Molecular regulation of platelet adhesion

Two platelet receptors, the glycoprotein (GP) Ib-IX-V complex and GPVI/FcRγ-chain, are pivotal in initiating signals that propagate both haemostasis and thrombosis. In the arterial circulation when an atherosclerotic plaque ruptures, these receptors initiate platelet adhesion in response to exposed thrombogenic materials by binding vessel wall von Willebrand Factor (VWF) and collagen, respectively. While these adhesive processes and subsequent events in thrombus formation have been the subject of intense investigation, the mechanisms that positively and negatively regulate the function of these and other receptors in activated platelets, and thus act to determine thrombus formation and stability, are only beginning to be understood. Platelet adhesion through the GPIb-IX-V complex is regulated at three distinct levels. First, the binding of VWF to the α-chain of GPIb is regulated by shear. Recent data from our laboratory exploiting analysis of canine-human chimaeras of GPIbα indicate that a negative-charge cluster involving leucine-rich repeats 2-4 plays an important role in the molecular regulation of shear-dependent VWF binding. Second, VWF affinity for the GPIb-IX-V complex is regulated by its association with the scaffolding protein, 14-3-3ζ, and phosphorylation of the α- and β-subunits of the GPIb-IX-V complex. Finally, calmodulin association with GPIbβ and GPV regulates the platelet activation dependent cleavage of GPIbα and GPV by the platelet membrane-associated metalloproteinase, ADAM17. Platelet adhesion through GPVI is primarily regulated by post-ligation-induced shedding of the GPVI ectodomain by ADAM10. Ligand-induced shedding is signaling-dependent and blocked by inhibitors of GPVI-dependent-signaling, Src, phosphoinositide 3-kinase (PI3-kinase) , or the ITAM-related Syk kinase. The calmodulin inhibitor W7 also induces shedding of GPVI, but independently of platelet activation. We have found that, like GPVI, another ITAM-containing receptor, the platelet Fc receptor, FcγRIIa, is cleaved in response to ligands of FcγRIIa (VM58, 14A2 or heparin-induced antibodies) or GPVI (convulxin) , or by W7. Conversely, ligands of FcγRIIa also induce GPVI shedding. FcγRIIa cleavage is blocked not only by Syk inhibition but also by the calpain inhibitor, E64d, and a cytoplasmic calpain-cleavage site was identified upstream of the ITAM domain. Thus, regulation of ligand binding or signaling by these unique platelet receptors through distinct proteolytic mechanisms may play a key role in limiting thrombus size and stability in response to vessel damage or immunological insult.