To cite this article: Djordjevic V, Kovac M, Miljic P, Murata M, Takagi A, Pruner I, Francuski D, Kojima T, Radojkovic D. A novel prothrombin mutation in two families with prominent thrombophilia -the first cases of antithrombin resistance in a Caucasian population. J Thromb Haemost 2013; 11: 1936-9.

Background The recently reported c.1787G>A mutation in the prothrombin gene leads to Arg596Gln replacement in the protein molecule (prothrombin Belgrade). This substitution impairs binding of antithrombin to thrombin and results in inherited thrombophilia, known as antithrombin resistance. Objectives We aimed to elucidate the clinical and biochemical characteristics of thrombophilia associated with antithrombin resistance in a large Serbian family with the prothrombin Belgrade mutation. Patients and methods Nineteen family members were investigated, among whom 10 were carriers of the c.1787G>A mutation. In all subjects the clinical phenotype was determined and laboratory investigations of hemostatic parameters were performed. Results Six out of the 10 mutation carriers developed thromboembolic events, mainly deep venous and mesenteric vein thrombosis. The median age of the first thrombotic event was 26.5 (12-41) years, whereas the incidence rate of first thrombosis was 2.2% per year. In all mutation carriers prothrombin activity was significantly decreased in comparison with non-carriers, clearly distinguishing each group. However, the presence of the mutation did not affect the prothrombin antigen level in plasma. The endogenous thrombin potential was significantly increased in all carriers in comparison with non-carriers, indicating the presence of blood hypercoagulability. Interestingly, levels of D-dimer and the F1+2 fragment were similar in both groups. Conclusions Although rare, the prothrombin Belgrade mutation represents strong thrombophilia with early onset of thrombosis in the investigated family. According to our results, decreased prothrombin activity may be a simple screening test for detection of this mutation in thrombotic patients.

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circulating microparticles (Mps) are procoagulant due to the surface containing phosphatidylserine (pS), which facilitates coagulation. We investigated if Mps improve hemostasis in HA plasma models. Mps isolated from pooled normal human plasma were added to severe, moderate and mild HA plasma models (0%, 2.5%, 20% FVIII). The MPs' effect on hemostasis was evaluated by calibrated automated thrombogram (CAT) and overall hemostasis potential (OHP) assays, while fibrin structure was imaged by standard confocal, stimulated emission depletion (SteD) microscopy and scanning electron microscopy (SEM). MPs partially restored thrombin generation and fibrin formation in all HA plasma models. The procoagulant effect of MPs requires PS exposure, to a less extent of contact pathway activation, but not tissue factor exposure or in vitro stimulation of Mps. Mps partially normalized the fibrin structure, and using super-resolution STED, MPs attached to fibrin were clearly resolved. in summary, our results demonstrate that pS positive Mps could improve hemostasis in HA plasma models. Hemophilia A (HA) is an inherited bleeding disorder, which is caused by a deficiency in coagulation factor VIII (FVIII) 1 and characterized by insufficient thrombin generation and fibrin formation 2. Patients with HA are characterized as having: severe (<1% of normal FVIII activity), moderate (1-5%) or mild (5-40%) HA. Prophylactic FVIII replacement therapy is recommended for severe and some moderate HA patients, in order to transform the bleeding phenotype from severe to non-severe 3. The major concerns of current therapy to HA are the development of FVIII inhibitors, and the need of frequent injections due to the short half-life time of drugs 4. Moreover, because of the high cost of therapy, HA patients in many developing countries do not have access to prophylaxis 5. Finding novel potential adjunctive therapies for HA is therefore of interest. Circulating microparticles (MPs) are small (0.1-1 µm) membrane vesicles originating from many different cells by membrane blebbing after activation, apoptosis, or high shear stress 6. MPs, as their parental cells, may provide cell surface component and participate in the coagulation process 7. In in vitro studies with plasma from healthy individuals, MPs enhance thrombin generation, fibrin clot structure and clot stability 8,9. Elevated levels of total MPs, especially tissue factor (TF) positive MPs, have been associated with cardiovascular disease and cancer 10. Few studies have investigated the role of MPs in HA. Levels of MPs in plasma have been found to be higher in untreated HA patients compared with healthy individuals 11. One previous clinical study of plasma from on-demand-treated severe HA patients showed that the level of MPs decreased after FVIII treatment, and was inversely correlated with thrombin generation and fibrin formation. These findings suggest that MPs may participate

Objectives This study was aimed to assess hemostatic disturbances in female patients with established rheumatoid arthritis (RA) in relation to menopausal status and disease activity. Method Ninety women were included in the study, 42 patients and 48 age-matched healthy controls. There were no differences between the investigated groups regarding the presence of traditional cardiovascular risk factors. Two global hemostatic assays were employed, namely endogenous thrombin potential (ETP) and overall hemostasis potential (OHP). The parameters of the ETP assay (ETP, C-max, t-lag, t-max) and OHP assay (overall coagulation potential (OCP) and overall fibrinolytic potential (OFP)) were assessed. Moreover, the parameters of the fibrin clot (lag time, Max Abs, and slope) were measured by clot turbidity and scanning electron microscopy (SEM). Both patients and controls were divided into four subgroups according to menopause status. Results The premenopausal controls differed significantly from all other subgroups in terms of diminished levels of ETP (p = 0.02), C-max (p = 0.01), OCP (p = 0.02), OHP (p = 0.001), and Max Abs (p = 0.008), while OFP (p = 0.0001) was increased. This tendency was not seen in the premenopausal RA patients compared with the postmenopausal RA patients. SEM images showed denser clots composed of thinner fibers in samples from RA patients. The disease activity measured by DAS28 correlated with OCP and OHP (r = 0.54; p = 0.001 and r = 0.44; p = 0.003, respectively) indicating persistent hypercoagulable condition in the whole group of RA patients. Conclusions Our results point towards coagulation activation in premenopausal women with established RA. The patients were well characterized, which enabled assessment in a real-life setting. Key Points • Extensive assessment points towards persistent coagulation activation in premenopausal women with established rheumatoid arthritis. • Impaired thrombin generation and fibrin formation are associated with menopause in healthy women, while rheumatoid arthritis closes the gap within patients regarding menopause. • Fibrin morphology is unfavorably altered and fibrinolysis is decreased in patients with established rheumatoid arthritis. • Increased activity of thrombin activatable fibrinolysis inhibitor (TAFI) may contribute to impaired fibrinolysis in patients with rheumatoid arthritis.