Abstract
A mutant derivative of human prothrombin in which active site aspartate at position 419 is replaced by an asparagine (D419N-prothrombin) has been designed, expressed in recombinant Chinese hamster ovary cells, and purified to homogeneity. D419N-prothrombin was converted to the related molecules D419N-meizothrombin and D419N-thrombin by limited proteolysis by Echis carinatus and Oxyuranus scutellatus venom protease, respectively, and affinity-purified using an immobilized modified C-terminal hirudin-derived peptide. Neither D419N-thrombin nor D419N-meizothrombin exhibited thrombin activity. Titration resulted in no detection of the active site, but binding to the most specific thrombin inhibitor, hirudin, was conserved in both proteins. In vitro examinations showed that D419N-thrombin and D419N-meizothrombin bind to immobilized hirudin, neutralize hirudin in human blood plasma as well as in the purified system, and reactivate the thrombin-hirudin complex. Animal model studies confirmed that D419N-thrombin and D419N-meizothrombin act as hirudin antagonist in blood circulation without detectable effects on the coagulation system. Thus, both D419N-thrombin and D419N-meizothrombin combine for the first time hirudin-neutralizing properties with the advantages of recombinant production of human coagulation factors.
Highlights
Hirudin, the 65-residue peptide anticoagulant from the salivary gland of the European leech Hirudo medicinalis, is the most specific and most effective inhibitor of the blood protease thrombin [1, 2]
A main concern in the use of such potent and specific thrombin inhibitors is the risk of bleeding associated with the potential effect of this drug on hemostasis, when the therapy is combined with invasive procedures, fibrinolytic treatment, or the predisposition of the patient to abnormal bleeding (1, 6 –10)
To prepare inactive thrombin but to preserve hirudin binding activity, active site aspartate at position 419 was changed to asparagine in the human prothrombin cDNA
Summary
Materials—Materials were purchased from the following companies: Oxyuranus scutellatus venom protease, Sigma; Echis carinatus venom protease and AcOH1⁄7HD-CHG-Ala-Arg-pNA, Pentapharm; r-hirudin, Rhein Biotech; partial thromboplastin reagent and coagulation factor II-deficient plasma, IMMUNO AG; NHS-activated Sepharose 4 Fast Flow, Pharmacia Biotech Inc.; microtiter plates (Maxisorb), Nunc; 3,3Ј,5,5Ј-tetramethylbenzidine and hydrogen peroxide, Bio-Rad. Construction of r-prothrombin and D419N-prothrombin Expression Vectors—The expression vectors used in this study essentially are based on pSV [11]. From pSV, -galactosidase insert and polylinker sequence were removed, and a new multiple cloning site was inserted upstream of the intron. FII cDNA from pTMemc-PT2, which represents pTM3 [12] with a FII cDNA insert from pTKgpt-PTHB [13], was inserted yielding wild-type FII expression vector pSV-FII. The EcoRV/DraIII fragment from pSV-FII was substi-
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