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Membrane binding properties of prothrombin and other gamma-carboxyglutamic acid-containing coagulation… Krisinger, Michael J. 2007

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Membrane Binding Properties of  Prothrombin and other Gamma-Carboxyglutamic Acid-Containing Coagulation Proteins by Michael J. Krisinger B.Sc., University of  Calgary, 2001 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Biochemistry and Molecular Biology) THE UNIVERSITY OF BRITISH COLUMBIA January 2007 © Michael J. Krisinger, 2007 Abstract Haemostasis is a highly regulated, fundamental,  physiological process featuring  numerous peripheral membrane proteins. Of  these, the membrane and calcium binding properties of  the vitamin K-dependent proteins are dependent on a common N-terminal y-carboxyglutamic acid (Gla)-containing domain. Previous work on Gla proteins has provided a wealth of  affinity  and kinetic membrane binding information.  These studies have employed a number of  biophysical techniques using artificial  phosphatidylserine-containing model membranes. However, many aspects of  the membrane binding interaction, in terms of  mechanism and modulation by protein cofactor  remain obscure. This thesis examines two methods for  studying the membrane binding properties of  human plasma derived Gla proteins with emphasis on prothrombin. In Chapter 3 differential  centrifugation  combined with immunoaffinity  detection was used to quantify  the effect  the cofactor  Factor Va had on the membrane binding affinity  of  prothrombin for  membrane. Factor Va bound to anionic phospholipid membrane undoubtedly enhanced the membrane binding affinity  of  prothrombin relative to prothrombin binding in the absence of  the cofactor.  Thus, these results indicate that Factor Va can recruit prothrombin or prethrombin 1, a Gla-domain less fragment  of  prothrombin, to the membrane surface,  plausibly contributing to its cofactor  function. In Chapters 4 and 5, surface  plasmon resonance (SPR) was used to evaluate the Ca2+-specific binding properties of  a number of  Gla proteins to immobilized membranes. Membrane affinity, molar binding preference  and kinetics controlling complex formation  and complex breakdown varied widely between Gla proteins. The comparative results obtained by SPR indicate that the majority of  homologous Gla proteins bind membranes with a complex mechanism which may involve membrane induced protein dimers. Unlike prothrombin, the binding profiles  for fragment  1 and fragment  1.2 fitted  closely to a one-site binding model. Apparent biphasic association and biphasic dissociation phases were observed for  prothrombin and commonly amongst the other Gla proteins at a wide range of  protein concentrations including physiological concentrations. For prothrombin, dimerization appears to be specific  to the protease domain as neither fragment  1 nor fragment  1.2 displays such binding complexities. It is possible that dimerization increases the half-life  of  membrane-bound Gla proteins thereby promoting their participation in complex assembly and function. Table of  Contents Abstract ii Table of  Contents iv List of  Tables vii List of  Figures viii List of  Abbreviations x Acknowledgements xiii Chapter 1. Introduction 1 1.1 Blood coagulation 1 1.2 Gla proteins ....3 1.2.1 Prothrombin 3 1.2.2 Other Gla proteins 5 1.3 Procoagulant membranes 8 1.4 Gla protein membrane interaction 11 1.5 Techniques to study protein-membrane interactions 15 1.5.1 Equlibrium binding 15 1.5.2 Kinetic analysis 17 1.6 Surface  plasmon resonance 18 1.6.1 Overview 18 1.6.2 Optical Configuration  and Detection Principles 20 1.7 Objectives and Overview 21 Chapter 2. Methods 24 2.1 Materials, proteins and miscellaneous reagents 24 2.2 Liposome preparation 25 2.3 SDS-PAGE electrophoresis, western blotting and densitometry 26 2.4 Differential  centrifugation  (FVa mediated binding) 28 2.5 Thrombin generation assay 29 2.6 Prothrombin enzymatic digestion 30 2.7 Amino-terminal sequence analysis 30 2.8 Surface  plasmon resonance: 31 2.8.1 Membrane immobilization 31 2.8.2 Protein binding experiments 31 2.8.3 Data analysis 32 Chapter 3. Effect  of  Factor Va on Prothrombin-Membrane Interaction 34 3.1 Rationale 34 3.2 LV characterization by prothrombinase activity 35 3.3 Factor Va mediated prothrombin binding: immunoaffinity  quantification  37 3.4 Determination of  -50 kDa species 46 3.5 Discussion 48 Chapter 4. Mechanism of  Prothrombin-Membrane Interaction 50 4.1 Rationale 50 4.2 Membrane immobilization and stability 51 4.3 Validation of  interactive membrane surface  prior to detailed kinetic analysis 54 4.3.1. BSA binding .. 54 4.3.2. Mass transport 56 4.3.3. Specificity  of  membrane interaction 57 4.4 Fl, F1.2 and prothrombin membrane binding: Affinity  and qualitative kinetic analysis.. 60 4.5 DOPS dependence of  prothrombin binding 63 4.5.1 Non-cooperative binding 67 4.6 On the binding mechanism of  prothrombin 69 4.6.1 Prothrombin membrane binding involves a linked reaction mechanism 69 4.6.2 Stability of  membrane bound species is dependent on prothrombin concentration.... 70 4.6.3 Analysis of  cross-linked prothrombin dimer membrane binding 71 4.6.4 Homogeneous Analyte 74 4.7 Estimation of  kinetic parameters 75 4.8 Inhibition of  prothrombin membrane binding by des-Gla prothrombin 78 4.9 Discussion 81 4.9.1 The prothrombin-membrane interaction is biphasic 81 4.9.2 Fast prothrombin-membrane phases are mediated by 82 fragment  1 82 4.9.3 Dimerization model for  slow prothrombin-membrane binding phases 82 4.9.4 Linked mechanism for  prothrombin-membrane interaction 83 4.9.5 Previous studies concur with membrane induced dimerization 85 4.9.6 Apparent discrepancies from  previous studies about the secondary kinetic event 86 4.9.7 Other models to explain prothrombin membrane binding 87 4.9.8 Significance  of  prothrombin dimerization 94 Chapter 5. Comparison of  Coagulation Gla Protein-Membrane Interactions 96 5.1 Rationale 96 5.2 Results 99 5.2.1 Purity analysis by SDS-PAGE 99 5.2.2 Gla protein binding profile  to 25% DOPS-containing membrane 101 5.3 Discussion 110 5.3.1 Binding comparison of  Gla proteins 110 5.3.2 Zymogen versus activated enzyme 113 5.3.3 Membrane binding is influenced  by regions outside of  Gla 115 5.3.4 Vitamin K-dependent protein dimerization 115 Chapter 6. Summary and General Discussion 118 Chapter 7. Future Directions 122 Bibliography 126 List of  Tables Table 1 Comparison of  vitamin K-dependent Gla proteins involved in blood coagulation 6 Table 2 Binding affinity  of  prothrombin, fragment  1, fragment  1.2 and xl-prothrombin to DOPS-containing membrane 65 Table 3 Parameters obtained from  global fitting  77 Table 4 Binding models for  peripheral membrane proteins 89 Table 5 Prothrombin membrane induced dimerization: Is there another model or process to explain the second kinetic event involved in membrane binding? 91 Table 6 Comparison of  membrane binding properties of  Gla proteins assessed by SPR 107 Table 7 Dissociation constants determined for  Protein C I l l List of  Figures Figure 1 Domains and fragments  of  prothrombin 3 Figure 2 Immobilization strategy and detection principle of  SPR 19 Figure 3 Prothrombinase activity of  LUV and LV phospholipid surfaces  as a function  of  total phospholipid concentration 36 Figure 4 Optimization of  differential  centrifugation  experiments: Effect  of  large vesicle concentration 39 Figure 5 Factor Va-dependent prothrombin binding to BPS/EPC (25%/75%) large vesicles.... 41 Figure 6 Prothrombin concentration standard curve 42 Figure 7 Prothrombin membrane binding: dependence on large vesicle and Factor Va 44 Figure 8 Factor Va mediated prothrombin binding to BPS/EPC (25/75) large vesicles assessed by differential  centrifugation  coupled with immunoaffinity  detection 45 Figure 9 Cleavage analysis of  prothrombin by thrombin 47 Figure 10 Liposome immobilization using the Biacore Sensor Chip LI 52 Figure 11 Baseline stability of  immobilized membrane and reference  surfaces  over time 54 Figure 12 BSA binding to immobilized DOPS-containing membrane 55 Figure 13 Prothrombin membrane interaction under variable flow  rate 57 Figure 14 Protein and DOPS dependent binding specificity  59 Figure 15 Fragment 1, fragment  1.2 and prothrombin binding profiles  to 25% DOPS-containing membrane 61 Figure 16 Prothrombin binding isotherms at different  percentage DOPS membrane concentration 64 Figure 17 Cooperativity analysis for  prothrombin interaction with DOPS-containing membrane. 68 Figure 18 Membrane dissociation of  prothrombin is dependent on duration of  association phase. 70 Figure 19 Initial extent of  membrane dissociation is dependent on prothrombin concentration. 71 Figure 20 xl-prothrombin dimer binding profile  to 25% DOPS-containing membrane 73 Figure 21 Characterization of  proteins by SDS-polyacrylamide gel electrophoresis 75 Figure 22 Determination of  kinetic parameters of  prothrombin and derivatives binding to 25% DOPS containing membrane using SPR 76 Figure 23 Des-Gla prothrombin inhibition of  prothrombin membrane interaction 80 Figure 24 Model for  prothrombin membrane binding based on a linked reaction mechanism involving membrane induced dimerization of  the Protease domain 84 Figure 25 Amino acid sequence alignment of  the Gla domain of  human Gla proteins involved in blood coagulation 97 Figure 26 Structural comparison of  three homologous Gla domains 98 Figure 27 Characterization of  proteins by SDS-polyacrylamide gel electrophoresis 101 Figure 28 Gla protein binding profiles  to 25% DOPS-containing membrane 105 List of  Abbreviations AP activation peptide APC activated protein C a-PT mouse monoclonal antibody against kringle 2 of  prothrombin BCA bicinchoninic acid BPS bovine brain phosphatidyl-serine BSA bovine serum albumin DOPS 1,2-dioleoyl-.s'tt-glycero-3-[phospho-L-serine] EDTA ethylenediaminetetraacetic acid EGF epidermal growth factor EPC egg L-lecithin-(phosphatidylcholine) F1 fragment  1 F1.2 fragment  1.2 FYa Factor Va FRET fluorescence  resonance energy transfer Gla y-carboxyglutamic acid HBS hepes buffered  saline HBC HBS plus BSA plus calcium HEPES 4-(2-Hydroxyethyl)piperazine-l -ethanesulfonic  acid HRP-2°Ab horseradish peroxidase conjugated goat anti-mouse IgG Kd dissociation constant rsapp ^d apparent dissociation constant LUV large unilamellar vesicle LV large vesicle MLV multilamellar vesicle Mr molecular weight PC phosphatidylcholine PEG polyethylene glycol POPC 1 -palmitoyl-2-oleoyl-.w-glycero-3-phosphocholine Prel pre thrombin 1 PS phosphatidylserine QCM-D quartz crystal microbalance with dissipation R m a x maximum response RU response unit S-2238 //-D-phenylalanyl-L-pipecolyl-L-arginine-P-nitroanilide SDS-PAGE sodium dodecyl sulphate - polyacrylamide gel electrophoresis SHBG sex hormone binding globulin SPR surface  plasmon resonance SUV small unilamellar vesicle TIR total internal reflection TIR-FM TIR-fluorescence  microscopy TIR-FPR TIR-fluorescence  photobleaching recovery TBS tris buffered  saline TBS-T TBS plus Tween-20 TRIS tris (Hydroxymethyl) aminomethane TSR thrombin sensitive region xl-prothrombin cross-linked prothrombin dimer ZPI protein Z dependent protease inhibitor Acknowledgements I would like to thank a number of  people that have helped me out during my time here at UBC. I greatly appreciated the scientific  discussions I had during my directed studies course with Drs. Dana Devine and Ed Pryzdial. They also provided great support on my project as committee members. A special thanks to Ed for  his help and guidance that helped make my project productive. And also, for  allowing me to take part at countless of  his lab meeting - an area where I was able to soak up a lot of  knowledge. I like to thank the numerous and enlightening conversations I have had with members of  the MacGillivray Lab, past and present, and for  their support and camaraderie. Special thanks to Iain, Cedric, Les, Dave, Mike P., Jeff,  Mark, and Ian T. that were especially frequent  at the Friday beer sessions in the MacGillivray lab. And the good times I had with you guys - you made this place a lot of  fun. I like to also thank my family,  friends  and Anne that have been there for  me at times when I needed support. Lastly, my supervisor, Ross MacGillivray, always understanding and patient. Thanks for  having me in your lab and sending me to several conferences  - the learning experiences I got from  them are irreplaceable and everlasting. I wish you all the best. Chapter 1. Introduction 1.1 Blood coagulation Blood coagulation is a highly regulated, fundamental,  physiological process required to stop blood from  leaking out of  the vasculature. Subsequent to vessel injury, platelets aggregate and activate setting off  a series of  membrane bound clotting reactions that result in a primary hemostatic plug and preventing further  blood loss. The end result of  this process is the production of  an insoluble fibrin  mesh or clot, which maintains the integrity of  the circulatory system. At sites of  vascular damage, initiation of  coagulation occurs when prothrombotic sub-endothelial tissue elements, usually hidden from  circulation, are exposed and platelets accumulate. Membranes containing anionic phospholipids promote the assembly of  coagulation factors  on their surface;  this in turn results in a dramatic acceleration of  the proteolytic reactions that result in thrombin formation.  The initiating pathway of  blood coagulation is known as the tissue factor  pathway or historically as the extrinsic pathway. The tissue factor  pathway is initiated when the integral membrane protein tissue factor  (extrinsic to blood) comes in contact with blood after  vascular injury (i). Tissue factor  interacts with the zymogen Factor VII, or with its activated form,  Factor Vila, which is continuously present at low levels in the circulation. The tissue factor-Factor  Vila complex converts the zymogens Factor IX, Factor X, and Factor VII itself  into active serine protease enzymes. Although assembly and catalytic activity of  the tissue factor-Factor  Vila complex is effective  in the absence of  anionic phospholipids, its activity is enhanced by phosphatidylserine (2). However, surface  exposure of phosphatidylserine on procoagulant membranes such as activated platelets is essential in promoting membrane assembly and efficient  catalytic activity of  two subsequent enzyme complexes, namely tenase and prothrombinase. The tenase complex forms  when the active protease Factor IXa binds to a high affinity  site created by the interaction of  the cofactor  Factor Villa with a phosphatidylserine-containing membrane surface  and calcium ions (3). Tenase rapidly converts the zymogen Factor X into its active protease Factor Xa in the presence of calcium ions. Likewise, Factor Xa binds to its high affinity  site created by the interaction of  the cofactor  Factor Va with a phosphatidylserine-containing membrane surface  and calcium ions (4). The prothrombinase complex rapidly converts prothrombin into the multifunctional  protease thrombin. Through its ability to promote further  platelet aggregation and fibrin  formation, thrombin ensures efficient  hemostatic plug formation  at the site of  injury. Thrombin also activates Factor XIII to Factor XHIa, which stabilizes the fibrin  mesh via its transglutaminase activity. Phosphatidylserine is equally important in cutting back the thrombin formation  by the anticoagulant protein C pathway (5). After  being activated by the thrombin-thrombomodulin complex on endothelial cells, protein C and its cofactor  protein S degrade Factors Villa and Va when bound to a phosphatidylserine-containing membrane surface.  This leads to disassembly of the tenase and prothrombinase complexes and in conjunction with a number of  serine protease inhibitors, down-regulates coagulation. 1.2 Gla proteins 1.2.1 Prothrombin Human prothrombin is comprised of  a single glycopolypeptide chain of  579 amino acids that circulates in blood at an average concentration of  1.4 jjM (6, 7). Post-translational modifications include vitamin K-dependent y-carboxylation of  10 glutamic acid residues (<§) and glycosylation at three sites (9). Prothrombin consists of  four  domains. Starting from  the N-terminus, these include the y-carboxyglutamic acid rich (Gla) domain, two Kringle domains and a C-terminal Protease domain which is homologous to the chymotrypsin/trypsin family  (Figure 1). Figure 1 Domains and fragments  of  prothrombin. Prothrombin consists of  four  domains. From the N-terminus there is a negatively charged y-carboxyglutamic acid (Gla) domain which mediates prothrombin binding to procoagulant phospholipid surfaces.  Following this domain are two kringle domains, which are thought to be involved in protein-protein interactions, and lastly the inactive Protease domain which is converted to the thrombin upon prothrombin activation. Disulfide  bonds and cleavage sites are also indicated. Nomenclatures of  prothrombin fragments  used throughout this study are also indicated. Protease domain prothrombin des-gla prothrombin prethrombin 1 prethrombin 2 fragment 1.2 fragment 1 A number of  different  fragments  of  prothrombin can be produced by enzymatic cleavages, which have been used successfully  in studies relating function  to the various domains. Thrombin auto-catalytically cleaves prothrombin at Arg155 and Arg284 forming  fragment  1/prethrombin 1 and fragment  1.2 + 13 residues/prethrombin 2 des-13 cleavage product pairs, respectively (10). Similarly, Factor Xa cleaves at two sites: Arg271 and Arg320 forming  fragment  1.2/prethrombin 2 and disulfide  linked 2-chained meizothrombin, respectively. Finally, in a non-physiological reaction, chymotrypsin cleaves at Tyr44 producing Gla and des-Gla prothrombin. In  vivo, prothrombin is activated by prothrombinase, an enzyme complex composed of  the membrane-bound protease Factor Xa, its membrane-bound cofactor  Factor Va, an anionic phospholipid membrane and calcium ions. Prothrombinase catalyzes the cleavage of  the zymogen prothrombin to the multifunctional  serine protease thrombin. The combined effect  of these interactions on prothrombin activation results in a 280,000-fold  increase in catalytic efficiency  compared to Factor Xa alone (11,  12). Activation of  prothrombin involves cleavage at Arg271 and Arg320 (Figure 1). Accordingly, two cleavage pathways exist; however, the activation mechanism in vivo proceeds via an ordered sequential reaction, with meizothrombin as the sole intermediate (13).  If  released during activation, meizothrombin has anticoagulant function,  as it has an active site and enzymatic activity towards protein C (14,  15). Thrombin generation is mainly concerned with procoagulant functions  including fibrinogen  cleavage, activation of Factors V, VIII & XIII and platelet aggregation (16). Prothrombin dimerization has been reported when bound to anionic phospholipids (17) or at very high concentration (14-200 jjM) in solution (18,  19). However, only the membrane-dependent dimers can form  at or below the physiological concentration of  prothrombin and are likely facilitated  by intermolecular protease domain interactions (17). Many prothrombin interactive sites have been identified  for  the prothrombin-Factor Xa and prothrombin-Factor Va binary complexes. Each of  the prothrombin domains has been implicated in Factor Va interactions. Kringle 1 has been implicated in Factor Va binding (20). Kringle 2 has also been shown to mediate an interaction between prothrombin and Factor Va by data derived from  prothrombin deletion mutants (21)  and a peptide inhibition study (22). One study also suggests that the Gla domain has an interactive site with the cofactor  (23).  Finally the Protease domain has been implicated in mediating prothrombin-Factor Va interaction through the anionic binding proexosite I (24,  25) and residues 473-487 (26). 1.2.2 Other Gla proteins Members of  the vitamin K-dependent Gla proteins involved in blood coagulation include the procoagulant factors;  prothrombin, Factor VII, Factor IX and Factor X and co-regulators of coagulation; protein C, protein S and protein Z. These plasma proteins involved in blood coagulation require vitamin K for  normal biosynthesis. Vitamin K is required for  carboxylation of  specific  glutamic acid residues to y-carboxyglutamic acid. Some properties of  these proteins are shown in Table 1. Table 1 Comparison of  vitamin K-dependent Gla proteins involved in blood coagulation®. post-translational modification plasma concentration (nM) molecular weight KDa carbohydrate p-hydroxylation Y -c a r b o x y | a t i on domain structure % # asp/asn residues # gla residues (N-term to C-term) prothrombin 1400 72 8 0 1 G l a - k r i n g l e - k r i n g l e - p r o t e a s e Factor VII 10 50 13 8 9 Gla-EGF-EGF-protease Factor IX 70-90 55 17 1 12 G la-EGF-EG F-AP-protease Factor X 170 59 15 1 11 Gla-EGF-EGF-AP-protease protein C 65-80 62 23 1 9 Gla-EGF-EGF-AP-protease protein S 145 (free) 360 (total) 69 7 3 10 Gla-TSR-EGF-EGF-EGF-EGF-SHBG like protein Z 45 62 ? ? ? Gla-EGF-EGF-pseudoprotease mature plasma protein" zymogen/cof actor Enzyme(cofactor) catalysis General Function prothrombin one chain zymogen FXa(FVa) to form thrombin numerous; converts fibrinogen to fibrin (pro/anti-coagulant) Factor VII one chain zymoqen FXa, FIXa, thrombin or FXIIa to form FVIIa initiation phase of coaqulation (procoaqulant) Factor IX one chain zymoqen FXIa or FVIIa(TF) to form FIXa propaqation phase of coaqulation (procoaqulant) Factor X two chain zymoqen FIXa(FVIIIa)  or FVIIa(rF) to form FXa propaqation phase of coagulation (procoagulant) protein C two chain zymoqen thrombin(tfirombomocMn)  to form APC APC cleaves Villa and Va (anticoagulant) protein S one chain cofactor - cofactor for APC (anticoagulant) - binds Factor Va and inhibits prothrombinase - binds and inhibits Factor Xa - 60% circulates in plasma in complex with C4b bindinq protein (complement) protein Z one chain cofactor - binds thrombin and directs to endothelial membrane - cofactor for ZPI involved in inhibition of FXa (coagulant property unknown) (a) references: www.haemtech.com, (7), (9), (27), (31), (34), (47), (133) supplementary information Gla - region containing y-carboxyglutamic acid residues EGF - region containing sequences homologous to human epidermal growth factor AP - activation peptide TSR - thrombin sensitive region Pseudoprotease - region which replaces the protease domain in vitamin K-dependent serine proteases SHGB - region which replaces the protease domain APC - activated protein C ZPI - protein Z dependent Protease Inhibitor, a serpin (b) two chained proteins are disulfide linked as Besides having a homologous Gla domain (discussed in section 1.4), the vitamin K dependent proteins possess a similar domain organization. With the exception of prothrombin, the Gla domain is followed  by regions homologous to epidermal growth factor  (EGF domains) for  all other Gla proteins. This 50-60 amino acid, disulfide-bonded, often  calcium bound domain is thought to be responsible for  mediating the many protein-protein interactions involved with these proteins (27). Like the kringle domains of  prothrombin, EGF domains likely provide spacers that allow the proper distance and positioning of  other parts of  the molecule for  optimal biological function  (28).  Thus all Gla proteins are believed to have an elongated structure. For example, prothrombin, having an overall length of  120 A , when bound to membrane has its FXa susceptible peptides bonds placed near the active site of  prothrombinase (29).  Finally, a large, carboxy-terminal domain makes up the other half  mass of  the Gla proteins. For the zymogens prothrombin, Factor VII, Factor X, Factor IX and protein C, this is the inactive serine protease domain. Upon activation via proteolysis, a new N-terminus is created, which inserts into a preformed  hydrophobic pocket by an internal salt bridge believed to trigger the conformational  change from  inactive to a functional  active site with a correctly shaped substrate binding region (30).  In protein Z, which is not a serine protease, the C-terminal region is a pseudoprotease domain since it lacks two of  the three characteristic catalytic triad residues, namely Ser and His (31,  32). Like protein Z, protein S is also a cofactor  lacking enzymatic activity. Its C-terminal domain is homologous to sex hormone binding globulin (SHBG) (33,  34). Finally, other Gla proteins not involved in clotting have been described including Gas 6 (growth factor  (35)),  osteocalcin & matrix Gla protein (bone development (36)),  and a family  of  four  transmembrane Gla proteins (putatively involved in signal transduction (37,  38)). Lastly a number of  Gla containing proteins have been described from  invertebrates (39). 1.3 Procoagulant membranes Membranes containing phosphatidylserine and other anionic phospholipids play an essential role in blood coagulation. In  vivo, suitable membranes for  the interaction of vitamin K-dependent Gla proteins are provided by activated platelets or other cells that have exposed anionic phospholipids to their outer leaflet  (40,  41). In resting platelets, an asymmetric bilayer is maintained with the majority of  phosphatidylserine and phosphatidylethanolamine hidden from  plasma and confined  to the membrane's inner cytoplasmic leaflet  (see (42)  and references  within). The regulation of  membrane lipid sidedness is controlled by a number of  specific  membrane proteins, referred  to as lipid transporters (reviewed in (43)).  Thus in a resting platelet, the bulk composition of  the outer leaflet  is phosphatidylcholine which is inefficient  to procoagulant complex assembly (44).  Exposure of  phosphatidylserine in platelets serves as a second messenger linking platelet activation to fibrin  clotting (45).  By binding to proteases and respective cofactors,  the membrane facilitates  the assembly of  macromolecular complexes which efficiently  convert circulating zymogens to active enzymes (46). Involving both K m and kcat, the precise enzymatic role of  the membrane is incompletely understood but has been attributed to conformation,  orientation and concentration effects resulting in co-localization of  the enzyme, cofactor  and substrate (47).  Binding of  the coagulation enzymes together with their nonenzymatic cofactors  and substrates to phosphatidylserine-containing membranes has been shown to cause conformational changes that serve to induce productive enzyme-substrate interactions. For example, the scissile bonds of  prothrombin and meizothrombin appear to be properly aligned with the Factor Xa active site due to membrane-induced conformational  changes in both substrates and enzyme (29,  48, 49). Moreover, the high local membrane-bound protein concentrations lead to a substantial decrease in apparent K m of  the substrates (e.g Factor X and prothrombin) from  far  above to far  below their respective plasma concentrations (46).  Efficient  membrane binding of  Gla proteins and cofactors  as well as catalysis by the enzyme complexes (e.g. tenase and prothrombinase) requires anionic phospholipids with phosphatidylserine being most effective  (50-52).  In addition, binding of  Factor Va and Factor Xa to naturally occurring phosphatidyl-L-serine is stereospecific  and occurs with lower affinity  to membranes containing phosphatidyl-D-serine (53), indicating specific  proteinaceous sites interact with the serine headgroup moiety. Furthermore, soluble phosphatidylserine molecules have been shown to support prothrombinase assembly and support catalytic activity comparable to membranes (54,  55). These recent findings  question the classical view the membrane surface  plays as often  thought to have "a surface  dimensionality reduction". Collapse of  membrane phospholipid asymmetry not only exposes phosphatidylserine but also leads to an increase of phosphatidylethanolamine in the outer membrane leaflet,  which has been shown to also have a procoagulant effect  (42).  Finally, it should be noted that specific  protein receptors in addition to phospholipid binding sites on the surface  of  activated platelets have also been shown to be important to promote the assembly of  the various coagulation enzyme-cofactor-substrate  complexes (56). Synthetic membranes used to study blood coagulation usually contain the phospholipids phosphatidylcholine, phosphatidylserine and/or more recently phosphatidylethanolamine and can be incorporated into diverse structures including small/large unilamellar vesicles, monolayers and immobilized bilayers. Historically, a composition of  20-25% phosphatidylserine and 75-80% phosphatidylcholine was chosen as a standard to study the enzymatic clotting reactions as this ratio produced optimal activity and most closely matched clotting times observed on platelet membranes (11).  Although the activated platelet outer leaflet  has been shown to contain only 4-10% phosphatidylserine (57), high experimental phosphatidylserine concentrations continue to be used to study binding and enzymatic function  of  clotting factors  (58-63).  Binding affinity  and activation rates are greatly dependent on the molar fraction  of  phosphatidylserine and at sub-optimal phosphatidylserine concentrations, phosphatidylethanolamine also has an enhancing effect.  Another variable which effects  membrane protein binding is the structure of  the membrane. Surface  curvature and accessibility to the hydrocarbon region of  the membrane varies between the different  membrane architectures and consequently has an impact on the binding dependent on hydrophobic interactions. For instance, prothrombin binding affinity  to membranes of  identical composition was dependent on vesicle size with 0.11, 0.23, 0.86 jiM for  liposome diameter of  27.5, 119, and 328 nm (64).  A similar binding preference  was observed with Factor V (65). 1.4 Gla protein membrane interaction The Gla domain has been solely implicated in the membrane contact process of  vitamin K-dependent proteins. Indeed, proteolytic fragments  of  prothrombin including fragment 1 (Fl; Gla and first  Kringle residues 1-155) and fragment  1.2 (F1.2; Gla and both Kringles, residues 1- 273) can interact with membranes, whereas prethrombin 1 (Pre 1; second Kringle and Protease domain, residues 156-579) cannot interact with membranes (66).  Prothrombin deletion studies, using a crude gel filtration  membrane binding assay, have indicated that neither kringle domain contributes to phospholipid binding (67).  Like prothrombin, anionic phospholipid membrane recognition by all of  the homologous Gla proteins has been solely attributed to the Gla domain (67-69). A prerequisite for  prothrombin membrane binding is the saturation of  seven Ca2+ sites in the Gla domain that changes its tertiary structure from  an unfolded  and non-functional conformation  to a tightly folded  domain facilitating  membrane binding (70,  71). This calcium binding requires Gla residues. The Gla domain within the various vitamin K-dependent proteins, comprised of  ~ 45 amino acids, contains between 9 to 12 Gla residues which mediate this calcium interaction. In protein C and prothrombin, a detailed analysis of  the function  of  each of  these Gla residues has been evaluated (68,  72). Of  the Gla residues, nine are strictly conserved throughout the Gla proteins. From crystal structures of  the Gla domain of  prothrombin and Factor Vila, the placement of  these calcium ions in relation to their Gla ligands is nearly identical in the two proteins (28). The conformational  transition induced by the cooperative binding of  calcium ions turns the N-terminal part of  the Gla domain inside out exposing the hydrophobic co-loop to solvent and burying the majority of  the Gla residues. Of  the 7 calcium ions, only 2 are accessible to solvent and may play a role in membrane binding. The other calcium ions are buried and are integral to maintaining the membrane binding conformation  (70,  73). Calcium binding to the Gla domain occurs at free  calcium concentrations (half  maximal binding: 0.5-0.7 mM) only slightly below those in blood (free  Ca2+ = 1.2 mM) (27). Thus, it has been speculated that calcium binding may serve as a regulatory function  in blood coagulation (28).  Another conserved feature  among the Gla proteins is a hexapeptide disulfide  loop which contributes to the overall Ca2+-dependent Gla conformation  (63).  However, it was shown not to be essential to phospholipid binding (63).  Thus, only part of  the Gla structure appears to be involved in binding to membrane. Other notable conserved features  of  the Gla domain are a solvent inaccessible N-terminus and an aromatic cluster at the C-terminal end (70).  Changes at these conserved sites result in the destabilization or alteration in the three-dimensional fold  of  the Gla domain and subsequent loss of  calcium and phospholipid binding (68,  74). Despite the conserved structural features  and sequence similarity, there is a wide range in affinity  of  the Gla proteins for  biological membranes. Although numerous studies focusing  on the various Gla domains have been conducted, no single site or single type of interaction appears to be exclusively responsible for  the affinity  of  the protein-membrane interaction. A hydrophobic  interaction  mediated by the small conformation-dependent ©-loop, was suggested by fluorescence  quenching of  a doxyl spin label within the bilayer (60)  and from  the derived crystal structure (75). However, this was considered insignificant  to prothrombin-membrane affinity  when studies were compared using small unilamellar vesicles (SUV) and large unilamellar vesicles (LUV) despite differences  in headgroup packing and accessibility to the hydrophobic membrane core (64).  Adding to the uncertainty, another study showed that an aberrant ©-loop in a bovine prothrombin molecule lacking the first  four  N-terminal residues caused a modest 5-fold  decrease in membrane affinity  (74),  indicating that other regions are also involved or exposed aberrantly in the mutant. Similarly, site directed mutagenesis of  various residues in the co-loop had only small impact on membrane affinity.  For example, replacement of  Phe4 of  protein C with Gin had no detected impact on membrane affinity  (76).  In addition to a hydrophobic contribution, an electrostatic  interaction  has been postulated, but is proposed to contribute only minimally to binding in a Ca2+ saturated system (77,  78). Specific  phosphatidylserine contact points have been identified.  Using a disulfide constrained co-loop peptide sequence of  Factor IX, Lys5, Leu6, Phe9 and VallO have been directly implicated in phosphatidylserine binding (58).  These findings  have been confirmed  by structural studies using human prothrombin Gla domain in complex with calcium ions and lysophosphatidylserine (75). The binding isotherms obtained by dynamic light scattering suggest the existence of  3-4 phosphatidylserine binding sites per prothrombin molecule (50). Others have speculated additional phosphatidylserine sites on the protein surface  (79,  80). Finally calcium ions in addition to the ones mentioned are bound during membrane contact as was shown for  prothrombin (81)  suggesting that the Gla domain crystal structures solved thus far  in the absence of  membrane are insufficient  to explain membrane binding. Membrane binding is undoubtedly complex and likely requires multiple synergistic interaction types between Gla protein and anionic phospholipid membrane. These multi-facetted  interactions complicate the study of  Gla domain membrane binding and inherently cause the interaction to deviate from  a simple binding model as observed by several other laboratories (82-87). Despite this progress, the mechanism of  the Gla protein-membrane binding interaction remains unclear. In addition, it is surprising that the Gla proteins bind membrane with a great range in affinity.  For instance, bovine protein C and bovine factor  X have only five major differences  in the amino terminal 34 residues, although the binding affinity  for membrane differs  by approximately 400-fold,  with K'' tpp > 15,000 and ~ 40 nM, respectively (79). The reason for  this difference  is still not obvious as mutagenesis studies have not been successful  at completely deciphering this discrepancy (76,  88). Although some non-conserved residues of  the Gla domain are related to affinity  (e.g. (89)),  there appear to be additional factors  that control membrane affinity.  The causal nature of  this complexity remains obscure. Prothrombin is known to have a number of  different  conformations  with unknown function.  A cis to trans isomerization of  Pro22 of  bovine prothrombin has been shown by fluorescence  due to Ca2+ binding (90)  but its impact on membrane binding remains controversial. Fluorescence studies suggest the trans conformer  binds membrane while the Ca2+ bound crystal structure data argues that the cis conformer  binds membrane. Furthermore, molecular dynamics simulations have shown that the cis form  is less likely to occur; however, it is permitted by the structure (91)  suggesting that during membrane binding a cis-trans equilibrium may result. Bovine prothrombin has also been shown to have membrane induced conformational  changes (29). In solution, prothrombin is an elongated molecule with an overall length of  120 A as determined by fluorescence resonance energy transfer  (FRET) measurements (66).  In the presence of  calcium and phosphatidylserine containing membranes, prothrombin was found  to undergo a shape change to a more tightly folded,  compressed, bent molecule with an overall length of  94 A (29). This compression shape change is not observed with meizothrombin, although both have the same shape in solution (66).  Membrane-induced changes in prothrombin secondary structure as well as alterations in interdomain interactions have also been implicated from  thermal denaturation studies (92). These studies together suggest that fragment  2 may act as a hinge region upon membrane binding. If  and how these conformations  of  prothrombin affect  membrane binding is not known. 1.5 Techniques to study protein-membrane interactions A number of  techniques have been used to study protein-membrane interactions. Separation assays generally provide only equilibrium binding data whereas direct assays provide additional information  on the kinetics of  an interaction. 1.5.1 Equlibrium binding Equilibrium separation assays commonly used to study the membrane affinity  of peripheral membrane proteins include differential  centrifugation  and chromatographic gel filtration.  These techniques offer  the advantage of  being inexpensive and require no labelling but, are labour intensive and often  have problems associated with the separation of  free  protein and membrane-bound protein. Centrifugation  assays can give reliable Kd measurements provided that the vesicle or phospholipid-coated bead centrifugation efficiency  is high (95%) which can be verified  by using a radiolabeled lipid. In additional, complexes must be relatively stable (small &0ff)  to allow accurate determination of  bound and free  protein. Gel filtration  chromatography has been used on the basis of  size exclusion to separate the large vesicle with associated protein from  the smaller free  protein (67).  However this technique is less sensitive, requires large amounts of  protein and is not amenable to interactions governed by fast  dissociation. Other less frequendy  used equilibrium binding techniques have also been used to determine peripheral protein-membrane dissociation constants. Requiring fluorescently labelled protein and liposome adsorbed latex beads, flow  cytometry can be used to derive protein ratios of  membrane bound to free.  A biophysical technique called quartz crystal microbalance has been used with prothrombin and other peripheral proteins to study binding to immobilized bilayers by monitoring dissipation and resonance frequency changes at the membrane surface  (87).  In addition to frequency  measurements at various bulk protein concentrations used to derive a dissociation constant, dissipation changes related to frictional  (viscous) losses at the bilayer give insight to rigidification  or intra-molecular protein interaction. Finally, ellipsometry, another biophysical technique requiring adsorbed mono or bi-layers, has also been used for  phospholipid affinity measurements for  a number of  Gla proteins. 1.5.2 Kinetic analysis Stopped flow  light scattering and FRET measurements at right angle are two conventional, frequently  used kinetic analysis methods that enable the direct measurement of  formation  or breakdown of  a peripheral protein - membrane complex. Fluorometric assays based on either intrinsic tryptophan residues in the protein or extrinsically introduced fluorescent  probes on either protein and/or lipid head group, measure changes in the microenvironment of  these reporter groups which are representative of  complex formation.  Membrane association can be followed  by mixing SUVs with peripheral protein or membrane dissociation can be followed  by mixing an equilibrated protein-SUV with excess unlabelled SUVs. The highest sensitivity for binding is obtained with the introduction of  both a donor and an acceptor fluorescent probe attached to either of  the interacting components. Binding is then observed by following  the change in FRET between donor and acceptor group which is proportional to complex formation.  Both light scattering and fluorescence  measurements require small liposomes and dilute protein concentrations (e.g. < 3 |J.M prothrombin) to maintain the vesicles in solution. Stopped flow  measurements allow the accurate determination of fast  kinetic events in the ms range as observed for  many Gla proteins. Equilibrium dissociation constants at surfaces  coated with monolayers have been determined from total internal reflection  (TIR) fluorescence  microscopy (TIR-FM) measurements of  the fluorescence  as a function  of  the concentration of  fluorescently  labelled protein in solution (e.g. prothrombin (93)).  TIR has been combined with fluorescence photobleaching recovery (TIR-FPR) to also provide kinetic binding constants (84,  85). 1.6 Surface  plasmon resonance 1.6.1 Overview Biacore instruments employ surface  plasmon resonance (SPR) to monitor the formation and breakdown of  complexes in real time. This optical biosensor technique provides detailed information  on the binding mechanisms and rate constants associated with macromolecular interactions. In a SPR experiment, one of  the molecules is immobilized on a surface  (referred  to as immobilized ligand) and the other is flowed  past the surface  in solution (referred  to as analyte). In the study of  peripheral protein - membrane interactions, the membrane is directly coupled to the biosensor surface.  This biosensor, known as an LI chip in Biacore terminology has been sequentially derivatized. First, a gold thiolate bond covalently links to a C-16 alkane containing a terminal hydroxyl group. Second, a carboxymethyl dextran (a carboxymethyl glucose polymer) is attached via two additional reactions. The carboxy groups are then derivatized with lipophilic alkyl chains capable of  capturing liposomes by interacting with the acyl chain moieties of phospholipids (Figure 2A). These captured bilayers are chemically and physically stable (94)  and can be used to probe for  peripheral protein interactions. When an analyte molecule interacts with the immobilized membrane ligand a surface  mass change is detected and a signal is generated in real time. Thus, the progress of  an interaction experiment is followed  directly in a sensorgram, a data plot of  response versus time. There are several advantages in using SPR to study interactions including lack of labelling, low sample consumption and high sensitivity. A dextran matrix thin gold coated glass surface lipophilic surface plasmon resonance In thin metal film light source exponentially decaying evanescent field bulk solution with analyte o detector e resonance intensity dip at resonance angle = f(n sur() Figure 2 Immobilization strategy and detection principle of  SPR. A. LI chip showing capture of  liposomes. Adaptedfrom www.biacore.com B. Schematic diagram illustrating a surface  plasmon resonance biosensor. TIR: total internal reflection;  nsurf:  refractive  index of  the bulk solution in the vicinity of  the sensor surface.  Adapted from  P. Schuck (95). 1.6.2 Optical Configuration  and Detection Principles Two basic requirements for  SPR are a source of  polarized light and a metal (e.g. gold) coated glass prism as illustrated in Figure 2B (96).  When polarized light is shone at a glass prism, at an appropriate angle, the incident light is totally reflected,  a principle known as total internal reflection  (TIR). At this critical angle, all photons are reflected and none pass through the prism. If  the prism is coated directly on top of  the TIR surface with a thin (50 nm) layer of  gold, then the set-up is known as the Kretchmann Configuration  (95).  In this configuration,  TIR of  light can be used to excite surface plasmons in the metal surface.  At a specific  angle of  incident light, 0 excitation occurs. The energy of  the incident photons are absorbed and converted to surface  plasmons traveling along the metal surface.  This resonance results in an electromagnetic field composed of  evanescent waves that decay exponentially with increasing distance perpendicular to the sensor surface.  The resonance causes an energy loss in the reflected light, which is visible as a sharp minimum in the angle-dependent reflectance,  an experimentally recorded quantity. The resonance angle (0) strongly depends on the refractive  index of  the sample within the evanescent field  above the sensor surface  (nsurf). Thus, adsorption or desorption of  analytes at the sensor surface  change the refractive index and produce a shift  in resonance angle that can be precisely measured. The shift  in resonance angle has been shown to be directly proportional to the mass that is bound to the sensor surface  (97). The Biacore Instrument consists of  a number of  integrated components that allow data collection under conditions of  continuous flow  (www.biacore.com). The sensor chip surface  forms  one wall of  the flow  cell (dimensions: 1 = 2.4 mm, w = 0.5 mm, h = 0.05 mm). The Biacore 3000 instrument has four  flow  cells in parallel which allow experiments and controls to be performed  with the same analyte sample simultaneously. Samples and reagents are delivered to the surface  through a microfluidic  system that ensures control over sample delivery times. Valves in the microfluidic  system can switch between buffer  and sample with high precision. This ensures that the sample is delivered as a defined  liquid segment with minimum mixing between sample and buffer.  A photo-detector precisely detects an array of  reflected  light angles dependent on surface  plasmon resonance. The shift  in angle required for  resonance, called the SPR-angle response is the quantitative measurement of  analyte binding to the surface.  SPR-angle response is then converted to the characteristic Response Unit (1 RU = 0.0001°) reported in the raw data sensorgrams (98)  which is equivalent to 1 pg/mm2 of  bound protein (96). 1.7 Objectives and Overview Although a wealth of  information  has been gathered, several questions remain about the overall mechanism of  phospholipid binding of  Gla proteins. In this thesis, prothrombin was chosen as the central molecule of  study as it services a pivotal role in coagulation. The impact of  Factor Va on the prothrombin-membrane interaction is addressed in Chapter 2. Although a number of  studies have suggested an interaction between Factor Va and prothrombin, none of  them have been conducted as a direct binding assay in the presence of  membrane. As a potential mechanism leading to prothrombinase assembly and substrate binding, the cofactor  may directly recruit prothrombin on the membrane surface.  Thus, it was my goal to address the question: Does Factor Va enhance the affinity  of  prothrombin for  membrane? Semi-quantitative binding data obtained by differential  centrifugation  experiments implicate the cofactor  in aiding the membrane affinity  of  prothrombin and also its non-Gla containing fragment  prethrombin 1. As outlined in previous introductory sections, the detailed kinetic events governing Gla protein binding have mainly been characterized using light scattering and FRET, and the results of  these studies make up the bulk of  known biophysical data describing the membrane interaction. In Chapter 3, the previously unexploited technique of  surface plasmon resonance is developed to further  probe the membrane interaction kinetics and affinity  of  prothrombin and derivatives of  prothrombin. The main objective of  this chapter is to determine if  the Gla domain - containing derivatives of  prothrombin (fragment  1 and fragment  1.2) behave similarly in membrane binding to prothrombin and, if  not, to determine the causative nature of  the observed differences.  Results of  these studies suggest that prothrombin membrane binding deviates from  the simple 1:1 membrane binding shown for  fragment  1.2, but rather follow  complex binding likely explained by a slow dimerization reaction on the membrane surface. As all Gla proteins involved in blood coagulation contain the highly conserved Gla domain (assessed by either sequence or available structure), it is surprising that the membrane binding properties of  these proteins vary widely in the literature. One problem has been in technique and membrane variability preventing valid comparisons between studies. To alleviate this discrepancy and on the basis of  the results obtained with prothrombin and its fragments,  a thorough membrane binding comparison of  kinetic and affinity  parameters is made of  the other Gla proteins, including zymogens, cofactors  and activated enzyme forms.  In doing so, I wished to address the question whether all Gla proteins have conserved membrane binding properties? As detailed in Chapter 5, results of  these studies indicate that the Gla proteins have adopted a wide range of  membrane binding properties that appear not to be solely caused by Gla domain differences  but also due to differences  in regions removed from  the membrane contact site. The significance of  these findings  and future  directions are also discussed. Chapter 2. Methods 2.1 Materials, proteins and miscellaneous reagents The synthetic lipids l,2-dioleoyl-sn-glycero-3-[phospho-L-serine] (DOPS) and 1-palmitoyl-2-oleoyl-.sn-glycero-3-phosphocholine (POPC) were purchased from  Northern Lipids, Inc. (Vancouver, BC) and Avanti Polar Lipids, Inc. (Alabaster, AL), respectively. The natural lipids L-lecithin-(phosphatidylcholine) from  egg (EPC) and phosphatidyl-serine from  bovine brain (BPS) were purchased from  Sigma-Aldrich (Oakville, ON). Polycarbonate filters  (100 nm diameter) were purchased from  Corning (Acton, MA). The peptidyl substrate, //-D-phenylalanyl-L-pipecolyl-L-arginine-P-nitroanilide (S-2238) was purchased from  Chromgenix (West Chester, OH). Pre-stained protein marker was obtained from  New England BioLabs (Pickering, ON). Human coagulation proteins (prothrombin, Pre 1, Fl, F1.2, a-thrombin, Factor Va, Factor X, Factor Xa, Factor Xa-DEGR, Factor IX, Factor XIa, Protein C, Activated Protein C, Protein S and Protein Z) were purchased from  Haematological Technologies (Essex Junction, VT). Prothrombin-Gla domainless was bought from  Enzyme Research Laboratories (South Bend, IN). Bovine serum albumin was purchased from  Sigma-Aldrich. Cross-linked prothrombin dimer (xl-prothrombin) was graciously provided by Dr. Peter Anderson (University of Ottawa) (77). Mouse monoclonal antibody against kringle 2 of  prothrombin (a-PT) was bought from  Haematological Technologies. Chemiluminescent reagents were purchased from  Pierce (Rockford,  IL) including horseradish peroxidase conjugated goat anti-mouse IgG (HRP-2°Ab) and luminal - peroxide solution. All coagulation proteins were judged to be greater than 98% pure from  an overloaded Coomassie blue-stained sodium dodecyl sulphate (SDS)-PAGE gel. Protein concentrations were determined by using the bicinchoninic acid assay, BCA (Pierce, Milwaukee, WI) and/or by absorbance using the following  extinction coefficients  (E280 1%, 1cm) and molecular weights (Mr) given by the supplier; prothrombin: 13.8, 72,000; prothrombin des-Gla: 14.5, 67,000; Prel: 17.8, 49,900; Fl: 11.9, 21,700; F1.2: 10.8, 34,600, a-thrombin: 18.3, 36,700; Factor Va: 17.4, 168,000; Factor X: 11.6, 58,900; Factor Xa and Xa-DEGR: 11.6, 46,000; Factor IX: 13.2, 55,000; Factor IXa: 14.0, 45,000; protein C: 14.5, 62,000; activated protein C 14.5, 56,200; protein S 9.5, 69,000; and protein Z: 12.0, 62,000. For ease of  comparison, the xl-prothrombin concentrations are given in terms of  prothrombin monomer. All surface plasmon resonance reagents, including LI sensor chips and HBS running buffer  were purchased from  Biacore (Biacore Inc., Piscataway, NJ). HEPES buffered  saline used for liposome immobilization experiments (HBS) consisted of  10 mM HEPES, pH 7.4 and 150 mM NaCl. Running buffer  used for  protein interaction experiments (HBC) consisted of  10 mM HEPES, pH 7.4, 150 mM NaCl, 0.1% bovine serum albumin (BSA) and 5 mM CaCb. All buffers  were filtered  through a 0.22 jim filter  and degassed before  use. All stock solutions were briefly  centrifuged  before  use to remove any potential insoluble material. All other chemicals were from  Sigma Chemical Co. and were of  the highest grade available. 2.2 Liposome preparation Liposomes were prepared as described previously (99) with minor revisions. Briefly, lyophilized lipids were dissolved in chloroform  to approximately 30-100 mM and concentrations were established by inorganic phosphate determination as described previously (100).  The appropriate molar ratios of  phospholipid in chloroform  was dried, first  under a stream of  argon and then under vacuum for  at least 3 hours. The resultant residue of  lipids was resolubilized in HEPES-buffered  saline (HBS) [10 mM HEPES, pH 7.4 and 150 mM NaCl] or HBS containing 15% sucrose (for  sucrose loaded vesicles) to a final  concentration of  approximately 10 mM. The resultant multilamellar vesicle (MLV) suspension was subjected to a rapid freeze-thaw  technique five  times by cycling in liquid nitrogen and warm water. Large unilamellar vesicles (LUV) were generated by extrusion of  MLVs under pressure through two stacked Nucleopore polycarbonate filters  with a 100 nm pore size (10 passes) using an extrusion device obtained from  Northern Lipids (Vancouver, BC). To prepare sucrose loaded large vesicles (LV), MLVs were extruded through an 800 nm pore size filter.  Non-encapsulated sucrose was replaced by HBS by adding equal volume HBS followed  by a 5 min, 14,000g centrifugation.  Sucrose loaded LV in the pellet were resuspended in HBS. Due to the large pore extrusion size LV remained multilamellar. Final liposome concentrations were determined by inorganic phosphate determination. Membrane composition is stated as the mole percentage of DOPS (or BPS) with the remainder of  the phospholipid being POPC (or EPC). The homogeneity and mean diameter of  LVs and LUVs was determined by dynamic light scattering using a NICOMP 370 particle sizer (Nicomp Particle Sizing Inc., Santa Barbara, CA) and found  to be > 1000 nm and 110 +/- 25 nm, respectively. 2.3 SDS-PAGE electrophoresis, western blotting and densitometry For differential  centrifugation  experiments, membrane bound prothrombin concentrations were determined by immunoaffinity  detection. Standard concentration samples along side experimental samples were denatured in Laemmli sample buffer  under reduced conditions for  10 min at 60 °C and subjected to SDS-PAGE on a 12% minigel. After electrophoresis, the separating gel and nitrocellulose paper were briefly  soaked in transfer buffer  (25 mM Tris.HCI/192 mM glycine/10% methanol, pH 8.3) and assembled in a transfer  chamber (Bio-Rad). Proteins were electroblotted onto the nitrocellulose in transfer  buffer  by application of  0.4 A for  2 hr. Nitrocellulose blots were soaked in SuperBlock (Pierce, Rockford,  EL) containing 0.05% Tween-20 for  1 h and then at 4 °C overnight in SuperBlock containing 4 |ig (saturating amounts) of  a-PT per mL, washed in Tris Buffered  Saline containing Tween-20 (TBS-T: 150 mM NaCl, 50 mM Tris.HCl, 0.05 % Tween-20, pH 7.4), and incubated for  1 hr in 40 ng HRP-2°Ab per mL SuperBlock. The blots were washed in TBS-T and for  detection incubated with a HRP chemiluminescent substrate - peroxide solution (Pierce) for  5 min. The image was directly captured on a Syngene GeneGenius Imager (Cambridge, U.K.) and the band density determined prior to pixel saturation using the integrated densitometry software. Experimental bands were quantified  using a prothrombin concentration standard curve. Experiments were performed  and analysed three times. The data are plotted as the mean +/- the standard deviation of  the three results. Prior to SPR analysis, analyte samples were examined for  homogeneity and total protein content by SDS-polyacrylamide gel electrophoresis. Samples were denatured under reducing or non-reducing conditions for  10 min at 95 °C and electrophoresed (120V for 45 min) in a pre-cast 4-20% gradient polyacrylamide SDS gel (Promega, Nepean, ON). Protein bands were visualized following  staining with EZBlue™ Coomassie Brilliant Blue G-250 (Sigma) and destaining in water. The destained gel was subsequently photographed over visible light. Products of  prothrombin proteolysis were examined for  cleavage products by SDS-PAGE (7.5 |ig/lane) or western blot analysis (10 ng/lane). Samples were denatured with SDS under reducing or non-reducing conditions for  10 min at 95 °C and electrophoresed in a 12% polyacrylamide SDS gel. Protein bands were visualized following  staining with Coomassie Blue R-250 (Sigma-Aldrich) and destaining in 40% methanol/5% acetic acid. Alternatively for  western analysis, procedures were the same as the immunoaffinity detection experiments (differential  centrifugation)  above with the exception that images were captured on x-ray film  (HyperFilm ECL, Amersham: Uppsala, Sweden). 2.4 Differential  centrifugation  (FVa mediated binding) In a 10 nL reaction volume, prothrombin (15 - 400 nM) was incubated in the presence and absence Factor Va (350 or 738 nM) with sucrose loaded LV (2400 |xM, 25% BPS) in HBS (20 mM Hepes, 150 mM NaCl, pH 7.4), 2 mM Ca2+ and 0.1% PEG 8000 at 22 °C for  25 min. The equilibrated sample was then carefully  layered on top of  a 200 |iL 15% sucrose sedimentation cushion without mixing the two aqueous solutions. The LV with associated proteins were separated from  unbound protein and pelleted by centrifugation  at 19,000g for  10 min and quantified  by immunoaffinity  detection (see Section 2.3). Experiments were also carried out in the absence of  LV to assess background signal and using a control protein (bacterial protein MT 1704) in place of  Factor Va to assess non-specific  binding. 2.5 Thrombin generation assay Different  membrane preparations were tested for  their ability to enhance thrombin generation by prothrombinase. Membrane preparations were incubated in a flat  bottom microtiter plate with a 5:1 or 3:1 molar ratio of  Factor Va to Factor Xa (at concentrations indicated in the figure  legends) along with excess prothrombin (1.4 jiM final).  Proteins were mixed at 22°C in HBS pH 7.4 supplemented with 0.01% PEG 8000 to minimize protein losses by adsorption. Prothrombinase assembly and subsequent prothrombin activation were initiated by the addition of  Ca2+ (2 mM final).  Continuous shaking during the reaction maintained the membrane preparation in suspension. The reaction was stopped after  an indicated time by the addition of  HBS pH 7.4 containing 0.01% PEG 8000 and 90 mM (final)  EDTA. An aliquot of  the assay solution was diluted in HBS containing 0.01% PEG 8000 and mixed with S-2238 to a final  concentration of  200 fiM at 22°C. The amount of  thrombin that formed  (progress curve) was measured spectrophotometrie ally by the absorbance change at 405 nm in a Spectramax kinetic microplate reader (Molecular Devices). From the change in absorbance at 405 nm the amount of  thrombin was calculated from  a calibration curve made with a purified thrombin standard. The calibration curve was determined with the assay conditions described above. A mean value of  the initial thrombin formation  rate (or Vmax) was determined from  three separate replicates. Experimental data were fit  by non-linear regression analysis using Graphpad Prism 4.0 software  (Graphpad). 2.6 Prothrombin enzymatic digestion Prothrombin (52.5 |ig = 5.4 jiM) was incubated for  5 min in HBS containing 0.01% PEG 8000 and 2 mM Ca2+. At time 0 s, thrombin (3.5 |ig = 675 nM) was added to start the proteolysis reaction a total reaction volume of  140 |iL. At indicated timed intervals 20 |iL aliquots (seven in total) of  the reaction mixture were removed and added to 95 °C sample loading buffer  containing 5% P-mercaptoethanol and analysed by SDS-PAGE analysis (see Section 2.3). For western blot analysis, the same conditions were used except prothrombin (70 ng = 6.94 nM) and thrombin (5 ng = 965 pM) concentrations were changed as indicated. 2.7 Amino-terminal sequence analysis Protein species in the prothrombin sample (10 ng total protein) from  the commercial supplier were separated by SDS-PAGE on 12% reduced gels, transfered  to Immobilon-P membranes (Millipore) in 25 mM Tris-base, 192 mM glycine, (pH 8.3) and 20% methanol, and stained with diluted Coomassie Brilliant Blue R-250. The 50 kDa protein band was cut from  the blots and subjected to protein sequencing using Edman chemistry and a Perkin-Elmer ABI automated sequencer model 476A by S.C. Perry of  the Nucleic Acid-Protein Service Unit (NAPS, University of  British Columbia). 2.8 Surface  plasmon resonance: 2.8.1 Membrane immobilization All SPR experiments were conducted on a Biacore 3000 (Uppsala, Sweden). Prior to lipid immobilization, the lipophilic LI sensor chip was washed with 50 mM octyl glucoside (1 min at 20 ^iL/min). LUV (500 |aM) composed of  either synthetic DOPS/POPC (or natural BPS/EPC) lipids were injected for  17 min at a 3 ^L/min flow rate in HBS running buffer.  LUV were immobilized to a reading of  5000-8500 RU depending on DOPS concentration, with higher DOPS concentrations resulting in lower immobilization values. The use of  new chips was crucial for  reproducibility of membrane immobilization values as used chips bound significantly  less phospholipid membrane. Weakly adhering LUV were removed with five  consecutive 10 mM EDTA pH 8.0 injections (2 min at 20 |iL/min) resulting in a stable membrane surface  as was indicated by an insignificant  loss in SPR signal for  the following  12 hours (data not shown). Prior to protein binding, running buffer  was changed to HBC and flow  cells were equilibrated until the baseline stabilized to less than 0.05 RU/min. An excess concentration of  Ca2+ (5 mM) was included to avoid limiting prothrombin membrane association (101)  and BSA (0.1%) was included to block any non-specific  protein-lipid and protein-protein interactions (102). 2.8.2 Protein binding experiments SPR experiments were performed  at 24 °C with a flow  rate of  20 |jL/min. Association times were typically between 4 and 25 min and dissociation was monitored as indicated on figure  legends. Controls for  bulk refractive  index changes between sample and HBC running buffer,  instrument drift  and non-specific  binding were performed  in parallel with an underivatized flow  cell. A 100% POPC membrane surface  was not used as a reference surface  since very low (< 2% binding compared to 25% DOPS), yet significant  binding was detected. The underivatized flow  cell showed no appreciable change in SPR response upon an injection of  prothrombin and was used as a reference  control. The immobilized membrane surface  was then regenerated by removing membrane bound protein with a 10 mM EDTA pH 8.0 injection, which returned the baseline to the value prior to introducing protein. The binding of  various peripheral proteins (prothrombin, Fl, F1.2, xl-prothrombin and other Gla proteins) was examined by multiple injections of  the analyte diluted in HBC running buffer  over a concentration series typically spanning a 10-fold  range above and below the apparent equilibrium dissociation constant (K%pp) of the interaction. All SPR experiments were carried out with new LI sensor chips, duplicate analyte concentration standards and replicate analyses. 2.8.3 Data analysis BIAevaluation v4.1 was used to make reference  corrections and to display sensorgram data (response units (RU) vs. time). All data was corrected for  non-specific  binding by subtracting the value from  the underivatized reference  flow  cell. A second reference correction was made by subtracting buffer  injections thereby eliminating any possible systematic artefacts  observed between the reaction and reference  surfaces.  The K a p p was determined by measuring the near steady state response units (Req) at several ligand concentrations. Graphpad Prism 4.0 was used to fit  binding data globally to a one-site binding hyperbola according to the relationship R e q = RmaxC/( Kf p +C), where Rmax is the response signal at saturation, C corresponds to the injected analyte concentration (Co), and Kj''p is the equilibrium dissociation constant. The dissociation constant is an approximation (hence Kf p) since the binding model is known to be more complex than the fitted  one-site binding model. Sensorgram data were globally fit  to various models made available through the BIAevaluation v4.1. They included a simple one-site (Langmur) model (A + B AB), a heterogeneous analyte-competition reaction model (A1 + B <-»• A1B; A2 + B A2B), a two-state reaction (conformation  change) model (A + B <-> AB <-> A*B), and a bivalent analyte (first  step: A + B <->• AB; second step: AB + B AB2) model. Together with a surface  dimerization model (first  step A + B <-+ AB; second step AB + AB <-> AAB) provided by Biacore, these models were used to determine which reaction mechanism dominated an interaction. Chapter 3. Effect  of  Factor Va on Prothrombin-Membrane Interaction 3.1 Rationale Previous binding studies between prothrombin and Factor Va have been based on experimental systems in solution lacking an anionic phospholipid membrane. Sedimentation equilibrium analysis has shown that prothrombin and Factor Va form  a weak 1:1 complex in solution with a Kf p = 10 (J.M (103).  Although informative,  this result seems insignificant  to complex formation  in vivo in light of  the prothrombin (1.4 jjM; (104))  and Factor V (21 nM; (105))  concentrations in plasma. Anionic phospholipids (especially phosphatidylserine) present in the outer leaflets  of  activated platelets and at sites where complex assembly occurs, have been shown to be crucial in the forming  of  a fully  active enzyme complex since they determine the proper conformation  and orientation of  prothrombin, Factor Va and Factor Xa (54).  Also, a tubular flow  enzymatic study has shown that prothrombin can directly contribute to the assembly of  the Factor Va-Factor Xa complex on phosphatidylserine-containing membranes (106).  There is also ample indirect evidence, from  prothrombinase kinetic studies using recombinant prethrombin 1 mutants (25), a proexosite 1 directed inhibitor (24),  Factor Va heavy chain directed inhibitors (107),  and a fast  dissociating form  of Factor Va (108)  that indicate a possible direct interaction between prothrombin and Factor Va on the membrane. It was my goal to elucidate the putative binding interaction between prothrombin and Factor Va in the presence of  an anionic phospholipid membrane in a simplified  Factor Xa-free  3-component system. It was anticipated that multiple regions on the prothrombin surface  contribute to the overall binding energy required to bind to the complementary exosites found  on Factor Va. By dissecting these binary protein interactions, I hoped to gain insight to the binding interaction of prothrombin to prothrombinase. Differential  centrifugation  of  large vesicles and associated proteins from  unbound proteins was used as a technique to assess the effect that membrane bound Factor Va had on the prothrombin membrane interaction. A similar method had been employed successfully  to measure cell receptor binding of specific  yet low-affinity  plasma proteins (109).  Since Factor Va forms  a tight complex with membrane with a IQ = 2.7 nM (110),  this technique was anticipated to be useful. 3.2 LV characterization by prothrombinase activity Prothrombin membrane binding was evaluated using sucrose loaded LV. To assess any deleterious effects  the sucrose may have on these liposomes, they were first  characterized in their ability to support prothrombinase activity. As expected, prothrombinase activity was supported by LUV composed of  25% BPS and 75% EPC, giving a saturation curve with half-maximal  activity at -0.95 pM phospholipid and a maximum velocity or kc&t of 80 +/- 5 nM of  thrombin min"1 (nM of  Factor Xa)"1 (Figure 3). 0.0 2.5 5.0 7.5 10.0 12.5 [phospholipid] (nM) 15.0 B c "c I <b E o (0 (0 X 0.00 0.05 0.10 0.15 0.20 0.25 [phospholipid] (nM) 0.30 Figure 3 Prothrombinase activity of  LUV and LV phospholipid surfaces  as a function  of  total phospholipid concentration. Factor Xa (0.17 nM), factor  Va (0.86 nM), and phospholipids were mixed with prothrombin and allowed to reach equilibrium. Reactions were initiated by addition of  2 mM Ca2+, and they were stopped after  4 min by addition of  excess EDTA. The amount of thrombin (Ha) formed  was measured by the absorbance change at 405 nm after  addition of  S-2238 to a final  concentration of  200 pM. A. Shows prothrombinase velocities at various concentrations of  BPS/EPC (25/75) LUV (•) or LV (A). B. Selected data from panel A, used to exemplify  lower concentration data at which phospholipids are rate limiting. Error bars represent the standard deviation from  3 experiments. The curve shape in Figure 3 was typical of  previous studies (11,  102). Sucrose loaded LV gave similar findings  with half-maximal  activity at -1.0 pM phospholipid and a kcat of  75 +/- 5 nM of  thrombin min"1 (nM of  Factor Xa)"1. Upon closer inspection of  the kinetic data at rate limiting phospholipid concentration (0.01 - 0.25 pM), velocities were determined to be 14.3 nM of  thrombin min"1 (nM of  Factor Xa)"1 and 10.2 nM of thrombin min"1 (nM of  Factor Xa)"1 per pM of  LUV and LV, respectively. LVs were 70% as efficient  at activating prothrombin compared to LUVs at sub-saturating concentrations. This difference  was anticipated and thought to be due to the lamellarity difference  between the two vesicle types. LVs were known to be multilamellar in structure and thus have a lower concentration of  catalytically participating phospholipids. LUVs were unilamellar and have an approximately 1:1 ratio of  interior to exposed lipids. Thus, at low phospholipid concentrations, velocity differences  were apparent but were overcome at increased phospholipid concentrations. These studies verify  that sucrose loaded LV retain prothrombinase activity but have a decreased percentage (70% relative to LUV) of  exposed phospholipids and consequently protein binding sites. LV structures were deemed suitable to use in prothrombin membrane binding studies. 3.3 Factor Va mediated prothrombin binding: immunoaffinity quantification Differential  centrifugation  coupled with immunoaffinity  detection was used to demonstrate Factor Va-dependent prothrombin binding to anionic phospholipid derived-vesicles. Large multilamellar vesicles loaded with a dense solution of  sucrose were defined  by size, concentration and composition (25:75 ratio of  phosphatidylserine: phosphatidylcholine), and bound with Factor Va. After  equilibrating the LV with prothrombin, vesicles along with bound protein were separated from  unbound protein in solution using centrifugation.  High concentration of  lipid (> 2000 pM) were required to observe prothrombin in the bound fraction  (Figure 4). 66 KDa — No LV (Background) B \ v HHHHHHHIH I Mmm 66 KDa — 1500 |JM LV /  * 66 KDa — J A " # #  A ' A o* ©• •BflSBHSnHHI  Sfil <y * * , * N  K  N  99 9} K* <v *v <v v I 2400 |JM LV 66 KDa — Figure 4 Optimization of  differential  centrifugation  experiments: Effect  of  large vesicle concentration. Representative western blots of  SDS-PAGE gel probed with saturating amounts of monoclonal anti-human prothrombin directed against fragment  2. Prothrombin (pM concentration indicated above gel lanes) was equilibrated for  25 minutes either in the absence (A), or presence of  1500 pM (B) or 2400 pM LV (C) that had been previously equilibrated with (+) or without 738 nM FVa (-) for  10 min in 20 mM HEPES, pH 7.4, 150 mM NaCl, 2 mM CaCl2, 0.1% PEG 8000. Prothrombin was detected in the bottom after  differential  centrifugation  assay (See Materials & Methods). Location of  66 kDa molecular weight marker indicated for  reference. Lower LV concentrations (< 2000 (iM) resulted in minimal, if  any, amounts of prothrombin in the bound fraction.  This was the case in the absence or presence of  Factor Va. The Factor Va concentrations used were well above the membrane dissociation constant to ensure plenty of  membrane bound Factor Va was present. At prothrombin concentrations over 1 pM, background detection increased and obscured any effect  the inclusion of  LV +/- Factor Va had on prothrombin membrane binding. Differential centrifugation  also revealed a degradation fragment  of  prothrombin of  approximately 50 kDa in the bound fraction.  The presence of  this band was observed exclusively in experiments containing both Factor Va and LV. It was determined that 1-5% (total protein) of  the commercial prothrombin contained this degradation fragment  as estimated from  an overloaded SDS-PAGE gel. Western blot analysis clearly showed an enhanced prothrombin membrane binding affinity  in the presence of  Factor Va compared to prothrombin self-mediated  membrane binding in the absence of  Factor Va. This enhanced affinity  for  LV was specific  to Factor Va as no enhancement was observed in the presence of  a bacterial protein, MT 1704 (Figure 5). Inclusion of  0.1% PEG 8000 in binding buffer  was essential as a protein stabilizing agent and to prevent non-specific  protein binding. 66 KDa 0.07 [jM Prothrombin Figure 5 Factor Va-dependent prothrombin binding to BPS/EPC (25%/75%) large vesicles. Representative western blots of  SDS-PAGE gel probed with saturating amounts of monoclonal anti-human prothrombin directed against fragment  2 subjected to centrifugation  assay (see Materials & Methods). Prothrombin at 0.07 joM (bottom) or 0.35 (iM (top) was equilibrated for  25 min either in the absence ( / -) or presence of  2400 fiM  LV ( / +) that had been previously equilibrated without ( - / ), or with 738 nM MT (MT / ) or with 738 nM FVa (FVa / ) for  10 min in 20 mM HEPES, pH 7.4, 150 mM NaCl, 2 mM CaCl2, 0.1% PEG 8000. Prothrombin was detected in pellets after differential  centrifugation.  Location of  66 KDa molecular weight marker indicated for reference. The amount of  prothrombin in bound fractions  from  the differential  centrifugation experiments was quantified  by densitometry protein bands from  western blots. A prothrombin concentration standard curve was obtained showing a linear relationship of chemiluminescence signal (pixel counts) to picogram quantities of  prothrombin (Figure 6). Assay conditions were optimized to accurately detect down to 10 pg prothrombin. Above 1700 pg, prothrombin concentrations were no longer proportional to pixel count due to signal saturation. A 66 KDa — "• -H H H H H H H H H h I prothrombin (pg) Figure 6 Prothrombin concentration standard curve. A. Concentration standards (triplicate) of  prothrombin (pg amounts indicated) were loaded onto 12% gels and subjected to SDS-PAGE and western blotting as described in Materials and Methods. B. Data points (mean +/- standard deviation) are densitometry integration units (pixel counts) obtained using a Syngene GeneGenius Imager and densitometry software.  Saturation of  enhanced chemiluminescence signal occurred >18 pixel counts (or > 1700 pg prothrombin). Over these protein concentrations, pixel count is no longer proportional to concentration. Western blot results show an enhanced prothrombin membrane binding affinity  compared to the experimental background (Figure 7). Prothrombin membrane binding and background levels were quantified  by performing  prothrombin band densitometry and shown graphically in Figure 8A. Results indicate an enhanced detection of  prothrombin with increasing reaction prothrombin concentrations regardless of  inclusion of  LV and/or Factor Va in reactions. Real prothrombin membrane binding was clearly obscured by background levels. Although with high error, reference  corrected membrane binding data show an increase in prothrombin binding in the presence of  Factor Va compared to without Factor Va (Figure 8B). Prothrombin binding data in the presence of  Factor Va was loosely fitted  to a 2-site binding model (r2 = 0.86). Data was further  corrected for self-mediated  binding illustrating prothrombin binding specifically  to protein site(s) on membrane bound Factor Va (Figure 8C). This data was fitted  to a 1-site binding model (r2 = 0.76) with an K« pp ~ 0.05 jjM. 66 KDa 66 KDa 66 KDa 66 KDa 66 KDa— 0.014 0.070 0.140 0.210 0.350 Figure 7 Prothrombin membrane binding: dependence on large vesicle and Factor Va. Representative western blots (triplicate) of  SDS-PAGE gel probed with saturating amounts of  monoclonal anti-human prothrombin directed against fragment  2 subjected to centrifugation  assay (see Materials & Methods). Prothrombin at indicated concentration (fiM;  right) was equilibrated for  25 min either in the absence ( - / -) or presence of  2400 ^M LV (- / LV) or presence of  both 185 nM Factor Va and 2400 jxM LV (FVa / LV) in 20 mM HEPES, pH 7.4, 150 mM NaCl, 2 mM CaCl2, 0.1% PEG 8000. Prothrombin was detected in the pellet after  differential  centrifugation.  Location of  molecular weight marker indicated for  size reference. 0.1 0.2 0.3 [prothrombin] (nM) 0.1 0.2 0.3 [prothrombin] (p.M) [prothrombin] (|iM) Figure 8 Factor Va mediated prothrombin binding to BPS/EPC (25/75) large vesicles assessed by differential  centrifugation  coupled with immunoaffinity detection. Prothrombin assayed by differential  centrifugation  in the absence of  LV (A), presence of 2000 pM LV (•) and presence of  both 2000 pM LV and 185 nM Factor Va (T) was subsequentiy detected by immunoaffinity  detection (see Materials & Methods). A. Detected prothrombin data was derived from  densitometry performed  on experimental samples and concentration standards from  western blots. Each data point was an average of  3 repeats and error bar represents the standard deviation. B. Background reference corrected prothrombin binding data. Binding data in the presence of  LV and FVa were fitted  to a 2-site binding model. C. Prothrombin binding to FVa-bound LV. Data corrected for  self-mediated  binding and fitted  to a 1 -site binding model. (Kd ~ 0.05 pM). 3.4 Determination of  ~so kDa species It was of  interest to determine the identity of  the -50 kDa degradation species that appeared in differential  centrifugation  experiments. Initial predictions were that this band was prethrombin 1 from  its size determined from  SDS-PAGE gels and reactivity to anti-prothrombin antibody confirming  the presence of  kringle 2 epitope. This hypothesis was confirmed  by cleavage analysis of  prothrombin by thrombin and amino-terminal sequence analysis. The time course of  product generation during proteolytic digestion of prothrombin by thrombin was followed  by SDS-PAGE and visualization of  protein bands by staining with Coomassie Blue (Figure 9A) or western blotting (Figure 9B). Considerable amounts of  prethrombin 1 were formed  in the early phase of  the incubation. Its concentration decreased after  prolonged incubation with formation  of  products with lower molecular weights, namely fragment  2 (not visible on the gel) and prethrombin 2. The appearance of  these bands is indicative for  thrombin-catalyzed cleavages in prethrombin 1. The prethrombin 1 fragment  electrophoresed at an identical location as the -50 kDa band in the commercial prothrombin sample. Amino-terminal sequence analysis of  the -50 kDa band present in prothrombin commercial sample yielded the following  sequence: Ser-Glu-Gly-Ser-Ser-Val-Asn-Leu. Comparison with the amino acid sequence of  human prothrombin {111)  indicates that this product results from thrombin-catalyzed cleavages at Argl55-Serl56 in prothrombin giving rise to prethrombin 1 (residues 156-581) and fragment  1 (residues 1-155). 10 60 120 240 ^ — — prothrombin • M * — prethrombin 1 X fr^*  » * M&m 31— 21.5— ''ff ej^' ^ ^ ^ ^ ^^^^^ ^^^^^^ thrombin / prethrombin 2 — fragment 1.2 i J <f  r f ragment 1 *m i • B r 0 1 3 10 60 120 240 prothrombin prethrombin 1 Figure 9 Cleavage analysis of  prothrombin by thrombin. The time course of  product generation during proteolytic cleavage of  prothrombin by thrombin was followed  by SDS-PAGE under reducing conditions and visualization of protein bands by A. staining with Coomassie Blue or B. western blotting probed with anti-human prothrombin. Prothrombin (52.5 pg ; Coomassie Blue, 70 ng; western) was incubated for  5 min in HBS containing 0.01% PEG 8000 and 2 mM Ca . At time 0 s thrombin (3.5 pg; Coomassie Blue, 5 ng; western) was added to start proteolysis reaction in 140 pL total reaction volume. At indicated timed intervals (min) 20 pL aliquots (seven in total) of  the reaction mixture were removed, stopped and analysed by SDS-PAGE. Molecular weight markers, prothrombin and thrombin standards as well as reaction intermediates are indicated. 3-5 Discussion Quantitative prothrombin binding results obtained using differential  centrifugation  are perhaps crude but nevertheless give an estimate of  the dissociation constant (-50 nM) to Factor Va bound to membrane for  the first  time. Unfortunately,  this method did not allow the dissociation constant for  prothrombin binding to anionic phospholipid membrane to be determined and thus a direct comparison between K<j in the presence and absence of  Factor Va was not possible. However, the results indicated that Factor Va bound to anionic phospholipid membrane enhanced the membrane binding affinity  of prothrombin by approximately 20-fold  relative to prothrombin binding in the absence of the cofactor  (using K^ = 1.1 p.M; determined by {67)).  Furthermore, membrane-bound Factor Va likely adopts a conformation  that binds prothrombin more tightly than the solution interaction described by Luckow and coworkers (Kd =10 (iM). My value of  50 nM must be used with caution as it was derived at Factor Va concentrations that were well above physiological. Lower Factor Va concentrations did not give any appreciable difference  in prothrombin membrane affinity.  Finally, the degradation fragment  of prothrombin, prethrombin 1 was localized to membrane by Factor Va and showed no association to membrane in the absence of  Factor Va. This finding  is not surprising as prethrombin 1 does not contain the membrane binding Gla domain. Thus, Factor Va can individually recruit prothrombin to the membrane surface,  plausibly contributing to its cofactor  function. Assessing protein binding to large vesicles by differential  centrifugation  used in this study presented several sources of  error that limited this method as a tool to study membrane binding. High background levels in experiments lacking the LV carrier were likely caused by mixing and subsequent diffusion  of  protein with the sucrose cushion. To try and overcome these limitations, several different  experimental set-ups in terms of reaction tubes and sucrose percentages in the LV and cushion were tested, but yielded limited success. In addition, reproducibility problems were likely attributed to the tedious layering of  sample onto cushion. Another source of  error was in the detection method using densitometry on western blots. In my hands, the transfer  variability between blots was also a significant  contributor to error. This variability was typified  by the prothrombin concentration standards. As upwards of  eight blots were required for experiments, transfer  variability added to a source of  quantification  error. Nevertheless differential  centrifugation  coupled with immunoaffinity  detection proved to be beneficial as a qualitative technique to assess protein - membrane interactions. Chapter 4. Mechanism of  Prothrombin-Membrane Interaction 4.1 Rationale The membrane-binding mechanism of  prothrombin has been studied extensively for  over 25 years using well established techniques. Techniques including, x-ray crystallography (70,  75), 90° light scattering (60,  64, 77-79,  82), fluorescence  resonance energy transfer (FRET) (64),  ellipsometry (83,  86), quartz crystal microbalance with dissipation monitoring (QCM-D) (87),  and total internal reflection  fluorescence  microscopy (TIR-FPR) (84,  85) have contributed to the current understanding of  membrane binding. Membranes of  simple, two-phospholipid systems composed of  phosphatidylcholine and phosphatidylserine have been considered to be quite uniform.  There is no evidence to suggest re-organization of  the membrane upon peripheral membrane protein binding (112),  indicating the characteristics of  the protein-membrane interaction is intrinsic to the protein and not the membrane in these model systems. Despite rigorous studies focusing on the membrane binding properties of  prothrombin, as was outlined in the introductory chapter, a binding mechanism describing the complete kinetic association and dissociation profile  has not been achieved. The interaction certainly deviates from  a simple binding model as predicted by several other laboratories (82-87).  The causal nature of  this protein-membrane binding complexity has not been resolved. In addition to this unexplained observation there is currently no justification  for  F1 or F1.2 having lower affinity  for  membranes compared to prothrombin, although all three proteins have identical membrane binding Gla domains. The current chapter addresses the membrane binding interaction of  prothrombin and derivatives of  prothrombin using surface  plasmon resonance. By providing real time association and dissociation measurements without requiring chemical modification  of the constituents for  detection, SPR has greatly furthered  the understanding of  other peripheral membrane proteins (113-118).  Here, I demonstrate that prothrombin binds to immobilized PS-containing membranes with a mechanism that deviates grossly from  a model for  a single type of  binding. The interaction between prothrombin and PS-membrane is consistent with a linked reaction mechanism, having fast  and slow phases during both association and dissociation. The protease-deficient  prothrombin fragments F1 and F1.2 bind to membranes with fast  association and dissociation phases, whereas a purified  cross-linked prothrombin dimer (xl-prothrombin) is representative of  the slow kinetic phases. Non-membrane binding des-Gla prothrombin can impede prothrombin membrane binding. Together, these data suggest that monomeric prothrombin dimerizes after  binding to the membrane in a Protease domain-dependent manner thereby increasing the membrane-bound half-life  by prolonging dissociation from  the membrane. 4.2 Membrane immobilization and stability To monitor the interaction kinetics of  prothrombin and derivatives to DOPS and POPC -containing membranes, I established an assay for  protein binding to immobilized membranes using SPR. Liposomes injected over the LI sensor chip surface  were immobilized and saturated the flow  cell surface  (Figure 10A). A 0 500 1000 1500 2000 2500 3000 Time (s) B 5 10 15 20 DOPS (%) Figure 10 Liposome immobilization using the Biacore Sensor Chip LI. A. Prior to lipid immobilization the sensor chip surface  was cleaned with 50 mM octyl glucoside (1 min at 20 pL/min). LUV (100 nm extruded pore size, 500 p.M) composed of DOPS and POPC were injected for  17 min at a 3 [il/min flow  rate in running buffer composed of  150 mM NaCl, 10 mM HEPES pH 7.4. Representative LUV composition were 0% (trace a), 10% (trace b) and 25% DOPS (trace c) or blank buffer  injection (trace d) as indicated on sensorgram. Five consecutive 10 mM EDTA pH 8.0 injections (2 min at 20 |iL/min) were used to generate stable membrane and stable non-immobilized surfaces.  B. Immobilized LUV response related to percentage DOPS in LUV. Error bar represents standard deviation from  4 separate experiments. Immobilization occurred by a tight interaction between the liposome hydrophobic outer leaflet  alkyl chains and the lipophilic alkyl groups of  the LI sensor chip. The amount of LUV immobilized at saturation in a flow  cell was dependent on the DOPS content with higher DOPS content resulting in lower immobilization levels (Figure 10B). The additional negative charge associated with DOPS likely caused a repulsion effect resulting in less LUV binding to the chip surface.  Analysis of  the structure of  the LI chip immobilized phospholipids has shown that injected liposomes, composed also of  DOPS and POPC, fuse  upon binding to form  a fluid  phospholipid bilayer on the sensor chip surface  (119).  In contrast, natural phospholipids have been shown to remain intact as discrete spherical structures (94).  Membrane-coated and underivatized control flow  cells were stable after  several brief  EDTA washes (Figure 10A). The initial EDTA wash step removed some loosely adhering membrane likely associated by weak electrostatic interactions. The EDTA treated artificial  (i.e. DOPS/POPC) membrane and underivatized control surfaces  were stable to continuous buffer  flow  for  the time required to run protein binding experiments as was shown by the insignificant  change (~1 RU/min) in SPR signal (Figure 11). There was a significant  loss in SPR signal (-83 RU/min) in flow  cells with naturally immobilized lipids (i.e. BPS/EPC) and thus these lipids were not used in protein interaction experiments. 1.01 D 0C (0 -600 § -400 0> (0 a -200 -800 0 0.74 83 0 1000 2000 3000 4000 5000 6000 Time (s) Figure 11 Baseline stability of  immobilized membrane and reference  surfaces  over time. Baseline stability response is shown over extended time of  immobilized membrane and reference  surfaces  to 20 (iL/min running buffer  (150 mM NaCl, 10 mM Hepes pH 7.4). Surfaces  characterized were 25% BPS (black trace), 25% DOPS (gray trace) and reference  or blank (light gray trace). Baseline stability is indicated next to trace in RU change per min. 4.3 Validation of  interactive membrane surface  prior to detailed kinetic analysis 4.3.1. BSA binding Prior to protein binding experiments, HEPES-buffered  saline (HBS) running buffer  was changed to HBC (HBS including bovine serum albumin (BSA) and Ca2+). The BSA bound to 0% DOPS (100% POPC) liposomes resulting in a SPR signal increase of  750 RU. Liposomes with DOPS (analyzed from  1 to 25% DOPS) allowed additional albumin adhesion to the membrane surface  with a 1000 RU signal increase at 25% DOPS (Figure 12). o (0 c o a. <0 3 a> cc cc < ffl 1000-9 0 0 -800 -700 DOPS Figure 12 BSA binding to immobilized DOPS-containing membrane. After  membrane immobilization running buffer  was changed from  150 mM NaCl, 10 mM HEPES pH 7.4, to 150 mM NaCl, 10 mM HEPES pH 7.4, 5 mM CaCl2 and 0.1% BSA. Stabilized response changes due to BSA were measured and indicated as a function  of DOPS membrane concentration. Error bar represent standard deviation from  4 separate experiments. BSA adsorption to membrane has been described previously and was shown to increase membrane permeability (120).  BSA is included in the running buffer  as it is an important additive to block any non-specific  protein-protein, protein-lipid and protein-underivatized LI chip interactions. If  unblocked, these unoccupied hydrophobic sites could give false binding of  the protein of  interest. After  BSA addition to buffer  and response stabilization (~ 30 min), no significant  change in SPR signal (<15 RU) was observed during 12 hours upon HBC running buffer  conditions, which vastly exceeded the time required to complete the protein binding experiments. The 0.1% concentration of  BSA used in experiments is below the physiological concentration of  albumin (3.5 - 5.5%) (121)  and was not expected to interfere  with subsequent protein binding analysis. 4>3»2* Mass transport The existence of  mass transport (the rate of  transport of  solution analyte to the sensor surface)  was tested by injecting prothrombin over the membrane surface  at variable flow rate. This was performed  to ensure that the sensorgram data collected was actually kinetic and not influenced  by a mass transport parameter. Inspection of  the variable flow rate sensorgrams show no difference  between the 20 jxL/min and the 62 pL/min data and thus no significant  mass transport term appears to be present in the data (Figure 13). Thus, mass transport of  the protein in solution to the surface  is not rate limiting and can be ignored. As a consequence, the data collected were not diffusion-limited,  but rather reaction-limited. Subsequent SPR experiments were performed  with a flow  rate of  20 (xL/min. Time (s) Figure 13 Prothrombin membrane interaction under variable flow  rate. Prothrombin at 0.1 pM was injected (4 min) over a stable 25% DOPS containing immobilized membrane surface  and dissociation phase was followed  for  approximately 7 min. Flow rate was varied: 20 nL/min (dark gray trace) and 62 fiL/min  (black trace). Experimental conditions: temperature = 25 °C, running buffer  =150 mM NaCl, 10 mM HEPES pH 7.4, 5 mM CaCl2 and 0.1% BSA. All data was reference  subtracted using a blank flow  cell followed  by a double reference  correction using a blank running buffer injection. 4.3.3. Specificity  of  membrane interaction To further  validate the application of  this method for  studying prothrombin-membrane interactions, I determined that binding was highly dependent on the DOPS concentration. The SPR signal increase during association of  an approximately physiological concentration of  prothrombin to the 25% DOPS membrane was much greater than the 4% DOPS membrane (Figure 14A). The 0% DOPS (100% POPC) membrane surface  had very low (< 2% binding compared to 25% DOPS) but significant  and specific prothrombin binding. The specificity  of  the prothrombin membrane binding interaction was shown by the lack of  detectable binding for  the Gla-domainless prothrombin molecules: prethrombin 1 and des-Gla prothrombin (Figure 14B). In addition, the interaction of  prothrombin was completely reversible and dependent on Ca2+, as any remaining prothrombin was completely removed from  the membrane surface with EDTA which restored the original baseline (Figure 14A). The phospholipid specificity  requirement of  the Gla-domain and Ca2+ dependence of  the prothrombin-phospholipid interaction verifies  that the binding of  prothrombin to the phospholipid-Ll biosensor chip resembled the well-characterized interaction of  prothrombin with phospholipid liposomes in solution. A => cc d> (ft c o Q, (ft V cc 2000 -1500 -1000 -500 -200 400 600 800 Time (s) 1000 B 3 cc a) (0 c o Q. (0 0) CC 200 400 600 Time (s) 800 1000 Figure 14 Protein and DOPS dependent binding specificity. A. Prothrombin at 1 pM was injected (4 min) over stable 0%, 4%, 10%, 14%, 19% and 25% DOPS (light gray to black traces at increasing DOPS concentration) containing immobilized membrane surface.  Dissociation phase was followed  for  7 min before  a 10 mM EDTA pH 8.0 injection (2 min) removing any remaining membrane adhering protein. Experimental conditions: flow  rate = 20 pL/min, temperature = 25 °C, running buffer  = 150 mM NaCl, 10 mM HEPES pH 7.4, 5 mM CaCl2 and 0.1% BSA. All data was reference  subtracted using a blank flow  cell followed  by a double reference correction using a blank running buffer  injection. B. Des-Gla prothrombin (black trace) and prethrombin 1 (gray trace) at 25 pM were injected over the 25% DOPS surface  under the same conditions. 4.4 Fi, F1.2 and prothrombin membrane binding: Affinity  and qualitative kinetic analysis Prothrombin and Gla-domain-containing fragments  of  prothrombin (Fl and Fl .2) were analyzed at various concentrations for  their interaction to a 25% DOPS membrane surface.  From the 340 s time-point indicating the amount bound within the plateau region of  the association phase (Figure 15A-C), the apparent equilibrium dissociation constants ( K f 9 ) for  Fl, F1.2 and prothrombin were calculated to be 2.20 +/- 0.23, 2.48 +/- 0.26 and 0.82 +/- 0.10 |iM, respectively, from  data fitted  to a simple rectangular hyperbola representing a single type of  binding (Figure 15A-C insets). 0 200 400 600 800 1000 Time (s) 0 200 400 600 800 1000 Time (») Time (s) Figure 15 Fragment 1, fragment  1.2 and prothrombin binding profiles  to 25% DOPS-containing membrane. LUV composed of  25% DOPS were stably immobilized to a Biacore LI sensor chip as described under "Materials & Methods". A titration series (indicated concentrations in )j.M) of  Fl (A), Fl .2 (B) and prothrombin (C) was analysed for  membrane binding. Data collection for  association phase (protein injection) occurred between 100 - 340 s (4 min) and dissociation phase from  340 - 775 s (~7 min). Experimental conditions: flow  rate = 20 (iL/min, temperature = 25 °C, running buffer  = 150 mM NaCl, 10 mM HEPES pH 7.4, 5 mM CaCb and 0.1% BSA. Each cycle of  protein data collection was followed  by a brief  10 mM EDTA, pH 8.0 injection to remove any remaining protein and returning the response signal to lipid baseline. All data was reference  subtracted using a blank flow cell followed  by a second reference  subtraction using a blank running buffer  injection. Above sensorgrams (Response vs Time) were obtained as a titration series overlay using BIAevaluation v4.1. Responses obtained at association phase end were used to generate a binding isotherm fitted  to a one-site binding hyperbola (see figure  insets) using GraphPad Prism v4. Binding isotherms were used to obtain an K f  and R m a x . I next used the kinetic data provided by SPR to relate the differences  in binding profile between Fl, F1.2 and prothrombin to on and off  rate parameters. The kinetic binding profiles  observed for  Fl and F1.2 differed  substantially from  prothrombin in both the association and dissociation phases. Fl and F1.2 membrane binding are dictated by a single extremely fast  on and off  rates as observed by the rapid response change that is completed within 3-5 s of  the start and end of  protein injection. Nearly complete dissociation is observed for  Fl and F1.2 in the 7 min dissociation phase as responses return to membrane baseline. Binding isotherms shown in insets of  Figure 15A (Fl) and Figure 15B (F1.2) were well represented by a mathematical model consistent with a single type of  binding (one-site model; r2 = 0.99). Remarkably different  than that of  Fl or F1.2, prothrombin association occurred by two independent processes characterized by an initial extremely fast  on rate (that was similar to the Fl and F1.2 rates) and a secondary slow on rate. This biphasic nature was absent in the Fl and F1.2 binding data. The secondary on rate was influenced  by prothrombin concentration, as higher prothrombin concentrations resulted in an increase in this rate. The more complex mechanism of  the prothrombin association phase was also reflected  in dissociation phase data characterized by an initial fast  off  rate and a secondary very slow off  rate. The initial fast  off  rate, complete within 3-5 s after  protein injection, appears similar to the fast  off  rate observed with Fl and F1.2. 4.5 DOPS dependence of  prothrombin binding Prothrombin binding affinity  was dependent on the membrane DOPS concentration as shown by binding isotherms (Figure 16) and is evident from  K f  and R m a x determined from  such plots (Table 2). 2.5 5.0 7.5 10.0 12.5 600 450 300 • 150-DC <D (0 C 1200-o 900-Q. 600-(/> 0 300-CC o!o 2.5 5.0 7.5 10.0 12.5 i.O 2.5 5.0 7.5 10.0 12.5 2.5 5.0 7.5 10.0 12.5 [prothrombin] (pM) Figure 16 Prothrombin binding isotherms at different  percentage DOPS membrane concentration. Data was collected as described in Figure 15 and Materials & Methods. Responses obtained at association phase end were used to generate a binding isotherm fitted  to a one-site binding hyperbola at indicated DOPS percentage using GraphPad Prism v4. Binding isotherms were used to obtain an Kf  and R m a x reported in Table 2. T a b l e 2 B i n d i n g aff inity  o f  p r o t h r o m b i n , f r a g m e n t  1, f r a g m e n t  1 .2 a n d x l - p r o t h r o m b i n to D O P S - c o n t a i n i n g m e m b r a n e 3 outer leaflet  DOPS maximum protein protein % DOPS immobilized (molecules)/mm R m a x ( R U ) r2 bound (molecules)/mm n prothrombin 4 8.94 x 1010 20.0 +/- 7.0 1036 0 . 9 9 3 9 0.87 x 1010 10.3 prothrombin 7 1.98 x 1011 9.1 +/- 1.8 1689 0 . 9 9 8 4 1.42 x 1010 13.9 prothrombin 10 2.33 x 1 0 u 5.0 +/-0.5 1995 0 . 9 9 8 7 1.67 x l O 1 0 14.0 prothrombin 14 3.36 x 1011 3.2 +/-0.2 2758 0 . 9 9 9 5 2.31 x 1010 14.5 prothrombin 19 4.14 x 1011 1.66+/-0.14 2856 0 . 9 9 9 5 2.39 x 1010 17.3 prothrombin 25 4.45 x 1011 0.82+/-0.10 3309 0 . 9 9 9 5 2.77 x 1010 16.1 F1.2 25 6.28 x 1011 2.48 +/- 0.26 879 0.9911 1.52 x 1010 41.3 F.l 25 6.28 x 1011 2.20 +/- 0.23 1122 0.9911 3.22 x 1010 19.5 xl-prothrombin 25 6.06 x 10" 0.28 +/- 0.07 3107 0 . 9 9 9 8 2.6 x 1010 23.3 (a) explanatory information The number (n) of  DOPS molecules bound per protein molecule can be estimated if  the following  assumptions are made: 1) all outer leaflet  phospholipids are accessible, 2) '/2 of  the DOPS molecules are present on the outer leaflet  and participate in protein binding, 3) 1000 RU correspond to 0.92 ng immobilized lipid/mm2 chip (Cooper etal.  (1998) Biochim Biophys Acta 1373, 101-11), 4) the value of  n is determined at R lrax, a value derived from  a direct fit  of  a rectangular hyperbola using a 1:1 binding model and 5) 1000 RU protein binding response is equivalent to 1 ng protein/mm2 (www.biacore.com). The trend observed in prothrombin binding affinity  due to DOPS concentration was similar to previous studies using bovine brain PS and SUV characterized by 90° light scattering (50).  Dissociation constants determined at the lower DOPS concentration (e.g. 4% and 7%) have higher associated uncertainty and must be considered with caution as they were obtained with prothrombin concentration below or approaching the determined Kf'  concentration. Prothrombin binding affinities  were not determined for DOPS concentration below 4% as vast amounts of  protein would have been required. Prothrombin binding profiles  for  various DOPS membrane concentrations showed the same binding profile  characterized by two apparent on rates and two apparent off  rates as described for  25% DOPS (Figure 15C). It is evident that the biphasic binding mechanism described for  prothrombin is intrinsic to the protein and is independent of  DOPS concentration. The phospholipid binding site on prothrombin and its derivates can be approximated in terms of  the number of  DOPS molecules that define  a binding site. Although the exact structure of  the immobilized membrane is not known, the number (n) of  DOPS molecules bound per prothrombin molecule can be estimated if  the following  assumptions are made a) all outer leaflet  phospholipids are accessible, b) half  of  the DOPS molecules are present on the outer leaflet  and are accessible to participate in protein binding, c) 1000 RU correspond to 0.92 ng immobilized lipid/mm2 chip (122),  d) the value of  n is determined at R m a x > a value derived from  a direct fit  of  a rectangular hyperbola using a 1:1 model and e) 1000 RU protein binding response is equivalent to 1 ng protein/mm2 (www.biacore.com). The estimated value of  n at different  DOPS membrane concentrations is consistently between 10-17 DOPS molecules bound per prothrombin molecule and appears to be independent of  DOPS concentration. This is consistent with the current view of  peripheral membrane protein binding, in which a given number of DOPS molecules are required to comprise a protein binding site that is stable on the membrane for  long enough time to be detected by SPR. Not necessarily are all DOPS molecules involved in direct contact with the protein 3-D structure but are required to make up a "binding site". This value of  n agrees with a previous report using natural phospholipid SUV and bovine prothrombin studied by light scattering which determined n = 10-20 (50). 4.5.1 Non-cooperative binding There was no evidence for  positive or negative cooperativity since Scatchard plots were linear (Figure 17 A) and a Hill plot of  binding data had a Hill coefficient  (h) of  ~1 (Figure 17B) regardless of  DOPS concentration analyzed. Data with cooperative binding have curved Scatchard plots and a Hill plot with a slope of  either >1 (positive cooperativity) or <1 (negative cooperativity). Both plots indicate that prothrombin membrane binding is a non-cooperative process. Thus complex binding process observed for  prothrombin to the DOPS-containing membrane must be explained by another process. 0.00 0.00 0.01 0.02 0.03 0.04 0.05 0.06 r B ® O) o -1.0 -0.5 0.0 0.5 1.0 1.5 log [free prothrombin] Figure 17 Cooperativity analysis for  prothrombin interaction with DOPS-containing membrane. Experimental data from  SPR experiments were fitted  to a Scatchard plot (A) and to a Hill plot (B). Scatchard analysis: r is expressed as bound prothrombin per total outer leaflet DOPS. 25% DOPS (•), 19% DOPS (•), 7% DOPS (•) are linear. Hill analysis: O is equal to Y/(l-Y), where Y is the fraction  of  DOPS binding sites occupied and defined  as r/n. At 25% DOPS; n = 0.060 prothrombin sites/DOPS; (•), 10% DOPS; n = 0.077 prothrombin sites/DOPS; ( • ) , 4% DOPS; n = 0.072 prothrombin sites/DOPS; (T). The Hill coefficient  (h) is the slope at log 9 = 0 and determined to be 0.956 +/- 0.039 at 25% DOPS (•), 0.995 +/- 0.024 at 10% DOPS (A) and 1.001 +/- 0.059 at 4% DOPS ( • ) . 4.6 On the binding mechanism of  prothrombin 4.6.1 Prothrombin membrane binding involves a linked reaction mechanism To investigate the complexity of  prothrombin-membrane binding further,  prothrombin was injected at a constant concentration (5 |oM) over an immobilized 25% DOPS membrane surface  for  variable periods of  time ranging from  4 to 25 min. After  the injection, dissociation of  bound prothrombin was followed  by flowing  only buffer  over the chip. The dissociation rate was much slower after  long association times (Figure 18). Clearly in the case of  prothrombin binding, the curves were not identical as the extent of initial dissociation decreased with increasing association time. The change in the stability of  the prothrombin-membrane complex over time indicates that the binding is controlled by an obligate sequential mechanism or linked reaction involving an intermediate. In case of  independent reactions the complex remains stable over time and the dissociation curves are identical for  short and long analyte injections (associations). This was clearly not observed verifying  that the reaction is indeed linked. o & -200 d) DC - 3 0 0 -0 200 4 0 0 Time (s) 600 800 Figure 18 Membrane dissociation of  prothrombin is dependent on duration of association phase. Each cycle of  protein data collection was obtained as described in Figure 15 with the following  exceptions: A fixed  concentration of  prothrombin (5 nM) was used for  each cycle and allowed to associate either for  4, 8, 12, 20 or 25 min. Data was transformed (using BIAevaluation v4.1) to 0 s and 0 RU at the end of  association phase resulting in the above displayed dissociation phase overlay. Only dissociation phase is shown. Time of  association phase in minutes is indicated on individual traces. 4.6.2 Stability of  membrane bound species is dependent on prothrombin concentration An interesting observation regarding the dissociation data is that at concentrations between 0.05 and 1.5 (iM, prothrombin dissociated 80%, whereas above 1.5 |iM, a trend is observed in which increasing prothrombin concentrations result in a decrease in percent dissociation from  the membrane (Figure 19). After  30s of  dissociation, the percentage of  prothrombin released was; 2.5 fiM,  76%; 5 jiM, 69%; 7.5 fiM,  64%; 10 pM, 61%; 20 fiM,  49%. These results indicate that a more stably bound form  of prothrombin becomes increasingly prevalent at higher prothrombin concentrations. This was not observed with either Fl or F1.2. The formed  prothrombin-membrane complex appears to be stabilized at higher prothrombin concentrations, plausibly via an intermolecular protein interaction not present in F1.2. Figure 19 Initial extent of  membrane dissociation is dependent on prothrombin concentration. Each cycle of  protein data collection was obtained as described in Figure 15. The amount of  prothrombin dissociated 30 s into the dissociation phase was quantified  (% of  R m a x ) and plotted against prothrombin concentration. 4.6.3 Analysis of  cross-linked prothrombin dimer membrane binding To test the hypothesis that the slow phase may be due to prothrombin dimerization, a purified,  cross-linked prothrombin dimer (xl-prothrombin) was used to determine if  the complex mechanism seen for  prothrombin-membrane binding could be explained by membrane-induced dimerization. xl-prothrombin was graciously prepared by Dr. Peter Anderson (University of  Ottawa) using an activated polyglutamate cross-linking agent (10 residues in length) in the presence of  unilamellar vesicles and 5 mM CaCl2 and purified  by gel filtration  as described previously (17).  Prothrombin could only be cross-linked in this reaction in the presence of  Ca2 + and PS -containing liposomes (17).  Purified xl-prothrombin was analyzed at several concentrations for  its interaction with a 25% DOPS surface.  From the overlaid dose-response binding curves (Figure 20A), the K ' f  was calculated to be 0.28 +/- 0.07 pM from  data fitted  to a simple rectangular hyperbola (Figure 20B). The interaction of  xl-prothrombin to membrane was Ca2+ dependent and completely reversible by EDTA, like the other prothrombin derivatives. From inspection of  dissociation phase data, it is apparent that cross linking two prothrombins had a profound  effect  on dissociation kinetics compared to Fl .2 or prothrombin. The off  rate was very slow and a resembled the second off  rate observed with prothrombin. A fast  off  rate was not seen as was observed for  both F1.2 and prothrombin. 0 200 400 600 800 1000 Time (s) B ^ 2500 0.2 0.4 0.6 [xl-prothrombin] (pM) Figure 20 xl-prothrombin dimer binding profile  to 25% DOPS-containing membrane. LUV (100 nm extruded pore size, composition: 25% DOPS 75% POPC) were stably immobilized to a Biacore LI sensor chip. A. A titration series (indicated concentrations in nM) of  xl-prothrombin was analysed for  membrane binding. Data collection for association phase (protein injection) occurred between 100 - 340 s (4 min) and dissociation phase from  340 - 775 s (~7 min). Experimental conditions: flow  rate = 20 H.L/min, temperature = 25 °C, running buffer  = 150 mM NaCl, 10 mM HEPES pH 7.4, 5 mM CaCl2 and 0.1% BSA. Each cycle of  protein data collection was followed  by a brief 10 mM EDTA, pH 8.0 injection to remove any remaining protein and returning the response signal to lipid baseline. All data was reference  subtracted using a blank flow cell followed  by a double reference  correction using a blank running buffer  injection. Above sensorgrams (Response vs Time) were obtained as a titration series overlay using BIAevaluation v4.1. Response obtained at association phase end were used to generate a binding isotherm fitted  to a one-site binding hyperbola (B) using GraphPad Prism v4. Binding isotherms were used to obtain an Kf p and R m a x . 4*6.4 Homogeneous Analyte To further  refine  my experimental system, I investigated the homogeneity of  our analyzed analyte molecules by SDS-PAGE. For heterogeneous protein mixtures, a phenomenon of  sequential binding has been described, in which a relatively abundant protein with low affinity  binds the membrane surface  and is replaced in time by a much less abundant protein with higher affinity  (123).  A protein mixture could explain the complex membrane binding seen with prothrombin. However, this was not the case as all proteins used were judged to be greater than 98% pure from  an overloaded Coomassie blue stained gel (Figure 21) and thus any type heterogeneous analyte model can likely be ruled out. Heterogeneity in terms of  prothrombin solution multimers (i.e. monomer, dimer, etc.) can also be ruled out as the concentration used in experiments (0.05-20 pM) are predominantly below the concentration where prothrombin is known to start dimerizing in solution (14 pM, (18,  19)) and 10 to 100-fold  below the Kd of  a dimerization reaction in solution. In addition, Sere et. al.(62)  showed that no multimeric forms  of  purified  prothrombin exist when analyzed by native PAGE in the absence of SDS. Taken together, these findings  rule out that the complex binding results for  the prothrombin-membrane interaction are caused by the existence of  prothrombin multimers in solution, prothrombin degradation species, or other contaminating molecules. xl-prothrombin prothrombin fragment 1.2 fragment 1 1 2 3 4 5 Figure 21 Characterization of  proteins by SDS-polyacrylamide gel electrophoresis. Proteins (5 pg) were applied to a gradient SDS gel (from  4 to 20% polyacylamide) under reducing conditions. Lane indicated are 2, Fl; 3, F1.2; 4, prothrombin; and 5, xl-prothrombin. Lane 1 contains the molecular weight markers with indicated protein sizes. Coomassie Brilliant Blue was used for  staining. 4.7 Estimation of  kinetic parameters Kinetic data were analysed by global fitting  of  multiple concentrations of  the various models made available by the BIAevaluation software.  Fl and F1.2 data gave mediocre fits  using a simple 1:1 model as indicated by obvious deviation from  the experimental data curves (Figures 22A and B) and high chi squared (x2) values (Table 3). 1:1 model 800 800 C xl-prothrombin bivalent model 2000 -800 D prothrombin 1:1 model 800 Figure 22 Determination of  kinetic parameters of  prothrombin and derivatives binding to 25 % DOPS containing membrane using SPR. Fitting of  kinetic data from  protein titration experiments using BIAevaluation v4.1. A representative data curve of  Fl (A, 1.5 pM, global fitting),  F1.2 (B, 1.5 pM, global fitting),  xl-prothrombin (C, 0.55 pM, global fitting)  and prothrombin (D, 1.5 pM, single concentration fitting)  is shown interacting with a 25% DOPS membrane surface. Continuous and dotted lines correspond to experimental and fitted  data, respectively. Indicated models gave best fits  from  experimental data. Table 3 Parameters obtained from  global fitting. F1a F1.2a xl-prothrombinb prothrombin all datac (2nd dissociation phase)" fronO^S-1) 36,600 42,000 8,460 nd — <foff(S-1) 0.18 0.23 0.0006 nd 0.0004 Kd kinetics (pM)° 4.87 5.48 0.07 nd — Kd steady state (nM) 2.20 2.48 0.28 0.82 — x2 390 172 124 nd  (very large) 88 explanatory information ( a ) data fitted to a 1:1 model ( b ) data fitted to a bivalent model ( c ) no model made available through BIAevaluation v4.1 gave acceptable fit ( d ) 500 to 750 s dissociation data fitted to 1:1 model ( e ) determined from k o n / k o n Other more complex models with additional float  parameters did not give better fits  to the experimental data and thus the 1:1 model was most appropriate to estimate kinetic parameters. The on rate (~ 40,000 M"1 s"1) and off  rate (~ 0.2 s"1) parameters for  Fl and F1.2 were at the detection limit of  the instrument as are indicative from  the steep sensorgram curves. In addition, better fits  were likely not obtained due to the inherent complexity of  the interaction comprising multiple small interactions at the Gla domain -membrane binding interface. Kinetic data for  xl-prothrombin fitted  best to a bivalent analyte binding model (Figure 22C). This was expected, as xl-prothrombin is a bivalent molecule with two membrane binding Gla domains per molecule. Inspection of  xl-prothrombin-membrane binding kinetics revealed single slow apparent on and off  rates resulting in a tightly associated membrane complex. From the dissociation phase (using data from  500 to 750 s), xl-prothrombin has a single apparent off  rate that was determined to be 6 x 10*4 s"1. A fit  of  a single prothrombin concentration sensorgram using a 1:1 model showed large deviations from  experimental data (Figure 22D). A global fit  of  prothrombin data was not possible with any of  the available models offered  by BIAevaluation. From prothrombin sensorgrams, specifically  using dissociation phase data from  500 to 750 s, the second slow off  rate was determined to be 4 x 10"4 s"1. The association and dissociation phases shown here suggest that the overall prothrombin membrane binding mechanism is consistent with a multi-step process as simpler binding models did not fit  to the data. 4.8 Inhibition of  prothrombin membrane binding by des-Gla prothrombin To provide more substantiating evidence for  the presence of  a membrane bound prothrombin dimer, a binding study was conducted in the presence of  des-Gla prothrombin. I hypothesized that this non-membrane binding prothrombin derivative would impede the formation  of  the membrane dimer by a competitive inhibition effect  at the dimerization site, thereby shifting  the kinetics to that resembling F1.2. Surprisingly this effect  was not seen but rather the inclusion of  excess des-Gla prothrombin caused an inhibition of  prothrombin membrane binding during the association phase. At 100-fold excess des-Gla prothrombin, prothrombin membrane binding was reduced by -50% (Figure 23A). At a constant des-Gla prothrombin concentration (10 pM), the inhibition effect  could be overcome with increasing prothrombin concentration (Figure 23B). The effect  on the affinity  was minimal but significant,  as prothrombin bound slightly tighter (Kf p = 0.90 +/- 0.11 pM) as compared to prothrombin in the presence of  constant des-Gla prothrombin (Kaf p (ICUlMdcs~Gla) = 1.36 +/- 0.06 |iM) again indicating that the inhibition effect  occurred only at excess des-Gla prothrombin concentrations. Consistent with this inhibition, the R m a x was unchanged for  prothrombin (3292 +/- 83 RU) as compared to prothrombin in the presence of  des-Gla prothrombin (3270 +/- 31 RU). Conflicting  with my hypothesis, the amount of  the stably bound membrane species was increased  by des-Gla prothrombin, when dissociation profiles  of  0.05 (J.M prothrombin and 0.1 pM prothrombin in the presence of  10 |iM des-Gla prothrombin are compared. A Time (s) B 3500i r 0.0 2.5 5.0 7.5 10.0 12.5 [prothrombin] (|xM) Figure 23 Des-Gla prothrombin inhibition of  prothrombin membrane interaction. A. Representative cycles of  prothrombin kinetic data collected as described in figure  15 pre-equilibrated for  20 min either in the absence of  des-Gla prothrombin (solid curves); 0.05 pM (light gray), 0.1 pM (gray) and 0.5 pM (black) prothrombin or in presence of  10 pM des-Gla prothrombin (dashed curves); 0.1 pM (gray) and 0.5 pM (black) prothrombin. B. Response obtained at association phase end were used to generate a binding isotherm in the absence (•) and presence (A) of  des-Gla prothrombin fitted  to a one-site binding hyperbola using GraphPad Prism v4. Binding isotherms were used to obtain an Kf p and R m a x . Inset shows near equilibrium responses at low prothrombin concentration. 4-9 Discussion 4.9.1 The prothrombin-membrane interaction is biphasic Blood coagulation is limited and localized by enzyme-cofactor  complexes that assemble on activated cell membranes (28).  Zymogen substrates, such as prothrombin, are in vast excess in plasma and their concentrations do not limit enzymatic rates. For physiologically-relevant proteolytic activation rates to occur, however, the substrate needs to be transported to membrane-bound enzyme complexes (11,  46). It is therefore fundamental  to understand precisely how prothrombin interacts with procoagulant PS-containing membranes for  subsequent presentation to the prothrombinase complex. In the current chapter, I demonstrate using SPR kinetic data that both association and dissociation of  prothrombin to and from  DOPS-containing membrane is biphasic, each involving very fast  and very slow kinetic components. Consequently, the interaction did not conform  to a simple one-site model between prothrombin and a single class of binding sites on the membrane surface.  The deviation from  the simple binding model was independent of  the fraction  of  DOPS in the membrane and is consequently an inherent property of  the prothrombin-membrane interaction, as implied previously by several other research groups (82,  83, 85-87). When prothrombin was allowed to associate with membrane for  increasingly long periods or was analyzed at increasingly higher concentrations, the fraction  of  stably-bound prothrombin correspondingly increased. These data suggest that after  membrane binding, prothrombin adopts a new configuration  that results in slow-dissociation from  the membrane. 4-9*2 Fast prothrombin-membrane phases are mediated by fragment  l To investigate which domains within prothrombin facilitate  the hypothesized fast-  or slow-dissociating configuration  I compared Fl and F1.2 to prothrombin binding to immobilized membranes. The binding profiles  for  Fl or F1.2 were clearly different  than prothrombin (Figure 15). The interaction between Fl and F1.2 and membrane were approximated by a simple one-site binding model characterized by a single very fast  on rate and very fast  off  rate that proceeded to complete dissociation (Figure 24A). Thus the portion of  prothrombin containing Gla and Kringle 1 with or without Kringle 2 accounted for  the fast  phases of  prothrombin binding to PS-containing membrane, but not the slow phases. These data therefore  suggest that the Protease domain mediates the slow association and dissociation binding phases I observed here. 4.9.3 Dimerization model for  slow prothrombin-membrane binding phases A plausible explanation for  the slow association and dissociation phases of  prothrombin-membrane interaction is the formation  of  membrane-bound dimers following  the initial fast  monomer-membrane interaction. Consistent with this hypothesis, dimers of prothrombin have been reported previously which were dependent on the availability of PS-membrane binding sites and calcium (17,  124). Furthermore, detection of  these dimers was dependent on the presence of  the Protease domain. To test whether dimerization may account for  the slow kinetic phases, I analyzed the membrane binding characteristics of  xl-prothrombin. Indeed, the association and dissociation kinetics of  xl-prothrombin were consistent with the slow phases of  prothrombin binding. The data were best fitted  to a model assuming bivalent binding (Figure 24B). Rather than both membrane-binding subunits of  the dimer acting in concert, likely one subunit of  the dimer interacts first  with the PS-containing membrane followed  by the second subunit providing an avidity effect.  Because the on rate is proportionally slow in comparison to the off  rate, the K ad pv for  xl-prothrombin is only approximately 3-fold  lower compared to prothrombin monomer (Table 2). 4.9.4 Linked mechanism for  prothrombin-membrane interaction Based on my observation that the biphasic prothrombin-membrane interaction can be broken down into fast  association and dissociation rates represented by Fl or F1.2 and slow phases represented by stabilized prothrombin dimer, I suggest a two-step model characterized by a linked reaction mechanism (Figure 24C). In this proposed model, prothrombin molecules in solution rapidly associate with the membrane surface  forming short-lived monomeric membrane-bound complexes due to their intrinsically fast dissociation rate. Molecules that remain bound long enough and diffuse  laterally on the membrane surface  may successfully  collide to form  high avidity membrane-bound prothrombin dimers. Thus, the fast  monomer-membrane association is linked to slow dimer formation,  which stabilizes the interaction of  prothrombin with membrane. A. F1.2 © o r © F . 1 ^paaooonoooQ^^ B. xl-prothrombin •low prothrombin Legend u Protease >•  Kringle Gla X cross- l inker • monomer-membrane complex dimer-membrane complex total bound: monomer + dimer complexes fa»t mL ^^ JC-ODf  ^OQQQoodc«, weakly bound (monomer-membrane complexes) A tightly bound (dimer-membrane complex) D. 5-1000 ae V g 500 a 8 « a. 1000 2000 Time (s) 3000 4000 Figure 24 Model for  prothrombin membrane binding based on a linked reaction mechanism involving membrane induced dimerization of  the Protease domain. A. Fl, F1.2, B. xl-prothrombin and C. prothrombin putative binding reaction schemes on immobilized DOPS-containing membranes. D. Prothrombin (5 pM) was injected for either 4 min (gray trace) or 25 min (black trace) over a 25% DOPS membrane immobilized surface  followed  by a 10 min dissociation phase. Experimental conditions and data analysis were as described in figure  2. At selected time points, traces marked at "x" were analysed using the linked reaction mechanism for  predicted contributions of monomer and dimer to total membrane bound prothrombin (black square-double gray triangle). Numerical calculations are based on the availability of  100 prothrombin membrane binding sites. Symbols used for  weakly membrane bound monomelic prothrombin and tightly membrane bound dimeric prothrombin are black square and double gray triangle, respectively. According to the proposed linked kinetic model, the prothrombin-membrane association would be followed  by a similar desorption process. Initial membrane dissociation is dominated by monomelic prothrombin molecules controlled by a fast  dissociation rate. Remaining membrane bound dimeric prothrombin has a very slow dissociation phase that may either directly dissociate from  the membrane as a dimer (not shown) or slowly separate into membrane bound monomers that dissociate rapidly from  the membrane individually (Figure 24C). The off  rates derived for  the second dissociation phase of prothrombin and the single dissociation phase of  xl-prothrombin are slow (4 and 6 x 10"4 s"1, respectively), whereas the dissociation rates for  Fl or F1.2 (-0.2 s"1) are comparatively much faster.  These observations argue that prothrombin dimerizes on the membrane to account for  the slow dissociation not observed for  Fl or F1.2. Consistent with the model a prolonged association time of  prothrombin with membrane would allow a more stable form  of  prothrombin (i.e. dimers) to assemble on the membrane, which I observed and illustrate schematically (Figure 24D). 4.9.5 Previous studies concur with membrane induced dimerization Numerous studies have been conducted to help understand the mechanism by which prothrombin interacts with PS-containing membranes. While several of  these have concluded that single exponential association and dissociation can occur (64,  82), others have predicted a biphasic mechanism using a variety of  experimental approaches: A) Biphasic association was observed by two independent laboratories (83,  86) following prothrombin binding to spread phospholipid monolayers using ellipsometry. The secondary kinetic event was not discussed (83)  or attributed to non-specific  adsorption (86).  B) Deviations from  a simple binding model were obtained from  prothrombin (1 fiM)  association (<2 min) binding data assessed by TTR-FPR fluorescence  recovery curves (85). In their discussion, Pearce et al. suggested the formation  of  dimers on the membrane surface  as a possible explanation. C) Very similar dissociation data as I report here has been noted using QCM-D. Richter et al. showed only 80% prothrombin dissociated from  supported lipid bilayers after  1 hr. (87);  complete dissociation was shown with EDTA. Their measurements of  dissipation deviated from  a one-site model and this deviation was attributed to membrane induced rigidification  of  prothrombin, which they hypothesized was due to lateral interactions on the membrane surface. Although these examples from  the literature support our biphasic model, there are also examples in the literature where a simpler mechanism was described as discussed in the following  section. 4.9.6 Apparent discrepancies from  previous studies about the secondary kinetic event Binding data from  stopped flow  intrinsic fluorescence,  stopped flow  FRET and light scattering experiments have concluded the prothrombin-membrane reaction proceeds by a simple model lacking a secondary kinetic step (64,  82) (also see introduction). Data fit well to one-site model under the conditions that were analyzed and gave good estimations of  the initial association and dissociation kinetic rates. Observations of  the secondary kinetic step were not made for  several reasons. Firstly, the prothrombin concentrations (less than 2.8 pM (82)  and 1 |iM (64))  used were below membrane binding site saturation. As a consequence the secondary kinetic step (a concentration dependent, protein - protein interaction dependent event) was minimal and thus not noted. At higher concentrations (12.5 fiM  (82))  the authors acknowledged that association became complex, but the cause of  this complexity was not addressed. Secondly, time of  analysis of  association (500 ms(S2) and < 1 s (64))  and dissociation (< 5 s (64))  were not long enough at concentrations used to observe a secondary kinetic step. Thirdly, dissociation experiments (Figure 4a in Wei et al.) confirmed  an absence of  complete prothrombin release after  5 sec of  data analysis indicating dissociation had not gone to completion. The prothrombin concentration (0.5 jjM (64))  used in this experiment gave a percent dissociation (-80% completion) that is in agreement with our results. Taken together these studies gave an accurate account of  the fast  kinetic event that is completely in line with our observations of  the fast  phase of  prothrombin membrane binding. By evaluating on and off  processes simultaneously, the dimerization model presented here is consistent with all of  the prior reports. 4.9.7 Other models to explain prothrombin membrane binding SPR experiments suggest that prothrombin membrane induced dimerization is likely the dominating process responsible for  the second kinetic event apparent in the biphasic prothrombin binding data. Much criticism has been directed for  attempting to define  a mechanistic model describing the experimental data for  prothrombin membrane binding. In order to address these criticisms, other models that can explain peripheral membrane binding reactions are introduced and outlined in Table 4. In these reactions peripheral membrane protein, P is in equilibrium with membrane, M. Rate designations (fast  or slow) and reaction descriptions are included that theoretically may produce data similar to the experimentally described prothrombin-membrane binding data. The reaction can be linked and thus involve an association time dependent event or not linked, as the case for  a simple bimolecular interaction of  a 1:1 (Langmuir) model. Reactions are presented in a way that could potentially result in the observed experimental prothrombin data. Thus, rates, binding types and strengths were chosen for each model to accommodate prothrombin-like data. Table 4 Binding models for  peripheral membrane proteins. Model Name Reaction Linked Reaction (Association Time Dependent Event) 1:1 (Langmuir) model P + M v. v MP No description:  P binds with a single proteinaceous site to a single site on the membrane. 1:2 heterogeneous immobilized P + Ma v PMa No ligand model P + Mb ^ PMb description:  P can bind one of two independent membrane sites; Ma or Mb. The two sites may represent different  PS clusterings 2:1 heterogeneous analyte model P0 + M f a s t s PaM yes linked slow P b + M s PbM description:  Pa is a fast, weak binder and Pb is a slow, tight binder. Pa and Pb can bind to a competing site on the membrane. Pa can be displaced by Pb over time. fast  slow 1:1 conformational change model P + M v v MP v s MP* yes description:  Two conformational states are present; P and P*. Only P can bind the membrane and then undergoes a slow conformational change producing a tightly bound species P*. The conformational change is much slower than the binding event. fast  slow 1:1 multivalent analyte model P + M ^ MP ^ MP* yes description:  P can bind membrane with 2 proteinaceous sites. The binding of the 1 -site, producing MP, facilitates the binding of the second proteinaceous site, producing a tightly bound molecule MP*. The second binding interaction is much slower than the initial binding event. fast  slow 2:1 membrane induced P + M s MP + MP N MPP yes dimerization model description:  P is a fast, weak binder. P can slowly dimerize with another membrane bound P forming a membrane bound dimer; MPP. P is peripheral membrane protein M is membrane For example, a 1:2 heterogeneous immobilized ligand model can be characterized by two independent binding sites (Ma and Mb). Each membrane site may have a kinetically distinctive binding rate constant designated simply as fast  and slow, making each site unique. Since each membrane site is independent the reactions at each site would not be linked, ruling out such a reaction to describe the prothrombin data. In addition it has been determined that mixed PS/PC membranes are homogeneous and do not contain acidic lipid domains or clusters prior to and after  prothrombin binding (112)  and thus only a single type of  binding site, M is available. Thus any theory of  membrane heterogeneity causing complex binding has to disagree with these previous studies and our linked reaction experiment. Finally, F1.2 is a useful  control to further  rule out such a model to explain prothrombin membrane binding mechanism, as F1.2, having an identical membrane contact site, showed simple binding kinetics. Other models and their descriptions in Table 4 include a 2:1 heterogeneous analyte model, 1:1 conformational change model, 1:1 multivalent analyte model and a 2:1 membrane induced dimerization model. The description of  these models is not discussed in further  detail here. Refuting  evidence against these models and other processes to explain the second kinetic event is outlined in Table 5. Table 5 Prothrombin membrane induced dimerization: Is there another model or process to explain the second kinetic event involved in membrane binding? Proposed model or process Refuting evidence 1) non-specific adsorption to chip surface • F1, F1.2, prethrombin 1 and des-gla prothrombin do not show second on/off  rate 2) multiple membrane bound species A) heterogeneity in analyte • SDS-PAGE analysis for proteolytic fragmentation (2:1 heterogeneous analyte model) • F1.2 would have the same minor heterogeneity such as carboxylation and glycosylation as prothrombin B) multi-step binding process via protein sub-sites • F1 and F1.2 do not show although Gla domain is the only membrane e.g. PS pocket(s), oj-loop, electrostatic interactions contact site and is identical to prothrombin (1:1 multivalent analyte model) C) conformers • None, No evidence for different  membrane bound conformers  either. (1:1 conformational change model) - Would be a Protease dependent Gla conformational change induced by membrane binding - Can not be ruled out 3) two dimensional surface causing binding anomalies • F1 & F1.2 do not show. Mass Transfer  experiments. e.g. crowding, rebinding, electrostatic or • prothrombin at low concentration has second kinetic event orientational effects • Surface is more representative of platelet membrane immobilized in a clot under flow compared to SUV in solution Most of  the models can be eliminated as an appropriate model by using F1.2 and other fragments  of  prothrombin as comparative controls. For example non-specific  adsorption to the LI sensor chip (e.g. to carboxymethyl dextran, unblocked lipophilic anchors, or Au surface)  could cause a slow association process and potentially result in a stable interaction. However, since none of  the other fragments  of  prothrombin show this type of interaction process and these fragments  together cover the entire primary amino acid sequence of  prothrombin such a process seems unlikely. Another model that should be addressed is the heterogeneous analyte model. Although SDS-PAGE analysis shows greater than 98% purity in the prothrombin preparations used in the analysis, prothrombin is well known to have multiple heterogeneous variants of similar molecular weight not distinguishable on SDS-PAGE. Variants in gamma-carboxylation (10 sites) and N-linked glycosylation (3 sites) have been described for plasma purified  prothrombin (9,  125). Again, the possibility of  two variants with different  membrane affinity;  one being a fast,  weak binder and the other a slow, tight binder could exist. Over time a linked reaction would ensue resulting in the replacement of  weak binders by tighter binders giving this slow kinetic phase. However, this situation seems unlikely in light of  the fact  that F1.2 is a product of  plasma purified  prothrombin and thus contains the same heterogeneity of  Gla residues and 2 of  3 carbohydrate sites (the 3 r d site being on the protease domain). Another concern that should be addressed here on the topic of  heterogeneous analyte model is the issue of  a prothrombin dimer contaminating the monomer in solution. A heterogeneous monomer/dimer mixture would undoubtedly complicate the analysis of  prothrombin kinetic data and would represent a form  of  the heterogeneous analyte model. As was apparent from  xl-prothrombin data, a natural solution dimer will have different  membrane kinetics compared to monomer. It has been shown that bovine prothrombin can dimerize in solution starting at a concentration of  14 pM as assessed by cross-linking experiments (19)  and sedimentation measurements (18).  The concentration used here for  prothrombin kinetic experiments were for  the most part well below this solution dimerization concentration. In addition, Sere et. al. (62)  showed that no multimeric forms  of  purified prothrombin exist when analyzed by native PAGE in the absence of  SDS. Taken together, these findings  rule out that the above described results for  prothrombin-membrane binding characterized by a linked reaction mechanism are caused by prothrombin degradation products, variants, solution phase dimers or other contaminating molecules. The binding of  prothrombin at different  sub-sites on the membrane contact site as described by the 1:1 multivalent analyte model could potentially also result in a biphasic binding mechanism. Specific  interactions between prothrombin and membrane have been under extensive investigation and considerable knowledge is now available. The presence of  multiple interactive sites mediated by PS pockets, hydrophobic and electrostatic interactions (for  details see introduction) is not disputed however these interactions likely occur too fast  and on a time scale indistinguishable by a SPR experiment. If  multivalent interactions were responsible for  the biphasic prothrombin-membrane interaction, then one would expect similar slow secondary kinetics to be seen with F1.2, which was not the case. One process in the prothrombin-membrane interaction that cannot be ruled out at this time is the existence of  more than one membrane-bound prothrombin conformer  with varying membrane affinities.  A membrane induced conformational  change does occur for  bovine prothrombin (29, 124, 126)',  however, multiple membrane-bound conformers have not been described. If  present, these membrane-bound conformers  would depend on the presence of  the Protease domain as F 1.2 lacking the Protease domain does not show the characteristic prothrombin biphasic kinetic data. At this time I can only speculate on these membrane bound conformers. 4-9-8 Significance  of  prothrombin dimerization The K'* p (0.82 +/- 0.10 pM) determined using SPR was similar to Kd values reported previously for  the interaction of  prothrombin with membranes composed of  25% PS using other techniques, including measurements with 90° light scattering and fluorescence  (0.11 - 0.86 pM dependent on liposome diameter (64,  68)) and gel filtration chromatography (1.1 pM) (67).  Also consistent with my observations, prothrombin membrane binding was determined to be 2-3 fold  higher in affinity  compared to Fl and F1.2 as was shown previously by other studies (67,  85, 127). Neither Kringle domain has been shown to affect  membrane binding, as prothrombin Kringle domain-deleted mutants have identical membrane affinity  compared to native prothrombin (67).  Other studies have also suggested that portions of  prothrombin that are C-terminal to Fl also contribute to membrane binding. Scanning calorimetry of  prothrombin suggested a difference  in denaturation patterns of  the C-terminal region of  prothrombin when it was associated with membranes compared to free  in solution (92,  126). These results indicate a role for the Protease domain in overall membrane binding. Although Fl, F1.2 and prothrombin make direct membrane contact through their identical Gla domains, only prothrombin forms  a bivalent, high avidity membrane interaction via our postulated dimerization binding mechanism. The dimerization site in the Protease domain is proposed to be near proexosite I of  membrane bound prothrombin, as the molecules can be cross-linked at proexosite I under similar experimental conditions (17).  To support the prothrombin membrane-induced dimerization hypothesis, we purified  a previously reported stable prothrombin dimer (xl-prothrombin) and demonstrated reduced K' cipp and slow kinetic properties compared to Fl and F1.2 binding to membranes. Thus, protease domain-dependent dimerization may account for  previously reported discrepancies between prothrombin and Fl or Fl .2 binding to membranes. At this time, I can only speculate on the physiological role of  prothrombin dimerization on procoagulant membrane in vivo. Membrane-induced dimerization will increase the half-life  of  prothrombin on the membrane. This may increase its availability to bind to the prothrombinase complex thereby increasing the subsequent production of  thrombin. Chapter 5. Comparison of  Coagulation Gla Protein-Membrane Interactions 5.1 Rationale To further  understand how the Gla domain and regions removed from  the Gla domain facilitate  membrane binding, other Gla proteins were analyzed for  their membrane binding properties. With the binding profile  differences  observed between F1.2 and prothrombin, it was anticipated that the other Gla proteins would also categorize themselves into one of  two types of  binders: simple 1:1 or multiphasic. Although many studies have characterized membrane binding of  individual Gla proteins, few  studies have attempted to characterize them in a single study making comparisons difficult  and unclear. Thus, many binding property comparisons that have been made between Gla proteins, were performed  with a variety of  parameters that made them difficult  to compare. These include the use of  a wide spectrum of  techniques (see introduction), protein origin (usually human versus bovine), membrane type (small unilamellar vesicles, large unilamellar vesicles, immobilized monolayer versus bilayer) and membrane composition (percentage and types of  phospholipids). Consequently at this time, it is difficult  to compare the membrane binding properties of  Gla proteins. In terms of  binding strength, the seven vitamin K-dependent clotting proteins vary tremendously in their affinity  for  PS-containing membranes; with protein S the strongest binder (mid nM range) and protein C/Factor VII the weakest binders (low-mid (iM range). An explanation for  this 100-1000-fold  difference  in binding affinity  is still under scrutiny. A most obvious reasoning for  this difference  would be attributed to differences in the protein regions involved in the membrane contact site. A sequence comparison of the Gla domains (Figure 25), however, shows a high sequence identity (55-68% amino acid identity for  any two sequences) with 16 of  44 residues of  the domain being strictly conserved. propeptidase chymotrypsin cleavage w _ | 0 0 p cleavage I prothrombin #: 1 10 30 40 prothrombin ANTFLXXVRKGNLXRXCVXXTCSYXXAFXALXSSTATDVFWAKY protein C ANSFLXXVRHSSLXRXCIXXICDFXXAKXIFQNVDDTLAFWSKH FIX NSQKLXXFVQGNLXRXCMXXKCSFXXARXVFXNTXRTTXFWKQY FX ANS F LXXM KKGH LX RX CMXXTC S YXXARXVFXD SD KTNX FWNKY FVII ANAFLXXLRPGSLXRXCKXXQCS FXXARXIFKDAXRTKLFWISY protein Z AQSYLLXXLFXGNLXKXCYXXICVYXXARXVFXNXVVTDXFWRRY protein S ANSLLXXTKQQNLXRXCIXXLCNKXXARXVFXNDPXTDYFYPKY * * * * * . * * * * * * * * * . . * * . Figure 25 Amino acid sequence alignment of  the Gla domain of  human Gla proteins involved in blood coagulation. Gla sequence is shown defined  between propeptidase and chymotrypsin cleavage sites. Strictly conserved residues are indicated with an asterisk (*). Highly conserved residues are indicated with a colon (:). Conserved residues are indicated with a period (.). Positions in the sequence at which gamma-carboxylation of  glutamic acid residues is either known to occur or may occur is indicated by X. The position of  the disulfide  loop within the Gla domain is also indicated. Amino acids are colored according to their chemical properties: non-polar amino acids (G,A,V,L,I,P,W,F,M) are red, polar (S,T,Y,C,Q,N) green, basic (K,R,H) purple, and acidic (D,X) amino acids are blue and black, respectively. The numbering at the top refers  to the prothrombin sequence. Prothrombin sequence 1 -44 was used as the query sequence to search the protein database using ClustalW multiple sequence alignment program (128). Furthermore, a structural comparison of  the Gla domains in the presence of  saturating calcium made possible from  x-ray crystallography data shows that the protein backbone orientation as well as position of  seven calcium ions required for  membrane binding are strictly maintained in these homologous proteins (Figure 26). Figure 26 Structural comparison of  three homologous Gla domains. Bovine prothrombin (2PF2, (70)) white trace (residues 1-45 and associated seven Ca2+ (white ball)) was used as reference  structure for  fitting  bovine Factor X (1IOD, (129))  in red trace (residues 1-44 and associated seven Ca2+ (red ball)) and human Factor IX (1CFI, (73))  in orange trace (residues 2-45). Factor IX associated Ca2 + are not shown for clarity. Hydrophobic residues (Phe4, Leu5 and Val8 from  left  to right) of  the W-loop known to interact with the membrane interior are also indicated on the prothrombin structure. Gla and other residues are not shown for  clarity. Crystal coordinates were obtained from  the Protein Data Bank using indicated accession numbers. Deep view/Swiss-PdbViewer 3.7 was used to make illustration. Many of  the strictly conserved residues undoubtedly are required for  domain structure and highly conserved residues are required for  the membrane interaction involving hydrophobic (e.g. co-loop as illustrated in Figure 26), Ca2+ bridging, electrostatic and specific  PS headgroup interactions as detailed earlier (see introduction). Again a most obvious reason for  the affinity  difference  would be in these minor differences  in sequence at the least conserved amino acid positions. However, numerous studies have tried to modulate membrane binding of  individual Gla proteins by altering individual or several, non-strictly conserved residues (altering these has profound  devastating effects  on membrane binding presumably by destabilizing the Gla domain) of  one Gla proteins to another. These studies have been unfulfilling  as they have failed  to drastically alter the affinity  of  the mutant protein. Thus it seems unlikely that several or (as often  proposed) a single residue in the Gla domain involved in membrane contact cause this astounding 100-1000-fold  difference  in binding affinity.  It seems apparent that sites removed from the membrane contact site may play a role in modulating the membrane binding process of  the Gla proteins. So the question remains in the field:  what really is the basis for  this difference  in membrane affinity  among the Gla proteins? It was hoped that in light of  the binding profile  differences  observed with prothrombin and F1.2, SPR data could shed light on this question. 5.2 Results 5.2.1 Purity analysis by SDS-PAGE In order to assess the binding properties of  all coagulation Gla proteins (except Factor VII - due to cost restrictions), proteins were first  analyzed for  purity by SDS-PAGE (Figure 27). From a heavily overloaded (in order to observe any heterogeneous protein) gradient gel electrophoresed under non-reducing conditions to maintain the integrity of  the disulfide  bonded two-chained proteins, it is apparent that the majority of  proteins migrate as a single band. These single band proteins (> 98%) included prothrombin, protein Z, protein S, Factor IX and Factor X. Factor Xa predominantly ran as a doublet equally represented by Factor Xaa (upper band) and Factor Xap (lower band) as a result of  a known autocatalytic cleavage of  a C-terminal fragment  of  its heavy chain (130).  Factor IXa predominantly ran as an expected single band (131)  (-90%); however, two smaller bands presumed to be degradation products were also apparent. Protein C ran as a known doublet (132)  equally represented by protein C a (upper band) and protein Cp (lower band) due to proteolytic cleavage of  a susceptible C-terminal peptide (Mr = 3000) of  the heavy chain. No functional  distinctions between a - and P-protein C have been observed. Activated protein C also had this heavy chain doublet in addition to a minor degradation fragment  of  low abundance (< -10%). I concluded that the proteins were of  expected homogeneity and were suitable as analytes for  SPR characterization. Figure 27 Characterization of  proteins by SDS-polyacrylamide gel electrophoresis. Proteins (7.5 (ig), as indicated in lanes were applied to a gradient SDS gel (from  4 to 20% polyacylamide) under non-reducing conditions as described in Materials & Methods. Coomassie Brilliant Blue was used for  staining. 5.2.2 Gla protein binding profile  to 25% DOPS-containing membrane Gla proteins of  human origin were analyzed over a wide concentration series for  their binding properties to an identical  25% DOPS membrane immobilized to a biosensor surface.  This allowed a direct comparison of  their binding properties in a non-biased fashion.  As expected, all Gla proteins bound to the 25% DOPS membrane surface  in a concentration dependent manner (Figure 28A1-I1). Response (RU) Ni w S § § 8 o © o o Response (RU) Response(RU) Response(RU) D1 Factor IXa [Factor IXa](uM) 200 400 600 800 1000 1200 1400 1600 Time (s) E18oo,PrPtqin,C 0.5 1.0 [protein C] (uM) 1000 1500 2000 2500 Time (s) F18oo activated protejn C 0.5 1.0 [APC] (nM) 1000 1500 Time (s) 2000 2500 G 1 p r o t e i n S => cc 0) tn c o o_ tn a> cc 4000 -3000 • 2000 1000 -G2 4500 -5.0 0.1 0.2 0.3 0.4 0.5 0.6 [protein S] (uM) Time (s) H ^ o o P r o t e i n Z => tr o tn c o a. <n <D DC 1500 -1 0 0 0 -500 • 1 2 3 [protein Z]  (jiM) Time (s) n .prQthrpmfrin,  , , I D DC a) to c o Q. to 0} DC 4000 3000 2000 1000 0 -200 400 600 800 1000 1200 1400 Time (s) 2.5 5.0 7.5 10.0 12.5 [prothrombin] (g.M) Figure 28 Gla protein binding profiles  to 25% DOPS-containing membrane. LUV composed of  25% DOPS were stably immobilized to a Biacore LI sensor chip as described under "Materials & Methods". A titration series of  Factor X (Al), Factor Xa (Bl), Factor IX (CI), Factor IXa (Dl), protein C (El), activated protein C (Fl), protein S (Gl), protein Z (HI) or prothrombin (II) was analysed for  membrane binding. Curves in each series are colored according to concentration (|xM): 0.005 green—, 0.001 cyan—, 0.0025 yellow--, 0.005 black—, 0.01 red<=, 0.015 blue—, 0.025 dark cyan—, 0.05 magenta=, 0.1 dark yellow—, 0.15 navy—, 0.25 wine—, 0.5 pink=, 1 olive—, 1.5 royal—, 2.5 orange=, 3 light gray=, 5 purple—, 10 violet—. A concentration curve corresponding to a near physiological concentration is marked with an x. Data collection for  association phase (protein injection) occurred for  6 to 12 min and dissociation phase for  12 to 25 min depending on the kinetics of  the protein under study. Experimental conditions: flow  rate = 20 (iL/min, temperature = 25 °C, running buffer  = 150 mM NaCl, 10 mM HEPES pH 7.4, 5 mM CaCl2 and 0.1% BSA. Each cycle of  protein data collection was followed  by a brief  10 mM EDTA, pH 8.0 injection to remove any remaining protein and returning the response signal to lipid baseline. All data was reference  subtracted using a blank flow  cell followed  by a double reference  correction using a blank running buffer  injection. Above sensorgrams (Response vs Time) were obtained as a titration series overlay using BIAevaluation v4.1. Responses obtained at association phase end were used to generate a binding isotherm fitted  to a one-site binding hyperbola (see figure  A2-I2) using GraphPad Prism v4. Binding isotherms were used to obtain an K'f  and R m a x . As was the case with prothrombin and derivatives thereof,  the interaction of  all Gla proteins was completely reversible and dependent on Ca2+, as any remaining protein was completely removed from  the membrane surface  with EDTA. Membrane binding site saturation was achieved using experimental concentrations for  Factor X, Factor Xa, Factor IX, Factor IXa and prothrombin. For the other Gla proteins protein C, activated protein C, protein S and protein Z, complete binding site saturation was not achieved at the highest experimental concentrations, as higher concentrations were not available due to cost considerations. For these proteins, the binding site saturation (Rmax) was accurately determined from  binding isotherms. From the association phase end point indicating the amount bound near equilibrium, the K f  for  the Gla proteins was calculated from  data fitted  to a simple rectangular hyperbola representing a single type of binding (Figure 28A2-I2) and is reported in Table 6. A comparative examination of  the kinetic binding profiles  of  the individual Gla proteins revealed some major differences  in the overall shape of  the kinetic curves. Such findings  were not anticipated and these differences  are discussed in detail below. Table 6 Comparison of  membrane binding properties of  Gla proteins assessed by SPR8 Gla protein [Plasma] (nM) K°pp(\iM) by SPR R,ras (RU) ? Max Binding" (%) pertinent binding observations prothrombin 1400 0.81 +/-0.07 3980+/- 100 0.9922 39 - linked reaction, biphasic kinetic properties - likely membrane induced dimerization Factor X 170 0.78 +/- 0.06 4290 +/- 100 0.9945 51 - similar profile  to prothrombin - saturation of  complex association phase - membrane induced dimerization/conformers? Factor Xa n/a 0.172+/-0.011 6530 +/- 110 0.9958 100 -1:1 binding model fits  best kon = 46,000 M'V , fc of f= 0.003 s"1 Factor IX 70-90 0.59 +/- 0.06 1085 +/-30 0.9920 14 - similar profile  to prothrombin - membrane induced dimerization/conformers? - distinct 2-phase association/dissociation Factor IXa n/a 0.277+/-0.017 3600 +/- 60 0.9957 56 - similar profile  to prothrombin - very similar profile  to Factor IX - membrane induced dimerization/conformers? protein C 65-80 2.49+/-0.31 1645 +/- 140 0.9990 19 - slow kinetics, weak binder - minor biphasic kinetic properties activated protein C n/a 0.50 +/- 0.03 794+/- 17 0.9990 14 - similar profile  as protein C - more biphasic character compared to protein C - higher affinity protein S 145 (free) 0.073 +/- 0.002 4334 +/- 55 1.0000 44 - very similar profile  to xl-prothrombin - stable membrane complex - known solution dimers present - bivalent model fit  best protein Z 45 1.07+/-0.07 2240+/- 181 0.9890 25 - similar profile  as protein C - slow kinetics, weak binder - minor biphasic kinetic properties (a) supplementary information 1) membrane DOPS content was 25%, 2) outer leaflet  DOPS immobilized = 4.21 x 1011 molecules, assuming 1000 RU correspond to 0.92 ng immobilized lipid/mm2 chip (Cooper et al. (1998) Biochim Biophys Acta 1373, 101-11), 3) 1000 RU protein binding response is equivalent to 1 ng bound protein/mm2 (www.biacore.com). (b) Max binding (%) relative to R ^ of  Factor Xa, values corrected for  differences  in protein molecular weight The kinetics of  binding of  the vitamin K-dependent coagulation Gla proteins are dramatically different  from  that of  prothrombin and F1.2 and also from  one another. With the exception of  Factor Xa, the kinetic data of  the other Gla proteins did not fit  to a simple 1:1 model and thus their binding had to be described by a more complex binding model. Factor Xa binding data lacked any biphasic kinetic properties and fitted  loosely to a 1:1 binding model although other complicating factors  are undoubtedly present in the binding data (x2 ~ 1000). Apparent biphasic association and dissociation were observed at all concentrations tested for  the other Gla proteins with the exception of  Factor Xa and protein S. As described for  prothrombin (see Chapter 4) this biphasic profile  is characterized by an initial extremely fast  on rate and a secondary slow on rate. The secondary on rate was influenced  by protein concentration, as higher protein concentrations resulted in an increase in this rate. Similarly, the dissociation phase data is characterized by an initial fast  off  rate and a secondary slow off  rate. The initial fast  on and off  rate, complete within 3-5 s of  association and dissociation phase respectively, varies significantly  in its contribution to overall association/dissociation between the Gla proteins that have this biphasic kinetic character. Whereas the initial on/off  rate for prothrombin made a major contribution to its overall association/dissociation profile,  the initial on/off  rate of  Factor IX, Factor IXa, and activated protein C each made a moderate contribution to their overall association/dissociation profile.  The effect  was even smaller for  protein C and protein Z, where the initial on/off  rate only made a minor contribution to their overall association/dissociation profile.  For these two proteins, the second association and dissociation rates contributed in a dominating way to their association/dissociation profile.  Coincidently, protein C and protein Z had the weakest membrane affinity  of  all Gla proteins tested. For Factor X, the initial on rate had a major contribution to association whereas the initial off  rate made only a minor contribution to dissociation and no contribution at the highest concentrations tested. In the most extreme case, protein S lacked biphasic kinetic properties in either association or dissociation phases as was evident from  the lack of  an initial fast  on or off  rate. The protein S kinetic data resembled that most closely of  xl-prothrombin characterized by a single slow apparent on and off  rate resulting in a tightly associated membrane complex. Kinetic data for  Protein S fitted  best to a bivalent analyte binding model (x2 = 141). Binding to such a model was appropriate, as Protein S forms  dimers in solution at even low concentrations (62)  and thus is a bivalent molecule with two membrane binding Gla domains. A comparison of  the kinetic binding profiles  of  the vitamin K-dependent Gla proteins by SPR has thus shown that these proteins have a highly variable membrane binding mechanism resulting in a wide range of  K a p p . The membrane affinity  varied by about 40-fold  between the human Gla proteins analyzed, with the expected extremes in range represented by protein S as the tightest binder at K ad pp = 73 +/- 2 nM and protein C as the weakest binder at K apv = 2.49 +/- 0.31 pM. The complete binding strength order for  the same 25% DOPS membrane surface from  tightest to weakest is protein S > FXa > FIXa > APC ~ FIX > FX ~ prothrombin > protein Z > protein C with determined K f  values indicated in Table 6. Another intriguing finding  is a comparison of  the total membrane binding capacities for  the individual Gla proteins (Table 6). Factor Xa has the highest surface  density for  all the Gla proteins ( R m a x = 6530 ry RU, 6.53 ng =141.9 fmol  bound per mm chip) analyzed. Relative to Factor Xa, the binding capacity from  highest to lowest is FXa > FIXa > FX > protein S > prothrombin > protein Z > protein C > FIX ~ APC with maximum binding percentage indicated in Table 6. Although these proteins have such high sequence similarity and conserved structure, the preferred  membrane analyte varies by almost a factor  of  10 when Factor Xa and Factor IX/activated protein C are compared. 5.3 Discussion 5.3.1 Binding comparison of  Gla proteins Using surface  plasmon resonance, I have investigated the detailed kinetic interaction between Gla proteins and a phosphatidylserine containing membrane surface.  Most studies focusing  on the Gla domain in relationship to membrane binding have evaluated single Gla proteins and a series of  mutants residing in the N-terminal half  of  the Gla domain (e.g. protein C/APC (133),  prothrombin (68),  Factor VII (134)),  particularly hydrophobic residues thought to insert into the membrane. These experiments primarily focused  on a single Gla protein and have provided detailed biochemical data describing the structure and functional  relationship between protein and membrane. However, data comparisons that are made between studies from  different  laboratories have large discrepancies and thus such comparisons are difficult  and often  invalid. Factors implicated in these data discrepancies between laboratories including technique, protein origin, membrane type, phospholipid composition and other experimental conditions (Ca2+, pH and temperature). For example, with protein C, a wide range of  Kj values to membranes have been reported: Table 7 Dissociation constants determined for  Protein C techniaue membrane protein source KH reference light scattering SUV (% PS = ?) bovine 0.23 pM (135) light scattering SUV (50% PS) human 1.9 pM (136) light scattering SUV (20% PS) human 7.3 pM (88) SPR immobilized human 3.5 pM (88) membrane (20% PS) light scattering SUV (25% PS) human 1.5 pM (79) light scattering SUV (25% PS) bovine 17 pM (79) light scattering SUV (25% PS) bovine 15 uM (137) Similarly for  prothrombin a IQj ranging from  0.11 to 1.1 pM were determined for  25% PS membranes of  various types by different  laboratories employing various techniques (see introduction for  details). To address this discrepancy, I have completed a thorough comparison of  binding affinity  and kinetic analysis of  human Gla proteins using the same membrane surface  and same technique. In the current Chapter, phospholipid membrane binding properties were determined using SPR for  all plasma derived human Gla proteins; zymogens, cofactors  and activated forms with the exception of  Factor VII over a wide concentration series using an identical membrane surface.  Results from  this study are comparable and are summarized in Table 6. To our surprise, there was great variation in all parameters investigated including membrane affinity  and binding preference  as well as kinetics controlling association and dissociation phases of  the membrane interaction. Unexpectedly, none of  the Gla proteins matched the binding profile  observed previously with prothrombin. With the exception of  Factor Xa and protein S, Gla protein - membrane binding was complex and could not be fit  to a simple 1:1 model or models made available through BIAevaluation describing a single additional process of  binding complexity (heterogeneity, conformers, multivalency - see Table 4). Thus, Gla-membrane binding is complex. A general trend seen throughout the Gla protein - membrane binding interaction is the involvement of apparent biphasic association and biphasic dissociation phases. This process was observed at all protein concentrations tested including physiological concentrations, with the exception of  protein Z which showed no detectable binding at physiological concentration. The comparative results obtained by SPR indicate that the homologous Gla proteins involved in different  enzymatic reactions of  coagulation have diverse membrane binding properties. The concept of  Gla interchangeability and similarity in membrane binding is often  thought to be a conserved feature  of  Gla proteins (132).  This is put into question with the current results indicating the vast differences  in membrane binding properties between these proteins. It is conceivable that the differences  in membrane binding observed here may actually facilitate  the different  roles and functions  these protein have in controlling coagulation. 5.3.2 Zymogen versus activated enzyme One would expect the membrane binding properties of  specific  zymogen and activated enzyme pair (with the exception of  prothrombin/thrombin) to be very similar if  not identical, as they have an identical Gla sequence responsible for  membrane contact. This is, however, clearly not the case. Striking membrane binding property differences  are observed between zymogen and respective activated enzyme. This was most dramatic for  Factor X and Factor Xa. Affinity  increased by over 4-fold  for  the activated enzyme with simultaneous 2-fold  increase in molar capacity for  membrane. Similar differences were observed for  Factor IX/IXa. Again the activated enzyme had a higher affinity  (2-fold)  and higher molar capacity for  membrane (3-fold)  relative to Factor IX. Finally, for the protein C/activated protein C pair, the activated enzyme form  showed a 5-fold increase in membrane affinity  with similar membrane binding capacity confirming  a previous light scattering study using bovine molecules and SUV (135).  Furthermore the mechanistic interaction also differed  between Factor X and Factor Xa as was evident from  the major shape differences  of  kinetic association and dissociation curves. This was not seen with two other zymogen-activated enzyme pairs studied (Factor IX/IXa and protein C/activated protein C). Clearly, zymogen activation results in membrane binding changes. Since the Gla sequence is unaltered, a sequence independent effect  must be the cause in this binding difference.  Conformational  changes in the Gla domain have not been described for  zymogen activation, but if  present could help explain such binding differences.  Alternatively, regions outside the Gla domain removed from  the membrane contact site may be responsible for  such binding differences.  Possible explanations for this behaviour include the existence of  membrane bound conformers  and the presence or absence between zymogen and activated enzyme of  a surface  induced dimerization site. These putative membrane binding modulators deserve more attention and are explained in more detail in sections 5.3.3 and 5.3.4. Interestingly, of  the Gla proteins examined, the membrane supported the highest molar binding capacity at saturation for  the activated procoagulant proteins; Factor Xa and Factor IXa. The significance  of  this finding  is unclear but implies that the activated procoagulant proteins bind negatively charged membranes favourably  in an environment where all proteins are present, such as the competing plasma environment at sites of procoagulant membrane. It makes physiological sense to have the activated procoagulant proteins out compete zymogen proteins for  binding sites at activated cell membranes. As enzymatic rates are controlled by the number of  activated enzyme complexes present, not substrate concentrations, then it follows  that it is more important to have enzymes bind these sites preferentially.  Of  course, this situation is complicated by the membrane affinity  enhancement effect  provided by their representative cofactors  (Factor Va and Factor Villa, respectively). Similarly, activated protein C has a higher membrane affinity compared to the zymogen, protein C. However, activated protein C was determined to have the lowest molar binding capacity for  membrane of  all Gla proteins analyzed. Not surprisingly, it relies on a 100-fold  enhancement of  membrane affinity  provided by its cofactor,  protein S (135)  to carry out its functions  localized to membrane surfaces. 5.3.3 Membrane binding is influenced  by regions outside of  Gla One study has shown that membrane binding can be influenced  by regions outside of  the Gla domain. The thrombin sensitive region (TSR) located between the Gla domain and the first  EGF domain is unique to protein S and was shown to be important to membrane binding (138).  In this study, a TSR deleted protein S molecule had a membrane affinity reduced by 15-fold  relative to intact protein S although the TSR peptide itself  was shown not to bind membranes, indicating that regions removed from  the membrane contact site influence  membrane binding. Unfortunately,  kinetic data for  the membrane interaction was not determined for  the two proteins. The study proposed a conformational stabilizing effect  by TSR on the Gla domain allowing for  an optimal interaction with phospholipids. Thus, the protein S molecule appears to have 2 membrane bound conformers  depending on the presence or absence of  TSR. It is conceivable then that upon zymogen activation (known to cause major Protease domain transitions) or initial membrane binding may result in conformational  changes that effect  the Gla - membrane interaction. 5.3.4 Vitamin K-dependent protein dimerization The effect  of  membrane binding of  Gla proteins may be complicated by surface  induced dimerization reactions. The biphasic kinetic data observed in the current study could be explained by such a reaction. Although no direct evidence exists for  such a reaction, Gla protein dimerization on membranes surfaces  should not be ignored but rather be considered as a potentially important event occurring on a membrane surface.  A number of  studies point to the existence of  such membrane dimers. It is known that prothrombin dimerizes in solution, although only at high concentration (between 15 - 230 |iM as assessed by cross-linking experiments (19)  and sedimentation measurements (18))  which indicates that an interactive site is present between two prothrombin molecules. This provides further  support that this intermolecular protein interaction on the membrane is likely to occur at much lower concentrations and as I propose physiological concentrations. Accordingly, Gla protein collisional events facilitating  dimer formation are much more probable to occur between membrane bound molecules as opposed to solution molecules, as diffusional  and rotational dynamics are restricted on the membrane surface. Additional supporting evidence comes from  Gla proteins described to dimerize only in the presence of  a membrane component. First, cross-linking studies using prothrombin (17)  and Factor VIlai (61)  an active site-inhibited form  of  Factor Vila cross-linked through the active sites (61)  at low protein concentrations were only possible in the presence of  phospholipid vesicles and calcium. This suggests that the membrane surface facilitates  the cross-linking reaction likely by a protein dimerization event prior to cross-linking. Second, cross-linked dimeric factor  VIlai is a bivalent membrane binding molecule with two Gla domains. The membrane binding characteristics of  this dimeric protein were shown to have an extremely slow off  rate compared to monomeric factor Vila, consistent with the xl-prothrombin studied here in Chapter 4, which greatly enhanced the dimer - membrane binding strength. Thus, any transient dimer formation  of a Gla protein on membrane would also result in an enhancement of  membrane affinity  by increasing the membrane resident time of  the protein. Third, non-covalently associated Protein S dimers and multimers in solution have been described previously to have greatly increased membrane affinity  compared to Protein S monomer (62).  Our data are consistent with these finding  as the protein S kinetic data was best described by a bivalent analyte model which assumes the presence of  solution dimers prior to membrane binding. Fourth, an observation was made with Factor IX crystallized on negatively charged phospholipid layers under near physiological conditions. The Factor IX molecules organized themselves as dimers in the 2-D crystals (139),  pointing to yet another example that these Gla proteins have a tendency to dimerize. Finally many heterodimeric Gla protein associations on membrane have been described. Protein S serves as a cofactor  to activated protein C by increasing the affinity  of  activated protein C for  membrane (135). Protein S binds to and inhibits Factor Xa (140).  These results in light of  the SPR data presented here indicate that membrane induced dimerization may be a common mechanism employed by the vitamin K-dependent Gla proteins which contributes to membrane binding. Chapter 6. Summary and General Discussion Blood coagulation depends on the effective  assembly of  a number of  multicomponent enzyme and regulatory complexes held together in the presence of  calcium ions by a plethora of  intermolecular, reversible interactions including protein-protein and protein-phospholipid. Of  these, the phosphatidylserine-containing membrane provided in vivo by a procoagulant surface  such as activated platelets, provides both allosteric and concentration effects  resulting in efficient  protein interactions. Assembly and subsequent activity of  these enzyme complexes depends on the binding properties of  these individual peripheral membrane proteins. As such, a sensitive and quantitative assay describing membrane affinity  and the kinetics governing association and dissociation is an essential tool for  understanding the function  and regulation of  these proteins. Equilibrium differential  centrifugation  and surface  plasmon resonance were employed to assess the membrane interaction of  prothrombin and other Gla protein involved in blood coagulation. In Chapter 3, the effect  Factor Va had on the affinity  of  prothrombin for  membrane was investigated. Taking advantage of  MLV that could be readily centrifuged  allowed separation of  bound and unbound peripheral protein. Combining this differential centrifugation  technique with immunoaffinity  quantification  allowed the basis of  some preliminary affinity  data collection. It has to be stressed, however, that the experimental errors associated with this technique were large, preventing the accurate determination of binding constants. Nevertheless, prothrombin-membrane binding results obtained gave a reasonable estimate of  the dissociation constant in the presence of  Factor Va (-50 nM) when bound to membrane. Factor Va bound to anionic phospholipid membrane undoubtedly enhanced the membrane binding affinity  of  prothrombin relative to prothrombin binding in the absence of  the cofactor.  Finally, prethrombin 1, a minor contaminant in the prothrombin preparation, was localized to membrane fractions  by Factor Va and showed no association to membrane in the absence of  Factor Va. Thus, Factor Va can individually recruit prothrombin or prethrombin 1 to the membrane surface,  plausibly contributing to its cofactor  function. Surface  plasmon resonance was proven to be a useful  method for  determining the detailed kinetics characterizing the membrane interaction of  Gla proteins. As detailed in Chapters 4 and 5, the technique provided further  insight into the nature of  the Gla protein-phosphatidylserine membrane interactions that had been previously characterized with a number of  other membrane binding assays. In Chapter 4, surface  plasmon resonance was used to evaluate the Ca2+-specific  binding of  prothrombin and proteolytic fragments  of  prothrombin to immobilized membranes composed of  mixtures of  phosphatidylserine (0-25%) and phosphatidylcholine . Equilibrium binding measurements showed that the apparent dissociation constants were 2-3 fold  lower for  prothrombin compared to fragment  1 or fragment  1.2, and these dissociation constants all increased with decreasing molar fraction  of  phosphatidylserine. Equilibrium binding profiles  for  fragment  1 and fragment  1.2 fitted  closely to a one-site binding model. Under the same conditions, however, prothrombin-membrane binding exhibited a biphasic association and dissociation process that deviated strongly from  the simple one-site binding model. This binding profile  was observed at all prothrombin concentrations tested (0.05 - 20 |xM) but was more pronounced at higher concentrations. Surface  plasmon resonance data fitted  best to a two step model characterized by an initial fast  kinetic step and a secondary slow kinetic step. In this model, the binding of prothrombin consists of  an obligate sequential mechanism involving a linked reaction because post-equilibrium dissociation profiles  varied with association phase time. A cross-linked dimer of  prothrombin formed  a high affinity  complex with membranes and had a similar off-rate  compared to the secondary slow off-rate  observed for  prothrombin. When combined, these results most likely are described by a model in which prothrombin forms  dimers on membranes. Dimerization may also explain the 2-3 fold  increase in membrane binding affinity  for  prothrombin compared to fragment  1 or fragment  1.2. Concurrent with these observations, dimerization appears to be specific  to the protease domain of  prothrombin as neither fragment  1 nor fragment  1.2 displays such binding complexities. The physiological significance  of  the observed prothrombin dimerization on procoagulant membranes is unclear at present. It is possible that dimerization increases the half-life  of  membrane-bound prothrombin. This increased availability may increase the participation of  prothrombin in the prothrombinase complex with subsequent thrombin production. The application of  SPR to study membrane binding was further  expanded to the other blood coagulation Gla proteins in Chapter 5. Membrane affinity  (40-fold),  molar binding preference  (10-fold)  and kinetics controlling complex formation  and complex breakdown varied widely between Gla proteins. Apparent biphasic association and biphasic dissociation phases were commonly observed amongst the Gla proteins at a wide range of protein concentrations including physiological concentrations. The comparative results obtained by SPR indicate that the majority of  homologous Gla proteins bind membranes with a complex mechanism which may involve membrane induced protein dimers (or multimers) and/or multiple membrane bound conformers.  Gla proteins appear to have a propensity to dimerize (17-19,  62, 102, 135, 139, 140). Thus, an explanation involving an intermolecular interaction between two Gla proteins on the membrane surface  after  initial membrane complex formation  appears to be the most likely explanation for  this binding complexity. Analysis and interpretation of  the underlying factors  governing the complex membrane kinetic data obtained by SPR is often  difficult  if  not impossible for  a given peripheral protein, as was the case for  many of  the experiments carried out in this thesis. This is often  described as major disadvantage of  this technique which is currently still in its infancy.  However, with further  experimentation, these complexities should be overcome and thereby provide significant  amount of  information.  A continuing objective in the field  of  membrane binding of  blood coagulation Gla proteins as well as other peripheral protein membrane is to decipher the mechanistic details of  the protein-membrane interaction and other effects  the membrane plays on bound proteins. For such a problem to be solved, a very sensitive kinetic techniques such as SPR will be required. Chapter 7. Future Directions Several experimental approaches are possible with the developments from  the experimental results in this thesis. Specific  molecules or reagents that will allow further investigation of  membrane binding properties of  Gla proteins include the use of  a physiological membrane, recombinant chimeric Gla proteins and prethrombin 1 as a Factor Va specific  membrane binding probe and as an inhibitor of  prothrombin membrane binding. For reasons of  simplicity, Gla protein - membrane binding studies focusing  on detailed kinetics have used artificial  membranes composed of  usually two and sometimes up to three types of  phospholipids. Compared to the physiological platelet membrane setting, these artificial  membranes clearly oversimplify  the membrane composition of  activated platelets which contain a number of  phospholipid types as well as integral and tightly associated proteins, many of  which have unknown function  (141).  Gla proteins have a tighter affinity  to platelets compared to artificial  liposome membranes. This may be caused by the presence of  specific  platelet receptor(s), reviewed in (56).  Thus, a binding study involving activated platelet membranes seems imperative. Platelet derived microparticles are prime candidates for  a SPR experiment as they are readily immobilized by a similar strategy used here for  liposomes. These microparticles are small membrane vesicles released from  activated platelets and provide a major catalytic surface  for  the assembly of  blood coagulation enzyme complexes (142,  143). Thus a study using platelet microparticles may give further  insight to the role physiological membranes have in Gla protein membrane binding. Vitamin K-dependent protein chimeras have been made for  protein C containing the Gla domain of  either Factor VII, Factor IX or prothrombin (132,144,145).  These studies have indicated an interchangeability of  the Gla domain for  protein C without dramatically altering its membrane binding properties under study. For example comparison of phospholipid binding of  prothrombin, protein C and the protein C containing the Gla domain of  prothrombin chimera showed very similar binding for  protein C and the chimera, whereas prothrombin was noticeably different  (132).  This was also reflected  in the Kf p obtained in this study; prothrombin (700 nM), protein C (430 nM) and chimera (520 nM). Similarly, membrane affinity  appears to be controlled by areas extrinsic to the Gla domain, as protein C and the protein C containing the Gla domain of  Factor IX chimera showed very similar binding affinities  that differed  significantly  from  Factor IX. Unfortunately,  only equilibrium binding data obtained by right angle light scattering were used to characterize membrane binding in these studies, as a detailed kinetic binding analysis was not performed.  This emphasizes that the unique binding characteristics observed for  individual Gla proteins by SPR (i.e. shape of  kinetic profiles  and affinity) are perhaps due to unique sites removed from  the membrane contact site. Thus an intriguing possibility exists that the characteristic SPR kinetic signature curves encompassing various complex mechanisms observed for  each Gla protein is a result of protein regions outside the Gla domain. For example, a chimeric study involving for instance prothrombin containing the Gla domain of  various other Gla proteins would be predicted to have membrane binding properties very similar to native/wild type prothrombin. Similarly, it should be feasible  to maintain the simpler kinetic profile  seen with Factor Xa by swapping its Gla domain with another Gla domain. Of  course there are some properties that appear to be intrinsic to the individual Gla domain, as was shown with the slighdy altered phospholipid specificities  of  protein C and prothrombin (132)\ however, these differences  are expected to be minor compared to the role the remainder of  the protein plays to overall membrane binding. Unlike the interaction between Factor Va and Factor Xa, the interaction between Factor Va and prothrombin on membrane is not well understood. In Chapter 3, prethrombin 1 was shown to bind specifically  to Factor Va-bound vesicles, as the Gla-less protein was unable to bind vesicles in the absence of  the cofactor.  It is apparent that when bound to vesicles prothrombin binds to Factor Va tighter (~ 50 nM, this study) then Factor Va in solution (1.9 (iM, (20)  and 1.3 (iM, (23)).  The use of  prethrombin 1 as a specific  probe for  Factor Va bound to membranes may have several applications. First prethrombin 1 could be used to determine the specific  site on the cofactor  mediating the interaction by using a number of  Factor Va derived peptides to inhibit binding. Furthermore, showing that prethrombin 1 has a higher affinity  to Factor Va when bound to vesicles compared to in solution would provide evidence that the membrane allosterically controls the configuration  of  the Factor Va binding site. Finally, prethrombin 1 was shown in Chapter 4 to clearly inhibit prothrombin membrane binding. The exact mechanism of  this inhibition remains unclear and also deserves further  study. One possible explanation is that prethrombin 1 interacts with prothrombin in solution, thereby changing the conformation  of  the membrane contact site on prothrombin. This prethrombin 1 - bound conformation  of  prothrombin may result in a slower, but tighter binder which may explain the SPR data observed in Chapter 4. It is thus intriguing if  one could potentially crosslink prothrombin and prethrombin 1 in solution. If  so, a crosslinked prothrombin - prethrombin 1 heterodimer may display simple 1:1 kinetics. Bibliography (1) Nemerson, Y. (1992) The tissue factor  pathway of  blood coagulation. Semin Hematol.  29, 170-6. (2) Bach, R., Gentry, R., and Nemerson, Y. (1986) Factor VII binding to tissue factor in reconstituted phospholipid vesicles: induction of  cooperativity by phosphatidylserine. Biochemistry 25, 4007-4020. (3) Ahmad, S. S„ Rawala-Sheikh, R„ and Walsh, P. N. (1992) Components and assembly of  the factor  X activating complex. Semin Thromb  Hemost.  18, 311-23. (4) Krishnaswamy, S. (1990) Prothrombinase complex assembly. Contributions of protein-protein and protein-membrane interactions toward complex formation.  J Biol Chem. 265, 3708-18. (5) Esmon, C. T., Ding, W., Yasuhiro, K., Gu, J. M., Ferrell, G., Regan, L. M., Stearns-Kurosawa, D. J., Kurosawa, S., Mather, T., Laszik, Z., and Esmon, N. L. (1997) The protein C pathway: new insights. Thromb  Haemost.  78, 70-4. (6) Degen, S. J., and Sun, W. Y. (1998) The biology of  prothrombin. Crit  Rev Eukaryot  Gene Expr. 8, 203-24. (7) Hijikata-Okunomiya, A. (1990) A new method for  the determination of prothrombin in human plasma. Thromb  Res. 57, 705-15. (8) Zytkovicz, T. H., and Nelsestuen, G. L. (1975) [3H]diborane reduction of  vitamin K-dependent calcium-binding proteins. Identification  of  a unique amino acid. J Biol Chem. 250, 2968-72. (9) Mizuochi, T., Fujii, J., Kisiel, W., and Kobata, A. (1981) Studies on the structures of  the carbohydrate moiety of  human prothrombin. J  Biochem (Tokyo).  90,1023-31. (10) Petrovan, R. J., Govers-Riemslag, J. W., Nowak, G., Hemker, H. C., Tans, G., and Rosing, J. (1998) Autocatalytic peptide bond cleavages in prothrombin and meizothrombin. Biochemistry 37, 1185-91. (11) Nesheim, M. E„ Taswell, J. B., and Mann, K. G. (1979) The contribution of bovine Factor V and Factor Va to the activity of  prothrombinase. J  Biol Chem. 254, 10952-62. (12) Krishnaswamy, S. (2005) Exosite-driven substrate specificity  and function  in coagulation. J  Thromb  Haemost.  3, 54-67. (13) Krishnaswamy, S., Mann, K. G., and Nesheim, M. E. (1986) The prothrombinase-catalyzed activation of  prothrombin proceeds through the intermediate meizothrombin in an ordered, sequential reaction. J  Biol Chem. 261, 8977-84. (14) Cote, H. C., Bajzar, L., Stevens, W. K, Samis, J. A., Morser, J., MacGillivray, R. T., and Nesheim, M. E. (1997) Functional characterization of  recombinant human meizothrombin and Meizothrombin(desFl). Thrombomodulin-dependent activation of  protein C and thrombin-activatable fibrinolysis  inhibitor (TAFI), platelet aggregation, antithrombin-III inhibition. J  Biol Chem. 272, 6194-200. (15) Cote, H. C., Stevens, W. K„ Bajzar, L., Banfield,  D. K, Nesheim, M. E., and MacGillivray, R. T. (1994) Characterization of  a stable form  of  human meizothrombin derived from  recombinant prothrombin (R155A, R271A, and R284A). J  Biol Chem. 269, 11374-80. (16) Walsh, P. N., and Ahmad, S. S. (2002) Proteases in blood clotting. Essays Biochem. 38. (17) Anderson, P. J. (1998) A dimeric form  of  prothrombin on membrane surfaces. Biochem J.  336, 631-8. (18) Jackson, C. M„ Peng, C. W„ Brenckle, G. M„ Jonas, A., and Stenflo,  J. (1979) Multiple modes of  association in bovine prothrombin and its proteolysis products. J  Biol Chem. 254, 5020-6. (19) Tarvers, R. C„ Noyes, C. M„ Tarvers, J. K„ and Lundblad, R. L. (1986) Mechanism of  the calcium-dependent self-association  of  bovine prothrombin. Use of  a covalent cross-linking reagent to study the reaction. J  Biol Chem. 261,4855-9. (20) Deguchi, H., Takeya, H„ Gabazza, E. C„ Nishioka, J., and Suzuki, K. (1997) Prothrombin kringle 1 domain interacts with factor  Va during the assembly of prothrombinase complex. Biochem J.  321, 729-35. (21) Kotkow, K. J., Deitcher, S. R„ Furie, B„ and Furie, B. C. (1995) The second kringle domain of  prothrombin promotes factor  Va-mediated prothrombin activation by prothrombinase. J  Biol Chem. 270, 4554-7. (22) Kim, B. J., Koo, S. Y„ and Kim, S. S. (2002) A peptide derived from  human prothrombin fragment  2 inhibits prothrombinase and angiogenesis. Thromb  Res. 106, 81-7. (23) Blostein, M. D„ Rigby, A. C., Jacobs, M„ Furie, B., and Furie, B. C. (2000) The Gla domain of  human prothrombin has a binding site for  factor  Va. J  Biol Chem. 275, 38120-6. (24) Anderson, P. J., Nesset, A., Dharmawardana, K. R„ and Bock, P. E. (2000) Role of  proexosite I in factor  Va-dependent substrate interactions of  prothrombin activation. J  Biol Chem. 275,16435-42. (25) Chen, L., Yang, L., and Rezaie, A. R. (2003) Proexosite-1 on prothrombin is a factor  Va-dependent recognition site for  the prothrombinase complex. J  Biol Chem. 278, 27564-9. (26) Yegneswaran, S„ Mesters, R. M„ Fernandez, J. A., and Griffin,  J. H. (2004) Prothrombin residues 473-487 contribute to factor  Va binding in the prothrombinase complex. J  Biol Chem. 279, 49019-25. (27) Stenflo,  J. (1999) Contributions of  Gla and EGF-like domains to the function  of vitamin K-dependent coagulation factors.  Crit  Rev Eukaryot  Gene Expr. 9, 59-88 review. (28) Nelsestuen, G. L„ Shah, A. M., and Harvey, S. B. (2000) Vitamin K-dependent proteins. Vitam  Horm.  58, 355-89. (29) Chen, Q., and Lentz, B. R. (1997) Fluorescence resonance energy transfer  study of  shape changes in membrane-bound bovine prothrombin and meizothrombin. Biochemistry 36, 4701-11. (30) Bode, W. (2005) The structure of  thrombin, a chameleon-like proteinase. Thromb Haemost.  3, 2379-88. (31) Broze, G. J. J. (2001) Protein Z-dependent regulation of  coagulation. Thromb Haemost.  86, 8-13. (32) Broze, G. J. J., and Miletich, J. P. (1984) Human Protein Z. J  Clin  Invest.  73, 933-8. (33) Edenbrandt, C. M„ Lundwall, A., Wydro, R., and Stenflo,  J. (1990) Molecular analysis of  the gene for  vitamin K dependent protein S and its pseudogene. Cloning and partial gene organization. Biochemistry 29, 7861-8. (34) Rigby, A. C., and Grant, M. A. (2004) Protein S: a conduit between anticoagulation and inflammation.  Crit  Care  Med.  32, S336-41. (35) Munoz, X., Sumoy, L., Ramirez-Lorca, R., Villar, J., de Frutos, P. G., and Sala, N. (2004) Human vitamin K-dependent GAS6: gene structure, allelic variation, and association with stroke. Hum  Mutat.  23, 506-12. (36) Price, P. A., Poser, J. W., and Raman, N. (1976) Primary structure of  the gamma-carboxyglutamic acid-containing protein from  bovine bone. Proc Natl  Acad  Sci U SA 73, 3374-5. (37) Kulman, J. D., Harris, J. E., Xie, L., and Davie, E. W. (2001) Identification  of  two novel transmembrane gamma-carboxyglutamic acid proteins expressed broadly in fetal  and adult tissues. Proc Natl  Acad  Sci U  S A 98, 1370-5. (38) Khazi, F. R., Chu, K. C., and High, K. A. in The  American Society  of  Hematology 48th Annual Meeting  and  Exposition December 9-12, 2006, Orlando, Florida. (39) Kulman, J. D., Harris, J. E., Nakazawa, N., Ogasawara, M., Satake, M., and Davie, E. W. (2006) Vitamin K-dependent proteins in Ciona intestinalis, a basal chordate lacking a blood coagulation cascade. Proc Natl  Acad  Sci USA  103, 15794-9. (40) Ahmad, S. S„ London, F. S„ and Walsh, P. N. (2003) The assembly of  the factor X-activating complex on activated human platelets. J  Thromb  Haemost.  1, 48-59. (41) McGee, M. P., Li, L. C., and Hensler, M. (1992) Functional assembly of  intrinsic coagulation proteases on monocytes and platelets. Comparison between cofactor activities induced by thrombin and factor  Xa. J  Exp Med.  176,  27-35. (42) Falls, L. A., Furie, B., and Furie, B. C. (2000) Role of  phosphatidylethanolamine in assembly and function  of  the factor  IXa-factor  Villa complex on membrane surfaces.  Biochemistry 39, 13216-22. (43) Zwaal, R. F., and Schroit, A. J. (1997) Pathophysiologic implications of membrane phospholipid asymmetry in blood cells. Blood  89, 1121-32. (44) Govers-Riemslag, J. W., Janssen, M. P., Zwaal, R. F., and Rosing, J. (1994) Prothrombin activation on dioleoylphosphatidylcholine membranes. Eur J Biochem. 220, 131-8. (45) Lentz, B. R. (2003) Exposure of  platelet membrane phosphatidylserine regulates blood coagulation. Prog Lipid  Res. 42, 423-38. (46) Mann, K. G., Nesheim, M. E., Church, W. R., Haley, P., and Krishnaswamy, S. (1990) Surface-dependent  reactions of  the vitamin K-dependent enzyme complexes. Blood  76,  1-16 review. (47) Furie, B., and Furie, B. C. (1988) The molecular basis of  blood coagulation. Cell 53, 505-18. (48) Husten, E. J., Esmon, C. T., and Johnson, A. E. (1987) The active site of  blood coagulation factor  Xa: its distance from  the phospholipid surface  and its conformational  sensitivity to components of  the prothrombinase complex. J  Biol Chem. 262, 12953-12961. (49) Armstrong, S. A., Husten, E. J., Esmon, C. T„ and Johnson, A. E. (1990) The active site of  membrane-bound meizothrombin. A fluorescence  determination of its distance from  the phospholipid surface  and its conformational  sensitivity to calcium and factor  Va. J  Biol Chem. 265, 6210-8. (50) Cutsforth,  G. A., Whitaker, R. N„ Hermans, J., and Lentz, B. R. (1989) A new model to describe extrinsic protein binding to phospholipid membranes of  varying composition: application to human coagulation proteins. Biochemistry 28, 7453-61. (51) Nelsestuen, G. L., and Broderius, M. (1977) Interaction of  prothrombin and blood-clotting factor  X with membranes of  varying composition. Biochemistry 16, 4172-7. (52) Bangham, A. D. (1961) A correlation between surface  charge and coagulant action of  phospholipids. Nature  192,1197-8. (53) Comfurius  P, S. E„ Willems GM, Bevers EM, Zwaal RF. (1994) Assembly of  the prothrombinase complex on lipid vesicles depends on the stereochemical configuration  of  the polar headgroup of  phosphatidylserine. Biochemistry 33, 10319-24. (54) Majumder, R„ Weinreb, G., Zhai, X., and Lentz, B. R. (2002) Soluble phosphatidylserine triggers assembly in solution of  a prothrombin-activating complex in the absence of  a membrane surface.  J  Biol Chem. 277,  29765-73. (55) Majumder, R., Weinreb, G., and Lentz, B. R. (2005) Efficient  thrombin generation requires molecular phosphatidylserine, not a membrane surface. Biochemistry 44, 16998-7006.. (56) Walsh, P. N. (2004) Platelet coagulation-protein interactions. Semin Thromb Hemost.  30, 461-71. (57) Bevers, E. M., Comfurius,  P., and Zwaal, R. F. (1983) Changes in membrane phospholipid distribution during platelet activation. Biochim Biophys Acta. 736, 57-66. (58) Grant, M. A., Baikeev, R. F., Gilbert, G. E„ and Rigby, A. C. (2004) Lysine 5 and phenylalanine 9 of  the factor  IX omega-loop interact with phosphatidylserine in a membrane-mimetic environment. Biochemistry 43, 15367-78. (59) McGee, M. P., Teuschler, H., and Liang, J. (1998) Effective  electrostatic charge of  coagulation factor  X in solution and on phospholipid membranes: implications for  activation mechanisms and structure-function  relationships of  the Gla domain. Biochem J.  330, 533-9. (60) Falls, L. A., Furie, B. C., Jacobs, M., Furie, B„ and Rigby, A. C. (2001) The omega-loop region of  the human prothrombin gamma-carboxyglutamic acid domain penetrates anionic phospholipid membranes. J  Biol Chem. 276,  23895-902. (61) Stone, M. D., Harvey, S. B., Martinez, M. B., Bach, R. R., and Nelsestuen, G. L. (2005) Large enhancement of  functional  activity of  active site-inhibited factor Vila due to protein dimerization: insights into mechanism of assembly/disassembly from  tissue factor.  Biochemistry 44, 6321-30. (62) Sere, K. M., Janssen, M. P., Willems, G. M., Tans, G., Rosing, J., and Hackeng, T. M. (2001) Purified  protein S contains multimeric forms  with increased APC-independent anticoagulant activity. Biochemistry 40, 8852-60. (63) Dai, Q., Prorok, M., and Castellino, F. J. (2005) Role of  the hexapeptide disulfide loop in the gamma-carboxyglutamic acid domain of  protein C in Ca2+-mediated structural and functional  properties. Biochemistry 37, 12508-14. (64) Lu, Y., and Nelsestuen, G. L. (1996) Dynamic features  of  prothrombin interaction with phospholipid vesicles of  different  size and composition: implications for protein—membrane contact. Biochemistry 35, 8193-200. (65) Abbott, A. J., and Nelsestuen, G. L. (1987) Association of  a protein with membrane vesicles at the collisional limit: studies with blood coagulation factor Va light chain also suggest major differences  between small and large unilamellar vesicles. Biochemistry 26, 7994-8003. (66) Lim, T. K„ Bloomfield,  V. A., and Nelsestuen, G. L. (1977) Structure of  the prothrombin- and blood clotting factor  X-membrane complexes. Biochemistry 16, 4177-81. (67) Kotkow, K. J., Furie, B., and Furie, B. C. (1993) The interaction of  prothrombin with phospholipid membranes is independent of  either kringle domain. J  Biol Chem. 268, 15633-9. (68) Ratcliffe,  J. V., Furie, B., and Furie, B. C. (1993) The importance of  specific gamma-carboxyglutamic acid residues in prothrombin. Evaluation by site-specific mutagenesis. J  Biol Chem. 268, 24339-45. (69) Pollock, J. S„ Shepard, A. J., Weber, D. J., Olson, D. L„ Klapper, D. G„ Pedersen, L. G., and Hiskey, R. G. (1988) Phospholipid binding properties of bovine prothrombin peptide residues 1-45. J  Biol Chem. 263, 14216-23. (70) Soriano-Garcia, M., Padmanabhan, K., de Vos, A. M., and Tulinsky, A. (1992) The Ca2+ ion and membrane binding structure of  the Gla domain of  Ca-prothrombin fragment  1. Biochemistry 31, 2554-66. (71) Sunnerhagen, M., Forsen, S., Hoffren,  A. M., Drakenberg, T., Teleman, O., and Stenflo,  J. (1995) Structure of  the Ca(2+)-free  Gla domain sheds light on membrane binding of  blood coagulation proteins. Nat  Struct  Biol. 2, 504-9. (72) Christiansen, W. T., Tulinsky, A., and Castellino, F. J. (1994) Functions of individual gamma-carboxyglutamic acid (Gla) residues of  human protein c. Determination of  functionally  nonessential Gla residues and correlations with their mode of  binding to calcium. Biochemistry 33, 14993-5000. (73) Freedman, S. J., Furie, B. C., Furie, B„ and Baleja, J. D. (1995) Structure of  the calcium ion-bound gamma-carboxyglutamic acid-rich domain of  factor  DC. Biochemistry 34, 12126-37. (74) Weber, D. J., Berkowitz, P., Panek, M. G„ Huh, N. W„ Pedersen, L. G„ and Hiskey, R. G. (1992) Modifications  of  bovine prothrombin fragment  1 in the presence and absence of  Ca(II) ions. Loss of  positive cooperativity in Ca(II) ion binding for  the modified  proteins. J  Biol Chem. 267,  4564-9. (75) Huang, M., Rigby, A., Morelli, X., Grant, M., Huang, G., Furie, B., Seaton, B„ and Furie, B. (2003) Structural basis of  membrane binding by Gla domains of vitamin K-dependent proteins. Nat  Struct  Biol. 10, 751-6. (76) Christiansen, W. T., Jalbert, L. R., Robertson, R. M., Jhingan, A., Prorok, M., and Castellino, F. J. (1995) Hydrophobic amino acid residues of  human anticoagulation protein C that contribute to its functional  binding to phospholipid Vesicles. Biochemistry 34, 10376-82. (77) Resnick, R. M., and Nelsestuen, G. L. (1980) Prothrombin-membrane interaction. Effects  of  ionic strength, pH, and temperature. Biochemistry 19, 3028-33. (78) McDonald, J. F., Evans, T. C. J., Emeagwali, D. B., Hariharan, M., Allewell, N. M., Pusey, M. L., Shah, A. M., and Nelsestuen, G. L. (1997) Ionic properties of membrane association by vitamin K-dependent proteins: the case for  univalency. Biochemistry 36, 15589-98. (79) McDonald, J. F„ Shah, A. M„ Schwalbe, R. A., Kisiel, W„ Dahlback, B., and Nelsestuen, G. L. (1997) Comparison of  naturally occurring vitamin K-dependent proteins: correlation of  amino acid sequences and membrane binding properties suggests a membrane contact site. Biochemistry 36, 5120-7. (80) Majumder, R„ Wang, J., and Lentz, B. R. (2003) Effects  of  water soluble phosphotidylserine on bovine factor  Xa: functional  and structural changes plus dimerization. Biophys J.  84, 1238-51. (81) Evans, T. C. J., and Nelsestuen, G. L. (1994) Calcium and membrane-binding properties of  monomeric and multimeric annexin II. Biochemistry 33, 13231-8. (82) Wei, G. J., Bloomfield,  V. A., Resnick, R. M„ and Nelsestuen, G. L. (1982) Kinetic and mechanistic analysis of  prothrombin-membrane binding by stopped-flow  light scattering. Biochemistry 21, 1949-59. (83) Kop, J. M„ Cuypers, P. A., Lindhout, T„ Hemker, H. C., and Hermens, W. T. (1984) The adsorption of  prothrombin to phospholipid monolayers quantitated by ellipsometry. J  Biol Chem. 259,13993-8. (84) Pearce, K. H., Hiskey, R. G„ and Thompson, N. L. (1992) Surface  binding kinetics of  prothrombin fragment  1 on planar membranes measured by total internal reflection  fluorescence  microscopy. Biochemistry 31, 5983-95. (85) Pearce, K. H., Hof,  M„ Lentz, B. R., and Thompson, N. L. (1993) Comparison of the membrane binding kinetics of  bovine prothrombin and its fragment  1. J  Biol Chem. 268, 22984-91. (86) Ellison, E. H., and Castellino, F. J. (1998) Adsorption of  vitamin K-dependent blood coagulation proteins to spread phospholipid monolayers as determined from combined measurements of  the surface  pressure and surface  protein concentration. Biochemistry 37, 7997-8003. (87) Richter, R. P., Maury, N., and Brisson, A. R. (2005) On the effect  of  the solid support on the interleaflet  distribution of  lipids in supported lipid bilayers. Langmuir 21, 299-304. (88) Sun, Y. H., Shen, L., and Dahlback, B. (2003) Gla domain-mutated human protein C exhibiting enhanced anticoagulant activity and increased phospholipid binding. Blood  101, 2277-84. (89) Shen, L„ Shah, A. M., Dahlback, B„ and Nelsestuen, G. L. (1998) Enhancement of  human protein C function  by site-directed mutagenesis of  the gamma-carboxyglutamic acid domain. J  Biol Chem. 273, 31086-91. (90) Evans, T. C. J., and Nelsestuen, G. L. (1996) Importance of  cis-proline 22 in the membrane-binding conformation  of  bovine prothrombin. Biochemistry 35, 8210-5. (91) Perera, L., Darden, T. A., and Pedersen, L. G. (1998) Trans-Cis Isomerization of Proline 22 in Bovine Prothrombin Fragment 1: A Surprising Result of  Structural Characterization. Biochemistry 37, 10920-7. (92) Lentz, B. R„ Zhou, C. M„ and Wu, J. R. (1994) Phosphatidylserine-containing membranes alter the thermal stability of  prothrombin's catalytic domain: a differential  scanning calorimetric study. Biochemistry 33, 5460-8. (93) Tendian, S. W„ Lentz, B. R„ and Thompson, N. L. (1991) Evidence from  total internal reflection  fluorescence  microscopy for  calcium-independent binding of prothrombin to negatively charged planar phospholipid membranes. Biochemistry 30, 10991-9. (94) Cooper, M. A., Hansson, A., Lofas,  S., and Williams, D. H. (2000) A vesicle capture sensor chip for  kinetic analysis of  interactions with membrane-bound receptors. Anal Biochem. 277,  196-205. (95) Schuck, P. (1997) Use of  surface  plasmon resonance to probe the equilibrium and dynamic aspects of  interactions between biological macromolecules. Anna Rev Biophys Biomol Struct.  26, 541-66. (96) Hall, D. (2001) Use of  optical biosensors for  the study of  mechanistically concerted surface  adsorption processes. Anal. Biochem. 288,109-25. (97) Stenberg, E., Persson, B., Roos, H., and Urbaniczky, C. (1991) Quantitative Determination of  surface  concentration of  protein with surface  plasmon resonance using radiolabeled proteins. J  Colloid  Interface  Sci.  143, 513-26. (98) Gizeli, E., and Lowe, C. R. (2002) In Biomolecular Sensors. Taylor and Francis Publishing, New York. (99) Mayer, L. D., Hope, M. J., and Cullis, P. R. (1986) Vesicles of  variable sizes produced by a rapid extrusion procedure. Biochim Biophys Acta 858, 161-8. (100) Chen, P. S„ JR. Toribara, T. Y., and Warner, H. (1956) Microdetermination of Phosphorus. Anal. Chem. 28,1756-8. (101) Nelsestuen, G. L., and Lim, T. K. (1977) Equilibria involved in prothrombin- and blood-clotting factor  X-membrane binding. Biochemistry 16, 4164-71. (102) Stone, M. D., and Nelsestuen, G. L. (2005) Efficacy  of  soluble phospholipids in the prothrombinase reaction. Biochemistry 44,4037-41. (103) Luckow, E. A., Lyons, D. A., Ridgeway, T. M., Esmon, C. T., and Laue, T. M. (1989) Interaction of  clotting factor  V heavy chain with prothrombin and prethrombin 1 and role of  activated protein C in regulating this interaction: analysis by analytical ultracentrifugation.  Biochemistry 28, 2348-54. (104) Mann, K. G. (1976) Prothrombin. Methods  Enzymology  45, 123-56. (105) Tracy PB, E. L., Bowie EJ, Mann KG.. (1982) Radioimmunoassay of  factor  V in human plasma and platelets. Blood  60, 59-63. (106) Billy, D„ Willems, G. M„ Hemker, H. C., and Lindhout, T. (1995) Prothrombin contributes to the assembly of  the factor  Va-factor  Xa complex at phosphatidylserine-containing phospholipid membranes. J  Biol Chem. 270, 26883-9. (107) Beck, D. O., Bukys, M. A., Singh, L. S„ Szabo, K. A., and Kalafatis,  M. (2004) The contribution of  amino acid region ASP695-TYR698 of  factor  V to procofactor  activation and factor  Va function.  J  Biol Chem. 279, 3084-95. (108) Sorensen, K. W., Nicolaes, G. A., Villoutreix, B. 0., Yamazaki, T„ Tans, G., Rosing, J., and Dahlback, B. (2004) Functional properties of  recombinant factor  V mutated in a potential calcium-binding site. Biochemistry 43, 5803-10. (109) Levesque, J. P., Hatzfeld,  A., and Hatzfeld,  J. (1985) A method to measure receptor binding of  ligands with low affinity.  Application to plasma proteins binding assay with hemopoietic cells. Exp. Cell  Res. 156, 558-562. (110) Krishnaswamy, S., and Mann, K. G. (1988) The binding of  factor  Va to phospholipid vesicles. J  Biol Chem. 263, 5714-23. (111) Degen, S. J., and Davie, E. W. (1987) Nucleotide sequence of  the gene for  human prothrombin. Biochemistry 26, 6165-77. (112) Lentz, B. R. (1995) Are acidic lipid domains induced by extrinsic protein binding to membranes? Mol  Membr  Biol. 12, 65-7. (113) Rich, R. L„ and Myszka, D. G. (2005) Survey of  the year 2004 commercial optical biosensor literature. J  Mol  Recognit.  18, 431-478. (114) Erb, E. M„ Stenflo,  J., and Drakenberg, T. (2002) Interaction of  bovine coagulation factor  X and its glutamic-acid-containing fragments  with phospholipid membranes. A surface  plasmon resonance study. Eur J  Biochem. 269, 3041-6. (115) Saenko, E., Sarafanov,  A., Ananyeva, N., Behre, E., Shima, M., Schwinn, H., and Josic, D. (2001) Comparison of  the properties of  phospholipid surfaces  formed  on HPA and LI biosensor chips for  the binding of  the coagulation factor  VIII. J ChromatogrA.  921, 49-56. (116) Saenko, E., Sarafanov,  A., Greco, N., Shima, M., Loster, K., Schwinn, H., and Josic, D. (1999) Use of  surface  plasmon resonance for  studies of  protein-protein and protein-phospholipid membrane interactions. Application to the binding of factor  VIII to von Willebrand factor  and to phosphatidylserine-containing membranes. J  Chromatogr  A. 852, 59-71. (117) Neuenschwander, P. F. (2004) Exosite occupation by heparin enhances the reactivity of  blood coagulation factor  IXa. Biochemistry 43, 2978-86. (118) Osterlund, M., Persson, E., Carlsson, U., Freskgard, P. O., and Svensson, M. (2005) Sequential coagulation factor  Vila domain binding to tissue factor. Biochem Biophys Res Commun. 337, 1276-82. (119) Erb, E. M„ Chen, X., Allen, S., Roberts, C. J., Tendler, S. J., Davies, M. C„ and Forsen, S. (2000) Characterization of  the surfaces  generated by liposome binding to the modified  dextran matrix of  a surface  plasmon resonance sensor chip. Anal Biochem. 280, 29-35. (120) Dimitrova, M. N., Matsumura, H„ Dimitrova, A., and Neitchev, V. Z. (2000) Interaction of  albumins from  different  species with phospholipid liposomes. Multiple binding sites system. Int  J  Biol Macromol.  27, 187-94. (121) Cirelli, N., Lebrun, P., Gueuning, C., Delogne-Desnoeck, J., Vanbellinghen, A. M., Graff,  G., and Meuris, S. (2001) Physiological concentrations of  albumin stimulate chorionic gonadotrophin and placental lactogen release from  human term placental explants. Hum  Reprod.  16, 441-8. (122) Cooper, M. A., Try, A. C„ Carroll, J., Ellar, D. J., and Williams, D. H. (1998) Surface  plasmon resonance analysis at a supported lipid monolayer. Biochim Biophys Acta 1373,101-11. (123) Willems, G. M., Janssen, M. P., Comfurius,  P., Galli, M„ Zwaal, R. F„ and Bevers, E. M. (2000) Competition of  annexin V and anticardiolipin antibodies for binding to phosphatidylserine containing membranes. Biochemistry 39, 1982-9. (124) Wu, J. R., and Lentz, B. R. (1991) Fourier transform  infrared  spectroscopic study of  Ca2+ and membrane-induced secondary structural changes in bovine prothrombin and prothrombin fragment  1. Biophys J.  60, 70-80. (125) Malholtra, O. P. (1991) Isolation and some properties of  11- and 9-Gla prothrombins from  normal plasma. Biochem Cell  Biol. 69, 404-8. (126) Lentz, B. R„ Wu, J. R„ Sorrentino, A. M„ and Carleton, J: N. (1991) Membrane binding induces lipid-specific  changes in the denaturation profile  of  bovine prothrombin. A scanning calorimetry study. Biophys J.  60, 942-51. (127) Mayer, L., Nelsestuen, G. L., and Brockman, H. L. (1983) Prothrombin association with phospholipid monolayers. Biochemistry 22, 316-21. (128) Chenna, R., Sugawara, H., Koike, T., Lopez, R., Gibson, T. J., Higgins, D. G., and Thompson, J. D. (2003) Multiple sequence alignment with the Clustal series of  programs Nucleic  Acids  Res 31, 3497-500. (129) Mizuno, H., Fujimoto, Z., Atoda, H., and Morita, T. (2001) Crystal structure of  an anticoagulant protein in complex with the Gla domain of  factor  X. Proc Natl  Acad Sci U  SA98,  7230-4. (130) Jesty, J., Spencer, A. K., and Y., N. (1974) The Mechanism of  Activation of Factor X. J  Biol Chem. 249, 5614-5622. (131) Rosenberg, J. S., McKenna, P. W„ and Rosenberg, R. D. (1975) Inhibition of human factor  IXa by human antithrombin. J  Biol Chem. 250, 8883-8. (132) Smirnov, M. D., Safa,  O., Regan, L„ Mather, T., Stearns-Kurosawa, D. J., Kurosawa, S., Rezaie, A. R., Esmon, N. L., and Esmon, C. T. (1998) A chimeric protein C containing the prothrombin Gla domain exhibits increased anticoagulant activity and altered phospholipid specificity.  J  Biol Chem. 273, 9031-40. (133) Castellino, F. J. (1995) Human protein C and activated protein C : Components of the human anticoagulation system Trends  in Cardiovascular  Medicine  5, 55-62. (134) Shah, A. M„ Kisiel, W., Foster, D. C„ and Nelsestuen, G. L. (1998) Manipulation of  the membrane binding site of  vitamin K-dependent proteins: enhanced biological function  of  human factor  VII. Proc Natl  Acad  Sci U  S A 95,4229-34. (135) Walker, F. J. (1981) Regulation of  activated protein C by protein S. The role of phospholipid in factor  Va inactivation. J  Biol Chem. 256,11128-31. (136) Colpitts, T. L., and Castellino, F. J. (1994) Calcium and phospholipid binding properties of  synthetic gamma-carboxyglutamic acid-containing peptides with sequence counterparts in human protein C. Biochemistry 33, 3501-8. (137) Nelsestuen, G. L., Kisiel, W., and Di Scipio, R. G. (1978) Interaction of  vitamin K dependent proteins with membranes. Biochemistry 17, 2134-8. (138) Borgel, D., Gaussem, P., Garbay, C., Bachelot-Loza, C., Kaabache, T., Liu, W. Q„ Brohard-Bohn, B„ Le Bonniec, B„ Aiach, M„ and Gandrille, S. (2001) Implication of  protein S thrombin-sensitive region with membrane binding via conformational  changes in the gamma-carboxyglutamic acid-rich domain. Biochem J.  360,499-506. (139) Stoylova, S., Gray, E., Barrowcliffe,  T. W., Kemball-Cook, G., and Holzenburg, A. (1998) Structural determination of  lipid-bound human blood coagulation factor IX. Biochim Biophys Acta. 1382, 175-8. (140) Heeb, M. J., Rosing, J., Bakker, H. M., Fernandez, J. A., Tans, G., and Griffin,  J. H. (1994) Protein S binds to and inhibits factor  Xa. Proc Natl  Acad  Sci USA  91, 2728-32. (141) Garcia, B. A., Smalley, D. M„ Cho, H„ Shabanowitz, J., Ley, K„ and Hunt, D. F. (2005) The platelet microparticle proteome. Journal  ofProteome  Research 4, 1516-21. (142) Sims, P. J., Faioni, E. M„ Wiedmer, T„ and Shattil, S. J. (1988) Complement proteins C5b-9 cause release of  membrane vesicles from  the platelet surface  that are enriched in the membrane receptor for  coagulation factor  Va and express prothrombinase activity. J  Biol Chem. 263, 18205-12. (143) Gilbert, G. E., Sims, P. J., Wiedmer, T„ Furie, B„ Furie, B. C., and Shattil, S. J. (1991) Platelet-derived microparticles express high affinity  receptors for  factor v m .J  Biol Chem. 266,  17261-8. (144) Christiansen, W. T., and Castellino, F. J. (1994) Properties of  recombinant chimeric human protein C and activated protein C containing the gamma-carboxyglutamic acid and trailing helical stack domains of  protein C replaced by those of  human coagulation factor  IX. Biochemistry 33, 5901-11. (145) Geng, J. P., and Castellino, F. J. (1997) Properties of  a recombinant chimeric protein in which the gamma-carboxyglutamic acid and helical stack domains of human anticoagulant protein C are replaced by those of  human coagulation factor VII. Thromb  Haemost.  77,  926-33. 

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