HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY Sequential cytoplasmic calcium signals in a 2-stage platelet activation process induced by the glycoprotein Ib mechanoreceptor

We found that the interaction of platelets with immobilized von Willebrand factor (VWF) under flow induces distinct elevations of cytosolic Ca concentration ([Ca ]i) that are associated with sequential stages of integrin IIb 3 activation. Fluid-dynamic conditions that are compatible with the existence of tensile stress on the bonds between glycoprotein Ib (GPIb ) and the VWF A1 domain led to Ca release from intracellular stores (type / peaks), which preceded stationary platelet adhesion. Raised levels of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate, as well as membrane-permeable calcium chelators, inhibited these [Ca ]i oscillations and prevented stable adhesion without affecting the dynamic characteristics of the typical platelet translocation on VWF mediated by GPIb . Once adhesion was established through the integrin IIb 3, new [Ca ]i oscillations (type ) of greater amplitude and duration, and involving a transmembrane ion flux, developed in association with the recruitment of additional platelets into aggregates. Degradation of released adenosine diphosphate (ADP) to AMP or inhibition of phosphatidylinositol 3-kinase (PI3-K) prevented this response without affecting stationary adhesion and blocked aggregation. These findings indicate that an initial signal induced by stressed GPIb -VWF bonds leads to IIb 3 activation sufficient to support localized platelet adhesion. Then, additional signals from ADP receptors and possibly ligand-occupied IIb 3, with the contribution of a pathway involving PI3-K, amplify platelet activation to the level required for aggregation. Our conclusions modify those proposed by others regarding the mechanisms that regulate signaling between GPIb and IIb 3 and lead to platelet adhesion and aggregation on immobilized VWF. (Blood. 2002;100:2793-2800)