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Kinetics of the reaction of thrombin and alpha 2-macroglobulin.

ABSTRACT: The kinetics of the reaction of alpha 2-macroglobulin (alpha 2M) with human thrombin were studied by recording the appearance of thiol groups spectrophotometrically and by measuring the distribution of protein species by denaturing non-reducing gel electrophoresis. The goals were to study the relation between the formation of various covalent enzyme-inhibitor complex species and the appearance of free thiol, and from the kinetic analysis, to try to characterize the chemical nature of the protein complexes. The kinetics of thiol-group release were observed to be biphasic, the early phase showing second-order behaviour, results consistent with previous reports in the literature. The observed second-order rate constant for thiol-group release was found to be faster than the second-order rate constant for the disappearance of the band corresponding to native alpha 2M on gel electrophoresis. This may be a reflection of the multiple products formed from the thioester. Alternatively, it is possible that covalent-bond formation is slower than some enzyme-induced change in the thioester centre, and this may be suggestive evidence for a reactive alpha 2M centre that does not contain an intact thioester. The kinetics of covalent-bond formation were found to be consistent with the internal cross-link of several alpha 2M chains by the bound proteinase, providing further evidence that the very-high-Mr species seen on gels may arise from dimers of the alpha 2M molecule held together by covalent bonds to the enzyme.

SUBMITTER: Feinman RD

PROVIDER: S-EPMC1152762 | biostudies-other | 1985 Oct

REPOSITORIES: biostudies-other

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Project description:Streptokinase-human plasmin complex (Sk-hPm) reacted rapidly with purified mouse alpha 2-macroglobulin (m alpha 2M) in vitro at 37 degrees C. Approx. 98% of the plasmin in Sk-hPm bound covalently to at least one m alpha 2M subunit. Most of the streptokinase dissociated (95%). The rate of Sk-hPm inactivation clearly depended on the m alpha 2M concentration. With 1.2 microM-m alpha 2M, 50% of the Sk-hPm (0.02 microM) reacted in less than 50 s. A double-reciprocal plot comparing pseudo-first-order rate constants (kapp.) and m alpha 2M concentration yielded a second-order rate constant of 2.3 x 10(4) M-1.s-1 (r = 0.97). This value is an approximation, since Sk-hPm preparations are heterogeneous. Sk-hPm reacted with human alpha 2M (h alpha 2M), forming alpha 2M-plasmin complex (98% covalent). More than 99% of the streptokinase dissociated. The rate of reaction of Sk-hPm with h alpha 2M did not clearly depend on inhibitor concentration. The kapp. values determined with 0.6-1.2 microM-h alpha 2M were decreased 10-20-fold compared with m alpha 2M. In order to study the effect of Sk-hPm heterogeneity on the reaction with alpha 2M, the proteinase was incubated for various amounts of time at 37 degrees C before addition of inhibitor. The enzyme amidase activity was maximal within 5 min; however, reaction of Sk-hPm with m alpha 2M or h alpha 2M was most extensive after 20 min and 2 h respectively. After incubation for more than 1 h, Sk-hPm acquired fibrinogenolytic activity, suggesting plasmin dissociation. Therefore the enhanced reaction of h alpha 2M with 'older' Sk-hPm preparations may have resulted in part from dissociated plasmin or 'plasmin-like' species. By contrast, the reaction of Sk-hPm with m alpha 2M was most rapid when the proteinase preparation was free of plasmin, indicating direct reaction of Sk-hPm with m alpha 2M as the only major mechanism. Finally, streptokinase-cat plasminogen complex reacted more extensively with m alpha 2M than with h alpha 2M, suggesting that m alpha 2M may be a superior inhibitor with this class of plasminogen activators in general.

Project description:The kinetics of plasmin inhibition by alpha 2-antiplasmin (alpha 2AP), alpha 2-macroglobulin (alpha 2M) and leupeptin were studied in the presence of fibrin monomer (Fn) and CNBr fragments of fibrinogen (Fg-CNBr). Active plasmin was detected in continuous and discontinuous assays using the chromogenic substrate D-Val-L-Leu-L-Lys p-nitroanilide hydrochloride (S-2251). The two 'fibrin-like' preparations functioned as hyperbolic mixed-type inhibitors of S-2251 hydrolysis. The dissociation constants (KF) for the binding of plasmin to Fn and Fg-CNBr were 22 nM and 17 nM respectively. Fn and Fg-CNBr inhibited the reaction of plasmin with alpha 2AP: the extent of inhibition depended on the fibrin concentration. In the presence of 800 nM-Fn or 800 nM-Fg-CNBr, the experimental second-order rate constant (K"app.) was decreased from 2.4 x 10(7) M-1.s-1 to 1.2 x 10(6) and 5.3 x 10(5) M-1.s-1 respectively. The effect of Fn and Fg-CNBr on the rate of plasmin inhibition by alpha 2M was even greater. The k"app. value was decreased from 4.0 x 10(5) M-1.s-1 to 8.0 x 10(2) and 1.3 x 10(3) M-1.s-1 in the presence of 800 nM-Fn and -Fg-CNBr respectively. By contrast, the fibrin preparations caused only a small change in the rate of plasmin inhibition by leupeptin. The maximum change in k"app. was 3-fold. All plasmin inhibition curves were linear, suggesting that free and fibrin-bound forms of plasmin remained in equilibrium during the course of reaction with proteinase inhibitors. Fn and Fg-CNBr had no effect on the reaction of miniplasmin with S-2251, alpha 2AP or alpha 2M. When 125I-plasmin was incubated with Fg-CNBr and then allowed to react with a premixed solution of alpha 2AP and alpha 2M, the Fg-CNBr did not significantly change the percentage of plasmin bound to alpha 2AP. These experiments demonstrate that the reaction of plasmin with alpha 2M is inhibited by the non-covalent binding of plasmin to fibrin. We propose that plasmin bound to the surface of a clot is protected from inhibition by alpha 2M as well as by alpha 2AP.

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Kinetics of the reaction of thrombin and alpha 2-macroglobulin.

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