Dual ITAM-mediated proteolytic pathways for irreversible inactivation of platelet receptors: de-ITAM-izing Fc gamma RIIa

Collagen binding to glycoprotein VI (GPVI) induces signals critical for platelet activation in thrombosis. Both ligand-induced GPVI signaling through its coassociated Fc-receptor gamma-chain (FcR gamma) immunoreceptor tyrosine-activation motif (ITAM) and the calmodulin inhibitor, W7, dissociate calmodulin from GPVI and induce metalloproteinase-mediated GPVI ectodomain shedding. We investigated whether signaling by another ITAM-bearing receptor on platelets, Fc gamma RIIa, also down-regulates GPVI expression. Agonists that signal through Fc gamma RIIa, the mAbs VM58 or 14A2, potently induced GPVI shedding, inhibitable by the metalloproteinase inhibitor, GM6001. Unexpectedly, Fc gamma RIIa also underwent rapid proteolysis in platelets treated with agonists for Fc gamma RIIa (VM58/14A2) or GPVI/FcR gamma (the snake toxin, convulxin), generating an approximate 30-kDa fragment. Immunoprecipitation/pull-down experiments showed that Fc gamma RIIa also bound calmodulin and W7 induced Fc gamma RIIa cleavage. However, unlike GPVI, the approximate 30-kDa Fc gamma RIIa fragment remained platelet associated, and proteolysis was unaffected by GM6001 but was inhibited by a membrane-permeable calpain inhibitor, E64d; consistent with this, mu-calpain cleaved an Fc gamma RIIa tail-fusion protein at (222)Lys/(223)Ala and (230)Gly/(231)Arg, upstream of the ITAM domain. These findings suggest simultaneous activation of distinct extracellular (metalloproteinase-mediated) and intracellular (calpain-mediated) proteolytic pathways irreversibly inactivating platelet GPVI/FcR gamma and Fc gamma RIIa, respectively. Activation of both pathways was observed with immunoglobulin from patients with heparin-induced thrombocytopenia (HIT), suggesting novel mechanisms for platelet dysfunction by Fc gamma RIIa after immunologic insult.