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Prothrombin Salakta: an abnormal prothrombin characterized by a defect in the active site of thrombin
Abstract:
An abnormal prothrombin has been detected in a 17 yr-old female originating from Tunisia. There was no history of excessive bleeding. Prothrombin time and activated partial thromboplastin time were moderately prolonged. Prothrombin activity was 15-18% when measured using either the classical one-stage and two-stage assays, or assays with Echis carinatus venom or staphylocoagulase, whereas prothrombin antigen was 100%. In keeping with current nomenclature practices, the abnormal molecule has been designated prothrombin Salakta. The electrophoretic behaviour and calcium binding properties of the abnormal prothrombin did not differ significantly from normal, as assessed by crossed immunoelectrophoresis. Prothrombin Salakta was isolated by chromatography on DEAE-Sephadex and Dextran sulphate sepharose. Electrophoretic migration of purified prothrombin Salakta on SDS polyacrylamide gels or alkaline disc gels was normal. Upon activation by either bovine factor Xa or Echis carinatus venom, thrombin activity produced by prothrombin Salakta was only 15% of normal, even when the incubation period was prolonged for 24 hours. The pattern of factor Xa-catalyzed proteolysis of prothrombin Salakta, investigated by SDS polyacrylamide gel electrophoresis, was found to be normal. These results indicated that prothrombin Salakta was characterized by a defective thrombin enzymatic activity. Thrombin Salakta was therefore isolated by heparin-sepharose chromatography. Affinity for heparin and molecular weight of thrombin Salakta were found to be normal. Biological activity of thrombin Salakta, determined by clotting assay, was 535 u/mg versus 3 200 u/mg for normal thrombin. Amidolytic activity of thrombin Salakta parallelled its clotting activity, suggesting that the defect resides either in the catalytic site or in the residues adjacent to the catalytic site and implicated as contact residues, rather than in the fibrinogen recognition site.