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 (rFXIII-A) showed a similar specific transglutaminase activity comparable to the human native FXIII-A from platelet. Using these rFXIII-A mutants, the activation kinetics by thrombin and the enzymatic properties of the activated rFXIII-A were characterized. rFXIII-A (V34L) and rFXIII-A (V34L/V35L) mutants were activated by thrombin much faster than those of wild type rFXIII-A and V35L variant. However, the activated rFXIII-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.
Blood Coagulation Tests ; Catalysis ; Enzyme Activation/genetics ; Escherichia coli/genetics ; Factor XIII/*genetics/*metabolism ; Fibrin/metabolism ; Human ; Immunoblotting ; Leucine/genetics ; Mutagenesis, Site-Directed ; *Polymorphism (Genetics) ; Recombinant Proteins/genetics/metabolism ; Thrombin/metabolism ; Valine/genetics

AIMTo study the effect of recombinant hirudin (rH) on tPA-induced fibrinolysis and the possible mechanism of its action.

METHODSThe effect of rH on thrombin-fibrin complex (Th-Fn) was detected by 99mTc labeled rH. In the in vitro clot lysis, tPA as plasminogen activator, and recalcified plasma as plasminogen resource were used to study the influence of rH on fibrinolysis by detecting TAFIa, D-Dimer and FXIII.

RESULTSIn a canine model of femoral artery thrombosis, a clear radioactivity strip was imaged in 30 - 60 min on a part image, and the femoral vein thrombosis developed at 30 min. rH efficiently inhibited clot regeneration. Addition of TM could inhibit clot lysis obviously, and CPI could shorten the delay of clot lysis which due to TAFIa. There was a dose-dependent relationship with TM concentration and TAFI activation. FXIII activation was inhibited by low concentration of rH ( < or = 0.2 u x mL(-1)), and the level of fibrinolysis product, D-Dimer, increased.

CONCLUSIONrH could inhibit the thrombin binding to fibrin. rH inhibited the activation of TAFI and FXIII by combining with thrombin which resulted in enhancement of thrombolysis.

Animals ; Blood Coagulation ; drug effects ; Carboxypeptidase B2 ; metabolism ; Carboxypeptidases ; antagonists & inhibitors ; Dogs ; Factor XIII ; metabolism ; Femoral Artery ; Femoral Vein ; Fibrinolysis ; drug effects ; Fibrinolytic Agents ; pharmacology ; Hirudins ; genetics ; pharmacology ; Male ; Plant Proteins ; pharmacology ; Protease Inhibitors ; Recombinant Proteins ; pharmacology ; Thrombomodulin ; metabolism ; Thrombosis ; metabolism ; Venous Thrombosis ; metabolism