Dimers of the platelet collagen receptor glycoprotein VI bind specifically to fibrin fibers during clot formation, but not to intact fibrinogen.

Masaaki Moroi, Isuru Induruwa, Richard W Farndale, Stephanie M Jung

Journal of thrombosis and haemostasis : JTH 2021 Aug

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The platelet collagen receptor glycoprotein VI (GPVI) has an independent role as a receptor for fibrin produced via the coagulation cascade. However, various reports of GPVI binding to immobilized fibrin(ogen) are not consistent. As a collagen receptor, GPVI-dimer is the functional form, but whether GPVI dimers or monomers bind to fibrin remains controversial. To resolve this, we analyzed GPVI binding to nascent fibrin clots, which more closely approximate physiological conditions. ELISA using biotinyl-fibrinogen immobilized on streptavidin-coated wells indicated that GPVI dimers do not bind intact fibrinogen. Clots were formed by adding thrombin to a mixture of near-plasma level of fibrinogen and recombinant GPVI ectodomain: GPVI dimer (GPVI-Fc2 or Revacept) or monomer (GPVI-His: single chain of Revacept GPVI domain, with His tag). Clot-bound proteins were analyzed by SDS-PAGE/immunoblotting. GPVI-dimer bound to noncrosslinked fibrin clots with classical one-site binding kinetics, with µM-level KD , and to crosslinked clots with higher affinity. Anti-GPVI-dimer (mFab-F) inhibited the binding. However, GPVI-His binding to either type of clot was nonsaturable and nearly linear, indicating very low affinity or nonspecific binding. In clots formed in the presence of platelets, clot-bound platelet-derived proteins were integrin αIIbβ3, present at high levels, and GPVI. We conclude that dimeric GPVI is the receptor for fibrin, exhibiting a similar KD to those obtained for its binding to fibrinogen D-fragment and D-dimer, suggesting that fibrin(ogen)'s GPVI-binding site becomes exposed after fibrin formation or cleavage to fragment D. Analysis of platelets bound to fibrin clots indicates that platelet GPVI binds to fibrin fibers comprising the clot. © 2021 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis.

Citation

Masaaki Moroi, Isuru Induruwa, Richard W Farndale, Stephanie M Jung. Dimers of the platelet collagen receptor glycoprotein VI bind specifically to fibrin fibers during clot formation, but not to intact fibrinogen. Journal of thrombosis and haemostasis : JTH. 2021 Aug;19(8):2056-2067