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

Abstract

The platelet receptors glycoprotein (Gp)VI, integrin α2β1 and GpIb/V/IX mediate platelet adhesion and activation during thrombogenesis. In-creases 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 re-mains 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 com-pared 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. Coengagement 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 synergise to generate [Ca2+] i signals that regulate platelet behaviour and thrombus formation. Antagonism of secondary signalling path-ways reveals distinct, separate roles for αIIbβ3 in stable platelet ad-hesion and aggregation.