Noncovalent Interaction of α_2-Antiplasmin with Fibrin(ogen): Localization of α_2-Antiplasmin-Binding Sites

摘要

Covalent incorporation (cross-linking) of plasmon inhibitor α_2-antiplasmin (α_2-AP) into fibrin clots increases their resistance to fibrinolysis. We hypothesized that α_2-AP may also interact noncovalently with fibrin prior to its covalent cross-linking. To test this hypothesis, we studied binding of α_2-AP to fibrin(ogen) and its fragments by an enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance. The experiments revealed that α_2-AP binds to polymeric fibrin and surfaceadsorbed fibrin(ogen), while no binding was observed with fibrinogen in solution. To localize the α_2-APbinding sites, we studied the interaction of α_2-AP with the fibrin(ogen)-derived D_1, D-D, and E_3 fragments, and the recombinant αC region and its constituents, αC connector and αC domain and its subdomains, which together encompass practically the whole fibrin(ogen) molecule. In the ELISA, α_2-AP bound to immobilized D_1, D-D, aC region, αC domain, and its C-terminal subdomain. The binding was Lys-independent and was not inhibited by plasminogen or IPA. Furthermore, the affinity of α_2-AP for D-D was significantly increased in the presence of plasminogen, while that to the αC domain remained unaffected. Altogether, these results indicate that the fibrin(ogen) D region and the C-terminal subdomain of the αC domain contain high-affinity α_2-AP-binding sites that are cryptic in fibrinogen and exposed in fibrin or adsorbed fibrinogen, and the presence of plasminogen facilitates interaction of α_2-AP with the D regions. The discovered noncovalent interaction of α_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 α_2-AP to the fibrin clot.