Dysregulated neutrophil extracellular traps formation in sepsis.

The migration and antimicrobial functions of neutrophils seem to be impaired during sepsis and contribute to the dysregulation of immune responses and disease pathogenesis. However, the role of neutrophil extracellular traps (NETs) remains to be clarified. The study aimed to analyse sequential phenotypic and functional changes of neutrophils during the time following the diagnosis of sepsis. We prospectively enrolled 49 septic and 18 non-septic patients from the intensive care unit (ICU) and emergency room (ER) and 20 healthy volunteers (HV). Baseline blood samples from septic and non-septic patients were collected within 12 h of admission to the hospital. Additional septic samples were drawn at 24, 48 and 72 h after baseline. Neutrophil phenotype and degranulation capacity were assessed by flow cytometry and NET formation was quantified by fluorescence. Neutrophils from septic patients exhibited increased CD66b, CD11b and CD177 expression but displayed reduced NET formation at baseline compared with non-septic patients and HV controls. Neutrophils expressing CD177 interacted less with platelets, were related to reduced NETosis and tended to indicate a worse sepsis outcome. In vitro experiments revealed that neutrophil function is compromised by the origin of sepsis, including the pathogen type and the affected organ. Assessing a decision tree model, our study showed that CD11b expression and NETosis values are useful variables to discriminate septic from non-septic patients. We conclude that sepsis induces changes in neutrophil phenotype and function that may compromise the effective capacity of the host to eliminate pathogens.

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.

Correlation between PlateletWorks® and PFA-100® for Measuring Platelet Function before Urgent Surgery in Patients with Chronic Antiplatelet Therapy.

Hemostasis is crucial for reducing bleeding during surgical procedures. The points-of-care based on the platelet function test could be useful to minimize the complications related to chronic antiplatelet therapy during surgery. The present study is aimed at comparing two point-of-care platelet function devices-Platelet Function Analyzer PFA-100® (Siemens Canada, Mississauga, ON, Canada) and Plateletworks®(Helena Laboratories, Beaumont, TX, USA). Our objective is to evaluate if they provide comparable and useful information to manage anti-aggregate patients before surgery. We included patients with a femoral fracture receiving chronic antiplatelet therapy and a median age of 89 years (range from 70 to 98). A platelet function evaluation was performed on all patients before surgery using both devices-Plateletworks® and PFA-100®. The correlation between Plateletworks® and PFA-100® was performed using Cohen's Kappa coefficient. Twenty consecutive patients participated in the trial; 16 patients were under treatment with 75 mg/day of clopidogrel, three with >300 mg/day of acetylsalicylic acid (ASA), and only one was in treatment with both antiplatelet agents. Cohen's Kappa coefficient was 0.327 comparing PFA-100®-ADP (adenosine diphosphate) and Plateletworks® and, 0.200 comparing PFA-100®-EPI (epinephrine) and Plateletworks®. In conclusion, we found a weak concordance comparing PFA-100® and Plateletworks®. This could partially be due to the advanced age of the included patients. However, given the limited sample size, more studies are necessary to confirm these results.

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.

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.

Influence of ABO Locus on PFA-100 Collagen-ADP Closure Time Is Not Totally Dependent on the Von Willebrand Factor. Results of a GWAS on GAIT-2 Project Phenotypes.

(1) Background: In a previous study, we found that two phenotypes related to platelet reactivity, measured with the PFA-100 system, were highly heritable. The aim of the present study was to identify genetic determinants that influence the variability of these phenotypes: closure time of collagen-ADP (Col-ADP) and of collagen-epinephrine (Col-Epi). (2) Methods: As part of the GAIT-2 (Genetic Analysis of Idiopathic Thrombophilia (2) Project, 935 individuals from 35 large Spanish families were studied. A genome-wide association study (GWAS) with ≈ 10 M single nucleotide polymorphisms (SNPs) was carried out with Col-ADP and Col-Epi phenotypes. (3) Results: The study yielded significant genetic signals that mapped to the ABO locus. After adjusting both phenotypes for the ABO genotype, these signals disappeared. After adjusting for von Willebrand factor (VWF) or for coagulation factor VIII (FVIII), the significant signals disappeared totally for Col-Epi phenotype but only partially for Col-ADP phenotype. (4) Conclusion: Our results suggest that the ABO locus exerts the main genetic influence on PFA-100 phenotypes. However, while the effect of the ABO locus on Col-Epi phenotype is mediated through VWF and/or FVIII, the effect of the ABO locus on Col-ADP phenotype is partly produced through VWF and/or FVIII, and partly through other mechanisms.

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.

Genetic Determinants of Thrombin Generation and Their Relation to Venous Thrombosis: Results from the GAIT-2 Project.

BACKGROUND: Venous thromboembolism (VTE) is a common disease where known genetic risk factors explain only a small portion of the genetic variance. Then, the analysis of intermediate phenotypes, such as thrombin generation assay, can be used to identify novel genetic risk factors that contribute to VTE. OBJECTIVES: To investigate the genetic basis of distinct quantitative phenotypes of thrombin generation and its relationship to the risk of VTE. PATIENTS/METHODS: Lag time, thrombin peak and endogenous thrombin potential (ETP) were measured in the families of the Genetic Analysis of Idiopathic Thrombophilia 2 (GAIT-2) Project. This sample consisted of 935 individuals in 35 extended families selected through a proband with idiopathic thrombophilia. We performed also genome wide association studies (GWAS) with thrombin generation phenotypes. RESULTS: The results showed that 67% of the variation in the risk of VTE is attributable to genetic factors. The heritabilities of lag time, thrombin peak and ETP were 49%, 54% and 52%, respectively. More importantly, we demonstrated also the existence of positive genetic correlations between thrombin peak or ETP and the risk of VTE. Moreover, the major genetic determinant of thrombin generation was the F2 gene. However, other suggestive signals were observed. CONCLUSIONS: The thrombin generation phenotypes are strongly genetically determined. The thrombin peak and ETP are significantly genetically correlated with the risk of VTE. In addition, F2 was identified as a major determinant of thrombin generation. We reported suggestive signals that might increase our knowledge to explain the variability of this important phenotype. Validation and functional studies are required to confirm GWAS results.