BH3 mimetics are a new class of proapo-ptotic anticancer agents that have shown considerable promise in preclinical animal models and early-stage human trials. These agents act by inhibiting the pro-survival function of one or more Bcl-2–related proteins. Agents that inhibit Bcl-x_L induce rapid platelet death that leads to thrombocytopenia; however, their impact on the function of residual circulating platelets remains unclear. In this study, we demonstrate that the BH3 mimetics, ABT-737 or ABT-263, induce a time- and dose-dependent decrease in platelet adhesive function that correlates with ectodomain shedding of the major platelet adhesion receptors, glycoprotein Ibα and glycoprotein VI, and functional down-regulation of integrin α_IIbβ₃. Analysis of platelets from mice treated with higher doses of BH3 mimetics revealed the presence of a subpopulation of circulating platelets undergoing cell death that have impaired activation responses to soluble agonists. Functional analysis of platelets by intravital microscopy revealed a time-dependent defect in platelet aggregation at sites of vascular injury that correlated with an increase in tail bleeding time. Overall, these studies demonstrate that Bcl-x_L–inhibitory BH3 mimetics not only induce thrombocytopenia but also a transient thrombocytopathy that can undermine the hemostatic function of platelets.

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.