MOLECULAR DEFECT IN PROTHROMBIN MADRID: SUBSTITUTION OF ARGININE 273 BY CYSTEINE PRECLUDES ACTIVATION

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Abstract
Prothrombin Madrid, a mutant prothrombin, was detected in a patient with a excessive bleeding history. The defect was characterized by a low coagulant activity contrasting with a normal level of prothrombin antigen in plasma. Activation of the purified protein was impaired by the absence of one of the two factor Xa catalyzed cleavages, generating meizothrombin which expressed a thrombin-like activity but was inactive on fibrinogen (Guillin et al., Ann. N.Y. Acad. Sci. 370:414, 1981). Prothrombin and prothrombin Madrid were isolated directly from plasma, with high yield, by immunoaffinity chromatography using conformation specific antibodies immobilized on Sepharose. After reduction and alkylation, purified proteins were hydrolyzed by trypsin. Resulting peptides were separated by reverse phase HPLC. Comparison of the two peptide maps showed that the prothrombin Madrid digest contained an additional peptide, identified by automated Edman degradation as residues 269 to 287 in prothrombin with the substitution of cysteine for arginine at position 273. Peptide 274—287, present in the prothrombin digest, was missing in the prothrombin Madrid digest. The mutation, precluding cleavage by factor Xa and normal generation of thrombin, is identical to the one described for prothrombin Barcelona. The two patients families are not related, raising the possibility that the gene coding for the cysteine 273 mutation in prothrombin is more common than anticipated. Of the seven mutants of vitamin E-dependant blood clotting proteins structurally characterized to date, three are functionally defective due to the presence of the propeptide on the mature amino-ternfinus (factor IX Cambridge, Oxford 3 and San Dimas) and three are due to an alteration that precludes zymogen activation (faotor TX Chapel Hill, prothrombin Barcelona and Madrid). This sample remains too small to anticipate the different classes of point mutations seen in the human population but functional abnormalities of protein processing, metal and lipid binding, zymogen activation, substrate recognition and enzyme catalysis will likely be important phenotypes. However genetic defects may be limited to a discrete group of point mutations that have significant functional implication for the proteins

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