RATIONAL DESIGN, RECOMBINANT PREPARATION, AND IN-VITRO AND IN-VIVO CHARACTERIZATION OF HUMAN PROTHROMBIN-DERIVED HIRUDIN ANTAGONISTS

A mutant derivative of human prothrombin in which active site aspartat e 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 re lated molecules D419N-meizothrombin and D419N-thrombin by limited prot eolysis by Echis carinatus and Oxyuranus scutellatus venom protease, r espectively, and affinity-purified using an immobilized modified C-ter minal hirudin-derived peptide, Neither D419N-thrombin nor D419N-meizot hrombin exhibited thrombin activity, Titration resulted in no detectio n of the active site, but binding to the most specific thrombin inhibi tor, hirudin, was conserved in both proteins, In vitro examinations sh owed that D419N-thrombin and D419N-meizothrombin bind to immobilized h irudin, neutralize hirudin in human blood plasma as well as in the pur ified system, and reactivate the thrombin-hirudin complex, Animal mode l 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-meizothro mbin combine for the first time hirudin-neutralizing properties with t he advantages of recombinant production of human coagulation factors.