Search

Abstract Background Dengue virus infected patients have high plasminogen activator inhibitor type I (PAI-1) plasma concentrations. Whether the insertion/deletion (4G/5G) polymorphism in the promotor region of the PAI-1 gene is associated with increased PAI-1 plasma concentrations and with death from dengue is unknown. We, therefore, investigated the relationship between the 4G/5G polymorphism and PAI-1 plasma concentrations in dengue patients and risk of death from dengue. Methods A total of 194 patients admitted to the Dr. Kariadi Hospital in Semarang, Indonesia, with clinical suspected severe dengue virus infection were enrolled. Blood samples were obtained on day of admission, days 1, 2 and 7 after admission and at a 1-month follow-up visit. Plasma concentrations of PAI-1 were measured using a sandwich ELISA kit. The PAI-1 4G/5G polymorphism was typed by allele-specific PCR analysis. Results Concentrations of PAI-1 on admission and peak values of PAI-1 during admission were higher than the values measured in healthy controls. Survival was significantly worse in patients with PAI-1 concentrations in the highest tertile (at admission: OR 4.7 [95% CI 0.9–23.8], peak value during admission: OR 6.3 [95%CI 1.3–30.8]). No association was found between the PAI-1 4G/5G polymorphism, and PAI-1 plasma concentrations, dengue disease severity and mortality from dengue. Conclusion These data suggest that the 4G/5G polymorphism has no significant influence on PAI-1 concentrations in dengue virus infected patients and is not associated with the risk of death from dengue. Other factors contributing to the variability of PAI-1 plasma concentrations in patients with dengue need to be explored.

Vitamin K epoxide, a product of gamma-carboxylation, must be rapidly recycled to its reduced form before it can be reused. The set of sequential reactions that guarantees vitamin K recycling is known as the vitamin K cycle. This review is focused on biochemical and genetic aspects of a recently characterized key enzyme of the cycle named vitamin K epoxide reductase complex subunit 1 (VKORC1). This 163-amino acid long protein (18 kDa) is responsible for vitamin K reduction. Recent studies also demonstrated the existence of a VKOR complex in which protein disulfide isomerase (PDI) and VKORC1 appear to be tightly associated. PDI is a thioredoxin-like oxidoreductase and chaperone that is present at high concentrations in the endoplasmic reticulum (ER) and that provides electrons for reduction of the CXXC center in VKORC1. Genetic studies have shown that the VKORC1 gene extends over 5126 base pairs on human chromosome 16 and comprises three exons. Mutations in the VKORC1 gene causes generalized defective vitamin K-dependent clotting factors (VKCFD2) and warfarin resistance (WR). More detailed pharmacogenetic studies have demonstrated a strong association between single nucleotide polymorphisms (SNPs) in the VKORC1 gene and the requirement of warfarin dosage. Screening for VKORC1 polymorphisms that affect warfarin dosage could be helpful to tailor dosage at the onset of oral anticoagulant treatment in order to lower thrombosis and bleeding risks. [ABSTRACT FROM AUTHOR]

Rationale: A runaway inflammatory response to systemic infection or severe trauma is characterized by the activation of a diversity of pathways, ultimately resulting in the development of disseminated intravascular coagulation (DIC) and multiorgan failure. Objectives: Despite increased fundamental knowledge of the pathogenesis of DIC, the exact molecular mechanisms remain elusive. We aimed therefore to improve our understanding of the molecular pathways underlying endotoxin-induced DIC. Methods: We performed large-scale gene expression profiling in the liver of mice during the onset of endotoxin-induced DIC. The relevance of an identified candidate gene involved in endotoxin-induced DIC was subsequently assessed in the generalized Shwartzman reaction. Measurements and Main Results: Approximately 5% of over 20,000 genes were differentially regulated. In addition to well-established sepsis-associated genes, such as macrophage inflammatory protein 1, plasminogen activator inhibitor 1, CD14, and A20, we identified several novel candidates for inflammatory disease of which the transcription factor C/EBPdelta (CAAT/enhancer binding protein delta) was studied further. Induction of DIC in C/EBPdelta-deficient mice decreased endotoxin-induced systemic inflammation as compared with wild-type mice, as evident from decreased plasma levels of tumor necrosis factor-alpha and IL-6. In addition, C/EBPdelta deficiency partly protected against DIC-induced mortality. Interestingly, C/EBPdelta deficiency seemed mainly protective by improving renal function. This latter notion was confirmed in an experimental model of renal ischemia/reperfusion injury in which C/EBPdelta deficiency reduced ischemia/reperfusion-induced creatinine and urea levels. Conclusions: Our results endorse the usefulness of gene expression profiling in identifying novel mediators of DIC by showing that C/EBPdelta regulates specific pathologic features of this endotoxin-induced syndrome. [ABSTRACT FROM AUTHOR]

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.)

Background: Major bleeding occurs annually in 1%-3% of patients on vitamin K antagonists (VKAs), despite close monitoring. Genetic variants in proteins involved in VKA response may affect this risk., Aim: To determine the association of genetic variants (cytochrome P450 enzymes 2C9 [CYP2C9] and 4F2 [CYP4F2], gamma-glutamyl carboxylase [GGCX]) with major bleeding in VKA users, separately and combined, including vitamin K epoxide reductase complex subunit-1 (VKORC1)., Methods: A case-cohort study was established within the BLEEDS cohort, which includes 16,570 patients who initiated VKAs between 2012 and 2014. We selected all 326 major bleeding cases that occurred during 17,613 years of follow-up and a random subcohort of 978 patients. We determined variants in CYP2C9, CYP4F2, GGCX, VKORC1 and evaluated the interaction between variant genotypes. Hazard ratios for major bleeding with 95% confidence intervals (95% CI) were estimated by weighted Cox regression., Results: Genotype was determined in 256 cases and 783 subcohort members. Phenprocoumon was the most prescribed VKA for both cases and the subcohort (78% and 75%, respectively). Patients with major bleeding were slightly older than subcohort patients. CYP4F2-TT carriership was associated with a 1.6-fold (95% CI 0.9-2.8) increased risk of major bleeding compared with CC-alleles, albeit not statistically significant. For the CYP2C9 and GGCX variants instead, the major bleeding risk was around unity. Carrying at least two variant genotypes in CYP2C9 (poor metabolizer), CYP4F2-TT, and VKORC1-AA was associated with a 4.0-fold (95%CI 1.4-11.4) increased risk, while carriers of both CYP4F2-TT and VKORC1-AA had a particularly increased major bleeding risk (hazard ratio 6.7, 95% CI 1.5-29.8) compared with carriers of CC alleles in CYP4F2 and GG in VKORC1. However, the number of major bleeding cases in carriers of multiple variants was few (8 and 5 patients, respectively)., Conclusions: CYP4F2 polymorphism was associated with major bleeding, especially in combination with VKORC1 genetic variants. These variants could be considered to further personalize anticoagulant treatment., (© 2024 The Authors. Pharmacotherapy: The Journal of Human Pharmacology and Drug Therapy published by Wiley Periodicals LLC on behalf of Pharmacotherapy Publications, Inc.)

OBJECTIVE: The Australian snake venom ptFV (Pseudonaja textilis venom-derived factor V) variant retains cofactor function despite APC (activated protein C)-dependent proteolysis. Here, we aimed to unravel the mechanistic principles by determining the role of the absent Arg306 cleavage site that is required for the inactivation of FVa (mammalian factor Va). APPROACH AND RESULTS: Our findings show that in contrast to human FVa, APC-catalyzed proteolysis of ptFVa at Arg306 and Lys507 does not abrogate ptFVa cofactor function. Remarkably, the structural integrity of APC-proteolyzed ptFVa is maintained indicating that stable noncovalent interactions prevent A2-domain dissociation. Using Molecular Dynamics simulations, we uncovered key regions located in the A1 and A2 domain that may be at the basis of this remarkable characteristic. CONCLUSIONS: Taken together, we report a completely novel role for uniquely adapted regions in ptFVa that prevent A2 domain dissociation. As such, these results challenge our current understanding by which strict regulatory mechanisms control FVa activity.

A wide variety of animal models on thrombosis and hemostasis are used in thrombosis and hemostasis research for the preclinical assessment of hemostatic agents. While the vertebrate coagulome is highly conserved, human and animal plasmas differ considerably when evaluated in coagulation assays such as prothrombin time (PT), activated partial thromboplastin time (APTT), and calibrated automated thrombography (CAT). Here, we have aimed to provide a reference framework for the evaluation of coagulation assays and inhibition of activated human FXa (hFXa) in various animal plasmas. To do so, a side-by-side evaluation of the extrinsic and intrinsic pathway of coagulation was performed by means of PT, APTT, and CAT measurements on (diluted) pooled plasmas from goats, pigs, rabbits, rats, mice, and humans. Plasma anti-FXa activity was assessed by determining the rate of recombinant hFXa inhibition through chromogenic activity analyses and immunoblotting. In general, rabbit, rat, and mouse plasmas exhibited robust clotting upon stimulation of both the extrinsic and intrinsic pathway, produced more thrombin during CAT upon plasma dilution, and displayed relatively high hFXa inhibitory activities. By comparison, goat, porcine, and human plasma displayed a similar profile in PT and APTT assays, produced less thrombin during CAT upon plasma dilution, and displayed comparable hFXa inhibitory activities. In conclusion, the observed differences in clotting parameters and anti-hFXa activity point to a higher anticoagulant threshold in plasma from rabbits, rats, and particularly in mice relative to human, goat, and porcine plasma. Finally, rat plasma was found to be more relevant to the preclinical assessment of human FX(a) in comparison to murine plasma., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background:  Major bleeding occurs in 1 to 3% of patients treated with oral anticoagulants per year. Biomarkers may help to identify high-risk patients. A proposed marker for major bleeding while using anticoagulants is soluble thrombomodulin (sTM)., Methods:  Plasma was available from 16,570 patients of the BLEEDS cohort that consisted of patients who started treatment with vitamin K antagonists between 2012 and 2014. A case-cohort study was performed including all patients with a major bleed ( n  = 326) during follow-up and a random sample of individuals selected at baseline ( n  = 652). Plasma sTM levels were measured and stratified by percentiles. Patients were also categorized by international normalized ratio (INR). Adjusted hazard ratios (for age, sex, hypertension, and diabetes) with 95% confidence intervals (CIs) were estimated by means of Cox regression., Results:  Plasma sTM levels were available for 263 patients with a major bleed and 538 control subjects. sTM levels were dose-dependently associated with risk of major bleeding, with a 1.9-fold increased risk (95% CI: 1.1-3.1) for levels above the 85th percentile versus the <25th percentile. A high INR (≥4) in the presence of high (≥70th percentile) sTM levels was associated with a 7.1-fold (95% CI: 4.1-12.3) increased risk of major bleeding, corresponding with a bleeding rate of 14.1 per 100 patient-years., Conclusion:  High sTM levels at the start of treatment are associated with major bleeding during vitamin K antagonist treatment, particularly in the presence of a high INR., Competing Interests: None declared., (Thieme. All rights reserved.)

The direct oral anticoagulants targeting coagulation factor Xa or thrombin are widely used as alternatives to vitamin K antagonists in the management of venous thromboembolism and nonvalvular atrial fibrillation. In case of bleeding or emergency surgery, reversal agents are helpful to counteract the anticoagulant therapy and restore hemostasis. While idarucizumab has been established as an antidote for the direct thrombin inhibitor dabigatran, reversal strategies for the direct factor Xa inhibitors have been a focal point in clinical care over the past years. In the absence of specific reversal agents, the off-label use of (activated) prothrombin complex concentrate and recombinant factor VIIa have been suggested as effective treatment options during inhibitor-induced bleeding complications. Meanwhile, several specific reversal agents have been developed. In this review, an overview of the current state of nonspecific and specific reversal agents for the direct factor Xa inhibitors is provided, focusing on the biochemistry and mechanism of action and the preclinical assessment of newly emerging therapies., Competing Interests: P. H. R. owns equity in VarmX. The remaining authors declare no competing financial interests., (Thieme. All rights reserved.)

The venom of the Australian snake Pseudonaja textilis comprises powerful prothrombin activators consisting of factor X (v-ptFX)- and factor V-like proteins. While all vertebrate liver-expressed factor X (FX) homologs, including that of P. textilis , comprise an activation peptide of approximately 45 to 65 residues, the activation peptide of v-ptFX is significantly shortened to 27 residues. In this study, we demonstrate that exchanging the human FX activation peptide for the snake venom ortholog impedes proteolytic cleavage by the intrinsic factor VIIIa-factor IXa tenase complex. Furthermore, our findings indicate that the human FX activation peptide comprises an essential binding site for the intrinsic tenase complex. Conversely, incorporation of FX into the extrinsic tissue factor-factor VIIa tenase complex is completely dependent on exosite-mediated interactions. Remarkably, the shortened activation peptide allows for factor V-dependent prothrombin conversion while in the zymogen state. This indicates that the active site of FX molecules comprising the v-ptFX activation peptide partially matures upon assembly into a premature prothrombinase complex. Taken together, the shortened activation peptide is one of the remarkable characteristics of v-ptFX that has been modified from its original form, thereby transforming FX into a powerful procoagulant protein. Moreover, these results shed new light on the structural requirements for serine protease activation and indicate that catalytic activity can be obtained without formation of the characteristic Ile 16 -Asp 194 salt bridge via modification of the activation peptide., Competing Interests: P.H.R. owns equity in VarmX B.V. The remaining authors declare no competing financial interests., (Thieme. All rights reserved.)

Deep vein thrombosis and pulmonary embolism, collectively defined as venous thromboembolism (VTE), are the third leading cause of cardiovascular death in the United States. Common genetic variants conferring increased varying degrees of VTE risk have been identified by genome-wide association studies (GWAS). Rare mutations in the anticoagulant genes PROC, PROS1 and SERPINC1 result in perinatal lethal thrombosis in homozygotes and markedly increased VTE risk in heterozygotes. However, currently described VTE variants account for an insufficient portion of risk to be routinely used for clinical decision making. To identify new rare VTE risk variants, we performed a whole-exome study of 393 individuals with unprovoked VTE and 6114 controls. This study identified 4 genes harboring an excess number of rare damaging variants in patients with VTE: PROS1, STAB2, PROC, and SERPINC1. At STAB2, 7.8% of VTE cases and 2.4% of controls had a qualifying rare variant. In cell culture, VTE-associated variants of STAB2 had a reduced surface expression compared with reference STAB2. Common variants in STAB2 have been previously associated with plasma von Willebrand factor and coagulation factor VIII levels in GWAS, suggesting that haploinsufficiency of stabilin-2 may increase VTE risk through elevated levels of these procoagulants. In an independent cohort, we found higher von Willebrand factor levels and equivalent propeptide levels in individuals with rare STAB2 variants compared with controls. Taken together, this study demonstrates the utility of gene-based collapsing analyses to identify loci harboring an excess of rare variants with functional connections to a complex thrombotic disease., (© 2020 by The American Society of Hematology.)

Thrombo-inflammation describes the complex interplay between blood coagulation and inflammation that plays a critical role in cardiovascular diseases. The third Maastricht Consensus Conference on Thrombosis assembled basic, translational, and clinical scientists to discuss the origin and potential consequences of thrombo-inflammation in the etiology, diagnostics, and management of patients with cardiovascular disease, including myocardial infarction, stroke, and peripheral artery disease. This article presents a state-of-the-art reflection of expert opinions and consensus recommendations regarding the following topics: (1) challenges of the endothelial cell barrier; (2) circulating cells and thrombo-inflammation, focused on platelets, neutrophils, and neutrophil extracellular traps; (3) procoagulant mechanisms; (4) arterial vascular changes in atherogenesis; attenuating atherosclerosis and ischemia/reperfusion injury; (5) management of patients with arterial vascular disease; and (6) pathogenesis of venous thrombosis and late consequences of venous thromboembolism., Competing Interests: None declared., (Georg Thieme Verlag KG Stuttgart · New York.)

Recently, our group reported that a small interfering RNA (siRNA) targeting coagulation factor XII (si F12 ) leads to an unexpected prothrombotic response in a mouse model where venous thrombosis follows inhibition of endogenous anticoagulants. In this study, we aimed to clarify this unexpected response by evaluating the effects of this si F12 (here, si F12 -A) on plasma coagulation through thrombin generation (TG). Besides a routine negative control siRNA (siNEG), we included extra siRNA controls: one siRNA similar to si F12-A except for positions 9-11 of the siRNA that are replaced with its complementary base pairs (si F12 -A C9/11 ), and a second siRNA against F12 (si F12 -B). Three days after injection, a significant increase in TG peak height was observed solely for animals injected with si F12 -A and si F12 -A C9/11 , which is considered prothrombotic. As this change in coagulation was unrelated to FXII we conclude that it was off-target. For siRNA studies we now recommend to include mismatch siRNA controls, such as the C9/11 mismatch control used in this study, and to consider plasma coagulation in off-target analysis.

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.)

Tissue factor, coagulation factor XII, platelets, and neutrophils are implicated as important players in the pathophysiology of (experimental) venous thrombosis (VT). Their role became evident in mouse models in which surgical handlings were required to provoke VT. Combined inhibition of the natural anticoagulants antithrombin ( Serpinc1 ) and protein C ( Proc ) using small interfering RNA without additional triggers also results in a venous thrombotic phenotype in mice, most notably with vessel occlusion in large veins of the head. VT is fatal but is fully rescued by thrombin inhibition. In the present study, we used this VT mouse model to investigate the involvement of tissue factor, coagulation factor XII, platelets, and neutrophils. Antibody-mediated inhibition of tissue factor reduced the clinical features of VT, the coagulopathy in the head, and fibrin deposition in the liver. In contrast, genetic deficiency in, and small interfering RNA-mediated depletion of, coagulation factor XII did not alter VT onset, severity, or thrombus morphology. Antibody-mediated depletion of platelets fully abrogated coagulopathy in the head and liver fibrin deposition. Although neutrophils were abundant in thrombotic lesions, depletion of circulating Ly6G-positive neutrophils did not affect onset, severity, thrombus morphology, or liver fibrin deposition. In conclusion, VT after inhibition of antithrombin and protein C is dependent on the presence of tissue factor and platelets but not on coagulation factor XII and circulating neutrophils. This study shows that distinct procoagulant pathways operate in mouse VT, dependent on the triggering stimulus., (© 2019 by The American Society of Hematology.)

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.)

Introduction: Altered levels of factor (F)VIII, prothrombin, or antithrombin have been associated with an increased risk for venous thromboembolism (VTE). However, the exact molecular mechanism by which these altered factor levels modulate the risk is incompletely understood. Here we hypothesize that elevated factor levels affect the pro- and anticoagulant balance in coagulation such that even minute amounts of tissue factor (TF) will initiate thrombin formation, thereby contributing to the VTE risk., Materials and Methods: To test this so-called TF-threshold hypothesis, we monitored thrombin generation initiated by very low TF concentrations in FXII-deficient plasma, to avoid any contact pathway-mediated thrombin formation. Furthermore, similar experiments were performed in the presence of increasing concentrations of pro- and anticoagulant proteins., Results: A TF-threshold was established in the FXII-deficient plasma, which is subject to inter-individual variation. Elevated plasma levels of procoagulant factors, such as FVIII or prothrombin, enhanced thrombin generation and reduced the amount of TF required for the initiation of thrombin formation. Conversely, elevated levels of the coagulation inhibitor antithrombin increased the TF-threshold., Conclusions: Our findings support a mediating role for the TF-threshold in the association between high procoagulant factor levels and the risk for VTE. Furthermore, elevated levels of anticoagulants may have a protective effect on the development of VTE., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Silencing of anticoagulant protein C using RNA interference (siProc) evokes low incident but spontaneous atherothrombosis in the aortic root of apolipoprotein E-deficient (Apoe -/- ) mice. The aims of the current study were (1) to analyze if plaque characteristics or circulating factors could be linked to atherothrombosis susceptibility, (2) to increase the incidence of atherothrombosis by transiently increasing blood pressure, and (3) to direct atherothrombosis to an additional predefined vascular site by applying a semi-constrictive collar around the carotid artery. siProc-driven spontaneous atherothrombosis in the aortic root of Apoe -/- mice was reproduced and occurred at an incidence of 23% (9 out of 39 mice), while the incidence of collar-induced atherothrombosis in the carotid artery was 2.6% (1 out of 39 mice). Treatment with phenylephrine, to transiently increase blood pressure, did not increase atherothrombosis in the aortic root of the Apoe -/- mice nor in the carotid arteries with collars. Plaques in the aortic root with an associated thrombus were lower in collagen and macrophage content, and mice with atherothrombosis had significantly more circulating platelets. Plasma protein C, white blood cell counts, total cholesterol, fibrinogen, serum amyloid A, and IL-6 were not different amongst siProc treated mice with or without thrombosis. Remarkably, our data revealed that thrombus formation preferably occurred on plaques in the right coronary sinus of the aortic root. In conclusion, there is a predilection of low protein C-induced spontaneous atherothrombosis in Apoe -/- mice for the right coronary sinus, a process that is associated with an increase in platelets and plaques lower in collagen and macrophage content.

Pulmonary embolism (PE) is caused by emboli, which have originated from venous thrombi, travelling to and occluding the arteries of the lung. PE is the most dangerous form of venous thromboembolism, and undiagnosed or untreated PE can be fatal. Acute PE is associated with right ventricular dysfunction, which can lead to arrhythmia, haemodynamic collapse and shock. Furthermore, individuals who survive PE can develop post-PE syndrome, which is characterized by chronic thrombotic remains in the pulmonary arteries, persistent right ventricular dysfunction, decreased quality of life and/or chronic functional limitations. Several important improvements have been made in the diagnostic and therapeutic management of acute PE in recent years, such as the introduction of a simplified diagnostic algorithm for suspected PE as well as phase III trials demonstrating the value of direct oral anticoagulants in acute and extended treatment of venous thromboembolism. Future research should aim to address novel treatment options (for example, fibrinolysis enhancers) and improved methods for predicting long-term complications and defining optimal anticoagulant therapy parameters in individual patients, and to gain a greater understanding of post-PE syndrome.

Background: Blood coagulation levels are associated with risk of venous thrombosis (VT). The role of factor (F)V is ambiguous since it plays a dual role in coagulation: it has a procoagulant role when it serves as a cofactor for the activation of thrombin and it has an anticoagulant role by enhancing the inactivation of activated FVIII., Objectives: To elucidate the association of FV levels with risk of VT., Patients/methods: We analyzed FV antigen levels in 2377 patients with VT and 2943 controls from the MEGA study. FV levels were categorized according using the 1st, 2.5th, 5th, 10th, 25th, 50th, 75th, 90th, and 95th percentiles of FV levels in controls as cut-off points. Odds ratios (ORs) were estimated using logistic regression models and adjusted for age and sex, liver disease, FVIII levels, FV Leiden, and TFPI., Results: The risk estimates were U-shaped with increased ORs for the lowest (<0.57 U/dL) levels (OR 1.46; 95% CI 0.87-2.43) as well as the highest (>1.22 U/dL) (OR 1.86; 95% CI 1.46-2.37) levels as compared with the reference group (25th-50th percentile). FVIII adjustment led to attenuation of the OR for high FV levels (OR 1.14; 95% CI 0.88-1.48), with little change for low FV levels (OR 1.68; 95% CI 0.97-2.91). Other adjustments had limited effects., Conclusions: We found high FV levels to be associated with increased risk for VT, which was explained by concurrently raised FVIII levels. For low levels of factor V, the increased risk for VT could not be explained by the mechanisms we explored.

Purpose: Multi-dose drug dispensing (MDD) is a dosing aid that provides patients with disposable bags containing all drugs intended for 1 dosing moment. MDD is believed to increase medication adherence, but studies are based on self-reported data, and results may depend on socially desirable answers. Therefore, our purpose was to determine the effect of MDD on medication adherence in non-adherent patients taking vitamin K antagonists (VKAs), and to compare with instructing patients on medication use., Methods: We conducted a before-after study in non-adherent patients where MDD was the exposure and change in adherence after MDD initiation was the outcome (within patient comparison). Time in therapeutic range (TTR) was selected as a measure for adherence, as this reflects stability of VKA treatment. To analyze whether MDD improved adherence as compared with standard care (ie, letters or calls from nurses of the anticoagulation clinic), non-adherent patients without MDD were also followed to estimate their TTR change over time (between patient comparison)., Results: Eighty-three non-adherent VKA patients started using MDD. The median TTR was 63% before MDD and 73% 6 months after MDD. The within patient TTR increased on average by 13% (95%CI 6% to 21%) within 1 month after starting MDD and remained stable during the next 5 months. The TTR of MDD-patients increased 10% (95%CI 2% to 19%) higher as compared with non-MDD patients within 1 month but was similar after 4 months (TTR difference 3%, 95%CI -2% to 9%)., Conclusions: Adherence improved after initiation of MDD. Compared with instructing patients, MDD was associated with better adherence within 1 month but was associated with similar improvement after 4 months., (Copyright © 2017 John Wiley & Sons, Ltd.)

Background: Family studies have shown a strong heritability component for venous thromboembolism (VTE), but established genetic risk factors are present in only half of VTE patients., Aim: To identify genetic risk factors in two large families with unexplained hereditary VTE., Methods: We performed whole exome sequencing in 10 affected relatives of two unrelated families with an unexplained tendency for VTE. We prioritized variants shared by all affected relatives from both families, and evaluated these in the remaining affected and unaffected individuals. We prioritized variants based on 3 different filter strategies: variants within candidate genes, rare variants across the exome, and SNPs present in patients with familial VTE and with low frequency in the general population. We used whole exome sequencing data available from 96 unrelated VTE cases with a positive family history of VTE from an affected sib study (the GIFT study) to identify additional carriers and compared the risk-allele frequencies with the general population. Variants found in only one individual were also retained for further analysis. Finally, we assessed the association of these variants with VTE in a population-based case-control study (the MEGA study) with 4,291 cases and 4,866 controls., Results: Six variants remained as putative disease-risk candidates. These variants are located in 6 genes spread among 3 different loci: 2p21 (PLEKHH2 NM&#95;172069:c.3105T>C, LRPPRC rs372371276, SRBD1 rs34959371), 5q35.2 (UNC5A NM&#95;133369.2:c.1869+23C>A), and 17q25.1 (GPRC5C rs142232982, RAB37 rs556450784). In GIFT, additional carriers were identified only for the variants located in the 2p21 locus. In MEGA, additional carriers for several of these variants were identified in both cases and controls, without a difference in prevalence; no carrier of the UNC5A variant was present., Conclusion: Despite sequencing of several individuals from two thrombophilic families resulting in 6 candidate variants, we were unable to confirm their relevance as novel thrombophilic defects.

The absence of an adequate reversal strategy to prevent and stop potential life-threatening bleeding complications is a major drawback to the clinical use of the direct oral inhibitors of blood coagulation factor Xa. Here we show that specific modifications of the substrate-binding aromatic S4 subpocket within the factor Xa active site disrupt high-affinity engagement of the direct factor Xa inhibitors. These modifications either entail amino-acid substitution of S4 subsite residues Tyr99 and/or Phe174 (chymotrypsinogen numbering), or extension of the 99-loop that borders the S4 subsite. The latter modifications led to the engineering of a factor Xa variant that is able to support coagulation in human plasma spiked with (supra-)physiological concentrations of direct factor Xa inhibitors. As such, this factor Xa variant has the potential to be employed to bypass the direct factor Xa inhibitor-mediated anticoagulation in patients that require restoration of blood coagulation.A major drawback in the clinical use of the oral anticoagulants that directly inhibit factor Xa in order to prevent blood clot formation is the potential for life threatening bleeding events. Here the authors describe factor Xa variants that are refractory to inhibition by these anticoagulants and could serve as rescue agents in treated patients.

Background: Use of ethinylestradiol, one of the active ingredients in combined oral contraceptives, affects the incidence of venous thrombosis. To explain why some women develop thrombosis when using oral contraceptives and others do not, we hypothesized a role for the first-pass metabolism of ethinylestradiol in the liver. We set out to determine the association between genetic variation in the first-pass metabolism of ethinylestradiol, venous thrombosis risk and the effect on Sex-hormone-binding-globulin (SHBG) levels., Methods: Premenopausal women were included from two case-control studies: LETS (103 cases; 159 controls) and MEGA (397 cases; 796 controls). Haplotype-tagging SNPs were selected in 11 candidate genes; COMT, CYP1A2, CYP2C9, CYP3A4, CYP3A5, SULT1A1, SULT1E1, UGT1A1, UGT1A3, UGT1A9, UGT2B7. Venous thrombosis risk was expressed as odds ratios (OR) with 95% confidence intervals (CI). For SHBG levels, mean differences with 95%CI were estimated in combined oral contraceptive-using control subjects from the MEGA study., Results: Two copies of haplotype D in the UGT2B7 gene increased venous thrombosis risk (OR LETS : 3.78; OR MEGA : 2.61) as well as SHBG levels (mean difference 27.6nmol/L, 95%CI: -61.7 to 116.9 compared with no copies) in oral contraceptive users and not in non-users. In oral contraceptive users, haplotype A and B in the CYP3A4 gene were associated with venous thrombosis risk, but not in non-users; however, the effect on SHBG levels was not directional with the risk. None of the other haplotypes were associated with venous thrombosis., Conclusion: Genetic variation in the UGT2B7 gene may, in part, explain venous thrombosis risk in combined oral contraceptive users., (Copyright © 2017 European Federation of Internal Medicine. Published by Elsevier B.V. All rights reserved.)

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.)

Objective: Murine atherosclerosis models do not spontaneously develop atherothrombotic complications. We investigated whether disruption of natural anticoagulation allows preexisting atherosclerotic plaques to progress toward an atherothrombotic phenotype., Approach and Results: On lowering of plasma protein C levels with small interfering RNA (si Proc ) in 8-week Western-type diet-fed atherosclerotic apolipoprotein E-deficient mice, 1 out of 4 mice displayed a large, organized, and fibrin- and leukocyte-rich thrombus on top of an advanced atherosclerotic plaque located in the aortic root. Although again at low incidence (3 in 25), comparable thrombi at the same location were observed during a second independent experiment in 9-week Western-type diet-fed apolipoprotein E-deficient mice. Mice with thrombi on their atherosclerotic plaques did not show other abnormalities and had equally lowered plasma protein C levels as si Proc -treated apolipoprotein E-deficient mice without thrombi. Fibrinogen and thrombin-antithrombin concentrations and blood platelet numbers were also comparable, and plaques in si Proc mice with thrombi had a similar composition and size as plaques in si Proc mice without thrombi. Seven out of 25 si Proc mice featured clots in the left atrium of the heart., Conclusions: Our findings indicate that small interfering RNA-mediated silencing of protein C in apolipoprotein E-deficient mice creates a condition that allows the occurrence of spontaneous atherothrombosis, albeit at a low incidence. Lowering natural anticoagulation in atherosclerosis models may help to discover factors that increase atherothrombotic complications., (© 2017 American Heart Association, Inc.)