Prothrombin Tokushima: Characterization of Dysfunctional Thrombin Derived From a Variant of Human Pro thrombin

A mutant prothrombin. designated prothrombin Tokushima. was purified from plasma of a proband with 12% of normal plasma clotting activity and 42% of normal prothrombin antigen. The purified preparation gave a single band with the same mobility as that of ‘�prothrombin” by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The factor Xa-catalyzed proteolysis of prothrombin Tokushima examined by SDS-PAGE was found to be identical to that of “prothrombin.” Subsequently thrombin Tokushima was prepared by CM-Sepharose CL-6B column chromatography after prothrombin activation by factor Xa. The molecular weight of thrombin Tokushima estimated by SDS-PAGE was identical to that of “thrombin.” Thrombin Tokushima exhibited less than 22% of normal clotting activity. and the value of kcat/Km (�mol/ C ONGENITAL dysprothrombinemia is a rare coagulation disorder. Only 16 variants have been reported,’’7 all of them being characterized by a decrease in the functional level of prothrombin relative to antigenic level of prothrombin. Five of the prothrombin variants thus far identified have been purified and characterized. In the case of prothrombin Barcelona2 and prothrombin Madrid,8 the functional defecthas been shown to be a specific impairment of one of the two factor Xa-catalyzed cleavages, whereas in the case of prothrombin Quick,6 prothrombin Metz,9 and prothrombin Salakta,’7 the defect is confined to the thrombin portion of the molecule. In 1983 we reported the first case of dysprothrombinemia in Japan as prothrombin Tokushima.’3’4 The maternaland paternal sides have heterozygotes, respectively, for dysprothrombinemia and hypoprothrombinemia. The proband was a double heterozygote for prothrombin Tokushima and hypoprothrombinemia and therefore synthesized only the abnormal molecule. In this paper the physicochemical and enzymatic properties of prothrombin Tokushima are presented. These results demonstrate that prothrombin Tokushima is a new variant having an abnormality in the thrombin portion of the molecule. L�1 second1) was less than one tenth of that of “thrombin” when Boc-Val-Pro-Arg-4-methylcoumaryl-7-amide was used as a substrate. However. active site titration using p-nitrophenyl-p’-guanidinobenzoate failed to detect any difference between the two. Thrombin Tokushima was 2.5% as effective as “thrombin” in inducing platelet aggregation. Interaction of thrombin Tokushima with antithrombin Ill was much slower than “thrombin” when followed by SDS-PAGE. Based on the residual thrombin activity. it was 33% as effective as “thrombin” in forming a complex with antithrombin Ill. These results indicate that the molecular defect resides in the thrombin portion of prothrombin Tokushima and that the binding sites for various substrates appear to be greatly impaired. a 1987 by Grune & Stratton. Inc. mol/L sodium phosphate, pH 6.5, and thrombin was eluted with a linear NaCI gradient (0 to 0.7 mol/L NaCI) in the same buffer. Titration of thrombin with p-nitrophenyl-p’-guanidinobenzoate . HCI (p-NPGB). The titration of thrombin with p-NPGB (Nutritional Biochemicals, Cleveland, OH) was performed by the method of Chase and Shaw.2’ Immediate burst of p-nitrophenol produced by the stoichiometric reaction of the catalytic site of