Kinetics of the reaction of streptokinase-plasmin complex with purified human and mouse alpha 2-macroglobulin. Implications for mechanism.
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.
Anonick
P K
PK
Department of Pathology, University of Virginia Health Sciences Center, Charlottesville 22908.
Vetter
W H
WH
Gonias
S L
SL
D000818:Animals; D004338:Drug Combinations / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007700:Kinetics; D051379:Mice; D008970:Molecular Weight; D010958:Plasminogen / Q000378:metabolism*; D011485:Protein Binding; D013300:Streptokinase / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism; D000511:alpha-Macroglobulins / Q000302:isolation & purification / Q000378:metabolism*
D004338:Drug Combinations; D000511:alpha-Macroglobulins; C049423:streptokinase-plasminogen complex; D010958:Plasminogen; D013300:Streptokinase; D014357:Trypsin