 2008; 29: 203-54.  86.  Di Cera E, Page MJ, Bah A, Bush-Pelc LA, Garvey LC. Thrombin allostery. Phys Chem Chem Phys. 2007; 9: 1291-306.  87.  Pozzi N, Chen R, Chen Z, Bah A, Di Cera E. Rigidification of the autolysis loop enhances Na(+) binding to thrombin. Biophys Chem.  88.  Gianni S, Ivarsson Y, Bah A, Bush-Pelc LA, Di Cera E. Mechanism of Na(+) binding to thrombin resolved by ultra-rapid kinetics. Biophys Chem. 2007; 131: 111-4.  89.  Page MJ, Di Cera E. Is Na+ a coagulation factor? Thromb Haemost. 2006; 95: 920-1.  90.  Bode W, Turk D, Karshikov A. The refined 1.9Å x-ray crystal structure of D-PhePro-Arg chloromethylketone-inhibited human alpha-thrombin: structure analysis, overall structure, electrostatic properties, detailed active-site geometry, and structure-function relationships. Protein Sci. 1992; 1: 426-71.  126  91.  Lechtenberg BC, Johnson DJ, Freund SM, Huntington JA. NMR resonance assignments of thrombin reveal the conformational and dynamic effects of ligation. Proc Natl Acad Sci U S A. 2010; 107: 14087-92.  92.  Bode W, Mayr I, Baumann U, Huber R, Stone SR, Hofteenge J. The refined 1.9 Å crystal structure of human alpha-thrombin: interaction with D-Phe-Pro-Arg chloromethylketone and significance of the Tyr-Pro-Pro-Trp insertion segment. EMBO J. 1989; 8: 3467-75.  93.  Davidson CJ, Tuddenham EG, McVey JH. 450 million years of hemostasis. J Thromb Haemost. 2003; 1: 1487-94.  94.  Doolittle RF. Coagulation in vertebrates with a focus on evolution and inflammation. J Innate Immun. 2011; 3: 9-16.  95.  Doolittle RF. Step-by-step evolution of vertebrate blood coagulation. Cold Spring Harb Symp Quant Biol. 2009; 74: 35-40.  96.  Banfield DK, Irwin DM, Walz DA, MacGillivray RTA. Evolution of prothrombin: isolation and characterization of the cDNAs encoding chicken and hagfish prothrombin. J Mol Evol. 1994; 38: 177-87.  97.  Adams TE, Li W, Huntington JA. Molecular basis of thrombomodulin activation of slow thrombin. J Thromb Haemost. 2009; 7: 1688-95.  98.  Frost C, Naud ÃR, Oelofsen W, Muramoto K, Naganuma T, Ogawa T. Purification and characterization of ostrich prothrombin. The Int J Biochem Cell Biol. 2000; 32: 1151.  99.  Banfield DK, MacGillivray RTA. Partial characterization of vertebrate prothrombin cDNAs: amplification and sequence analysis of the B chain of thrombin from nine different species. Proc Natl Acad Sci USA. 1992; 89: 277983.  100.  Irwin DM, Robertson KA, MacGillivray RTA. Structure and evolution of the bovine prothrombin gene. J Mol Biol. 1988; 200: 31-45.  101.  Page MJ, Di Cera E. Evolution of peptidase diversity. J Biol Chem. 2008; 283: 30010-4.  102.  Geppert AG, Binder BR. Allosteric regulation of tPA-mediated plasminogen activation by a modifier mechanism: evidence for a binding site for plasminogen on the tPA A-chain. Arch Biochem Biophys. 1992; 297: 205-12.  103.  Summaria L, Robbins KC. Isolation of a human plasmin-derived, functionally active, light (B) chain capable of forming with streptokinase an equimolar light  127  (B) chain-streptokinase complex with plasminogen activator activity. J Biol Chem. 1976; 251: 5810-13. 104.  Sinha D, Marcinkiewicz M, Navaneetham D, Walsh PN. Macromolecular substrate-binding exosites on both the heavy and light chains of factor XIa mediate the formation of the Michaelis complex required for factor IX-activation. Biochemistry. 2007; 46: 9830-9.  105.  Castro HC, Zingali RB, Albuquerque MG, Pujol-Luz M, Rodrigues CR. Snake venom thrombin-like enzymes: from reptilase to now. Cell Mol Life Sci. 2004; 61: 843-56.  106.  Maroun RC. Molecular basis for the partition of the essential functions of thrombin among snake venom serine proteinases: the case of thrombin-like enzymes. Haemostasis. 2001; 31: 247-56.  107.  Castro HC, Silva DM, Craik C, Zingali RB. Structural features of a snake venom thrombin-like enzyme: thrombin and trypsin on a single catalytic platform? Biochim Biophys Acta. 2001; 1547: 183-95.  108.  Itoh N, Tanaka N, Mihashi S, Yamashina I. Molecular cloning and sequence analysis of cDNA for batroxobin, a thrombin-like snake venom enzyme. J Biol Chem. 1987; 262: 3132-5.  109.  Serrano SM, Maroun RC. Snake venom serine proteinases: sequence homology vs. substrate specificity, a paradox to be solved. Toxicon. 2005; 45: 1115-32.  110.  Lancellotti S, De Cristofaro R. Congenital prothrombin deficiency. Semin Thromb Hemost. 2009; 35: 367-81.  111.  Akhavan S, Mannucci PM, Lak M, Mancuso G, Mazzucconi MG, Rocino A, et al. Identification and three-dimensional structural analysis of nine novel mutations in patients with prothrombin deficiency. Thromb Haemost. 2000; 84: 989.  112.  De Cristofaro R, Akhavan S, Altomare C, Carotti A, Peyvandi F, Mannucci PM. A natural prothrombin mutant reveals an unexpected influence of A-chain structure on the activity of human alpha-thrombin. J Biol Chem. 2004; 279: 13035-43.  113.  De Cristofaro R, Carotti A, Akhavan S, Palla R, Peyvandi F, Altomare C, et al. The natural mutation by deletion of Lys9 in the thrombin A-chain affects the pKa value of catalytic residues, the overall enzyme's stability and conformational transitions linked to Na+ binding. Febs J. 2006; 273: 159-69.  114.  Lefkowitz JB, Haver T, Clarke S, Jacobson L, Weller A, Nuss R, et al. The prothrombin Denver patient has two different prothrombin point mutations  128  resulting in Glu-300-->Lys and Glu-309-->Lys substitutions. Br J Haematol. 2000; 108: 182. 115.  Sun WY, Burkart MC, Holahan JR, Degen SJF. Prothrombin San Antonio: a single amino acid substitution at a Factor Xa activation site (Arg320 to His) results in dysprothrombinemia. Blood. 2000; 95: 711-4.  116.  Akhavan S, Luciani M, Lavoretano S, Mannucci PM. Phenotypic and genetic analysis of a compound heterozygote for dys- and hypoprothrombinaemia. Br J Haematol. 2003; 120: 142-4.  117.  Côté HCF, Stevens WK, Banfield DK, Nesheim ME, MacGillivray RTA. Characterization of a stable form of human meizothrombin derived from recombinant prothrombin (R155A, R271A, R284A). J Biol Chem. 1994; 269: 11374-80.  118.  Akhavan S, Rocha E, Zeinali S, Mannucci PM. Gly319 --> arg substitution in the dysfunctional prothrombin Segovia. Br J Haematol. 1999; 105: 667-9.  119.  Magnusson S, Peterson TE, Sottrup-Jensen L, Claeys H. Complete primary structure of prothrombin: structure and reactivity of ten carboxylated glutamic acid residues and regulation of prothrombin activation by thrombin. In: Reich E, Rifkin DB, Shaw E, editors. Proteases and Biological Control. 0 ed. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratories; 1975. p. 123-49.  120.  Endres GF, Swenson MK, Scheraga HA. Structural aspects of thrombin specificity. Arch Biochem Biophys. 1975; 168: 180-7.  121.  Fujikawa K, Legaz ME, Kato H, Davie EW. The mechanism of activation of bovine factor IX (Christmas factor) by bovine factor XIa (activated plasma thromboplastin antecedent). Biochemistry. 1974; 13: 4508-16.  122.  Hageman TC, Endres GF, Scheraga HA. Mechanism of action of thrombin on fibrinogen. On the role of the A chain of bovine thrombin in specificity and in differentiating between thrombin and trypsin. Arch Biochem Biophys. 1975; 171: 327-36.  123.  Pirkle H, Theodor I, Christofferson M, Vukasin P, Miyada D. On the location in the thrombin B chain of substrate recognition sites for fibrinopeptide release and factor XIII activation. Thromb Res. 1989; 55: 737-46.  124.  Rajesh Singh R, Chang JY. Structural stability of human alpha-thrombin studied by disulfide reduction and scrambling. Biochim Biophys Acta. 2003; 1651: 85-92.  125.  Roberts PS, Burkat RK, Braxton WE. Thrombin's esterase activity in the presence of anticoagulant and other salts. Thromb Diath Haemorrh. 1969; 21: 103-10.  129  126.  Workman EF, Jr., Lundblad RL. The effect of monovalent cations on the catalytic activity of thrombin. Arch Biochem Biophys. 1978; 185: 544-8.  127.  Orthner CL, Kosow DP. Evidence that human alpha-thrombin is a monovalent cation-activated enzyme. Arch Biochem Biophys. 1980; 202: 63-75.  128.  Landis BH, Koehler KA, Fenton JW, 2nd. Human thrombins. Group IA and IIA salt-dependent properties of alpha-thrombin. J Biol Chem. 1981; 256: 4604-10.  129.  Di Cera E, De Cristofaro R, Albright DJ, Fenton JW, 2nd. Linkage between proton binding and amidase activity in human alpha-thrombin: effect of ions and temperature. Biochemistry. 1991; 30: 7913-24.  130.  De Cristofaro R, Di Cera E. Effect of protons on the amidase activity of human alpha-thrombin. Analysis in terms of a general linkage scheme. J Mol Biol. 1990; 216: 1077-85.  131.  De Cristofaro R, Di Cera E. Modulation of thrombin-fibrinogen interaction by specific ion effects. Biochemistry. 1992; 31: 257-65.  132.  De Cristofaro R, Fenton JW, 2nd, Di Cera E. Linkage between proton binding and amidase activity in human gamma-thrombin. Biochemistry. 1992; 31: 1147-53.  133.  Tsiang M, Jain AK, Dunn KE, Rojas ME, Leung LL, Gibbs CS. Functional mapping of the surface residues of human thrombin. J Biol Chem. 1995; 270: 16854-63.  134.  Papaconstantinou ME, Bah A, Di Cera E. Role of the A chain in thrombin function. Cell Mol Life Sci. 2008; 65: 1943-7.  135.  Derian CK, Damiano BP, D'Andrea MR, Andrade-Gordon P. Thrombin regulation of cell function through protease-activated receptors: implications for therapeutic intervention. Biochemistry (Mosc). 2002; 67: 56-64.  136.  Ebert MP, Lamer S, Meuer J, Malfertheiner P, Reymond M, Buschmann T, et al. Identification of the thrombin light chain a as the single best mass for differentiation of gastric cancer patients from individuals with dyspepsia by proteome analysis. J Proteome Res. 2005; 4: 586-90.  137.  Chang PC, Wu HL, Lin HC, Wang KC, Shi GY. Human plasminogen kringle 1-5 reduces atherosclerosis and neointima formation in mice by suppressing the inflammatory signaling pathway. J Thromb Haemost. 2010; 8: 194-201.  130  138.  Kim SR, Chung ES, Bok E, Baik HH, Chung YC, Won SY, et al. Prothrombin kringle-2 induces death of mesencephalic dopaminergic neurons in vivo and in vitro via microglial activation. J Neurosci Res. 2010; 88: 1537-48.  139.  Stepanova VV, Beloglazova IB, Gursky YG, Bibilashvily RS, Parfyonova YV, Tkachuk VA. Interaction between kringle and growth-factor-like domains in the urokinase molecule: possible role in stimulation of chemotaxis. Biochemistry (Mosc). 2008; 73: 252-60.  140.  Papareddy P, Rydengard V, Pasupuleti M, Walse B, Morgelin M, Chalupka A, et al. Proteolysis of human thrombin generates novel host defense peptides. PLoS Pathog. 2010; 6: e1000857.  141.  Wells CM, Di Cera E. Thrombin is a Na(+)-activated enzyme. Biochemistry. 1992; 31: 11721-30.  142.  Di Cera E. Thrombin interactions. Chest. 2003; 124: 11S-7S.  143.  Liu CC, Brustad E, Liu W, Schultz PG. Crystal structure of a biosynthetic sulfohirudin complexed to thrombin. J Am Chem Soc. 2007; 129: 10648-9.  144.  The PyMOL Molecular Graphics System. Version 1.10, Schrödinger, LLC.  145.  Akhavan S, Miteva MA, Villoutreix BO, Venisse L, Peyvandi F, Mannucci PM, et al. A critical role for Gly25 in the B chain of human thrombin. J Thromb Haemost. 2005; 3: 139.  146.  Le Bonniec BF, MacGillivray RTA, Esmon CT. Thrombin Glu-39 restricts the P'3 specificity to nonacidic residues. J Biol Chem. 1991; 266: 13796-2803.  147.  Palmiter RD, Behringer RR, Quaife CJ, Maxwell F, Maxwell IH, Brinster RL. Cell lineage ablation in transgenic mice by cell-specific expression of a toxin gene. Cell. 1987; 50: 435-43.  148.  Sun YM, Jin DY, Camire RM, Stafford DW. Vitamin K epoxide reductase significantly improves carboxylation in a cell line overexpressing factor X. Blood. 2005; 106: 3811-5.  149.  Johnson DJ, Adams TE, Li W, Huntington JA. Crystal structure of wild-type human thrombin in the Na+-free state. Biochem J. 2005; 392: 21-8.  150.  Soejima K, Mimura N, Yonemura H, Nakatake H, Imamura T, Nozaki C. An efficient refolding method for the preparation of recombinant human prethrombin2 and characterization of the recombinant-derived alpha-thrombin. J Biochem. 2001; 130: 269-77.  131  151.  Kisiel W, Hanahan DJ. Purification and characterization of human Factor II. Biochim Biophys Acta. 1973; 304: 103-13.  152.  Fenton JW, 2nd, Fasco MJ, Stackrow AB. Human thrombins. Production, evaluation, and properties of alpha-thrombin. J Biol Chem. 1977; 252: 3587-98.  153.  Olson ST. Heparin and ionic strength-dependent conversion of antithrombin III from an inhibitor to a substrate of alpha-thrombin. J Biol Chem. 1985; 260: 10153-60.  154.  Bush LA, Nelson RW, Di Cera E. Murine thrombin lacks Na+ activation but retains high catalytic activity. J Biol Chem. 2006; 281: 7183-8.  155.  Conway EM, Nowakowski B, Steiner-Mosonyi M. Human neutrophils synthesize thrombomodulin that does not promote thrombin-dependent protein C activation. Blood. 1992; 80: 1254-63.  156.  Griffith MJ, Kingdon HS, Lundblad RL. The interaction of heparin with human alpha-thrombin: effect on the hydrolysis of anilide tripeptide substrates. Arch Biochem Biophys. 1979; 195: 378-84.  157.  Bychkova VE, Dujsekina AE, Klenin SI, Tiktopulo EI, Uversky VN, Ptitsyn OB. Molten globule-like state of cytochrome c under conditions simulating those near the membrane surface. Biochemistry. 1996; 35: 6058-63.  158.  Rosell FI, Mauk MR, Mauk AG. pH- and metal ion-linked stability of the hemopexin-heme complex. Biochemistry. 2005; 44: 1872-9.  159.  Guntert P, Salzmann M, Braun D, Wuthrich K. Sequence-specific NMR assignment of proteins by global fragment mapping with the program MAPPER. J Biomol NMR. 2000; 18: 129-37.  160.  Iwahana H, Yoshimoto K, Shigekiyo T, Shirakami A, Saito S, Itakura M. Molecular and genetic analysis of a compound heterozygote for dysprothrombinemia of prothrombin Tokushima and hypoprothrombinemia. Am J Hum Genet. 1992; 51: 1386-95.  161.  Bloom JW, Mann KG. Prothrombin domains: circular dichroic evidence for a lack of cooperativity. Biochemistry. 1979; 18: 1957-61.  162.  Bloom JW, Mann KG. Metal ion induced conformational transitions of prothrombin and prothrombin fragment 1. Biochemistry. 1978; 17: 4430-8.  163.  Hampton RY. ER stress response: getting the UPR hand on misfolded proteins. Curr Biol. 2000; 10: R518-21.  132  164.  Chen Z, Pelc LA, Di Cera E. Crystal structure of prethrombin-1. Proc Natl Acad Sci U S A. 107: 19278-83.  165.  Debnath DK, Mukhopadhyay K, Basak S. Acid-induced denaturation and refolding of prothrombin. Biophys Chem. 2005; 116: 159-65.  166.  Cavallini L, Alexandre A. Ca2+ efflux from platelets. Control by protein kinase C and the filling state of the intracellular Ca2+ stores. Eur J Biochem. 1994; 222: 693-702.  167.  Leslie AG. The integration of macromolecular diffraction data. Acta Crystallogr D Biol Crystallogr. 2006; 62: 48-57.  168.  The CCP4 suite: programs for protein crystallography. Acta Crystallogr D Biol Crystallogr. 1994; 50: 760-3.  169.  Emsley P, Cowtan K. Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr. 2004; 60: 2126-32.  170.  McCoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD, Storoni LC, Read RJ. Phaser crystallographic software. J Appl Crystallogr. 2007; 40: 658-74.  171.  Guillaume YC, Guinchard C, Berthelot A. Affinity chromatography study of magnesium and calcium binding to human serum albumin: pH and temperature variations. Talanta. 2000; 53: 561-9.  172.  Sachs CE, Bourdeau AM. Bovine serum albumin (BSA)-calcium binding studies with a calcium selective liquid membrane electrode. Preliminary report. Clin Orthop Relat Res. 1971; 78: 24-9.  173.  Thomas WR, Seegers WH. Terminal amino acids of bovine prothrombin and thrombin preparations. Biochim Biophys Acta. 1960; 42: 556-7.  174.  Marchler-Bauer A, Anderson JB, Chitsaz F, Derbyshire MK, DeWeese-Scott C, Fong JH, et al. CDD: specific functional annotation with the Conserved Domain Database. Nucleic Acids Res. 2009; 37: D205-10.  175.  Walz DA, Seegers WH. Amino acid sequence of human thrombin A chain. Biochem Biophys Res Commun. 1974; 60: 717-22.  176.  Marino F, Chen ZW, Ergenekan CE, Bush-Pelc LA, Mathews FS, Di Cera E. Structural basis of Na+ activation mimicry in murine thrombin. J Biol Chem. 2007; 282: 16355-61.  177.  Rippon WB, Hiltner WA. The 225-240-nm circular dichroism band in disordered and charged polypeptides. Macromolecules. 1973; 6: 282-5.  133  178.  DiBella EE, Maurer MC, Scheraga HA. Expression and folding of recombinant bovine prethrombin-2 and its activation to thrombin. J Biol Chem. 1995; 270: 163-9.  179.  Sheehan JP, Sadler JE. Molecular mapping of the heparin-binding exosite of thrombin. Proc Natl Acad Sci USA. 1994; 91: 5518-22.  180.  Horne MK, 3rd, Gralnick HR. The oligosaccharide of human thrombin: investigations of functional significance. Blood. 1984; 63: 188-94.  181.  Chang JY. The structures and proteolytic specificities of autolysed human thrombin. Biochem J. 1986; 240: 797-802.  182.  Yang L, Manithody C, Rezaie AR. Activation of protein C by the thrombinthrombomodulin complex: cooperative roles of Arg-35 of thrombin and Arg-67 of protein C. Proc Natl Acad Sci U S A. 2006; 103: 879-84.  183.  Gandhi PS, Chen Z, Di Cera E. Crystal structure of thrombin bound to the uncleaved extracellular fragment of PAR1. J Biol Chem. 2010; 285: 15393-8.  184.  Carter WJ, Cama E, Huntington JA. Crystal structure of thrombin bound to heparin. J Biol Chem. 2005; 280: 2745-9.  185.  Sheehan JP, Sadler JE. Molecular mapping of the heparin-binding exosite of thrombin. Proc Natl Acad Sci U S A. 1994; 91: 5518-22.  186.  Sheehan JP, Wu Q, Tollefsen DM, Sadler JE. Mutagenesis of thrombin selectively modulates inhibition by serpins heparin cofactor II and antithrombin III. Interaction with the anion-binding exosite determines heparin cofactor II specificity. J Biol Chem. 1993; 268: 3639-45.  187.  Stone SR, Hofsteenge J. Effect of heparin on the interaction between thrombin and hirudin. Eur J Biochem. 1987; 169: 373-6.  188.  Arosio D, Ayala YM, Di Cera E. Mutation of W215 compromises thrombin cleavage of fibrinogen, but not of PAR-1 or protein C. Biochemistry. 2000; 39: 8095-101.  189.  Gandhi PS, Page MJ, Chen Z, Bush-Pelc L, Di Cera E. Mechanism of the anticoagulant activity of thrombin mutant W215A/E217A. J Biol Chem. 2009; 284: 24098-105.  190.  Berny-Lang MA, Hurst S, Tucker EI, Pelc LA, Wang RK, Hurn PD, et al. Thrombin mutant W215A/E217A treatment improves neurological outcome and  134  reduces cerebral infarct size in a mouse model of ischemic stroke. Stroke. 42: 1736-41. 191.  Vogt AD, Bah A, Di Cera E. Evidence of the E*-E equilibrium from rapid kinetics of Na+ binding to activated protein C and factor Xa. J Phys Chem B. 114: 16125-30.  192.  Ye J, Liu LW, Esmon CT, Johnson AE. The fifth and sixth growth factor-like domains of thrombomodulin bind to the anion-binding exosite of thrombin and alter its specificity. J Biol Chem. 1992; 267: 11023-8.  193.  Cunningham MT, Nelsestuen GL. Comparison of the kinetic behavior of human and bovine proteins in the heparin-catalyzed antithrombin III/thrombin reaction. Biochim Biophys Acta. 1987; 911: 66-70.  194.  MacGillivray RTA, Degen SJF, Chandra T, Woo SLC, Davie EW. Cloning and analysis of a cDNA encoding bovine prothrombin. Proc Natl Acad Sci USA. 1980; 77: 5153-7.  195.  Wells CM, Di Cera E. Thrombin is a Na+-activated enzyme. Biochemistry. 1992; 31: 11721-30.  196.  Briet E, Noyes CM, Roberts HR, Griffith MJ. Cleavage and activation of human prothrombin by Echis carinatus venom. Thromb Res. 1982; 27: 591-600.  197.  Speijer H, Govers-Riemslag JW, Zwaal RF, Rosing J. Prothrombin activation by an activator from the venom of Oxyuranus scutellatus (Taipan snake). J Biol Chem. 1986; 261: 13258-67.  198.  Bowman LJ, Anderson CD, Chapman WC. Topical recombinant human thrombin in surgical hemostasis. Semin Thromb Hemost. 2010; 36: 477-84.  199.  Greenhalgh DG, Gamelli RL, Collins J, Sood R, Mozingo DW, Gray TE, et al. Recombinant thrombin: safety and immunogenicity in burn wound excision and grafting. J Burn Care Res. 2009; 30: 371-9.  200.  Lundblad RL, Bradshaw RA, Gabriel D, Ortel TL, Lawson J, Mann KG. A review of the therapeutic uses of thrombin. Thromb Haemost. 2004; 91: 851-60.  201.  Lew WK, Weaver FA. Clinical use of topical thrombin as a surgical hemostat. Biologics. 2008; 2: 593-9.  202.  Diesen DL, Lawson JH. Bovine thrombin: history, use, and risk in the surgical patient. Vascular. 2008; 16 Suppl 1: S29-36.  135  203.  Lawson JH. The clinical use and immunologic impact of thrombin in surgery. Semin Thromb Hemost. 2006; 32 Suppl 1: 98-110.  204.  Heffernan JK, Ponce RA, Zuckerman LA, Volpone JP, Visich J, Giste EE, et al. Preclinical safety of recombinant human thrombin. Regul Toxicol Pharmacol. 2007; 47: 48-58.  205.  Boissel JP, Le Bonniec B, Rabiet MJ, Labie D, Elion J. Covalent structures of beta and gamma autolytic derivatives of human alpha-thrombin. J Biol Chem. 1984; 259: 5691-7.  206.  Bell R, Stevens WK, Jia Z, Samis J, Cote HC, MacGillivray RT, et al. Fluorescence properties and functional roles of tryptophan residues 60d, 96, 148, 207, and 215 of thrombin. J Biol Chem. 2000; 275: 29513-20.  207.  De Filippis V, De Dea E, Lucatello F, Frasson R. Effect of Na+ binding on the conformation, stability and molecular recognition properties of thrombin. Biochem J. 2005; 390: 485-92.  208.  Petrera NS, Stafford AR, Leslie BA, Kretz CA, Fredenburgh JC, Weitz JI. Long range communication between exosites 1 and 2 modulates thrombin function. J Biol Chem. 2009; 284: 25620-9.  209.  Isetti G, Maurer MC. Employing mutants to study thrombin residues responsible for factor XIII activation peptide recognition: a kinetic study. Biochemistry. 2007; 46: 2444-52.  210.  Papaconstantinou ME, Gandhi PS, Chen Z, Bah A, Di Cera E. Na+ binding to meizothrombin desF1. Cell Mol Life Sci. 2008; 65: 3688-97.  211.  Hwang HS, Kim DW, Kim SS. Structure-activity relationships of the human prothrombin kringle-2 peptide derivative NSA9: anti-proliferative activity and cellular internalization. Biochem J. 2006; 395: 165-72.  212.  Amara U, Rittirsch D, Flierl M, Bruckner U, Klos A, Gebhard F, et al. Interaction between the coagulation and complement system. Adv Exp Med Biol. 2008; 632: 71-9.  213.  Kjellberg M, Ikonomou T, Stenflo J. The cleaved and latent forms of antithrombin are normal constituents of blood plasma: a quantitative method to measure cleaved antithrombin. J Thromb Haemost. 2006; 4: 168-76.  214.  Danckwardt S, Gehring NH, Neu-Yilik G, Hundsdoerfer P, Pforsich M, Frede U, et al. The prothrombin 3'end formation signal reveals a unique architecture that is sensitive to thrombophilic gain-of-function mutations. Blood. 2004; 104: 428-35.  136  215.  Danckwardt S, Hartmann K, Gehring NH, Hentze MW, Kulozik AE. 3' end processing of the prothrombin mRNA in thrombophilia. Acta Haematol. 2006; 115: 192-7.  216.  Lincz LF, Scorgie FE, Enjeti A, Seldon M. Variable plasma levels of Factor V Leiden correlate with circulating platelet microparticles in carriers of Factor V Leiden. Thromb Res. 2011 [Epub ahead of print].  217.  Zakai NA, McClure LA. Racial Differences in Venous Thromboembolism. J Thromb Haemost. 2011 [Epub ahead of print].  218.  Emeis JJ, Jirouskova M, Muchitsch EM, Shet AS, Smyth SS, Johnson GJ. A guide to murine coagulation factor structure, function, assays, and genetic alterations. J Thromb Haemost. 2007; 5: 670-9.  219.  Mullins ES, Kombrinck KW, Talmage KE, Shaw MA, Witte DP, Ullman JM, et al. Genetic elimination of prothrombin in adult mice is not compatible with survival and results in spontaneous hemorrhagic events in both heart and brain. Blood. 2009; 113: 696-704.  220.  Sun WY, Coleman MJ, Witte DP, Degen SJ. Rescue of prothrombin-deficiency by transgene expression in mice. Thromb Haemost. 2002; 88: 984-91.  221.  Westrick RJ, Winn ME, Eitzman DT. Murine models of vascular thrombosis (Eitzman series). Arterioscler Thromb Vasc Biol. 2007; 27: 2079-93.  137  

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.24.1-0072069/manifest

Comment

Related Items