Studies on prothrombin.
thesisposted on 19.11.2015, 09:06 by Linda Jane. Beecroft
Clotting and chromogenic assays utilising Echis carinatus venom were set up to determine the total prothrombin-related material (PRM) in plasma and liver fractions. The chromogenic assay revealed an increase in the PRM of microsomes prepared from warfarin-treated rats, reflecting a build-up of prothrombin precursors. A clear relationship between warfarin dosage and the increase in PRM in the microsomal fraction was demonstrated. The chromogenic assay was not only useful for the determination of PRM in turbid samples. It was also used to assess specific activity at each stage in the purification of prothrombin, in the kinetic analysis of ecarin- generated thrombin, and as a functional test for anti-prothrombin specificity of anti sera, raised against the prothrombin concentrate. It was hoped that the anticoagulant, PP888, could be used to investigate post-translational modification of prothrombin. However, PP888 was found to be much less active than warfarin in vivo and was inactive in the in vitro vitamin K-dependent carboxylation assay. This indicated that a metabolite of PP888 was the active anticoagulant. The structurally related compound, PP493, was found to inhibit vitamin K-dependent carboxylation in both solubilised and non-solubilised microsomes. The processing of prothrombin precursors in the rat liver is both complicated (five different isoelectric forms of precursor have been identified) and poorly understood. It was decided that the best way to investigate post-translational modification was to isolate the primary, unmodified translation product. To accomplish this, messenger RNA, isolated from rat liver, was translated in vitro, using a rabbit reticulocyte translation system. Prothrombin-related translation products were then identified by immunoprecipitation, using antisera raised against the prothrombin concentrate. Two prothrombin-related translation products were obtained in this way. They were characterised biochemically and their properties compared with those of prothrombin. Finally, attempts were made to modify the primary translation products and mimic in vivo processing. The potential of a system where proteins were translated in the presence of rat liver microsomes is discussed.