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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2004;24:1727.)
© 2004 American Heart Association, Inc.

Thrombosis

Principal Role of Glycoprotein VI in 2ß1 and IIbß3 Activation During Collagen-Induced Thrombus Formation

Christelle Lecut; Anne Schoolmeester; Marijke J.E. Kuijpers; Jos L.V. Broers; Marc A.M.J. van Zandvoort; Karen Vanhoorelbeke; Hans Deckmyn; Martine Jandrot-Perrus; Johan W.M. Heemskerk

From the Departments of Biochemistry (C.L., M.J.E.K., J.W.M.H.), Molecular Cell Biology and Genetics (J.L.V.B.), and Biophysics (M.A.M.J.V.), CARIM, Maastricht University, The Netherlands; the Laboratory for Thrombosis Research (A.S., K.V., H.D.), KU Leuven, Campus Kortrijk, Belgium; and E348 Institut National de la Santé et de la Recherche Médicale (C.L., M.J.-P.), Faculté Xavier Bichat, Université Paris, France.

Correspondence to J.W.M. Heemskerk, PhD, Department of Biochemistry, Maastricht University, PO Box 616, 6200 MD Maastricht, The Netherlands. E-mail jwm.heemskerk{at}bioch.unimaas.nl

Objective— High-shear perfusion of blood over collagen results in rapid platelet adhesion, aggregation, and procoagulant activity. We studied regulation of 2ß1 and IIbß3 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 2ß1 and IIbß3, 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 2ß1 and IIbß3 activation. Adhesion was fully inhibited only with simultaneous blocking of GPVI and 2ß1, indicating that the integrin can support platelet–collagen binding in the absence of its activation. Blockade or absence of GPIb only moderately influenced integrin activation and adhesion unless GPVI was inhibited.

Conclusions— GPVI- and autocrine-released ADP induce affinity changes of 2ß1 and IIbß3 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 2ß1 and IIbß3 integrin, respectively. Glycoprotein VI blockade by 9O12 antibody or P2Y ADP receptors reduced integrin activation along with aggregate formation and fibrinogen binding but not 2ß1-dependent adhesion.

Key Words: ADP • collagen • glycoprotein VI • integrins • platelets • thrombus

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