Stopped-flow fluorescence kinetics of bovine α2-antiplasmin inhibition of bovine midiplasmin

Abstract

In the conversion of bovine plasminogen to bovine plasmin not only the expected urokinase-catalysed cleavage of Arg-557-Val-558, and the following autocatalytic cleavage separating the N-terminal peptide 1-77 from the heavy chain of plasmin, but also a cleavage at Arg-342-Met-343 between kringles 3 and 4 is seen. Here, kinetic studies of the interaction of bovine alpha 2-antiplasmin with bovine plasmin were performed on isolated bovine midiplasmin (lacking kringles 1-3) and on bovine plasmin containing all of the activation products from the bovine plasminogen. A series of experiments using stopped-flow fluorescence fast kinetics as well as conventional techniques suggests a reaction model in accordance with the one known for the human system. First, a tight complex (K1 in the nanomolar range) is formed in a fast reaction step; and second, a tightening of this complex occurs in a slow reaction step. The final complex is indeed so tight (Ki < or = pM), that the reaction for many practical purposes is legitimately considered irreversible. The stopped-flow method allows for the determination of reliable values of the second-order rate constant for the fast association step. At pH 7.4 and 25 degrees C, k+1 = 1.7 x 10(6) M-1 s-1 was obtained in the absence and k+1 = 0.9 x 10(6) M-1.s-1 in the presence of the kringles 1-3 domain of bovine plasmin. In contrast to this, substantial reductions of k+1 were seen in the presence of concentrations of 6-amino-hexanoic acid corresponding to lysine-binding-site interactions and far too low to be attributed to active-site interactions with the bovine plasmins (for each, Ki = 42 mM). All in all, the data indicated that the lysine-binding site(s) not of kringle 1, but of midiplasmin (those of kringles 4 and 5) are regulating the inhibition reaction.

Reaction of human α2‐antiplasmin and plasmin Stopped‐flow fluorescence kinetics

“…The possibility of a more prominent role of the LBSs of kringles 4 and 5 rather than of the kringle I-LBS as regulatory domains in the human system has recently received attention [l 1,12,15,33,46,47].…”

Effects of Extracellular DNA on Plasminogen Activation and Fibrinolysis

“…Finally, the effect of dsDNA (0 -10 g/ml) and oligo(dAT) 33 (0 -10 M) on the rate constant for the second slow step of the reaction (k ϩ2 ; Scheme 2) was studied by monitoring the residual PL amidolytic activity after mixing high concentrations of PL (0.1-0.5 M) with equimolar amounts of ␣ 2 AP in the presence of the fluorogenic substrate, as described previously (63,77). Although the values of k ϩ2 did not change with an increase in the dsDNA concentration and slightly decreased with an increase in the [oligo(dAT) 33 ] (Fig.…”

PA-PG-PL System in Dairy Animals and its Significance: A Review

“…Surprisingly, a further hydrolysis occurs in the bovine system, as the Arg342-Met343 bond is susceptible to cleavage by plasmin, resulting in a truncated form containing kringle 4, kringle 5 and the serine proteinase part (Benfeldt, 1992). This form has been called midiplasmin (Christensen et al, 1995).…”