[en] OBJECTIVE: High-shear perfusion of blood over collagen results in rapid platelet adhesion, aggregation, and procoagulant activity. We studied regulation of alpha2beta1 and alphaIIbbeta3 integrin activation during thrombus formation on collagen. METHODS AND RESULTS: Blockade of glycoprotein (GP) VI by 9O12 antibody or of P2Y purinergic receptors permitted platelet adhesion but reduced aggregate formation, fibrinogen binding, and activation of alpha2beta1 and alphaIIbbeta3, as detected with antibodies IAC-1 and PAC1 directed against activation-dependent epitopes of these integrins. Combined blockade of GPVI and P2Y receptors and thromboxane formation abolished integrin activation but still allowed adhesion of morphologically unstimulated, nonprocoagulant platelets. Exogenous ADP partly restored the suppressive effect of GPVI blockade on integrin alpha2beta1 and alphaIIbbeta3 activation. Adhesion was fully inhibited only with simultaneous blocking of GPVI and alpha2beta1, indicating that the integrin can support platelet-collagen binding in the absence of its activation. Blockade or absence of GPIbalpha only moderately influenced integrin activation and adhesion unless GPVI was inhibited. CONCLUSIONS: GPVI- and autocrine-released ADP induce affinity changes of alpha2beta1 and alphaIIbbeta3 during thrombus formation on collagen under flow. These integrin changes are dispensable for adhesion but strengthen platelet-collagen interactions and thereby collagen-induced platelet activation. Integrin activation during thrombus formation on collagen was studied using fluorescent-labeled antibodies IAC-1 and PAC1 directed against activation-dependent epitopes of alpha2beta1 and alphaIIbbeta3 integrin, respectively. Glycoprotein VI blockade by 9O12 antibody or P2Y ADP receptors reduced integrin activation along with aggregate formation and fibrinogen binding but not alpha2beta1-dependent adhesion.
Disciplines :
Hematology
Author, co-author :
Lecut, Christelle ; INSERM (France) et CARIM (The Netherlands) > E348, Faculté de Médecine Xavier Bichat, Université Paris 7, France et Department of Biochemistry, University of Maastricht, The Netherlands
Schoolmeester, Anne
Kuijpers, Marijke J E
Broers, Jos L V
van Zandvoort, Marc A M J
Vanhoorelbeke, Karen
Deckmyn, Hans
Jandrot-Perrus, Martine
Heemskerk, Johan W M
Language :
English
Title :
Principal role of glycoprotein VI in alpha2beta1 and alphaIIbbeta3 activation during collagen-induced thrombus formation.
Publication date :
2004
Journal title :
Arteriosclerosis, Thrombosis and Vascular Biology
ISSN :
1079-5642
eISSN :
1524-4636
Publisher :
Lippincott Williams & Wilkins, Baltimore, United States - Maryland
Savage B, Ginsberg MH, Ruggeri ZM. Influence of fibrillar collagen structure on the mechanisms of platelet thrombus formation under flow. Blood. 1999;94:2704-2715.
Nieswandt B, Brakebusch C, Bergmeier W, Schulte V, Bouvard D, Mohtari-Nejad R, Lindhout T, Heemskerk JWM, Zirngibl H, Fässler R. Glycoprotein VI but not α2β1 integrin is essential for platelet interaction with collagen. EMBO J. 2001;20:2120-2130.
Shattil SJ, Kashiwagi H, Pampori N. Integrin signaling, the platelet paradigm. Blood. 1998;91:2645-2657.
Savage B, Sixma JJ, Ruggeri ZM. Functional self-association of von Willebrand factor during platelet adhesion under flow. Proc Natl Acad Sci U S A. 2002;99:425-430.
Kasirer-Friede A, Ware J, Leng L, Marchese P, Ruggeri ZM, Shattil SJ. Lateral clustering of platelet GP Ib-IX complexes leads to up-regulation of adhesive function of integrin αIIbβ3. J Biol Chem. 2002;277:11949-11956.
Daniel JL, Dangelmaier C, Jin JG, Kim YB, Kunapuli SP. Role of intracellular signaling events in ADP-induced platelet aggregation. Thromb Haemost. 1999;82:1322-1326.
Eckly A, Gendrault JL, Hechler B, Ceazenave JP, Cachet C. Differential involvement of P2Y1 and P2YT receptors in morphological changes of platelet aggregation. Thromb Haemost. 2001;85:694-701.
Wang Z, Leisner TM, Parise LV. Platelet α2β1 integrin activation: contribution of ligand internalization and the α2-cytoplasmic domain. Blood. 2003;102:1307-1315.
Jung SM, Moroi M. Platelet collagen receptor integrin α2β1 activation involves differential participation of ADP-receptor subtypes P2Y 1 and P2Y12. Eur J Biochem. 2001;268:3513-3522.
Takagi J, Petre BM, Walz T, Springer TA. Global conformational rearrangements in integrin extracellular domains in outside-in and inside-out signaling. Cell. 2002;110:599-511.
Goto S, Tamura N, Handa S, Arai M, Kodama K, Takayama H. Involvement of glycoprotein VI in platelet thrombus formation on both collagen and von Willebrand factor surfaces under flow conditions. Circulation. 2002;106:266-272.
Nieswandt B, Watson SP. Platelet collagen interaction: is GPVI the central receptor? Blood. 2003;102:449-461.
Massberg S, Gawaz M, Grüner S, Schulte V, Konrad I, Zohlhöfer D, Heinzmann U, Nieswandt B. A crucial role of glycoprotein VI for platelet recruitment to the injured arterial wall in vivo. J Exp Med. 2003;197:41-49.
Chen H, Locke D, Liu Y, Liu C, Kahn ML. The platelet receptor GPVI mediates both adhesion and signaling responses to collagen in receptor density-dependent fashion. J Biol Chem. 2002;277:3011-3019.
Best D, Senis YA, Jarvis GE, Eagleton HJ, Roberts DJ, Saito T, Jung SM, Moroi M, Harrison P, Green FR, Watson SP. GPVI levels in platelets: relationship to platelet function at high shear. Blood. 2003;102:2811-2818.
Kato K, Kanaji T, Russell S, Kunicki TJ, Furihata K, Kanaji S, Marchese P, Reininger A, Ruggeri ZM, Ware J. The contribution of glycoprotein VI to stable platelet adhesion and thrombus formation illustrated by targeted gene deletion. Blood. 2003;102:170-1707.
Kuijpers MJE, Schulte V, Bergmeier W, Lindhout T, Brakebusch C, Offermanns S, Fässler R, Heemskerk JWM, Nieswandt B. Complementary roles of glycoprotein VI and α2β1 integrin in collagen-induced thrombus formation in flowing whole blood ex vivo. FASEB J. 2003;17:685-687.
Chen H, Kahn ML. Reciprocal signaling by integrin and nonintegrin receptors during collagen activation of platelets. Mol Cell Biol. 2003;23:4764-4777.
Suzuki-Inoue K, Inoue O, Frampton J, Watson SP. Murine GPVI stimulates weak integrin activation in PLCγ2 -/- platelets. Blood. 2003;102:1367-1373.
Turner NA, Moake JL, McIntire LV. Blockade of ADP receptors P2Y 12 and P2Y1 is required to inhibit platelet aggregation in whole blood under flow. Blood. 2001;98:3340-3345.
Remijn JA, Wu YP, Heninga EH, Ijsseldijk MJW, van Willigen G, de Groot PG, Sixma JJ, Nurden AT, Nurden P. Role of ADP receptor P2Y12 in platelet adhesion and thrombus formation in flowing blood. Arterioscler Thromb Vasc Biol. 2002;22:686-691.
Heemskerk JWM, Bevers EM, Lindhout T. Platelet activation and blood coagulation. Thromb Haemost. 2002;88:186-193.
Gu MY, Xi XD, Englund GD, Berndt MC, Du XP. Analysis of the roles of 14-3-3 in platelet glycoprotein Ib-IX-mediated activation of integrin αIIbβ3 using a reconstituted mammalian cell expression model. J Cell Biol. 1999;147:1085-1096.
Yap CL, Hughan SC, Cranmer SL, Nesbitt WS, Rooney MM, Giuliano S, Kulkarni S, Dopheide SM, Yuan YP, Salem HH, Jackson SP. Synergistic adhesive interactions and signaling mechanisms operating between platelet glycoprotein Ib/IX and integrin αIIbβ3. J Biol Chem. 2000;275:41377-41388.
Lecut C, Feeney LA, Kingsbury G, Hopkins J, Lanza F, Cachet C, Villeval JL, Jandrot-Perrus M. Human platelet glycoprotein VI function is antagonised by monoclonal antibody-derived Fab fragments. J Thromb Haemost. 2003;1:2653-2662.
Heemskerk JWM, Vuist WMJ, Feijge MAH, Reutelingsperger CPM, Lindhout T. Collagen but not fibrinogen surfaces induce bleb formation, exposure of phosphatidylserine and procoagulant activity of adherent platelets. Blood. 1997;90:2615-2625.
Schoolmeester A, Vanhoorelbeke K, Depraetere H, Feys HB, Heemskerk JWM, Hoylaerts M, Deckmyn H. Monoclonal antibody IAC-1 is specific for activated α2β1 and binds to amino acid 199-201 of the integrin α2 I-domain. Blood 2004;in press.
Abrams C, Deng YJ, Steiner B, O'Toole T, Shattil SJ. Determinants of specificity of a baculovirus-expressed antibody Fab fragment that binds selectively to activated form of integrin αIIbβ3. J Biol Chem. 1994;269:18781-18788.
Cauwenberghs N, Vanhoorelbeke K, Vauterin S, Westra DF, Romo G, Huizinga EG, Lopez JA, Berndt MC, Harsfalvi J, Deckmyn H. Epitope mapping of inhibitory antibodies against platelet glycoprotein Ibα reveals interaction between the leucine-rich repeat N-terminal and C-terminal flanking domains of glycoprotein Ibα. Blood. 2001;98:652-660.
Inoue O, Suzuki-Inoue K, Dean WL, Frampton J, Watson SP. Integrin α2β1 mediates outside-in regulation of platelet spreading on collagen through activation of Src kinases and PLCγ2. J Cell Biol. 160;769-780.