Thrombin binding to GPIbα induces platelet aggregation and fibrin clot retraction supported by resting αIIbβ3 interaction with polymerized fibrin

Summary

We have investigated the mechanisms leading to platelet aggregation following thrombin interaction with the glycoprotein (GP) Ib-IX-V complex. We show that platelets desensitized for the two thrombin receptors, PAR-1 and PAR-4, are still able to aggregate in response to thrombin and that this aggregation can be inhibited by a monoclonal antibody (VM16d) that blocks thrombin binding to GPIbα, or by pretreatment of platelets with Mocarhagin, a protease that specifically cleaves GPIbα. The thrombin/GPIbα-initiated signaling cascade induces platelet shape change through activation of the Rho kinase p160ROCK, independent of calcium mobilization, transient MEK-1 phosphorylation as well as the cleavage of talin through a calcium-independent mechanism. This signaling cascade does not induce the exposure of high affinity αIIbβ3 integrin receptors, nor does it lead to µ-calpain cleavage of filamin or the integrin cytoplasmic tail. In contrast, we provide evidence that binding of thrombin to GPIbα induces fibrin binding to resting αIIbβ3 leading to fibrin-dependent platelet aggregation and clot retraction, that can be selectively inhibited by αIIbβ3 antagonists such as RGDS, the dodecapeptide or lamifiban, as well as by the fibrin polymerization inhibitor GPRP-amide.

Keywords

Platelets - GPIb-IX-V - thrombin - intracellular signaling - aggregation

• References

• 1 Weiss HJ.. Platelet physiology and abnormalities of platelet function (first of two parts). N Engl J Med 1975; 293: 531-41.
• 2 Kahn ML, Zheng YW, Huang W, Bigornia V, Zeng D, Moff S, Farese RVJ, Tam C, Coughlin SR.. A dual thrombin receptor system for platelet activation. Nature 1998; 394: 690-94.
• 3 Okumura T, Hasitz M, Jamieson GA.. Platelet glycocalicin. Interaction with thrombin and role as thrombin receptor of the platelet surface. J Biol Chem 1978; 253: 3435-43.
• 4 Wu Y, Suzuki-Inoue K, Satoh K, Asazuma N, Yatomi Y, Berndt MC, Ozaki Y.. Role of Fc receptor gamma-chain in platelet glycoprotein Ib-mediated signaling. Blood 2001; 97: 3836-45.
• 5 De Marco L, Girolami A, Russell S, Ruggeri ZM.. Interaction of asialo von Willebrand factor with glycoprotein Ib induces fibrinogen binding to the glycoprotein IIb/IIIa complex and mediates platelet aggregation. J Clin Invest 1985; 75: 1198-203.
• 6 Vu TK, Hung DT, Wheaton VI, Coughlin SR.. Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell 1991; 64: 1057-68.
• 7 Vassallo Jr. RR, Kieber-Emmons T, Cichowski K, Brass LF.. Structure-function relationships in the activation of platelet thrombin receptors by receptor-derived peptides. J Biol Chem 1992; 267: 6081-85.
• 8 Coughlin SR.. How the protease thrombin talks to cells. Proc Natl Acad Sci 1999; 96: 11023-27.
• 9 Xu WF, Andersen H, Whitmore TE, Presnell SR, Yee DP, Ching A, Gilbert T, Davie EW, Foster DC.. Cloning and characterization of human protease-activated receptor 4. Proc Natl Acad Sci U S A 1998; 95: 6642-6.
• 10 Macfarlane SR, Seatter MJ, Kanke T, Hunter GD, Plevin R.. Proteinase-activated receptors. Pharmacol Rev 2001; 53: 245-82.
• 11 Shapiro MJ, Weiss EJ, Faruqi TR, Coughlin SR.. Protease-activated receptors 1 and 4 are shut off with distinct kinetics after activation by thrombin. J Biol Chem 2000; 275: 25216-21.
• 12 Covic L, Gresser AL, Kuliopulos A.. Biphasic kinetics of activation and signaling for PAR1 and PAR4 thrombin receptors in platelets. Biochemistry 2000; 39: 5458-67.
• 13 Yamamoto N, Kitagawa H, Tanoue K, Yamazaki H.. Monoclonal antibody to glyco-protein Ib inhibits both thrombin- and ristocetin-induced platelet aggregations. Thromb Res 1985; 39: 751-9.
• 14 Ware J.. Molecular analyses of the platelet glycoprotein Ib-IX-V receptor. Thromb Haemost 1998; 79: 466-78.
• 15 Sixma JJ, Schiphorst ME, Verweij CL, Pannekoek H.. Effect of deletion of the A1 domain of von Willebrand factor on its binding to heparin, collagen and platelets in the presence of ristocetin. Eur J Biochem 1991; 196: 369-75.
• 16 Nurden AT, Didry D, Rosa JP.. Molecular defects of platelets in Bernard-Soulier syndrome. Blood Cells 1983; 9: 333-58.
• 17 De Marco L, Mazzucato M, Masotti A, Fenton JWn, Ruggeri ZM.. Function of glycoprotein Ib alpha in platelet activation induced by alpha-thrombin. J Biol Chem 1991; 266: 23776-83.
• 18 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: 21173-83.
• 19 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 2001; 98: 1823-8.
• 20 Chow TW, Hellums JD, Moake JL, Kroll MH.. Shear stress-induced von Willebrand factor binding to platelet glycoprotein Ib initiates calcium influx associated with aggregation. Blood 1992; 80: 113-20.
• 21 Phillips DR, Charo IF, Scarborough RM.. GPIIb-IIIa: the responsive integrin. Cell 1991; 65: 359-62.
• 22 Yuan Y, Dopheide SM, Ivanidis C, Salem HH, Jackson SP.. Calpain regulation of cytoskeletal signaling complexes in von Willebrand factor-stimulated platelets. Distinct roles for glyco-protein Ib-V-IX and glycoprotein IIb-IIIa (integrin alphaIIbbeta3) in von Willebrand factor-induced signal transduction. J Biol Chem 1997; 272: 21847-54.
• 23 Kouns WC, Hadvary P, Haering P, Steiner B.. Conformational modulation of purified glyco-protein (GP) IIb-IIIa allows proteolytic generation of active fragments from either active or inactive GPIIb-IIIa. J Biol Chem 1992; 267: 18844-51.
• 24 Steiner B, Trzeciak A, Pfenninger G, Kouns WC.. Peptides derived from a sequence within beta 3 integrin bind to platel, et al.pha IIb beta 3 (GPIIb-IIIa) and inhibit ligand binding. J Biol Chem 1993; 268: 6870-3.
• 25 Du X, Saido TC, Tsubuki S, Indig FE, Williams MJ, Ginsberg MH.. Calpain cleavage of the cytoplasmic domain of the integrin beta 3 subunit. J Biol Chem 1995; 270: 26146-51.
• 26 Otey C, Griffiths W, Burridge K.. Characterization of monoclonal antibodies to chicken gizzard talin. Hybridoma 1990; 9: 57-62.
• 27 Cong J, Thompson VF, Goll DE.. Effect of monoclonal antibodies specific for the 28-kDa subunit on catalytic properties of the calpains. J Biol Chem 1993; 268: 25740-7.
• 28 Hartwig JH.. Mechanisms of actin rearrangements mediating platelet activation. J Cell Biol 1992; 118: 1421-42.
• 29 Fox JE, Goll DE, Reynolds CC, Phillips DR.. Identification of two proteins (actin-binding protein and P235) that are hydrolyzed by endogenous Ca2+-dependent protease during platelet aggregation. J Biol Chem 1985; 260: 1060-6.
• 30 Mazurov AV, Vinogradov DV, Vlasik TN, Repin VS, Booth WJ, Berndt MC.. Characterization of an antiglycoprotein Ib monoclonal antibody that specifically inhibits platelet-thrombin interaction. Thromb Res 1991; 62: 673-84.
• 31 Meyer S, Kresbach G, Haring P, SchumppVonach B, Clemetson KJ, Hadvary P, Steiner B.. Expression and characterization of functionally active fragments of the platelet glyco-protein (GP) Ib-IX complex in mammalian cells. Incorporation of GP Ib alpha into the cell surface membrane. J Biol Chem 1993; 268: 20555-62.
• 32 Fox JE, Reynolds CC, Boyles JK.. Studying the platelet cytoskeleton in Triton X-100 lysates. Methods Enzymol 1992; 215: 42-58.
• 33 Grynkiewicz G, Poenie M, Tsien RY.. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 1985; 260: 3440-50.
• 34 Kahn ML, Nakanishi-Matsui M, Shapiro MJ, Ishihara H, Coughlin SR.. Protease-activated receptors 1 and 4 mediate activation of human platelets by thrombin. J Clin Invest 1999; 103: 879-87.
• 35 Schoenwaelder SM, Yuan Y, Jackson SP.. Calpain regulation of integrin alpha IIb beta 3 signaling in human platelets. Platelets 2000; 11: 189-98.
• 36 Bauer M, Retzer M, Wilde JI, Maschberger P, Essler M, Aepfelbacher M, Watson SP, Siess W.. Dichotomous regulation of myosin phosphorylation and shape change by Rho-kinase and calcium in intact human platelets. Blood 1999; 94: 1665-72.
• 37 Klages B, Brandt U, Simon MI, Schultz G, Offermanns S.. Activation of G12/G13 results in shape change and Rho/Rho-kinase-mediated myosin light chain phosphorylation in mouse platelets. J Cell Biol 1999; 144: 745-54.
• 38 Uehata M, Ishizaki T, Satoh H, Ono T, Kawahara T, Morishita T, Tamakawa H, Yamagami K, Inui J, Maekawa M, Narumiya S.. Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension. Nature 1997; 389: 990-4.
• 39 Schoenwaelder SM, Kulkarni S, Salem HH, Imajoh-Ohmi S, Yamao-Harigaya W, Saido TC, Jackson SP.. Distinct substrate specificities and functional roles for the 78- and 76-kDa forms of mu-calpain in human platelets. J Biol Chem 1997; 272: 24876-84.
• 40 Fox JE, Taylor RG, Taffarel M, Boyles JK, Goll DE.. Evidence that activation of platelet calpain is induced as a consequence of binding of adhesive ligand to the integrin, glycoprotein IIb-IIIa. J Cell Biol 1993; 120: 1501-7.
• 41 Li Z, Xi X, Du X.. A mitogen-activated protein kinase-dependent signaling pathway in the activation of platelet integrin alpha IIbbeta3. J Biol Chem 2001; 276: 42226-32.
• 42 Borsch-Haubold AG, Kramer RM, Watson SP.. Inhibition of mitogen-activated protein kinase kinase does not impair primary activation of human platelets. Biochem J 1996; 318: 207-12.
• 43 Pasquet JM, Noury M, Nurden AT.. Evidence that the platelet integrin alphaIIb beta3 is regulated by the integrin-linked kinase, ILK, in a PI3-kinase dependent pathway. Thromb Haemost 2002; 88: 115-22.
• 44 Schoenwaelder SM, Yuan Y, Cooray P, Salem HH, Jackson SP.. Calpain cleavage of focal adhesion proteins regulates the cytoskeletal attachment of integrin alphaIIbbeta3 (platelet glycoprotein IIb/IIIa) and the cellular retraction of fibrin clots. J Biol Chem 1997; 272: 1694-702.
• 45 Azam M, Andrabi SS, Sahr KE, Kamath L, Kuliopulos A, Chishti AH.. Disruption of the mouse mu-calpain gene reveals an essential role in platelet function. Mol Cell Biol Mar 2001; 21: 2213-20.
• 46 Andrieux A, Hudry-Clergeon G, Ryckewaert JJ, Chapel A, Ginsberg MH, Plow EF, Marguerie G.. Amino acid sequences in fibrinogen mediating its interaction with its platelet receptor, GPIIbIIIa. J Biol Chem 1989; 264: 9258-65.
• 47 Lewis JC, Johnson C, Ramsamooj P, Hantgan RR.. Orientation and specificity of fibrin protofibril binding to ADP-stimulated platelets. Blood 1988; 72: 1992-2000.
• 48 Alemany M, Concord E, Garin J, Vincon M, Giles A, Marguerie G, Gulino D.. Sequence 274-368 in the beta 3-subunit of the integrin alpha IIb beta 3 provides a ligand recognition and binding domain for the gamma-chain of fibrinogen that is independent of platelet activation. Blood 1996; 87: 592-601.
• 49 Lam SC, Plow EF, Smith MA, Andrieux A, Ryckwaert JJ, Marguerie G, Ginsberg MH.. Evidence that arginyl-glycyl-aspartate peptides and fibrinogen gamma chain peptides share a common binding site on platelets. J Biol Chem 1987; 262: 947-50.
• 50 Santoro SA, Lawing Jr WJ. Competition for related but nonidentical binding sites on the glycoprotein IIb-IIIa complex by peptides derived from platelet adhesive proteins. Cell 1987; 48: 867-73.
• 51 Ward CM, Kestin AS, Newman PJ.. A Leu262Pro mutation in the integrin beta(3) subunit results in an alpha(IIb)-beta(3) complex that binds fibrin but not fibrinogen. Blood 2000; 96: 161-9.