Phosphatidylserine positive microparticles improve hemostasis in in-vitro hemophilia A plasma models

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

The Silence Speaks, but We Do Not Listen: Synonymous c.1824C gt T Gene Variant in the Last Exon of the Prothrombin Gene as a New Prothrombotic Risk Factor

BACKGROUND: Thrombosis is a major global disease burden with almost 60% of cases related to underlying heredity and most cases still idiopathic. Synonymous single nucleotide polymorphisms (sSNPs) are considered silent and phenotypically neutral. Our previous study revealed a novel synonymous FII c.1824C gt T variant as a potential risk factor for pregnancy loss, but it has not yet been associated with thrombotic diseases. METHODS: To determine the frequency of the FII c.1824C gt T variant we have sequenced patients' DNA. Prothrombin RNA expression was measured by quantitative PCR. Functional analyses included routine hemostasis tests, western blotting and ELISA to determine prothrombin levels in plasma, and global hemostasis assays for thrombin and fibrin generation in carriers of the FII c.1824C gt T variant. Scanning electron mi- croscopy was used to examine the structure of fibrin clots. RESULTS: Frequency of the FII c.1824C gt T variant was significantly increased in patients with venous thromboembolism and cerebrovascular insult. Examination in vitro demonstrated increased expression of prothrombin mRNA in FII c.1824C gt T transfected cells. Our ex vivo study of FII c.1824C gt T carriers showed that the presence of this variant was associated with hyperprothrom-binemia, hypofibrinolysis, and formation of densely packed fibrin clots resistant to fibrinolysis. CONCLUSION: Our data indicate that FII c.1824C gt T, although a synonymous variant, leads to the development of a prothrombotic phenotype and could represent a new prothrombotic risk factor. As a silent variant, FII c.1824C gt T would probably be overlooked during genetic screening, and our results show that it could not be detected in routine laboratory tests

Effects of rivaroxaban and dabigatran on global hemostasis in patients with atrial fibrillation

The study was aimed to evaluate the effects of two standard doses of rivaroxaban and dabigatran on global hemostatic assays in patients with atrial fibrillation. The study included 52 patients treated with rivaroxaban (15/20 mg), 50 on dabigatran (110/150 mg) and 20 healthy individuals. Platelet-poor plasma was used for determination of three global hemostatic assays, namely endogenous thrombin potential (ETP), calibrated automated thrombogram (CAT) and overall hemostasis potential (OHP). Rivaroxaban and dabigatran reduced ETP (P lt 0.01) although OHP (P lt 0.05) was diminished only by dabigatran. Strong correlations were noticed between ETP parameters and the plasma concentrations of rivaroxaban (ETP, rUS0.51; c-max, rUS0.85; t-lag, rU0.83; t-max, rU0.66) as well as with plasma concentration of dabigatran (ETP, rUS0.75; c-max, rUS0.74; t-lag, rU0.73; t-max, rU0.52). Analysis of dabigatran concentrations under 50 ng/ml showed that ETP parameter has area under the concentration-time curvereceiver operating characteristic value of 0.879 (95% confidence interval 0.776-0.980). Dabigatran treatment paradoxically increased area under the concentration-time curve and peak values although rivaroxaban decreased peak values (P lt 0.01). However, significant correlation between CAT parameters and plasma concentration of both direct oral anticoagulants was not observed. We confirmed that the CAT assay is inappropriate for estimation of dabigatran effects and is not fully sensitive as regards rivaroxaban. The ETP assay can potentially be the appropriate method for estimation of global hemostatic capacity as regards both direct oral anticoagulants. The role of OHP needs to be confirmed in additional studies. ETP parameter of chromogenic assay has promising potential in exclusion of high plasma concentrations of dabigatran