| Abstract: | Prothrombin Barcelona has been isolated from a patient with a normal prothrombin antigen level but low prothrombin coagulant activity. The activation of this protein is impaired by the absence of one of the two factor Xa-catalyzed cleavages that normally lead to the formation of thrombin. Prothrombin Barcelona and prothrombin were isolated from patient plasma and normal plasma, respectively, in a single-step, high-yield immunoaffinity purification using conformation-specific antibodies immobilized on Sepharose. After reduction and alkylation, the purified proteins were subjected to trypsin hydrolysis. The resulting peptides were separated by reverse-phase high performance liquid chromatography. Comparison of the peptide maps of prothrombin Barcelona and prothrombin demonstrated that a peptide, identified as fragment 274-287 in prothrombin by automated Edman degradation, was missing in the prothrombin Barcelona digest. In the chromatogram derived from prothrombin Barcelona, an additional peptide was observed. The amino acid sequence of this peptide was Ala-Ile-Glu-Gly-Cys-Thr-Ala-Thr-Ser-Glu-Tyr-Gln-Thr-Phe-Phe-Asn-Pro-Arg, corresponding to residues 269-287 in prothrombin except for the substitution of cysteine for arginine at residue 273. The substitution of cysteine for arginine was confirmed by tryptic digestion of 14C-carboxymethylated prothrombin Barcelona. Edman degradation of fragment 269-287 indicated the association of 14C with the cysteine at residue 273. The replacement of arginine by cysteine at residue 273, adjacent to the known factor Xa cleavage site, precludes normal activation of prothrombin Barcelona by factor Xa and the generation of thrombin. |
