Differential integrin activity mediated by platelet collagen receptor engagement under flow conditions

The platelet receptors glycoprotein (Gp)VI, integrin α2β1 and GpIb/V/IX mediate platelet adhesion and activation during thrombogenesis. Increases of intracellular Ca2+ ([Ca2+]i) are key signals during platelet activation; however, their relative importance in coupling different collagen receptors to functional responses under shear conditions remains unclear. To study shear-dependent, receptor-specific platelet responses, we used collagen or combinations of receptor-specific collagen-mimetic peptides as substrates for platelet adhesion and activation in whole human blood under arterial flow conditions and compared real-time and endpoint parameters of thrombus formation alongside [Ca2+]i measurements using confocal imaging. All three collagen receptors coupled to [Ca2+]i signals, but these varied in amplitude and temporal pattern alongside variable integrin activation. GpVI engagement produced large, sustained [Ca2+]i signals leading to real-time increases in integrins α2β1– and αIIbβ3-mediated platelet adhesion. αIIbβ3-dependent platelet aggregation was dependent on P2Y12 signalling. Co-engagement of α2β1 and GpIb/V/IX generated transient [Ca2+]i spikes and low amplitude [Ca2+]i responses that potentiated GpVI-dependent [Ca2+]i signalling. Therefore α2β1, GpIb/V/IX and GpVI synergize to generate [Ca2+]i signals that regulate platelet behaviour and thrombus formation. Antagonism of secondary signalling pathways reveals distinct, separate roles for αIIbβ3 in stable platelet adhesion and aggregation.