HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY Continuous signaling via PI3K isoforms and is required for platelet ADP receptor function in dynamic thrombus stabilization

Signaling from collagen and G protein– coupled receptors leads to platelet adhesion and subsequent thrombus formation. Paracrine agonists such as ADP, thromboxane, and Gas6 are required for platelet aggregate formation. We hypothesized that thrombi are intrinsically unstable structures and that their stabilization requires persistent paracrine activity and continuous signaling, maintaining integrin IIb 3 activation. Here, we studied the disassembly of human and murine thrombi formed on collagen under high shear conditions. Platelet aggregates rapidly disintegrated (1) in the absence of fibrinogen-containing plasma; (2) by blocking or inhibiting IIb 3; (3) by blocking P2Y12 receptors; (4) by suppression of phosphoinositide 3-kinase (PI3K) . In murine blood, absence of PI3K led to formation of unstable thrombi, leading to dissociation of multiplatelet aggregates. In addition, blocking PI3K delayed initial thrombus formation and reduced individual platelet-platelet contact. Similarly without flow, agonist-induced aggregation was reversed by late suppression of P2Y12 or PI3K isoforms, resulting in single platelets that had inactivated IIb 3 and no longer bound fibrinogen. Together, the data indicate that continuous outside-in signaling via P2Y12 and both PI3K and PI3K isoforms is required for perpetuated IIb 3 activation and maintenance of a platelet aggregate. This novel concept of intrinsic, dynamic thrombus instability gives possibilities for the use of antiplatelet therapy. (Blood. 2006;108:3045-3052)