Coagulation-induced shedding of platelet glycoprotein VI mediated by factor Xa

Abstract

This study evaluated shedding of the platelet collagen receptor, glycoprotein VI (GPVI) in human plasma. Collagen or other ligands induce metalloproteinasemediated GPVI ectodomain shedding, generating approximately 55-kDa soluble GPVI (sGPVI) and approximately 10-kDa platelet-associated fragments. In the absence of GPVI ligands, coagulation of platelet-rich plasma from healthy persons induced GPVI shedding, independent of added tissue factor, but inhibitable by metalloproteinase inhibitor, GM6001. Factor Xa (FXa) common to intrinsic and tissue factor-mediated coagulation pathways was critical for sGPVI release because (1) shedding was strongly blocked by the FXa-selective inhibitor rivaroxaban but not FIIa (thrombin) inhibitors dabigatran or hirudin; (2) Russell viper venom that directly activates FX generated sGPVI, with complete inhibition by enoxaparin (inhibits FXa and FIIa) but not hirudin; (3) impaired GPVI shedding during coagulation of washed platelets resuspended in FX-depleted plasma was restored by adding purified FX; and (4) purified FXa induced GM6001-inhibitable GPVI shedding from washed platelets. In 29 patients with disseminated intravascular coagulation, mean plasma sGPVI was 53.9 ng/mL (95% confidence interval, 39.9-72.8 ng/mL) compared with 12.5 ng/mL (95% confidence interval, 9.0-17.3 ng/mL) in thrombocytopenic controls (n ‫؍‬ 36, P < .0001), and 14.6 ng/mL (95% confidence interval, 7.9-27.1 ng/mL) in healthy subjects (n ‫؍‬ 25, P ‫؍‬ .002). In conclusion, coagulation-induced GPVI shedding via FXa down-regulates GPVI under procoagulant conditions. FXa inhibitors have an unexpected role in preventing GPVI down-regulation. (Blood. 2011;117(14):3912-3920) IntroductionIn atherothrombotic disease, thrombus formation at arterial shear rates involves initial platelet adhesion and activation at sites of vascular injury/collagen exposure, and activation of coagulation that generates active thrombin and stabilizes the thrombus by fibrin. 1 Bidirectional regulation of platelet function and coagulation is important in both the pathology of atherothrombotic diseases, such as heart attack or acute ischemic stroke, and in the clinical use of antiplatelet or anticoagulant drugs. [2][3][4][5] The platelet collagen receptor, glycoprotein (GP)VI, is a promising target of antithrombotic therapy because it is not only critical for initiating platelet activation and thrombus formation at arterial shear rates but also promotes thrombus growth and stability because of effects on coagulation. [6][7][8][9] In experimental models, depletion of platelet GPVI results in decreased responsiveness to collagen, impaired procoagulant activity and thrombin generation ex vivo and in vivo, and protection from tissue factor (TF)-induced thromboembolism. 9-11 GPVI agonists also increase phosphatidylserine exposure and microparticle formation, whereas stimulation of platelets with a GPVI ligand (convulxin) and thrombin increase active factor X (FXa) and thrombin, regulated by a subpopulation of platelets with increased binding of c...

New Fundamentals in Hemostasis

“…Once activated, GPVI is easily shed from the platelet surface by the extracellular proteases ADAM-10 and -17, which abolishes GPVI-induced responses (24). Interestingly, under clotting conditions this proteolytic cleavage appears to be regulated by FXa, but not by thrombin (4). This finding demonstrates a novel negative-feedback mechanism of the coagulation process on a platelet activation pathway.…”

Pathologic shear triggers shedding of vascular receptors: a novel mechanism for down-regulation of platelet glycoprotein VI in stenosed coronary vessels

“…Recently, we have shown that active Factor X can promote ADAM10-mediated shedding of GPVI independent of platelet activation. 49 Shear may unfold GPVI into a conformer able to be cleaved by ADAM10, consistent with the "memory of shear" in which GPVI continues to be shed after the discontinuance of shear ( Figures 1E and 5) and the presence of only mature ADAM10 on platelets ( Figure 1G), an effect possibly elicited via shearinduced changes to actin and cytoskeletal networks, 50 which are directly linked to GPIb-IX-V and coassociated GPVI. 26 The finding that shear influences GPVI cleavage warrants further research into shear-dependent shedding of other ADAM substrates on platelets and other cell types in vivo.…”

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

“…Exposure to elevated shear or FXa induces shedding that is also independent of platelet activation. 62,63 Fibrin binds to multiple proteins on the platelet surface; 1 or more of these interactions may contribute to GPVI shedding, possibly by clustering and juxtaposing GPVI to ADAM10 and/or other metalloproteinases. Further, soluble fibrin can also reduce GPVI ligand-induced function in platelets, presumably by blockade or steric hindrance of the ligand binding site, as the effect was not blocked by GM6001.…”