Venous thromboembolism (VTE), the third leading cause of cardiovascular mortality, is a complex thrombotic disorder with environmental and genetic determinants. Although several genetic variants have been found associated with VTE, they explain a minor proportion of VTE risk in cases. We undertook a meta-analysis of genome-wide association studies (GWASs) to identify additional VTE susceptibility genes. Twelve GWASs totaling 7,507 VTE case subjects and 52,632 control subjects formed our discovery stage where 6,751,884 SNPs were tested for association with VTE. Nine loci reached the genome-wide significance level of 5 × 10(-8) including six already known to associate with VTE (ABO, F2, F5, F11, FGG, and PROCR) and three unsuspected loci. SNPs mapping to these latter were selected for replication in three independent case-control studies totaling 3,009 VTE-affected individuals and 2,586 control subjects. This strategy led to the identification and replication of two VTE-associated loci, TSPAN15 and SLC44A2, with lead risk alleles associated with odds ratio for disease of 1.31 (p = 1.67 × 10(-16)) and 1.21 (p = 2.75 × 10(-15)), respectively. The lead SNP at the TSPAN15 locus is the intronic rs78707713 and the lead SLC44A2 SNP is the non-synonymous rs2288904 previously shown to associate with transfusion-related acute lung injury. We further showed that these two variants did not associate with known hemostatic plasma markers. TSPAN15 and SLC44A2 do not belong to conventional pathways for thrombosis and have not been associated to other cardiovascular diseases nor related quantitative biomarkers. Our findings uncovered unexpected actors of VTE etiology and pave the way for novel mechanistic concepts of VTE pathophysiology.

IntroductionAn increased or disturbed activation and aggregation of platelets plays a major role in the pathophysiology of thrombosis and haemostasis and is related to cardiovascular disease processes. In addition to qualitative disturbances of platelet function, changes in thrombopoiesis or an increased elimination of platelets, (e. g., in autoimmune thrombocytopenia), are also of major clinical relevance. Flow cytometry is increasingly used for the specific characterisation of phenotypic alterations of platelets which are related to cellular activation, haemostatic function and to maturation of precursor cells. These new techniques also allow the study of the in vitro response of platelets to stimuli and the modification thereof under platelet-targeted therapy as well as the characterisation of platelet-specific antibodies. In this protocol, specific flow cytometric techniques for platelet analysis are recommended based on a description of the current state of flow cytometric methodology. These recommendations are an attempt to promote the use of these new techniques which are at present broadly evaluated for diagnostic purposes. Furthermore, the definition of the still open questions primarily related to the technical details of the method should help to promote the multi-center evaluation of procedures with the goal to finally develop standardized operation procedures as the basis of interlaboratory reproducibility when applied to diagnostic testing.

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SummaryActivated platelets can be detected by measuring platelet-bound fibrinogen in a whole blood, flow cytometric assay, using a fluorescently-conjugated polyclonal antibody.Fibrinogen binding to unstimulated platelets from normal subjects was low in this assay, as was expression of the CD63 antigen. Single cell counting of samples prepared for flow cytometric analysis showed platelet aggregates do not form during the assay procedure. Immune complexes were not seen, and fibrinogen binding to the platelets was unaffected by the CD32 MAb, IV.3. Artefactual activation of the unfixed samples could be minimised by control of phlebotomy, time and temperature of incubation. Variations in platelet count in the range 140–430 × 109 1-1 and in plasma fibrinogen in the range 2–6 g 1-1 did not affect the assay results.Comparison of fibrinogen binding with expression of CD63 antigen on normal platelets, stimulated with agonists in vitro, demonstrated that fibrinogen binding detects an earlier stage of platelet activation.Platelet bound fibrinogen was shown to be sensitive in detecting small numbers of activated platelets in clinical samples in twelve patients on intensive care, four undergoing haemofiltration. The patients had a significantly higher median percentage of circulating platelets with bound fibrinogen (p <0.005), but fibrinogen binding was significantly lower (p <0.02) in response to 10-5 M ADP, compared to twelve age-matched normal Controls.

SummaryPre-eclampsia is a common complication of pregnancy, in which platelets may have an early pathogenetic role. In this prospective study a whole blood flow cytometric method has been used to detect circulating activated platelets in pregnant women prior to the development of pre-eclampsia. Activated platelets were identified by bound fibrinogen or by CD63 antigen expression. Of 121 healthy primiparous women studied at 28 weeks of pregnancy, 18 (15%) developed clinical pre-eclampsia six to thirteen weeks later. The platelets of these women showed increased fibrinogen binding ex vivo (5.1% platelets positive, compared with 3.4% in those who completed a normal pregnancy, p <0.02), and increased CD63 antigen expression (0.73% positive compared to 0.45%, p = 0.01). In contrast, no differences between the women with different outcomes were detected at 28 weeks in platelet counts, or plasma ß-thromboglobulin levels. These findings confirm that whole blood flow cytometry is a sensitive technique for investigating platelet activation in a clinical setting and support the hypothesis that platelets have a critical role in the pathogenesis of pre-eclampsia

SummaryThe monoclonal antibody RFF-VIII:R/1 recognises an epitope on von Willebrand factor involved in its interaction with GPIbα. A two-site, solid phase ELISA has been established using RFF-VIII:R/1 as the solid-phase, capture antibody and an enzyme-conjugated, polyclonal antibody to human VWF, which provides an assay for VWF functional activity with a detection limit of 0.5 U/dl VWF and an interassay %CV<10. Plasma from 192 VWD patients (48 studied retrospectively; 144 prospectively) showed VWF levels of <50 U/dl in type 1 patients (n = 156), <25 U/dl in type 2A (n = 26) and <35 U/dl in type 2B (n = 8) which, in type 1 and 2A patients, correlated with RiCoF activity (r >0.82). In plasma from patients with type 1 VWD values of VWF in the Mab-based ELISA were similar to levels of VWF:Ag measured in a polyclonal antibody-based ELISA (r >0.87) but were significantly lower than VWF:Ag in type 2A and 2B plasmas (p <0.0005), allowing discrimination of variant VWD. The Mab-based ELISA has advantages of sensitivity and reproducibility over the RiCoF assay to measure VWF activity and can be used to analyse stored samples. In conjunction with an ELISA for VWF:Ag and VWF multimer analysis, it provides a reliable method, for the laboratory diagnosis of VWD.

The increased tendency of platelets from patients with the Pro33 form of GPIIIa may predispose patients with this allele to a higher risk of acute thrombotic events, and argues for selective use of therapeutic agents that inhibit ADP-mediated platelet activation in occlusive vascular disease states.

The intake of (n-3) long-chain PUFA is associated with a decreased risk of fatal myocardial infarction. Whether this effect is attributable to the effects of docosahexaenoic acid [22:6(n-3) (DHA)] on vascular function, particularly at intakes <1 g/d, is unknown. We report a randomized, double-blind, crossover, placebo controlled trial of 0.7 g DHA/d as a purified algal derived triacylglycerol (1.5 g/d) vs. placebo (1.5 g olive oil/d) on vascular function and biochemical indices of endothelial dysfunction in 38 healthy men and women, aged 40-65 y. Each treatment phase lasted 3 mo, separated by a 4 mo washout period. Supplementation increased the proportion of DHA in erythrocytes lipids by 58%, compared with placebo. Arterial compliance and endothelium independent and dependent responses, plasma concentrations of C-reactive protein, soluble thrombomodulin, E-selectin, von Willebrand factor antigen, and urinary microalbumin and isoprostane excretion were unaffected by treatment. Diastolic blood pressure decreased by 3.3 mm Hg (95% CI -6.1 to -0.6; P = 0.01). Heart rate tended to be 2.1 beats/min lower after DHA treatment than after the placebo period (P = 0.15). The results indicate that a moderate increase in the daily intake of DHA to approximately 0.7 g DHA lowers diastolic BP but does not influence indices of endothelial function or arterial stiffness in the short term.