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.