BACKGROUND: In the fibrin-forming process, thrombin cleaves fibrinogen to fibrin, which form fibrils and then fibers, producing a gel-like clot. Thrombin also activates coagulation factor XIII (FXIII), which crosslinks fibrin γ-chains and α-chains, stabilizing the clot. Many proteins bind to fibrin, including FXIII, an established regulation of clot structure, and platelet glycoprotein VI (GPVI), whose contribution to clot function is largely unknown. FXIII is present in plasma, but the abundant FXIII in platelet cytosol becomes exposed to the surface of strongly activated platelets. OBJECTIVES: We determined if GPVI interacts with FXIII and how this might modulate clot formation. METHODS: We measured interactions between recombinant proteins of the GPVI extracellular domain: GPVI-dimer (GPVI-Fc2) or monomer (GPVIex) and FXIII proteins (nonactivated and thrombin-activated FXIII, FXIII subunits A and B) by ELISA. Binding to fibrin clots and fibrin γ-chain crosslinking were analyzed by immunoblotting. RESULTS: GPVI-dimer, but not GPVI-monomer, bound to FXIII. GPVI-dimer selectively bound to the FXIII A-subunit, but not to the B-subunit, an interaction that was decreased or abrogated by the GPVI-dimer-specific antibody mFab-F. The GPVI-dimer-FXIII interaction decreased the extent of γ-chain crosslinking, indicating a role in the regulation of clot formation. CONCLUSIONS: This is the first report of the specific interaction between GPVI-dimer and the A-subunit of FXIII, as determined in an in vitro system with defined components. GPVI-dimer-FXIII binding was inhibitory toward FXIII-catalyzed crosslinking of fibrin γ-chains in fibrin clots. This raises the possibility that GPVI-dimer may negatively modulate fibrin crosslinking induced by FXIII, lessening clot stability.