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Bioactive polymers : a comparative study on the antithrombotic properties of soluble polymers and surface… Lai, Benjamin Fook Lun 2010

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Bioactive Polymers: A Comparative Study on the Antithrombotic Properties of Soluble Polymers and Surface Grafted Polymers by Benjamin Fook Lun Lai B.M.L.Sc., University of British Columbia, 2006  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Pathology and Laboratory Medicine) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  February 2010 © Benjamin Fook Lun Lai, 2010  Abstract Use of synthetic materials in medical applications is one of the most common practices in modern medicine. Yet occurrence of surface-induced thrombus formation on these materials, especially those associated with cardiovascular applications, generates a need for surface modifications. Limiting thrombus formation on a biomaterial surface represents the ultimate success for blood contacting devices. One interesting approach is to enhance fibrinolysis before the blood clot becomes stabilized. Herein, two synthetic polymers, poly-N- [(2, 2-dimethyl-1, 2-dioxolane) methyl] acrylamide (PDMDOMA) and poly- (N-isopropylacrylamide) (PNIPAm), were tested for this particular antithrombotic property. Surface-grafted PNIPAm samples, brush-PNIPAm and starPNIPAm, were also tested for the biological activity. We evaluated the influence of these synthetic polymers on blood hemostasis by studying the fibrin polymerization process, the three-dimensional clot structure, and the mechanical properties of blood clot such as its clot strength, clot elasticity and clot fibrinolysis.  Both linear PDMDOMA and PNIPAm altered the normal fibrin  polymerization by changing the rate of protofibril aggregation and resulting in a 5-fold increase in the overall turbidity. Fibrin clots formed in presence of these synthetic polymers exhibited thinner fibers with less branching and resulted in a more porous and heterogeneous clot structure in scanning electron micrographs. The structural changes in these clots led to significant difference to their mechanical properties.  Lower clot  strength and clot elasticity were recorded from the thromboelastography study. More interestingly, enhanced clot lysis was measured by thromboelastography when whole blood was clotted in presence of PDMDOMA or PNIPAm. Further evidence of the altered clot structure and clot cross-linking was obtained from the significant decrease in  ii  D-dimer levels measured from degraded plasma clot. Similar results were obtained when star-form of PNIPAm was used but not for brush-form PNIPAm. The antithrombotic activity of soluble PDMDOMA and PNIPAm could potentially lead to the development of novel antithrombotic agents that could enhance endogenous fibrinolytic activity by modulating the fibrin clot structure. In the exploratory analysis of surface grafted PNIPAm (brush-PINPAm), brush-PNIPAm showed that the biological activity of attached chains is quite different from soluble polymers and several parameters need to be optimized to generate an antithrombotic coating for biomaterials.  iii  Table of Contents Abstract.............................................................................................................................. ii Table of Contents ............................................................................................................. iv List of Figures.................................................................................................................. vii Abbreviations ................................................................................................................... ix Acknowledgements .......................................................................................................... xi Co-authorship Statement ............................................................................................... xii 1  Introduction................................................................................................................. 1 1.1 Coagulation cascade............................................................................................... 1 1.2 Fibrin polymerization and three dimensional clot structure .................................. 5 1.2.1 Fibrin polymerization process......................................................................... 6 1.2.2 Fibrinolysis: systematic control for fibrin mass size ...................................... 9 1.3 Antithrombotic therapy........................................................................................ 10 1.3.1 Current anticoagulants .................................................................................. 11 1.3.1.1 Direct inhibitors ..................................................................................... 11 1.3.1.2 Indirect inhibitors................................................................................... 14 1.3.2 Current antiplatelet agents ............................................................................ 18 1.3.3 Current fibrinolytic agents ............................................................................ 19 1.3.4 Problems associated with current antithrombotic therapies.......................... 20 1.4 biomaterials and associated complications .......................................................... 21 1.4.1 Mechanisms for adsorption and activation of proteins and cells .................. 22 1.4.2 Current surface modifications strategies....................................................... 23 1.5 Novel functional polymers................................................................................... 25 1.6 Thesis overview ................................................................................................... 26 1.6.1 Specific aims................................................................................................. 27 1.7 References............................................................................................................ 28  2  Influence of poly-N- [(2, 2-dimethyl-1, 2-dioxolane) methyl] acrylamide on the normal fibrin clot structure ..................................................................................... 35 2.1 Introduction.......................................................................................................... 35 2.2 Experimental ........................................................................................................ 38 2.2.1 Materials ....................................................................................................... 38 2.2.1.1 Chemical ................................................................................................ 38 2.2.1.2 Biological............................................................................................... 38 2.2.2 Methods......................................................................................................... 39 2.2.2.1 Measurement of clotting time: prothrombin time (PT) and activated partial thromboplastin time (APTT) ...................................................... 39 2.2.2.2 Fibrin polymerization assay................................................................... 39 2.2.2.3 Analysis of fibrin cross-linking in clot by SDS-PAGE ......................... 40 2.2.2.4 Analysis of fibrin cross-linking in clot by d-dimer enzyme immunoassay (EIA) ............................................................................... 41 iv  2.2.2.5 Analysis of fibrin clot structure by scanning electron microscopy........ 41 2.2.2.6 Analysis of overall strength of fibrin clot .............................................. 42 2.2.2.7 Statistical analysis.................................................................................. 43 2.3 Results.................................................................................................................. 43 2.3.1 In vitro blood coagulation assay ................................................................... 44 2.3.2 Kinetics of fibrin polymerization.................................................................. 46 2.3.3 Fibrin clot structure studied by scanning electron microscopy .................... 48 2.3.4 Influence of fibrin cross-linking by factor XIIIa in the presence of PDMDOMA. ................................................................................................. 51 2.3.5 Analysis of clot structure by d-dimer EIA.................................................... 55 2.3.6 Blood clot analyses by thromboelastograph ................................................. 56 2.4 Discussion ............................................................................................................ 64 2.4.1 Conclusions................................................................................................... 68 2.5 References............................................................................................................ 70 3  A comparative study on the influence of soluble and grafted poly (Nisopropylacrylamide) on fibrin clot structure ........................................................ 73 3.1 Introduction.......................................................................................................... 73 3.2 Experimental ........................................................................................................ 75 3.2.1 Materials ....................................................................................................... 75 3.2.1.1 Chemical ................................................................................................ 75 3.2.1.2 Biological............................................................................................... 76 3.2.2 Methods......................................................................................................... 77 3.2.2.1 Measurement of clotting time: prothrombin time (PT) and activated partial thromboplastin time (APTT) ...................................................... 77 3.2.2.2 Fibrin polymerization assay................................................................... 77 3.2.2.3 Analysis of fibrin clot structure by scanning electron microscopy........ 78 3.2.2.4 Analysis of cross-linking in fibrin clot by SDS-PAGE ......................... 78 3.2.2.5 Analysis of overall strength of whole blood clot ................................... 79 3.2.2.6 Statistical analysis.................................................................................. 80 3.3 Results.................................................................................................................. 80 3.3.1 In vitro blood coagulation assays.................................................................. 80 3.3.2 Kinetics of fibrin polymerization.................................................................. 82 3.3.3 Analysis of factor XIIIa-induced cross-linking of fibrin in the presence of PNIPAm ........................................................................................................ 83 3.3.4 Fibrin clot structure studied by scanning electron microscopy .................... 86 3.3.5 Blood clot analyses by thromboelastograph ................................................. 89 3.4 Discussion ............................................................................................................ 93 3.4.1 Conclusions................................................................................................... 98 3.5 References.......................................................................................................... 100  v  4  Summary and future directions............................................................................. 102 4.1 Summary ............................................................................................................ 102 4.2 Future work........................................................................................................ 106 4.3 References.......................................................................................................... 108  Appendix........................................................................................................................ 109  vi  List of Figures Figure 1.1 Blood coagulation cascade ..............................................................................1 Figure 1.2 Coagulation inhibition by endogenous anticoagulant ..................................4 Figure 1.3 Fibrinogen molecule ........................................................................................6 Figure 1.4 Fibrin polymerization process ........................................................................7 Figure 1.5 Factor XIIIa catalyzed crosslink ....................................................................8 Figure 1.6 Fibrinolysis system ..........................................................................................9 Figure 1.7 Fibrin degradation products.........................................................................10 Figure 1.8 Bivalent method .............................................................................................12 Figure 1.9 Common disaccharides in heparin...............................................................14 Figure 1.10 Heparin inhibition pentasaccharide sequence ..........................................15 Figure 1.11 Warfarin acts against recycle of vitamin K...............................................17 Figure 2.1 Structures and characteristics of the polymers tested for antithrombotic activity...............................................................................................................................44 Figure 2.2 Coagulation cascade analysis by APTT and PT assays .............................45 Figure 2.3 Turbidity analysis of fibrin-formation in presence of polymers ...............47 Figure 2.4 SEM analysis of fibrin clot structure...........................................................49 Figure 2.5 Evaluation of cross-linking in fibrin clot using reducing SDS-PAGE......52 Figure 2.6 Analysis of band intensity .............................................................................55 Figure 2.7 Quantitative analysis of fibrin clot structure: d-dimer EIA......................56 Figure 2.8 Evaluation of overall clot properties measured by TEG ...........................57 Figure 2.9 Overall clot strength and clot elasticity recorded by TEG (polymer comparison) ......................................................................................................................60 Figure 2.10 Overall clot strength and clot elasticity recorded by TEG (MW comparison) ......................................................................................................................61 Figure 2.11 Fibrinolysis recorded by TEG ....................................................................62 vii  Figure 3.1 Structure and characteristics of the polymers tested .................................76 Figure 3.2 Coagulation cascade analysis by APTT and PT assays .............................81 Figure 3.3 Turbidity analysis of fibrin-formation in presence of polymers ...............83 Figure 3.4 Evaluation of cross-linking in PPP clot by reducing SDS-PAGE .............86 Figure 3.5 SEM analysis of fibrin clot structure...........................................................87 Figure 3.6 Evaluation of overall clot properties measured by TEG ...........................89 Figure 3.7 Evaluation of overall clot strength, clot elasticity and percent of fibrinolysis measured by TEG ........................................................................................92 Figure 3.8: Fibrinolysis recorded by TEG.....................................................................93 Figure 4.1: Summary diagram......................................................................................105 Appendix S1: Final concentration of soluble polymers in terms of molarity...........109 Appendix S2: Evaluation of undiluted cross-linking in fibrin clot using reducing SDS-PAGE......................................................................................................................110 Appendix S3: TEG trace of whole blood clot incubated with PDMDOMA-PDHPA or PDHPA .......................................................................................................................111  viii  Abbreviations APC: activated protein C APTT: activated partial thromboplastin time ASA: acetylsalicylic acid AT: antithrombin ATRP: atom transfer radical polymerization DTT: dithiothieitol FEU: fibrinogen equivalent units FV: factor V FVa: activated factor V FVII: factor VII FVIIa: activated factor VII FVIII: factor VIII FVIIIa: activated factor VIII FIX: factor IX FIXa: activated factor IX FX: factor X FXa: activated factor X FXI: factor XI FXIa: activated factor XI FXII: factor XII FXIIa: activated factor XII FXIII: factor XIII FXIIIa: activated factor XIII  ix  G’: elastic shear modulus GP: glycoprotein HEPES: 4-(2-Hydroxyethyl) piperazine-1-ethanesulfonic acid sodium salt HIT: heparin-induced thrombocytopenia HMW: high molecular weight HMWK: high molecular weight kininogen Mn: number average molecular weight MA: maximum amplitude PC: protein C PDI: polydispersity index PDMA: poly (N,N-dimethyl acrylamide) PDMDOMA: poly (N- [(2,2-dimethyl-1, 3dioxolane) methyl] acrylamide) PEG: poly (ethylene glycol) PK: prekallilrein PNIPAm: poly (N-isopropylacrylamide) PPP: platelet poor plasma PT: prothrombin time SDS-PAGE: sodium dodecyl sulfate- polyacrylamide gel electrophoresis TEG: thromboelastograph TF: tissue factor tPa: tissue plasminogen activator uPa: urokinase plasminogen activator  x  Acknowledgements I would like to first thank my supervisor Dr. Jayachandran Kizhakkedathu for taking me on as a graduate student and for supporting me unconditionally in every aspect of my graduate life. I couldn’t ask for a better environment to be in and a better teacher to learn from. I would also like to thank my co-supervisor Dr. Donald Brooks who has been a vast source of information and direction. Without their constant ideas and them believing in me, I doubt this project and this thesis could have been done so smoothly. Secondly, I would like to give special thanks to Dr. Yuquan Zou and Dr. Nicholas Rossi for their assistance, and helpful insights, especially Yuquan for synthesizing all of the polymers for me. You guys are great friends. Much appreciation goes to Dr. Johan Janzen for his Microsoft expertise and for his vast knowledge in different fields. A big thank you to past and current members of Brooks/Kizhakkedathu Lab: Carla, Guangzheng, Kai, Iren, Irina, Janice, Rajesh K., Rajesh S., Sammy, Samuel, Sonja, Xifei and all of the summer students. Thank you for all of your support and humors. I absolutely enjoyed these past three years! Lastly I thank Canadian Blood Services Graduate Fellowship Program and UBC Centre for Blood Research Strategic Training Program in Transfusion Science for the research fellowship during my studies.  xi  Co-authorship Statement In collaboration with my supervisors, Dr. Kizhakkedathu and Dr. Brooks, I performed research, analyzed data and wrote the manuscripts in the thesis. Dr. Yuquan Zou was responsible for the preparation and analyses of polymers PDMDOMA and linear PNIPAm and Dr. Krishnan Ranganathan was responsible for the preparation and analyses of polymers star-PNIPAm and brush PNIPAm. Dr. Kizhakkedathu designed the work, analyzed data and edited the manuscripts.  xii  1 Introduction 1.1 Coagulation cascade Blood coagulation is part of a complex physiological process that is responsible for maintaining the normal structural integrity of the vascular system [1]. Upon injury to the blood vessel wall, this host defense process is initiated to restore the proper fluidity of blood in an intact environment. Therefore the ultimate goal of blood coagulation is forming an insoluble plug at the vascular injury site. The blood coagulation cascade is composed of two separate pathways: tissue factordependent (extrinsic) pathway and contact activation (intrinsic) pathway (Figure 1.1).  Figure 1.1: Blood coagulation cascade. A diagrammatic summary of blood coagulation cascade is shown. Blood coagulation involves two distinctive pathways: TF-initiated and contact activation initiated. Both pathways converge to a common step, conversion of prothrombin to thrombin. Thrombin is central figure of the coagulation as it cleaves fibrinogen to form fibrin clot, provides positive feedback to the cascade, and activates cascade inhibition mechanism. Green dotted arrow illustrates thrombin feedback mechanism.  1  The coagulation mechanism is a result of a series of proteolytic reactions that leads to the activation of a variety of inactive clotting factors. All of these clotting factors are zymogens in plasma and most of these zymogens are serine proteases that contain catalytic domains. Once converted to their active form, they exert protease activity on the following clotting factors as shown in the blood coagulation cascade (Figure. 1.1). The coagulation cascade is triggered upon injury to the endothelium of the blood vessel walls, exposing tissue factor (TF) to circulating blood [2]. In the circulating blood, there is a trace amount of activated form of clotting factor VII (FVIIa) [3;4]. Exposed tissue factors will interact with circulating FVIIa to form the FVIIa-TF complex. This complex converts the zymogens, factor IX (FIX) and factor X (FX) to their active serine proteases form, activated factor IX (IXa) and X (FXa) respectively. The newly activated FX alone can activate small amount of thrombin that activates platelets, and activates factor V (FV) and FVIII to factor Va (FVa) and FVIIIa [5]. Free FXa with its cofactor FVa can then assemble on the surface of an activated platelet, in presence of calcium ions, to form the prothrombinase complex [6]. This complex is responsible for the conversion of prothrombin to thrombin. The conversion is done by the hydrolysis of two internal peptide bonds, Arg271-Thr and Arg320-Ile [7;8]. The resulting thrombin, a serine protease, is then responsible for activation of platelets, FV, factor VIII (FVIII) and factor XI (FXI). The activation of these clotting factors is an amplification mechanism for the generation of more thrombin. The activated FVIII (FVIIIa) is a co-factor of FIXa and together these factors form the “tenase” complex that is responsible for activation of FX [9]. The FXa can now form the prothrombinase complex with thrombin-activated FV on a phospholipids surface to convert prothrombin to thrombin. This prothrombinase complex  2  generates thrombin at a rate 300,000 times faster than FXa [5;7;10]. The activated factor XI enhances FIXa generation, which activates FX, and more prothrombinase complex is formed. As a result the generation of thrombin continues. Other than the TF-initiated pathway, blood coagulation can also be initiated by means of the contact activation pathway. This is when circulating factor XII (FXII), prekallikrein, and high molecular weight kininogen (HMWK) bind to an artificial surface. The binding of FXII on the surface leads to its auto-activation to its active serine protease form, FXIIa [1]. The FXIIa can then activate pre-kallikrein and HMWK by limited proteolysis. Activated HMWK serves as the co-factor of FXIIa for the initiation of other clotting factors. FXIIa-HMWK complex on surface is responsible for triggering the series of activation of FXI, FIX, FX, and prothrombin to FXIa, FIXa, FXa, and thrombin. Thus, as shown in Figure 1.1, both pathways of the coagulation cascade leads to the formation of thrombin. Thrombin serves as the central serine protease of the coagulation cascade because of its ability in the production of a stable fibrin clot formation. It is responsible for the conversion of soluble fibrinogen into insoluble fibrin through cleavage of fibrinogen peptide bonds. Thrombin has two sites responsible for fibrin conversion: an active site targets fibrinopeptide A/B for proteolytic cleavage of fibrinogen and an exosite enhances thrombin binding on fibrinogen.  This conversion of fibrinogen to fibrin monomer  promotes the polymerization process of fibrin into a fibrin gel mesh. The gel mesh is important in strengthening the platelet plug formed at the vascular injury site [1]. Thrombin is also responsible for the conversion of factor XIII (FXIII) to its active form FXIIIa.  FXIIIa is a transglutaminase that catalyzes the covalent cross-linking  3  between Gln and Lys residues of adjacent fibrin molecules [11]. This cross-linking creates strong fibrin mesh that supports the overall structural integrity of fibrin clot. A control mechanism is needed to regulate this highly self-proliferating clotting process.  In the normal physiological conditions, there are three major endogenous  anticoagulants that govern the normal formation of thrombin: (A) antithrombin, (B) tissue factor pathway inhibitor, and (C) activated protein C (Figure 1.2). (A)  (B)  (C)  Figure 1.2: Coagulation inhibition by endogenous anticoagulant. Blood coagulation cascade is inhibited by three endogenous anticoagulants: (A) antithrombin (AT), (B) tissue factor pathway inhibitor (TFPI), and (C) activated protein C (APC). AT forms 1:1 stoichiometric complex with thrombin at the active site. TFPI forms quaternary complex with FXa and FVIIa-TF complex. APC inhibits phospholipids (PC)-bound FVa and FVIIIa and degrades the two serine proteases.  4  The main inhibitor of thrombin generation is antithrombin (AT). AT is a serine protease inhibitor and is able to inhibit FXIIa, FXIa, FIXa, FXa, and thrombin. AT covalently binds to the activated thrombin’s active site to form a stable one-to-one ATprotease complex [12]. Tissue factor pathway inhibitor (TFPI) directly inhibits FXa by forming a quaternary complex with FXa and the FVIIa-TF complex [13;14].  This  inhibits the sequential activation reaction of FXa. TFPI exerts its anticoagulant function as early as the initiation of the coagulation cascade. Lastly, the activated protein C (APC), with its co-factor protein S, inactivates FVa and FVIIIa. APC is formed by thrombin when thrombin is bound to thrombomodulin on the vascular endothelium. APC itself is a serine protease and exerts its anticoagulant effect on FVa and FVIIIa by proteolytic cleavage that positively destroying the catalytic activities of FVa and FVIIIa [15]. In addition, APC can also exert its proteolytic inactivation to the inactive form of FV [16] and FVIII.  1.2 Fibrin polymerization and three dimensional clot structure Fibrinogen is a glycoprotein molecule made up of three pairs of polypeptide chains, Aα, Bβ, and γ (Figure 1.3). It is roughly 45 nm in length and has a molecular mass of 340 kDa [17]. The amino-terminal ends of the three pairs of polypeptide chains are joined together by five symmetrical disulfide bridges in the central E domain of the molecule. The E domain is linked to two flanking outer D domains by a three-chain αhelical coiled coil. The two globular D domains contain the carboxyl-terminal end of the Bβ and γ chains. The carboxyl-terminal end of Aα chains consists of a globular αC domain that is folded back to associate with the central E domain. Both the E and D domains participate as binding sites that are responsible for the conversion of fibrinogen  5  to fibrin, fibrin monomer polymerization, crosslinking between fibrils, and as attachment sites for substrates like calcium ions, fibrionectin, plasminogen and platelets [18;19;20].  Figure 1.3: Fibrinogen molecule. A cartoon model of the fibrinogen is shown. It is made up of 3 pair of polypeptide chains, Aα, Bβ, and γ.  1.2.1  Fibrin polymerization process  Fibrinogen is present in plasma at a concentration of ~ 3 mg/ml. Upon thrombin generation from the coagulation cascade, circulating fibrinogen is converted to fibrin when thrombin cleaves fibrinopeptide A (FPA) from the amino terminal of the Aα chain and followed by a slower release of fibrinopeptide B (FPB) from the Bβ chain. The cleavage of fibrinopeptides at the E domain initiates the fibrin clot polymerization process [17]. Cleavage of FPA not only creates a new amino-terminal beginning with a sequence Gly-Pro-Arg [21;22] but also exposes a binding site at the central E domain. This binding site subsequently interacts with a complementary site in the D domain of the  6  carboxyl-terminal of γ chain of an adjacent fibrin molecule [17;21]. The association of these two sites results in the formation of a double-stranded protofibril of which the fibrin monomers are half-staggered and lined up in an end-to-end fashion (Figure 1.4). Protofibrils associate with each other laterally to form longer and wider fibrin fibers, and the fibers form branches that result in space-filling complex three-dimensional network [23]. Degree of lateral aggregation and branching enhance the overall strength and rigidity to the fibrin clot. The thickness of fibers and the extent of branching of a clot vary greatly depending on the conditions of polymerization. Concentration of fibrinogen and thrombin, ionic and pH conditions, and different plasma proteins can affect the fibrin clot overall structure and mechanical properties [24;25].  Figure 1.4: Fibrin polymerization process. Thrombin cleavage of the fibrinopeptide A/B (FPA/ FPB) of fibrinogen leads to the non-covalent half staggering interaction with adjacent fibrin to form protofibrils. The gray lines between fibrin molecules indicate the linkage.  7  The fibrin gel mesh is further supported by cross-links formed between the fibrin molecules. Thrombin-activated plasma factor XIII (FXIIIa) is a transglutaminase and is responsible for catalyzing amide crosslinks between glutamine (Gln) and lysine (Lys) residues between proteins (Figure 1.5). On the carboxyl-terminal end of the γ-chains, there is a cross-linking site that FXIIIa can form intermolecular Nε-(γ-glutamyl) lysine covalent cross-link between fibrin molecules [26;27]. This γ-chain cross-link can occur between adjacent or neighbor fibrin molecules, thus forming γ-dimers at the D domains. In addition to γ-dimers, higher order of γ-chain cross-links exist. Similar cross-linking occurs between amine and lysine residue in the α-chains, forming α-polymers. But this cross-linking occurs much more slowly [28]. Overall, the covalent cross-links produced  Figure 1.5: Factor XIIIa catalyzed crosslink. The cross-links between adjacent or neighbor fibrin molecules is shown (red circle). This crosslinking reaction (as shown in inset) is between glutamine and lysine amino acid on γ-chain of adjacent/ neighbor fibrin molecules and is catalyzed by FXIIIa, a transglutaminase.  8  dramatic stabilization of the fibrin gel mesh. Fibrin molecules with crosslinking greatly increase the elasticity of an individual fibrin fiber in a clot [29;30]. In addition to stiffening the fibrin clot, FXIIIa is also responsible for attaching blood constituents like α2-plasmin inhibitor and plasminogen activator inhibitor (PAI-2) to the Aα-chain of the fibrinogen. The Gln residues on both inhibitors, Gln2 of α2-plasmin inhibitor and Gln83/86 of PAI-2, can be cross-linked to the Lys303 residue on the fibrinogen through the trans-amide exchange catalyzed by FXIIIa [31]. This greatly increases the resistance of fibrin clot to degradation by the fibrinolytic system. 1.2.2  Fibrinolysis: systematic control for fibrin mass size  Degradation of the stable fibrin clot is also a part of the physiological process in restoring the normal blood flow of the vascular system (Figure 1.6).  Figure 1.6: Fibrinolysis system. A diagrammatic summary of the fibrinolysis system is shown. Plasmin is the central figure that is responsible for degrading the fibrin mass (gray mass). tPA (blue) initiates plasminogen to plasmin (yellow). Plasminogen activator inhibitor (PAI, light blue) and α2-plasmin inhibitor inhibit the fibrinolysis system.  The central molecule of fibrinolysis system is plasmin. Plasmin is an active cleavage enzyme that is responsible for degrading fibrin into soluble fibrin degradation products [32]. Active plasmin is converted from its proenzyme plasminogen by two plasminogen  9  activators: tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA). Plasminogen has domain structure referred to as “kringles” which have binding sites specific for lysine and aminohexyl. These sites facilitate the binding of plasminogen to fibrin [33]. Circulating tPA will then form a ternary complex with fibrin-bound plasminogen and converts plasminogen to plasmin. The cleavage of fibrin occurs mainly between the D and the E regions of a fibrin molecule and the αC domain on fibrin strand surface. These cleavages generate fibrin degradation products of varying molecular weight: D-D:E1 complex, the FXIIIa-cross-linked D-D dimer, and E3 fragment (Figure 1.7) [34].  Figure 1.7: Fibrin degradation products. Three FDP after fibrin mass is cleaved by plasmin are shown. D-D:E1 is made up of 2 D domains (blue) and a 1 E domain (green). D-D dimer is two D domains linked by the Nε-(γ-glutamyl) lysine crosslinks.  Like the coagulation cascade, fibrinolytic system is also regulated. It is regulated at PA-level by plasminogen activator inhibitor 1 and 2 (PAI 1/ 2), and at plasmin-level by α2-plasmin inhibitor.  Both PAI 1/ 2 and α2-plasmin inhibitor inhibit their target  proteinase by forming a one-to-one complex with tPA and plasmin, respectively [32].  1.3 Antithrombotic therapy Antithrombotic therapy is one of the most common practices in modern medicine. It serves the purpose of treating and preventing thrombus formation in diverse clinical  10  scenarios like atrial fibrillation [35] and venous thromboembolism [36] or various types of invasive surgeries. Inhibition of thrombosis can be achieved by three therapeutic approaches: inhibit at the coagulation cascade, increase endogenous fibrinolytic activity, and alter platelet activation and aggregation [37;38]. 1.3.1  Current anticoagulants  The success of an anticoagulant drug lies in its specificity to target a clotting enzyme or a step in coagulation. Therefore, for decades, the design for new anticoagulant drugs was focused on the two most prominent serine proteases of the coagulation cascade, thrombin and FXa. The mechanism of action of these anticoagulants has been classified as either direct inhibition or indirect inhibition. Direct inhibitors bind to the enzyme and disrupt its catalytic ability. Indirect inhibitors do not bind to the enzyme but utilize the endogenous anticoagulant systems (i.e. antithrombin) and amplify their anticoagulant effect. 1.3.1.1 Direct inhibitors Hirudin This 65-amino acid polypeptide, extracted from the salivary gland of Hirudo medicinalis, a medicinal leech, is the most potent natural inhibitor of thrombin [39]. It forms a one-to-one complex with thrombin and its bioactivity against the serine protease is bivalent (Figure 1.8).  11  Figure 1.8: Bivalent method. Left: An illustration shows thrombin (yellow) with its active site and its substrate recognition site (exosite I/II). Right: An illustration shows how hirudin or bivalirudin interacting with and inhibiting thrombin. The amino-terminal interacts with the active site of thrombin and carboxylterminal interacts with exosite I (fibrin binding site).  Hirudin’s globular amino-terminal binds to the active site of thrombin and its anion carboxyl-terminal interacts with the fibrinogen-binding domain [40]. Thus, hirudin is highly selective for thrombin over other serine proteases [41]. Recombinant hirudin is commercially available, as lepirudin and desirudin. However, because of the lack of specific antidote, the hirudin/ thrombin complex is irreversible, a potential drawback of this anticoagulant. Bivalirudin This 20-amino acid synthetic polypeptide is an analog of hirudin. Similar to hirudin, the bioactivity of bivalirudin is bivalent, with the amino-terminal region, D-Phe-Pro-ArgPro, binding to the active site of thrombin and the anionic carboxyl-terminal region exhibiting specificity for the anion-binding exosite in thrombin [42]. The bivalirudin also forms a one-to-one stoichiometric complex with thrombin.  Unlike hirudin, the  bivalirudin/thrombin is reversible. The active site of thrombin eventually is able to  12  cleave off the Pro-Arg bond at the amino terminus of bivalirudin and thus thrombin can regenerate its activity [43]. Argatroban This is a small synthetic molecule of molecular mass 527 Da that displays good selectivity for the active site of thrombin [44]. Unlike hirudin and bivalirudin, argatroban is univalent, with only specificity for active site of thrombin. Argatroban has rapid onset of anti-thrombin action and rapid reversibility. In addition, because of its small size, argatroban is able to exert its anticoagulant effect to fibrin clot-bounded thrombin [45]. Argatroban has also been shown with ability to affect platelet aggregation in thrombus. The only drawback of this anticoagulant is its parenteral route of administration. Dabigatran This is a synthetic, potent direct thrombin inhibitor. Similar to argatroban, dabigatran is a univalent direct thrombin inhibitor, as it binds specifically and reversibly to the active site of thrombin. The mechanism of inhibition is due to salt bridge formation between the amidine groups of dabigatran with the carboxylate of aspartate residue, Asp189 [46]. Furthermore, because of its small size, dabigatran is able to inhibit fibrin clot-bounded thrombin. However, due to its high polarity, in clinic, dabigatran is orally administered as the prodrug dabigatran etexilate [47]. Administration of dabigatran as its prodrug form would facilitate gastrointestinal absorption. Because of its display in highly selective and reversible binding to thrombin and route of administration, dabigatran shows promise as a replacement for warfarin in clinical settings [48].  13  1.3.1.2 Indirect inhibitors Heparin and its LMW derivatives Heparin is a glycosaminoglycan, with repeated sequence of highly sulfated disaccharide units [49]. It is a linear polymer and varies in sizes, ranging from 3 to 50 kDa. It is produced naturally in mast cell and stored in the granules of basophil, and is usually isolated from mammalian sources, for example porcine intestine. The most common repeating disaccharide unit is a 2-O-sulfated iduronic acid and 6-O-sulfated, Nsulfated glucosamine (IdoA(2S)-GlcNS(6S)), connected in a 1-4 linkage (Figure 1.9) [50]. Within these sequences of repeating disaccharide units, a specific motif of pentasaccharide (Figure 1.10) gives heparin its anticoagulant activity [51;52].  Figure 1.9: Common disaccharides in heparin. Six common disaccharides seen in heparin are shown. The carbohydrates are connected in 1-4 linkage. The anticoagulant effect of heparin is an indirect effect, in which it enhances the binding and reaction of antithromin (AT) against thrombin or other coagulant factors by 2000-fold [51]. Heparin binds to ATIII through a specific pentasaccharide sequence  14  (Figure 1.10).  This binding can be greatly affected with a chemical alteration of  tryptophan residue (Trp49) on AT peptide [53;54], showing the importance of Trp being  Figure 1.10: Heparin inhibition pentasaccharide sequence. Heparin (black) has a pentasaccharide sequence that can bind to antithrombin III (ATIII, blue) and facilitate ATIII binding to thrombin (light blue) by 2000 fold. This pentasaccharide sequence is (shown in inset) made up of GlcNS(6S)-GlcA-GlcNS(3S,6S)-IdoA(2S)-GlcNS(6S)OMe, all are connected in 1-4 linkage.  at or near the heparin-binding site. Once bound, heparin accelerates AT inhibition on thrombin and the active form factor X (FXa). The mechanism of inhibition varies among the serine proteases. This is because the binding of heparin would modify the AT conformation slightly. The conformational change mediates the AT inhibition of FXa. The AT inhibition of thrombin, however, requires the highly anionic chain of heparin to bind with thrombin and to draw thrombin close to form a ternary complex between AT, thrombin and heparin. If the heparin fragment is too short, then the heparin bridging mechanism and the activity of heparin bound AT will be affected [55]. This ternary complex of AT: thrombin: heparin can be reversed with the use of a protamine sulfate antidote [51]. In addition to its ability to bind to AT, heparin can also bind with platelet factor 4 (PF4), a chemokine released from α-granules of activated platelets [56]. This binding affects heparin anticoagulant ability by the inability to form the AT: thrombin: 15  heparin ternary complex and generates adverse effect with antibody being formed against the heparin-PF4 complex. Because of its effectiveness and ease of manufacture, heparin is the most common anticoagulant used in clinical settings [57].  There are two types of heparin:  unfractionated heparin (UFH), directly from porcine source, and low molecular weight heparin (LMWH), filtered and de-polymerized to concentrate the active pentasaccharide sequence. UFH exhibits unstable pharmacokinetics and has undesirable side effects. Thus LMWH is the preferred choice of heparin anticoagulant. However, the drawbacks for this particular anticoagulant include its inability to limit the activity of the clot-bound thrombin and it is difficult to neutralize [49;50]. Fondaparinux It is a synthetic version of the specific pentassaccharide sequence found in heparin. Like heparin, fondaparinux is an indirect inhibitor of the coagulation cascade, as it binds with AT and accelerates AT inhibition activity towards coagulation factors [58;59]. However, fondaparinux is only able to inhibit FXa and not thrombin because of its inability to form the bridging mechanism between thrombin and AT. Fondaparinux is safer than heparin derivatives as there is no production of auto- antibodies because of the lack of recognition for PF4, but it does not have a suitable antidote like heparin does [60]. Warfarin This is a synthetic derivative of a naturally occurring chemical compound coumarin [61]. It is an indirect anticoagulant by affecting the vitamin K cycle (Figure 1.11). Warfarin exerts its anticoagulant effect by inhibiting vitamin K epoxide reductase [62], and thus inhibits the recycling of oxidized vitamin K back to its reduced form. The  16  reduced state of vitamin K is the coenzyme of γ-carboxylase that is responsible for the carboxylation of coagulation factors VII, IX, X and prothrombin [63]. Without the necessary carboxyl group on the glutamic acid residue in thrombin and FVIIa, calcium – assisted binding of thrombin and FVIIa to phospholipids is inhibited. The partially decarboxylated forms of these coagulant factors are inert during the coagulation process. Warfarin also affects other vitamin K dependent factors in the coagulation cascade, for example protein C, and protein S.  Figure 1.11: Warfarin acts against recycle of vitamin K. Warfarin inhibits recycle of oxidized form of vitamin K to its reduced form. Warfarin exerts it effect against vitamin K epoxide reductase. This mechanism affects vitamin K dependent clotting factors.  17  1.3.2  Current antiplatelet agents  The success of an antiplatelet agent is based on its success in inhibiting platelet activation and aggregation. Therefore, these agents are likely to focus on attenuating biochemical formation of mediators that are responsible for platelet activation and aggregation or targeting for ligands on platelet surface that are responsible for aggregation. Acetylsalicylic acid Acetylsalicylic acid (ASA), or commonly known as aspirin, induces an irreversible effect on platelet activation and results in prolongation of bleeding time [64]. The mechanism of action of ASA involves the inactivation of prostaglandin-endoperoxide synthase 1, a member of the cyclooxgenase family. This synthase is responsible for the metabolism of prostaglandin (the conversion of arachidonic acid to prostaglandin G2 to prostaglandin H2). The prostaglandin is important for the synthesis of thromboxane A2 (TXA2), a biological mediator responsible for platelet activation. ASA exerts its effect by selectively acetylating a hydroxyl group at Ser529 in the polypeptide chain of prostaglandin-endoperoxide synthase 1 [65;66].  Thus, this leads to a decrease in  biosynthesis of prostaglandin and thromboxane, thereby preventing platelet activation and aggregation via TXA2 stimulation. Clopidogrel Clopidogrel is a novel synthetic pro-drug that inhibits adenosine diphosphate (ADP) receptor on platelet surface (P2Y12). ADP is an important agonist for platelet activation and aggregation. After its release from thrombin, activated platelet and collagen, ADP binds to its receptor P2Y12 and initiates the activation of GPIIb/IIIa, which is important  18  for platelet aggregation [67;68].  Administration of clopidogrel leads to a quick  adsorption and metabolism by cytochrome P450 enzyme in liver to the drug’s active chemical metabolite, a carboxylic acid derivative.  This metabolite selectively and  covalently binds to P2Y12, thereby blocking ADP binding to its receptor [69;70]. Therefore, clopidogrel attenuates all the normal P2Y12 mediation of platelet activation and aggregation. Some studies have shown that clopidogrel has a more prolific effect on platelet aggregation than ASA [71]. Platelet GPIIb/IIIa Antagonists Glycoprotein IIb/IIIa (GPIIb/IIIa) is an integrin on platelet surface. GPIIb/IIIa is responsible for the aggregation of activated platelet to form the platelet plug in the damaged vascular area [72]. Therefore, the development of antagonists that specifically block GPIIb/IIIa can inhibit platelet aggregation. Three GPIIb/IIIa blockers are available commercially: abciximab, tirofiban, and eptifibatide [57]. 1.3.3  Current fibrinolytic agents  As mentioned previously, the fibrinolytic system plays a part in the regulation of normal integrity of the vascular system by controlling the mass of fibrin clot formed. This system is initiated by means of tissue type plasminogen activator (tPA) or urokinase type plasminogen activator (uPA). tPA and uPA initiates the conversion of plasminogen to plasmin which is important in the lyzing of the fibrin clot. Thus agents that induce the conversion of plasminogen to plasmin are invaluable for the antithrombotic therapy. Currently recombinant forms of plasminogen activator are available.  Reteplase,  tenecteplase, and saruplase are all point-and-deletion mutant of tPA or uPA [57]. In addition to recombinant plasminogen activator, streptokinase is a naturally occurring  19  bacterial protein displaying ability to convert plasminogen to plasmin. Streptokinase itself does not process the protease ability to cleave plasminogen to plasmin. Instead, streptokinase forms a cleaving complex with plasminogen that is responsible for the conversion of free circulating plasminogen to plasmin [57;73]. 1.3.4  Problems associated with current antithrombotic therapies  All the antithrombotic agents mentioned are commonly used in clinical settings and have shown great success in attenuating thrombus formation either working alone or with other agents. They, however, suffer drawbacks that somewhat overshadow their success. The major determinants of antithrombotic-induced complications are the intensity of the drug effect, patient characteristics, the concomitant use of drugs that interfere with hemostasis, and the length of therapy. One major complication is excessive bleeding in patients. This occurs because the drug has impaired the normal hemostasis, leading to a delayed onset of the coagulation cascade, an alteration of platelet function, and a lack of control on fibrin clot mass. The sites of bleeding are usually in the gastrointestinal tract, and the intracranial cavity. Patients who are older and suffering from a recent myocardial infarction, and history of stroke and liver problems are likely to have increased risk of antithrombitic agent related bleeding [74;75]. In addition, the risk of bleeding increases when more than one type of antithrombitic agents is administrated. Some other adverse effects of antithrombotic therapy are patient dependent. For example, heparin-induced thrombocytopenia (HIT) is a devastating complication that occurs to 1-3% of patients administrated with unfractionated heparin. HIT is an autoimmunological response that leads to low platelet count in patients. This complication  20  arises when self-generated antibodies (usually of the IgG class) binds to the heparinplatelet factor 4 complexes. These antibodies can then bind with platelets leading to platelet activation and aggregation and as a result there is a decrease in platelet count [76]. A similar thrombocytopenia disorder is seen when patients are administered with clopidogrel. Another example of patient dependent complication is skin necrosis, as seen when patients are administrated with warfarin [77]. It has been proposed that warfarin necrosis is related to a hypersensitivity reaction in the body [78]. Drug toxicity, the non-availability of antidotes to reverse the drug activity, and the inability to inactivate clot-bound thrombin are problems that also render some of the antithrombotics problematic for common medicinal use. Liver toxicity seen in patients led to decrease administration of ximelagatran as a first-choice antithrombotic therapy and due to the dependence of AT for anticoagulant effect, heparin requires other antithrombotics to attenuate activity of clot-bound thrombin [48;79].  1.4 Biomaterials and associated complications Synthetic materials are frequently used in clinical applications, for example blood catheters, blood storage bags, cardiovascular bypass machines, drug delivery vesicles and other blood-contacting devices. However, thrombus generation on the surface of these materials or generation of an embolus is a major complication that renders current materials incompatible with their biomedical application [80;81;82] and this, in turn, affects both short term and long term performance of these materials. The surface induced blood clot formation is mainly due to uncharacteristic plasma protein, particularly the coagulation factors and fibrinogen, adsorption on the artificial surfaces, and partly due to subsequent platelet and leukocytes adhesion and activation on  21  the surface [80]. The rate of adsorption and adhesion of proteins and cells is greatly related to the surface chemistry of the biomaterial: charge, wettability and topography [83]. 1.4.1  Mechanisms for adsorption and activation of proteins and cells  Adsorption of plasma proteins and cells on the surface is rapid and is a highly competitive process. It involves adsorption and exchange of different types of plasma proteins and cells on the biomaterial surface. Both proteins and cells may bind to the surface via ionic or electronic interactions, hydrophobic interactions and hydrogen bonding. The spontaneity of protein and cell adsorption to the artificial biomaterial surface is dependent on Gibbs free energy exchange equation: ∆G = ∆H − T∆S < 0 ∆G = change in Gibbs free energy ∆H = change in enthalpy ∆S = change in entropy The adsorption occurs spontaneously either when there is a favorable interaction between the surface and protein/ cell (a change in enthalpy) or when there is a disorderly change in water structure near the surface (a change in entropy) [84;85]. Proteins interacting with the surface through ionic or electrostatic interactions or hydrogen bonding would lead to change in enthalpy. Proteins interacting to surfaces with hydrophobic interactions correlate to a change in entropy. Upon adsorption onto the surface, proteins and cells undergo transformations that lead to the activation of the coagulation cascade,  22  complement activation, and platelet/ leukocytes activation [86]. It has been reported that the activation of these bodily defensive mechanisms are highly interconnected [80]. Speculations have been made that adsorption of FXII on biomaterial surfaces may result with auto-activation of FXII to FXIIa. This in turn would initiate the contact activation pathway of the coagulation cascade and ultimately activate platelets. There are also reports suggesting that TF released from surface-activated monocytes may initiate the coagulation cascade [80].  There are studies showing the necessity of platelet  activation on biomaterial surface leading to the initiation of the coagulation cascade [80]. This is because significant thrombin formation is greatly enhanced through the surface of an activated platelet. 1.4.2  Current surface modifications strategies  Many approaches have been applied to improve blood compatibility of the materials used for various applications.  Surface modifications with synthetic polymers and  immobilization of anticoagulant agents like heparin onto biomaterials are the most common methods to prevent thrombus formation [87;88]. The purpose of grafting hydrophilic polymers such as poly (ethylene glycol) and poly (N, N-dimethylacrylamide) onto biomaterial surfaces is to reduce plasma protein adsorption in expectation to lowering thrombus formation. The hydrophilic nature of the grafted polymer should reduce protein interactions with surface which commonly occur via hydrophobic interactions [89]. In addition, the tethered hydrophilic polymers are able to repel protein from its surface because protein adsorption creates an energetically unfavorable compression to the grafted polymers in their conformational restrained state [90;91]. Other factors such as the surface coverage of grafted polymer and the molecular  23  weight of the grafted chains play an important role in limiting protein adsorption on surface. Grafting hydrophilic polymer chains at a closer distance increases the high conformational entropy in these anchored chains and thus provides a higher tendency to repel proteins from surfaces.  The grafted polymer chains act as a barrier layer to  minimize the interaction between the surface and plasma proteins. However, varying degree of success have been seen using this particular approach, as plasma proteins can still be adsorbed onto surfaces modified with hydrophilic polymers and thrombus formation remains a significant problem. Another approach to prevent thrombus formation is to immobilize anticoagulant or clot lysis-enhancing compounds on surfaces. Heparin is frequently the choice for this application because of its clinical success as an antithrombotic agent. Heparin-bound surface modification has shown significant improvement of blood compatibility in the in vitro studies [92;93]. However, variation of heparin’s bioactivity after covalent bonding to surface, and degradation of heparin proved to be the cause of inconsistency seen in the in vivo studies for these heparin-bound surface modifications [94;95]. Furthermore, as fore mentioned, heparin needs AT to inhibit thrombin. Thus success of the heparinimmobilized surfaces could be more diverse, as the highly anionic charge of heparin provides a good protein adsorption site for circulating plasma proteins.  Therefore,  current approaches are designed to resolve this problem by covalently attaching antithrombin-heparin complex (ATH) to surface [96;97]. ATH has been shown to be higher in bioavailability than heparin alone and increases attraction for plasma AT [98]. Although surfaces coated with ATH may significantly improve the anticoagulant  24  properties, it does not resolve the problem with protein adsorption onto surfaces, as the bound AT can act as a platform for other proteins to bind to the surface of the biomaterial.  1.5 Novel functional polymers One of the major focuses of our laboratory is to design novel synthetic polymers having biological activity and to incorporate them into current biomaterials.  These  studies vary from grafting polymer brushes on surfaces to modify their surface morphology [99;100] and to synthesizing a novel heparin antidote, as well as to synthesizing polymer-based iron chelators [101]. To synthesize novel polymers with biological functions, our laboratory utilizes a well-characterized and well-controlled polymerization method, atom transfer radical polymerization (ATRP). Using ATRP, we are able to control the composition of the polymers synthesized. Poly (N-[2,2-dimethyl1,2-dioxolane)methyl]acrylamide) (PDMDOMA) is a novel synthetic polymer that was recently synthesized in our laboratory [102]. Recently we have discovered PDMDOMA and poly (N-isopropylacrylamide) PNIPAm produced a significant reduction in both clot strength and clot elasticity as measured by a clinical coagulation machine, thromboelastograph (TEG). Since there was no prior knowledge of such bioactivity by these polymers, we wish to further examine the biological properties of PDMDOMA and PNIPAm.  In addition to studying the  antithrombotic properties of PDMDOMA and PNIPAm as a soluble agent, we further examined the activity of these polymers when grafted onto model surfaces.  25  1.6 Thesis overview Administration of antithrombotic agents is one the most widely practiced procedure in medicine. However, owing to an increased risk of excessive bleeding and other undesirable complications such as HIT and skin necrosis, these clinically used antithrombotic agents possess certain degree of vulnerability to patients.  Thus,  development of novel antithrombotic agents that is free of complications could provide significant improvement to the current antithrombotic treatments. There are many different approaches to attenuate thrombus formation. One possible approach is to enhance fibrinolysis of the fibrin clot. This can be done by means of altering the fibrin polymerization process or affecting the FXIIIa-catalyzed cross-linking reaction between fibrin monomers. Both of these processes would render the resulting fibrin gel mesh less rigid and much easier to dissolve during fibrinolysis. The inability of synthetic materials to avoid surface-induced thrombus generation has resulted in the use of antithrombotic agents when these materials are administered. There have been advances in surface engineering and chemistry in developing biocompatible surfaces by immobilizing antithrombotic agents. However, mixed results are seen with these attempts in both in vitro and in vivo studies. Therefore, thrombus generation on these surfaces remain a significant problem.  Thus, the development of novel  antithrombotic agents that can reduce uncharacteristic thrombus generation on biomaterial surfaces could significantly improve their biocompatibility. The general scope of this thesis is to investigate the potential of two synthetic polymers, poly-N-[(2, 2-Dimethyl-1, 2-Dioxolane) Methyl] Acrylamide (PDMDOMA) and poly (N-isopropylacrylamide) (PNIPAm), to act as novel antithrombotic agents. The  26  antithrombotic properties of these polymers in soluble and surface grafted forms are investigated. 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Rossi, N.A.A., Mustafa, I., Jackson, J.K., Burt, H.M., Horte, S.A., Scott, M.D., and Kizhakkedathu, J.N. In vitro chelating, cytotoxicity, and blood compatibility of degradable poly(ethylene glycol)-based macromolecular iron chelators. Biomaterials 30, 638- 648 (2008). 102. Zou, Y., Brooks, D.E., and Kizhakkedathu, J.N. A novel functional polymer with tunable LCST. Macromolecules 41, 5393- 5404 (2008).  34  2 Influence of poly-N- [(2, 2-dimethyl-1, 2-dioxolane) methyl] acrylamide on the normal fibrin clot structure1 2.1 Introduction Antithrombotics are an essential part of modern clinical practice. They serve the purpose of treating and preventing thrombus formation in diverse clinical conditions like atrial fibrillation [1] and venous thromboembolism [2], or various types of invasive surgeries. Inhibition of thrombosis can be achieved by three therapeutic approaches: inhibit at the coagulation cascade, increase endogenous fibrinolytic activity, and alter platelet activation and aggregation [3;4]. It is well known that the success of an anticoagulant drug lies in its specificity to either directly or indirectly inhibit a target involved in the coagulation cascade [5]. A major target of anticoagulants in the coagulation cascade is thrombin. For example, two widely available and effective anticoagulant agents used today are heparin and warfarin: both of which indirectly inhibit thrombin. Direct thrombin inhibitors such as hirudin and argatroban, and anticoagulants that act against different steps of the coagulation cascade are also available.  Other examples include low molecular weight heparin and  fondaparinux, which are indirect inhibitors of coagulation factor Xa. Another example is the recombinant form of activated protein C which targets coagulation factors Va and VIIIa. Two further approaches of antithrombotic therapy involve the administration of fibrinolytic agents and antiplatelet agents. Fibrinolytic agents are drugs that promote the conversion of plasminogen to plasmin, which then proteolytically degrades the arterial or venous thrombus; examples include tissue plasminogen activator (tPA) and streptokinase. 1  A version of this chapter will be submitted for publication. Lai, B.F.L., Zou, Y., Brooks, D.E., and Kizhakkedathu, J.N. A Neutral Synthetic Polymer Influences the Normal Fibrin Clot Structure by Altering the Fibrin Polymerization and Fibrin Cross-linking. 35  Antiplatelet agents, such as ASA and clopidogrel [4], are drugs that interfere with the ability of platelets to aggregate to form a platelet plug.  Despite their success in  preventing thrombosis, current therapies have limitations that range from bleeding complications to allergic responses [6;7;8].  Thus the development of novel  antithrombotic agents without adverse effects is very important. One target for novel antithrombotic agents in the blood coagulation cascade is the fibrin clot, the final product of coagulation. Upon cleavage of fibrinogen by thrombin, fibrin monomers polymerize to give a fibrin clot with highly diverse structural properties [9]. This polymerization process initially involves fibrin monomers interacting with each other in a half-staggering, end-to-end fashion to form double stranded protofibril. The protofibrils then aggregate in a lateral manner to form fibers and finally fibers branching in various directions to produce the overall structural network of a fibrin clot [10]. With the three-dimensional gel mesh, the fibrin clot is then stabilized by formation of intermolecular Nε-(γ-glutaml) lysine covalent cross-links between adjacent or neighbor fibrin molecules [11] and catalyzed by coagulation factor XIIIa. The overall stability of a fibrin clot is therefore dependent on both the threedimensional network branching between fibrin fibers and the cross-linking of gamma chains between fibrin monomers [12;13]. An alteration to the branching network or the stabilizing cross-links in the fibrin clot would render the clot less rigid and less elastic, and more prone to lysis by plasmin. Thus designing an agent that specifically affects the normal fibrin clot polymerization and the cross-linking process offers a novel target in the attenuation of thrombosis [14]. A few such inhibitors have already been reported. Compounds derived from natural sources, Tridegin (leech) [15], cysteine proteinase  36  inhibitor (potatoes) [16] and alutacenoic acids (fungi) [17], and synthetic compounds such as, 2-[(2-q-oxopropyl) thio] imidazolium [18;19], 1,2,4-thiadiazole [20], and tyrosine melanin [21;22] have demonstrated ability in modulating cross-links within fibrin clots and enhancing fibrinolysis. In the current work, we report a novel observation that a synthetic neutral polymer poly (N- [(2,2-dimethyl-1, 3dioxolane) methyl] acrylamide) (PDMDOMA) influences the normal overall fibrin clot structure and clot properties through abnormal lateral aggregation of the protofibrils and poorer cross-linking between the monomers. This is the first report of a synthetic neutral macromolecule showing such effects. Experiments modeling the anticipated in vivo PDMDOMA exposure showed that the blood clot formed in presence of PDMDOMA in whole blood is less rigid and elastic. This result is further supported by the change in overall fibrin clot structure imaged by scanning electron microscope (SEM), the protofibril formation by turbidity measurement and fibrin cross-links presented on 1-D gel electrophoresis and D-dimer ELISA analysis. We have also shown that the chemical structure of the PDMDOMA is responsible for its action. Taken together, these observations indicate that PDMDOMA is a promising synthetic polymer-based anticoagulant that affects normal fibrin clot structure, and viscoelastic properties of fibrin clot.  37  2.2 Experimental 2.2.1  Materials  2.2.1.1 Chemical Poly (N-[(2,2-dimethyl-1, 3dioxolane)methyl]acryl amide) (PDMDOMA) and poly (N,N-dimethyl acryl amide) (PDMA) were synthesized in our laboratory by following previous reports [23].  The polymers were purified by dialysis against water after  incubated with EDTA solution (0.1 M) to remove any adsorbed copper species. The molecular weights of the polymers were determined by gel permeation chromatography (GPC) system coupled with a multi angle light scattering detector (Wyatt Technology Corp., US).  Partially de-protected, PDMDOMA-diol (PDMDOMA-co-PDHPA) and  100% dioxolane de-protected PDMDOMA-diol (PDHPA) was prepared by the acid cleavage of the dioxolane groups. Poly (ethylene glycol) (PEG) of molecular weight 35,000 Da was purchased from Fluka. 4-(2-Hydroxyethyl) piperazine-1-ethanesulfonic acid sodium salt (HEPES), CaCl2 (99.99%) and NaCl2 (99%) were purchased from Aldrich (Ontario, Canada) of the highest purity. Distilled water used in all experiments was further purified using a Milli-Q Plus water purification system (Millipore Corp., Bedford, MA). 2.2.1.2 Biological Blood from healthy donors was collected into 3.8% sodium citrated tube with a blood/anticoagulant ratio of 9:1 after consenting informed healthy volunteer donors at Centre for Blood Research, University of British Columbia. Platelet-poor plasma (PPP) was prepared by centrifuging whole blood samples at 1200 X g for 20 min in an Allegra X-22R Centrifuge (Beckman Coulter, Canada).  38  Human plasma proteins such as  fibrinogen (> 80% of clottable protein), plasminogen (≥ 2 units/mg protein), tissue plasminogen activator (≥ 500,000 IU/mg), and thrombin (≥ 2,000 NIH units/mg protein) were all purchased from Sigma (Ontario, Canada). Human plasma factor XIII (FXIII) and activated plasma factor XIII (FXIIIa) were purchased from Haematologic Technologies Inc. (VT, USA). All of the lyophilized plasma proteins were made into solution by HEPES-saline buffer (10 mM HEPES, 150 mM NaCl, pH 7.4). The reagents were kept at -80°C until use. Reagents for conventional coagulation assays activated partial thromboplastin time and prothrombin time, actin FSL and recombinant thromboplastin, were purchased from Dade Behring (Marburg, Germany). 2.2.2  Methods  2.2.2.1 Measurement of clotting time: prothrombin Time (PT) and activated partial thromboplastin time (APTT) Sodium citrate anticoagulated platelet poor plasma (PPP) is used for PT and APTT analysis. The effect of each polymer solution in HEPES-saline (10 mM HEPES, 150 mM NaCl, pH 7.4) on coagulation was examined by mixing PPP with the polymer solution (9:1 v/v) for a final concentration of 1 mg/ml in plasma in the cuvette-strips at 37°C for the required time before addition of the coagulation reagents. Control experiments were performed with identical volumes of HEPES-saline solution.  Each experiment was  repeated in triplicates on a STart®4 coagulometer (Diagnostica Stago, France). Detailed experimental procedure was as described by our group previously [24]. 2.2.2.2 Fibrin polymerization assay 180 µl of Human fibrinogen (3 mg/ml) and 20 µl of HEPES-buffered polymer solutions (1 mg/ml final concentration) were pre-incubated at 37°C for 10 min. Human fibrinogen in 20 µl of HEPES-saline buffer was also tested as a control for the analysis.  39  The mixture was added to 96-well microtitre plate. Fibrin polymerization was then initiated by the addition of 20 µl of 3 NIHU/ml human thrombin, 20 µl of 25 µg/ml activated plasma factor XIII (FXIIIa), and 20 µl of 40 mM CaCl2 (3 mM final concentration) to each well. The optical density (OD) was monitored at 405 nm using a SpectraMax plate reader (Molecular Devices, USA). Each measurement was carried out in triplicates for 1 h at 37°C. 2.2.2.3 Analysis of fibrin cross-linking in clot by SDS-PAGE The cross-linking of fibrin by activated human plasma factor XIII (FXIIIa) was analyzed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) [15;18]. The fibrin clot was either formed from PPP or purified human fibrinogen (3 mg/ml). In the case of PPP, the 40 µl of polymer solutions in HEPES-saline buffer was mixed with 360 µl of plasma in a 1:9 ratio and clotting was initiated by the addition of 20 µl of 1 NIHU/ml human thrombin and 20 µl of 0.2 M CaCl2 (10 mM final concentration). Final polymer concentration was maintained at 1 mg/ml.  In the case of purified  fibrinogen, we followed similar protocol except the addition of 20 µl of FXIII (30 µg/ml) along with 20 µl of 3 NIHU/ml thrombin and 20 µl of 40 mM CaCl2 (1.67 mM final concentration) for the initiation of clot. The samples were incubated at 37°C water bath for 2 h and a stable fibrin gel was formed. The fibrin gel was then washed with HEPES buffer and centrifuged at 2300 X g for 2 min to remove excess clotting solution and procedure was repeated 5 times. The fibrin gel was dissolved by the addition of a reducing loading buffer (9 M urea, 40 mM DTT, 0.1 M Tris-HCl, 2% SDS, 0.2 % bromophenol blue, and 20% glycerol, pH 6.8), followed by overnight incubation at 37°C. Pure fibrinogen and PPP without the addition of polymer samples were prepared in the  40  same manner used as control samples. Equal volumes of the samples were loaded onto the gel. After electrophoresis, the gel was stained with Coomassie brilliant blue (BioRad). Images of the SDS-PAGE gel were captured by Odyssey Infrared Imaging system (LICOR® Biosciences, USA). Band intensity was reported as the integrated intensity value (sum of the intensity values for all pixels enclosed by a feature, multiplied by the area of the feature: count mm2) of each protein band. 2.2.2.4 Analysis of fibrin cross-linking in clot by d-dimer enzyme immunoassay (EIA) D-dimer is the cleavage product of fibrin clot when the fibrin is degraded by plasmin. In this analysis, d-dimer was quantified by an anti-human d-dimer F(ab’)2 fragment (2F7) by an micro-latex enzyme immunoassay (Diagnostica Stago, France).  Platelet-poor  plasma was clotted in the presence of various polymer samples at a final concentration of 1 mg/ml by adding 20 µl of 1 NIHU/ml human thrombin and 20 µl of 0.2 M CaCl2 (10 mM final concentration), for 1 h at 37°C. The reaction was stopped by the addition of 40 µl of tissue plasminogen activator (65 µg/ml) and 40 µl of plasminogen (10 µg/ml). Degraded fibrin samples were collected after 2 h of incubation with tPa and plasminogen and were stored at -80°C until analysis. D-dimer levels are expressed in initial fibrinogen equivalent units (FEU): the actual amount of D-dimer is approximately half of an FEU. 2.2.2.5 Analysis of fibrin clot structure by scanning electron microscopy For SEM analysis, fibrin clot was prepared from 360 µl of 3mg/ml HEPES-buffered fibrinogen, 40 µl of 3 NIHU/ml of thrombin, 20 µl of 30 µg/ml FXIIIa, 20 µl of 40 mM CaCl2 (1.67 mM final concentration) in presence or absence of the 40 µl of polymer samples. The mixtures were thoroughly premixed and were incubated for 1 h at 37°C to  41  allow clotting to proceed. Specimens were first washed 3 times with 50 mM phosphate buffer (pH 7.4) to remove excess salt and fixed with 2% glutaraldehyde. We followed a method reported by Weisel et al. [25] with a slight modification for the preparation of the clots for SEM imaging. Clots were frozen and fractured for 1 h, and critical point-dried with CO2 in a DCP-1 critical point drying apparatus (Denton Vacuum Co., USA). The specimens were mounted and sputter-coated with gold-palladium, and then examined with a Philips XL30 scanning electron microscope (Philips Electronics Co., USA). The electron micrographs were taken at magnifications between 5k and 10k. From the images, the size of each of the fibrin fiber strands was determined using Image J (NIH, USA), by drawing a line to bisect foreground fibers to obtain a pixel value and relating to pixel value obtained for the scale bar on the image. Fifteen different fibrin strands on each image were analyzed. At least two images were analyzed for each sample. The diameter of individual fibrin strands on the images was reported as an average for all fiber strands measured. 2.2.2.6 Analysis of overall strength of fibrin clot The Thromboelastograph® (TEG) Hemostasis System 5000 series (Haemoscope Corporation, USA) was used to examine the viscoelastic properties of whole blood clots. Citrated human whole blood was first incubated with HEPES-saline buffered polymer samples at ratio 9:1 v/v. Two separate analyses were performed. One investigation was studying the change in clot viscoelastic properties when different polymer samples were used. The final concentration of the polymer was 1 mg/ml. The second study was to identify dose dependent effect on clot viscoelastic properties. The final concentrations of the HMW PDMDOMA samples were at 0.1 and 1 mg/ml. Whole blood added with  42  HEPES buffer without polymer was used as control. Aliquot of the reaction mixtures (340 µl) were then added to TEG cup and the clotting was initiated by the addition of 20 µl of 0.2 M CaCl2 (10 mM final concentration). The TEG analysis was allowed to proceed to a 2 h preset completion time or until the analysis was prematurely terminated by TEG system itself. The overall coagulation profile was recorded by TEG system. The overall clot strength (maximum amplitude (MA)), clot elasticity (G`) and fibrinolysis at 30 min were measured and analyzed. Both MA and G` recorded are direct representation of the dynamic properties between fibrin and platelet bonding via GP IIb/IIIa. The percentage of fibrinolysis was also measured and analyzed by TEG. 2.2.2.7 Statistical analysis Statistical analyses of data to include 95% confidence intervals, two-tailed Student’s t-tests were performed on the Microsoft Office 2003 Excel software. Due to the volume of data, only relevant comparisons were made. Only comparisons between data from HMW PDMDOMA to buffer control and data from LMW PDMDOMA to buffer control were made.  2.3 Results The structures and characteristics of the polymers studied are given in Figure 2.1. H2 C  H C  n O  HN  O O  PDMDOMA  PDMA  43  PEG  n 1-x  x  O  O  O  HN  HN  HN  O  OH  OH  O 70%  OH 30%  OH  PDMDOMA-co-PDHPA  PDHPA  Polymer PDMA (control)  Mn 6,400  PDI 1.74  PEG (control  35,000  1.22  HMW PDMDOMA  45,000  1.51  LMW PDMDOMA  6,300  1.14  PDMDOMA-co-PDHPA PDHPA  42,000 34,800  1.51 1.51  Figure 2.1 Structures and characteristics of the polymers tested for antithrombotic activity. PDMA is used as “control” model due to its structural similarity to PDMDOMA and PEG is used as “control” model because of its proven biocompatibility. The table shows the number average molecular weight (Mn) and polydispersity (PDI) of each polymer.  A high molecular weight PDMDOMA (HMW-PDMDOMA), a low molecular weight PDMDOMA (LMW-PDMDOMA), a partially cleaved PDMDOMA (PDMDOMA-coPDHPA) containing 30% diol content, a completely cleaved PDMDOMA (100% diol content, PDHPA), PEG and PDMA were used in this study (Figure 2.1). PEG and PDMA are used as controls as they are known to be neutral to blood coagulation cascade. 2.3.1  In vitro blood coagulation assay  The effect of PDMDOMA and the control polymers, PDMA and PEG, on the coagulation cascade was investigated by conventional blood coagulation assays APTT  44  and PT. The time taken for the PPP to form a clot after the addition of APTT or PT reagents was recorded as the time required for thrombin formation via either extrinsic or intrinsic pathways of the coagulation cascade. As shown in Figure 2.2A and 2.2B, the HMW-PDMDOMA sample at 1 mg/ml concentration does not have significant effects on the initial coagulation of plasma when compared to buffer, PDMA or PEG samples. APTT PT  35  30  Time (seconds)  25  20  15  10  5  0 Buffer Control  1 mg/ml PDMA  1 mg/ml PEG  1 mg/ml HMW PDMDOMA  Polymer Samples  Figure 2.2: Coagulation cascade analysis by APTT and PT assays. (A) APTT: Platelet poor plasma (PPP) was incubated with APTT reagent (actin FSL), CaCl2 (25 mM), and soluble polymer samples (1 mg/ml) and the plasma hemostasis was measured with at 37°C by ST4 hemostasis analyzer. (B) PT: Platelet poor plasma (PPP) was incubated with PT reagent (tissue factor), and soluble polymer samples (1 mg/ml) and the plasma hemostasis was measured with at 37°C by ST4 hemostasis analyzer.  The PDMDOMA samples slightly increased the APTT values but these differences are not significant (p=0.15) compared to control samples. According to these routine clinical  45  coagulation assays, PDMDOMA does not appear to influence the steps involved in the clotting cascade that would lead to the delay in thrombin generation and fibrin formation. 2.3.2  Kinetics of fibrin polymerization  The kinetics of fibrin formation was characterized by measuring the change in turbidity over a 1 h time frame when thrombin and calcium were added to pure fibrinogen solution. It has been well documented that the change in absorbance with time is directly relate to overall fibrin polymerization [25;26]. A turbidity curve has three phases: (A) lag time, (B) maximum rate of change in optical density (the steepest part of the curve), and (C) final turbidity (Figure 2.3). The lag time is a measure of the rate of protofibril formation, the rate of change in optical density is referred to the rate of protofibril lateral aggregation, and the final turbidity is an indication of the number and size of fiber formed. As shown in Figure 2.3, no significant difference can be detected in the rate of protofibrils formation (A) in the fibrinogen clot assembled in presence of HMWPDMDOMA and control samples (both PDMA and PEG, and buffer). This result further supports the argument that the thrombin activity is not affected by the addition of PDMDOMA. In this case, fibrinopeptide A is still being cleaved off from fibrinogen by thrombin, while fibrin monomers still polymerize to form a double stranded protofibril. However, significant differences were observed for both the rate of protofibrils lateral aggregation (B) and the final optical density (C) in the case of HMW-PDMDOMA compared to the buffer control. In the case of HMW-PDMDOMA these values are much higher than the buffer control and the control polymer samples PDMA and PEG. PDMA and PEG showed a slight increase in the overall optical density when compared to buffer;  46  however, these were much lower than the HMW-PDMDOMA increases. Fibrin monomer polymerized in the presence of HMW-PDMDOMA at a much faster rate of lateral aggregation resulted compared to buffer control (0.008629 OD/min vs. 0.002743 OD/min). A 4-fold increase in the final optical density of the clot was observed (Figure 2.3) in the case of HMW-PDMDOMA compared to the buffer control.  C  0.2  0.18  PDMDOMA  PDMDOMA-coPDHPA  0.16  Optical Density (Abs 405nm)  0.14  0.12  PEG  B  0.1  PDHPA 0.08  PDMA 0.06  Buffer  0.04  0.02  A  0 0 -0.02  10  20  30  40  50  60  Time (minutes)  Figure 2.3: Turbidity analysis of fibrin-formation in presence of polymers. Fibrinogen (3 mg/ml) was clotted with 3 NIHU/ml thrombin, 30 µg/ml FXIIIa, and 3 mM CaCl2 in presence of soluble polymer samples (1 mg/ml). A: Lag Time; B: lateral aggregation or protofibrils; C: Final Optical density.  In addition, the presence of HMW-PDMDOMA resulted in longer lateral aggregation time, while the time for the turbidity curve to reach the final optical density also  47  increased. These results indicate that the HMW-PDMDOMA significantly alters the normal lateral aggregation of protofibrils. As a comparison, the turbidity values for fibrin clots formed in presence of PDMDOMA-co- PDHPA (30% diol) and PDHPA (100% diol content) are also shown (Figure 2.3). Like HMW-PDMDOMA, the PDMDOMA-coPDHPA also showed a change in the assembly of the fibrin clot.  However, after  complete removal of dioxolane groups from the PDMDOMA, the resulting PDHPA polymer showed similar turbidity values to those of the control polymers. As had been shown for the control polymers (PEG and PDMA), PDHPA also caused a slight increase in the final turbidity. 2.3.3  Fibrin clot structure studied by scanning electron microscopy  SEM was used to investigate the effect of PDMDOMA (LMW and HMW) on the overall fibrin clot structure.  Fibrin clots prepared from purified fibrinogen in the  presence of PDMDOMA samples was compared with clots formed in the presence of control PEG and in the absence of any polymer (buffer control). As is evident from the micrographs shown in Figure 2.4A, the fibrin clot formed in the absence of (i) polymer or in presence of (ii) PEG showed a highly branched fibrin network. In addition, the clot architecture was homogenous with only few compact areas of fiber. The mean diameters of fibers measured from the micrographs of these control clots were 122 ± 12 nm for buffer and 130 ± 21 nm for the PEG sample (Figure 2.4B). However, fibrin clot structure formed in presence of either (iv) HMW-PDMDOMA (at final concentration 1 mg/ml) or (v) LMW-PDMDOMA (at final concentration 0.25 mg/ml) sample showed significant difference in appearance. Although individual fibers making up the overall clot structure could still be seen, the fiber strands were significantly smaller in diameter (87 ± 13 nm  48  for HMW PDMDOMA and 86 ± 17 nm for LMW PDMDOMA). In addition, these thinner fibers favored bundling together to form large aggregates instead of branching in every direction. Aggregations of fiber strands led to the formation of networks with larger pores than in control clots, and resulting in a heterogeneous architecture. For comparison, fibrin clots formed in the presence of PDHPA (100% diol content) are also given. The PDHPA showed similar fibrin clot structure to the buffer and polymer controls, suggesting that the particular chemical structure of the PDMDOMA is responsible for the change in clot structure. Figure 2.4: SEM analysis of fibrin clot structure. Purified human fibrinogen (3 mg/ml) was incubated with specific synthetic testing materials, 3 NIHU/ml human thrombin, and 1.67 mM CaCl2 for 1 h at 37°C. The samples then underwent fixing, cyro-freezing, critical point drying and gold sputter coating. (A) SEM images of fibrin clot formed in (i) Buffer control, (ii) 1 mg/ ml PEG control, (iii) 1 mg/ml PDHPA, (iv) 1 mg/ ml HMW PDMDOMA, and (v) 0.25 mg/ ml LMW PDMDOMA. The scale bar indicates 5 µm. (B) Comparison of diameters of individual fiber strands formed in presence of various polymer samples calculated from SEM micrographs.  (A)  (i) Control Sample  (ii) PEG Sample  49  (iii) PDHPA Sample  (iv) HMW PDMDOMA Sample  (v) LMW PDMDOMA Sample  50  (B) 160  140  Fiber Size (nm)  120  100  80  60  40  20  0 Buffer Control  PEG  PDHPA  HMW PDMDOMA LMW PDMDOMA  Polymer Samples  2.3.4  Influence of fibrin cross-linking by factor XIIIa in the presence of PDMDOMA.  The cross-linking of γ-chains of fibrin monomer by FXIIIa in the presence of PDMDOMA and other control samples (buffer, PEG and PDMA) was investigated by SDS-PAGE. Two sets of experiments were performed: fibrin clot formed from i) PPP with the addition of thrombin and calcium, and ii) from purified fibrinogen with the addition of human thrombin and factor XIII. As seen from the SDS-PAGE gel image from PPP (Figure 2.5A), the clot formed in buffer (lane 4) showed a more prominent level of cross-linking on the γ-chain, resulting with higher intensity in γ-γ dimer band. It should be noted that the loading of control (buffer, PDMA and PEG) samples have been  51  diluted by 10-fold, whereas no dilution was done for both the HMW and LMW PDMDOMA samples.  A gel obtained from the undiluted samples is given in the  appendix. In control PDMA or PEG clots (lane 5 and 6) samples, a similar high intensity of γ-γ dimer was observed as in the case of buffer. However, in presence of HMW or LMW-PDMDOMA (lane 7 and 8), the γ-γ dimer formation was strikingly reduced, compared to control samples. The amount of clot formed in presence of PDMDOMA was less than the control samples. A similar study was carried out for the fibrin clot formed from purified fibrinogen (Figure 2.5B). However, it must be noted that the difference between γ-γ dimer formed in the purified fibrinogen samples do not have such striking difference in intensity between the tested samples. Significant fibrin crosslinking (higher molecular weight band in the gel) in presence of control samples (buffer and PEG) compared to PDMDOMA was observed, supporting the notion that PDMDOMA limits cross-linking of fibrin.  This is highlighted by the fact that the  integrated bands intensity of γ-γ dimer formed in the presence of PDMDOMA is less than half the value observed for the controls (Figure 2.6). Figure 2.5: Evaluation of cross-linking in fibrin clot using reducing SDS-PAGE. The clotting was initiated in citrated platelet poor plamsa or purified fibrinogen, thrombin (1 NIHU/ml for PPP clot and 3 NIHU/ml for fibrinogen clot), plasma factor XIIIa (25 µg/ml), and CaCl2 (10mM final concentration for PPP clot and 1.67 mM final concentration for fibrinogen clot), in presence of buffer control, control polymers (PEG and PDMA) and PDMDOMA (HMW and LMW) for 2 h in 37°C water bath. The clots were cleaned by washing with buffer to remove the uncross-linked proteins and the clots were then dissolved in 9 M urea. The samples were reduced using DTT and loaded onto gel. The 10% gels of the two clots are shown (A) platelet poor plasma, (B) purified fibrinogen. (lane 1: MW standard, lane 2: fibrinogen control, lane 3: thrombin + FXIIIa, lane 4: buffer control, lane 5: 1 mg/ml PEG control, lane 6: 1 mg/ml PDMA control, lane 7: 1 mg/ml HMW PDMDOMA, and lane 8: 0.25 mg/ml LMW PDMDOMA).  52  (A) 1  2  3  4  5  250  6  7  8 α−polymer α− olymer  130 95  γ−γ dimer  72 α chain β γ  55  36  53  (B) 1  2  3  4  5  250 130 95 72  6  7  8 α polymer γ−γ dimer α β γ  55  54  Integrated Intensity of γ−γ band (Pixel)  80  70  60  50  40  30  20  10  0 Buffer Control  PEG  PDMA  HMW PDMDOMA  LMW PDMDOMA  Polymer Samples  Figure 2.6: Analysis of band intensity. The protein band intensity analysis of γ-γ cross-links (Figure 2.5A) in fibrin clot formed in blood plasma.  2.3.5  Analysis of clot structure by d-dimer EIA  The γ-chain crosslink was further analyzed using enzyme immuno assay (EIA) for ddimer. D-dimers are degradation products of a fibrin mesh that have been stabilized by factor XIIIa through the γ-chain of the D-domain of fibrinogen molecule. After two hours of fibrinolysis of the clot by the added plasmin, the amount of d-dimer formed in presence of PDMDOMA samples is lower than that of the control samples (buffer and PEG) (Figure 2.7). The LMW-PDMDOMA gave less of d-dimer compared to HMWPDMDOMA. The results indicate that the amount of cross-linking by FXIIIa is less in  55  presence of PDMDOMA samples compared to control samples. This analysis further supports the γ-γ dimer analysis, TEG data and heterogeneous clot structure as shown by SEM images. 500 450  Level of D-Dimer (ug/ml)  400 350 300 250 200 150 100 50 0 Buffer Control  PEG  HMW PDMDOMA  LMW PDMDOMA  Polymer Samples Figure 2.7: Quantitative analysis of fibrin clot structure: d-dimer EIA. Platelet-poor plasma incubated with HEPES buffer saline, PEG, HMW PDMDOMA and LMW PDMDOMA was coagulated with the addition of 1 NIHU/ml human thrombin and 10 mM CaCl2. Fibrin clot was allowed to form for 2 h at 37°C. The final concentration of polymer samples is 1 mg/ml and the final concentration for LMW-PDMDOMA is 0.25 mg/ml. After clot formation, tPA (65 µg/ml) and plasminogen (10 µg/ml) were added to degrade clot. Samples of degraded products were collected after 2 h.  2.3.6  Blood clot analyses by thromboelastograph  The overall strength (MA) and overall rigidity (G`) of a whole blood clot formed over a 2 h time period were investigated using a clinical hemodynamic analyzer known as Thromboelastography (TEG).  Typical TEG traces showing the clot formation in  56  presence and absence of PDMDOMA samples are given in Figure 2.8. The buffer control and the polymer controls gave characteristic TEG traces (Figure 2.8A). However, the clot formed in presence of HMW and LMW PDMDOMA samples showed significant decrease in values for MA and G`, indicating that the clot structure is significantly affected (Figure 2.8Ai). The addition of either HMW or LMW PDMDOMA resulted in a whole blood clot that was not stable enough to maintain in a state that can be measured by TEG. From the data show in Figure 2.8A, there is no significant difference in the initial fibrin formation when whole blood is incubated with either PDMDOMAs or the control samples. In addition to the effect of the polymers, different concentrations of the HMW PDMDOMA sample also had an influence on the overall clot structure, i.e. the higher concentration of the polymer used, the greater the decrease in the clot strength and rigidity (Figure 2.8B). Figure 2.8: Evaluation of overall clot properties measured by TEG. Citrated whole blood was re-calcified with 10 mM CaCl2 and was allowed to clot in the presence of HMW-PDMDOMA (1 mg/ml), LMW-PDMDOMA (0.25 mg/ml) and control polymer samples: PEG and PDMA, and buffer control for 2 h at 37°C in the Thromboelastograph® analyzer. Thromboelastograph traces of whole blood clot are given: (A) Effect of different polymers on whole blood clot properties (i) effect of HMW-PDMDOMA (1 mg/ml) and LMWPDMDOMA (0.25 mg/ml) samples and buffer control samples and (ii) effect of control polymers (1 mg/ml) and buffer control samples; and (B) Effect of the concentration of HMW-PDMDOMA on whole blood clot properties. The concentrations of the HMW-PDMDOMA used are given in figure itself.  57  (A) (i)  (ii)  58  (B)  The calculated values of MA and G` obtained from the TEG traces are in given Figure 2.9A and 2.9B, respectively. The MA values and G` values of the clot formed in presence of PDMDOMA samples are significantly lower than that of the control samples (buffer, PEG and PDMA). The overall clot strength and rigidity have shown at least a 10-fold decrease (p<0.000005) in presence of HMW-PDMDOMA at 1 mg/ml concentration when compared to control samples. The change in the overall clot strength and rigidity of the blood clot in presence of HMW-PDMDOMA is also dose dependent. Furthermore, a similar change in the whole blood clot rheological properties were recorded when whole blood was clotted with the partially de-protected PDMDOMA-coPDHPA (30% diol content) (appendix S3). When the dioxolane groups were completely removed from the polymer (PDHPA), the TEG trace was similar to the control samples. The recorded values of MA and G` for PDMDOMA-co-PDHPA (also given in Figure 2.9A and 2.9B) show similar decrease in overall clot strength and clot elasticity. The recorded MA and G` values of PDHPA sample were similar to those values of control 59  whole blood clot. PDMDOMA.  This further supports the influence of the specific chemistry of  It must be noted that there is no difference in the bioactivity of  PDMDOMA on whole blood clotting process when different molecular weight of PDMDOMA samples are used. Similar to HMW-PDMDOMA, LMW-PDMDOMA is effective in decreasing the strength (MA) as well as rigidity (G`) of the whole blood clot with a slight variation (Figure 2.10A and 2.10B). Figure 2.9: Overall clot strength and blood clot elasticity recorded by TEG (polymers comparison). Blood Clots were formed in TEG at 37°C from solutions of citrated whole blood, 10 mM CaCl2, and were incubated with buffer control, control polymers (PEG and PDMA), PDMDOMA (HMW-), and partially de-protected PDMDOMA-diol (PDMDOMA-co-PDHPA). The final concentration for the control polymers was 1 mg/ml and the final concentration for PDMDOMA varies from 0 to 1 mg/ml. (A) Maximum Amplitude and (B) Shear elasticity of the whole blood clot were measured. The t-test shows significance difference of MA and G’ values between the 1 mg/ml HMW PDMDOMA to the control (for MA: * p<0.000005 and for G`: * p<0.0005).  (A) 60  *  Maxi mum Ampl itude (mm )  50  40  30  20  10  *  0 Bu ff er C o nt ro l  1 m g /ml PE G  1 mg /m l P D M A  0 .1 mg /ml HM W PD M D O MA  P o ly m er S a m p le s  60  1 m g /m l HM W PD MD O MA  1 m g/ m l P D M D O MA -c o P DHP A  1 m g/ m l P D HP A  7  (B) 6  *  G' (kdynes/cm2)  5  4  3  2  1  * 0 Buffer C ontrol  1 mg/ml PEG  1 mg/ml PDMA  0.1 mg/ml HMW PD MD OMA  1 mg/ ml HMW PD MD OMA  1 mg/ml PD MD OMA-c oPD HPA  1 mg/ml PD HPA  P o ly m e r Sa m p les  Figure 2.10: Overall clot strength and blood clot elasticity recorded by TEG (MW comparison). Similar TEG study comparing the effect of different molecular weight of PDMDOMA sample to whole blood clotting was performed. The final concentrations for the PDMDOMA samples were 0.25 mg/ml. (A) Maximum Amplitude and (B) Shear elasticity of the whole blood clot was measured. The t-test shows significance difference of MA and G’ values between the 0.25 mg/ml HMW- and LMW-PDMDOMA compare to the control (for MA: * p<0.0000004 and ** p<0.0003 and for G’: * p<0.00001 and ** p<0.000004)  (A) 60  */ **  Maximum Amplitude (mm)  50  40  30  **  20  * 10  0  Buffer control  HMW PDMDOMA (0.25 mg/ml)  Polymer Samples  61  LMW PDMDOMA (0.25 mg/ml)  (B) 7  6  */ **  G' (kdynes/cm2)  5  4  3  2  **  1  *  0 Buffer control  HMW PDMDOMA (0.25 mg/ml)  LMW PDMDOMA (0.25 mg/ml)  Polymer Samples  In addition to the change in both clot strength and clot elasticity, whole blood clot formed in the presence of either HMW or LMW PDMDOMA had a higher rate of clot lysis (Figure 2.10). However, no clot lysis was observed for the buffer control or the polymer sample controls.  This difference in percent of clot lysis is statistically  significant (for 1 mg/ml HMW PDMDOMA: p<0.02). A similar rate of clot lysis was observed for PDMDOMA-co-PDHPA sample. 2.11: Fibrinolysis recorded by TEG (fibrinolysis at 30 minutes). (A) Whole blood clots were formed in TEG at 37°C from citrated whole blood, 10 mM CaCl2, and were incubated with buffer control, control polymers (PEG, and PDMA), PDMDOMA (HMW), and partially de-protected PDMDOMA-diol (PDMDOMA-co-PDHPA). The final concentration of the control polymers was 1 mg/ml and the final concentration for PDMDOMA varies from 0 to 1 mg/ml. The t-test shows significance difference of %LY30 values between the HMW-PDMDOMA and LMW-PDMDOMA to the control (* p<0.02). (B) Similar TEG study comparing the effect of different molecular weight of PDMDOMA sample to whole blood clotting was performed. The final concentration for HMW and LMW PDMDOMA was 0.25 mg/ml. The t-test shows significance difference of %LY30 between HMW-PDMDOMA and buffer control and between LMW-PDMDOMA and buffer control (* p<0.02 and ** p<0.003).  62  (A) 100  90  *  80  % Fibrinolysis (%LY30)  70  60  50  40  30  20  10  * 0 Buffer Control  1 mg/ml PEG  1 mg/ml PDMA  0.1 mg/ml HMW PDMDOMA  1 mg/ml HMW PDMDOMA  1 mg/ml PDMDOMA-coPDHPA  Polymer Samples  (B) 70  *  % Fibrinolysis (%LY30)  60  **  50 40  30 20 10  */ ** 0 Buffer control  HMW PDMDOMA (0.25 mg/ml)  Polymer Samples  63  LMW PDMDOMA (0.25 mg/ml)  1 mg/ml PDHPA  2.4 Discussion During blood coagulation, the conversion of soluble fibrinogen to insoluble fibrin is triggered by thrombin. The fibrin monomers interact in a half-staggered end-to-end fashion to form double-stranded protofibrils. Lateral aggregation and branching between the protofibrils leads to the final fibrin clot structure. The initial fibrin clot is then stabilized by factor XIIIa through the formation of intermolecular Nε-(γ-glutaml) lysine cross-links between the γ-chains in the D-domain of a fibrin monomer in the fibrin network. The covalent cross-links within the fibrin gel produce drastic changes to the viscoelastic properties of the clot [27]. As the clot becomes more rigid, it becomes less soluble and less susceptible to fibrinolysis. In addition to the strengthening through branching and cross-links of fibrin molecules, the clot structure is further supported by platelet aggregation through the binding of the platelet protein receptor integrin GPIIb/IIIa to the C-terminal domain of fibrinogen [28;29]. Any alterations to either the structure of the fibrin mesh in the blood clot or the platelet support would render the clot less rigid and making it more susceptible to fibrinolysis [30;31;32;33]. Thus, this feature offers the opportunity to develop novel antithrombotic agents that target the structural basis of the fibrin clot. The results presented here illustrate how a novel synthetic polymer, PDMDOMA, can interrupt the formation of a normal branched and cross-linked fibrin network. The results are compared with two proven biocompatible polymers, PEG and PDMA [34;35]. The absence of any desired biological activity of PDHPA (all the dioxolane groups of PDMDOMA are removed) points to the fact that the amide-linked dioxolane pendent groups on the PDMDOMA are responsible for its bioactivity. The data shows that the  64  dioxolane pendent groups of PDMDOMA have a significant effect in altering the lateral aggregation of fibrin fibers and cross-linking of the fibrin molecules by factor XIIIa. As a result, PDMDOMA affected fibrin clot polymerization and clot stabilization and leading to a dramatic change in the clot rheological properties. Unlike traditional anticoagulants, such as heparin, PDMDOMA does not have a specific target in the coagulation cascade. This is evidenced by its neutral activity in conventional coagulation assays. The measurement of thrombin-induced clotting of PPP also showed that PDMDOMA has had no direct influence on fibrin formation (Figure 2.2A and 2.2B) via either the intrinsic or extrinsic coagulation pathway. However, it is important to stress that both APTT and PT assays stop the measurement when a fibrin mesh has formed. Thus these coagulation assays by no means refer to the significance of whole blood hemostasis, where the roles played by platelets and plasma proteins, such as factor XIII, are more prominent in forming a stable and rigid fibrin clot [36;37]. However, no information on the kinetics of fibrin polymerization can be obtained from the APTT and PT assays. The effect of PDMDOMA on the fibrin monomer assembly process is shown by the kinetic profile of turbidity development (Figure 2.3). The parameters obtained from turbidity analysis (Figure 2.3) correspond to an important step in fibrin polymerization, i.e. lateral aggregation of protofibril into fibrin chains [25]. A faster rate of lateral aggregation of protofibrils in presence of PDMDOMA is evident from the longer time taken to reach the maximum turbidity and the higher final optical density. The dramatic increase in both the rate of protofibril aggregation and final turbidity are strong indicators for the change in rate of lateral aggregation. Typically, an increase in the turbidity end  65  point is equivalent to an increase in the final fiber diameter [38].  However, a  contradictory result was observed in SEM analysis in the case of PDMDOMA polymers. SEM analysis of fibrin clots formed in presence of PDMDOMA showed that the overall spatial organization of the fibrin network differed and thin fibers formed. Heterogeneous clot architecture, with compact areas of fibers alternating with looser areas giving a more disorganized appearance was formed in the presence of PDMDOMA compared to control samples. In addition, the individually formed fibers are highly twisted with sharply truncated ends, indicating limited lateral growth [31]. The incorporation of PDMDOMA during fibrin monomer assembly may have led to a “capping” effect on the growing ends of the protofibrils. Therefore, instead of forming an extension to the overall length of each fibrin fiber strand, the polymerization process was altered to form short thin fibers with many free ends. Such fibers assembled to generate a heterogeneous fibrin clot structure compared to the control samples. The bundles of short thin fibers may scatter light similar to thick individual fibers that account for the increase in the final turbidity in the present case. Vadseth et al. reported a similar increase in turbidity values when the fibrin diameter was smaller than that of the control samples [39]. The clot formed in presence of PEG has higher fiber diameter and is consistent with increase in turbidity. In addition, the “capping” effect by PDMDOMA may also influence the cross-linking between fibrin molecules by FXIIIa, as shown by SDS-PAGE (Figure 2.5A) and d-dimer EIA analyses (Figure 2.7). Although very little information can be obtained from SEM analysis about the physical changes to the fibrin structures in presence of PDMDOMA, it provided overall changes in clot structures. Highly ordered fibrin clot structure is related to the increase in rigidity and resistance to fibrinolysis. Thus, the thinner fiber that  66  aggregates to form a non-uniform branching network, as seen with both HMW and LMW PDMDOMA samples, results in a decrease of overall clot strength and resistance to fibrinloysis.  The TEG data on clot lysis (Figure 2.10) also support this argument.  However, one limitation of the current SEM study was the lack of analysis on the abundance of fiber ends and branching points between the fibrin strands among clots formed in presence of PDMDOMA or the control samples. Such a comparison would provide more information about the change in overall clot structure produced by PDMDOMA. The  measurement  of  the  overall  clot  viscoelastic  properties  using  thromboelastography proved invaluable. A significant decrease in both strength and elasticity was recorded for the whole blood clot formed in presence of PDMDOMA. This confirms conclusions extracted from the SEM and irregular lateral protofibril aggregation as measured by turbidity measurements. The change in the structure of the clot was further confirmed by 1-D SDS-PAGE. A decrease in intensity of γ-chain crosslinks between fibrin molecules was observed while a lower level of d-dimer was detected by EIA analysis. These results support and explain the decreased elasticity and clot strength observed in TEG analysis. The change in lateral aggregation to side-to-side aggregation between the γC-domain of fibrinogen may have an effect on the binding sites of platelet integrins αIIbβ3 and α5β1 for fibrinogen γC-domain [28]. These changes can also influence the overall clot strength by affecting normal platelet binding to the clot. Thus, in addition to the possible affect on the overall lateral aggregation of protofibrils in the fibrin clot, a disruption of binding between fibrin and platelet in presence of the PDMDOMA may also be a reason for the decrease in overall clot strength in the present  67  case. Due to the changes in the overall structure of the clot, the whole blood clot formed in the presence of PDMDOMA is more likely to degrade before reaching its maximal strength. The enhanced lysis rate was observed using TEG analysis (Figure 2.10). It must be noted that for gel electrophoresis the amount of isolated clot formed (washed) in presence of different PDMDOMA samples was smaller than the control samples. This might be due to either a decreased amount of protofibril incorporation in the clot or to the enhanced lysis. Based on the evidence from the clot formation using purified fibrinogen (Figure 2.5B), the latter is more plausible. This is also supported by the TEG lysis data. The increase in lysis may be due to the change in elasticity of the clot, caused by a decrease in γ-γ cross-links. In addition to the altered 3-dimensional clot structure in the presence of PDMDOMA as previously mentioned, cross-links between individual fibrin molecules might also have been affected. This will influence the incorporation of new protofibril to the existing fibrin fiber strand due to its inability to stretch and recover during the fibrin polymerization process [12]. This may also explain why a lower level of d-dimer degradation product is seen for PDMDOMA samples, even though the lysis rate is higher. In addition, lower amounts of protofibrils might also be incorporated to the growing fibrin fibers. 2.4.1  Conclusions  To the best of our knowledge, this is the “first detailed study” of a synthetic polymer that shows antithrombotic properties by influencing the normal fibrin polymerization in the blood clot formation. The change in the fibrin network by a synthetic polymer has particular biological and engineering significance. Our data show that the PDMDOMA  68  influences the lateral aggregation of protofibrils as well as it alters the FXIIIa mediated cross-linking support of fibrin clot.  Therefore, in the presence of PDMDOMA, a  heterogeneous clot with thinner fiber structure and reduced mechanical strength was formed. We have shown that this alteration of fibrin clot structure by PDMDOMA is due to the amide linked dioxolane pendent groups along the polymer backbone. The antithrombotic activity is also dose dependent. Thus, we anticipate that this new polymer may have applications as a novel antithrombotic agent, and in the development of novel antithrombotic surfaces for various vascular applications.  69  2.5 References 1. Albers, G.W., Dalen, J.E., Laupacis, A., Manning, W.J., Petersen, P., and Singer, D.E. Antithrombotic therapy in atrial fibrillation. Chest 119, 194S- 206S (2001). 2. Hyers, T.M, Agnelli, G., Hull, R.D., Morris, T.A., Samama, M., Tapson, V., and Weg, J.G. Antithrombotic therapy for venous thromboembolic disease. Chest 119, 176S- 193S (2001). 3. Albers, G.W., Amarenco, P., Easton, J.D., Sacco, R.L., and Teal, P. Antithrombotic and thrombolytic therapy, 8th edition: ACCP guidelines. Chest 133, 630S- 669S (2008). 4. Fernandex, J.S., Sadaniantz, B.T., and Sadaniantz, A. Review of antithrombotic agents used for acute coronary syndromes in renal patients. American Journal of Kidney Diseases 42, 446- 455 (2003). 5. Weitz, J.I., Hirsh, J., and Samama, M. New anticoagulant drugs. Chest 126, 265S-286S (2004). 6. Warkentin, T.E., and Greinacher, A. Heparin-induced thrombocytopenia: recognition, treatment, and prevention. Chest 126, 311S- 337S (2004). 7. Chan, Y.C., Valenti, D., Mansfield, A.O., and Stansby, G. Warfarin induced skin necrosis. British Journal of Surgery 87, 266- 272 (2002). 8. Bircher, B.J., Harr, T., Hohenstein, L., and Tsakiris, D.A. Hypersensitiity reactions to anticoagulant drugs: diagnosis and management options. Allergy 61, 1432- 1440 (2006). 9. Ryan, E.A., Mockros, L.F., Weisel, J.W., and Lorand, L. Structural origins of fibrin clot rheology. Biophysical Journal 77, 2813- 2826 (1999). 10. Mosesson, M., Siebenlist, K.R., and Meh, D.A. The structure and biological features of fibrinogen and fibrin. Annals of the New York Academy Science 936, 11- 30 (2001). 11. Ariens, R.A.S., Lai, T.S., Weisel, J.W., Greenberg, C.S., and Grant, P.J. Role of factor XIII in fibrin clot formation and effects of genetic polymorphisms. Blood 100, 743- 754 (2002). 12. Weisel, J.W. Structure of fibrin: impact on clot stability. Journal of Thrombosis and Haemostasis 5, 116- 124 (2007). 13. Weisel, J.W., and Medved, L. The structure and function of the αC domains of fibrinogen. Annals of the New York Academy Science 936, 312- 327 (2001). 14. Lorand, L. Sol Sherry lecture in thrombosis: research on clot stabilization provides clues for improving thrombolytic therapies. Arteriosclerosis, Thrombosis, and Vascular Biology 20, 2-9 (2000). 15. Finney, S., Seale, L., Sawyer, R.T., and Wallis, R.B. Tridegin, a new peptidic inhibitor of factor XIIIa, from the blood-sucking leech Haementeria ghilianii. Biochemical Journal 324, 797- 805 (1997). 16. Kostanova, E.A., Roxenfel’d, M.A., Revina, T.A., and Valueva, T.A. Protein inhibitors of fibrin stabilizing factor FXIII. Biology Bulletin 34, 230- 235 (2007). 17. Kogen, H., Kiho, T., Tago, K., Miyamoto, S., Fujioka, T., Otsuka, N., SuzukiKonagi, K., and Ogita, T.J. Alutacenoic acids A and B, rare naturally occurring cyclopropenone derivatives isolated from fungi: potent non-peptide factor XIIIa inhibitor. Journal American Chemical Society 122, 1842- 1843 (2000).  70  18. Freund, K.F., Doshi, K.P., Gaul, S.L., Claremon, D.A., Remy, D.C., Baldwin, J.J., Pitzenberger, S.M., and Stern, A.M. Transglutaminase inhibition by 2-[(2oxopropyl) thio]imidazolium derivatives: mechanism of factor XIIIa inactivation. Biochemistry 33, 10109- 10119 (1994). 19. Ryan, E.A., Mockros, L.F., Stern, A.M., and Lorand, L. Influence of a natural and a synthetic inhibitor of factor XIIIa on fibrin clot rheology. Biophysical Journal 77, 2827- 2836 (1999). 20. Marrano, C., de Macedo, P., Gagnon, P., Lapierre, D., Gravel, C., and Keillor, J.W. Synthesis and evaluation of novel dipeptide-bound 1,2,4-thiadizoles as irreversible inhibitors of guinea pig liver transglutaminase. Bioorganic & Medicinal Chemistry 9, 3231- 3241 (2001). 21. Standeven, K.F., Ariens, R.A.S., Whitaker, P., Ashcroft, A.E., Weisel, J.W., and Grant, P.J. The effect of dimethylbiguanide on thrombin activity, FXIII activation, fibrin polymerization, and fibrin clot formation. Diabetes 51, 189- 197 (2002). 22. Ikura, K., Otomo, C., Natsuka, S., Ichikawa, A., Wakamatsu, K., Ito, S., and Taoguchi, S. Inhibition of transglutaminase by synthetic tyrosine melanin. Bioscience, Biotechnology, and Biochemistry 66, 1412- 1414 (2002). 23. Zou, Y., Brooks, D.E., and Kizhakkedathu, J.N. A novel functional polymer with tunable LCST. Macromolecules 41, 5393-5405 (2008). 24. Rossi, N.A.A., Mustafa, I., Jackson, J.K., Burt, H.M., Scott, M.D., and Kizhakkedathu, J.N. In vitro chelating, cytoxocity, and blood compatibility of degradable poly(ethylene glycol)-based macromolecular iron chelators. Biomaterials 30, 638- 648 (2009). 25. Weisel, J.W., and Nagaswami, C. Computer modeling of fibrin polymerization kinetics correlated with electron microscope and turbidity observations: clot structure and assembly are kinetically controlled. Biophysical Journal 63, 111128 (1992). 26. Wolberg, A.S., Gabriel, D.A., and Hoffman, M. Analyzing fibrin clot structure using a microplate reader. Blood Coagulation and Fibrinolysis 13, 533- 539 (2002). 27. Standeven, K.F., Carter, A.M., Grant, P.J., Weisel, J.W., Chernysh, I., Masova, L., Lord, S.T., and Ariens, R.A.S. Functional analysis of fibrin γ-chain cross-linking by activated factor XIII: determination of a cross-linking pattern that maximizes clot stiffness. Blood 110, 902- 907 (2007). 28. Podolnikova, N.P., Yakubenko, V.P., Volkov, G.L., Plow, E.F., and Ugarova, T.P. Identification of a novel binding site for platelet integrins αIIbβ3 (GPIIb/IIIa) and α5β1 in the γC-domain of fibrinogen. Journal of Biological Chemistry 278, 32251- 32258 (2003). 29. Dardik, R., Shenkman, B., Tamarin, I., Eskaraev, R., Harsfalvi, J., Varon, D., and Inbal, A. Factor XIII mediates adhesion of platelets to endothelial cells through αVβ3 and glycoprotein IIb/IIIa integrins. Thrombosis Research 105, 317-323 (2002). 30. Gabriel, D.A., Muga, K., and Boothroyd, E.M. The effect of fibrin structure on fibrinolysis. The Journal of Biological Chemistry 267, 24259- 24263 (1992).  71  31. Weisel, J.W., Nagaswami, C., and Makowski, L. Twisting of fibrin fibers limits their radial growth. Proceedings of the National Academy of Sciences 84, 89918995 (1987). 32. Scheiner, T., Jirouskova, M., Nagaswami, C., Coller, B.S., and Weisel, J.W. A monoclonal antibody to the fibrinogen γ-chain alters fibrin clot structure and its properties by producing short, thin fibers arranged in bundles. Journal of Thrombosis and Hawmostasis 1, 2594- 2602 (2003). 33. Veklich, Y., Francis, C.W., White, J., and Weisel, J.W. Structural studies of fibrinolysis by electron microscopy. Blood 92, 4721- 4729 (1998). 34. Alcantar, N.A., Aydil, E.S., and Israelachvili, J.N. Polyethylene glycol-coated biocompatible surfaces. Journal of Biomedical Materials Research Part A 51, 343- 351 (2000). 35. Neugebauer, D., and Matyjaszewski, K. Copolymerization of N,NDimethylacrylamide with n-butyl acrylate via atom transfer radical polymerization. Macromolecules 36, 2598- 2603 (2003). 36. Collet, J.P., Shuman, H., Ledger, R.E., Lee, S., and Weisel, J.W. The elasticity of an individual fibrin fiber in a clot. Proceedings of the National Academy of Sciences of USA 102, 9133- 9137 (2005) 37. Liu, W., Jawerth, L.M., Sparks, E.A., Fakvo, M.R., Hantgan, R.R., Superfine, R., Lord, S.T., and Guthold, M. Fibrin fibers have extraordinary extensibility and elasticity. Science 313, 634 (2006). 38. Carr, M.E., and Gabriel, D.A. Dextran-induced changes in fibrin fiber size and density based on wavelength dependence of gel turbidity. Macromolecules 13, 1473- 1477 (1980). 39. Vadseth C., Souza, J.M., Thomson, L., Seagraves, A., Nagaswami, C., Scheiner, T., Torbe, J., Vilaire, G., Bennett, J.S., Murcian, J.C., Muzykantov, V., Penn, M.S., Hazen, S.L., Weisel, J.W., and Ischiropoulos, H. Pro-thrombotic state induced by post-translational modification of fibrinogen by reactive nitrogen species. The Journal of Biological Chemistry 279, 8820-8826 (2004).  72  3 A comparative study on the influence of soluble and grafted poly (N-isopropylacrylamide) on fibrin clot structure2 3.1 Introduction Surface induced thrombus generation considerably impact the performance of biomaterials used in blood contacting applications. Examples include catheters, blood storage bags, cardiovascular bypass machines, cardiovascular implants, biosensors and drug delivery vehicles. Thrombus formation is partly due to uncharacteristic adsorption of plasma proteins on the surface the devices, and partly due to subsequent platelet adhesion and activation [1;2;3]. Different approaches have been investigated to improve the blood compatibility of biomaterials which include surface modification using synthetic polymers [4;5] and immobilization of anticoagulant agents like heparin [6] with limited success. Heparin coated surfaces has shown significant improvements of blood compatibility in in vitro studies [7;8]. But the variation of bioactivity of heparin after its covalent immobilization and degradation of heparin are proved to be the cause of the inconsistency seen in in vivo studies [9;10]. Thus more effort is needed in the search for surface coatings to biomaterials exhibiting excellent hemocompatibility. Stable blood clot formation occurs through a series of steps. Under physiological conditions, fibrin network assembly begins when thrombin catalyzes the conversion of fibrinogen to fibrin by cleaving fibrinopeptides from the domain of the molecule. This initiates the end-to-end interaction between individual fibrin monomers to form doublestranded protofibrils. The protofibrils aggregate to form fiber and branch to form the 2  A version of this chapter will be submitted for publication. Lai, B.F.L., Zou, Y., Brooks, D.E., and Kizhakkedathu, J.N. A Comparative Study on the Influence of Soluble and Grafted Poly(N-Isopropylacrylamide) on Fibrin Clot Structure. 73  three-dimensional clot structure. The soft fibrin clot structure is then further stabilized by the formation of intermolecular Nε-(γ-glutaml) lysine covalent cross-links between fibrin monomers [11;12]. This process is catalyzed by the coagulation factor XIIIa, a cysteine protransglutaminase that becomes activated through combined actions of thrombin cleavage and Ca2+-mediated conformational change. The covalent cross-linking within the fibrin networks produces dramatic stiffening of the fibrin clot [13;14]. Furthermore, the interaction between the platelets and fibrinogen through platelet integrin GPIIb/IIIa and the γC-terminal domain of fibrinogen further tightens the overall clot structure by enhancing the platelet adhesion, platelet aggregation and fibrin clot retraction [15;16]. Thus the overall clot stability and resistance to enzyme-initiated fibrinolysis is depended on the fibrin polymerization process, stabilization of cross-links and the interaction of platelets with fibrin clot. A tighter fibrin structure compared to a looser structure and increase in γ-γ crosslinks can greatly increase a fibrin clot resistance to fibrinolysis. In this chapter, we investigated the influence of linear poly (N-isopropylacrylamide) (PNIPAm) on blood clot formation and its stability using a series of in vitro experiments. We have shown that PNIPAM directly affecting the normal overall fibrin clot structure and clot properties through unusual lateral aggregation of the protofibrils and reduced cross-linking between the fibrin monomers. We have also studied the behavior of surface grafted PNIPAM (PNIPAM brushes) as well as star shaped PNIPAM (Star-PNIPAM). Experiments are designed to understand the changes in the bioactivity of the PNIPAM when it is conformational-restricted and would give a clear indication whether such type of coatings can be used to develop antithrombotic surfaces.  74  3.2 Experimental 3.2.1  Materials  3.2.1.1 Chemical Soluble poly (N-isopropylacrylamide) (PNIPAm) and poly (N,N-dimethylacrylamide) (PDMA) were synthesized by atom transfer radical polymerization (ATRP).  Poly  (ethylene glycol) (PEG) of molecular weight 35,000 Da was bought from Fluka and used as such. The development of PNIPAM brushes on polystyrene microspheres [17] and star-PNIPAM on hyperbranched polyglycerol [18] was reported previously.  The  characteristics of the grafted surfaces and polymers are given in Figure 3.1. The soluble polymers were characterized by NMR and gel permeation chromatography. PNIPAM brushes were characterized by determining the molecular weight of the grafted chains, graft density and hydrodynamic thickness.  4-(2-Hydroxyethyl) piperazine-1-  ethanesulfonic acid sodium salt (HEPES), CaCl2 (99.99%) and NaCl2 (99%) were purchased from Aldrich (Ontario, Canada) of the highest purity. Distilled water used in all experiments was further purified using a Mili-Q Plus water purification system (Millipore Corp., Bedford, MA). (A)  PNIPAm  PDMA  75  PEG  (B)  (C) Polymer  Mn  PDI  Brush Graft Density  PDMA PEG Linear PNIPAm Star-PNIPAm 1 Star-PNIPAm 2 Star-PNIPAm 3 Brush-PNIPAm 1 Brush-PNIPAm 2 Brush-PNIPAm 3  6400 35,000 32,000 282,000 299,000 108,000  1.74 1.22 1.43 1.39 1.5 1.34  N/A N/A N/A N/A N/A N/A  796,700  1.26  0.105  837,600  1.28  0.133  126,300  1.22  0.015  Figure 3.1: Structure and characteristics of the polymers tested. (A) Structure of PNIPAm and the control polymers: PDMA (structural control) and PEG (biocompatible control). (B) A cartoon showing different architectures of the PNIPAm studied (linear PNIPAm, star-PNIPAm, and brush-PNIPAm). (C) Molecular weight and PDI values of the PNIPAm samples and control polymers.  3.2.1.2 Biological Whole blood from healthy donors was collected into 3.8% sodium citrated tube with a blood/anticoagulant ratio of 9:1. Platelet-poor plasma was prepared by centrifuging whole blood samples at 1500 X g for 20 min in Allegra X-22R Centrifuge (Beckman Coulter, Canada).  Human plasma proteins such as fibrinogen (> 80% of clottable  protein), plasminogen (≥ 2 units/mg protein), tissue plasminogen activator (≥ 500,000  76  IU/mg), and thrombin (≥ 2,000 NIH units/mg protein) were all purchased from Sigma (Ontario, Canada). Human plasma factor XIII (FXIII) and activated plasma factor XIII (FXIIIa) were purchased from Haematologic Technologies Inc. (VT, USA). All of the lyophilized plasma proteins were made into solution by HEPES-saline buffer (10 mM HEPES, 150 mM NaCl, pH 7.4). The reagents were kept at -80°C until use. 3.2.2  Methods  3.2.2.1 Measurement of clotting time: prothrombin time (PT) and activated partial thromboplastin time (APTT) For activated partial thromboplastin time (APTT) analysis and prothrombin time (PT) analysis PPP was used. Linear PNIPAm, and star-PNIPAM in HEPES buffer was added to PPP at a volume ratio (1:9) to obtain a final concentration of PNIPAM 1 mg/ml. HEPES-saline buffer, PEG and PDMA were used as control samples at a similar dilution. The time required for the formation of a clot was measured with the STart®4 coagulometer (Diagnostica Stago, France). The detailed experimental procedure was reported previously [19]. 3.2.2.2 Fibrin polymerization assay Human fibrinogen (3 mg/ml) and HEPES-buffered polymer solutions (1 mg/ml final concentration) were pre-incubated at 37°C for 10 min. Human fibrinogen in HEPESsaline buffer was also tested as a control for the analysis. The mixture was added to 96well microtiter plate. Fibrin polymerization was then initiated by the addition of 20 µl of 3 NIHU/ml human thrombin, 20 µl of 25 µg/ml activated plasma factor XIII (FXIIIa), and 20 µl of 40 mM CaCl2 (3 mM final concentration) to each well. Changes in optical density (OD) were monitored at 405 nm using a SpectraMax plate reader (Molecular Devices, USA). Each measurement was carried out in triplicate for 1 h at 37°C. 77  3.2.2.3 Analysis of fibrin clot structure by scanning electron microscopy Fibrin clots were prepared for scanning electron microscopy (SEM) imaging from mixtures of 360 µl of 3 mg/ml fibrinogen, 40 µl of 2 NIHU/ml thrombin, 20 µl of 30 µg/ml FXIIIa, 20 µl of 40 mM CaCl2 (1.67 mM final concentration), and 40 µl of 10  mg/ml linear PNIPAM, star-PNIPAM, PNIPAM brushes or control samples.  The  mixtures were thoroughly premixed and were incubated for 1 h at 37°C to allow the fibrinogen to form a clot. Specimens were first washed 3 times with 50 mM phosphate buffer (pH 7.4) to remove excessive salt and fixed with 2% glutaraldehyde. We followed a method reported by Weisel et al. [20] with a slight modification for the preparation of the clots for SEM imaging. Clots were frozen and fractured for 1 h, and critical pointdried with CO2 in a DCP-1 critical point drying apparatus (Denton Vacuum Co., USA). The specimens were mounted and sputter-coated with gold-palladium, and then examined with a Philips XL30 scanning electron microscope (Philips Electronics Co., USA). The electron micrographs were taken at magnifications between 5k and 10k. From the images, the size of each of the fibrin fiber strands was determined using Image J (NIH, USA), by drawing a line to bisect foreground fibers to obtain a pixel value and relating to pixel value obtained for the scale bar on the image. Fifteen different fibrin strands on each image were analyzed. At least two images were analyzed for each sample. The diameter of individual fibrin strands on the images was reported as an average for all fiber strands measured. 3.2.2.4 Analysis of cross-linking in fibrin clot by SDS-PAGE The cross-linking of fibrin by activated human plasma factor XIII (FXIIIa) was analyzed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE)  78  [21;22]. The fibrin clot was either formed from PPP or purified human fibrinogen (3 mg/ml). In the case of PPP, the polymer solutions in HEPES-saline buffer (40 µl) was mixed with plasma (360 µl) at 1:9 ratio and clotting was initiated by the addition of 20 µl of 1 NIHU/ml human thrombin and 20 µl of 0.2 M CaCl2 (10 mM final concentration). Final polymer concentration was maintained at 1 mg/ml.  In the case of purified  fibrinogen, we followed similar protocol except the addition of 20 µl of FXIII (30 µg/ml) along with 20 µl of thrombin (3NIHU/ml) and 20 µl of 40 mM CaCl2 (1.67 mM final concentration) for the initiation of clot. The samples were incubated at 37°C water bath for 1 h and a stable fibrin gel was formed. The fibrin gel was then washed with HEPES buffer and centrifuged at 2300 X g for 2 min to remove excess clotting solution and procedure was repeated 5 times. The fibrin gel was dissolved by the addition of a reducing loading buffer (9 M urea, 40 mM DTT, 0.1 M Tris-HCl, 2% SDS, 0.2 % bromophenol blue, and 20% glycerol, pH 6.8), followed by overnight incubation at 37°C. Pure fibrinogen and PPP without the addition of polymer samples were prepared in the same manner used as control samples. Equal volumes of the samples were loaded onto the gel. After the electrophoresis, the gel was stained with Coomassie brilliant blue (BioRad). Images of the SDS-PAGE gel were captured by Odyssey Infrared Imaging system (LI-COR® Biosciences, USA). Band intensity was reported as the integrated intensity value (sum of the intensity values for all pixels enclosed by a feature, multiplied by the area of the feature: count mm2) of each protein band. 3.2.2.5 Analysis of overall strength of whole blood clot Whole blood clot viscoelastic properties were analyzed in a Thromboelastograph® (TEG) Hemostasis System 5000 series analyzer (Haemoscope Corporation, USA).  79  Citrated human whole blood was first incubated with HEPES-buffered testing materials: linear PNIPAM, star-PNIPAM, PNIPAM brushes and control samples.  Final  concentrations of polymers were at 1 mg/ml. In the case of brush-PNIPAm, the amount of grafted PNIPAm on the surface of the polystyrene particles was used for calculating the concentration of PNIPAm used. 360 µl aliquots of the whole blood mixture were then added to TEG cup and 20 µl of 0.2 M CaCl2 (10 mM final concentration) was added to initiate the clotting process. The TEG analysis was allowed to a 2 h preset completion time or until the analysis was prematurely terminated by TEG system itself. The overall coagulation profile was recorded. The overall clot strength (maximum amplitude), clot elasticity (G`) and fibrinolysis at 30 min were determined. Both maximum amplitude and G` recorded are the direct representation of the dynamic properties between fibrin and platelet bonding via GP IIb/IIIa. 3.2.2.6 Statistical analysis Statistical analyses of data to include 95% confidence intervals, two-tailed Student’s t-tests were performed on the Microsoft Office 2003 Excel software. Due to the volume of data, only relevant comparisons were made. Only comparisons between data from linear PNIPAm to buffer control, data from star-PNIPAm to buffer control and data from brush-PNIPAm to buffer control were made.  3.3 Results 3.3.1  In vitro blood coagulation assays  The effect of linear PNIPAm, star-PNIPAm and the control samples, PDMA, PEG and buffer on coagulation cascade were investigated by conventional blood coagulation assays, activated partial thromboplastin time (APTT) and prothrombin time (PT). The  80  time taken for the platelet poor plasma to form a clot after the addition of APTT or PT reagents was recorded as a measure of time required for thrombin formation via either extrinsic or intrinsic pathways of the coagulation cascade.  Linear PNIPAm at  concentration 1 mg/ml does not have a significant effect on the coagulation cascade when compared to control samples, shown in Figure 3.2. APTT PT  35  30  Time (seconds)  25  20  15  10  5  0  Buffer  1 mg/ml PEG  1 mg/ml PDMA  1 mg/ml Linear PNIPAm  1 mg/ml StarPNIPAm 1  1 mg/ml StarPNIPAm 2  Polymer Samples  Figure 3.2: Coagulation cascade analysis by APTT and PT assays. APTT: Platelet poor plasma was incubated with APTT reagent (actin FSL), CaCl2 (25 mM), and soluble polymer samples (1 mg/ml). The plasma hemostasis was measured with at 37°C by ST4 hemostasis analyzer. PT: Platelet poor plasma was incubated with PT reagent (tissue factor), and soluble polymer samples (1 mg/ml) and the plasma hemostasis was measured with at 37°C by ST4 hemostasis analyzer.  Similar APTT and PT values were seen for star-PNIPAm. APTT and PT measurements were not carried out for brush-PNIPAm. Our results show that the changes in both APTT and PT values for different PNIPAm samples do not appear to affect any step in the  81  clotting cascade that would lead to an extended delay in thrombin formation and subsequent fibrinogen conversion to fibrin monomers. 3.3.2  Kinetics of fibrin polymerization  To investigate the effects of PNIPAm samples on fibrin clot polymerization process, a turbidity analysis of fibrin formation was performed.  Analysis of the change in  turbidity has been well documented as a functional measurement of the kinetics of fibrin polymerization process. There are three phases in a turbidity curve: (A) lag time, (B) rate of change in turbidity (the steepest part of the curve), and (C) final turbidity (Figure 3.3). The lag time is a measure of the rate of protofibril formation, the rate of change in optical density is referred to the rate of protofibrils lateral aggregation, and the final turbidity is an indication of the number and size of fiber formed. This analysis of pure fibrinogen clot polymerization was done over 1 h time period. As shown in Figure 3.3, both the rate of protofibrils lateral aggregation (B) and final turbidity (C) exhibited no significant difference in fibrin polymerization in presence of linear PNIPAm compared to buffer or polymer controls. A slightly higher final turbidity was observed in presence of control PEG compared to buffer control. However, in the lag phase (A) of the turbidity curve, a significant dip in turbidity value was seen for the linear PNIPAm sample. This change in turbidity directly correlates to two parameters of initial fibrin clot polymerization process: the rate of which fibrinopeptide A is being cleaved off by thrombin and the rate of initial fibrin monomer association. This decrease in initial part of the turbidity curve of PNIPAm sample may suggest that PNIPAm may be affecting the number of fibrin monomers being added to the protofibril.  82  0.11  C  PEG  0.09  PDMA  Optical Density (Abs 405nm)  0.07  B PNIPAm Buffer  0.05  0.03  0.01  A 0  10  20  30  40  50  60  Time (Minutes)  -0.01  Figure 3.3: Turbidity analysis of fibrin-formation in presence of soluble polymers. Fibrinogen (3 mg/ml) was clotted with 3 NIHU/ml thrombin, 30 µg/ml FXIIIa, and 3 mM CaCl2 in presence of soluble polymer samples (1 mg/ml). A: Lag Time; B: lateral aggregation or protofibrils; C: Final Optical density.  3.3.3  Analysis of factor XIIIa-induced cross-linking of fibrin in the presence of PNIPAm  Examination of fibrin clots by SDS-PAGE analysis showed obvious differences in the levels of cross-linking in fibrin clots formed in platelet-poor plasma (PPP) with the incubation of linear PNIPAm and star-PNIPAm compared to brush-PNIPAm, buffer and polymer-controls (Figure 3.4A).  It must be noted that the amount of isolated clot  (washed) from the linear-PNIPAm and star-PNIPAm was smaller than that of control samples. In a control experiment (lane 3), when thrombin and FXIII was added to the fibrinogen, the γ band disappeared and γ-γ dimer cross-links appeared. In addition, high  83  molecular weight α-polymer bands were also generated. The appearance of γ-γ dimer and high molecular weight α-polymer bands in the case of control samples (buffer, PEG and PDMA), indicating that fibrinogen in PPP was cleaved by thrombin and was able to form high degree of cross-links induced by FXIIIa in the fibrin clot. These control samples were run in lanes 4, 5 and 6, and were at 1:10 dilution before loading into the gel. Brush-PNIPAm samples (diluted 1:10) (lane 7-9) also showed no significant change in the fibrin cross-linking of either γ-γ dimer or α-polymer compared to the control samples. However, the addition of linear PNIPAm, and star-PNIPAm samples produced significant change to the degree of cross-linking in the PPP fibrin clots; the intensity of both the γ-γ dimer band and the α-polymer bands were much lower than that of the control samples (lane 10-13). The integrated intensity analysis of both γ-γ dimer and α-polymer bands confirmed the significant differences in the degree of cross-links seen in the fibrin clot of control samples and the linear PNIPAm and star-PNIPAm samples were significant. The γ-γ dimer band of linear PNIPAm and star-PNIPAm samples have at least a 2-fold decrease in intensity compared to that of control samples (Figure 3.4B). Similarly, up to a 10-fold decrease in α-polymer cross-links intensity value was seen for both linear PNIPAm and star-PNIPAm compared to that of control samples. Figure 3.4: Evaluation of cross-linking in PPP clot by reducing SDS-PAGE. Development of cross-links in reaction mixtures contained citrated PPP, thrombin (1 NIHU/ml), CaCl2 (10 mM), and testing samples (1 mg/ml) for 2 h at 37°C. (A) 10% gel of samples formed from platelet poor plasma. (B) Integrated intensity comparison between γ-γ dimer crosslinks and α-polymer bands formed in fibrin clots incubated with the different samples.  84  (A)  Lane No.  Sample  1 2 3 4 5 6  Protein Size Marker Pure Fibrinogen Thrombin + FXIII 1:10 diluted Buffer 1:10 diluted PEG 1:10 diluted PDMA 1: 10 diluted Brush-PNIPAm 1 1:10 diluted Brush-PNIPAm 2 1: 10 diluted Brush-PNIPAm 3 Linear PNIPAm Star-PNIPAm 1 Star-PNIPAm 2 Star-PNIPAm 3  7 8 9 10 11 12 13  85  (B) 120  gamma crosslinks alpha-polymer  Integrated Intensity (Pixel)  100  80  60  ` 40  20  0 Buffer  PEG  PDMA  BrushPNIPAm 1  BrushPNIPAm 2  BrushPNIPAm-3  Linear PNIPAm  StarPNIPAm 1  StarPNIPAm 2  StarPNIPAm 3  Polymer Samples  3.3.4  Fibrin clot structure studied by scanning electron microscopy  The effect of various PNIPAm polymers on fibrin clot formation, in particular the morphological difference of the fibrin clots, was investigated using SEM. As shown in Figure 3.5A, the SEM micrographs of fibrin clot formed in (i) buffer control and (ii) control PEG sample displayed homogeneous clot architecture. The fibrin clots obtained from these samples had a highly branching fibrin fiber network with uniform pore sizes. The average diameters of individual fibrin strand were 122 ± 12 nm and 130 ± 21 nm for buffer and PEG control respectively (Figure 3.5B). However, in the presence of linear PNIPAm (iii), the clot formed has a different overall spatial organization in the fibrin fiber network.  A more heterogeneous architecture with compact areas of fibers  86  alternating with looser areas was also seen produced a more disorganized appearance with varying pore sizes. In addition, the clot formed in presence of PNIPAM contained much thinner fibers (88 ± 20 nm) compared to the control samples (Figure 3.5B). The star-PNIPAm has much disorganized clot structure compared to linear PNIPAm and control samples with the size of individual fiber 82 ± 12 nm. Although the brushPNIPAm (v) also appeared to have some influence in the clot architecture, this difference is less significant than linear and star-PNIPAM samples and the size of the fiber is larger (97 ± 13 nm) compared to the other PNIPAm samples.  Figure 3.5: SEM analysis of fibrin clot structure. Purified human fibrinogen (3 mg/ml) was incubated with specific synthetic testing materials (1 mg/ml), human thrombin (3 NIHU/ml), and CaCl2 (1.67 mM) for 1 h at 37°C. The samples then underwent fixing, cyro-freezing, critical point drying and gold sputter coating. (A) SEM images of fibrin clot formed in (i) Buffer, (ii) PEG, (iii) linear PNIPAm, (iv) star-PNIPAm 1, and (v) brush-PNIPAm 1. (B) A Comparison of diameter of individual fiber strands in fibrin clot formed in presence of different polymer samples.  87  (A)  (i) Buffer control  (ii) PEG samples  (iii) Linear PNIPAm sample  (iv) Star-PNIPAm sample  (v) Brush PNIPAm sample  88  (B) 160  140  120  Fiber Size (nm)  100  80  60  40  20  0 Control  PEG  Linear PNIPAm  Star-PNIPAm 1  Brush-PNIPAm 1  Polymer Sample  3.3.5  Blood clot analyses by thromboelastograph  The rheological properties were analyzed and compared using thromboelastograph when whole blood was allowed to clot in the presence of the various PNIPAm samples and control samples. The overall strength (MA), rigidity (G`) and fibrinolysis at 30 minutes for whole blood clot formed over a 2 h time period were investigated. Compared to controls, different PNIPAm samples have different influence on the overall clot strength and clot stability as shown in the recorded thromboelastograph trace (Figure 3.6). Figure 3.6: Evaluation of overall clot properties measured by TEG. Citrated whole blood was re-calcified with 10 mM CaCl2 and was allowed to clot with linear PNIPAm, star-PNIPAM, brush-PNIPAM, and controls for 2 h at 37°C in the Thromboelastograph (TEG) analyzer. The final concentration of polymer samples is 1 mg/ml. TEG traces for different samples are shown.  89  (i)  (ii)  With the addition of linear PNIPAm and star-PNIPAm, the whole blood clot formed were not stable enough to maintain in a state that can be measured by TEG system compared to the control samples. Consistent results were obtained with whole blood donated from eight different donors.  90  Quantitative assessments of the overall clot strength (Figure 3.7A) and rigidity (Figure 3.7B) calculated from TEG traces show that at least a 10-fold decrease in clot strength (MA) and rigidity (G`) for whole blood clots formed with these two types of PNIPAm samples compared to values for the control samples (p < 0.000005). In the case of brush-PNIPAm, however, no significant influence on either clot strength or rigidity was detected by the thromboelastograph.  Even when the concentration of brush-  PNIPAm was increased to 5 to 10 fold compared to the 1 mg/ml linear PNIPAm and 1 mg/ml star-PNIPAm samples, the overall blood clot strength and blood clot elastic modulus for brush-PNIPAm were similar to those of the control blood clots. Taken together, these TEG results demonstrate that the architecture of the PNIPAm samples is important in modulating the fibrin clot overall strength and elasticity. Furthermore, the percent of clot lysis was measured after 30 minutes. Figure 3.8 shows the percent of lysis of the clot when whole blood was incubated with different samples. In presence of linear-PNIPAm and star-PNIPAm, the blood clot lysed much faster than the control samples. However, the brush-PNIPAM did not enhance the lysis rate of the clot produced compared to the control samples.  91  (A) 60  Maximum Amplitude (MA)  50  40  30  20  10  0 Buffer Control  PEG (1 mg/ml) PDMA (1 mg/ml) Linear PNIPAM (1 mg/ml)  Star-PNIPAm 1 Star-PNIPAM 2 Star-PNIPAM 3 (1 mg/ml) (1 mg/ml) (1 mg/ml)  Brush-PNIPAm 3 (1 mg/ml)  Polymer Samples  (B) 7  6  G` (kdynes/cm2)  5  4  3  2  1  0 Buffer Control  PEG (1 mg/ml)  PDMA (1 mg/ml)  Linear PNIPAM Star-PNIPAm 1 Star-PNIPAM 2 Star-PNIPAM 3 Brush-PNIPAm (1 mg/ml) (1 mg/ml) (1 mg/ml) (1 mg/ml) 3 (1 mg/ml)  Polymer Samples  Figure 3.7: Evaluation of overall clot strength, and clot elasticity measured by TEG. Whole blood clots were formed in TEG at 37°C from solutions of citrated whole blood, CaCl2 (10 mM), and were incubated with buffer control, control polymers (PEG and PDMA), linear PNIPAm and star- and brush- form of PNIPAm. Maximum Amplitude (A), and Shear elasticity (B) of the whole blood clot was measured.  92  100  Percent of Fibrinolysis (% LY30)  90  80  70  60  50  40  30  20  10  0 Buffer Control  PEG (1 mg/ml)  PDMA (1 mg/ml)  Linear PNIPAM Star-PNIPAm 1 Star-PNIPAM 2 Star-PNIPAM 3 Brush-PNIPAm (1 mg/ml) (1 mg/ml) (1 mg/ml) (1 mg/ml) 3 (1 mg/ml)  Polymer Samples  Figure 3.8: Fibrinolysis recorded by TEG. Clots were formed in TEG at 37°C from solutions of citrated whole blood, CaCl2 (10 mM), and were incubated with buffer control, control polymers (PEG, and PDMA), linear PNIPAm and star- and brush- form of PNIPAm.  3.4 Discussion In the present study, we report for the first time that soluble linear PNIPAm can modulate the overall structure of fibrin clot, as well as its mechanical properties such as overall strength and elasticity by affecting the fibrin polymerization process. Furthermore, we show that this effect can vary when different architectures of PNIPAm (linear, star and brush from) are used. The linear and star-PNIPAm represent the soluble forms of the PNIPAm, whereas the brush-PNIPAm represents a surface coating on an implant or surface chemically modified with linear PNIPAm. The data revealed that with a closer resemblance to linear architecture, star-PNIPAm also has a significant effect in altering the initial polymerization properties of fibrin fibers and lowering the  93  strengthening cross-links formed between fibrin molecules by factor XIIIa. As a result, the blood clot formed with the star- PNIPAm has a dramatic change in the clot overall mechanical properties unlike the brush PNIPAM. The results are compared with two proven biocompatible polymers PEG and PDMA [23;24]. Results from the conventional blood coagulation assays show that both linear and star-PNIPAm do not have a specific target in the coagulation cascade to inhibit thrombus formation. Corresponding values for normal re-calcified plasma is reported to be 31 s for APTT and 10.3 s for PT assay. Slightly higher values were seen in both assays, when plasma was clotted with either linear or star-PNIPAm. However, both APTT and PT assays are only measuring the rate of thrombus formation through initiating the coagulation cascade. The significant roles played by platelets and plasma proteins such as factor XIII in forming a stable and rigid fibrin clot cannot be measured with these conventional coagulation assays [13;14].  They therefore by no means give any  information of the overall structure and the stability of the blood clot. We demonstrate that the effect of PNIPAm on the fibrin clot structure is directly correlated with the initial process of the fibrin monomer assembly.  The turbidity  measurement is directly related measure of the kinetics of fibrin polymerization process [20]. Using the change in turbidity analysis of fibrin clot formation, we show that PNIPAm does not affect the rate of lateral aggregation of protofibrils or the final turbidity value seen for the individual fiber strand. Instead, PNIPAm exerts its effect on the rate of protofibril formation. From the initial phase of the turbidity curve (Figure 3.3), we observed a decrease in turbidity value. Both the rate of fibrinopeptide A cleavage and the rate of initial fibrin monomer association can directly affect this portion of the curve.  94  Based on the on data presented, the presence of PNIPAm slows the protofibril formation, however, it remains unclear whether PNIPAm is affecting the formation of fibrin monomers or inhibiting the growth of protofibrils before its lateral aggregation. One speculation based on the conventional coagulation assay is that the bioactivity of thrombin is not altered in the presence of PNIPAm and thus fibrinopeptides A would still be cleaved by thrombin and fibrinogen is getting converted to fibrin monomers. However, instead of undergoing half staggered side-to-side interaction with other fibrin monomers, the fibrin monomers are quickly “capped” in presence of PNIPAm chains. This leads to a decrease of double stranded protofibrils with the minimal length required for lateral aggregation, as reflected by the negative turbidity value seen in the initial phase of the turbidity curve. We observed an alteration to the overall spatial organization of fibrin network in presence of linear PNIPAm and star-PNIPAm compared to the control samples (buffer and PEG polymer) from SEM analysis.  There are two significant morphological  differences between the control and PNIPAm samples; the arrangement of the fibers in the fibrin clot and size of the fibers. Fibrin clots formed in presence of linear and starPNIPAm resulted in a more disorganized appearance; instead of forming a highly branching network of individual fibrin fibers, the presence of soluble PNIPAm resulted in bundles with obvious truncated ends. The clot formed therefore is more porous with alternating areas of compact fiber bundles to areas of fewer fibers. Similar structural alteration to fibrin clot was observed from a novel synthetic polymer poly (N-[(2,2dimethyl-1, 3dioxolane)methyl]acryl amide) (PDMDOMA) reported by our group previously [25]. The second morphological difference is the size of the individual fibers  95  formed in presence of PNIPAm and star-PNIPAm compared to that of the control samples. Thinner fiber is generated in presence of PNIPAM with different architectures. This, however, is contrary to our observations from the turbidity analysis. From the turbidity analysis, we expect similar fiber diameters for fiber strands formed with the linear PNIPAm sample as those of the control buffer. But turbidity is related to fiber diameter only if the fibers are uniformly distributed in the clot, as this is not the case as visualized from SEM micrographs. Therefore, the SEM analysis further support the previous speculation that the “capping” of fibrin monomer by PNIPAm produced lower amount of minimal length of protofibrils required for normal lateral aggregation and thus the overall structure of the fibrin clot is affected. As a result, the clot formed in presence of PNIPAm exhibit shorter individual fiber strands with higher affinity to form bundles of fibers. In this case, large bundle of fibers generated in these clots could account for the increase in turbidity for linear PNIPAm sample compared to buffer control. Similar structural change to the fibrin clot is seen with brush-PNIPAm; however, its effect is not as strong as both linear PNIPAm and star-PNIPAm. This may be due to the fact that brush-PNIPAm is conformationally restricted and its interaction with fibrin monomers may be restricted compared to soluble PNIPAm samples. From the SDS-PAGE analysis, we observed a significant decrease in intensity of both γ-γ dimer and α-polymer cross-links from fibrin clots formed with linear PNIPAm and star-PNIPAm. This change in FXIIIa-mediated cross-linking would make the clot less stable, as both γ-γ dimer and α-polymer are responsible for strengthening the overall structure of fibrin clot. There has been evidence to suggest that the degree of stiffening produced by cross-linking may be dependent on network structure [21;26].  96  This  disorganized structure may be the reason for the lower level of cross-linking seen in clots formed in presence of PNIPAm and star-PNIPAm. As for clots formed with brushPNIPAm samples, there is no difference between the γ-γ dimer and α-polymer cross-links generated compared to both buffer and polymer controls. Although there is alteration to the overall structure of fibrin clot formed with brush-PNIPAm, it does not significantly affect the FXIIIa catalyzed cross-linking process between γ chains and α chains of adjacent or neighboring fibrin molecules. Thus we did not observe as significant change in the fibrin cross-links formed in presence of brush-PNIPAm compared to other PNIPAm samples. Using the TEG analyzer, we were able to study the change in mechanical properties of whole blood fibrin clot − both overall clot strength and clot elastic modulus. A significant decrease in both strength and elasticity were recorded from whole blood clots formed in presence of linear PNIPAm and star-PNIPAm. This further supports the erratic overall clot structure observed from SEM studies and the lower level of fibrin monomer cross-linking observed by SDS-PAGE analysis. Moreover, because of the insufficient length of the protofibrils produced in presence of PNIPAm, shorter fiber strands formed and the higher likelihood of fibers forming bundles may have an effect on the binding site of platelet integrins αIIbβ3 and α5β1 for fibrinogen γC-domain [15;16]. This may also support the poor mechanical properties of the whole blood clot formed in presence of linear PINPAm and star-PNIPAm compared to the controls.  Furthermore, we have  shown that the change in clot strength and clot elasticity produced in presence of PNIPAm samples is directly dependent on the architecture of PNIPAm used.  The  mechanical properties of the clot did not change when the clot was formed in presence of  97  brush-PNIPAm as measured by TEG analysis. This is pointing to the fact that although brush-PNIPAm is affecting the overall structure of fibrin clot (evident from SEM analysis and SDS-PAGE), it does not translate to a change in the overall clot strength and clot elasticity.  Furthermore, we have shown that there is also an increase in clot lysis  recorded for whole blood clot formed in the presence of linear PNIPAm and starPNIPAm. This further suggests that the poor mechanical properties of the clot along with its heterogeneous structure increase the rate of clot lysis. As a result these whole blood clots are more likely to degrade overtime. The lack of significant difference in fibrin clot structure and mechanical properties formed in presence of brush-PNIPAm can be correlated to the structure of the grafted PNIPAm. The effect of the grafted PNIPAm is not pronounced as the soluble form. But the change in the clot structure based on the SEM analysis indicates that the brush-form is influencing the clot formation. We anticipate that when the graft density of the chains on the surface is optimal, it may interact with the fibrin monomers similar to the soluble polymers. 3.4.1  Conclusions  In this study, we have identified a novel biological activity of PNIPAm in disrupting the normal fibrin polymerization in the blood clot formation. Through a series of analysis, we have shown that linear PNIPAm influences the initial protofibril formation in the fibrin clot formation. This leads to a decrease in length and size of fibrin fiber. In addition, we have shown that the change in fibrin clot structure influenced FXIIIa mediated cross-linking; the overall mechanical strength and elasticity of the blood clot were reduced. Furthermore, we proved that the biological activity of activity of PNIPAm  98  is dependent on architecture of the PNIPAm used, i.e. linear, star and brush form. We have shown that star-PNIPAm has a higher influence on the fibrin clot overall structure and overall mechanical properties whereas the brush-PNIPAm only having a minimal effect on the clot structure and no direct influence to the clot overall strength or elasticity. The poor mechanical properties of the clot and heterogeneous nature of the clot resulted in increased clot lysis.  99  3.5 References 1. Gorbet, M.B., and Sefton, M. Biomaterial-associated thrombosis: roles of coagulation factors, complement, platelets and leukocytes. Biomaterials 25, 5681- 5703 (2004). 2. Courtney, J.M., Sundaram, S., Matata, B.M., Gaylor, J.D.S., and Forbes, C.D. Biomaterials in cardiopulmonary bypass. Perfusion 9, 3-10 (1994). 3. Courtney, J.M. and Forbes, C.D. Thrombosis on foreign surfaces. British Medical Bulletin 50, 966- 981 (1994). 4. Uyama, Y., Kato, K., and Ikada, Y. Surface modification of polymers by grafting. Advances in Polymer Science 137, 1- 39 (1997). 5. Jo, S. and Park, K. Surface modification using silanated poly(ethylene glycol)s. Biomaterials 21, 605- 616 (2000). 6. Lin, W.C., Liu, T.Y., and Yang, M.C. Hemocompatibility of polyacrylonitrile diaslysis membrane immobilized with chitosan and heparin conjugate. Biomaterials 25, 1947- 1957 (2004). 7. Tsai, C.C., Chang, Y., Sung, H.W., Hsu, J.C., and Chen, C.N. Effects of heparin immobilization of the surface characteristics of a biological tissue fixed with a naturally occurring crosslinking agent (genipin): an in vitro study. Biomaterials 22, 523- 533 (2001). 8. Chen, H., Chen, Y., Sheardown, H., and Brook, M.A. Immobilization of heparin on a silicone surface through a heterobifunctional PEG spacer. Biomaterials 26, 7418- 7424 (2005). 9. Gorman, R.C., Ziats, N.P., Rao, A.K., Gikakis, N., Sun, L., Khan. M.M.H., Stenach, N., Sapatnekar, S., Chouhan, V., Gorman, J.H., Niewiarowski, S., Colman, R.W., Anderson, J.M., and Edmunds, L.H. Surface-bound heparin fails to reduce thrombin formation during clinical cardiopulmonary bypass. Journal of Thoracic and Cardiovascular Surgery 111, 1- 12 (1996). 10. Mangoush, O., Purkayastha, S., Haj-Yahia, S., Kinross, J., Hayward, M., Bartolozzi, F., Darzi, A., Athanasiou, T. Hepari-bonded circuits versus nonheparin-bonded circuits: an evaluation of their clinical outcomes. European Journal of Cardio-Thoracic Surgery 31, 1058- 1069 (2007). 11. Lorand, L. Factor XIII: structure, activation, and interactions with fibrinogen and fibrin. Annals of New York Academy of Sciences 936, 291- 311 (2001). 12. Mosesson, M.W., Siebenlist, K.R., Amrani, D.L., and DiOprio, J.P. Identification of covalently linked trimeric and tetrameric D domains in crosslinked fibrin. Proceedings of the National Academy of Sciences of USA 86, 1113- 1117 (1989). 13. Collet, J.P., Shuman, H., Ledger, R.E., Lee, S., and Weisel, J.W. The elasticity of an individual fibrin fiber in a clot. Proceedings of the National Academy of Sciences of USA 102, 9133- 9137 (2005). 14. Liu, W., Jawerth, L.M., Sparks, E.A., Falvo, M.R., Hantgan, R.R., Superfine, R., Lord, S.T., and Guthold, M. Fibrin fibers have extraordinary extensibility and elasticity. Science 313, 634 (2006). 15. Podolnikova, N.P., Yakubenko, V.P., Volkov, G.L., Plow, E.F., and Ugarova, T.P. Identification of a novel binding site for platelet integrins αIIbβ3 (GPIIb/IIIa) and  100  α5β1 in the γC-domain of fibrinogen. Journal of Biological Chemistry 278, 32251- 32258 (2003). 16. Dardik, R., Shenkman, B., Tamarin, I., Eskaraev, R., Harsfalvi, J., Varon, D., and Inbal, A. Factor XIII mediates adhesion of platelets to endothelial cells through αVβ3 and glycoprotein IIb/IIIa integrins. Thrombosis Research 105, 317-323 (2002). 17. Kizhakkedathu, J.N., Norris-Jones, R., and Brooks, D.E. Synthesis of welldefined environmentally responsive polymer brushes by aqueous ATRP. Macromolecules 37, 734- 743 (2004). 18. Ranganathan, K., Deng, R., Kainthan, R.K., Wu, C., Brooks, D.E., and Kizhakkedathu, J.N. Synthesis of thermoresponsive mixed arm star polymers by combination of RAFT and ATRP from a multifunctional core and its selfassembly in water. Macromolecules 41, 4226- 4234 (2008). 19. Rossi, N.A.A., Mustafa, I., Jackson, J.K., Burt, H.M., Scott, M.D., and Kizhakkedathu, J.N. In vitro chelating, cytoxocity, and blood compatibility of degradable poly(ethylene glycol)-based macromolecular iron chelators. Biomaterials 30, 638- 648 (2009). 20. Weisel, J.W., and Nagaswami, C. Computer modeling of fibrin polymerization kinetics correlated with electron microscope and turbidity observations: clot structure and assembly are kinetically controlled. Biophysical Journal 63, 111128 (1992). 21. Ryan, E.A., Mockros, L.F., Stern, A.M., and Lorand, L. Influence of a natural and a synthetic inhibitor of factor XIIIa on fibrin clot rheology. Biophysical Journal 77, 2827- 2836 (1999). 22. Standeven, K.F., Ariens, R.A.S., Whitaker, P., Ashcroft, A.E., Weisel, J.W., and Grant, P.J. The effect of dimethylbiguanide on thrombin activity, FXIII activation, fibrin polymerization, and fibrin clot formation. Diabetes 51, 189- 197 (2002). 23. Alcantar, N.A., Aydil, E.S., and Israelachvili, J.N. Polyethylene glycol-coated biocompatible surfaces. Journal of Biomedical Materials Research Part A 51, 343- 351 (2000). 24. Neugebauer, D., and Matyjaszewski, K. Copolymerization of N,NDimethylacrylamide with n-butyl acrylate via atom transfer radical polymerization. Macromolecules 36, 2598- 2603 (2003). 25. Lai, B.F.L., Zou, Y., Brooks, D.E., and Kizhakkedathu, J.N. Journal of Biologcial Chemistry (To be submitted). 26. Ryan, E.A., Mockros, L.F., Weisel, J.W., and Lorand, L. Structural origins of fibrin clot rheology. Biophysical Journal 77, 2813- 2826 (1999).  101  4 Summary and future directions 4.1 Summary The development of novel antithrombotic agents free of complications is a challenging yet burgeoning field of study [1;2;3]. One promising approach is to augment the degradation of blood clot by enhancing the fibrinolysis before the blood clot is strengthened. The basis of this approach is to alter the overall fibrin clot structure which will make the clot less rigid and may become more susceptible to fibrinolysis. In this study, we have revealed a novel biological activity of two synthetic polymers. From the results presented, we confirm that the soluble forms of poly-N-[(2,2-dimethyl1,2-dioxolane)methyl]acrylamide  (PDMDOMA)  and  poly(N-isopropylacrylamide)  (PNIPAm) have significant influence in the physiological fibrin polymerization process during blood clot formation. This greatly alters the normal fibrin clot structure and translates to a drastic change in fibrin clot strength and fibrin clot elasticity. PDMDOMA influences the lateral aggregation of protofibrils as detected by turbidity measurements, and scanning electron microscopy analysis. As a result of enhanced lateral protofibril aggregation, thinner individual fibers with truncated ends are formed. In addition, the individual fibers in fibrin clots formed in presence of PDMDOMA have higher affinity for each other and aggregate together as a bundle. The bundling effect leads  to  porous  heterogeneous  appearance  of  the  overall  clot  structure.  Thromboelastograph analysis shows decreased clot strength, elasticity and enhanced fibrinolysis of the whole blood clot possibly due to the decrease in cross-linking of the individual fibers and heterogeneous clot structure.  SDS-PAGE analysis confirms a  decrease in the fibrin cross-linking in presence of PDMDOMA.  102  The antithrombotic activity of PDMDOMA is dose dependent and is not modulated by the size of the individual polymer.  Furthermore, this antithrombotic effect of  PDMDOMA appears to be mediated by the amide linked dioxolane pendent groups in the polymers. Through acid cleavage of the dioxolane group, the partially de-protected PDMDOMA-PDHPA retains some of the bioactivity and however, completely deprotected PDHPA (non dioxolane groups) does not show the similar effect in altering fibrin clot structure. The exact mechanism of effect of PDMDOMA on fibrin clot polymerization is not confirmed but our results (APTT and PT) show that it is not a biomolecular inhibition targeting at the coagulation cascade.  Based on the current  evidences, PDMDOMA interfere with the fibrin polymerization and assembly of fibrin clot structure. The decreased mechanical stability of the fibrin clot structure formed in presence of PDMODMA enhanced fibrinolysis of the fibrin clot by endogenous fibrinolysis agents.  Thus we anticipate PDMDOMA having applications as a new  antithrombotic agent and in the development of novel antithrombotic surfaces. In the second part of the thesis, we investigated the antithrombotic property of a different synthetic polymer, PNIPAM and compared the effect of different polymer architectures.  We also tried to address the changes in antithrombotic property of  PNIPAm when it is attached to solid surface.  The activity of linear PINIPAm is  compared to that of a star PNIPAm and a brush PNIPAm (surface grafted PNIPAm). To the best of our knowledge, this study represents the first investigation reveals the alteration of fibrin clot structure and clot strength by PNIPAm. Both linear- and star-PNIPAm show similar characteristics as an effective antithrombotic agent. Similar to PDMDOMA, linear PNIPAm does not have a direct  103  inhibitory effect at the coagulation cascade. SEM study reveals that thinner individual fiber strands formed in presence of PNIPAM have higher tendency to bundle and produce highly heterogeneous clot structure. This structural change leads to a decrease in the cross-linking of fibrin monomers, γ-γ dimers and α-polymers, as evident from SDSPAGE analysis. Furthermore, TEG study confirms a decreased mechanical stability of the whole blood clot with enhanced fibrinolysis possibly due to the changes in the clot structure. Our results also show that the activity of PNIPAm was significantly reduced when it is grafted to a surface, although we observe slightly disorganized clot structure in presence of grafted polymers. The conformational restriction of grafted PNIPAm may be preventing its interaction with fibrin monomers and we need to optimize the both graft density and molecular weight of PNIPAm on the surface. Our results confirms that the architecture of the PNIPAm have a profound effect on their biological activity and these finding has important implications in design of novel antithrombotic surfaces as conventional modifications of surfaces such heparin coating fail to eliminate fibrin clot formation on surfaces. It is interesting to note that although both linear PDMDOMA and linear PNIPAm affect the fibrin polymerization process; both apparently exert their biological activity at different stages of the fibrin polymerization process. From the turbidity analysis we confirmed that linear PDMDOMA limits the level of lateral aggregation between protofibrils and linear PNIPAm affects at an earlier stage by affecting the number of fibrin monomers being added to the protofibril. This is summarized in Figure 4.1.  104  Figure 4.1: Summary diagram. Normal fibrinogen polymerization is initiated by thrombin cleavage of the FPA/FPB and cross-links between protofibrils are established by FXIIIa via γ-glutamyl-ε-lysine amide of the fibrin molecules (1). However, fibrin clot formation is altered by PDMDOMA/PNIPAm when the novel synthetic material is involved with the polymerization process. PDMDOMA/PNIPAm may interact with the newly formed protofibrils (2). This affects the overall network by altering the binding affinity of the new amino-terminal end of the FPA/FPB to their respective binding site. Furthermore this affects the cross-link formed between the fibrin molecules (3). As a result, the fibrin clot is much more prone to lysis when plasminogen is activated (4).  105  4.2 Future work One area that needs further examination is how the biological activity of linear PDMDOMA changes when it is presented in different architectures, for example as a star-PDMDOMA and brush-PDMDOMA. From the PNIPAm study, one would expect a similar effect but the graft density and molecular weight of PDMDOMA on the surface needs to be optimized. In addition, we need to optimize the assay for evaluating the antithrombotic activity of surface grafted polymers. With the brush-PDMDOMA and brush-PNIPAm, we can also evaluate the amount and types protein adsorbed onto the surfaces which can possibility address the difference in bioactivity of grafted polymers. This would also demonstrate the importance of designing a polymer brush coated surface with reduced or even eliminate the surface induced thrombus formation. In addition to the studies carried in the present work, we can expand on how PDMDOMA interact with other blood components. One proposed study is to look at the level of platelet activation and adhesion. Platelets play an important role during normal whole blood hemostasis. A lower level of platelet activation can greatly affect the clot overall strength. It would be also interesting to study how the resting and activated platelets interact with the fibrin clot formed in presence of PDMDOMA. Another study may focus on whether or not the complement cascade is initiated in presence of these polymers. This is of particular importance if we were to use PDMDOMA for modifying biomaterial surfaces or as a soluble antithrombotic agent. Lastly, from this study, we were able to determine that the bioactivity of PDMDOMA is solely dependent on the amide linked dioxolane pendent groups in the polymer. Thus  106  it would be interesting to study whether this antithrombotic effect can be mimicked when this group is transferred either to a small carrier or other polymers.  107  4.3 References 1. Gorbet, M.B., and Sefton, M. Biomaterial-associated thrombosis: roles of coagulation factors, complement, platelets and leukocytes. Biomaterials 25, 5681- 5703 (2004). 2. Courtney, J.M., Sundaram, S., Matata, B.M., Gaylor, J.D.S., and Forbes, C.D. Biomaterials in cardiopulmonary bypass. Perfusion 9, 3-10 (1994). 3. Courtney, J.M. and Forbes, C.D. Thrombosis on foreign surfaces. British Medical Bulletin 50, 966- 981 (1994).  108  Appendix Final Concentration (mg/ml) HMWPDMDOMA  Final Molarity (M=mol/L)  0.25 1  LMWPDMDOMA PNIPAm PEG PDMA  0.25 1 1 1  Appendix S1: Final concentration of soluble polymers in terms of molarities  109  Appendix S2: Evaluation of undiluted cross-linking in fibrin clot using reducing SDS-PAGE. This is a 10% gel of the clots from platelet poor plasma. The clots for the buffer control and polymer controls were undiluted by a factor of 10 (lane 1: MW standard, lane 2: fibrinogen control, lane 3: thrombin + FXIIIa, lane 4: buffer control, lane 5: 1 mg/ml PEG control, lane 6: 1 mg/ml PDMA control, lane 7: 1 mg/ml HMW-PDMDOMA, and lane 8: 0.25 mg/ml LMWPDMDOMA).  110  Appendix S3: TEG trace of whole blood clot incubated with PDMDOMA-PDHPA or PDHPA. Citrated whole blood was re-calcified with 0.2 M CaCl2 and was allowed to clot with PDMODOMA-PDHPA or PDHPA (1 mg/ml) and buffer control for 2 h at 37°C in the Thromboelastograph® analyzer. Thromboelastograph trace of whole blood clot is given.  111  

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