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

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, GPIb alpha (the major ligand-binding subunit) and GPV (that regulates thrombin-dependent activation via GPIb alpha). Methods and results: Immunoblotting human platelet lysates using affinity-purified antibodies against cytoplasmic domains of GPVI, GPIb alpha or GPV allowed simultaneous analysis of intact and cleaved receptor, and revealed (i) that a significant fraction of GPIb alpha, 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 GPIb alpha 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 boolean AND Q(243)YY) as analyzed by MALDI-TOF-MS. In contrast, ADAM17, but not ADAM10, cleaved within the GPIb alpha-based peptide (LRG boolean AND V 465 LQ). Both ADAM10 and ADAM17 cleaved within a GPV-based peptide (AQP boolean AND(VTT)-T-494). Metalloproteinase-mediated shedding of GPIb alpha 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, GPIb alpha and GPV may be controlled by distinct mechanisms involving ADAM10 and/or ADAM17.