Soluble fibrin preparations inhibit the reaction of plasmin with alpha 2-macroglobulin. Comparison with alpha 2-antiplasmin and leupeptin.
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.
Anonick
P K
PK
Department of Pathology, University of Virginia Health Sciences Center, Charlottesville 22908.
Gonias
S L
SL
D002863:Chromogenic Compounds; D003488:Cyanogen Bromide; D005337:Fibrin / Q000378:metabolism; D005338:Fibrin Fibrinogen Degradation Products / Q000494:pharmacology*; D005340:Fibrinogen / Q000494:pharmacology; D005341:Fibrinolysin / Q000037:antagonists & inhibitors* / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D007976:Leupeptins / Q000494:pharmacology; D009842:Oligopeptides / Q000378:metabolism; D000511:alpha-Macroglobulins / Q000494:pharmacology*
D002863:Chromogenic Compounds; D005338:Fibrin Fibrinogen Degradation Products; D007976:Leupeptins; D009842:Oligopeptides; D000511:alpha-Macroglobulins; C011468:fibrinmonomer; C024668:plasmin-alpha(2)-macroglobulin complex; C018788:valyl-leucyl-lysine 4-nitroanilide; D005337:Fibrin; D005340:Fibrinogen; D005341:Fibrinolysin; C032854:leupeptin; D003488:Cyanogen Bromide