Noncovalent Interaction of α2-Antiplasmin with Fibrin(ogen): Localization of α2-Antiplasmin-Binding Sites

Covalent incorporation (cross-linking) of plasmin inhibitor alpha(2)-antiplasmin (alpha(2)-AP) into fibrin clots increases their resistance to fibrinolysis. We hypothesized that alpha(2)-AP may also interact noncovalently with fibrin prior to its covalent cross-linking. To test this hypothesis, we studied binding of alpha(2)-AP to fibrin(ogen) and its fragments by an enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance. The experiments revealed that alpha(2)-AP binds to polymeric fibrin and surface-adsorbed fibrin(ogen), while no binding was observed with fibrinogen in solution. To localize the alpha(2)-AP-binding sites, we studied the interaction of alpha(2)-AP with the fibrin(ogen)-derived D1, D-D, and E-3 fragments, and the recombinant alpha C region and its constituents, alpha C connector and alpha C domain and its subdomains, which together encompass practically the whole fibrin(ogen) molecule. In the ELISA, alpha(2)-AP bound to immobilized D-1, D-D, alpha C region, alpha C domain, and its C-terminal subdomain. The binding was Lys-independent and was not inhibited by plasminogen or tPA. Furthermore, the affinity of alpha(2)-AP for D-D was significantly increased in the presence of plasminogen, while that to the alpha C domain remained unaffected. Altogether, these results indicate that the fibrin(ogen) D region and the C-terminal subdomain of the alpha C domain contain high-affinity alpha(2)-AP-binding sites that are cryptic in fibrinogen and exposed in fibrin or adsorbed fibrinogen, and the presence of plasminogen facilitates interaction of alpha(2)-AP with the D regions. The discovered noncovalent interaction of alpha(2)-AP with fibrin may contribute to regulation of the initial stage of fibrinolysis and provide proper orientation of the cross-linking sites to facilitate covalent cross-linking of alpha(2)-AP to the fibrin clot.