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. 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 α_IIbβ₃-mediated platelet adhesion. α_IIbβ₃-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 synergise to generate [Ca²⁺]_i signals that regulate platelet behaviour and thrombus formation. Antagonism of secondary signalling pathways reveals distinct, separate roles for α_IIbβ₃ in stable platelet adhesion and aggregation.