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• W2099971136 abstract "HomeArteriosclerosis, Thrombosis, and Vascular BiologyVol. 28, No. 2New Direction for WE Thrombin Free AccessEditorialPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessEditorialPDF/EPUBNew Direction for WE Thrombin Michael C. Berndt and Robert K. Andrews Michael C. BerndtMichael C. Berndt From the Department of Immunology, Alfred Medical Research and Education Precinct (AMREP), Melbourne, Victoria, Australia. Search for more papers by this author and Robert K. AndrewsRobert K. Andrews From the Department of Immunology, Alfred Medical Research and Education Precinct (AMREP), Melbourne, Victoria, Australia. Search for more papers by this author Originally published1 Feb 2008https://doi.org/10.1161/ATVBAHA.107.159301Arteriosclerosis, Thrombosis, and Vascular Biology. 2008;28:205–207An appealing strategy for developing therapeutic agents is to modify the design of human proteins, thereby taking advantage of target-specificity, multifunctionality of many proteins, and human compatibility—that is, provided any undesirable functional effects of the parent molecule can be selectively engineered away. In 2000, Cantwell & Di Cera1 reported a mutant form of the human serine protease, α-thrombin, a multifunctional enzyme involved in both pro- and antithrombotic pathways. This mutant, termed WE thrombin, containing Trp215Ala and Glu217Ala substitutions, still promoted formation of activated protein C (APC) that inhibits coagulation factors Va and VIIIa (antithrombotic), but inefficiently converted fibrinogen to fibrin (prothrombotic), and was an effective antithrombotic agent in primate models of thrombosis.2,3 But a number of observations suggested this was not the full story. For instance, at low WE thrombin concentrations, there was a greater antithrombotic effect than expected based on circulating APC levels and minimal systemic anticoagulation, and in addition, labeled WE thrombin was incorporated into a developing thrombus, suggesting it might be interacting with platelets. In this issue of Arteriosclerosis, Thrombosis, and Vascular Biology, Berny and colleagues4 have provided further information (see Figure), revealing an unanticipated additional antithrombotic effect of WE thrombin, that is, binding to platelet glycoprotein (GP)Ibα (the major ligand-binding subunit of the GPIb-IX-V complex),5–7 and inhibiting platelet adhesion to von Willebrand factor (vWF) under hydrodynamic flow and thrombus formation on a collagen matrix. The interaction of both wild-type and WE thrombin with GPIbα involves the N-terminal ligand-binding region of GPIbα (residues 1 to 282), but with profoundly different functional consequences. Download figureDownload PowerPointFigure. An antithrombotic form of mutant thrombin, WE, is ineffective at converting fibrinogen to fibrin, but is able to generate APC, which inhibits activation of coagulation factors V and VIII (to FVa and FVIIIa, respectively). Berny and colleagues4 now show that WE thrombin also binds to GPIbα and inhibits its interaction with the vWF A1 domain, as an additional antithrombotic mechanism. Unlike wild-type thrombin, WE thrombin does not activate platelets via GPIbα or PAR-1.See accompanying article on page 329The Role of Thrombin in ThrombosisThrombin has multiple roles in hemostasis and thrombosis.8 Control of these processes involves regulation of active thrombin generation at the business end of intrinsic (FXII-dependent) or extrinsic (tissue factor [TF]-dependent) coagulation pathways, thrombin inhibitors, and regulators of proteins downstream of thrombin activity. This extensive system of positive and negative feedback loops is essential for fine-tuning of thrombin-dependent events. The effect of thrombin on platelets is closely linked to its activity toward noncellular targets associated with thrombus formation. Thrombin activity is involved in converting fibrinogen to fibrin, activation of FXIII to FXIIIa (involved in fibrin cross-linking), activation of thrombin-activatable fibrinolysis inhibitor (TAFI), activating FXI (consolidation pathway), converting FV to FVa, and when associated with thrombomodulin, converting protein C to APC.1–4,8 FIXa-FVIIIa and TF-FVIIa generate FXa-FVa that converts prothrombin to thrombin.Thrombin also activates platelets by different mechanisms. First, thrombin activates the G protein–coupled 7 transmembrane protease-activated receptor-1 or -4 (PAR-1 or PAR-4). PAR-1 activation is accelerated by thrombin binding to GPIbα, the major ligand-binding subunit of the GPIb-IX-V complex.5,6,8–10 Interestingly, thrombin can activate platelets by a second mechanism involving binding to GPIbα, a process facilitated by proteolytic removal of GPV (a substrate for thrombin or metalloproteinases, ADAM-10 and -17).11–13 Platelets promote intrinsic and extrinsic coagulation pathways, and binding of thrombin to the ligand-binding N-terminal region of GPIbα provides a mechanism for increasing the effects of thrombin activity, for example, by orientation of active thrombin with platelet-bound substrates including fibrinogen or FXI.5 The way in which thrombin recognizes different substrates, cofactors, or binding partners involves distinct exosites. Exosite I, or the fibrinogen recognition site, is centered on loop Arg67-Ile82, whereas exosite II, or the heparin-binding site, includes residues 93/97/101/173/174/175/233/236/240.10 How these sites control thrombin activity in thrombus formation is being revealed by structure-function analysis.1–5,8,10 Alanine scanning of thrombin also revealed residues outside these sites conferring binding partner recognition selectivity,14,15 opening the way for assessing the role of individual thrombin interactions in vivo and investigating pretherapeutic applications of mutant thrombin.1–4,16 Trp215Ala or Glu217Ala mutations of thrombin vastly decreased efficiency of fibrin production relative to near-normal binding to thrombomodulin and activation of protein C, with this effect maximized in a combined Trp215Ala plus Glu217Ala (WE) form of mutant thrombin.1–3,16 To date, unanswered, however, was how WE thrombin interacted with GPIbα, either activating platelets via GPIbα or PAR-1, or affecting interaction of GPIbα with vWF or other ligands.Current StudyThe current study of Berny et al4 describes for the first time that mutant WE thrombin can bind platelet GPIbα, but unlike wild-type thrombin, not activate platelets either directly via GPIbα,11,12 or indirectly by proteolysis of PAR-1.9,10 WE thrombin also inhibited platelet tethering and rolling of platelets on immobilized vWF when whole blood was passed over the surface under shear-flow conditions. Another significant finding involves platelet spreading on immobilized wild-type thrombin. Human platelets or platelets from WAVE-1–deficient mice (WAVE-1 is an actin scaffolding protein) adhered and formed lamellipodia over 45 minutes at 37°C, whereas platelets from mice deficient in the cytoskeletal regulatory protein, Rac1, adhered and formed filopodia, but remained alamellipodic. Platelet adhesion to wild-type thrombin was also unaffected by soluble WE thrombin under static conditions. These experiments not only showed a dependence on Rac1 for platelet spreading on thrombin, but also showed a lack of platelet interaction with immobilized WE thrombin, prothrombin, PPACK-inhibited thrombin, or catalytically-inactive thrombin mutant, Ser195Ala. Active thrombin is emphatically essential for supporting platelet adhesion under static conditions or under flow conditions (300 s−1), however in the latter case, soluble WE thrombin and the anti-GPIbα antibody, SZ2, significantly blocked platelet adhesion to wild-type thrombin.4 SZ2 has previously been mapped to the anionic, sulfated tyrosine-containing sequence of GPIbα (Asp269-Glu282) within the N-terminal ligand-binding domain (residues 1 to 282), and this sulfated region makes a major contribution to thrombin recognition, and interacts with thrombin exosite II.5–10Together, these results suggest that WE thrombin is a novel antagonist for GPIbα binding to vWF. Crystal structures of a complex of wild-type thrombin and the N-terminal fragment of GPIbα show that thrombin can interact with this receptor in 2 orientations, involving different thrombin exosites, allowing 1 thrombin molecule to potentially cross-link (and activate) 2 GPIbα receptors on the same or adjacent platelets.8,17,18 Precisely how the mutant WE form of thrombin recognizes GPIbα is unclear. The thrombin-binding faces of GPIbα are distinct but partially overlap interactive surfaces of GPIbα N-terminal fragment and the vWF-A1 domain determined from cocrystal structures under static conditions.5–7,19–21 However, functional studies using cross-species chimeras show a discrete anionic region of GPIbα becomes increasingly important for tethering to vWF as shear rate increases.7 This implies shear-dependent conformational changes could alter the functional binding state of GPIbα (or vWF-A1), but it remains to be seen whether the functional exposure of the anionic patch within the leucine-rich repeat domain5–7 regulates the interaction with thrombin or other ligands. In this respect, SZ2, which blocks binding of thrombin to GPIbα, also inhibits binding of P-selectin, and given WE thrombin is a GPIbα antagonist toward vWF ligand-binding A1 domain, it would also be interesting to assess its potential antiinflammatory role in blocking GPIbα-dependent platelet adhesion to activated endothelial cells (mediated by endothelial P-selectin) or leukocytes (mediated by the leukocyte integrin, αMβ2, expressing an αM ligand-binding I-domain homologous to vWF-A1).5Conclusions and Future PerspectivesThe study showing WE thrombin is antithrombotic by acting as an antagonist of GPIbα-dependent adhesion to vWF, as well promoting APC but not fibrin production, enhances the potential use of WE thrombin for controlling thrombosis.1–4 Interestingly, the patho/physiological importance of the GPIbα-vWF interaction in thrombus formation at arterial shear rates in vivo or ex vivo/in vitro is addressed in many past and recent studies.21–24 For example, depleting mouse platelets of the extracellular domain of GPIbα (in interleukin 4 [IL-4]-GPIbα cytoplasmic domain transgenic mice, where macrothrombocytopenia associated with GPIbα knockout is rescued), suggests a vital role for GPIbα in thrombus formation, but far less contribution from vWF (shown using vWF-deficient mice).24 This suggests that ligands for GPIbα other than vWF may be essential for stable thrombus formation. In this regard, in vivo or ex vivo studies with mouse and human platelets are consistent with vWF mediating initial, reversible platelet tethering at high shear rates preceding platelet activation and aggregation involving GPIb-IX-V associated receptors such as GPVI, Fc receptors, and integrins (mainly αIIbβ3).4,5,21–24 With this in mind, future analysis should address whether WE thrombin associated with platelets/GPIbα is comparably efficient at generating APC, or more importantly, beneficially localizes this process to the developing thrombus surface.4 The combined antithrombotic effects of WE thrombin in coagulation, localized platelet-mediated coagulation, and platelet adhesion to vWF is likely to make a significant advance in understanding the functions and antithrombotic potential of this reagent in particular, and the approach of using engineered human proteins more broadly, including the unexpected (advantageous) consequences of this mutagenesis.Sources of FundingWe thank the National Health and Medical Research Council of Australia, and Monash University for financial support.DisclosuresNone.FootnotesCorrespondence to Professor Michael C. Berndt, Department of Immunology, Monash University, Alfred Medical Research and Education Precinct (AMREP), Commercial Road, Melbourne, Victoria, Australia 3004. E-mail [email protected] References 1 Cantwell AM, Di Cera E. Rational design of a potent anticoagulant thrombin. J Biol Chem. 2000; 275: 39827–39830.CrossrefMedlineGoogle Scholar2 Gruber A, Cantwell AM, Di Cera E, Hanson SR. The thrombin mutant W215A/E217A shows safe and potent anticoagulant and antithrombotic effects in vivo. J Biol Chem. 2002; 277: 27581–27584.CrossrefMedlineGoogle Scholar3 Gruber A, Marzec UM, Bush L, Di Cera E, Fernández JA, Berny MA, Tucker EI, McCarty OJ, Griffin JH, Hanson SR. Relative antithrombotic and antihemostatic effects of protein C activator versus low-molecular-weight heparin in primates. Blood. 2007; 109: 3733–3740.CrossrefMedlineGoogle Scholar4 Berny MA, White TC, Tucker EI, Bush-Pelc LA, Di Cera E, Gruber A, McCarty OJ. Thrombin mutant W215A/E217A acts as a platelet GPIb antagonist. Arterioscler Thromb Vasc Biol. 2008; 28: 329–334.LinkGoogle Scholar5 Andrews RK, Berndt MC, López JA. The glycoprotein Ib-IX-V complex. In: Platelets. (2nd edition) A.Michelson (ed), San Diego: Academic Press, 2006; 145–163.Google Scholar6 Andrews RK, Gardiner EE, Shen Y, Whisstock JC, Berndt MC. Glycoprotein Ib-IX-V. Int J Biochem Cell Biol. 2003; 35: 1170–1174.CrossrefMedlineGoogle Scholar7 Shen Y, Cranmer SL, Aprico A, Whisstock JC, Jackson SP, Berndt MC, Andrews RK. Leucine-rich repeats 2–4 (Leu60-Glu128) of platelet glycoprotein Ibα regulate shear-dependent cell adhesion to von Willebrand factor. J Biol Chem. 2006; 281: 26419–26423.CrossrefMedlineGoogle Scholar8 Adams TE, Huntington JA. Thrombin-cofactor interactions. Structural insights into regulatory mechanisms. Arterioscler Thromb Vasc Biol. 2006; 26: 1738–1745.LinkGoogle Scholar9 De Candia E, Hall SW, Rutella S, Landolfi R, Andrews RK, De Cristofaro R. Binding of thrombin to glycoprotein Ib accelerates the hydrolysis of Par-1 on intact platelets. J Biol Chem. 2001; 276: 4692–4698.CrossrefMedlineGoogle Scholar10 De Cristofaro R, De Candia E. Thrombin domains: structure, function and interaction with platelet receptors. J Thromb Thrombolysis. 2003; 15: 151–163.CrossrefMedlineGoogle Scholar11 Ramakrishnan V, DeGuzman F, Bao M, Hall SW, Leung LL, Phillips DR. A thrombin receptor function for platelet glycoprotein Ib-IX unmasked by cleavage of glycoprotein V. Proc Natl Acad Sci U S A. 2001; 98: 1823–1828.CrossrefMedlineGoogle Scholar12 Soslau G, Class R, Morgan DA, Foster C, Lord ST, Marchese P, Ruggeri ZM. Unique pathway of thrombin-induced platelet aggregation mediated by glycoprotein Ib. J Biol Chem. 2001; 276: 21173–21183.CrossrefMedlineGoogle Scholar13 Gardiner EE, Karunakaran D, Shen Y, Arthur JF, Andrews RK, Berndt MC. Controlled shedding of platelet glycoprotein (GP)VI and GPIb-IX-V by ADAM family metalloproteinases. J Thromb Haemost. 2007; 5: 1530–1537.CrossrefMedlineGoogle Scholar14 Tsiang M, Jain AK, Dunn KE, Rojas ME, Leung LL, Gibbs CS. Functional mapping of the surface residues of human thrombin. J Biol Chem. 1995; 270: 16854–16863.CrossrefMedlineGoogle Scholar15 Hall SW, Nagashima M, Zhao L, Morser J, Leung LL. Thrombin interacts with thrombomodulin, protein C, and thrombin-activatable fibrinolysis inhibitor via specific and distinct domains. J Biol Chem. 1999; 274: 25510–25516.CrossrefMedlineGoogle Scholar16 Gibbs CS, Coutré SE, Tsiang M, Li WX, Jain AK, Dunn KE, Law VS, Mao CT, Matsumura SY, Mejza SJ, Paborsky LR, Leung LL. Conversion of thrombin into an anticoagulant by protein engineering. Nature. 1995; 378: 413–416.CrossrefMedlineGoogle Scholar17 Dumas JJ, Kumar R, Seehra J, Somers WS, Mosyak L. Crystal structure of the GPIbα-thrombin complex essential for platelet aggregation. Science. 2003; 301: 222–226.CrossrefMedlineGoogle Scholar18 Celikel R, McClintock RA, Roberts JR, Mendolicchio GL, Ware J, Varughese KI, Ruggeri ZM. Modulation of α-thrombin function by distinct interactions with platelet glycoprotein Ibα. Science. 2003; 301: 218–221.CrossrefMedlineGoogle Scholar19 Dumas JJ, Kumar R, McDonagh T, Sullivan F, Stahl ML, Somers WS, Mosyak L. Crystal structure of the wild-type von Willebrand factor A1-glycoprotein Ibα complex reveals conformation differences with a complex bearing von Willebrand disease mutations. J Biol Chem. 2004; 279: 23327–23334.CrossrefMedlineGoogle Scholar20 Huizinga EG, Tsuji S, Romijn RA, Schiphorst ME, de Groot PG, Sixma JJ, Gros P. Structures of glycoprotein Ibα and its complex with von Willebrand factor A1 domain. Science. 2002; 297: 1176–1179.CrossrefMedlineGoogle Scholar21 Chen J, López JA. Interactions of platelets with subendothelium and endothelium. Microcirculation. 2005; 12: 235–246.CrossrefMedlineGoogle Scholar22 Ruggeri ZM, Mendolicchio GL. Adhesion mechanisms in platelet function. Circ Res. 2007; 100: 1673–1685.LinkGoogle Scholar23 Maxwell MJ, Westein E, Nesbitt WS, Giuliano S, Dopheide SM, Jackson SP. Identification of a 2-stage platelet aggregation process mediating shear-dependent thrombus formation. Blood. 2007; 109: 566–576.CrossrefMedlineGoogle Scholar24 Bergmeier W, Piffath CL, Goerge T, Cifuni SM, Ruggeri ZM, Ware J, Wagner DD. The role of platelet adhesion receptor GPIbα far exceeds that of its main ligand, von Willebrand factor, in arterial thrombosis. Proc Natl Acad Sci U S A. 2006; 103: 16900–16905.CrossrefMedlineGoogle Scholar Previous Back to top Next FiguresReferencesRelatedDetails February 2008Vol 28, Issue 2 Advertisement Article InformationMetrics https://doi.org/10.1161/ATVBAHA.107.159301PMID: 18216328 Originally publishedFebruary 1, 2008 Keywordsthrombus formationGPIb-IX-VplateletsthrombinPDF download Advertisement" @default.
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