Characterization of recombinant human .alpha.2-antiplasmin and of mutants obtained by site-directed mutagenesis of the reactive site

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Abstract
Human .alpha.2-antiplasmin (.alpha.2AP) has been expressed in Chinese hamster ovary cells and purified from conditioned media. The recombinant protein (r.alpha.2AP) is immunologically identical with natural .alpha.2AP and indistinguishable with respect to plasmin(ogen) binding properties. Second-order rate constants (k1) for the interaction of .alpha.2AP and r.alpha.2AP with plasmin are both (1-2) .times. 107 M-1 s-1. In order to examine the effects of alterations within the reactive site of .alpha.2AP, deletions of the P1 residue Arg-364(r.alpha.2AP-.DELTA.Arg364) or the P''1 residue Met-365 (r.alpha.2AP-.DELTA.Met365) were introduced by in vitro site-directed mutagenesis. r.alpha.2AP-.DELTA.Met365 completely retains its ability to inhibit both plasmin and trypsin, indicating that .alpha.2AP has no absolute requirement for Met in the P''1 position. Unexpectedly, no increase in antithrombin activity was observed. r.alpha.2AP-.DELTA.Arg364 has lost the ability to inhibit plasmin, trypsin, and thrombin, but unlike the wild-type protein, this variant is an effective elastase inhibitor (k1 = 1.5 .times. 105 M-1 s-1).