Controlled shedding of platelet glycoprotein (GP)VI and GPIb-IX-V by ADAM family metalloproteinases

Elizabeth Ellen Gardiner, Denuja Karunakaran, Yang Shen, Jane Frances Arthur, Robert Keith Andrews, Michael Claude Berndt

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Abstract

BACKGROUND: Platelet glycoprotein (GP)VI that binds collagen, and GPIb-IX-V that binds von Willebrand factor, initiate thrombus formation. OBJECTIVES: In this study, we investigated the mechanisms of metalloproteinase-mediated ectodomain shedding that regulate the surface expression of GPVI, GPIbalpha (the major ligand-binding subunit) and GPV (that regulates thrombin-dependent activation via GPIbalpha). METHODS AND RESULTS: Immunoblotting human platelet lysates using affinity-purified antibodies against cytoplasmic domains of GPVI, GPIbalpha or GPV allowed simultaneous analysis of intact and cleaved receptor, and revealed (i) that a significant fraction of GPIbalpha, but not GPVI, exists in a cleaved state on platelets, even when isolated in the presence of metalloproteinase inhibitor (GM6001) or EDTA; (ii) the same-sized membrane-associated fragments of GPVI or GPIbalpha are generated by phorbol-ester (PMA), the mitochondrial-targeting reagent CCCP, the calmodulin inhibitor W7, or the thiol-modifying reagent, N-ethylmaleimide, that directly activates ADAM10/ADAM17; and (iii) GPV is shed by both metalloproteinase- and thrombin-dependent mechanisms, depending on the concentration of thrombin. Based on the predicted cleavage area defined by these studies, ADAM10, but not ADAM17, cleaved a GPVI-based synthetic peptide within the extracellular membrane-proximal sequence (PAR;Q(243)YY) as analyzed by MALDI-TOF-MS. In contrast, ADAM17, but not .....
Original languageEnglish
Pages (from-to)1530 - 1537
Number of pages8
JournalJournal of Thrombosis and Haemostasis
Volume5
Issue number7
DOIs
Publication statusPublished - 2007

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