Platelet Glycoprotein VI Dimerization, an Active Process Inducing Receptor Competence, Is an Indicator of Platelet Reactivity

Abstract

Objective-The immune receptor homologue glycoprotein VI (GPVI)/FcR receptor ␥ chain complex is primarily responsible for platelet activation by collagen. There is growing evidence that optimal binding of GPVI to collagen depends on the assembly of GPVI dimers. The valence of GPVI on resting platelets needs to be clearly established because platelet avidity for collagen would be greater if GPVI is constitutively expressed as a dimer than as a monomer. Methods and Results-Using a monoclonal antibody (9E18) that preferentially binds to GPVI dimers, we found that GPVI was maintained in a monomeric form on human resting platelets under the control of intraplatelet cAMP concentration. Activation by soluble agonists or von Willebrand factor induced a shift toward GPVI dimerization related to increased platelet adhesion to collagen. A correlation between platelet binding of 9E18 and P-selectin exposure was observed in patients experiencing coronary artery disease, and antagonists of the ADP receptor P2Y 12 limited ADP-induced GPVI dimerization. Key Words: antiplatelet drugs Ⅲ collagen Ⅲ monoclonal antibodies Ⅲ platelets Ⅲ glycoprotein VI P latelet adhesion at sites of vascular damage is required for normal hemostasis. Circulating platelets adhere to proteins of the subendothelial matrix exposed by vessel injury in a process involving several receptors. Collagen fibers are highly thrombogenic, and the platelet glycoprotein VI (GPVI) predominantly mediates collagen-induced platelet responses. Conclusion-TheGPVI is a platelet-specific receptor of the immunoglobulin (Ig) superfamily containing 2 extracellular Ig domains, a single transmembrane domain, and a short cytoplasmic tail. [1][2][3] GPVI shares with other receptors of the same family the particularity that the extracellular ligand-binding domain and the intracellular signaling domain are on separate subunits. GPVI signals through the noncovalently associated immune receptor adaptor FcR␥. 4 The receptor is assembled via a transmembrane interaction between Asp11 in the FcR␥ homodimer and Arg273 of GPVI. On stimulation, the Tyr residues of the immunoreceptor tyrosine-based activation motif of FcR␥ are phosphorylated in an early, obligatory event triggering the signaling cascade. See accompanying article on page 552There is growing evidence that optimal binding of GPVI to collagen depends on the formation of GPVI dimers at the platelet surface. Miura et al, 5 using recombinant proteins, were the first to report that collagen binds to the dimeric form but not to the monomeric form of GPVI and that only the former was able to inhibit collagen-induced platelet activation. Crystallographic data showing dimerization of GPVI ectodomains, 6 studies using synthetic peptides with differentially spaced GPVI binding motifs to activate the receptor in platelets, 7 and studies using chemical cross-linking agents 8 have strongly reinforced the notion that GPVI functions as a dimer. However, the valence of GPVI on resting and on activated platelets is still a matter of debate. It was ...

Tetraspanin Tspan9 regulates platelet collagen receptor GPVI lateral diffusion and activation

“…Moreover, GPVI lateral mobility on the cell surface was reduced by approximately 50% in the absence of Tspan9. Since GPVI clustering is important for its activation and signalling [10, 11], a reduction in its lateral diffusion could explain the observed platelet aggregation and secretion defects. No defects in GPVI clustering on collagen were observed by super-resolution imaging, but these experiments were done on fully spread platelets and would not detect a delay in the kinetics of clustering.…”

“…This is followed by downstream phosphorylation and assembly of a signalosome nucleated by the adapter protein LAT, phospholipase Cγ2 (PLCγ2) activation, Ca 2+ mobilisation, platelet shape change, granule secretion, integrin activation and platelet spreading and aggregation [1, 4]. GPVI recognises collagen as a dimer, and a proportion of GPVI exists as a dimer on resting platelets, with dimer levels increasing upon platelet activation [10, 11]. GPVI has been reported to be lipid raft-associated [12–14] and more recently to be a component of a distinct type of microdomain formed by tetraspanin transmembrane proteins [15].…”

The ABCC4 membrane transporter modulates platelet aggregation

“…34 Elevation of cAMP can also attenuate GPVI signaling by decreasing surface expression of GPVI. 35 These results suggest that balancing the intracellular level of cAMP is vital to GPVI localization.…”

Soluble GPVI is elevated in injured patients: shedding is mediated by fibrin activation of GPVI

“…14 On resting platelets, GPVI is predominantly monomeric, but clusters and dimerizes on activation, [15][16][17] triggering ITAM signaling involving Src family kinases. 17,18 In hemostasis and thrombosis, GPVI supports platelet adhesion and aggregation.…”