Reaction of human alpha(2)-antiplasmin and plasmin - Stopped-flow fluorescence kinetics

The interaction of human plasmin with human alpha(2)-antiplasmin was measured in the presence and absence of lysine-binding ligands using the corresponding active site fluorescence changes. The stopped-flow method allows for direct determination of reliable values of the second order rate constant for the fast association step of plasmin and alpha(2)-antiplasmin in the absence of another interacting compound, e.g. a plasmin substrate. At pH 7.4, 25 degrees C, k(1) = 2.2 x 10(7) M(-1) s(-1) was obtained. Substantial reductions in k(1) were seen in the presence of trans-4-(aminomethyl)cyclohexane-1-carboxylic acid at concentrations corresponding to lysine-binding site interactions at kringle 4 of plasmin; at saturation the rate constant is reduced 20-fold, whereas the effect of saturation of kringle 1 is only a 2-fold reduction. It is thus found that the interaction of alpha(2)-antiplasmin with the lysine-binding site of kringle 1 is of little importance compared with that of kringle 4 in regulating the inhibition reaction of plasmin with alpha(2)-antiplasmin. Similar results were recently obtained for the bovine plasmin-bovine alpha(2)-antiplasmin reaction (Christensen ct al. (1995) Biochem. J. 305, 97-102).