Dual ITAM-mediated proteolytic pathways for irreversible inactivation of platelet receptors: de-ITAM-izing FcγRIIa

E.E. Gardiner, D. Karunakaran, J.F. Arthur, F.T. Mu, M.S. Powell, R.I. Baker, P.M. Hogarth, M.L. Kahn, R.K. Andrews, M.C. Berndt

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    71 Citations (Scopus)


    Collagen binding to glycoprotein VI (GPVI) induces signals critical for platelet activation in thrombosis. Both ligand-induced GPVI signaling through its coassociated Fc-receptor γ-chain (FcRγ) 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, FcγRIIa, also down-regulates GPVI expression. Agonists that signal through FcγRIIa, the mAbs VM58 or 14A2, potently induced GPVI shedding, inhibitable by the metalloproteinase inhibitor, GM6001. Unexpectedly, FcγRIIa also underwent rapid proteolysis in platelets treated with agonists for FcγRIIa (VM58/14A2) or GPVI/FcRγ (the snake toxin, convulxin), generating an approximate 30-kDa fragment. Immunoprecipitation/pull-down experiments showed that FcγRIIa also bound calmodulin and W7 induced FcγRIIa cleavage. However, unlike GPVI, the approximate 30-kDa FcγRIIa fragment remained platelet associated, and proteolysis was unaffected by GM6001 but was inhibited by a membrane-permeable calpain inhibitor, E64d; consistent with this, μ-calpain cleaved an FcγRIIa tail-fusion protein at 222Lys/223Ala and 230Gly/231Arg, 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/FcRγ and FcγRIIa, respectively. Activation of both pathways was observed with immunoglobulin from patients with heparin-induced thrombocytopenia (HIT), suggesting novel mechanisms for platelet dysfunction by FcγRIIa after immunologic insult. © 2008 by The American Society of Hematology
    Original languageEnglish
    Pages (from-to)165-174
    Issue number1
    Publication statusPublished - 2008


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