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Background: Direct oral factor (F)Xa inhibitors are widely used as alternatives to conventional vitamin K antagonists in managing venous thromboembolism and nonvalvular atrial fibrillation. Unfortunately, bleeding-related adverse events remain a major concern in clinical practice. In case of bleeding or emergency surgery, rapid-onset reversal agents may be required to counteract the anticoagulant activity., Objectives: The ability of FXa variants to bypass the direct oral FXa inhibitors was assessed., Methods: Human FXa variants were generated through substitution of phenylalanine 174 (F174) for either alanine, isoleucine, or serine. FXa variants were stably expressed in HEK293 cells and purified to homogeneity using ion-exchange chromatography., Results: F174-substituted human FX variants demonstrated efficacy in restoring thrombin generation in plasma containing direct FXa inhibitors (apixaban, rivaroxaban, edoxaban). Their ability to bypass the anticoagulant effects stems from a significantly reduced sensitivity for the direct FXa inhibitors due to a decrease in binding affinity determined using molecular dynamics simulations and free energy computation. Furthermore, F174 modification resulted in a partial loss of inhibition by tissue factor pathway inhibitor, enhancing the procoagulant effect of F174-substituted FX. Consequently, the F174A- and F174S-substituted FX variants effectively counteracted the effects of 2 widely used anticoagulants, apixaban and rivaroxaban, in plasma of atrial fibrillation and venous thromboembolism patients., Conclusion: These human FX variants have the potential to serve as a rescue reversal strategy to overcome the effect of direct FXa inhibitors in case of life-threatening bleeding events or emergency surgical interventions., Competing Interests: Declaration of competing interests P.H.R. and D. Verhoef own equity in VarmX B.V. T.M.H. and S.T. own equity in Coagulation Profile B.V. M.H.A.B. has received consultancy fees from VarmX B.V. (all fees to the institution) and is inventor on intellectual property related to this work. The remaining authors declare no competing financial interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Blood coagulation factor Va serves an indispensable role in hemostasis as cofactor for the serine protease factor Xa. In the presence of an anionic phospholipid membrane and calcium ions, factors Va and Xa assemble into the prothrombinase complex. Following formation of the ternary complex with the macromolecular zymogen substrate prothrombin, the latter is rapidly converted into thrombin, the key regulatory enzyme of coagulation. Over the years, multiple binding sites have been identified in factor Va that play a role in the interaction of the cofactor with factor Xa, prothrombin, or the anionic phospholipid membrane surface. In this review, an overview of the currently available information on these interactive sites in factor Va is provided, and data from biochemical approaches and 3D structural protein complex models are discussed. The structural models have been generated in recent years and provide novel insights into the molecular requirements for assembly of both the prothrombinase and the ternary prothrombinase-prothrombin complexes. Integrated knowledge of functionally important regions in factor Va will allow for a better understanding of factor Va cofactor activity., (© 2019 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis.)

Essentials Elevated procoagulant levels are associated with an increased risk of venous thrombosis (VT). The dependency on concurrent increased factor levels and VT was analyzed in a large study. Factor VIII (FVIII) and von Willebrand factor (VWF) were associated with the highest VT risk. The risks for other procoagulant factor levels were largely explained by FVIII and VWF. SUMMARY: Background Coagulation factors are essential for robust clot formation. However, elevated levels of procoagulant factors are associated with an increased risk of venous thrombosis (VT). The precise contribution of these factors to the development of VT is not yet understood. Objectives We determined the thrombosis risk for the highest levels of eight selected coagulation factors. Furthermore, we analyzed which of these coagulation factors had the strongest impact on the supposed association. Methods We used data of 2377 patients with a first VT and 2940 control subjects in whom fibrinogen, von Willebrand factor (VWF), factor II, FVII, FVIII, FIX, FX and FXI levels were measured. Results The odds ratios (ORs) for the various coagulation factor levels (> 99th percentile versus ≤ 25th percentile) varied between 1.8 and 4, except for FVIII (OR 23.0; 95% confidence interval [CI] 14.7-36.0) and VWF (OR 24.0; 95% CI 15.3-37.3). Adjustment for FVIII and VWF in a mediation analysis reduced the risks of the other factors to unity, with the exception of FIX and FXI (remaining ORs between 1.7 and 1.9). Conversely, the ORs for FVIII and VWF levels remained high after adjustment for all other procoagulant factors (FVIII: 16.0; 95% CI 9.7-26.3; VWF: 17.6; 95% CI 10.7-28.8). Conclusions Our results imply that the observed relationship between VT and coagulation factor levels can be largely explained by FVIII and VWF. FVIII and VWF levels were also associated with the highest VT risk., (© 2018 International Society on Thrombosis and Haemostasis.)

Essentials Low-molecular-weight-heparins (LMWH) kinetics differ which may result in different bleeding risks. A cohort of 12 934 venous thrombosis patients on LMWH was followed until major bleeding. The absolute major bleeding risk was low among patients registered at the anticoagulation clinic. Once-daily dosing was associated with a lower bleeding risk as compared with twice-daily., Summary: Background Low-molecular-weight heparins (LMWHs) are considered members of a class of drugs with similar anticoagulant properties. However, pharmacodynamics and pharmacokinetics between LMWHs differ, which may result in different bleeding risks. As these agents are used by many patients, small differences may lead to a large effect on numbers of major bleeding events. Objectives To determine major bleeding risks for different LMWH agents and dosing schedules. Methods A cohort of acute venous thrombosis patients from four anticoagulation clinics who used an LMWH and a vitamin K antagonist were followed until they ceased LMWH treatment or until major bleeding. Exposures were classified according to different types of LMWHs and for b.i.d. and o.d. use. Cumulative incidences for major bleeding per 1000 patients and risk ratios were calculated and adjusted for study center. Results The study comprised 12 934 patients with a mean age of 59 years; 6218 (48%) were men. The cumulative incidence of major bleeding was 2.5 per 1000 patients (95% confidence interval [CI], 1.7-3.5). Enoxaparin b.i.d. or o.d. was associated with a relative bleeding risk of 1.7 (95% CI, 0.2-17.5) compared with nadroparin o.d. In addition, a nadroparin b.i.d. dosing schedule was associated with a 2.0-fold increased major bleeding risk (95% CI, 0.8-5.1) as compared with a nadroparin o.d. dosing schedule. Conclusions Absolute major bleeding rates were low for all LMWH agents and dosing schedules in a large unselected cohort. Nevertheless, twice-daily dosing with nadroparin appeared to be associated with an increased major bleeding risk as compared with once-daily dosing, as also suggested in a meta-analysis of controlled clinical trials., (© 2017 International Society on Thrombosis and Haemostasis.)

Unlabelled: Essentials Genetic architecture of venous thromboembolism (VTE) remains to be fully disentangled. 11 newly discovered candidate polymorphisms were genotyped in 3019 VTE cases and 2605 controls. None of the 11 polymorphisms were significantly associated with VTE risk. Additional major efforts are needed to identify VTE-associated genetic variants., Summary: Background Through a meta-analysis of 12 genome-wide association studies, the International Network against VENous Thrombosis (INVENT) consortium identified two novel susceptibility loci for venous thromboembolism (VTE). This project has also generated other candidates that need to be confirmed. Objectives To assess the association with VTE of common single-nucleotide polymorphisms (SNPs) that demonstrated strong statistical, but not genome-wide, significance in the INVENT cohorts. Patients/methods Eleven SNPs were genotyped and tested for association with VTE in three case-control studies totaling 3019 patients and 2605 healthy individuals. Results and conclusions None of the tested SNPs showed evidence for association with VTE. Different strategies are needed to decipher the whole spectrum of common and rare genetic variations associated with VTE risk., (© 2016 International Society on Thrombosis and Haemostasis.)

Unlabelled: Essentials Statins, especially rosuvastatin, may reduce venous thrombosis risk, but the mechanism is unclear. We performed a randomized trial investigating the effect of rosuvastatin on platelet reactivity. Thromboxane-A2 mediated platelet aggregation was measured before and after rosuvastatin therapy. Rosuvastatin did not inhibit thromboxane-mediated platelet aggregation in venous thrombosis patients., Summary: Background Statins may exert a protective effect against the risk of venous thrombosis (VT), but the mechanism is unclear. Objectives In this open-label, randomized clinical trial (www.clinicaltrials.gov NCT01613794), we aimed to determine the ex vivo effect of rosuvastatin on platelet reactivity in patients with a history of VT. Methods Platelet reactivity, in platelet reaction units (PRUs), was measured at baseline and after 28 days with VerifyNow, which uses arachidonic acid to determine thromboxane-mediated platelet aggregation, in 50 consecutive patients included in our study (25 receiving rosuvastatin and 25 without intervention). Results Forty-seven of 50 (94.0%) consecutively enrolled patients had two valid PRU measurements. The mean PRUs in rosuvastatin users were 609 at baseline and 613 at the end of the study (mean change 5; 95% confidence interval [CI] - 18 to 27). The mean PRUs in non-users were 620 at baseline and 618 at the end of the study (mean change - 2; 95% CI - 15 to 12). The mean difference in PRU change between users and non-users was 6 (95% CI - 20 to 33). After exclusion of patients who used antiplatelet medication, or had thrombocytopenia, similar results were obtained, i.e. no apparent effect of rosuvastatin on PRUs, with a mean difference in PRU change between users and non-users of - 1 (95% CI - 20 to 19). Conclusions Rosuvastatin does not affect platelet reactivity when arachidonic acid is used as an agonist in patients with a history of VT., (© 2016 International Society on Thrombosis and Haemostasis.)

Unlabelled: Essentials Minor bleeding is associated with subsequent major bleeding in patients treated with vitamin K antagonists. This study confirms that patients with minor bleeds have a 2.5-fold increased risk of major bleeds. A case-crossover analysis revealed that the increased risk is due to fixed underlying risk factors. Future research may unveil these unknown fixed risk factors and improve major bleeding risk scores., Summary: Background Patients who have a minor bleed during treatment with vitamin K antagonists (VKAs) have a 3-fold increased risk of subsequent major bleeding. The nature of the underlying risk factors is largely unknown. Objectives To indicate why patients with minor bleeds are at increased risk of subsequent major bleeds (e.g. are risk factors of a transient or a fixed nature). Methods Patients who started VKA treatment between 2003 and 2013 were included. Exposure was from the minor bleed until 3 months later. We used two analyses: a Cox model which we adjusted for several known risk factors, and a case-crossover (CCO) design, which corrects for all fixed risk factors (such as chronic diseases and genes) as patients are compared with themselves. The combination of both analyses gives insight into whether the association of minor with major bleeds is a result of fixed or transient risk factors. Results Out of 26 130 patients who were included and followed for '61 672 patient years', 7194 experienced a minor bleed and 913 a major bleed. The Cox model indicated that patients with minor bleeds had a 2.5-fold increased risk of experiencing subsequent major bleeding after adjustment for known risk factors, whereas the CCO gave risk estimates around unity (odds ratio, 0.9; 95% confidence interval, 0.5-1.5). Conclusions The combination of both analyses indicates that minor bleeds are markers for fixed and currently unknown risk factors for major bleeding events., (© 2016 International Society on Thrombosis and Haemostasis.)

Background: Endothelial von Willebrand factor (VWF) inhibits angiogenesis. Accordingly, blood outgrowth endothelial cells (BOECs) isolated from von Willebrand disease (VWD) patients showed enhanced in vitro angiogenesis when compared with healthy control BOECs. Characterization of the angiogenic response of VWD BOECs is limited and differences between the different types of VWD have not been investigated in detail., Objectives: The aim of this study was to further explore the potential pathogenic effect of VWF mutations on angiogenesis., Methods: BOECs were isolated from four healthy individuals, 10 patients with VWD and one heterozygous carrier of a type 2N mutation. Cell migration and tube formation were measured., Results: Migration velocity and total tube formation were similar between VWD patients and controls in general. BOECs from the type 3 VWD patient and one type 2B patient showed increased migratory velocity and tube formation compared with BOECs from other patients and healthy controls. Directional migration was impaired in eight out of 10 VWD BOECs and the ability to form tubes was limited to early passage numbers, but not for BOECs from healthy controls., Conclusion: BOECs can be a useful tool for ex vivo assessment of endothelial cell function in patients with different types of VWD, but possible limitations, such as early loss of angiogenic capacity, should be recognized. BOECs from most VWD patients consistently showed impairment in the directionality of migration. This is the first report on angiogenic properties of a type 3 VWD BOEC, which showed increased in vitro angiogenesis., (© 2015 International Society on Thrombosis and Haemostasis.)

Background: Procoagulant full-length tissue factor (flTF) and its minimally coagulant alternatively spliced isoform (asTF), promote breast cancer (BrCa) progression via different mechanisms. We previously showed that flTF and asTF are expressed by BrCa cells, resulting in autoregulation in a cancer milieu. BrCa cells often express hormone receptors such as the estrogen receptor (ER), leading to the formation of hormone-regulated cell populations., Objective: To investigate whether TF isoform-specific and ER-dependent pathways interact in BrCa., Methods: Tissue factor isoform-regulated gene sets were assessed using ingenuity pathway analysis. Tissues from a cohort of BrCa patients were divided into ER-positive and ER-negative groups. Associations between TF isoform levels and tumor characteristics were analyzed in these groups. BrCa cells expressing TF isoforms were assessed for proliferation, migration and in vivo growth in the presence or absence of estradiol., Results: Ingenuity pathway analysis pointed to similarities between ER- and TF-induced gene expression profiles. In BrCa tissue specimens, asTF expression was associated with grade and stage in ER-positive but not in ER-negative tumors. flTF was only associated with grade in ER-positive tumors. In MCF-7 cells, asTF accelerated proliferation in the presence of estradiol in a β1 integrin-dependent manner. No synergy between asTF and the ER pathway was observed in a migration assay. Estradiol accelerated the growth of asTF-expressing tumors but not control tumors in vivo in an orthotopic setting., Conclusion: Tissue factor isoform and estrogen signaling share downstream targets in BrCa; the concomitant presence of asTF and estrogen signaling is required to promote BrCa cell proliferation., (© 2015 International Society on Thrombosis and Haemostasis.)

Background: One of the major determinants of von Willebrand factor (VWF) plasma levels is ABO blood group status, and individuals with blood group O have ~ 25% lower plasma levels. The exact mechanism behind this relationship remains unknown, although effects on clearance have been postulated., Objectives: To determine whether clearance of VWF is directly dependent on the presence of ABH antigens on VWF., Methods: Three type 3 von Willebrand disease (VWD) patients were infused with Haemate-P, and the relative loading of VWF with ABH antigens at different time points was measured. VWF-deficient mice were injected with purified plasma-derived human VWF obtained from donors with either blood group A, blood group B, or blood group O., Results: In mice, we found no difference in clearance rate between plasma-derived blood group A, blood group B and blood group O VWF. Faster clearance of the blood group O VWF present in Haemate-P infused in type 3 VWD patients would have resulted in a relative increase in the loading of VWF with A and B antigens over time. However, we observed a two-fold decrease in the loading with A and B antigens in two out of three patients, and stable loading in the third patient., Conclusion: There is no direct effect of ABH antigens on VWF in VWF clearance. We demonstrate that, in a direct comparison within one individual, blood group O VWF is not cleared faster than blood group A or blood group B VWF. Clearance differences between blood group O and non-blood group O individuals may therefore be related to the blood group status of the individual rather than the ABH antigen loading on VWF itself., (© 2015 International Society on Thrombosis and Haemostasis.)