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Fibrinolytic compromise by simultaneous administration of site-directed inhibitors of thrombin
1Department of Pharmacology, Loyola University Chicago, Stritch School of Medicine, Maywood, Illinois.
Abstract:
Newly developed synthetic and recombinant thrombin inhibitors possess strong anticoagulant effects. Despite these effects, interactions of these agents with enzymes in the fibrinolytic network result in the modulation of such proteases as t-PA, u-PA and streptokinase. The inhibitory spectrum of several thrombin inhibitors [D-Phe-Pro-Arg-H(GYKI 14166), D-MePhe-Pro-Arg-H(GYKI 14766), Boc-D-Phe-Pro-Arg-H (GYKI 14451), Ac-D-Phe-Pro-boroArg-OH (DuP 714), recombinant hirudin (r-Hir) and unfractionated porcine mucosal heparin complexed with antithrombin III (Heparin/AT-III)] was studied towards various serine proteases such as tissue plasminogen activator (t-PA), plasmin, plasminogen/streptokinase complex, urokinase and kallikrein. Aprotinin was also studied in the same systems as the thrombin inhibitors. All four tripeptide derivatives were found to inhibit t-PA, plasmin and plasminogen/streptokinase complex at micromolar concentrations (IC50: 0.57 mM-3.3 microM). Boc-D-Phe-Pro-Arg-H and Ac-D-Phe-Pro-boroArg-OH also inhibited urokinase, while Ac-D-Phe-Pro-boroArg-OH inhibited kallikrein as well (IC50: 0.15 mM-16 microM). In contrast, r-Hir and Heparin/AT-III did not inhibit any of these enzymes at millimolar concentrations (IC50 > or = 1 mM). Aprotinin inhibited plasmin, plasminogen/streptokinase complex and kallikrein at micromolar concentrations (IC50: 3.1-0.85 microM). In a rabbit thrombolysis model, where pre-formed clots are lysed by streptokinase, simultaneous administration of D-MePhe-Pro-Arg-H or Ac-D-Phe-Pro-boroArg-OH, at concentrations approximately 1 mumol/kg, i.v. resulted in complete inhibition of the fibrinolytic process. Aprotinin at 0.1 mumol/kg, i.v. produced similar inhibition. These results demonstrate that thrombin inhibitors may exert significant antiprotease actions against various fibrinolytic enzymes.
Insights
Newly developed thrombin inhibitors show significant antiprotease activity against fibrinolytic enzymes like t-PA and plasmin. These potent anticoagulant agents can modulate the fibrinolytic network, impacting clot lysis processes.
Area of Science:
- Biochemistry
- Pharmacology
- Molecular Biology
Background:
- Synthetic and recombinant thrombin inhibitors are effective anticoagulants.
- These inhibitors may interact with enzymes in the fibrinolytic system, potentially affecting clot breakdown.
- Understanding these interactions is crucial for evaluating their overall therapeutic profile.
Purpose of the Study:
- To investigate the inhibitory effects of various thrombin inhibitors on key fibrinolytic enzymes.
- To determine the spectrum of protease inhibition for selected tripeptide derivatives, recombinant hirudin, and heparin complexed with antithrombin III.
- To assess the impact of these inhibitors on a rabbit thrombolysis model.
Main Methods:
- Enzyme inhibition assays were performed using purified serine proteases including tissue plasminogen activator (t-PA), plasmin, urokinase, and kallikrein.
- The inhibitory activity of thrombin inhibitors [D-Phe-Pro-Arg-H, D-MePhe-Pro-Arg-H, Boc-D-Phe-Pro-Arg-H, Ac-D-Phe-Pro-boroArg-OH], recombinant hirudin (r-Hir), and Heparin/AT-III was quantified (IC50 values).
- A rabbit thrombolysis model utilizing streptokinase was employed to evaluate the in vivo effects of selected inhibitors.
Main Results:
- Four tripeptide thrombin inhibitors demonstrated significant inhibition of t-PA, plasmin, and plasminogen/streptokinase complex at micromolar concentrations.
- Boc-D-Phe-Pro-Arg-H and Ac-D-Phe-Pro-boroArg-OH also inhibited urokinase, with Ac-D-Phe-Pro-boroArg-OH additionally inhibiting kallikrein.
- Recombinant hirudin and Heparin/AT-III showed no significant inhibitory activity against these fibrinolytic enzymes at millimolar concentrations.
- In vivo, D-MePhe-Pro-Arg-H and Ac-D-Phe-Pro-boroArg-OH completely inhibited streptokinase-induced thrombolysis in rabbits.
- Aprotinin also exhibited inhibitory effects on plasmin, plasminogen/streptokinase complex, and kallikrein, and demonstrated similar in vivo inhibition of thrombolysis.
Conclusions:
- Thrombin inhibitors, particularly tripeptide derivatives, possess significant antiprotease activity against multiple fibrinolytic enzymes.
- These findings highlight potential off-target effects of thrombin inhibitors on the fibrinolytic system.
- The observed modulation of fibrinolysis by these agents warrants further consideration in their clinical application and development.