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

The platelet receptors glycoprotein (Gp)VI, integrin α 2 β 1 and GpIb/V/IX mediate platelet adhesion and activation during thrombogenesis. In-creases of intracellular Ca 2+ ([Ca 2+ ] 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 [Ca 2+ ] i measurements using confocal imaging. All three collagen receptors coupled to [Ca 2+ ] i signals, but these varied in amplitude and temporal pattern alongside variable integrin activation. GpVI engagement produced large, sustained [Ca 2+ ] i signals leading to real-time increases in integrins α 2 β 1 - and α IIb β 3 -mediated platelet adhesion. α IIb β 3 -dependent platelet aggregation was dependent on P 2 Y 12 signalling. Coengagement of α 2 β 1 and GpIb/V/IX generated transient [Ca 2+ ] i spikes and low amplitude [Ca 2+ ] i res ponses that potentiated GpVI-dependent [Ca 2+ ] i signalling. Therefore α 2 β 1 , GpIb/V/IX and GpVI synergise to generate [Ca 2+ ] i signals that regulate platelet behaviour and thrombus formation. Antagonism of secondary signalling path-ways reveals distinct, separate roles for α IIb β 3 in stable platelet ad-hesion and aggregation.