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Characterization of a stable form of human meizothrombin derived from recombinant prothrombin (R155A, R271A, and
H C Côté1, W K Stevens, L Bajzar
1Department of Biochemistry, University of British Columbia, Vancouver, Canada.
Abstract:
Meizothrombin is a transient intermediate produced during the activation of prothrombin by the prothrombinase complex. Because meizothrombin is very sensitive to further activation and autolysis, its isolation is possible only in the presence of active site thrombin inhibitors. This complicates studies of the activities and functions of meizothrombin. As a model, we have expressed a mutant human prothrombin cDNA (R155A, R271A, R284A) with three of the cleavage sites modified so that they are no longer cleaved by factor Xa or thrombin. Several stable baby hamster kidney cell lines were isolated that secreted up to 20 micrograms/ml of carboxylated mutant prothrombin. After purification, the mutant prothrombin was activated by the prothrombinase complex or by ecarin, resulting in the formation of a meizothrombin-like molecule. Electrophoretic analysis and NH2-terminal sequence analysis were consistent with cleavage of a single bond between Arg320-Ile321 and proper processing of the prepropeptide. The meizothrombin was stable for weeks at 4 degrees C. Activation in the presence of dansylarginine N-(3-ethyl-1,5-pentanediyl) amide confirmed the conversion of prothrombin via meizothrombin. Compared with human plasma-derived thrombin, recombinant meizothrombin demonstrated approximately 7% clotting activity, 100% p-toluene-sulfonylarginine methyl ester esterase activity, and approximately 35% S2238 amidolytic activity, and could attenuate fibrinolysis.
Insights
Researchers created a stable meizothrombin model by modifying human prothrombin. This breakthrough allows for detailed study of meizothrombin
Area of Science:
- Biochemistry
- Molecular Biology
- Hemostasis
Background:
- Meizothrombin is a crucial but unstable intermediate in prothrombin activation.
- Its transient nature and sensitivity to degradation hinder functional studies.
- Understanding meizothrombin is key to comprehending thrombin generation and coagulation disorders.
Purpose of the Study:
- To develop a stable, recombinant meizothrombin model for detailed functional analysis.
- To overcome the limitations of studying native, transient meizothrombin.
- To characterize the enzymatic activities of the novel meizothrombin construct.
Main Methods:
- Engineered a mutant human prothrombin cDNA (R155A, R271A, R284A) to prevent cleavage at specific sites.
- Expressed and purified the carboxylated mutant prothrombin in baby hamster kidney cells.
- Activated the mutant prothrombin using the prothrombinase complex or ecarin to generate a stable meizothrombin-like molecule.
- Analyzed the resulting molecule using electrophoresis, N-terminal sequencing, and enzymatic activity assays.
Main Results:
- Successfully produced and isolated a stable meizothrombin-like molecule from recombinant prothrombin.
- Confirmed the correct cleavage site (Arg320-Ile321) and processing.
- The recombinant meizothrombin exhibited distinct enzymatic properties compared to plasma-derived thrombin: 7% clotting activity, 100% esterase activity, and 35% amidolytic activity.
- Demonstrated the ability of recombinant meizothrombin to attenuate fibrinolysis.
Conclusions:
- The engineered mutant prothrombin provides a robust system for generating stable meizothrombin.
- This stable model facilitates in-depth investigation of meizothrombin's enzymatic functions and biological roles.
- Recombinant meizothrombin possesses unique enzymatic characteristics and modulates fibrinolysis, offering new insights into coagulation pathways.