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Summary. 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 (L13GNQEPGGQTALKSPPGVCS32) 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. |
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