Abstract
Change in fibrin stabilizing activity of factor XIII A subunit (FXIII-A) caused by a specific mutation, Val34Leu, is recently implicated to incidences of pathophysiology of thrombosis. In an effort to understand the effect of Val34Leu on enhanced catalytic role of FXIII-A, wild type human factor XIII A (HFXIII-A) and mutant HFXIII-A: HFXIII-A (V34L), HFXIII-A (V35L) and HFXIII-A (V34L/V35L) cDNA were expressed in E.coli system where the purified recombinant FXIII-A (gammaFXIII-A) showed a similar specific transglutaminase activity comparable to the human native FXIII-A from platelet. Using these gammaFXIII-A mutants, the activation kinetics by thrombin and the enzymatic properties of the activated gammaFXIII-A were characterized. gammaFXIII-A (V34L) and gammaFXIII-A (V34L/V35L) mutants were activated by thrombin much faster than those of wild type gammagFXIII-A and V35L variant. However, the activated gammaFXIII-A and mutants showed the identical catalytic efficiency as measured by in vitro assay. These results suggest that ready activation caused by a specific mutation of neighboring thrombin cleavage site(s) in the activation peptide of FXIII-A like V34L resulted in the real-time amount of the activated factor XIII-A that could influence the outcome of fibrin stabilization in vivo such as alpha2-plasmin inhibitor crosslinking to fibrin, a reaction known to be dependent on the initial concentration of active factor-XIII-A.
Highlights
The cross-linking of fibrin by activated blood coagulation factor XIII (FXIII) is the last enzymatic step in the cascade of blood coagulation
In order to confirm the rFXIII-A expressed in E.coli retain their intrinsic catalytic properties, transglutaminase activities of the FXIII A subunit (FXIII-A) and mutants were compared with factor XIII-A from platelet in vitro amine incorporation assay as described in the method
HFXIII-A subunit is synthesized as a monomeric form and exists in a dimeric zymogen where the 37 N-terminal amino acid activation peptide (AP) of each subunit crosses over dimer interface and partially occludes the opening of the catalytic cavity in the second subunit and preclude substrate binding to the zymogen (Arg-11 of first monomer binds to Asp-343 of core domain in second monomer) (Sadasivan and Yee, 2000)
Summary
The cross-linking of fibrin by activated blood coagulation factor XIII (FXIII) is the last enzymatic step in the cascade of blood coagulation. FXIII A subunit (FXIII-A), transglutaminase (EC 2.3.2.13) produced by thrombin activation of the protransglutaminase, catalyzes the covalent intermolecular polymerization of fibrin, through formation of ε-(γ-glutamyl) lysine crosslinks during blood clotting (Mandel, 1971; Folk and Chung, 1973; Fork and Finlayson, 1973; 1977; Losowsky and Miloszewiski, 1977). The biochemical and physiological effects exerted by a specific mutation, Val34Leu, which is earlier reported to have greater transglutaminase activity (Kangsadalampai and Board, 1998) and associated with incidences with deep vein thrombosis and myocardial infarction are somewhat perplexing there is reports that the Val34Leu polymorphism affects the function of FXIII by increasing the rate of FXIII activation by thrombin (Ariaens et al, 2000; Wartiovaara et al, 2000). Structural properties (primary, secondary structure and 3D structure coordinates) of each enzymes from rabbit and human were quite similar except greater variances in amino acid sequences in the activation peptide and found residues near the thrombin cleavage site (Arg37) as Leu and Leu in rabbit enzyme in place of Val and Val of human enzyme
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