Single-Molecule Force Spectroscopy of the Interactions Between Platelet Integrin αIIbβ3 and Monomeric Fibrin

Abstract

Platelet-fibrin interactions under hydrodynamic blood shear are mediated by the integrin αIIbβ3, but the mechanism of αIIbβ3 binding to fibrin is largely unknown, although interactions with fibrinogen have been extensively studied. We used the optical trap to measure forces required to separate a laser-trappedbead coated with monomeric fibrin from a pedestal coatedwith purified αIIbβ3. Experiments were performed with recombinant fibrin obtained from thrombin-treated fibrinogen, either wild-type or variants lacking putative integrin-binding sites. Integrin-fibrin interactions manifested as a bimodal force histogram with rupture forces from 20 pN to 140 pN, similar to αIIbβ3-fibrinogen but with somewhat higher binding probability. To test a role of the γ-chain C-terminal 400-411 dodecapeptide, the major αIIbβ3-binding site in fibrinogen, the most abundant fibrin (γA/γA) was replaced with a splicing variant (γ’/γ’), in which the γC-terminus has new amino acids from 408 to 427. Unexpectedly, the lack of the γC400-411 motif did not affect the ability of fibrin to interact with αIIbβ3, suggesting that this structure may not be a major integrin-binding site in fibrin. At the same time, fibrin-integrin interactions were partially inhibited by the γC-dodecapeptide, indicating that the γC400-411 motif still may be involved, perhaps indirectly. Two RGD motifs, one located in the αC region and the other in the coiled-coil connector, were tested as the potential binding sites by using fibrin(ogen) variants αD574E and αD97E. Both of them had a reduced integrin-binding strength and displayed the cumulative binding probability about 2/3 of that of the wild-type fibrin, suggesting that the RGD motifs play a role in the αIIbβ3-fibrin interactions. Free γC-dodecapeptide did not affect the reactivity of the D574E and D97E mutants. The results suggest that the αIIbβ3-fibrin interactions involve the RGD sites rather than the γC400-411 motif.