Search

Objectives: Previous research has suggested beneficial heterologous effects of the Bacillus Calmette-Guérin (BCG) vaccine on non-mycobacterial infections and other immune-mediated diseases. During the COVID-19 pandemic, randomized controlled trials BCG-PRIME (n = 5349) and BCG-CORONA-ELDERLY (n = 1907) investigated the impact of BCG on SARS-CoV-2 infections in older individuals. We extended the follow-up in these studies by one year (BCG-Long Term study), to assess the overall effects of BCG vaccination on infectious and immune-mediated diseases in individuals aged over 60., Methods: Prior participants were invited to complete a one-year follow-up survey after their completion of the original trial. Data on vaccinations, hospital admissions, infectious episodes, and new medical diagnoses were collected and compared between BCG- and placebo-vaccinated participants. Variables of interest were combined with the previous trial databases and analyzed using relative risks (RR) and an adjusted Cox regression model accounting for participation probability., Results: The response in the follow-up survey was 60%, including 4238 individuals in the final analysis (2317 had received BCG and 1921 placebo). Incidence and severity of infectious diseases and other diagnoses, including cardiovascular diseases and cancer, did not differ between the groups. The proportion of individuals hospitalized for cardiac arrhythmias after BCG was two-fold higher than reported after placebo (1.6% versus 0.8%, RR 2.0 (95% confidence interval 1.1-3.6)). Cardiac arrhythmia-related hospitalizations were primarily due to exacerbation of pre-existing arrhythmias., Conclusion: The results of the present study confirm that BCG has no relevant effect on non-mycobacterial infectious diseases and other immune-mediated diseases in a population of generally mycobacteria-naïve older Dutch individuals in the two years following vaccination. However, our study suggests that BCG may aggravate pre-existing cardiac arrhythmia, which warrants further investigation., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: MGN is a scientific founder of TTxD, Lemba and BioTrip, and a member of the TTxD scientific advisory board. MGN has received research grants from TTxD and GSK., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: Factor V (FV) is a key molecular player in the coagulation cascade. FV plasma levels have been associated with several human diseases, including thrombosis, bleeding, and diabetic complications. So far, 2 genes have been robustly found through genome-wide association analyses to contribute to the inter-individual variability of plasma FV levels: structural F5 gene and PLXDC2., Methods and Results: The authors used the underestimated Brown-Forsythe methodology implemented in the QuickTest software to search for non-additive genetic effects that could contribute to the inter-individual variability of FV plasma activity. QUICKTEST was applied to 4 independent genome-wide association studies studies (LURIC [Ludwigshafen RIsk and Cardiovascular Health Study], MARTHA [Marseille Thrombosis Association], MEGA [Multiple Environmental and Genetic Assessment], and RETROVE [Riesgo de Enfermedad Tromboembolica Venosa]) totaling 4505 participants of European ancestry with measured FV plasma levels. Results obtained in the 4 cohorts were meta-analyzed using a fixed-effect model. Additional analyses involved exploring haplotype and gene×gene interactions in downstream investigations. A genome-wide significant signal at the PSKH2 locus on chr8q21.3 with lead variant rs75463553 with no evidence for heterogeneity across cohorts was observed ( P =0.518). Although rs75463553 did not show an association with mean FV levels ( P =0.49), it demonstrated a robust significant ( P =3.38x10 -9 ) association with the variance of FV plasma levels. Further analyses confirmed the reported association of PSKH2 with neutrophil biology and revealed that rs75463553 likely interacts with two loci, GRIN2A and POM121L12 , known for their involvement in smoking biology., Conclusions: This comprehensive approach identifies the role of PSKH2 as a novel molecular player in the genetic regulation of FV, shedding light on the contribution of neutrophils to FV biology.

The aim of this study was to investigate the associations among sex hormone-binding globulin (SHBG), visceral adipose tissue (VAT), liver fat content, and risk of type 2 diabetes (T2D). In the Netherlands Epidemiology of Obesity study, 5,690 women (53%) and men (47%) without preexisting diabetes were included and followed for incident T2D. SHBG concentrations were measured in all participants, VAT was measured using MRI, and liver fat content was measured using proton magnetic resonance spectroscopy in a random subset of 1,822 participants. We examined associations between SHBG and liver fat using linear regression and bidirectional Mendelian randomization analyses and between SHBG and T2D using Cox regression adjusted for confounding and additionally for VAT and liver fat to examine mediation. Mean age was 56 (SD 6) years, mean BMI was 30 (SD 4) kg/m2, median SHBG was 47 (interquartile range [IQR] 34-65) nmol/L in women and 34 (26-43) nmol/L in men, and median liver fat was 3.4% (IQR 1.6-8.2%) in women and 6.0% (2.9-13.5%) in men. Compared with the highest SHBG quartile, liver fat was 2.9-fold (95% CI 2.4, 3.4) increased in women and 1.6-fold (95% CI 1.3, 1.8) increased in men, and the hazard ratio of T2D was 4.9 (95% CI 2.4, 9.9) in women and 1.8 (1.1, 2.9) in men. Genetically predicted SHBG was associated with liver fat content (women: SD -0.45 [95% CI -0.55, -0.35]; men: natural logarithm, -0.25 [95% CI -0.34, -0.16]). VAT and liver fat together mediated 43% (women) and 60% (men) of the SHBG-T2D association. To conclude, in a middle-aged population with overweight, the association between low SHBG and increased risk of T2D was, for a large part, mediated by increased VAT and liver fat., (© 2024 by the American Diabetes Association.)

Hypercoagulability is characterized by abnormal elevations of coagulation factor levels and increased thrombin generation potential. Prior studies demonstrated links between impaired glucose metabolism, endothelial dysfunction, and hypercoagulability. However, the associations between hypercoagulability and incident type 2 diabetes as well as its underlying mechanism remain unclear. We aimed to assess the associations between coagulation parameters including coagulation factor (F) VIII, FIX, FXI, fibrinogen, thrombin generation potential (lag time, endogenous thrombin potential [ETP], peak, time-to-peak, velocity) and incident type 2 diabetes, and to study the underlying mechanism by examining the mediating role of glycoprotein acetylation (GlycA). In the Netherlands Epidemiology of Obesity study, we applied a Cox Proportional-Hazards Model in 5718 participants after adjustment for confounders. We further conducted a mediation analysis investigating the mediation effect of GlycA on the observed associations. During a median follow-up of 6.7 years, 281 incident type 2 diabetes diagnoses were reported. Compared with the lowest quartile, hazard ratio (95% confidence interval) of the highest quartile was 2.47 (1.48-4.14) for FIX, 1.37 (0.85-2.20) for FVIII, 1.11 (0.76-1.63) for FXI, 0.98 (0.65-1.48) for fibrinogen, 1.56 (1.07-2.28) for ETP, 1.84 (1.23-2.74) for peak, 1.59 (1.08-2.33) for velocity, 0.92 (0.62-1.38) for lag time, and 1.21 (0.86-1.70) for time-to-peak. GlycA mediated only a small proportion of all observed associations. In conclusion, elevated levels of coagulation factor and thrombin generation potential are associated with incident type 2 diabetes, suggesting the involvement of hypercoagulability in the pathogenesis of type 2 diabetes., (© 2024. The Author(s).)

Genetic studies have identified numerous regions associated with plasma fibrinogen levels in Europeans, yet missing heritability and limited inclusion of non-Europeans necessitates further studies with improved power and sensitivity. Compared with array-based genotyping, whole genome sequencing (WGS) data provides better coverage of the genome and better representation of non-European variants. To better understand the genetic landscape regulating plasma fibrinogen levels, we meta-analyzed WGS data from the NHLBI's Trans-Omics for Precision Medicine (TOPMed) program (n=32,572), with array-based genotype data from the Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) Consortium (n=131,340) imputed to the TOPMed or Haplotype Reference Consortium panel. We identified 18 loci that have not been identified in prior genetic studies of fibrinogen. Of these, four are driven by common variants of small effect with reported MAF at least 10 percentage points higher in African populations. Three signals (SERPINA1, ZFP36L2, and TLR10) contain predicted deleterious missense variants. Two loci, SOCS3 and HPN, each harbor two conditionally distinct, non-coding variants. The gene region encoding the fibrinogen protein chain subunits (FGG;FGB;FGA), contains 7 distinct signals, including one novel signal driven by rs28577061, a variant common in African ancestry populations but extremely rare in Europeans (MAFAFR=0.180; MAFEUR=0.008). Through phenome-wide association studies in the VA Million Veteran Program, we found associations between fibrinogen polygenic risk scores and thrombotic and inflammatory disease phenotypes, including an association with gout. Our findings demonstrate the utility of WGS to augment genetic discovery in diverse populations and offer new insights for putative mechanisms of fibrinogen regulation., (Copyright © 2024 American Society of Hematology.)

Background: Extensive evidence is available on hormonal contraceptive (HC) use and the risk of a first venous thromboembolism (VTE) event. Despite recommendations to discontinue combined HC (CHC) use, some women continue or start its use after a first VTE., Objectives: We aimed to evaluate the VTE recurrence risk associated with HC use in premenopausal women., Methods: Premenopausal women with a first VTE included in the Multiple Environmental and Genetic Assessment of Venous Thrombosis study between 1999 and 2004 were followed for a recurrence until 2010. Data on HC use were available through linkage to the Dutch Foundation for Pharmaceutical Statistics. The risk of recurrence was assessed 1) during anticoagulant therapy and 2) after cessation of anticoagulant therapy. Time-dependent Cox proportional hazards models were used to estimate hazard ratios (HRs) with 95% CIs adjusted for age and body mass index at baseline and thromboprophylaxis use during follow-up., Results: Six hundred fifty women were uniquely linked and followed for a total of 3538 person-years (median, 6.1 years), during which 57 VTE recurrences occurred. Five occurred (8.8%) during anticoagulation treatment, with no clear risk difference for CHC use vs nonuse (HR, 0.8; 95% CI, 0.1-8.2). After anticoagulation cessation, CHC use was associated with a 2.4-fold higher risk of recurrence (HR, 2.4; 95% CI, 1.2-5.0) compared with nonuse. Recurrence risk for levonorgestrel-releasing intrauterine device use was similar to that for nonuse (HR, 0.9; 95% CI, 0.3-3.1)., Conclusion: CHC use after a first VTE is safe during anticoagulant use but substantially increases the risk of a recurrent VTE event in absence of anticoagulant use. This study adds to the evidence regarding the use of a levonorgestrel-releasing intrauterine device as a safe alternative., Competing Interests: Declaration of competing interests The authors declare no competing interests. B.H.S. is currently employed at the Knowledge Institute of the Dutch Association of Medical Specialists, Utrecht, The Netherlands. J.F.T. is currently employed at ZonMw, The Hague, The Netherlands. L.E.F. is currently employed at Nivel, Utrecht, The Netherlands., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Increased risk of venous thromboembolism (VTE) is a life-threatening side effect for users of oral contraceptives (OCs) or hormone therapy (HT)., Objectives: To investigate the potential for genetic predisposition to VTE in OC or HT users, we conducted a gene-by-environment case-only meta-analysis of genome-wide association studies (GWAS)., Methods: Use or nonuse of OCs (7 studies) or HT (8 studies) at the time of the VTE event was determined by pharmacy records or self-report. A synergy index (SI) was modeled for each variant in each study and submultiplicative/supramultiplicative gene-by-environment interactions were estimated. The SI parameters were first meta-analyzed across OC and HT studies and subsequently meta-analyzed to obtain an overall estimate. The primary analysis was agnostic GWAS and interrogated all imputed genotypes using a P value threshold of <5.0 × 10 -8 ; secondary analyses were candidate-based., Results: The VTE case-only OC meta-analysis included 2895 OC users and 6607 nonusers; the case-only HT meta-analysis included 2434 HT users and 12 793 nonusers. In primary GWAS meta-analyses, no variant reached genome-wide significance, but the smallest P value approached statistical significance: rs9386463 (P = 5.03 × 10 -8 ). We tested associations for 138 candidate variants and identified 2 that exceeded statistical significance (0.05/138 = 3.62 × 10 -4 ): F5 rs6025 (P = 1.87 × 10 -5 ; SI, 1.29; previously observed) and F11 rs2036914 (P = 2.0 × 10 -4 ; SI, 0.91; new observation)., Conclusion: The candidate variant approach to identify submultiplictive/supramultiplicative associations between genetic variation and OC and HT use identified a new association with common genetic variation in F11, while the agnostic interrogations did not yield new discoveries., Competing Interests: Declaration of competing interests T.N. receives speaking honoraria from Servier Finland and AstraZeneca. All other authors have no relevant disclosures., (Copyright © 2024 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

Abstract: Coagulation factor VIII (FVIII) and its carrier protein von Willebrand factor (VWF) are critical to coagulation and platelet aggregation. We leveraged whole-genome sequence data from the Trans-Omics for Precision Medicine (TOPMed) program along with TOPMed-based imputation of genotypes in additional samples to identify genetic associations with circulating FVIII and VWF levels in a single-variant meta-analysis, including up to 45 289 participants. Gene-based aggregate tests were implemented in TOPMed. We identified 3 candidate causal genes and tested their functional effect on FVIII release from human liver endothelial cells (HLECs) and VWF release from human umbilical vein endothelial cells. Mendelian randomization was also performed to provide evidence for causal associations of FVIII and VWF with thrombotic outcomes. We identified associations (P < 5 × 10-9) at 7 new loci for FVIII (ST3GAL4, CLEC4M, B3GNT2, ASGR1, F12, KNG1, and TREM1/NCR2) and 1 for VWF (B3GNT2). VWF, ABO, and STAB2 were associated with FVIII and VWF in gene-based analyses. Multiphenotype analysis of FVIII and VWF identified another 3 new loci, including PDIA3. Silencing of B3GNT2 and the previously reported CD36 gene decreased release of FVIII by HLECs, whereas silencing of B3GNT2, CD36, and PDIA3 decreased release of VWF by HVECs. Mendelian randomization supports causal association of higher FVIII and VWF with increased risk of thrombotic outcomes. Seven new loci were identified for FVIII and 1 for VWF, with evidence supporting causal associations of FVIII and VWF with thrombotic outcomes. B3GNT2, CD36, and PDIA3 modulate the release of FVIII and/or VWF in vitro.

Background: Fractional-dosed intradermal (i.d.) vaccination produces antibody concentrations above the proposed proxy for protection against severe disease as compared with intramuscular (i.m.) vaccination and may be associated with a decreased prothrombotic effect., Objectives: To assess changes in coagulation following standard dosed i.m. or fractional-dosed i.d. (one-fifth of i.m.) mRNA-1273 SARS-CoV-2 vaccine and to determine the association between the inflammatory response and coagulation., Methods: This study was embedded in a randomized controlled trial assessing the immunogenicity of an i.d. fractional-dosed mRNA-1273 vaccine. Healthy participants, aged 18 to 30 years, were randomized (2:1) to receive either 2 doses of i.d. or i.m. vaccine. Blood was drawn prior to first and second vaccination doses and 1 and 2 weeks after the second dose. The outcomes were changes in coagulation parameters (primary endpoint peak height of the thrombin generation curve) and inflammation (high-sensitivity C-reactive protein [hs-CRP])., Results: One hundred twenty-three participants were included (81 i.d.; 42 i.m.). Peak height increased after vaccination (i.m., 28.8 nmol; 95% CI, 6.3-63.8; i.d., 17.3 nmol; 95% CI, 12.5-47.2) and recovered back to baseline within 2 weeks. I.m. vaccination showed a higher inflammatory response compared with i.d. vaccination (extra increase hs-CRP, 0.92 mg/L; 95% CI, 0.2-1.7). Change in endogenous thrombin potential was associated with change in hs-CRP (beta, 28.0; 95% CI, 7.6-48.3)., Conclusion: A transient increase in coagulability after mRNA-1273 SARS-CoV-2 vaccination occurred, which was associated with the inflammatory response. While i.d. administration showed antibody concentrations above the proposed proxy for protection against severe disease, it was associated with less systemic inflammation. Hence, i.d. vaccination may be safer., (© 2024 The Author(s).)

Background: The incidence rate of venous thrombosis (VT) in women switching combined oral contraceptives (COCs) is unknown., Objectives: We hypothesize that women switching COCs may have a similar increased incidence rate of VT as women who start COCs. Switching means starting with a new COC, which may biologically approximate starting., Methods: We conducted a cohort study with data from the Netherlands and Denmark. First, we identified starters who were defined as women who did not use COCs in the 2 years prior to the start of their first COC prescription within the study period. Switchers were a subset of COC starters who redeemed a COC formulation different from their initial COC during follow-up but not longer than 12 months after starting. We estimated incidence rate ratios (adjusted incidence rate ratio [aIRR]) of VT with 95% CIs among COC switchers as compared with COC starters using Poisson regression adjusted for age, COC progestogen generation, and preexisting obesity., Results: In both countries, we found an increased risk of VT among switchers as compared with starters during the first 3 months of the follow-up (aIRR = 1.77; 95% CI, 1.22-2.56 in the Netherlands and aIRR = 1.50; 95% CI, 1.04-2.16 in Denmark)., Conclusion: Switchers, particularly in the first 3 months after switching, may experience a renewed starter effect thereby increasing the risk of VT., (© 2024 The Author(s).)

Recently three large meta-analyses of genome-wide association studies for venous thromboembolism (VTE) identified over 130 genetic variants. However, mechanisms by which newly identified and therefore underexplored VTE-associated genetic variants influence VTE remain unclear. To elucidate the mechanism, we investigated the association between 61 newly identified VTE-associated genetic variants and the levels of coagulation factor (F) VIII, FIX, FXI, and fibrinogen as well as thrombin generation parameters (lag time, peak, endogenous thrombin potential, time-to-peak, and velocity), which are well-known biological traits associated with VTE. This study was conducted on 5341 participants of the Netherlands Epidemiology of Obesity study. The associations between VTE-associated genetic variants and coagulation factor levels and thrombin generation parameters were examined using linear regression analyses, adjusted for age, sex, body mass index, oral contraceptive use, hormone replacement therapy, and menopausal status. Of 61 genetic variants, 33 were associated with one or more of the coagulation factor levels and thrombin generation parameters. Following multiple testing corrections, five genetic variants remained significant, of which MAP1A rs55707100 exhibited the most robust association with thrombin generation parameters and FXI levels (β = -5.33%, 95% confidence interval: -8.44, -2.22). Our findings shed light on the underlying mechanisms by which these genetic variants influence the risk of VTE., Competing Interests: Ruifang Li‐Gao is a part‐time consultant for Metabolon Inc. The other authors declare no conflict of interest., (© 2024 The Authors. eJHaem published by British Society for Haematology and John Wiley & Sons Ltd.)

Venous thromboembolism (VTE) refers to abnormal blood clots in veins occurring in 1 to 2 per 1000 individuals every year. While anticoagulant treatment can prevent VTE, it increases the risk of bleeding. This emphasizes the importance of identifying individuals with a high risk of VTE and providing prophylactic interventions to these individuals to reduce both VTE and bleeding risks. Current risk assessment of VTE is based on the combination of mainly clinical risk factors. With the identification of an increasing number of genetic variants associated with the risk of VTE, the addition of genetic findings to clinical prediction models can improve risk prediction for VTE. Especially for individuals in high-risk situations, the added value of genetic findings to clinical prediction models may have benefits such as better prophylaxis of VTE and the reduced side effects of bleeding from unnecessary treatment. Nevertheless, the question of whether these models will eventually have clinical utility remains to be proven. Here, we review the current state of knowledge on genetic risk factors for VTE, explore genetic prediction models for VTE, and discuss their clinical implications and challenges., Competing Interests: Declaration of competing interests There are no competing interests to declare., (Copyright © 2023 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

Background: Microvascular dysfunction is a growing determinant of sex differences in coronary heart disease (CHD). Dysregulation of the coagulation system is involved in CHD pathogenesis and can be induced by endothelial glycocalyx (EG) perturbation. However, little is known about the link between EG function and coagulation parameters in population-based studies on sex specificity., Objectives: We sought to examine the sex differences in the relationship between EG function and coagulation parameters in a middle-aged Dutch population., Methods: Using baseline measurements of 771 participants from the Netherlands Epidemiology of Obesity study (age, 56 years [IQR, 51-61 years]; 53% women; body mass index, 27.9 kg/m 2 [IQR, 25.1-30.9 kg/m 2 ]), associations between glycocalyx-related perfused boundary region (PBR) derived using sidestream dark-field imaging and coagulation parameters (factor [F]VIII/IX/XI; thrombin generation parameters; and fibrinogen) were investigated using linear regression analyses, adjusting for possible confounders (including C-reactive protein, leptin, and glycoprotein acetyls), followed by sex-stratified analyses., Results: There was a sex difference in the associations between PBR and coagulation parameters. Particularly in women, 1-SD PBR (both total and feed vessel, indicating poorer glycocalyx status) was associated with higher FIX activity ([1.8%; 95% CI, 0.3%-3.3%] and [2.0%; 95% CI, 0.5%-3.4%], respectively) and plasma fibrinogen levels ([5.1 mg/dL; 95% CI, 0.4-9.9 mg/dL] and [5.8 mg/dL; 95% CI, 1.1-10.6 mg/dL], respectively). Furthermore, 1-SD PBR capillary was associated with higher FVIII activity (3.5%; 95% CI, 0.4%-6.5%) and plasma fibrinogen levels (5.3 mg/dL; 95% CI, 0.6-10.0 mg/dL)., Conclusion: We revealed a sex-specific association between microcirculatory health and procoagulant status, which suggests that microvascular health be considered during early development of CHD in women., Competing Interests: Declaration of competing interests H.V. works for MicroVascular Health Solutions LLC. R.L.-G. works for Metabolon Inc. The remaining authors, including L.Y., J.H., A.I.M.v.d.V., R.d.M., F.R.R., A.v.H.V., T.J.R., and B.M.v.d.B., have no competing interests to disclose., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Introduction: The associations of plasma factor VIII (FVIII) and factor IX (FIX) levels with risk of venous thromboembolism (VTE) are not well defined. We performed a systematic review and meta-analysis of these associations., Methods: Random effects inverse-variance weighted meta-analysis was used to estimate pooled odds ratios for comparisons across equal quartiles of the distributions and 90 % thresholds (higher versus lower), and for testing linear trends., Results: Among 15 studies (5327 cases) the pooled odds ratio of VTE for the fourth versus first quarter was 3.92 (95 % confidence interval 1.61, 5.29) for FVIII level; and among 7 studies (3498 cases) 1.57 (1.32, 1.87) for FIX level. Comparing factor levels above, versus below, the 90th percentile, the estimated pooled odds ratios were 3.00 (2.10, 4.30) for FVIII; 1.77 (1.22, 2.56) for FIX; and 4.56 (2.73, 7.63) for both FVIII and FIX considered jointly., Conclusions: We confirm increases in risk of VTE across population distributions of FVIII and FIX levels. Levels above the 90th percentile have almost twice the risk for FIX level compared to levels below; three-fold risk for FVIII level; and almost five-fold risk for both FVIII and FIX levels elevated., Competing Interests: Declaration of competing interest GDOL, OW and MW declare consultancies with Freeline Therapeutics, London. AvHV, FRR and AF declare that they have no known competing interests that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background and Aims: Non-alcoholic fatty liver disease (NAFLD) is characterized by the pathological accumulation of triglycerides in hepatocytes and is associated with insulin resistance, atherogenic dyslipidaemia and cardiometabolic diseases. Thus far, the extent of metabolic dysregulation associated with hepatic triglyceride accumulation has not been fully addressed. In this study, we aimed to identify metabolites associated with hepatic triglyceride content (HTGC) and map these associations using network analysis., Methods: To gain insight in the spectrum of metabolites associated with hepatic triglyceride accumulation, we performed a comprehensive plasma metabolomics screening of 1363 metabolites in apparently healthy middle aged (age 45-65) individuals (N = 496) in whom HTGC was measured by proton magnetic resonance spectroscopy. An atlas of metabolite-HTGC associations, based on univariate results, was created using correlation-based Gaussian graphical model (GGM) and genome scale metabolic model network analyses. Pathways associated with the clinical prognosis marker fibrosis 4 (FIB-4) index were tested using a closed global test., Results: Our analyses revealed that 118 metabolites were univariately associated with HTGC (p-value <6.59 × 10 -5 ), including 106 endogenous, 1 xenobiotic and 11 partially characterized/uncharacterized metabolites. These associations were mapped to several biological pathways including branched amino acids (BCAA), diglycerols, sphingomyelin, glucosyl-ceramide and lactosyl-ceramide. We also identified a novel possible HTGC-related pathway connecting glutamate, metabolonic lactone sulphate and X-15245 using the GGM network. These pathways were confirmed to be associated with the FIB-4 index as well. The full interactive metabolite-HTGC atlas is provided online: https://tofaquih.github.io/AtlasLiver/., Conclusions: The combined network and pathway analyses indicated extensive associations between BCAA and the lipids pathways with HTGC and the FIB-4 index. Moreover, we report a novel pathway glutamate-metabolonic lactone sulphate-X-15245 with a potential strong association with HTGC. These findings can aid elucidating HTGC metabolomic profiles and provide insight into novel drug targets for fibrosis-related outcomes., (© 2023 The Authors. Liver International published by John Wiley & Sons Ltd.)

Background: There is limited information on short- and long-term effects of venous thromboembolism (VTE) on health-related quality of life (HRQoL) in the elderly., Objectives: To assess change in generic HRQoL and disease-specific HRQoL in patients 1 year after the VTE., Methods: The Age and Thrombosis, Acquired and Genetic risk factors in the elderly (AT-AGE) study is a 2-center case-control study performed in Leiden, the Netherlands, and Vermont, United States, among individuals aged ≥70 years. We measured generic HRQoL using the 36-item Short Form Health Survey (SF-36) and disease-specific HRQoL using the Venous Insufficiency Epidemiological and Economic Study-Quality of Life/Symptoms Questionnaire (VEINES-QoL/Sym) and the Pulmonary Embolism-Specific Quality of Life Questionnaire (PEmb-QoL). All patients completed these questionnaires shortly after their VTE and 1 year later, while controls completed the 36-item Short Form Health Survey questionnaire once. Linear regression for change in quality of life scores was performed and adjusted for potential confounders., Results: For the current analysis, we included patients who were visited twice ( n  = 316) and controls ( n  = 427) with HRQoL information. Mean age of patients and controls was similar (78.8 vs 75.5 years). In patients who survived at least 1 year after the VTE, generic HRQoL improved for both summary scores, but it did not reach the level of the age-matched controls: physical and mental summary scores increased by 5.6 and 5.5 points, respectively, but compared with controls, remained 8.2 and 6.4 points lower. For disease-specific HRQoL, the Pulmonary Embolism-Specific Quality of Life Questionnaire overall score decreased from 21.7% to 15.2%, indicating improved HRQoL. Venous Insufficiency Epidemiological and Economic Study-Quality of Life/Symptoms Questionnaire scores did not change over time., Conclusion: Overall, the quality of life of patients with VTE was worse than that of controls after 1 year, indicating a long-term impact of VTE diagnosis in the elderly., (© 2023 The Author(s).)

Tandem cytosine-adenine-guanine (CAG) repeat sizes of 36 or more in the huntingtin gene (HTT) cause Huntington's disease (HD). Apart from neuropsychiatric complications, the disease is also accompanied by metabolic dysregulation and weight loss, which contribute to a progressive functional decline. Recent studies also reported an association between repeats below the pathogenic threshold (<36) for HD and body mass index (BMI), suggesting that HTT repeat sizes in the non-pathogenic range are associated with metabolic dysregulation. In this study, we hypothesized that HTT repeat sizes < 36 are associated with metabolite levels, possibly mediated through reduced BMI. We pooled data from three European cohorts (n = 10 228) with genotyped HTT CAG repeat size and metabolomic measurements. All 145 metabolites were measured on the same targeted platform in all studies. Multilevel mixed-effects analysis using the CAG repeat size in HTT identified 67 repeat size metabolite associations. Overall, the metabolomic profile associated with larger CAG repeat sizes in HTT were unfavorable-similar to those of higher risk of coronary artery disease and type 2 diabetes-and included elevated levels of amino acids, fatty acids, low-density lipoprotein (LDL)-, very low-density lipoprotein- and intermediate density lipoprotein (IDL)-related metabolites while with decreased levels of very large high-density lipoprotein (HDL)-related metabolites. Furthermore, the associations of 50 metabolites, in particular, specific very large HDL-related metabolites, were mediated by lower BMI. However, no mediation effect was found for 17 metabolites related to LDL and IDL. In conclusion, our findings indicate that large non-pathogenic CAG repeat sizes in HTT are associated with an unfavorable metabolomic profile despite their association with a lower BMI., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: There is room for improvement of prevention of venous thromboembolism (VTE) after lower-leg cast application or knee arthroscopy. Information about the mechanism of clot formation in these patients may be useful to identify new prophylaxis targets. We aimed to study the effect of 1) lower-leg injury and 2) knee arthroscopy on thrombin generation., Methods: A cross-sectional study was conducted using plasma samples of POT-(K)CAST trials to measure ex vivo thrombin generation (Calibrated Automated Thrombography [CAT]) and plasma levels of prothrombin fragment 1 + 2 (F1 + 2), thrombin-antithrombin (TAT), fibrinopeptide A (FPA). Plasma was obtained shortly after lower-leg trauma or before and after (< 4 h) knee arthroscopy. Participants were randomly selected from those who did not develop VTE. For aim 1, samples of 88 patients with lower-leg injury were compared with 89 control samples (i.e., preoperative samples of arthroscopy patients). Linear regression was used to obtain mean differences (or ratios if ln-retransformed because of skewedness) adjusted for age, sex, body mass index, comorbidities. For aim 2, pre- and postoperative samples of 85 arthroscopy patients were compared, for which mean changes were obtained., Results: In patients with lower-leg injury (aim 1), endogenous thrombin potential, thrombin peak, velocity index, FPA and TAT were increased as compared with controls. In arthroscopy patients (aim 2), pre- and postoperative levels were similar for all parameters., Conclusion: Lower-leg trauma increases thrombin generation both ex vivo and in vivo, in contrast to knee arthroscopy. This may imply that the pathogenesis of VTE is different in both situations., (© 2023. The Author(s).)

Background: In studies of time-to-events, it is common to collect information about events that occurred before the inclusion in a prospective cohort. When the studied risk factors are independent of time, including both pre- and post-inclusion events in the analyses, generally referred to as relying on an ambispective design, increases the statistical power but may lead to a selection bias. In the field of venous thromboembolism (VT), ABO blood groups have been the subject of extensive research due to their substantial effect on VT risk. However, few studies have investigated their effect on the risk of VT recurrence. Motivated by the study of the association of genetically determined ABO blood groups with VT recurrence, we propose a methodology to include pre-inclusion events in the analysis of ambispective studies while avoiding the selection bias due to mortality., Methods: This work relies on two independent cohorts of VT patients, the French MARTHA study built on an ambispective design and the Dutch MEGA study built on a standard prospective design. For the analysis of the MARTHA study, a weighted Cox model was developed where weights were defined by the inverse of the survival probability at the time of data collection about the events. Thanks to the collection of information on the vital status of patients, we could estimate the survival probabilities using a delayed-entry Cox model on the death risk. Finally, results obtained in both studies were then meta-analysed., Results: In the combined sample totalling 2,752 patients including 993 recurrences, the A1 blood group has an increased risk (Hazard Ratio (HR) of 1.18, p = 4.2 × 10 -3 ) compared with the O1 group, homogeneously in MARTHA and in MEGA. The same trend (HR = 1.19, p = 0.06) was observed for the less frequent A2 group., Conclusion: The proposed methodology increases the power of studies relying on an ambispective design which is frequent in epidemiologic studies about recurrent events. This approach allowed to clarify the association of ABO blood groups with the risk of VT recurrence. Besides, this methodology has an immediate field of application in the context of genome wide association studies., (© 2023. The Author(s).)

Men seem to have a higher intrinsic risk of venous thromboembolism (VTE) than women, regardless of age. To date, this difference has not been explained. By integrating state-of-the-art research presented at the International Society on Thrombosis and Haemostasis Congress of 2021 with the available literature, we address potential explanations for this intriguing risk difference between men and women. We discuss the role of exogenous and endogenous sex hormones as the most important known sex-specific determinants of VTE risk. In addition, we highlight clues on the role of sex hormones and VTE risk from clinical scenarios such as pregnancy and the polycystic ovary syndrome. Furthermore, we address new potential sex-specific risk factors and unanswered research questions, which could provide more insight in the intrinsic risk difference between men and women, such as body height and differences in body fat distribution, leading to dysregulation of metabolism and inflammation., (© 2022 The Authors. Research and Practice in Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis (ISTH).)

Importance: Previous venous thrombosis (VT) is associated with risk of future VT, but quantification of risk over the life course is poorly understood. More information is needed for clinicians to understand the association of remote history of VT with the risk of VT in older patients., Objective: To assess the association between a remote history of VT and the development of VT in older age., Design, Setting, and Participants: The Age and Thrombosis, Acquired and Genetic Risk Factors in the Elderly case-control study enrolled patients 70 years and older with VT and control individuals 70 years and older without VT between June 2008 and August 2011. The Age and Thrombosis, Acquired and Genetic Risk Factors in the Elderly study is a 2-center, population-based case-control study that was conducted in Burlington, Vermont, in the US and in Leiden, the Netherlands. Consecutively diagnosed patients with an objectively proven episode of VT (deep vein thrombosis of the leg or pulmonary embolism) were included. Control individuals were identified in the same geographic areas as the patients and were randomly selected. Data were analyzed between May 2021 and October 2021., Exposures: Self-reported remote VT (occurring >10 years before to enrollment)., Main Outcomes and Measures: The main outcome was the risk of VT at older age. The association of self-reported history of remote VT with VT at older age was assessed by calculating odds ratios (ORs) as estimates of relative risk with 95% CIs., Results: A total of 460 patients with VT and 456 control participants were included. There were slightly more women than men in both groups (60.2% of patients [n = 277] were women and 52.4% of control participants [n = 239] were women), and the mean (range) age of patients was 78.7 (70.0-100.9) years, which was similar to the control participants. Compared with individuals without remote VT, those with a remote history of VT had an increased risk of VT (OR, 2.54; 95% CI, 1.56-4.13). The crude risk estimate was robust to adjustment and time since remote VT, that is, individuals with a VT 10 to 30 years ago (OR, 2.74; 95% CI, 1.34-5.57) and those with a VT more than 30 years ago (OR, 2.42; 95% CI, 1.21-4.84) had a an increased risk of VT. The population-attributable fraction of a remote history of VT was 7.7%., Conclusions and Relevance: In this study, a remote history of VT was associated with risk of VT in older individuals. This quantification could assist clinicians in advising patients on VT prevention.

Background: The preponderance of the evidence supports no association between traditional cardiovascular risk factors and venous thromboembolism (VTE), other than obesity. There are limited data in older people., Objectives: To investigate whether cardiovascular risk factors (body mass index, smoking, alcohol intake, hypertension, and diabetes) are associated with the risk of VTE in elderly and to assess the combined effect between cardiovascular risk factors and genetic risk factors for VTE (factor V Leiden/prothrombin 20210A, positive family history of VTE, and non-O blood group)., Methods: The Age and Thrombosis, Acquired and Genetic risk factors in the Elderly study is a multicenter case-control study performed in Vermont, USA and Leiden, the Netherlands, comprising 401 cases with first VTE and 431 control subjects, all aged ≥70 years. To assess the risk of VTE, odds ratios (OR) with 95% confidence intervals (CIs) were calculated, adjusting for potential confounders., Results: Both height and weight were positively associated with VTE risk: the ORs were 2.2 (95% CI, 1.2-3.9) and 1.5 (95% CI, 1.0-2.4) in the top quartile for height and weight separately. This risk was more pronounced for unprovoked VTE. Smoking, alcohol intake, and diabetes were not associated with VTE. Higher systolic and diastolic blood pressure and hypertension were associated with a decreased risk of VTE. In the presence of a genetic predisposition, height and weight further increased the risk of VTE., Conclusions: In the elderly, height and weight are positively associated with the risk of VTE. With genetic predisposition, higher levels of height and weight further increase the risk of VTE., (© 2022 The Authors. Research and Practice in Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis (ISTH).)

In thrombin generation (TG) assays, regarded as global coagulation tests, contact activation is considered a major problem which can be eliminated by adding Corn Trypsin Inhibitor (CTI). In previous studies, however, venous thrombosis risk prediction using TG assays did not improve after CTI addition. However, it is unknown whether CTI addition could help to detect subtle but relevant nuances in determinants of TG, making the assay more suitable to detect disturbances in the coagulation system. This study's objective was to assess whether the addition of CTI is associated with a broader contribution of individual coagulation factors to the total amount of thrombin formed in Calibrated Automated Thrombogram (CAT) and Technoclone Thrombin Generation Assay (TGA). Thrombin generation was measured in 326 healthy individuals from THE VTE study at very low tissue factor concentrations, with and without addition of CTI prior to blood sampling. The influence of several coagulation factors on total amount of thrombin formed, i.e. area under the curve (AUC) or endogenous thrombin potential (ETP), was analysed using multiple linear regression with standardisation of all values resulting in Z-scores with 95% confidence intervals (95%CI). Association between coagulation factors and TG changed minimally after addition of CTI. Largest changes after CTI addition were found for following factors: for CAT: free protein S (from 0.00 (95%CI -0.12 to 0.12) to -0.29 (95%CI -0.43 to -0.15)) and protein S (from -0.05 (95%CI -0.18 to 0.08) to -0.21 (95%CI -0.37 to -0.05)); for TGA: antithrombin (from -0.11 (-0.23 to 0.02) to -0.19 (-0.30 to -0.07)), factor VIII (from 0.15 (0.03 to 0.27) to 0.24 (0.13 to 0.36)) and fibrinogen (from 0.12 (-0.01 to 0.26) to 0.19 (0.06 to 0.32)). In conclusion, there is no clear trend towards a broader contribution of coagulation factors in samples handled with CTI compared with those handled without CTI., Competing Interests: The authors have declared that no competing interests exist.

Background: Oral postmenopausal hormone therapy (HT) increases the risk of venous thrombosis (VT). We postulated that activated protein C (APC) resistance induced by HT is one of the mechanisms causing VT, and also assessed the role of one of the main determinants of APC resistance (i.e., tissue factor pathway inhibitor [TFPI])., Methods: We performed a nested case-control study embedded within two Women's Health Initiative hormone trials. Women were randomized to hormone therapy or placebo. Biomarkers were measured at baseline and after 1 year in 217 cases and 817 controls., Results: Increased APC resistance and decreased TFPI at baseline were associated with VT (odds ratio 1.20-2.06). However, women with such prothrombotic profile at baseline did not have further increased risk of VT when randomized to HT compared with placebo. Although there was no change in APC resistance or TFPI in placebo group after 1 year, HT group showed prothrombotic changes in the biomarkers (i.e., an increase in APC resistance) (mean [standard deviation] 0.39 [0.54]) and decrease in TFPI (-0.21 [0.50]: free TFPI, -0.24 [0.22]: TFPI activity -0.22 [0.20]: total TFPI). However, HT induced prothrombotic change in biomarkers did not increase risk of VT., Conclusion: Women with prothrombotic levels of APC resistance and TFPI at baseline were not at increased risk of VT when randomized to HT compared with placebo. This suggests that testing for these biomarkers before starting HT is not required. HT led to prothrombotic change in these biomarkers after one year, but this did not relate to increased risk of VT., (© 2021 International Society on Thrombosis and Haemostasis.)

Background: A high D-dimer level and parameters of the thrombin generation (TG) potential are associated with the risk of a first venous thrombosis (VT) in young and middle-aged populations., Objectives: To investigate whether D-dimer and TG potential (lag-time, time-to-peak [ttPeak], peak thrombin, endogenous thrombin potential [ETP], and velocity index), are associated with the risk of a first VT in those aged 70 years and older., Methods: We included 215 patients with a first VT and 358 controls, all aged >70 years, from the Age and Thrombosis, Acquired and Genetic Risk Factors in the Elderly (AT-AGE) study. To assess the risk of VT, odds ratios with 95% confidence intervals (CIs) were estimated using logistic regression analysis., Results: D-dimer and all TG parameters except lag time were associated with an increased risk of VT in a dose-response manner. Comparing the fourth with the first quartile (for ttPeak comparing the first with the fourth quartile), risk estimates were: 7.8 (95% CI, 4.0-15.0) for peak, 2.0 (95% CI, 1.2-3.3) for ttPeak, 9.1 (95% CI, 4.4-18.9) for ETP, and 11.5 (95% CI, 5.7-23.3) for velocity index. Comparing the highest quartile of D-dimer with the lowest, the risk was 7.7-fold increased (95% CI, 4.0-14.8). Furthermore, all factors also increased the risk of VT after dichotomizing at more extreme cutoff values. The risk of VT was further increased in the presence of multiple prothrombotic TG parameters and elevated D-dimer level or in combination with prothrombotic mutations., Conclusions: D-dimer and TG parameters (except lag time) are associated with the risk of first VT in elderly population., (© 2021 The Authors. Research and Practice in Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis (ISTH).)

Glycemic traits are used to diagnose and monitor type 2 diabetes and cardiometabolic health. To date, most genetic studies of glycemic traits have focused on individuals of European ancestry. Here we aggregated genome-wide association studies comprising up to 281,416 individuals without diabetes (30% non-European ancestry) for whom fasting glucose, 2-h glucose after an oral glucose challenge, glycated hemoglobin and fasting insulin data were available. Trans-ancestry and single-ancestry meta-analyses identified 242 loci (99 novel; P < 5 × 10 -8 ), 80% of which had no significant evidence of between-ancestry heterogeneity. Analyses restricted to individuals of European ancestry with equivalent sample size would have led to 24 fewer new loci. Compared with single-ancestry analyses, equivalent-sized trans-ancestry fine-mapping reduced the number of estimated variants in 99% credible sets by a median of 37.5%. Genomic-feature, gene-expression and gene-set analyses revealed distinct biological signatures for each trait, highlighting different underlying biological pathways. Our results increase our understanding of diabetes pathophysiology by using trans-ancestry studies for improved power and resolution.

Background: Venous thromboembolism (VTE) is a complex disease with an incidence rate of about 1 in 1000 per year. Despite the availability of validated biomarkers for VTE, unprovoked events account for 50% of first events. Therefore, emerging high-throughput proteomics are promising methods for the expansion of VTE biomarkers. One such promising high-throughput platform is SomaScan, which uses a large library of synthetic oligonucleotide ligands known as aptamers to measure thousands of proteins., Objective: The aim of this study was to evaluate the viability of the aptamer-based SomaScan platform for VTE studies by examining its agreement with standard laboratory methods., Methods: We examined the agreement between eight established VTE biomarkers measured by SomaScan and standard laboratory immunoassay and viscosity-based instruments in 54 individuals (27 cases and 27 controls) from the Thrombophilia, Hypercoagulability and Environmental Risks in Venous Thromboembolism study. We performed the agreement analysis by using a regression model and predicting the estimates and the 95% prediction interval (PI) of the laboratory instrument values using SomaScan values., Results: SomaScan measurements exhibited overall poor agreement, particularly for D-dimer (average fit, 492.7 ng/mL; 95% PI, 110.0-1998.2) and fibrinogen (average fit, 3.3 g/L; 95% PI, 2.0-4.7)., Conclusion: Our results indicate that SomaScan measurement had poor agreement with the standard laboratory measurements. These results may explain why some genome-wide association studies with VTE proteins measured by SomaScan did not confirm previously identified loci. Therefore, SomaScan should be considered with caution in VTE studies., (© 2021 The Authors. Research and Practice in Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis (ISTH).)

Genetic risk score (GRS) analysis is a popular approach to derive individual risk prediction models for complex diseases. In venous thrombosis (VT), such type of analysis shall integrate information at the ABO blood group locus, which is one of the major susceptibility loci. However, there is no consensus about which single nucleotide polymorphisms (SNPs) must be investigated when properly assessing association between ABO locus and VT risk. Using comprehensive haplotype analyses of ABO blood group tagging SNPs in 5425 cases and 8445 controls from 6 studies, we demonstrate that using only rs8176719 (tagging O1) to correctly assess the impact of ABO locus on VT risk is suboptimal, because 5% of rs8176719-delG carriers do not have an increased risk of developing VT. Instead, we recommend the use of 4 SNPs, rs2519093 (tagging A1), rs1053878 (A2), rs8176743 (B), and rs41302905 (O2), when assessing the impact of ABO locus on VT risk to avoid any risk misestimation. Compared with the O1 haplotype, the A2 haplotype is associated with a modest increase in VT risk (odds ratio, ∼1.2), the A1 and B haplotypes are associated with an ∼1.8-fold increased risk, whereas the O2 haplotype tends to be slightly protective (odds ratio, ∼0.80). In addition, although the A1 and B blood groups are associated with increased von Willebrand factor and factor VIII plasma levels, only the A1 blood group is associated with ICAM levels, but in an opposite direction, leaving additional avenues to be explored to fully understand the spectrum of biological effects mediated by ABO locus on cardiovascular traits.

Background: Von Willebrand factor (VWF) levels are regulated by genetic and acquired factors. The acquired factors are mostly related to age and could be mediators of the age effect on VWF levels., Objectives: To disentangle the role of genetic (sex, blood group) and acquired factors (comorbidities, body mass index, reduced kidney function, hormone use, and inflammation) in regulating von Willebrand factor antigen (VWF:Ag) and factor VIII activity (FVIII:C) levels in the normal population., Methods: Analysis were performed in a large population sample (2923 individuals) from the Multiple Environmental and Genetic Assessment of risk factors for venous thrombosis (MEGA study), after exclusion of individuals with active cancer and women who were pregnant or within nine months postpartum. The increase of VWF:Ag and FVIII:C with age was evaluated by linear regression after the age of 40 years. Analyses were adjusted for acquired factors and stratified for sex and blood group., Results: VWF:Ag and FVIII:C increased with age: increase per decade of age for VWF:Ag 18 IU/dL (95%CI 15-20) and for FVIII:C 12 IU/dL (95%CI 10-14). After adjustment for acquired factors, the increase per decade was 13 IU/dL (95%CI 10-16) for VWF:Ag and 9 IU/dL (95%CI 6-11) for FVIII:C. The stratified analysis for blood group showed higher increase in the non-O group, but these differences were annulled after adjustment for acquired factors., Conclusions: VWF:Ag and FVIII:C increase with age. Carriers of blood group non-O present a steeper increase of VWF:Ag and FVIII:C with age, that is mediated by acquired factors., (Copyright © 2020. Published by Elsevier Ltd.)

Essentials VWF and FVIII increase with age in patients affected by VWD. VWF and FVIII increase in type 1 and in low levels of VWF patients. VWF and FVIII do not increase in type 1 Vicenza. FVIII increases in type 2 VWD patients. ABSTRACT: Background Increasing levels of von Willebrand factor (VWF) and factor VIII (FVIII:C) was associated with age in type 1 von Willebrand disease (VWD). Objectives To evaluate VWF and FVIII:C increase with age in a large group of patients with VWD and low levels of VWF, in whom levels were repeatedly measured. Methods Clinical charts from all patients evaluated at the A. Bianchi Bonomi Center between 1970 and 2018 were reviewed and data on VWF and FVIII:C collected. Patients affected by type 3, severe type 1 and 2N VWD were excluded. The repeated measurements were evaluated by linear mixed-effects models. A linear association between age and VWF/FVIII:C was shown after the age of 40 years in the linear mixed models and analyzed by calculating the regression slope coefficient (β). Results A total of 617 patients were included in the study (314 type 2, 112 type 1, 181 low VWF levels), with a median age at first measurement of 28 years (interquartile range 14/42) and a mean follow-up of 16 years (standard deviation 11). VWF and FVIII:C increased with age in the whole group. The increase became linear after the age of 40 years (3.68 and 7.44 IU/dL per decade for VWF:activity and FVIII:C). In type 2, FVIII:C increased with age, whereas an increase of both VWF:activity and FVIII:C were shown in patients with type 1 VWD and low levels of VWF. Conclusions A differential increase of VWF and FVIII:C with age was shown among in different ages and types of VWD., (© 2020 International Society on Thrombosis and Haemostasis.)

Essentials Venous thrombosis (VT) risk and coagulation factor levels increase with age. We studied the association between levels of procoagulant factors and the risk of a first VT in the elderly. Higher levels of factors VIII, IX, and XI, but not prothrombin, were associated with the risk of VT. Similar risk patterns were observed for provoked and unprovoked VT and for deep vein thrombosis and pulmonary embolism separately. ABSTRACT: Background Venous thrombosis (VT) incidence increases markedly with age. Coagulation factors are also positively associated with age. Objective To study whether higher levels of coagulation factors II (prothrombin), VIII, IX, and XI are associated with risk of a first VT in the elderly. Methods Four hundred and one patients and 431 control subjects aged 70 and older were included in the Age and Thrombosis, Acquired and Genetic risk factors in the Elderly (AT-AGE) study. Blood was collected 1 year after the event in patients and in all control subjects for measurement of coagulation factors. To assess the risk of VT, odds ratios (ORs) were calculated after stratification of coagulation factors in quartiles and at the 90th percentile, adjusting for potential confounders (age, sex, body mass index, and study center). Results Mean age was 78 years (range: 70-100 years). The ORs of VT for factors in the top quartile compared with the lowest quartile were 4.5 (95% confidence interval [CI]:2.7-7.3) for factor VIII, 2.4 (95% CI:1.1-5.2) for factor IX, and 1.7 (95% CI:1.0-2.9) for factor XI. High prothrombin was not associated with an increased VT risk. There was no dose-response association between the number of high coagulation factors and VT risk. The population attributable risk (PAR) of VT was 37.6%, 23.3%, and 12.4% for factor VIII, IX, and XI, respectively. Conclusion In this study of the elderly, higher factors VIII, IX, and XI but not prothrombin, were positively associated with the risk of VT., (© 2020 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis.)

Objective: Whether hepatic triglyceride content (HTGC) contributes to hypercoagulability beyond total body fat (TBF) and visceral adipose tissue (VAT) is unclear. We, therefore, aimed to investigate the association between HTGC and coagulation factors (F)I (fibrinogen), VIII, IX, and XI while adjusting for TBF and VAT. Approach and Results: In this cross-sectional analysis of the NEO study (Netherlands Epidemiology of Obesity; n=6671), a random subset of participants underwent magnetic resonance imaging and magnetic resonance spectroscopy to assess VAT and HTGC (n=2580). We excluded participants without complete imaging and coagulation assessment, and with history of liver disease, venous thrombosis, or on anticoagulation. Mean differences in coagulation factor levels across HTGC quartiles were estimated by linear regression adjusted for age, sex, ethnicity, education, alcohol intake, physical activity, smoking, estrogen, and menopause, in addition to TBF and VAT. Among the 1946 participants included, median HTGC was 2.66% (interquartile range: 1.34%-6.27%). Coagulation factor levels increased dose-dependently across HTGC quartiles. Mean differences between the fourth and first quartiles were 14.7 mg/dL (95% CI, 2.1-27.2) for fibrinogen, 6.7 IU/dL (95% CI, 0.5-12.9) for FVIII, 26.1 IU/dL (95% CI, 22.4-29.8) for FIX, and 8.6 IU/dL (95% CI, 4.6-12.6) for FXI. With further adjustment for TBF and VAT, the dose-response association of HTGC with FIX persisted, whereas associations with other factors disappeared., Conclusions: HTGC was associated with various coagulation factors, of which FIX remained associated with HTGC after adjustment for TBF and VAT. HTGC might contribute to venous thrombosis risk beyond total body and visceral fat through FIX levels.

Metabolomics studies have seen a steady growth due to the development and implementation of affordable and high-quality metabolomics platforms. In large metabolite panels, measurement values are frequently missing and, if neglected or sub-optimally imputed, can cause biased study results. We provided a publicly available, user-friendly R script to streamline the imputation of missing endogenous, unannotated, and xenobiotic metabolites. We evaluated the multivariate imputation by chained equations (MICE) and k-nearest neighbors (kNN) analyses implemented in our script by simulations using measured metabolites data from the Netherlands Epidemiology of Obesity (NEO) study ( n = 599). We simulated missing values in four unique metabolites from different pathways with different correlation structures in three sample sizes (599, 150, 50) with three missing percentages (15%, 30%, 60%), and using two missing mechanisms (completely at random and not at random). Based on the simulations, we found that for MICE, larger sample size was the primary factor decreasing bias and error. For kNN, the primary factor reducing bias and error was the metabolite correlation with its predictor metabolites. MICE provided consistently higher performance measures particularly for larger datasets ( n > 50). In conclusion, we presented an imputation workflow in a publicly available R script to impute untargeted metabolomics data. Our simulations provided insight into the effects of sample size, percentage missing, and correlation structure on the accuracy of the two imputation methods.

It is not known whether the synergistic effect of genetic markers, increasing the risk of venous thrombosis (VT), and combined oral contraceptives (COC) use varies between different types of progestogens in these preparations. We investigated the joint effect of genetic risk factor, that is, F5 rs6025, F2 rs1799963, and FGG rs2066865 mutations, and different progestogens on the risk of VT. The constrained maximum likelihood estimation (CMLE) method was used to calculate joint effects, expressed as odds ratio (OR) with 95% confidence intervals [CI]. As the dose of estrogen is known to be a risk factor for VT, analyses were restricted to COC with 30 µg estrogen and each progestogen. Overall, the joint effect of COC and genetic variants was lowest for COC containing the progestogen levonorgestrel, albeit CIs were wide. The OR (95% CI) of the four different analyses (i.e. joint effect with F5 rs6025, F2 rs1799963, F5 rs6025 or F2 rs1799963 and FGG rs2066865) ranged between 7·4 (5·4-10·2) and 24·8 (12·3-50·0) for levonorgestrel. For gestodene the joint effect ranged between 11·7 (7·2-19·1) and 30·9 (10·6-89·9). Desogestrel and cyproterone acetate had the highest risk estimates: 14·6 (9·7-21·9) and 32·6 (13·2-80·6) and 15·5 (9·7-24·9) and 44·4 (16·9-116·3) respectively. In women with inherited thrombophilia, COC containing levonorgestrel were associated with the lowest risk of VT, albeit the CIs were wide., (© 2020 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

Bleeding disorders and thrombotic complications are major causes of morbidity and mortality with many cases being unexplained. Thrombus formation involves aberrant expression and activation of tissue factor (TF) in vascular endothelial and smooth muscle cells. Here, we sought to identify factors that modulate TF gene expression and activity in these vascular cells. The LIM-only protein FHL2 is a scaffolding protein that modulates signal transduction pathways with crucial functions in endothelial and smooth muscle cells. However, the role of FHL2 in TF regulation and thrombosis remains unexplored. Using a murine model of venous thrombosis in mesenteric vessels, we demonstrated that FHL2 deficiency results in exacerbated thrombus formation. Gain- and loss-of-function experiments revealed that FHL2 represses TF expression in endothelial and smooth muscle cells through inhibition of the transcription factors nuclear factor κB and activating protein-1. Furthermore, we observed that FHL2 interacts with the cytoplasmic tail of TF. In line with our in vivo observations, FHL2 decreases TF activity in endothelial and smooth muscle cells whereas FHL2 knockdown or deficiency results in enhanced TF activity. Finally, the FHL2 single nucleotide polymorphism rs4851770 was associated with the risk of venous thrombosis in a large population of venous thrombosis cases and control subjects from 12 studies (INVENT consortium). Altogether, our results highlight functional involvement of FHL2 in TF-mediated coagulation and identify FHL2 as a novel gene associated with venous thrombosis in humans., (Copyright© 2020 Ferrata Storti Foundation.)

Objectives: We investigated combined hormonal contraceptives (CHC) prescribing patterns (focusing on combined oral contraceptives; COC) in three countries (Netherlands, Denmark, United Kingdom) in a time period preceding and in a time period following the European Commission's decision to update product information, and we estimated changes in incidence of venous thromboembolism (VTE) between the two periods., Study Design: We conducted a drug utilization analysis and a cohort study using routinely collected data. We calculated number, proportion and incidence rate of new users, switchers, and stoppers of COC in both time periods. VTE incidence was calculated in new users of COC and in all women aged 18-49 years., Results: In all countries, the largest proportion (> 75%) of new users used COC containing levonorgestrel, norethisterone, or norgestimate, (i.e., indicated by European Medicines Agency (EMA) as the safest preparations) in both time periods. Switching did not demonstrate a clear pattern towards these types of COC and distribution of stoppers was similar in both time periods. While the proportion of new users initiating COC containing levonorgestrel, norethisterone, or norgestimate increased slightly, this did not translate to a decrease in the overall VTE incidence., Conclusion: All three countries had the greatest proportion of women initiating a COC containing levonorgestrel, norethisterone, or norgestimate, and this proportion increased in the period after the European Commission decision albeit the increase was small due to the high percentage of use before the decision. This did not translate into a measureable change in the incidence of VTE., Implications: Both before and after the European Commission's decision, the largest proportion of new users started with combined oral contraceptives containing levonorgestrel, norethisterone, or norgestimate. Earlier studies had already indicated an increased risk of VTE associated with COC containing other progestogens compared with these preparations, so it is possible that physicians were already preferentially prescribing COC containing levonorgestrel, norethisterone, or norgestimate to new users., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:, (© 2020 The Authors.)

Early-phase insulin secretion is a determinant of postprandial glucose homeostasis. In this study, we aimed to identify novel genetic variants associated with the early-phase insulin response to a liquid mixed meal by a genome-wide association study using a discovery and replication design embedded in the Netherlands Epidemiology of Obesity (NEO) study. The early-phase insulin response was defined as the difference between the natural logarithm-transformed insulin concentrations of the postprandial state at 30 min after a meal challenge and the fasting state (Δinsulin). After Bonferroni correction, rs505922 (β: -6.5% [minor allele frequency (MAF) 0.32, P = 3.3 × 10 -8 ]) located in the ABO gene reached genome-wide significant level ( P < 5 × 10 -8 ) and was also replicated successfully (β: -7.8% [MAF 0.32, P = 7.2 × 10 -5 ]). The function of the ABO gene was assessed using in vitro shRNA-mediated knockdown of gene expression in the murine pancreatic β-cell line MIN6. Knocking down the ABO gene led to decreased insulin secretion in the murine pancreatic β-cell line. These data indicate that the previously identified elevated risk of type 2 diabetes for carriers of the ABO rs505922:C allele may be caused by decreased early-phase insulin secretion., (© 2019 by the American Diabetes Association.)

Venous thromboembolism (VTE) is a significant contributor to morbidity and mortality. To advance our understanding of the biology contributing to VTE, we conducted a genome-wide association study (GWAS) of VTE and a transcriptome-wide association study (TWAS) based on imputed gene expression from whole blood and liver. We meta-analyzed GWAS data from 18 studies for 30 234 VTE cases and 172 122 controls and assessed the association between 12 923 718 genetic variants and VTE. We generated variant prediction scores of gene expression from whole blood and liver tissue and assessed them for association with VTE. Mendelian randomization analyses were conducted for traits genetically associated with novel VTE loci. We identified 34 independent genetic signals for VTE risk from GWAS meta-analysis, of which 14 are newly reported associations. This included 11 newly associated genetic loci (C1orf198, PLEK, OSMR-AS1, NUGGC/SCARA5, GRK5, MPHOSPH9, ARID4A, PLCG2, SMG6, EIF5A, and STX10) of which 6 replicated, and 3 new independent signals in 3 known genes. Further, TWAS identified 5 additional genetic loci with imputed gene expression levels differing between cases and controls in whole blood (SH2B3, SPSB1, RP11-747H7.3, RP4-737E23.2) and in liver (ERAP1). At some GWAS loci, we found suggestive evidence that the VTE association signal for novel and previously known regions colocalized with expression quantitative trait locus signals. Mendelian randomization analyses suggested that blood traits may contribute to the underlying risk of VTE. To conclude, we identified 16 novel susceptibility loci for VTE; for some loci, the association signals are likely mediated through gene expression of nearby genes.

Background: It is insufficiently understood if there is an association between diabetes and VT, and what the underlying mechanism would be., Objectives: We aimed to study the association between glucose concentrations with several coagulation factors in the general population., Methods: This is a cross-sectional analysis of baseline measurements within 5778 participants of the Netherlands Epidemiology of Obesity (NEO) study, a population-based cohort study of individuals 45 to 65 years. Associations between fasting glucose and HbA1c concentrations, and postprandial glucose response and factor (F) VIII, FIX, FXI, and fibrinogen levels were examined using linear regression analyses and by calculating mean levels per category of glucose concentrations while adjusting for confounding factors., Results: Per each mmol/L higher fasting glucose concentration we observed higher levels of fasting FVIII (5.33%, 95% CI: 4.00-6.65), FIX (6.19%, 95% CI: 5.15-7.23), and FXI (2.11%, 95% CI: 1.20-3.02). Results for fasting HbA1c and postprandial glucose response were similar. Participants with an impaired fasting glucose, high fasting glucose, and diabetes mellitus had higher mean levels of FVIII, FIX, and FXI than those with a normal glucose metabolism, with the highest differences in the levels of FVIII, FIX, and FXI between a high fasting glucose and a normal glucose metabolism. All associations attenuated after adjustment for total body fat, yet all of the above associations remained after adjustment for the confounding factors, except for fibrinogen when contrasted to glucose., Conclusion: Concentrations of fasting glucose and HbA1c and postprandial glucose response were positively associated with FVIII, FIX, and FXI, and to some extent also with fibrinogen., (© 2019 International Society on Thrombosis and Haemostasis.)

Background: Recurrent venous thromboembolism (VTE) is common. Current guidelines suggest that patients with unprovoked VTE should continue anticoagulants unless they have a high bleeding risk, whereas all others can stop. Prediction models may refine this dichotomous distinction, but existing models apply only to patients with unprovoked first thrombosis. We aimed to develop a prediction model for all patients with first VTE, either provoked or unprovoked., Methods and Findings: Data were used from two population-based cohorts of patients with first VTE from the Netherlands (Multiple Environment and Genetic Assessment of Risk Factors for Venous Thrombosis [MEGA] follow-up study, performed from 1994 to 2009; model derivation; n = 3,750) and from Norway (Tromsø study, performed from 1999 to 2016; model validation; n = 663). Four versions of a VTE prediction model were developed: model A (clinical, laboratory, and genetic variables), model B (clinical variables and fewer laboratory markers), model C (clinical and genetic factors), and model D (clinical variables only). The outcome measure was recurrent VTE. To determine the discriminatory power, Harrell's C-statistic was calculated. A prognostic score was assessed for each patient. Kaplan-Meier plots for the observed recurrence risks were created in quintiles of the prognostic scores. For each patient, the 2-year predicted recurrence risk was calculated. Models C and D were validated in the Tromsø study. During 19,201 person-years of follow-up (median duration 5.7 years) in the MEGA study, 507 recurrences occurred. Model A had the highest predictive capability, with a C-statistic of 0.73 (95% CI 0.71-0.76). The discriminative performance was somewhat lower in the other models, with C-statistics of 0.72 for model B, 0.70 for model C, and 0.69 for model D. Internal validation showed a minimal degree of optimism bias. Models C and D were externally validated, with C-statistics of 0.64 (95% CI 0.62-0.66) and 0.65 (95% CI 0.63-0.66), respectively. According to model C, in 2,592 patients with provoked first events, 367 (15%) patients had a predicted 2-year risk of >10%, whereas in 1,082 patients whose first event was unprovoked, 484 (45%) had a predicted 2-year risk of <10%. A limitation of both cohorts is that laboratory measurements were missing in a substantial proportion of patients, which therefore were imputed., Conclusions: The prediction model we propose applies to patients with provoked or unprovoked first VTE-except for patients with (a history of) cancer-allows refined risk stratification, and is easily usable. For optimal individualized treatment, a management study in which bleeding risks are also taken into account is necessary., Competing Interests: The authors have declared that no competing interests exist. SCC is a member of the Editorial Board of PLOS Medicine.

The measurement of homocysteine is still part of routine thrombosis or thrombophilia work-up in many thrombosis centres in the world. Previous observational studies have shown that hyperhomocysteinaemia is associated with an increased risk of first and recurrent venous thrombosis (VT). Randomised trials, however, showed no benefit of homocysteine-lowering therapy on the risk of first or recurrent VT. This discrepancy could be explained by incomplete adjustment for confounders in the observational studies. We investigated in a large population-based follow-up study whether if the levels of homocysteine and its metabolites, methionine and cysteine, were associated with recurrent VT. Approximately three months after discontinuation of anticoagulant treatment, homocysteine, methionine and cysteine concentrations were measured in 2210 patients with VT. During a median follow-up of 6·9 years, 340 patients developed a recurrence (incidence rate, 2·8/100 patient-years). We found that elevated homocysteine concentrations were not associated with an increased risk of recurrent VT, neither as a continuous variable per 5 μmol/l increase (hazard ratio [HR] 0·98 (95% confidence interval [CI], 0·90-1·04)) nor when levels were >95th (>23·0 μmol/l) percentile (HR 1·03 (95% CI, 0·65-1·64)). Similar results were obtained for cysteine and methionine values. We conclude that hyperhomocysteinaemia is not associated with an increased risk of recurrent VT., (© 2019 The Authors British. Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

Background: Cholesteryl ester transfer protein (CETP) plays an important role in lipoprotein metabolism. Previous studies have suggested that the CETP TaqI B1/B2 allele is associated with the risk of venous thrombosis (VT)., Aim: To investigate the associations between genetically determined CETP concentrations and 22 hemostatic factors in healthy individuals, and the risk of a first VT event, in a large VT case-control study., Methods: Analyses were performed in the Multiple Environmental and Genetic Assessment of Risk Factors for Venous Thrombosis (MEGA) case-control study. CETP unweighted/weighted genetic risk scores (GRSs) were derived from three single-nucleotide polymorphisms that were identified from a recent genome-wide association study on serum CETP concentrations. The associations between CETP GRSs and 22 hemostatic factors (procoagulant/anticoagulant and fibrinolytic factors) were assessed by linear regression from an additive model in controls (n = 2813). The associations between CETP GRSs and the risk of a first VT were assessed by logistic regression analyses in 3950 VT cases and 4765 controls., Results: In the controls (median age, 49 years; 53% women), both unweighted and weighted GRSs showed that factor VII activity was negatively associated with the genetically determined CETP concentration (weighted GRS β -3.08 IU/dL per μg/mL genetically determined CETP, 95% confidence interval -5.73 to -0.42). No association was observed with the risk of a first VT., Conclusions: Genetically determined CETP concentrations only showed a weak negative association with factor VII activity. However, this did not lead to an association with the risk of a first VT., (© 2019 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis.)

Background: The risk of venous thromboembolism (VTE) in young women can predominantly be attributed to exogenous hormone use. The influence of (abnormalities in) endogenous sex hormones, as in polycystic ovary syndrome (PCOS) or primary ovarian insufficiency (POI), on VTE risk is uncertain., Objectives: Th assess the association between endogenous sex hormone levels and VTE risk., Methods: Women aged ≤45 years from the MEGA case-control study who provided a blood sample in the absence of exogenous hormone exposure or pregnancy were included. Sex hormone-binding globulin (SHBG), estradiol, follicle-stimulating hormone (FSH) and testosterone were measured. The free androgen index (FAI) and estradiol to testosterone ratio (E:T) were calculated. VTE risk was assessed according to quartiles (Qs) of levels and clinical cut-offs as proxies for PCOS (FAI > 4.5) and POI (FSH > 40 U/L). Logistic regression models were used to estimate adjusted odds ratios (ORs) with 95% confidence intervals (CIs)., Results: Six hundred and sixty-five women (369 cases; 296 controls) were eligible for the analyses. Testosterone and FSH levels, E:T and POI (FSH > 40 U/L vs FSH ≤ 40 U/L) were not associated with VTE risk. For estradiol, VTE risk was increased with levels in Q4 vs Q1 (OR 1.6; 95% CI 1.0-2.5). There was a dose-response relationship between SHBG levels and VTE risk, with the highest OR at Q4 vs Q1: 2.0 (95% CI 1.2-3.3). FAI > 4.5 (PCOS proxy) vs FAI ≤ 4.5 was associated with increased VTE risk (OR 3.3; 95% CI 0.9-11.8)., Conclusions: Estradiol, SHBG and FAI were associated with VTE risk, suggesting a role for endogenous sex hormones in the pathophysiology of VTE in young women., (© 2019 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis.)

Factor VII (FVII) is an important component of the coagulation cascade. Few genetic loci regulating FVII activity and/or levels have been discovered to date. We conducted a meta-analysis of 9 genome-wide association studies of plasma FVII levels (7 FVII activity and 2 FVII antigen) among 27 495 participants of European and African ancestry. Each study performed ancestry-specific association analyses. Inverse variance weighted meta-analysis was performed within each ancestry group and then combined for a trans -ancestry meta-analysis. Our primary analysis included the 7 studies that measured FVII activity, and a secondary analysis included all 9 studies. We provided functional genomic validation for newly identified significant loci by silencing candidate genes in a human liver cell line (HuH7) using small-interfering RNA and then measuring F7 messenger RNA and FVII protein expression. Lastly, we used meta-analysis results to perform Mendelian randomization analysis to estimate the causal effect of FVII activity on coronary artery disease, ischemic stroke (IS), and venous thromboembolism. We identified 2 novel ( REEP3 and JAZF1-AS1 ) and 6 known loci associated with FVII activity, explaining 19.0% of the phenotypic variance. Adding FVII antigen data to the meta-analysis did not result in the discovery of further loci. Silencing REEP3 in HuH7 cells upregulated FVII, whereas silencing JAZF1 downregulated FVII. Mendelian randomization analyses suggest that FVII activity has a positive causal effect on the risk of IS. Variants at REEP3 and JAZF1 contribute to FVII activity by regulating F7 expression levels. FVII activity appears to contribute to the etiology of IS in the general population.

Essentials The role of statins in hemostasis and venous thromboembolism (VTE) prophylaxis is not clear. This trial assessed whether rosuvastatin use affects thrombin generation in patients with VTE. Endogenous thrombin potential and peak were decreased by 10% and 5% with rosuvastatin therapy. These results provide basis for trials on the efficacy of statins in reducing recurrent VTE risk. SUMMARY: Background Statin therapy could form an alternative prophylactic treatment for venous thromboembolism (VTE) if statins are proven to downregulate hemostasis and prevent recurrent VTE, without increasing bleeding risk. Objectives The STAtins Reduce Thrombophilia (START) trial investigated whether statin affects coagulation in patients with prior VTE. Patients/methods After anticoagulation withdrawal, patients were randomized to rosuvastatin 20 mg day -1 for 4 weeks or no intervention. Plasma samples taken at baseline and at the end of the study were analyzed employing thrombin generation assay. Results and conclusions The study comprised 126 rosuvastatin users and 119 non-users. Mean age was 58 years, 61% were men, 49% had unprovoked VTE and 75% had cardiovascular (CV) risk factors. Endogenous thrombin potential (ETP) increased from baseline to end of study in non-statin users (mean 97.22 nm*min; 95% CI, 40.92-153.53) and decreased in rosuvastatin users (mean -24.94 nm*min; 95% CI, -71.81 to 21.93). The mean difference in ETP change between treatments was -120.24 nm*min (95% CI, -192.97 to -47.51), yielding a 10.4% ETP reduction by rosuvastatin. The thrombin peak increased in both non-statin (mean 20.69 nm; 95% CI, 9.80-31.58) and rosuvastatin users (mean 8.41 nm; 95% CI -0.86 to 17.69). The mean difference in peak change between treatments was -11.88 nm (95% CI, -26.11 to 2.35), yielding a 5% peak reduction by rosuvastatin. Other thrombin generation parameters did not change substantially. The reduction in ETP and peak by rosuvastatin was more pronounced in the subgroups of participants with CV risk factors and with unprovoked VTE. We conclude that rosuvastatin reduces thrombin generation potential in patients who had VTE., (© 2018 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis.)