Assessment of hemostatic disturbances in women with established rheumatoid arthritis

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

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

Diagnostic Accuracy in Acute Venous Thromboembolism: Comparing D-Dimer, Thrombin Generation, Overall Hemostatic Potential, and Fibrin Monomers

Introduction For acute venous thromboembolism (VTE), a biomarker with higher specificity than D-dimer would be of great clinical use. Thrombin generation and overall hemostatic potential (OHP) reflect the hemostatic balance by globally assessing multiple coagulation factors and inhibitors. These tests discriminate between healthy controls and patients with a prothrombotic tendency but have yet to be established as clinical biomarkers of VTE. Objective This study compares endogenous thrombin potential (ETP) and OHP to D-dimer and fibrin monomers (FM) in outpatients with suspected VTE. Methods A cross-sectional diagnostic study where 954 patients with suspected pulmonary embolism or deep venous thrombosis were recruited consecutively from the medical emergency department at Karolinska University Hospital. D-dimer, FM, OHP, and ETP were analyzed in a subpopulation of 60 patients with VTE and 98 matched controls without VTE. VTE was verified either by ultrasonography or computed tomography and clinical data were collected from medical records. Results Compared with healthy controls, both VTE and non-VTE patients displayed prothrombotic profiles in OHP and ETP. D-dimer, FM, ETP area under the curve (AUC), and ETP T lag were significantly different between patients with VTE and non-VTE. The largest receiver-operating characteristic AUCs for discrimination between VTE and non-VTE, were found in D-dimer with 0.94, FM 0.77, and ETP AUC 0.65. No useful cutoff could be identified for the ETP or the OHP assay. Conclusion Compared with D-dimer, neither ETP nor OHP were clinically viable biomarkers of acute venous thrombosis. The data indicated that a large portion of the emergency patients with suspected VTE were in a prothrombotic state.Funding agencies: Foundation for Coagulation Research at Karolinska Institutet, the ScandinavianResearch Foundation for Varicose Veins and other Venous Diseases, FoU Region Stockholm, the Swedish Society onThrombosis and Haemostasis with Leo Pharma.</p

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

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

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

Clinical Implications of Discrepancy between One-stage Clotting and Chromogenic Factor IX Activity in Haemophilia B

BACKGROUND: Discrepancy in factor IX activity (FIX:C) between one-stage assay (OSA) and chromogenic substrate assay (CSA) in patients with haemophilia B (PwHB) introduces challenges for clinical management.AIM: To study the differences in FIX activity using OSA and CSA in moderate and mild haemophilia B (HB), their impact on classification of severity, and correlation with genotype.METHODS: Single-centre study including 21 genotyped and clinically characterised PwHB. FIX:C by OSA was measured using ActinFSL (Siemens) and CSA by Biophen (Hyphen). In addition, in-vitro experiments with wild-type FIX were performed. Reproducibility of CSA was assessed between three European coagulation laboratories.RESULTS: FIX:C by CSA was consistently lower than by OSA, with 10/17 PwHB having a more severe haemophila type by CSA. OSA displayed a more accurate description of the clinical bleeding severity, compared with CSA. A two-fold difference between OSA:CSA FIX:C was present in 12/17 PwHB; all patients had genetic missense variants in the FIX serine protease domain. Discrepancy was also observed with healthy control FIX, most significant for values below 0.10 IU/mL. Assessment of samples with low FIX:C showed excellent reproducibility of the CSA results between the laboratories.CONCLUSIONS: FIX activity was consistently higher by OSA compared to the CSA. Assessing FIX by CSA alone would have led to diagnosis of a more severe haemophilia type in a significant proportion of patients. Our study suggests using both OSA and CSA FIX:C together with genotyping to classify haemophilia B severity and provide essential information for clinical management