Genome-Wide Search for Nonadditive Allele Effects Identifies PSKH2 as Involved in the Variability of Factor V Activity.

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.

Prediction model for major bleeding in anticoagulated patients with cancer-associated venous thromboembolism using machine learning and natural language processing.

PURPOSE: We developed a predictive model to assess the risk of major bleeding (MB) within 6 months of primary venous thromboembolism (VTE) in cancer patients receiving anticoagulant treatment. We also sought to describe the prevalence and incidence of VTE in cancer patients, and to describe clinical characteristics at baseline and bleeding events during follow-up in patients receiving anticoagulants. METHODS: This observational, retrospective, and multicenter study used natural language processing and machine learning (ML), to analyze unstructured clinical data from electronic health records from nine Spanish hospitals between 2014 and 2018. All adult cancer patients with VTE receiving anticoagulants were included. Both clinically- and ML-driven feature selection was performed to identify MB predictors. Logistic regression (LR), decision tree (DT), and random forest (RF) algorithms were used to train predictive models, which were validated in a hold-out dataset and compared to the previously developed CAT-BLEED score. RESULTS: Of the 2,893,108 cancer patients screened, in-hospital VTE prevalence was 5.8% and the annual incidence ranged from 2.7 to 3.9%. We identified 21,227 patients with active cancer and VTE receiving anticoagulants (53.9% men, median age of 70 years). MB events after VTE diagnosis occurred in 10.9% of patients within the first six months. MB predictors included: hemoglobin, metastasis, age, platelets, leukocytes, and serum creatinine. The LR, DT, and RF models had AUC-ROC (95% confidence interval) values of 0.60 (0.55, 0.65), 0.60 (0.55, 0.65), and 0.61 (0.56, 0.66), respectively. These models outperformed the CAT-BLEED score with values of 0.53 (0.48, 0.59). CONCLUSIONS: Our study shows encouraging results in identifying anticoagulated patients with cancer-associated VTE who are at high risk of MB.

Polygenic risk of major depressive disorder as a risk factor for venous thromboembolism.

Major depressive disorder (MDD), bipolar disorder (BD), and schizophrenia (SCZ) are associated with an increased risk of cardiovascular diseases, including venous thromboembolism (VTE). The reasons for this are complex and include obesity, smoking, and use of hormones and psychotropic medications. Genetic studies have increasingly provided evidence of the shared genetic risk of psychiatric and cardiometabolic illnesses. This study aimed to determine whether a genetic predisposition to MDD, BD, or SCZ is associated with an increased risk of VTE. Genetic correlations using the largest genome-wide genetic meta-analyses summary statistics for MDD, BD, and SCZ (Psychiatric Genetics Consortium) and a recent genome-wide genetic meta-analysis of VTE (INVENT Consortium) demonstrated a positive association between VTE and MDD but not BD or SCZ. The same summary statistics were used to construct polygenic risk scores for MDD, BD, and SCZ in UK Biobank participants of self-reported White British ancestry. These were assessed for impact on self-reported VTE risk (10 786 cases, 285 124 controls), using logistic regression, in sex-specific and sex-combined analyses. We identified significant positive associations between polygenic risk for MDD and the risk of VTE in men, women, and sex-combined analyses, independent of the known risk factors. Secondary analyses demonstrated that this association was not driven by those with lifetime experience of mental illness. Meta-analyses of individual data from 6 additional independent cohorts replicated the sex-combined association. This report provides evidence for shared biological mechanisms leading to MDD and VTE and suggests that, in the absence of genetic data, a family history of MDD might be considered when assessing the risk of VTE.

Development of a predictive model of venous thromboembolism recurrence in anticoagulated cancer patients using machine learning.

INTRODUCTION: Patients with cancer and venous thromboembolism (VTE) show a high risk of VTE recurrence during anticoagulant treatment. This study aimed to develop a predictive model to assess the risk of VTE recurrence within 6 months at the moment of primary VTE diagnosis in these patients. MATERIALS AND METHODS: Using the EHRead® technology, based on Natural Language Processing (NLP) and machine learning (ML), the unstructured data in electronic health records from 9 Spanish hospitals between 2014 and 2018 were extracted. Both clinically- and ML-driven feature selection were performed to identify predictors for VTE recurrence. Logistic regression (LR), decision tree (DT), and random forest (RF) algorithms were used to train different prediction models, which were subsequently validated in a hold-out data set. RESULTS: A total of 16,407 anticoagulated cancer patients with diagnosis of VTE were identified (54.4 % male and median age 70). Deep vein thrombosis, pulmonary embolism and metastases were observed in 67.2 %, 26.6 %, and 47.7 % of the patients, respectively. During the study follow-up, 11.4 % of the patients developed a recurrent VTE, being more frequent in patients with lung cancer. Feature selection and model training based on ML identified primary pulmonary embolism, deep vein thrombosis, metastasis, adenocarcinoma, hemoglobin and serum creatinine levels, platelet and leukocyte count, family history of VTE, and patients' age as predictors of VTE recurrence within 6 months of VTE diagnosis. The LR model had an AUC-ROC (95 % CI) of 0.66 (0.61, 0.70), the DT of 0.69 (0.65, 0.72) and the RF of 0.68 (0.63, 0.72). CONCLUSIONS: This is the first ML-based predictive model designed to predict 6-months VTE recurrence in patients with cancer. These results hold great potential to assist clinicians to identify the high-risk patients and improve their clinical management.

Antithrombin, Protein C, and Protein S: Genome and Transcriptome-Wide Association Studies Identify 7 Novel Loci Regulating Plasma Levels.

BACKGROUND: Antithrombin, PC (protein C), and PS (protein S) are circulating natural anticoagulant proteins that regulate hemostasis and of which partial deficiencies are causes of venous thromboembolism. Previous genetic association studies involving antithrombin, PC, and PS were limited by modest sample sizes or by being restricted to candidate genes. In the setting of the Cohorts for Heart and Aging Research in Genomic Epidemiology consortium, we meta-analyzed across ancestries the results from 10 genome-wide association studies of plasma levels of antithrombin, PC, PS free, and PS total. METHODS: Study participants were of European and African ancestries, and genotype data were imputed to TOPMed, a dense multiancestry reference panel. Each of the 10 studies conducted a genome-wide association studies for each phenotype and summary results were meta-analyzed, stratified by ancestry. Analysis of antithrombin included 25 243 European ancestry and 2688 African ancestry participants, PC analysis included 16 597 European ancestry and 2688 African ancestry participants, PSF and PST analysis included 4113 and 6409 European ancestry participants. We also conducted transcriptome-wide association analyses and multiphenotype analysis to discover additional associations. Novel genome-wide association studies and transcriptome-wide association analyses findings were validated by in vitro functional experiments. Mendelian randomization was performed to assess the causal relationship between these proteins and cardiovascular outcomes. RESULTS: Genome-wide association studies meta-analyses identified 4 newly associated loci: 3 with antithrombin levels (GCKR, BAZ1B, and HP-TXNL4B) and 1 with PS levels (ORM1-ORM2). transcriptome-wide association analyses identified 3 newly associated genes: 1 with antithrombin level (FCGRT), 1 with PC (GOLM2), and 1 with PS (MYL7). In addition, we replicated 7 independent loci reported in previous studies. Functional experiments provided evidence for the involvement of GCKR, SNX17, and HP genes in antithrombin regulation. CONCLUSIONS: The use of larger sample sizes, diverse populations, and a denser imputation reference panel allowed the detection of 7 novel genomic loci associated with plasma antithrombin, PC, and PS levels.

Pseudothrombocytopenia, beyond a laboratory phenomenon: study of 192 cases.

The platelet antibodies that cause pseudothrombocytopenia (PTCP) act only in vitro and do not produce clinical bleeding. Most studies on PTCP have focused on improving differential diagnosis with true thrombocytopenia but studies on the characteristics of patients with PTCP are limited. In this study, we aimed to evaluate the clinical and biological characteristics of 192 patients with PTCP. In addition to general variables, we evaluated automated and microscopic platelet counts, platelet clumps, platelet diameters, immature platelet fraction (IPF), and platelet antibodies. Adult women accounted for the largest subgroup of patients (n=82; 42.7%) and 67 patients (34.9%) were grouped into families. Forty-four patients (22.9%) had one or more associated autoimmune disorders (ADs); 39 relatives of these patients (19.8%) had ADs and 45 relatives (23.4%) had immune thrombocytopenia (ITP) or unspecified thrombocytopenia. Platelet cryptantibodies and/or autoantibodies were positive in 56 patients (30.1%). Most patients (n=169; 80%) had automated platelet counts >80×109/L. In all patients, microscopic platelet counts were ≥150×109/L. The platelet clump index (% increase in microscopic platelet count compared to automatic count) ranged from 30 to >7000%. Platelet diameters and IPF parameters were significantly greater in the PTCP versus healthy controls (p<0.001). A total of 17 patients (8.8%) had had previous ITP or the PTCP evolved into ITP. Our data suggest that PTCP should be considered a situation of autoimmunity; the assessment of platelet clumps has a high diagnostic value; the close association between ITP and PTCP suggests that these conditions could be different phases of the same process.

Clinical outcomes in patients with atrial fibrillation treated with DOACs in a specialized anticoagulation center: Critical appraisal of real-world data.

AIMS: Direct oral anticoagulants (DOAC) are progressively replacing vitamin K antagonists in the prevention of thromboembolism in patients with atrial fibrillation. However, their real-world clinical outcomes appear to be contradictory, with some studies reporting fewer and others reporting higher complications than the pivotal randomized controlled trials. We present the results of a clinical model for the management of DOACs in real clinical practice and provide a review of the literature. METHODS: The MACACOD project is an ongoing, observational, prospective, single-center study with unselected patients that focuses on rigorous DOAC selection, an educational visit, laboratory measurements, and strict follow-up. RESULTS: A total of 1,259 patients were included. The composite incidence of major complications was 4.93% py in the whole cohort vs 4.49% py in the edoxaban cohort. The rate of all-cause mortality was 6.11% py for all DOACs vs 5.12% py for edoxaban. There weren't differences across sex or between Edoxaban reduced or standard doses. However, there were differences across ages, with a higher incidence of major bleeding complications in patients >85 years (5.13% py vs 1.69% py in <75 years). CONCLUSIONS: We observed an incidence of serious complications of 4.93% py, in which severe bleeding predominated (3.65% py). Considering our results, more specialized attention seems necessary to reduce the incidence of severe complications and also a more critical view of the literature. Considering our results, and our indirect comparison with many real-world studies, more specialized attention seems necessary to reduce the incidence of severe complications in AF patients receiving DOACs.

Multiallelic Copy Number Variation in ORM1 is Associated with Plasma Cell-Free DNA Levels as an Intermediate Phenotype for Venous Thromboembolism.

Venous thromboembolism (VTE) is a common disease with high heritability. However, only a small portion of the genetic variance of VTE can be explained by known genetic risk factors. Neutrophil extracellular traps (NETs) have been associated with prothrombotic activity. Therefore, the genetic basis of NETs could reveal novel risk factors for VTE. A recent genome-wide association study of plasma cell-free DNA (cfDNA) levels in the Genetic Analysis of Idiopathic Thrombophilia 2 (GAIT-2) Project showed a significant associated locus near ORM1. We aimed to further explore this candidate region by next-generation sequencing, copy number variation (CNV) quantification, and expression analysis using an extreme phenotype sampling design involving 80 individuals from the GAIT-2 Project. The RETROVE study with 400 VTE cases and 400 controls was used to replicate the results. A total of 105 genetic variants and a multiallelic CNV (mCNV) spanning ORM1 were identified in GAIT-2. Of these, 17 independent common variants, a region of 22 rare variants, and the mCNV were significantly associated with cfDNA levels. In addition, eight of these common variants and the mCNV influenced ORM1 expression. The association of the mCNV and cfDNA levels was replicated in RETROVE (p-value = 1.19 × 10-6). Additional associations between the mCNV and thrombin generation parameters were identified. Our results reveal that increased mCNV dosages in ORM1 decreased gene expression and upregulated cfDNA levels. Therefore, the mCNV in ORM1 appears to be a novel marker for cfDNA levels, which could contribute to VTE risk.

Integrated GWAS and Gene Expression Suggest ORM1 as a Potential Regulator of Plasma Levels of Cell-Free DNA and Thrombosis Risk.

Plasma cell-free DNA (cfDNA) is a surrogate marker of neutrophil extracellular traps (NETs) that contribute to immunothrombosis. There is growing interest about the mechanisms underlying NET formation and elevated cfDNA, but little is known about the factors involved. We aimed to identify genes involved in the regulation of cfDNA levels using data from the Genetic Analysis of Idiopathic Thrombophilia (GAIT-2) Project.Imputed genotypes, whole blood RNA-Seq data, and plasma cfDNA quantification were available for 935 of the GAIT-2 participants from 35 families with idiopathic thrombophilia. We performed heritability and GWAS analysis for cfDNA. The heritability of cfDNA was 0.26 (p = 3.7 × 10-6), while the GWAS identified a significant association (rs1687391, p = 3.55 × 10-10) near the ORM1 gene, on chromosome 9. An eQTL (expression quantitative trait loci) analysis revealed a significant association between the lead GWAS variant and the expression of ORM1 in whole blood (p = 6.14 × 10-9). Additionally, ORM1 expression correlated with levels of cfDNA (p = 4.38 × 10-4). Finally, genetic correlation analysis between cfDNA and thrombosis identified a suggestive association (ρ g = 0.43, p = 0.089).All in all, we show evidence of the role of ORM1 in regulating cfDNA levels in plasma, which might contribute to the susceptibility to thrombosis through mechanisms of immunothrombosis.

Platelet hyperaggregability and venous thrombosis risk: results from the RETROVE project.

The aim of the study was to determine whether platelet hyperaggregability correlates with short closure times (PFA-100) and if hyperaggregability is associated with the risk of venous thrombosis in a Spanish population. Case--control study (RETROVE project) involving 400 patients with venous thrombosis and 400 healthy controls. We determined platelet aggregation in platelet-rich plasma (PRP) by light transmission aggregometry. Various concentrations of two aggregation agonists [ADP and epinephrine (EPI)] were tested to determine the percentage of maximal aggregation and the percentage area under the curve (AUC). Venous thrombosis risk associated with platelet hyperaggregability was calculated by logistic regression. We estimated the crude and adjusted (by sex and age) odds ratios (OR) for venous thrombosis risk. An agonist concentration of 0.5 µmol/l differentiated between hypo-responders and hyper-responders at the following AUC cut-off values: EPI: the 50th percentile for aggregation with 0.5 µmol/l of EPI (EPI_AUC) was 22.53% (>22.53% = hyper-EPI); the crude risk for venous thrombosis was statistically significant (OR = 1.37; 95% CI 1.03-1.82); ADP: the 75th percentile for aggregation with 0.5 µmol/l of ADP (ADP_AUC) was 29.6% (>29.6% = hyper-ADP), with a significant crude risk for venous thrombosis (OR = 1.44; 95% CI 1.05-1.98). However, after adjustment for confounders (age), the ORs for EPI or ADP aggregation were no longer significant. EPI_AUC and PFA-100 values with the EPI agonist were significantly correlated (R = -0.342, P < 0.01). Only 12% of the PFA-100 values were explained by platelet aggregation. In this case--control study, platelet hyperaggregability was not associated with the risk of developing venous thrombosis.

Expression of microRNAs in human platelet-poor plasma: analysis of the factors affecting their expression and association with proximal genetic variants.

To translate circulating microRNAs (miRNAs) into the clinic, a deeper understanding of the factors affecting their expression is needed. In this study, we explored the features affecting the expression of miRNAs and their genetic regulation using the expression data of 103 miRNAs obtained by qPCR in the platelet-poor plasma of 104 subjects. The principal components (PCs) of the expression of the miRNAs were associated with technical and biological features (e.g., synthetic controls or sex) and with blood cell counts. Also, the associations with proximal genetics variants were analysed. We found that haemolysis marker (dCt hsa-miR-23a-3p-hsa-miR-451a) was correlated strongly (ß = 0.84, p = 2.07x10-29) with the second PC, which explained 10.1% of the overall variability. Thus, we identified haemolysis as a source of variability for miRNA expression even in mild hemolyzed samples (haemolysis marker dCt <5). In addition to hsa-miR-23a-3p and hsa-miR-451a, the miRNAs most stable and most susceptible to haemolysis were identified. Then, we discovered that the expression of miRNAs in platelet-poor plasma was not biased by any blood cell count, and thus, our results supported their role as biomarkers of tissue-specific conditions. Finally, we identified 1,323 genetic variants that corresponded to 158 miRNA expression quantitative trait loci for 14 miRNAs (FDR <0.2), which were enriched in promoter regions (p = 0.03). This enrichment corresponded to a range of specific tissues (e.g., breast or fat) although not to blood tissue, supporting the concept that the expression of circulating miRNAs is under the genetic control of different tissues.

Identification of a Plasma MicroRNA Profile Associated With Venous Thrombosis.

OBJECTIVE: Venous thrombosis (VT) is a complex condition with a highly heritable genetic component that predisposes one to its development. Certain microRNAs (miRNAs) might be used as biomarkers of VT, but few studies have examined miRNA expression in this respect. The aim of the present work was to identify a plasma miRNA profile associated with VT. Approach and Results: miRNAs were analyzed by quantitative polymerase chain reaction in plasma samples from members of the GAIT-2 (Genetic Analysis of Idiopathic Thrombophilia 2) population (n=935). A discovery phase involving the screening of 752 miRNAs from a subset of 104 GAIT-2 subjects was followed by an internal validation phase in which the selected miRNAs were quantified in the whole GAIT-2 population. In the discovery phase, 16 miRNAs were selected, including 9 associated with VT and 7 that correlated with an intermediate phenotype of VT. In the next phase, 4 miRNAs were validated as differentially expressed (false discovery rate, <0.1) in VT: hsa-miR-126-3p, hsa-miR-885-5p, hsa-miR-194-5p, and hsa-miR-192-5p. The 4 miRNAs each returned a significant (P<0.05) odds ratio for VT (range of 1.3-1.8). A risk model including the 4 miRNAs, age, and sex returned an area under the receiver operating characteristic curve of 0.77. Moreover, all 4 miRNAs showed significant correlations with intermediate phenotypes of VT (eg, protein S and factor VII). The targets of the miRNAs in the blood coagulation pathway and their interactions are also discussed. CONCLUSIONS: The present results suggest a 4-miRNA plasma profile associated with VT is of potential use in predicting the risk of this condition.

VAMP8 and serotonin transporter levels are associated with venous thrombosis risk in a Spanish female population. Results from the RETROVE Project.

INTRODUCTION: Platelet hyper-reactivity has been associated with thrombosis and high levels of human vesicle-associated membrane protein 8 (VAMP8) and serotonin transporter (SERT). Two polymorphisms (rs1010 of VAMP8 gene and in SERT gene (SLC6A4)) are associated with arterial thrombosis. AIM: To determine if levels of serotonin, SERT and/or VAMP8 and these polymorphisms are associated with the risk of venous thrombosis. MATERIAL AND METHODS: A total of 324 individuals were included in the RETROVE Study (Riesgo de Enfermedad TROmboembólica VEnosa). VAMP8, SERT and serotonin were determined by ELISA; polymorphisms of SLC6A4 and VAMP8 by polymerase chain reaction (PCR) and real time PCR. The venous thrombotic risk was calculated by a logistic regression method to estimate the crude and adjusted OR (adjusted for sex, age, body mass index and venous thrombosis risk co-factors). RESULTS: Statistically significant high levels of VAMP8 and SERT were found in patients, but not in controls. In contrast, serotonin showed lower levels in patients than in controls. When individuals were studied by gender, only women exhibited a statistically significant difference: the OR for VAMP8 was 3.25 (1.61-6.56 95% CI). The adjusted OR did not change. The OR for SERT was 2.76 (1.36-5.60 95% CI), the adjusted OR was maintained also. For serotonin with OR of 2.62 (1.40-4.92 95% CI), the adjusted OR was not significant. In contrast males did not show significant differences. No statistically differences between patients and controls were found for both polymorphisms. CONCLUSIONS: VAMP8 and SERT levels are associated with venous thrombosis in a female Spanish population.

A genome-wide association study identifies new loci for factor VII and implicates factor VII in ischemic stroke etiology.

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.

Study of tumor growth indicates the existence of an "immunological threshold" separating states of pro- and antitumoral peritumoral inflammation.

BACKGROUND: Peritumoral inflammation-a response mainly involving polimorphonuclear neutrophils-has traditionally been thought protumoral in its effects. In recent years, however, a number of studies have indicated that it may play an important antitumoral role. This discrepancy has been difficult to explain. METHODS AND FINDINGS: This work describes a tool for simulating tumor growth that obeys the universal model of tumor growth dynamics, and shows through its use that low intensity peritumoral inflammation exerts a protumoral effect, while high intensity inflammation exerts a potent antitumoral effect. Indeed, the simulation results obtained indicate that a sufficiently strong antitumoral effect can reverse tumor growth, as has been suggested several times in the clinical literature. CONCLUSIONS: The present result indicate that an 'immunological threshold' must exist, marking the boundary between states in which peritumoral inflammation is either harmful or beneficial. These findings lend support to the idea that stimulating intense peritumoral inflammation could be used as a treatment against solid tumors.

Short closure time values in PFA-100® are related to venous thrombotic risk. Results from the RETROVE Study.

INTRODUCTION: Platelets play a role in the pathophysiology of venous thromboembolism (VTE). Some studies have not found an association between VTE and platelet aggregation. The PFA-100® analyser is an in vitro assay for assessing primary haemostasis. But, there are no studies to evaluate its association with VTE. We investigated the contribution of the global platelet function and aggregation in the development of VTE. MATERIAL AND METHODS: We analysed 800 individuals who were included in the RETROVE Study (Riesgo de Enfermedad TROmboembólica VEnosa). Global platelet function was evaluated as closure times (CT) with the agonists ADP and epinephrine using a PFA-100® analyser. Platelet aggregation was evaluated by Multiplate™ analyser. The VTE risk for all the parameters was calculated by unconditional logistic regression analyses considering the potential confounders: age, gender, body mass index (BMI), factor VIII (FVIII), the von Willebrand factor (vWF) and the ABO blood group system. RESULTS: The unadjusted odds ratio (OR) values ≤10th percentile for the PFAadp and PFAepi were 4.02 (95% CI, 2.76-5.95) and 3.33 (2.27-4.97). Also, after adjusting for vWF, we obtained lower OR for the PFAadp and for PFAepi: 2.24 (1.44-3.49) and 1.63 (1.04-2.59). But, the whole blood aggregation parameters did not shown an association with VTE risk. CONCLUSION: We demonstrated an association between short CT and VTE risk. Although, the whole blood aggregation parameters did not show an association with the VTE risk. This striking contrast suggests that there are other platelet function mechanisms (e.g. adhesion) that are responsible of VTE risk.

Next generation sequencing to dissect the genetic architecture of KNG1 and F11 loci using factor XI levels as an intermediate phenotype of thrombosis.

Venous thromboembolism is a complex disease with a high heritability. There are significant associations among Factor XI (FXI) levels and SNPs in the KNG1 and F11 loci. Our aim was to identify the genetic variation of KNG1 and F11 that might account for the variability of FXI levels. The KNG1 and F11 loci were sequenced completely in 110 unrelated individuals from the GAIT-2 (Genetic Analysis of Idiopathic Thrombophilia 2) Project using Next Generation Sequencing on an Illumina MiSeq. The GAIT-2 Project is a study of 935 individuals in 35 extended Spanish families selected through a proband with idiopathic thrombophilia. Among the 110 individuals, a subset of 40 individuals was chosen as a discovery sample for identifying variants. A total of 762 genetic variants were detected. Several significant associations were established among common variants and low-frequency variants sets in KNG1 and F11 with FXI levels using the PLINK and SKAT packages. Among these associations, those of rs710446 and five low-frequency variant sets in KNG1 with FXI level variation were significant after multiple testing correction and permutation. Also, two putative pathogenic mutations related to high and low FXI levels were identified by data filtering and in silico predictions. This study of KNG1 and F11 loci should help to understand the connection between genotypic variation and variation in FXI levels. The functional genetic variants should be useful as markers of thromboembolic risk.

Genome-wide association study with additional genetic and post-transcriptional analyses reveals novel regulators of plasma factor XI levels.

Coagulation factor XI (FXI) has become increasingly interesting for its role in pathogenesis of thrombosis. While elevated plasma levels of FXI have been associated with venous thromboembolism and ischemic stroke, its deficiency is associated with mild bleeding. We aimed to determine novel genetic and post-transcriptional plasma FXI regulators.We performed a genome-wide association study (GWAS) for plasma FXI levels, using novel data imputed to the 1000 Genomes reference panel. Individual GWAS analyses, including a total of 16,169 European individuals from the ARIC, GHS, MARTHA and PROCARDIS studies, were meta-analysed and further replicated in 2,045 individuals from the F5L family, GAIT2 and MEGA studies. Additional association with activated partial thromboplastin time (aPTT) was tested for the top SNPs. In addition, a study on the effect of miRNA on FXI regulation was performed using in silico prediction tools and in vitro luciferase assays.Three loci showed robust, replicating association with circulating FXI levels: KNG1 (rs710446, P-value = 2.07 × 10-302), F11 (rs4253417, P-value = 2.86 × 10-193), and a novel association in GCKR (rs780094, P-value = 3.56 ×10-09), here for the first time implicated in FXI regulation. The two first SNPs (rs710446 and rs4253417) also associated with aPTT. Conditional and haplotype analyses demonstrated a complex association signal, with additional novel SNPs modulating plasma FXI levels in both the F11 and KNG1 loci. Finally, eight miRNAs were predicted to bind F11 mRNA. Over-expression of either miR-145 or miR-181 significantly reduced the luciferase activity in cells transfected with a plasmid containing FXI-3'UTR.These results should open the door to new therapeutic targets for thrombosis prevention.