Met-α₂AP and Asn-α₂AP cross-linking to fibrin and their plasmin-inhibitory activity. (A) Time-dependent cross-linking of Met-α₂AP and Asn-α₂AP to fibrin by FXIIIa catalysis. Solutions of purified human α₂AP, fibrinogen, and FXIII were clotted with thrombin and CaCl₂, and at the indicated times above each gel lane, clotting was terminated by solubilizing in urea-SDS-DTT. Non–cross-linked α₂AP and α₂AP-fibrin cross-linked complexes were detected by immunoblot analysis. (B) Densitometric analysis of the percent incorporation of α₂AP into fibrin. Each data point is the mean ± SD of 3 experiments. (C) Plasmin-inhibitory activity of Met-α₂AP and Asn-α₂AP. In control sample, α₂AP was replaced with reaction buffer. Each α₂AP was incubated with an equimolar amount of plasmin for selected incubation periods, and then using a plasmin chromogenic substrate (S-2251) residual plasmin activity was assayed by a published method.10 Each data point is the average of 2 experiments.