Signaling Role for Phospholipase Cγ2 in Platelet Glycoprotein Ibα Calcium Flux and Cytoskeletal Reorganization

Abstract

Interaction of the platelet GPIb-V-IX complex with surface immobilized von Willebrand factor (vWf) is required for the capture of circulating platelets and their ensuing activation. In previous work, it was found that GPIb/vWf-mediated platelet adhesion triggers Ca 2 + release from intracellular stores, leading to cytoskeletal reorganization and filopodia extension. Despite the potential functional importance of GPIb-induced cytoskeletal changes, the signaling mechanisms regulating this process have remained ill-defined. The studies presented here demonstrate an important role for phospholipase C (PLC)-dependent phosphoinositide turnover for GPIb-dependent cytoskeletal remodeling. This is supported by the findings that the vWf-GPIb interaction induced a small increase in inositol 1,4,5-triphosphate (IP 3) and that treating platelets with the IP 3 receptor antagonist APB-2 or the PLC inhibitor U73122 blocked cytosolic Ca 2 + flux and platelet shape change. Normal shape change was observed in Gα q –/– mouse platelets, excluding a role for PLCβ isoforms in this process. However, decreased shape change and Ca 2 + mobilization were observed in mice lacking PLCγ2, demonstrating that this isotype played an important, albeit incomplete, role in GPIb signaling. The signaling pathways utilized by GPIb involved one or more members of the Src kinase family as platelet shape change and Ca 2 + flux were inhibited by the Src kinase inhibitors PP1 and PP2. Strikingly, shape change and Ca 2 + release occurred independently of immunoreceptor tyrosine-based activation motif (ITAM)-containing receptors, because these platelet responses were normal in human platelets treated with the anti-FcγRIIA blocking monoclonal antibody IV.3 and in mouse platelets deficient in the FcRγ chain. Taken together, these studies define an important role for PLCγ2 in GPIb signaling linked to platelet shape change. Moreover, they demonstrate that GPIb-dependent calcium flux and cytoskeletal reorganization involves a signaling pathway distinct from that utilized by ITAM-containing receptors.