Fibrinolysis with des-kringle derivatives of plasmin and its modulation by plasma protease inhibitors.
Quantitative characterization of the interaction of des-kringle1-5-plasmin (microplasmin) with fibrin(ogen) and plasma protease inhibitors may serve as a tool for further evaluation of the role of kringle domains in the regulation of fibrinolysis. Comparison of fibrin(ogen) degradation products yielded by plasmin, miniplasmin (des-kringle1-4-plasmin), microplasmin, and trypsin on SDS gel electrophoresis indicates that the differences in the enzyme structure result in different rates of product formation, whereas the products of the four proteases are very similar in molecular weight. Kinetic parameters show that plasmin is the most efficient enzyme in fibrinogen degradation, and the kcat/KM ratio decreases in parallel with the loss of the kringle domains. The catalytic sites of the four proteases have similar affinities for fibrin (KM values between 0.12 and 0.21 microM). Trypsin has the highest catalytic constant for fibrin digestion (kcat = 0.47 s-1), and among plasmins with different kringle structures, the loss of kringle5 results in a markedly lower catalytic rate constant (kcat = 0.0076 s-1 for microplasmin vs 0.048 s-1 for miniplasmin and 0.064 s-1 for plasmin). In addition, microplasmin is inactivated by plasmin inhibitor (k" = 3.9 x 10(5) M-1 s-1) and antithrombin (k" = 1.4 x 10(3) M-1 s-1) and the rate of inactivation decreases in the presence of fibrin(ogen). Heparin (250 nM) accelerates the inactivation of microplasmin by antithrombin (k" = 10.5 x 10(3) M-1 s-1 ), whereas that by plasmin inhibitor is not affected (k" = 4.2 x 10(5) M-1 s-1).
Komorowicz
E
E
Department of Medical Biochemistry, Semmelweis University of Medicine, Budapest, Hungary.
Kolev
K
K
Machovich
R
R
D000818:Animals; D000933:Antifibrinolytic Agents / Q000097:blood; D000990:Antithrombin III / Q000378:metabolism; D001780:Blood Coagulation Tests; D002417:Cattle; D000431:Ethanol; D005338:Fibrin Fibrinogen Degradation Products / Q000378:metabolism; D005341:Fibrinolysin / Q000378:metabolism*; D005342:Fibrinolysis / Q000187:drug effects; D006801:Humans; D007700:Kinetics; D018082:Kringles / Q000187:drug effects; D010446:Peptide Fragments / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000097:blood* / Q000494:pharmacology; D012995:Solubility; D013379:Substrate Specificity; D014357:Trypsin / Q000097:blood
D000933:Antifibrinolytic Agents; D005338:Fibrin Fibrinogen Degradation Products; D010446:Peptide Fragments; D015842:Serine Proteinase Inhibitors; C019493:miniplasmin; D000431:Ethanol; C054561:microplasmin; D000990:Antithrombin III; D014357:Trypsin; D005341:Fibrinolysin