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, GPIbα (the major ligand-binding subunit) and GPV (that regulates thrombin-dependent activation via GPIbα).Methods and results: Immunoblotting human platelet lysates using affinity-purified antibodies against cytoplasmic domains of GPVI, GPIbα or GPV allowed simultaneous analysis of intact and cleaved receptor, and revealed (i) that a significant fraction of GPIbα, 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 GPIbα 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^Q243YY) as analyzed by MALDI-TOF-MS. In contrast, ADAM17, but not ADAM10, cleaved within the GPIbα-based peptide (LRG^V465LQ). Both ADAM10 and ADAM17 cleaved within a GPV-based peptide (AQP^V494TT). Metalloproteinase-mediated shedding of GPIbα from GPIb-IX-transfected or GPVI-transfected cells induced by W7 or N-ethylmaleimide was inhibited by mutagenesis of sequences identified from peptide analysis.Conclusions: These findings suggest surface levels of GPVI, GPIbα and GPV may be controlled by distinct mechanisms involving ADAM10 and/or ADAM17.