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.

We have identified platelet glycoprotein (GP) Ibα as a counterreceptor for P-selectin. GP Ibα is a component of the GP Ib-IX-V complex, which mediates platelet adhesion to subendothelium at sites of injury. Cells expressing P-selectin adhered to immobilized GP Ibα, and GP Ibα–expressing cells adhered to and rolled on P-selectin and on histamine-stimulated endothelium in a P-selectin–dependent manner. In like manner, platelets rolled on activated endothelium, a phenomenon inhibited by antibodies to both P-selectin and GP Ibα. Unlike the P-selectin interaction with its leukocyte ligand, PSGL-1 (P-selectin glycoprotein ligand 1), the interaction with GP Ibα required neither calcium nor carbohydrate core-2 branching or α(1,3)-fucosylation. The interaction was inhibited by sulfated proteoglycans and by antibodies against GP Ibα, including one directed at a tyrosine-sulfated region of the polypeptide. Thus, the GP Ib-IX-V complex mediates platelet attachment to both subendothelium and activated endothelium.

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.

Reactive oxygen species (ROS) are generated within activated platelets and play an important role in regulating platelet responses to collagen and collagen-mediated thrombus formation. As a major collagen receptor, platelet-specific glycoprotein (GP)VI is a member of the immunoglobulin (Ig) superfamily, with two extracellular Ig domains, a mucin domain, a transmembrane domain and a cytoplasmic tail. GPVI forms a functional complex with the Fc receptor γ-chain (FcRγ) that, following receptor dimerization, signals via an intracellular immunoreceptor tyrosine-based activation motif (ITAM), leading to rapid activation of Src family kinase signaling pathways. Our previous studies demonstrated that an unpaired thiol in the cytoplasmic tail of GPVI undergoes rapid oxidation to form GPVI homodimers in response to ligand binding, indicating an oxidative submembranous environment in platelets after GPVI stimulation. Using a redox-sensitive fluorescent dye (H2DCF-DA) in a flow cytometric assay to measure changes in intracellular ROS, we showed generation of ROS downstream of GPVI consists of two distinct phases: an initial Syk-independent burst followed by additional Syk-dependent generation. In this review, we will discuss recent findings on the regulation of platelet function by ROS, focusing on GPVI-dependent platelet activation and thrombus formation.