Reconstitution of non-carrier, heterozygous and homozygous prothrombin belgrade mutation carrier plasma using recombinant proteins

Abstract

Introduction: The prothrombin Belgrade variant (c.1787G>A, p.Arg596Gln) is a rare mutation found in Serbia, Japan, China, America, India and leads to antithrombin resistance. Prothrombin Belgrade mutation influencesthrombin-antithrombin interactions and leadsto impaired inactivation of mutated thrombin. Also, it affectssodium binding site in thrombin, which isimportant forswitching from fast thrombin configuration (coagulant properties) to slow configuration (anticoagulant properties). It has only been found in a heterozygous state, which could mean that homozygous carriers are incompatible with life. By using prothrombin (FII) deficient plasma, we could reconstitute plasma of wild type, heterozygous and homozygous carrier, which could give more insight into the mechanism of this mutation. Methods: Recombinant wild type and mutated prothrombin were generated by transient transfection in HEK293T cell line. Western blot analysis was performed to test the efficiency of transfection. Human Prothrombin ELISA (Nordic BioSite, Sweden) was used in order to measure recombinant prothrombin concentration. Overall Hemostasis Potential (OHP) assay was performed to assess recombinant protein activity. Recombinant wild type and mutated prothrombin were added to FII deficient plasma (Siemens, Germany) in order to create reconstituted plasma, in the final concentration of 0.1 mg/mL, as it is approximately the level of prothrombin in human plasma. Results: Reconstituted plasma samples that correspond to non-carrier, heterozygous carrier, and homozygous mutation carrier plasma were reconstructed. Recombinant proteinstested by OHP assay were functional. Conclusion: Reconstituted plasma samples allow us to examine the mechanism of prothrombin Belgrade mutation in various assays and in homozygous form as well