MOLECULAR CHARACTERIZATION OF NEW PROTHROMBIN MUTATIONS RESPONSIBLE FOR THROMBOPHILIA

Background: The Arg596Trp prothrombin variant, also known as prothrombin Padua 2, was first described by Bulato et al. (2016) in two unrelated Italian families with a history of venous thromboembolism. Unlike most prothrombin defects, which are typically associated with bleeding, this mutation confers a prothrombotic phenotype. Functional studies showed that the mutant thrombin is resistant to inhibition by antithrombin, resulting in prolonged thrombin activity and a significantly increased risk of thrombosis. Accordingly, this variant represents a rare but clinically significant form of hereditary thrombophilia. Aims: Although Bulato et al. (2016) primarily investigated the impaired interaction between prothrombin Padua 2 and antithrombin, other functional effects of the variant have not been explored. The Arg596 residue is critical for thrombin’s interaction with protein C, and substitutions at this site can compromise thrombin-mediated activation of protein C, a key anticoagulant mechanism. Based on these considerations, the present PhD project had two main objectives: (1) to evaluate the impact of the Arg596Trp mutation on the protein C anticoagulant pathway, and (2) to identify laboratory assays able to detect the defect more accurately, as conventional coagulation tests fail to reveal the underlying functional abnormality. Methods: All carriers of the Arg596Trp prothrombin variant underwent comprehensive coagulation screening. The functional status of the protein C anticoagulant system was evaluated using global coagulation assays, including thrombin generation tests performed on platelet-poor plasma and whole blood, both in the absence and presence of thrombomodulin. Increased residual ETP and peak ratios compared to normal controls indicated impaired protein C pathway function. Additionally, rotational thromboelastometry (ROTEM) was used to assess clot formation and stability in whole blood using INTEM, EXTEM, and FIBTEM assays, to provide complementary information on the overall hemostatic profile. Results: Platelet-poor plasma thrombin generation without thrombomodulin showed a prolonged start-to-tail in one of the carriers studied (41.3 min) compared to normal controls (21.7 min), reflecting delayed thrombin inactivation. In the presence of thrombomodulin, residual ETP and peak ratios in this individual were comparable to normal controls. In the whole blood thrombin generation assay, thrombomodulin markedly reduced ETP and peak in the normal control (to 51% and 57% of baseline), whereas in the proband the reduction was moderate (68% and 78%), consistent with impaired protein C anticoagulant function. ROTEM analysis of whole blood from the proband using INTEM, EXTEM, and FIBTEM showed CT, CFT, and MCF within normal ranges, indicating normal coagulation function. Conclusions: Our findings suggest that Prothrombin Padua 2 leads to a prothrombotic phenotype in carriers by two complementary mechanisms: resistance of thrombin to antithrombin and partial impairment of protein C activation. Traditional coagulation tests, including PT, aPTT, and prothrombin activity, fail to detect the defect. Thrombin generation assays in platelet-poor plasma and whole blood, performed both in the absence and presence of thrombomodulin, revealed two key abnormalities: a prolonged start-to-tail, indicating resistance to antithrombin, and increased residual ETP and peak ratios, reflecting impaired protein C activation. In conclusion, thrombin generation, together with antithrombin resistance assay and thrombin–antithrombin complex measurement, could represent useful diagnostic tools, guiding clinicians toward F2 gene sequencing to confirm the mutation.