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Expansion of human megakaryoblasts from peripheral blood-derived CD34+ cells is commonly used to characterize inherited or acquired thrombocytopenia and evaluate defects in megakaryocyte (MK) differentiation, MK maturation and proplatelet formation. We applied single-cell RNA sequencing to understand local gene expression changes during megakaryopoiesis (days 6 and 11 of differentiation) in peripheral CD34+ cells from healthy controls and patients with ETV6-related thrombocytopenia.Analysis of gene expression and regulon activity revealed distinct clusters partitioned into seven major cell stages: hematopoietic stem/progenitor cells (HSPC), common-myeloid progenitors (CMP), MK-primed CMP, granulocyte-monocyte progenitors, megakaryocyte-erythroid progenitors (MEP), MK progenitor /mature MK (MKP/MK) and platelets. We observed a subpopulation of MEP that arose directly from HSPC, deviating from the canonical MK differentiation pathway.ETV6 deficiency was characterized by an increase in HSPC, a decrease in MKP/MK, and a lack of platelets. ETV6 deficiency also led to the development of aberrant MEP and MKP/MK cell populations. Genes involved in “mitochondrial” and “DNA repair” pathways were downregulated, while genes involved in “translation” pathways were upregulated. Analysis of patient samples and hematopoietic cell lines transduced with an ETV6 variant revealed increased translation in MK. Ribosomal protein small 6 (RPS6) levels in MK, platelets and peripheral blood mononuclear cells was consistent with the translation findings.Our results provide a framework to understand peripheral CD34+ cell-derived megakaryocytic cultures. Our observations also shed light on ETV6-variant pathology and reveal potential targets for diagnostic and therapeutic purposes.Key points-scRNAseq gain insight into in vitro megakaryopoiesis, identify MK-primed CMP, and a differentiation trajectory that bypasses the CMP.-ETV6 variants led to the development of aberrant MEP and MK cell populations.

International audience; Background Transfusion of defective platelets could contribute to the inefficiency of platelet transfusion in preventing or stopping bleeding. Study Design and Methods This single-center prospective study aimed to determine the prevalence of functional platelet abnormalities in a population of blood donors with a clinical history of bleeding diathesis or with history of hematoma (>4 cm) during blood donation. Donors with positive bleeding screening questionnaire were referred to the reference center for rare platelet diseases at La Timone University Hospital (Marseille) to confirm the bleeding tendency using a more extensive bleeding questionnaire (MCMDMscore) and to assess hemostasis, including a comprehensive platelet analysis. Results One hundred and ninety-five donors identified based on a history of hematoma and 2434 blood donors were included in the study. Eighty-eight donors (3.6%) had a bleeding score indicating a potential bleeding disorder. Five donors with a history of hematoma (2.5%) and 15 (17%) donors with a confirmed bleeding score underwent hemostatic analysis, including two men and 18 women with average age of 33.9 years. Minor hemostatic abnormalities were observed in three donors. Two donors exhibited accelerated fibrinolysis with reduced euglobulin lysis time and increased D-dimer levels in serum. Two donors had a platelet granule defect, without identification of genetic abnormality. Conclusion The bleeding questionnaire proved to be a valuable tool to screen blood donors for potential platelet defects. Platelet dysfunction was rare in the blood donor population assessed. Additional studies are necessary to understand the clinical impact that the transfusion of platelets with qualitative defects has on recipients.

Venous thromboembolism (VTE), comprising both deep vein thrombosis (DVT) and pulmonary embolism (PE) 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. We used multiplex proteomics profiling to screen plasma from patients with suspected acute VTE, and a case-control study of patients followed up after ending anticoagulant treatment for a first VTE. With replication in 5 independent studies, together totalling 1137 patients and 1272 controls, we identify Complement Factor H Related Protein (CFHR5), a regulator of the alternative pathway of complement activation, as a novel VTE associated plasma biomarker. Using GWAS analysis of 2967 individuals we identified a genome-wide significant pQTL signal on chr1q31.3 associated with CFHR5 levels. We showed that higher CFHR5 levels are associated with increased thrombin generation in patient plasma and that recombinant CFHR5 enhances platelet activationin vitro. Thrombotic complications are a frequent feature of COVID-19; in hospitalised patients we found CFHR5 levels at baseline were associated with short-time prognosis of disease severity, defined as maximum level of respiratory support needed during hospital stay. Our results indicate a clinically important role for regulation of the alternative pathway of complement activation in the pathogenesis of VTE and pulmonary complications in acute COVID-19. Thus, CFHR5 is a potential diagnostic and/or risk predictive plasma biomarker reflecting underlying pathology in VTE and acute COVID-19.

In studies of time-to-events, it is common to collect information about events that occurred before the inclusion in a prospective cohort. In an ambispective design, when the risk factors studied are independent of time, including both pre- and post-inclusion events in the analyses increases the statistical power but may lead to a selection bias. To avoid such a bias, we propose a survival analysis weighted by the inverse of the survival probability at the time of data collection about the events.This method is applied to the study of the association of ABO blood groups with the risk of venous thromboembolism (VT) recurrence in the MARTHA and MEGA cohorts. The former relying on an ambispective design and the latter on a standard prospective one. In the combined sample totalling 2,752 patients including 993 recurrences, compared with the O1 group, A1 has an increased risk (Hazard Ratio (HR) of 1.18, p=4.2×10−3), homogeneously in MARTHA and in MEGA. The same trend (HR=1.19, p=0.06) was observed for the less frequent A2 group.In conclusion, this work clarified the association ofABOblood groups with the risk of VT recurrence. Besides, the methodology proposed here to analyse time-independent risk factors of events in an ambispective design has an immediate field of application in the context of genome wide association studies.

Venous thromboembolism (VTE) is the third most common cardiovascular disease, affecting ∼ 1,000,000 individuals each year in Europe. VTE is characterized by an annual recurrent rate of ∼ 6%, and ∼ 30% of patients with unprovoked VTE will face a recurrent event after a six-month course of anticoagulant treatment. Even if guidelines recommend life-long treatment for these patients, about ∼ 70% of them will never experience a recurrence and will receive unnecessary lifelong anti-coagulation that is associated with increased risk of bleeding and is highly costly for the society. There is then urgent need to identify biomarkers that could distinguish VTE patients with high risk of recurrence from low-risk patients. Capitalizing on a sample of 913 patients followed up for the risk of VTE recurrence during a median of ∼ 10 years and profiled for 376 plasma proteomic antibodies, we here develop an artificial neural network (ANN) based strategy to identify a proteomic signature that helps discriminating patients at low and high risk of recurrence. In a first stage, we implemented a Repeated Editing Nearest Neighbors algorithm to select a homogeneous sub-sample of VTE patients. This sub-sample was then split in a training and a testing sets. The former was used for training our ANN, the latter for testing its discriminatory properties. In the testing dataset, our ANN led to an accuracy of 0.86 that compared to an accuracy of 0.79 as provided by a random forest classifier. We then applied a Deep Learning Important FeaTures (DeepLIFT) – based approach to identify the variables that contribute the most to the ANN predictions. In addition to sex, the proposed DeepLIFT strategy identified 6 important proteins (DDX1, HTRA3, LRG1, MAST2, NFATC4 and STXBP5) whose exact roles in the etiology of VTE recurrence now deserve further experimental validations. © 2021, Springer Nature Switzerland AG.

International audience; Background Coagulation factor V (FV), present in plasma and platelets, has both pro- and anticoagulant functions.Objective We investigated an FV-deficient patient (FV:C 3%, FV:Ag 4%) paradoxically presenting with recurrent venous thrombosis (11 events) instead of bleeding.Methods/Results Thrombophilia screening revealed only heterozygosity for the F2 20210G>A mutation. Although thrombin generation in the patient's platelet-poor plasma was suggestive of a hypocoagulable state, thrombin generation in the patient's platelet-rich plasma (PRP) was higher than in control PRP and extremely resistant to activated protein C (APC). This was partially attributable to the complete abolition of the APC-cofactor activity of FV and a marked reduction of plasma tissue factor pathway inhibitor antigen and activity. The patient was homozygous for a novel missense mutation (Ala2086Asp, FVBesancon) that favors a "closed conformation" of the C2 domain, predicting impaired binding of FV(a) to phospholipids. Recombinant FVBesancon was hardly secreted, indicating that this mutation is responsible for the patient's FV deficiency. Model system experiments performed using highly diluted plasma as a source of FV showed that, compared with normal FVa, FVa(Besancon) has slightly (

International audience; Introduction In daily practice in haematology laboratories, red blood cell (RBC) abnormalities are frequent and their management is a real challenge. The aim of this study is to establish a "decision tree" using RBC and reticulocyte parameters from the SYSMEX XN-10 analyser to distinguish between patients with a hereditary RBC disease from iron deficiency anaemia and other patients. Methods We analysed results of complete RBC counts in a cohort composed of 8217 adults divided into 5 different groups: iron deficiency anaemia (n = 120), heterozygous haemoglobinopathy (n = 92), sickle cell disease syndrome (n = 56), hereditary spherocytosis (n = 18) and other patients (n = 7931). A Classification And Regression Tree (CART) analysis was used to obtain a two-step decision tree in order to predict these previous groups. Results Five parameters and the calculated RBC score were selected by the CART method: mean corpuscular haemoglobin concentration, percentage of microcytes, distribution width of the RBC histogram, percentage of nucleated red blood cells, immature reticulocytes fraction and finally RBC Score. When applying the tree and recommended flowchart, 158/166 of the RBC hereditary disease patients and 114/120 iron deficiency anaemia patients are detected. Overall, the correct classification rate reached 99.4%. Sensitivity and specificity for RBC disease detection were 95.2% and 99.9%, respectively. These results were confirmed in an independent validation cohort. Conclusion Based on the XN-10 RBC and reticulocyte parameters, we propose a two-step decision tree delivering a good prediction and classification of hereditary RBC diseases. These results can be used to optimize additional reticulocyte analysis and microscopy review.

Platelets play a major role in primary hemostasis, where activated platelets form plugs to stop hemorrhaging in response to vessel injuries. Defects in any step of the platelet activation process can cause a variety of platelet dysfunction conditions associated with bleeding. To make an accurate diagnosis, constitutional platelet dysfunction (CPDF) should be considered once von Willebrand disease and drug intake are ruled out. CPDF may be associated with thrombocytopenia or a genetic syndrome. CPDF diagnosis is complex, as no single test enables the analysis of all aspects of platelet function. Furthermore, the available tests lack standardization, and repeat tests must be performed in specialized laboratories especially for mild and moderate forms of the disease. In this review, we provide an overview of the laboratory tests used to diagnose CPDF, with a focus on light transmission platelet aggregation (LTA), flow cytometry (FC), and granules assessment. Global tests, mainly represented by LTA, are often initially performed to investigate the consequences of platelet activation on platelet aggregation in a single step. Global test results should be confirmed by additional analytical tests. FC represents an accurate, simple, and reliable test to analyze abnormalities in platelet receptors, and granule content and release. This technique may also be used to investigate platelet function by comparing resting- and activated-state platelet populations. Assessment of granule content and release also requires additional specialized analytical tests. High-throughput sequencing has become increasingly useful to diagnose CPDF. Advanced tests or external research laboratory techniques may also be beneficial in some cases.

Venous thromboembolism is the third common cardiovascular disease and is composed of two entities, deep vein thrombosis (DVT) and its fatal form, pulmonary embolism (PE). While PE is observed in ∼40% of patients with documented DVT, there is limited biomarkers that can help identifying patients at high PE risk.To fill this need, we implemented a two hidden-layers artificial neural networks (ANN) on 376 antibodies and 19 biological traits measured in the plasma of 1388 DVT patients, with or without PE, of the MARTHA study. We used the LIME algorithm to obtain a linear approximate of the resulting ANN prediction model. As MARTHA patients were typed for genotyping DNA arrays, a genome wide association study (GWAS) was conducted on the LIME estimate. Detected single nucleotide polymorphisms (SNPs) were tested for association with PE risk in MARTHA. Main findings were replicated in the EOVT study composed of 143 PE patients and 196 DVT only patients.The derived ANN model for PE achieved an accuracy of 0.89 and 0.79 in our training and testing sets, respectively. A GWAS on the LIME approximate identified a strong statistical association peak (p = 5.3×10−7) at the PLXNA4 locus, with lead SNP rs1424597 at which the minor A allele was further shown to associate with an increased risk of PE (OR = 1.49 [1.12 – 1.98], p = 6.1×10−3). Further association analysis in EOVT revealed that, in the combined MARTHA and EOVT samples, the rs1424597-A allele was associated with increased PE risk (OR = 1.74 [1.27 – 2.38, p = 5.42×10−4) in patients over 37 years of age but not in younger patients (OR = 0.96 [0.65 – 1.41], p = 0.848).Using an original integrated proteomics and genetics strategy, we identified PLXNA4 as a new susceptibility gene for PE whose exact role now needs to be further elucidated.Author SummaryPulmonary embolism is a severe and potentially fatal condition characterized by the presence of a blood clot (or thrombus) in the pulmonary artery. Pulmonary embolism is often the consequence of the migration of a thrombus from a deep vein to the lung. Together with deep vein thrombosis, pulmonary embolism forms the so-called venous thromboembolism, the third most common cardiovascular disease, and its prevalence strongly increases with age. While pulmonary embolism is observed in ∼40% of patients with deep vein thrombosis, there is currenly limited biomarkers that can help predicting which patients with deep vein thrombosis are at risk of pulmonary embolism. We here deployed an Artificial Intelligence based methodology integrating both plasma proteomics and genetics data to identify novel biomarkers for PE. We thus identified the PLXNA4 gene as a novel molecular player involved in the pathophysiology of pulmonary embolism. In particular, using two independent cohorts totalling 1,881 patients with venous thromboembolism among which 467 experienced pulmonary embolism, we identified a genetic polymorphism in the PLXNA4 gene that associates with ∼2 fold increased risk of pulmonary embolism in patients aged more than ∼40 years.

International audience

Rare variants outside the classical coagulation cascade might cause rare inherited thrombosis. We aimed to identify the variant(s) causing venous thromboembolism (VTE) in a family with multiple relatives affected with unprovoked VTE and no thrombophilia defects. We identified by whole exome sequencing an extremely rare Arg to Gln variant (R89Q) in the Microtubule Associated Serine/Threonine Kinase 2 (MAST2) gene that segregates with VTE in the family. Free-tissue factor pathway inhibitor (f-TFPI) plasma levels were significantly decreased in affected family members compared to healthy relatives. Conversely, plasminogen activator inhibitor-1 (PAI-1) levels were significantly higher in affected members than in healthy relatives. RNA sequencing analysis of RNA interference experimental data conducted in endothelial cells revealed that, of the 13,387 detected expressed genes, 2,354 have their level of expression modified by MAST2 knockdown, including SERPINE1 coding for PAI-1 and TFPI. In HEK293 cells overexpressing the MAST2R289Q variant, TFPI and SERPINE1 promoter activities were respectively lower and higher than in cells overexpressing the MAST2 wild type allele. This study identifies a novel thrombophilia-causing R89Q variant in the MAST2 gene that is here proposed as a new molecular player in the etiology of VTE by interfering with hemostatic balance of endothelial cells. Author Summary Venous thromboembolism (VTE) is a multifactorial disease in which the genetic burden. We here present the case of a French family with multiple relatives affected with unprovoked VTE (i.e. that occurred in the absence of clinical risk factors) in which no thrombophilia defects had been identified. Adopting a whole exome sequencing approach, we identified an extremely rare variant located in the Microtubule-associated serine/threonine-protein kinase-2 (MAST2) gene that perfectly segregates with the VTE phenotype and that interferes with hemostatic balance of endothelial cells. Our results pave the way for adding MAST2 to the list of genes to be sequenced and looked for in thrombophilia families with unprovoked VTE.

SummaryVariants in 5’UTR regions that create upstream translation initiation AUG codons are a class of neglected non coding variations. When they associate with a premature stop codon and create upstream open reading frames (uORFs) whose translation competes with that of natural proteins, they can have strong impact on human diseases. We here describe MORFEE, a new bioinformatics tool that detects, annotates and predicts, from a standard VCF file, the creation of uORF by any 5’UTR variants on uORF creation. MORFEE was applied to two genomic resources and identified candidate functional variants that could explain statistical association signals observed in the context of Genome Wide Association Studies or could be responsible for rare forms of diseases. In conclusion MORFEE is an easy-to-use tool complementary to existing ones that can help resolving genetic investigations that remained so far unfruitful.Availability and implementationMORFEE is written in R with code and package available at https://github.com/daissi/MORFEE.Contactdavid-alexandre.tregouet@inserm.fr; david-alexandre.tregouet@u-bordeaux.fr

IntroductionABO blood group influence the risk of venous thrombosis (VT) by modifying A and B glycosyltransferases (AGT and BGT) activities that further modulates Factor VIII (FVIII) and von Willebrand Factor (VWF) plasma levels. The aim of this work was to evaluate the association of plasma GTs activities with VWF/FVIII plasma levels and VT risk in a case-control study.Materials and Methods420 cases were matched with 420 controls for age and ABO blood group. GT activities in plasma were measured using the quantitative transfer of tritiated N-acetylgalactosamine or galactose to the 2’-fucosyl-lactose and expressed in disintegration per minute/30µL of plasma and 2 hours of reaction (dpm/30µL/2H). FVIII and VWF plasma levels were respectively measured using human FVIII-deficient plasma in a 1-stage factor assay and STA LIATEST VWF (Diagnostica Stago).ResultsA and B GT activities were significantly lower in cases than in controls (8119±4027 vs 9682±4177 dpm/30µL/2H, p=2.03 × 10−5, and 4931±2305 vs 5524±2096 dpm/30µL/2H, p=0.043 respectively). This association was observed whatever the ABO blood groups. The ABO A1 blood group was found to explain∼80% of AGT activity. After adjusting for ABO blood groups, AGT activity was not correlated to VWF/FVIII plasma levels. Conversely, there was a moderate correlation (ρ∼0.30) between BGT activity and VWF/ FVIII plasma levels in B blood group carriers.ConclusionThis work showed, for the first time, that GT activities were decreased in VT patients in comparison to controls with the same ABO blood group. The biological mechanisms responsible for this association remained to be determined.

International audience; Heparin induced thrombocytopenia (HIT) is a life and limb-threatening complication of heparin exposure. The misdiagnosis of this disease can have major consequences on the patients. The objective of this study was to evaluate a diagnostic strategy that combines the 4Ts score with the result of HemosIL (R) AcuStar HIT-IgG (PF4-H) to confirm the diagnosis of HIT. Citrated plasmas from 1300 patients with suspicion of HIT were analyzed with a fully automated quantitative chemiluminescent immunoassay (HemosIL (R) AcuStar HIT-IgG (PF4/H)). If the IgG anti-PF4/H antibodies were positive (cut-off, 1 U/mL), HIT diagnosis was confirmed using functional tests. In total, 1300 samples of consecutive patients were enrolled, 94 (7.2%) of which gave positive results in HemosIL (R) AcuStar-IgG. HIT was diagnosed in 65 out of these patients, corresponding to a prevalence of 5%. Using ROC curve analysis, patients were divided into three groups according to their titer of antibodies. Higher values of the IgG (PF4-H) were associated with increased probability of HIT, and the diagnostic specificity was greatly increased using the combination of a 4Ts score > 3 and a positive titer >= 3.25 U/mL. Importantly, the diagnostic specificity is 100% when the titer is > 12.40 U/mL. We demonstrated that higher values of Anti PF4/H Antibodies were associated with a high probability of having HIT. A titer of HemosIL (R) IgG (PF4-H) > 12.40 U/mL has a specificity of 100% which should no require a functional test to confirm the diagnosis of HIT.

International audience; Background and aims: Genetic partial lipodystrophies are rare heterogeneous disorders characterized by abnormalities of fat distribution and associated metabolic complications including a predisposition for atherosclerotic cardiovascular disease. We hypothesized that the milder forms of these diseases might be under-diagnosed and might result in early acute coronary syndrome (ACS) as the first sign of the pathology. Methods: We performed targeted sequencing on a panel of 8 genes involved in genetic lipodystrophy for 62 patients with premature ACS, and selected heterozygous missense variations with low frequency. To confirm those results, we analyzed a second independent group of 60 additional patients through Sanger sequencing, and compared to a control group of 120 healthy patients. Results: In the first cohort, only PLIN1 exhibited variants in more than 1 patient. In PLIN1, 3 different variants were found in 6 patients. We then analyzed PLIN1 sequence in the second cohort with premature ACS and found 2 other patients. Altogether, 8 patients were carriers of 4 different mutations in PLIN1. The variant frequencies in the total cohort of 122 patients were compared to frequencies observed in a local control cohort and in 2 different public databases showing a significant difference between patient vs control group frequencies for two mutations out of 4 (c.245C > T p = 10(-4); c.839G > A p = 0.014). Discussion: This is the first study that identifies a high frequency of potential pathogenic mutations in PLIN1 related to early onset ACS. These findings could contribute to the prevention and care of precocious ACS in families carrying those mutations.

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, Los micro-ARN (miARN) son pequeñas moléculas de ARN reguladoras que participan en varios procesos biológicos y están implicados en diversas patologías. Mensurables en los líquidos corporales, se ha planteado que los miARN pueden ser biomarcadores eficaces para el diagnóstico de enfermedades y/o características asociadas. Aquí hemos llevado a cabo un análisis de miARN plasmático con tecnología de secuenciación de última generación en 344 pacientes con trombosis venosa (TV) y hemos evaluado la asociación de los niveles de miARN con distintas características hemostáticas y el riesgo de recidiva de TV. Entre los hallazgos más significativos, hemos detectado una asociación entre hsa-miR-199b-3p y los niveles de hematocritos (p = 0,0016); dos marcadores que se habían asociado de forma independiente con el riesgo de sufrir TV. Asimismo, hemos observado una evidencia indicativa de asociación entre hsa-miR-370-3p (p = 0,019), hsa-miR-27b-3p (p = 0,016) y hsa-miR-222-3p (p = 0,049) y la recidiva de TV; los resultados los dos últimos miARN confirman los hallazgos recientes de Wang et al. (Clin Epigenetics, 2019). Además, al efectuar estudios de asociación del genoma completo sobre los niveles de miARN y al metaanalizar nuestros resultados con otros disponibles públicamente, hemos identificado 21 asociaciones nuevas de polimorfismos de un solo nucleótido (PSN) con niveles de miARN plasmático con un umbral de significación estadística de p

International audience; 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.

Factor V serves an important role in the regulation of blood coagulation. The rs6025 (R534Q) and rs4524 (K858R) polymorphisms in the F5 gene, are known to influence the risk of venous thrombosis. While the rare Q534 (factor V Leiden) allele is associated with an increased risk of venous thrombosis, the minor R858 allele is associated with a lower risk of disease. However, no study has deeply examined the cumulative impact of these two variations on venous thrombosis risk. We study the association of these polymorphisms with the risk of venous thrombosis in 4 French case-control populations comprising 3719 patients and 4086 controls. We demonstrate that the Q534 allele has a dominant effect over R858. Besides, we show that in individuals not carrying the Q534 allele, the protective effect of the R858 allele acts in a dominant mode. Thrombin generation-based normalized activated protein C sensitivity ratio was lower in the 858R/R homozygotes than in the 858K/K homozygotes (1.92 ± 1.61 vs 2.81 ± 1.57, p = 0.025). We demonstrate that the R858 allele of the F5 rs4524 variant protects from venous thrombosis only in non-carriers of the Q534 allele of the F5 rs6025. Its protective effect is mediated by reduced factor VIII levels and reduced activated protein C resistance.