Dimeric Glycoprotein VI Binds to Collagen but Not to Fibrin

Abstract

Platelet glycoprotein VI (GPVI) acts as a decisive collagen receptor in atherothrombosis. Besides collagen, injured atherosclerotic plaques expose tissue factor (TF) that triggers fibrin formation. Two recent studies reported that platelet GPVI also functions as fibrin receptor, which would importantly widen the mode of action of GPVI-targeted antithrombotic drugs. We studied the binding of two GPVI fusion proteins to fibrin under static and arterial flow conditions. Fibrin was prepared from purified fibrinogen or generated more physiologically from endogenous fibrinogen by coagulating plasma with thrombin. Fibrin formation was also triggered by exposing TF-coated surfaces or human atherosclerotic plaque slices to arterially flowing blood. By binding studies and advanced optical imaging, we found that recombinant dimeric GPVI-Fc fusion proteins with Fc from either IgG1 (GPVI-Fc1) or IgG2 (GPVI-Fc2) bound to collagen fibres, but neither to fibrin prepared from purified fibrinogen obtained from three suppliers, nor to physiological fibrin formed by thrombin in plasma or triggered by exposing TF or atherosclerotic plaque slices to arterially flowing blood. Our findings do not support a role of dimeric platelet GPVI as receptor for fibrin. This is important for the understanding of plaque-triggered platelet thrombus formation and is clinically relevant for future GPVI-targeting therapies with recombinant GPVI-Fc and anti-GPVI antibodies.

Functional significance of the platelet immune receptors GPVI and CLEC-2

“…In comparison, CLEC-2 has a minor-to-negligible role in hemostasis, as shown by in vitro flow studies on collagen and by (34)(35)(36)(37), although interestingly, fibrinogen only activates human GPVI (37). There are contrasting reports on whether the binding to monomeric or dimeric GPVI occurs, possibly due to use of different forms of recombinant or shed GPVI (35,36,38,39). The binding site for fibrin(ogen) resides in the D-region, with D-dimer inhibiting platelet activation by collagen at concentrations that lie at the upper end of those reached in vivo (36,40).…”

Platelets as Modulators of Cerebral Ischemia/Reperfusion Injury

“…A second phase II study in patients with coronary artery disease has been initiated and is in the phase of patient recruitment (NCT 03312855). Revacept binds to immobilized collagen and thereby indirectly prevents platelet adhesion and activation at sites of collagen exposure whereas it does not interact with fibrin (60). ACT017 (9O12), a humanized Fab fragment against GPVI, which was designed to directly inhibit GPVI on the platelet surface, was shown to inhibit collagen-induced platelet aggregation ex vivo and there were no signs of thrombocytopenia or excessive bleeding (61).…”

Proteolytic processing of platelet receptors

“…The fibrin interaction with GPVI is mediated by the D‐dimer region of fibrin and for GPVI shedding to occur, fibrin must be polymerized . Whether fibrin can bind platelet GPVI monomer or dimer remains a matter for debate; however, dimeric GPVI‐Fc fusion proteins do not engage fibrin . Similarly, the fibrin‐binding site within GPVI is contentious.…”

“…109 Whether fibrin can bind platelet GPVI monomer or dimer 33,108 remains a matter for debate; however, dimeric GPVI-Fc fusion proteins do not engage fibrin. 110 Similarly, the fibrin-binding site within GPVI is contentious.…”