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Purpose: Platelets are key mediators in cardiovascular disease (CVD). Low cardiorespiratory fitness (CRF) is a risk factor for CVD. The purpose of our study was to assess if CRF associates with platelet function., Methods: Platelet assays and cardiopulmonary exercise testing were conducted in the Framingham Heart Study ( N = 3014). Linear mixed effects models estimated associations between CRF (assessed by peak oxygen uptake [V̇O 2 ]) and multiple platelet reactivity assays. Models were adjusted for multiple medications, risk factors, relatedness, and prevalent CVD., Results: Nineteen associations passed the significance threshold in the fully adjusted models, all indicating higher CRF associated with decreased platelet reactivity. Significant traits spanned multiple platelet agonists. Strongest associations were observed in multiplate whole blood testing after TRAP-6 (e.g., velocity, beta = -0.563, 95% CI = -0.735 to -0.391, P = 1.38E-10), ADP (e.g., velocity, beta = -0.514, 95% CI = -0.681 to -0348, P = 1.41E-09), collagen (e.g., velocity, beta = -0.387, 95% CI = -0.549 to -0.224, P = 3.01E-06), ristocetin (e.g., AUC, beta = -0.365, 95% CI = -0.522 to -0.208, P = 5.17E-06) and arachidonic acid stimulation of platelets (e.g., velocity, beta = -0.298, 95% CI = -0.435 to -0.162, P = 3.39E-04), and light transmission aggregometry (LTA) after ristocetin stimulation (e.g., max aggregation, beta = -0.362, 95% CI = -0.540 to -0.184, P = 6.64E-05). One trait passed significance threshold in the aspirin subsample (LTA ristocetin primary slope, beta = -0.733, 95% CI = -1.134 to -0.333, P = 3.30E-04) and another in a model including von Willebrand Factor levels as a covariate (U46619, a thromboxane receptor mimetic, AUC in the Optimul assay, beta = -0.36, 95% CI = -0.551 to -0.168, P = 2.35E-04). No strong interactions were observed between the associations and sex, age, or body mass index in formal interaction analyses., Conclusions: Our findings build on past work that shows CRF to be associated with reduced CVD by suggesting decreased platelet reactivity may play a mechanistic role. We found significant associations with multiple platelet agonists, indicating higher CRF may globally inhibit platelets; however, given multiple strong associations after TRAP-6 and ADP stimulation, PAR-1 and purinergic signaling may be most heavily involved. This is notable because each of these receptor pathways are tied to anticoagulant (DOAC/thrombin inhibitors) and antiplatelet therapies (P2Y12/PAR1/PAR4 inhibitors) for CVD prevention., (Copyright © 2024 by the American College of Sports Medicine.)

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.)

Venous thromboembolism (VTE) is a significant contributor to morbidity and mortality, with large disparities in incidence rates between Black and White Americans. Polygenic risk scores (PRSs) limited to variants discovered in genome-wide association studies in European-ancestry samples can identify European-ancestry individuals at high risk of VTE. However, there is limited evidence on whether high-dimensional PRS constructed using more sophisticated methods and more diverse training data can enhance the predictive ability and their utility across diverse populations. We developed PRSs for VTE using summary statistics from the International Network against Venous Thrombosis (INVENT) consortium genome-wide association studies meta-analyses of European- (71 771 cases and 1 059 740 controls) and African-ancestry samples (7482 cases and 129 975 controls). We used LDpred2 and PRS-CSx to construct ancestry-specific and multi-ancestry PRSs and evaluated their performance in an independent European- (6781 cases and 103 016 controls) and African-ancestry sample (1385 cases and 12 569 controls). Multi-ancestry PRSs with weights tuned in European-ancestry samples slightly outperformed ancestry-specific PRSs in European-ancestry test samples (e.g. the area under the receiver operating curve [AUC] was 0.609 for PRS-CSx_combinedEUR and 0.608 for PRS-CSxEUR [P = 0.00029]). Multi-ancestry PRSs with weights tuned in African-ancestry samples also outperformed ancestry-specific PRSs in African-ancestry test samples (PRS-CSxAFR: AUC = 0.58, PRS-CSx_combined AFR: AUC = 0.59), although this difference was not statistically significant (P = 0.34). The highest fifth percentile of the best-performing PRS was associated with 1.9-fold and 1.68-fold increased risk for VTE among European- and African-ancestry subjects, respectively, relative to those in the middle stratum. These findings suggest that the multi-ancestry PRS might be used to improve performance across diverse populations to identify individuals at highest risk for VTE., (© The Author(s) 2024. Published by Oxford University Press.)

Background: Our prior genome-wide association study of thrombin-induced platelet aggregation identified a G protein-coupled receptor kinase 5 (GRK5) noncoding variant (rs10886430-G) that is strongly associated with increased platelet reactivity to thrombin. This variant predisposes to increased risk of stroke, pulmonary embolism, and venous thromboembolism., Objectives: To determine role of platelet specific GRK5 in platelet responses to agonists and injury., Methods: Platelets from GRK5 mutant mice have been shown to have increased thrombin sensitivity, indicating that GRK5 may be a negative regulator of platelet activation. However, this has not been studied in a platelet-specific manner. We therefore used platelet-specific GRK5 mutant mice and models of thrombosis and pulmonary embolism., Results: We now demonstrate that mice lacking GRK5 specifically in platelets had a mild increase in thrombin responses in vitro and a shortened time to arterial thrombosis in vivo . In addition, platelet GRK5 mutant mice had increased thrombin but not collagen-induced thrombus burden in a mouse model of pulmonary embolism., Conclusion: These data indicate that platelet GRK5 has a significant role in limiting platelet responses to thrombin., (© 2024 The Author(s).)

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: Assessment of platelet function is key in diagnosing bleeding disorders and evaluating antiplatelet drug efficacy. However, there is a prevailing "one-size-fits-all" approach in the interpretation of measures of platelet reactivity, with arbitrary cutoffs often derived from healthy volunteer responses., Objectives: Our aim was to compare well-used platelet reactivity assays., Methods: Blood and platelet-rich plasma obtained from the Framingham Heart Study ( N  = 3429) were assayed using a range of agonists in 5 platelet assays: light transmission aggregometry, Optimul aggregometry, Multiplate impedance aggregometry (Roche Diagnostics), Total Thrombus-Formation Analysis System, and flow cytometry. Using linear mixed-effect models, we determined the contribution of preanalytical and technical factors that modulated platelet reactivity traits., Results: A strong intra-assay correlation of platelet traits was seen in all assays, particularly Multiplate velocity ( r  = 0.740; ristocetin vs arachidonic acid). In contrast, only moderate interassay correlations were observed ( r  = 0.375; adenosine diphosphate Optimul E max vs light transmission aggregometry large area under the curve). As expected, antiplatelet drugs strongly reduced platelet responses, with aspirin use primarily targeting arachidonic acid-induced aggregation, and explained substantial variance (β = -1.735; P  = 4.59 × 10 -780 ; variance proportion = 46.2%) and P2Y 12 antagonists blocking adenosine diphosphate responses (β = -1.612; P  = 6.75 × 10 -27 ; variance proportion = 2.1%). Notably, female sex and older age were associated with enhanced platelet reactivity. Fasting status and deviations from standard venipuncture practices did not alter platelet reactivity significantly. Finally, the agonist batch, phlebotomist, and assay technician (more so for assays that require additional sample manipulation) had a moderate to large effect on measured platelet reactivity., Conclusion: Caution must be exercised when extrapolating findings between assays, and the use of standard ranges must be medication-specific and sex-specific at a minimum. Researchers should also consider preanalytical and technical variables when designing experiments and interpreting platelet reactivity measures.

Background: Platelets play a key role in hemostasis, inflammation, and cardiovascular diseases. Platelet reactivity is highly variable between individuals. The drivers of this variability in populations from Sub-Saharan Africa remain largely unknown., Objectives: We aimed to investigate the nongenetic and genetic determinants of platelet reactivity in healthy adults living in a rapidly urbanizing area in Northern Tanzania., Methods: Platelet activation and reactivity were measured by platelet P-selectin expression and the binding of fibrinogen in unstimulated blood and after ex vivo stimulation with adenosine diphosphate and PAR-1 and PAR-4 ligands. We then analyzed the associations of platelet parameters with host genetic and nongenetic factors, environmental factors, plasma inflammatory markers, and plasma metabolites., Results: Only a few associations were found between platelet reactivity parameters and plasma inflammatory markers and nongenetic host and environmental factors. In contrast, untargeted plasma metabolomics revealed a large number of associations with food-derived metabolites, including phytochemicals that were previously reported to inhibit platelet reactivity. Genome-wide single-nucleotide polymorphism genotyping identified 2 novel single-nucleotide polymorphisms (rs903650 and rs4789332) that were associated with platelet reactivity at the genome-wide level (P < 5 × 10 -8 ) as well as a number of variants in the PAR4 gene (F2RL3) that were associated with PAR4-induced reactivity., Conclusion: Our study uncovered factors that determine variation in platelet reactivity in a population in East Africa that is rapidly transitioning to an urban lifestyle, including the importance of genetic ancestry and the gradual abandoning of the traditional East African diet., Competing Interests: Declaration of competing interests There are no competing interests to disclose., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: People with cancer experience high rates of venous thromboembolism (VTE). Risk of subsequent cancer is also increased in people experiencing their first VTE. The causal mechanisms underlying this association are not completely understood, and it is unknown whether VTE is itself a risk factor for cancer., Methods: We used data from large genome-wide association study meta-analyses to perform bidirectional Mendelian randomization analyses to estimate causal associations between genetic liability to VTE and risk of 18 different cancers., Results: We found no conclusive evidence that genetic liability to VTE was causally associated with an increased incidence of cancer, or vice versa. We observed an association between liability to VTE and pancreatic cancer risk [odds ratio for pancreatic cancer: 1.23 (95% confidence interval: 1.08-1.40) per log-odds increase in VTE risk, P = 0.002]. However, sensitivity analyses revealed this association was predominantly driven by a variant proxying non-O blood group, with inadequate evidence to suggest a causal relationship., Conclusions: These findings do not support the hypothesis that genetic liability to VTE is a cause of cancer. Existing observational epidemiological associations between VTE and cancer are therefore more likely to be driven by pathophysiological changes which occur in the setting of active cancer and anti-cancer treatments. Further work is required to explore and synthesize evidence for these mechanisms., (© The Author(s) 2023. Published by Oxford University Press on behalf of the International Epidemiological Association.)

Background: Alcohol consumption is linked to decreased platelet function. Whether this link is dependent on sex or type of beverage remains unclear., Methods: Cross-sectional data were obtained from the Framingham Heart Study (N = 3427). Alcohol consumption was assessed by using standardized medical history and Harvard semi-quantitative food frequency questionnaires. Five bioassays measured 120 platelet reactivity traits across agonists in whole-blood and platelet-rich plasma samples. Linear mixed-effects models adjusted for age, sex and aspirin use, hypertension, body mass index, cholesterol, high-density lipoprotein, triglycerides, smoking and diabetes evaluated associations between platelet reactivity and alcohol consumption. Beta effects, the regression coefficients that estimate the amount of change in each unit of the predictor variable whereas all other predictor variables remain fixed, for heavy alcohol consumption were compared with effects of aspirin use., Results: Alcohol consumption was associated with decreased platelet reactivity, with more associations among wine and liquor compared with beer. Many platelet-alcohol associations in the full sample (86%, P < 0.01) had larger effect sizes in females. Lower light transmission aggregometry adenosine diphosphate (1.82 µM) maximum aggregation (P = 2.6E-3, 95% CI = -0.07, -0.02, β = -0.042) and area under the curve (P = 7.7E-3, 95% CI = -0.07, -0.01, β = -0.039) were associated with white wine consumption; however, red wine had no associations with platelet reactivity. The effect of aspirin use was on average 11.3 (±4.0) times greater than that of heavy drinking in our full sample., Conclusions: We confirm associations between alcohol consumption and decreased platelet reactivity. Effects appeared larger for liquor and wine intake and in our female cohort. Red wine consumption is not associated with lower platelet function, contrasting with prior population studies. Although we report an inhibitory relationship between alcohol intake and platelet function, these effects appear much smaller than that of aspirin use., (Published by Oxford University Press on behalf of the International Epidemiological Association 2023.)

Arterial tonometry and vascular calcification measures are useful in cardiovascular disease (CVD) risk assessment. Prior studies found associations between tonometry measures, arterial calcium, and CVD risk. Activated platelets release angiopoietin-1 and other factors, which may connect vascular structure and platelet function. We analyzed arterial tonometry, platelet function, aortic, thoracic and coronary calcium, and thoracic and abdominal aorta diameters measured in the Framingham Heart Study Gen3/NOS/OMNI-2 cohorts ( n  = 3,429, 53.7% women, mean age 54.4 years ±9.3). Platelet reactivity in whole blood or platelet-rich plasma was assessed using 5 assays and 7 agonists. We analyzed linear mixed effects models with platelet reactivity phenotypes as outcomes, adjusting for CVD risk factors and family structure. Higher arterial calcium trended with higher platelet reactivity, whereas larger aortic diameters trended with lower platelet reactivity. Characteristic impedance (Zc) and central pulse pressure positively trended with various platelet traits, while pulse wave velocity and Zc negatively trended with collagen, ADP, and epinephrine traits. All results did not pass a stringent multiple test correction threshold ( p  < 2.22e-04). The diameter trends were consistent with lower shear environments invoking less platelet reactivity. The vessel calcium trends were consistent with subclinical atherosclerosis and platelet activation being inter-related.

Platelets are anucleated cells produced by megakaryocytes, from which they inherit all the components necessary to carry their functions. They circulate in blood vessels where they play essential roles in coagulation, wound repair or inflammation, and have been implicated in various pathological conditions such as thrombosis, viral infection or cancer progression. The importance of these cells has been established over a century ago, and effective anti-platelet medications with different mechanisms of action have since been developed. However, these therapies are not always effective and can incur adverse events, thus a better understanding of platelets molecular processes is needed to address these issues and improve our understanding of platelet functions. In recent years, an increasing number of studies have leveraged OMICs technologies to analyze their content and identify molecular signatures and mechanisms associated with platelet functions and platelet related disorders. In particular, the increased accessibility of microarrays and RNA sequencing opened the way for studies of the platelet transcriptome under a wide array of conditions. These studies revealed distinct expression profiles in diverse pathologies, which could lead to the discovery of novel biomarkers and therapeutic targets, and suggests a dynamic transcriptome that could influence platelet mechanisms. In this review, we highlight the different sources of transcript level variability in platelets while summarizing recent advances and discoveries from this emerging field., Competing Interests: Declaration of competing interest 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., (Published by Elsevier Ltd.)

Venous thromboembolism (VTE) is a common, multi-causal disease with potentially serious short- and long-term complications. In clinical practice, there is a need for improved plasma biomarker-based tools for VTE diagnosis and risk prediction. Here we show, using proteomics profiling to screen plasma from patients with suspected acute VTE, and several case-control studies for VTE, how Complement Factor H Related 5 protein (CFHR5), a regulator of the alternative pathway of complement activation, is a VTE-associated plasma biomarker. In plasma, higher CFHR5 levels are associated with increased thrombin generation potential and recombinant CFHR5 enhanced platelet activation in vitro. GWAS analysis of ~52,000 participants identifies six loci associated with CFHR5 plasma levels, but Mendelian randomization do not demonstrate causality between CFHR5 and VTE. Our results indicate an important role for the regulation of the alternative pathway of complement activation in VTE and that CFHR5 represents a potential diagnostic and/or risk predictive plasma biomarker., (© 2023. The Author(s).)

Background: Hypertriglyceridemia is an independent risk factor for major adverse cardiovascular events, though the mechanisms linking triglycerides and platelet function with thrombosis, remain elusive. The aim of this study was to assess the association between platelet function and triglyceride levels., Methods: We included participants from the Framingham Heart Study Third Generation cohort, OMNI, and New Offspring Spouse cohort who attended the third examination cycle (2016-2019). Eligible participants were categorized into four triglyceride subgroups., Results: The study comprised a total of 1897 (55.53 %) participants with normal TG levels; 883 (25.85 %) participants with high-normal TGs; 378 (11.07 %) with borderline high TGs; and 258 (7.55 %) participants with hypertriglyceridemia. After adjusting for age, sex, alcohol consumption, aspirin, statin and P2Y 12 inhibitors, the levels of ADP-induced platelet aggregation were inversely associated with total cholesterol levels (P < 0.0001). Platelet disaggregation was associated with low-density lipoprotein and high-density lipoprotein cholesterol levels (P < 0.0001). Lastly, in a shear-stress chamber assay mimicking arterial flow velocities, TG levels in the normal-high group were associated with increased levels of collagen-dependent thrombogenicity (β = 24.16, SE = 6.65, P < 0.0001)., Conclusion: Triglyceride levels are associated with altered platelet activation and aggregation. Furthermore, increased platelet-driven thrombogenicity is directly associated with triglyceride levels after adjusting for medications and other covariates., Competing Interests: Declaration of competing interest The authors declare no conflict of interest. The funders had no role in the design of the study, collection, analyses or interpretation of the data, writing of the manuscript or in the decision to publish the reported results., (Copyright © 2023. Published by Elsevier Ltd.)

Background: Fibrinogen plays an essential role in blood coagulation and inflammation. Circulating fibrinogen levels may be determined by inter-individual differences in DNA methylation at CpG sites, and vice versa., Methods: We performed an epigenome-wide association study (EWAS) of circulating fibrinogen levels in 18,037 White, Black, American Indian, and Hispanic participants representing 14 studies from the CHARGE consortium. Circulating leukocyte DNA methylation was measured in 12,904 participants using the Illumina 450K array, and in 5,133 participants using the EPIC array. Each study performed an EWAS of fibrinogen using linear mixed models adjusted for potential confounders. Study-specific results were combined using array-specific meta-analysis, followed by cross-replication of epigenome-wide significant associations. We compared models with and without C-reactive protein (CRP) adjustment to examine the role of inflammation., Results: We identified 208 and 87 significant CpG sites associated with fibrinogen from the 450K (p-value<1.03×10 -7 ) and EPIC arrays (p-value<5.78×10 -8 ), respectively. There were 78 associations from the 450K array that replicated in the EPIC array and 26 vice versa. After accounting for the overlapping sites, there were 83 replicated CpG sites located in 61 loci, of which only 4 have been previously reported for fibrinogen. Examples of genes located near these CpG sites were SOCS3 and AIM2, which are involved in inflammatory pathways. The associations for all 83 replicated CpG sites were attenuated after CRP adjustment, although many remained significant., Conclusion: We identified 83 CpG sites associated with circulating fibrinogen levels. These associations are partially driven by inflammatory pathways shared by both fibrinogen and CRP., (Copyright © 2023 International Society on Thrombosis and Haemostasis. All rights reserved.)

Background: Venous thromboembolism (VTE) is a life-threatening vascular event with environmental and genetic determinants. Recent VTE genome-wide association studies (GWAS) meta-analyses involved nearly 30 000 VTE cases and identified up to 40 genetic loci associated with VTE risk, including loci not previously suspected to play a role in hemostasis. The aim of our research was to expand discovery of new genetic loci associated with VTE by using cross-ancestry genomic resources., Methods: We present new cross-ancestry meta-analyzed GWAS results involving up to 81 669 VTE cases from 30 studies, with replication of novel loci in independent populations and loci characterization through in silico genomic interrogations., Results: In our genetic discovery effort that included 55 330 participants with VTE (47 822 European, 6320 African, and 1188 Hispanic ancestry), we identified 48 novel associations, of which 34 were replicated after correction for multiple testing. In our combined discovery-replication analysis (81 669 VTE participants) and ancestry-stratified meta-analyses (European, African, and Hispanic), we identified another 44 novel associations, which are new candidate VTE-associated loci requiring replication. In total, across all GWAS meta-analyses, we identified 135 independent genomic loci significantly associated with VTE risk. A genetic risk score of the significantly associated loci in Europeans identified a 6-fold increase in risk for those in the top 1% of scores compared with those with average scores. We also identified 31 novel transcript associations in transcriptome-wide association studies and 8 novel candidate genes with protein quantitative-trait locus Mendelian randomization analyses. In silico interrogations of hemostasis and hematology traits and a large phenome-wide association analysis of the 135 GWAS loci provided insights to biological pathways contributing to VTE, with some loci contributing to VTE through well-characterized coagulation pathways and others providing new data on the role of hematology traits, particularly platelet function. Many of the replicated loci are outside of known or currently hypothesized pathways to thrombosis., Conclusions: Our cross-ancestry GWAS meta-analyses identified new loci associated with VTE. These findings highlight new pathways to thrombosis and provide novel molecules that may be useful in the development of improved antithrombosis treatments.

Host genetic variants influence the susceptibility and severity of several infectious diseases, and the discovery of genetic associations with coronavirus disease 2019 (COVID-19) phenotypes could help to develop new therapeutic strategies to decrease its burden. Between May 2020 and June 2021, we used COVID-19 data released periodically by UK Biobank and performed 65 genome-wide association studies in up to 18 releases of COVID-19 susceptibility (n = 18,481 cases in June 2021), hospitalization (n = 3,260), severe outcomes (n = 1,244), and deaths (n = 1,104), stratified by sex and ancestry. In coherence with previous studies, we observed two independent signals at the chr3p21.31 locus (rs73062389-A, odds ratio [OR], 1.21 (P = 4.26 × 10 -15 ) and rs71325088-C, OR, 1.62 [P = 2.25 × 10 -9 ]) modulating susceptibility and severity, respectively, and a signal influencing susceptibility at the ABO locus (rs9411378-A; OR, 1.10; P = 3.30 × 10 -12 ), suggesting an increased risk of infection in non-O blood groups carriers. Additional signals at the APOE (associated with severity and death) LRMDA (susceptibility in non-European) and chr2q32.3 (susceptibility in women) loci were also identified, but did not replicate in independent datasets. We then devised an approach to extract variants suggestively associated (P < 10 -5 ), exhibiting an increase in significance over time. When applied to the susceptibility, hospitalization and severity analyses, this approach revealed the known RPL24 , DPP9 , and MAPT loci, respectively, among hundreds of other signals. These results, freely available on the GRASP portal, provide insights on the genetic mechanisms involved in COVID-19 phenotypes., Competing Interests: The authors declare no competing interests., (© 2022.)

Depression is an independent risk factor of cardiovascular disease morbidity. Serotonin is a key neurotransmitter in depressive pathology, contained within platelets, and is a weak activator of platelets. Our study assessed the link between platelet reactivity traits, depression, and antidepressant (AD) use in a large population sample. Our study was conducted in the Framingham Heart Study (n = 3,140), and AD use (n = 563) and aspirin use (n = 681) were noted. Depression was measured using the Center for Epidemiological Studies-Depression (CES-D) survey. Platelet reactivity traits were measured across multiple agonists using five distinct assays. We utilized a linear mixed effects model to test associations between platelet traits and depression, adjusting for age, sex, aspirin use, and AD use. Similarly, we analyzed trait associations with any AD use, serotonin-affecting ADs, and norepinephrine-affecting ADs, respectively. There were strong associations with reduced platelet function and AD use, particularly with serotonin-affecting medications. This included lower Optimul epinephrine maximal aggregation (P = 4.87E-13), higher U46619 half maximal effective concentration (P = 9.09E-11), lower light transmission aggregometry (LTA) adenosine diphosphate (ADP) final aggregation (P = 1.03E-05), and higher LTA ADP disaggregation (P = 2.28E-05). We found similar associations with serotonin-affecting ADs in an aspirin-taking subset of our sample. There were no significant associations between platelet traits and depression. In the largest study yet of AD use and platelet function we show that antidepressants, particularly serotonin-affecting ADs, inhibit platelets. We did not find evidence that depressive symptomatology in the absence of medication is associated with altered platelet function. Our results are consistent with AD use leading to platelet serotonin depletions, decreased stability of platelet aggregates, and overall decreased aggregation to multiple agonists, which may be a mechanism by which ADs increase risk of bleeding and decrease risk of thrombosis., (© 2021 The Authors. Clinical Pharmacology & Therapeutics published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics. This article has been contributed to by US Government employees and their work is in the public domain in the USA.)

Platelets play a key role in thrombosis and hemostasis. Platelet count (PLT) and mean platelet volume (MPV) are highly heritable quantitative traits, with hundreds of genetic signals previously identified, mostly in European ancestry populations. We here utilize whole genome sequencing (WGS) from NHLBI's Trans-Omics for Precision Medicine initiative (TOPMed) in a large multi-ethnic sample to further explore common and rare variation contributing to PLT (n = 61 200) and MPV (n = 23 485). We identified and replicated secondary signals at MPL (rs532784633) and PECAM1 (rs73345162), both more common in African ancestry populations. We also observed rare variation in Mendelian platelet-related disorder genes influencing variation in platelet traits in TOPMed cohorts (not enriched for blood disorders). For example, association of GP9 with lower PLT and higher MPV was partly driven by a pathogenic Bernard-Soulier syndrome variant (rs5030764, p.Asn61Ser), and the signals at TUBB1 and CD36 were partly driven by loss of function variants not annotated as pathogenic in ClinVar (rs199948010 and rs571975065). However, residual signal remained for these gene-based signals after adjusting for lead variants, suggesting that additional variants in Mendelian genes with impacts in general population cohorts remain to be identified. Gene-based signals were also identified at several genome-wide association study identified loci for genes not annotated for Mendelian platelet disorders (PTPRH, TET2, CHEK2), with somatic variation driving the result at TET2. These results highlight the value of WGS in populations of diverse genetic ancestry to identify novel regulatory and coding signals, even for well-studied traits like platelet traits., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: Use of targeted exome-arrays with common, rare variants and functionally enriched variation has led to discovery of new genes contributing to population variation in risk factors. Plasminogen activator-inhibitor 1 (PAI-1), tissue plasminogen activator (tPA), and the plasma product D-dimer are important components of the fibrinolytic system. There have been few large-scale genome-wide or exome-wide studies of PAI-1, tPA, and D-dimer., Objectives: We sought to discover new genetic loci contributing to variation in these traits using an exome-array approach., Methods: Cohort-level analyses and fixed effects meta-analyses of PAI-1 (n = 15 603), tPA (n = 6876,) and D-dimer (n = 19 306) from 12 cohorts of European ancestry with diverse study design were conducted, including single-variant analyses and gene-based burden testing., Results: Five variants located in NME7, FGL1, and the fibrinogen locus, all associated with D-dimer levels, achieved genome-wide significance (P < 5 × 10 -8 ). Replication was sought for these 5 variants, as well as 45 well-imputed variants with P < 1 × 10 -4 in the discovery using an independent cohort. Replication was observed for three out of the five significant associations, including a novel and uncommon (0.013 allele frequency) coding variant p.Trp256Leu in FGL1 (fibrinogen-like-1) with increased plasma D-dimer levels. Additionally, a candidate-gene approach revealed a suggestive association for a coding variant (rs143202684-C) in SERPINB2, and suggestive associations with consistent effect in the replication analysis include an intronic variant (rs11057830-A) in SCARB1 associated with increased D-dimer levels., Conclusion: This work provides new evidence for a role of FGL1 in hemostasis., (© 2021 International Society on Thrombosis and Haemostasis. This article has been contributed to by US Government employees and their work is in the public domain in the USA.)

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.

MicroRNAs (miRNAs) are small regulatory RNAs participating to several biological processes and known to be involved in various pathologies. Measurable in body fluids, miRNAs have been proposed to serve as efficient biomarkers for diseases and/or associated traits. Here, we performed a next-generation-sequencing based profiling of plasma miRNAs in 344 patients with venous thrombosis (VT) and assessed the association of plasma miRNA levels with several haemostatic traits and the risk of VT recurrence. Among the most significant findings, we detected an association between hsa-miR-199b-3p and haematocrit levels ( P  = 0.0016), these two markers having both been independently reported to associate with VT risk. We also observed suggestive evidence for association of hsa-miR-370-3p ( P  = 0.019), hsa-miR-27b-3p ( P  = 0.016) and hsa-miR-222-3p ( P  = 0.049) with VT recurrence, the observations at the latter two miRNAs confirming the recent findings of Wang et al . Besides, by conducting Genome-Wide Association Studies on miRNA levels and meta-analyzing our results with some publicly available, we identified 21 new associations of single nucleotide polymorphisms with plasma miRNA levels at the statistical significance threshold of P  < 5 × 10 -8 , some of these associations pertaining to thrombosis associated mechanisms. In conclusion, this study provides novel data about the impact of miRNAs' variability in haemostasis and new arguments supporting the association of few miRNAs with the risk of recurrence in patients with venous thrombosis., (Published on behalf of the European Society of Cardiology. © The Author(s) 2020.)

Motivation: MicroRNAs (miRNAs) are small RNA molecules (∼22 nucleotide long) involved in post-transcriptional gene regulation. Advances in high-throughput sequencing technologies led to the discovery of isomiRs, which are miRNA sequence variants. While many miRNA-seq analysis tools exist, the diversity of output formats hinders accurate comparisons between tools and precludes data sharing and the development of common downstream analysis methods., Results: To overcome this situation, we present here a community-based project, miRNA Transcriptomic Open Project (miRTOP) working towards the optimization of miRNA analyses. The aim of miRTOP is to promote the development of downstream isomiR analysis tools that are compatible with existing detection and quantification tools. Based on the existing GFF3 format, we first created a new standard format, mirGFF3, for the output of miRNA/isomiR detection and quantification results from small RNA-seq data. Additionally, we developed a command line Python tool, mirtop, to create and manage the mirGFF3 format. Currently, mirtop can convert into mirGFF3 the outputs of commonly used pipelines, such as seqbuster, isomiR-SEA, sRNAbench, Prost! as well as BAM files. Some tools have also incorporated the mirGFF3 format directly into their code, such as, miRge2.0, IsoMIRmap and OptimiR. Its open architecture enables any tool or pipeline to output or convert results into mirGFF3. Collectively, this isomiR categorization system, along with the accompanying mirGFF3 and mirtop API, provide a comprehensive solution for the standardization of miRNA and isomiR annotation, enabling data sharing, reporting, comparative analyses and benchmarking, while promoting the development of common miRNA methods focusing on downstream steps of miRNA detection, annotation and quantification., Availability and Implementation: https://github.com/miRTop/mirGFF3/ and https://github.com/miRTop/mirtop., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Factor V (FV) is a circulating protein primarily synthesized in the liver, and mainly present in plasma. It is a major component of the coagulation process., Objective: To detect novel genetic loci participating to the regulation of FV plasma levels., Methods: We conducted the first Genome Wide Association Study on FV plasma levels in a sample of 510 individuals and replicated the main findings in an independent sample of 1156 individuals., Results: In addition to genetic variations at the F5 locus, we identified novel associations at the PLXDC2 locus, with the lead PLXDC2 rs927826 polymorphism explaining ~3.7% (P = 7.5 × 10 -15 in the combined discovery and replication samples) of the variability of FV plasma levels. In silico transcriptomic analyses in various cell types confirmed that PLXDC2 expression is positively correlated to F5 expression. SiRNA experiments in human hepatocellular carcinoma cell line confirmed the role of PLXDC2 in modulating factor F5 gene expression, and revealed further influences on F2 and F10 expressions., Conclusion: Our study identified PLXDC2 as a new molecular player of the coagulation process., (© 2019 International Society on Thrombosis and Haemostasis.)

Next-generation sequencing is an increasingly popular and efficient approach to characterize the full set of microRNAs (miRNAs) present in human biosamples. MiRNAs' detection and quantification still remain a challenge as they can undergo different posttranscriptional modifications and might harbor genetic variations (polymiRs) that may impact on the alignment step. We present a novel algorithm, OPTIMIR, that incorporates biological knowledge on miRNA editing and genome-wide genotype data available in the processed samples to improve alignment accuracy. OPTIMIR was applied to 391 human plasma samples that had been typed with genome-wide genotyping arrays. OPTIMIR was able to detect genotyping errors, suggested the existence of novel miRNAs and highlighted the allelic imbalance expression of polymiRs in heterozygous carriers. OPTIMIR is written in python, and freely available on the GENMED website (http://www.genmed.fr/index.php/fr/) and on Github (github.com/FlorianThibord/OptimiR)., (© 2019 Thibord et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Summary: Predicted deleteriousness of coding variants is a frequently used criterion to filter out variants detected in next-generation sequencing projects and to select candidates impacting on the risk of human diseases. Most available dedicated tools implement a base-to-base annotation approach that could be biased in presence of several variants in the same genetic codon. We here proposed the MACARON program that, from a standard VCF file, identifies, re-annotates and predicts the amino acid change resulting from multiple single nucleotide variants (SNVs) within the same genetic codon. Applied to the whole exome dataset of 573 individuals, MACARON identifies 114 situations where multiple SNVs within a genetic codon induce an amino acid change that is different from those predicted by standard single SNV annotation tool. Such events are not uncommon and deserve to be studied in sequencing projects with inconclusive findings., Availability and Implementation: MACARON is written in python with codes available on the GENMED website (www.genmed.fr)., Supplementary Information: Supplementary data are available at Bioinformatics online.

Background and Purpose- Arterial vasospasm is a well-known delayed complication of aneurysmal subarachnoid hemorrhage (aSAH). However, no validated biomarker exists to help clinicians discriminating patients with aSAH who will develop vasospasm (VSP + ) and identifying those who then deserve aggressive preventive therapy. We hypothesized that whole-blood miRNAs could be a source of candidate biomarkers for vasospasm. Methods- Using a next-generation sequencing approach, we performed whole-blood miRNA profiling between VSP + patients with aSAH and patients who did not develop vasospasm (VSP - ) in a prospective cohort of 32 patients. Profiling was performed on the admission day and 3 days before vasospasm. Results- Four hundred forty-two miRNAs were highly expressed in whole blood of patients with aSAH. Among them, hsa-miR-3177-3p demonstrated significant ( P=5.9×10 -5 ; P Bonferroni corrected =0.03) lower levels in VSP - compared with VSP + patients. Looking for whole-blood mRNA correlates of hsa-miR-3177-3p, we observed some evidence that the decrease in hsa-miR-3177-3p levels after aSAH was associated with an increase in LDHA mRNA levels in VSP - ( P<10 -3 ) but not in VSP + ( P=0.66) patients. Conclusions- Whole-blood miRNA levels of hsa-miR-3177-3p could serve as a biomarker for vasospasm. Clinical Trial Registration- URL: https://www.clinicaltrials.gov . Unique identifier: NCT01779713.