Fibrinolysis is amplified by converting α₂-antiplasmin from a plasmin inhibitor to a substrate

α₂-antiplasmin (α₂-AP) is the fast serpin inhibitor of plasmin and appears to limit the success of treatment for thrombosis. We examined the mechanisms through which monoclonal antibodies (mAbs) against α₂-AP amplify fibrinolysis. The mAbs RWR, 49 and 77 interfered with the ability of α₂-AP to inhibit plasmin, microplasmin and trypsin. In solution, mAbs 49 and 77 bound to α₂-AP with 5-fold to 10-fold higher relative affinity than mAb-RWR, while mAb-RWR bound with greater avidity to immobilized or denatured α₂-AP. Binding studies with chimeric α₂-APs revealed that none of the mAbs bound to sites in α₂-AP that form putative contacts with plasmin, namely the carboxy terminal lysines of α₂-AP, or the reactive center loop in the serpin domain of α₂-AP. Rather, mAb-RWR recognized an epitope in the amino-terminus of α₂-AP (L 13 GNQEPGGQTALKSPPGVCS 32) near the site at which α₂-AP cross-links to fibrin. mAbs 49 and 77 bound to another conformational epitope in the serpin domain of α₂-AP. mAbs 49 and 77 markedly increased the stoichiometry of plasmin inhibition by α₂-AP (from 1.1 ± 0.1 to 51 ± 4 and 67 ± 7) indicating that they convert α₂-AP from an inhibitor to a substrate of plasmin. This was confirmed by sodium dodecylsulfate polyacrylamide gel electrophoresis analysis showing cleavage of α₂-AP by plasmin in the presence of these mAbs. In summary, these mAbs appear to act at sites distinct from known α₂-AP-plasmin contacts to enhance fibrinolysis by converting α₂-AP from an inhibitor to a plasmin substrate.