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Multiple active forms of thrombin. IV. Relative activities of meizothrombins
1Department of Biochemistry, University of Vermont College of Medicine, Burlington 05405-0068.
The Journal of Biological Chemistry
|June 25, 1990
Summary
Meizothrombin intermediates show significantly lower procoagulant activity than alpha-thrombin but retain substantial anticoagulant function, particularly in protein C activation. This suggests distinct roles in hemostasis.
Area of Science:
- Biochemistry
- Hemostasis and Thrombosis
- Enzymology
Background:
- Prothrombin activation generates thrombin, a key enzyme in blood coagulation.
- Intermediates like meizothrombin are formed during prothrombin activation.
- Understanding the distinct enzymatic properties of these intermediates is crucial for comprehending hemostasis regulation.
Purpose of the Study:
- To characterize the enzymatic activity of purified prothrombin activation intermediates, meizothrombin and meizothrombin(desF1).
- To compare the procoagulant and anticoagulant functions of meizothrombin intermediates with alpha-thrombin.
- To elucidate the role of thrombomodulin and phospholipids in modulating these activities.
Main Methods:
- Purification of active meizothrombin and meizothrombin(desF1) with minimal autolysis.
- Enzymatic assays comparing activity towards macromolecular substrates (factor V, fibrinogen, platelets) and chromogenic substrates.
- Protein C activation assays in the presence and absence of thrombomodulin and phospholipid vesicles.
- Determination of kinetic parameters (Km, catalytic efficiency) and binding affinities (dissociation constants).
Main Results:
- Meizothrombin and meizothrombin(desF1) exhibited significantly lower procoagulant activity (1-10%) compared to alpha-thrombin (100%) towards factor V, fibrinogen, and platelets.
- Catalytic efficiency towards a chromogenic substrate (S2238) was similar across all tested enzymes, indicating intact catalytic triads.
- Meizothrombin intermediates retained substantial anticoagulant activity, activating protein C at 75% the rate of alpha-thrombin.
- Thrombomodulin binding was observed for all enzymes, but meizothrombin-thrombomodulin complex showed reduced protein C activation rates, partly due to higher Km, which was normalized by phospholipids.
Conclusions:
- Meizothrombin generation is primarily directed towards anticoagulant functions, such as protein C activation.
- Alpha-thrombin generation is predominantly involved in procoagulant functions, including fibrinogen clotting and platelet aggregation.
- The distinct substrate specificities and regulatory mechanisms highlight the finely tuned roles of thrombin species in hemostasis.