Haemostatic effects of a new combined oral contraceptive, nomegestrol acetate/17ß-estradiol, compared with those of levonorgestrel/ethinyl estradiol. A double-blind, randomised study.

Use of oral contraceptives (OC) that combine a progestogen with synthetic ethinyl estradiol (EE) is associated with increased risk of venous thromboembolism. NOMAC/E2 is a new monophasic OC that combines nomegestrol acetate (NOMAC), a highly selective progestogen, with 17ß-estradiol (E2). The study objective was to compare the effects on markers of haemostasis of NOMAC/E2 (2.5 mg/1.5 mg) versus the second-generation OC, levonorgestrel (LNG)/EE (100 µg/20 µg). Healthy women (age 18-38 years) received once-daily treatment for three consecutive 28-day cycles in a double-blind, randomised study: either NOMAC/E2 for 24 days with a four-day placebo interval (n=45) or LNG/EE for 21 days with a seven-day placebo interval (n=45) per cycle. Mean changes from baseline to end-of-treatment in coagulation markers, including prothrombin fragment 1+2 (primary endpoint), fibrinolysis markers and platelet functions were assessed. Mean prothrombin fragment 1+2 levels (primary endpoint) did not increase with NOMAC/E2 compared with LNG/EE ( -0.02 vs. +0.08 nM, p<0.01). Other significant differences between NOMAC/E2 and LNG/EE were mean changes in antithrombin (+0.3% vs. -4.4%, p<0.001), activated protein C resistance - normalised ratio (+0.20 vs. +0.46, p<0.01), D-dimer ( -53 vs. +43 ng/ml, p<0.001), plasminogen (+6% vs. +30%, p<0.0001) and plasminogen activator inhibitor-1 ( -3.1 vs. -8.0 ng/ml, p<0.001). There was no effect of either treatment on platelet aggregation. The NOMAC/E2 pill regimen has fewer adverse effects on blood biological coagulation and fibrinolysis markers than LNG/EE. This suggests that NOMAC/E2 could have a more favourable venous thromboembolism risk profile than LNG/EE; further epidemiological data are required to confirm this.

Detection and characterisation of large SERPINC1 deletions in type I inherited antithrombin deficiency.

Methods routinely used for investigating the molecular basis of antithrombin (AT) deficiency do not detect large SERPINC1 rearrangements. Between 2000 and 2008, 86 probands suspected of having AT-inherited type I deficiency were screened for SERPINC1 mutations in our laboratory. Mutations causally linked to the deficiency were identified by sequencing analysis in 63 probands. We present here results of multiplex ligation-dependent probe amplification (MLPA) analysis performed in 22 of the 23 remaining probands, in whom sequencing had revealed no mutation. Large deletions, present at the heterozygous state, were detected in 10 patients: whole gene deletions in 5 and partial deletions removing either exon 6 (n = 2), exons 1-2 (n = 1) or exons 5-7 (n = 2) in 5 others. Exon 6 partial deletions are a 2,769-bp deletion and a 1,892-bp deletion associated with a 10-bp insertion, both having 5' and/or 3' breakpoints located within Alu repeat elements. In addition, we identified the 5' breakpoint of a previously reported deletion of exons 1-2 within an extragenic Alu repeat. Distinct mutational mechanisms explaining these Alu sequence-related deletions are proposed. Overall, in this series, large deletions detected by MLPA explain almost half of otherwise unexplained type I AT-inherited deficiency cases.

Interleukin 8 is differently expressed and modulated by PAR-1 activation in early and late endothelial progenitor cells.

The proinflammatory chemokine interleukin 8 exerts potent angiogenic effects on endothelial cells by interacting with its receptors CXCR1 and CXCR2. As thrombin is also a potent inflammatory factor, and as endothelial progenitor cells (EPC) express functional PAR-1 thrombin receptor, we examined whether PAR-1 stimulation interferes with the IL-8 pathway in EPC. EPC were obtained from adult blood (AB) and cord blood (CB). The effect of PAR-1 stimulation by the peptide SFLLRN on IL-8, CXCR1 and CXCR2 expression was examined by RTQ-PCR and at the protein level in AB and CB late EPC and in AB early EPC. Specific siRNA was used to knock down PAR-1 expression. The IL-8 gene was expressed strongly in AB early EPC and moderately in late EPC. In contrast, CXCR1 and CXCR2 gene expression was restricted to AB early EPC. The IL-8 level in AB early EPC conditioned medium was high in basal conditions and did not change after PAR-1 activation. By contrast, IL-8 secretion by late EPC was low in basal conditions and strongly up-regulated upon PAR-1 activation. PAR-1 activation induced a number of genes involved in activating protein-1 (AP-1) and nuclear factor (NF)-kappaB pathways. Conditioned medium of PAR-1-activated late EPC enhanced the migratory potential of early EPC, and this effect was abrogated by blocking IL-8. Target-specific siRNA-induced PAR-1 knockdown, and fully inhibited PAR-1-induced IL-8 synthesis. In conclusion, PAR-1 activation induces IL-8 synthesis by late EPC. This could potentially enhance cooperation between late and early EPC during neovascularization, through a paracrine effect.

Bone morphogenetic proteins 2 and 4 are selectively expressed by late outgrowth endothelial progenitor cells and promote neoangiogenesis.

OBJECTIVE: Endothelial progenitor cells are currently identified either by their surface antigen expression or by their generation of early colonies in culture (CFU-Hill). Another population, endothelial colony-forming cells (ECFCs), has strong vessel-forming capacity but is less well characterized. Given the potential usefulness of CFU-Hill and ECFCs as cell therapy products, their thorough characterization is of major importance. METHODS AND RESULTS: CFU-Hill and ECFCs were expanded from human cord and adult blood. Bone morphogenetic proteins 2 and 4 (BMP2/4) were selectively expressed by ECFCs but not by CFU-Hill. The BMP pathway was involved in ECFC commitment and angiogenic potential in vitro. In vivo, BMP inhibition strongly reduced plug vascularization in bFGF-containing Matrigel plugs implanted in C57/Bl6 mice. Moreover, ECFC exposure to BMP increased their therapeutic potential in a nude mouse model of hindlimb ischemia. In amputation specimens from patients with critical leg ischemia who had received a local therapeutic injection of bone marrow mononuclear cells, newly formed vessels were strongly positive for BMP2/4, suggesting that endothelial cells involved in neovascularization have an ECFC-like phenotype. CONCLUSIONS: BMP2/4 are a marker of ECFCs and play a key role in ECFC commitment and outgrowth during neovascularization.

Thrombin bound to a fibrin clot confers angiogenic and haemostatic properties on endothelial progenitor cells.

Recent data suggest that endothelial progenitor cells (EPCs) are involved in recanalizing venous thrombi. We examined the impact of a fibrin network, and particularly of adsorbed thrombin, on EPCs derived from cord blood CD34(+) cells. Fibrin networks generated in microplates by adding CaCl(2) to platelet-depleted plasma retained adsorbed thrombin at the average concentration of 4.2 nM per well. EPCs expressed high levels of endothelial cell protein C receptor and thrombomodulin, allowing the generation of activated protein C on the fibrin matrix in the presence of exogenous human protein C. The fibrin matrix induced significant EPC proliferation and, when placed in the lower chamber of a Boyden device, strongly enhanced EPC migration. These effects were partly inhibited by hirudin by 41% and 66%, respectively), which suggests that fibrin-adsorbed thrombin interacts with EPCs via the thrombin receptor PAR-1. Finally, spontaneous lysis of the fibrin network, studied by measuring D-dimer release into the supernatant, was inhibited by EPCs but not by control mononuclear cells. Such an effect was associated with a 10-fold increase in plasminogen activator inhibitor-1 (PAI-1) secretion by EPCs cultivated in fibrin matrix. Overall, our data show that EPCs, in addition to their angiogenic potential, have both anticoagulant and antifibrinolytic properties. Thrombin may modulate these properties and contribute to thrombus recanalization by EPCs.

Bone marrow-derived mononuclear cell therapy induces distal angiogenesis after local injection in critical leg ischemia.

Critical leg ischemia is associated with a high risk of amputation when revascularization is not possible. Cell therapy based on bone marrow-derived mononuclear cells or with peripheral mononuclear cells, collected after stimulation with G-CSF has been used in an attempt to stimulate angiogenesis. Although several studies have raised the hope that such cell therapy may be effective in critical leg ischemia, no direct demonstration of angiogenesis induced by bone marrow-derived mononuclear cell/peripheral mononuclear cell injection has been reported in man. The aim of this study was to identify and to evaluate the extent of the angiogenic process associated with cell therapy in critical leg ischemia in man. To address this question, this pathological study was conducted in patients enrolled in the OPTIPEC clinical trial (Optimization of Progenitor Endothelial Cells in the Treatment of Critical leg ischemia), an interventional cell therapy study in critical leg ischemia. Amputation specimens from these patients were submitted to a standardized dissection protocol. In three patients, an active angiogenesis was observed in the distal part of the ischemic limb but not in the gastrocnemius muscle, the site of bone marrow cell injection. All the newly formed vessels were positive for endothelial cell markers (CD31, CD34, von Willebrand factor) and negative for markers of lymphatic vessels (podoplanin). Immunohistochemical staining for Ki-67 and c-kit showed extensive endothelial cell proliferation within the new vessels. Bone marrow-derived mononuclear cell therapy in patients with critical leg ischemia induces an active, substained angiogenesis in the ischemic and distal parts of the treated limb, although this may not prevent amputation in some patients with very severe ischemia.

Thrombomodulin gene polymorphisms in brain infarction and mortality after stroke.

BACKGROUND AND PURPOSE: Thrombomodulin is expressed at the surface of endothelial cells and controls thrombin generation and thrombin-induced platelets and vascular cell activation. Several thrombomodulin gene polymorphisms have been associated with coronary events and brain infarction. In a previous analysis from the Etude du Profil Génétique de l'Infarctus Cérébral (GENIC) study, we found that soluble thrombomodulin (sTM) concentration modulated the risk of and prognosis for brain infarction. METHODS: In 474 brain infarction cases and 483 controls from the GENIC study, we investigated the relationship between three thrombomodulin gene polymorphisms (-1748G/C, -1208/-1209delTT, +1418C/T) and sTM levels, brain infarction risk and 5-year mortality after stroke. RESULTS: The three polymorphisms were in linkage disequilibrium and defined three major haplotypes with no influence on sTM concentration (all P values > 0.16). Single locus and haplotype analyses found no significant association with brain infarction, even when the analysis was restricted to individuals without a vascular history. After 5 years of follow-up, we found no relationship with vascular or total mortality (all P values > 0.64). CONCLUSION: Our results suggest that these three thrombomodulin gene polymorphisms do not contribute to sTM level variations and are not associated with risk of brain infarction and mortality after stroke.

Purinergic receptor P2Y, G-protein coupled, 12 gene variants and risk of incident ischemic stroke, myocardial infarction, and venous thromboembolism.

Recent data have implicated a haplotype of the purinergic receptor P2Y, G-protein coupled, 12 gene (P2RY12), as potential risk determinant for atherothrombosis. However, to date, no prospective, genetic-epidemiological data are available. Using DNA samples collected at baseline in a prospective cohort of 14,916 initially healthy American men, we examined the possible association of P2RY12 genetic variants, in particular a haplotype H2 (constituted by dbSNP rs10935838, rs2046934, rs5853517, and rs6809699) amongst 708 white males who subsequently developed a thromboembolic event (incident myocardial infarction (MI), ischemic stroke, or deep venous thromboembolism/pulmonary embolism (DVT/PE)) and amongst an equal number of age- and smoking-matched white males who remained free of reported vascular disease during follow-up (controls). The P2RY12 gene variants tested were in linkage disequilibrium. The haplotype H2 distribution was significantly different between the DVT/PE cases (12%) and their matched controls (21%), p-permuted=0.02. In an adjusted conditional logistic regression analysis, the haplotype H2 was significantly associated with a lower risk of incident DVT/PE as compared to the reference haplotype H1 (odds ratio=0.50, 95% CI=0.27-0.93, p=0.028). However, we found no evidence for an association of the P2RY12 variants or the haplotype H2 with incident MI or ischemic stroke. The present investigation provides evidence for an association of the P2RY12 haplotype H2 with lower risk of DVT/PE; however these findings require replication in other well-designed studies.

Alternative mRNA is favored by the A3 haplotype of the EPCR gene PROCR and generates a novel soluble form of EPCR in plasma.

The endothelial cell protein C receptor also exists in soluble form in plasma (sEPCR), resulting from ADAM17 cleavage. Elevated sEPCR levels are observed in subjects carrying the A3 haplotype, which is characterized by a Ser219Gly substitution in the transmembrane domain, rendering the receptor more sensitive to cleavage. Because sEPCR production is not completely blocked by metalloprotease inhibition, we looked for another mechanism. Comparing mRNA expression patterns and levels in A3 and non-A3 cells from 32 human umbilical cord veins, we detected a truncated mRNA in addition to the full-length mRNA. This truncated mRNA was 16 times more abundant in A3 human umbilical vein endothelial cells than in non-A3 human umbilical vein endothelial cells and encoded a protein lacking the transmembrane domain. We stably expressed a recombinant form of this protein (rEPCRisoform) and a protein mimicking the plasma sEPCR (rEPCRsol). Functional studies of the purified recombinant proteins revealed that the rEPCRisoform bound to recombinant protein C with similar affinity than rEPCRsol and that it also inhibited the anticoagulant activity of APC. Trace amounts of the EPCR isoform were found in the plasma of A3 subjects. These results suggest that the sEPCRisoform could contribute to the regulatory effect of sEPCR in plasma.

Increased VEGFR2 expression during human late endothelial progenitor cells expansion enhances in vitro angiogenesis with up-regulation of integrin alpha(6).

In vitro expansion of late endothelial progenitor cells (EPCs) might yield a cell therapy product useful for myocardial and leg ischaemia, but the influence of EPC expansion on the angiogenic properties of these cells is unknown. In the present study, we investigated the effect of in vitro EPC expansion on vascular endothelial growth factor (VEGF) receptor expression. EPCs were obtained from CD34(+) cord blood cells and expanded for up to 5 weeks. Real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR) showed that VEGFR2 expression, contrary to VEGFR1 and VEGFR3 expression, was significantly higher on expanded EPCs than on freshly isolated CD34(+) cells or on human umbilical vein endothelial cells (HUVECs). Quantitative flow cytometry confirmed that VEGFR2 density on EPCs increased during the expansion process and was significantly higher than on HUVECs. The impact of VEGFR2 increase was studied on the three theoretical steps of angiogenesis, i.e., EPC proliferation, migration and differentiation. VEGFR2 up-regulation had no effect on VEGF-induced cell proliferation, but significantly enhanced EPC migration and pseudotubes formation dependent on integrin alpha(6) subunit overexpression. In vitro expansion of late EPCs increases the expression of VEGFR2, the main VEGF receptor, with possible implications for EPC-based angiogenic therapy.

A comparative study of the protein C pathway in septic and nonseptic patients with organ failure.

RATIONALE: Severe sepsis is associated with an exacerbated procoagulant state with protein C (PC) system impairment. In contrast, the inflammatory and coagulation status of nonseptic patients with organ failure (OF) is less documented. OBJECTIVES: To compare coagulation activation, focusing on the PC system, and inflammatory status in septic and nonseptic patients with OF. METHODS: Thirty patients with severe sepsis and 30 nonseptic patients were recruited at the onset of OF and compared with 30 matched healthy subjects. We performed an extensive analysis of the PC pathway, including plasma protein measurements and quantification of leukocyte expression of PC system receptors. In addition, we analyzed the inflammatory status, based on inflammation-related gene leukocyte expression. MEASUREMENTS AND MAIN RESULTS: We observed coagulation activation, reflected by a similar increase in tissue factor mRNA expression, in the two patient groups when compared with the healthy subjects. Soluble thrombomodulin levels were higher in septic patients than in healthy control subjects, whereas PC, protein S, and soluble endothelial cell PC receptor levels were lower. Similar results were obtained in nonseptic patients with OF. Monocyte thrombomodulin overexpression, together with increased circulating levels of activated PC, suggests that the capacity for PC activation is at least partly preserved in both settings. No difference in the inflammatory profile was found between septic and nonseptic patients. CONCLUSIONS: The pathogenesis of OF in critical care patients is characterized by an overwhelming systemic inflammatory response and by exacerbated coagulation activation, independently of whether or not infection is the triggering event. Clinical trial registered with www.clinicaltrials.gov (NCT 00361725).

Development of a novel immunoassay for the assessment of plasma Gas6 concentrations and their variation with hormonal status.

BACKGROUND: Gas6 is a vitamin K-dependent antiapoptotic protein that has been implicated in cardiovascular pathophysiology. We report the development and validation of an ELISA for Gas6, and the variation of plasma Gas6 with hormonal status in a study designed to evaluate the effect of oral contraception on plasma markers. METHODS: After validation of the main stages of the ELISA assay, we measured plasma Gas6 concentrations in 94 male and 88 female healthy volunteers ages 18 to 38 years. Forty-five of the women then received an oral contraceptive, which contained ethinylestradiol and levonorgestrel, for 3 months before a new measurement was performed at the same time point in their menstrual cycles. RESULTS: Interassay imprecision was 5.8%-11.8%, and the detection limit was 5.9 microg/L. Mean Gas6 plasma concentrations were significantly lower in men (52.0 microg/L) than in women not receiving oral contraceptives (63.8 microg/L, P <0.001). In the women who received oral contraceptives, Gas6 concentrations decreased after 3 months of therapy from 63.6 microg/L to 51.9 microg/L (P <0.001). CONCLUSIONS: We have developed a simple and reproducible ELISA assay for measuring plasma Gas6 concentrations, which vary with sex and are decreased by oral contraceptive use. These results suggest regulation of plasma Gas6 concentrations by sex hormones. Future clinical studies may require participants to be stratified by sex.

[Coagulation factor mutations and thrombosis]. / Mutations des protéines de la coagulation et thromboses.

The coagulation system is governed by a subtle balance between clotting activators and inhibitors. Many genes can contribute to the overall phenotype, and polymorphisms may act to up regulate or down regulate the generation of thrombin, the coagulation-key enzyme. An increase in coagulation factor (gain function) or/and a decrease in coagulation inhibitors (loss of function) may favor venous thromboembolism (VTE). It has been observed since a long time that VTE may be a familial disease, but it was only in 1965 that Egeberg published the first case of inherited antithrombin (AT) deficiency. This was followed by similar reports of protein C (PC) and protein S (PS) deficiencies. Hereditary thrombophilia was thus initially considered as a rare monogenic disorder with incomplete penetrance. AT, PC and PS deficiencies are due to multiple and mostly private mutations of the corresponding genes. Most patients are heterozygous and experience VTE at adult age. Homozygosity associated with severe thrombosis at birth has been observed in newborns with undetectable PC or PS concentrations. The discovery of factor (F) V Leiden and F2 g.20210 G>A, two gain of function mutations, challenged the view of thrombophilia as a rare monogenic disorder. FV Leiden and F2 g.20210 G>A are due to a founder effect and affect populations of European descent with frequencies at 5% and 3% respectively. These two mutations are moderate of risk factor for thrombosis and paved the way for gene-gene and gene-environment interactions. Patients carrying more than one genetic risk factor are at higher risk to develop VTE. The exposition to acquired risk factors such as estrogen based oral contraception may also have a synergistic effect favoring thrombosis in patients with FV Leiden or other genetic risk factors.

Identification of functional polymorphisms of the thromboxane A2 receptor gene in healthy volunteers.

Thromboxane A2 receptor (TP) is an important actor in vascular physiology and plays a crucial role in the platelet activation process. Genetic polymorphisms of the gene coding for the TP have been described, but their impacts on platelet function tests are unknown. The aim of this study was to investigate the relationship between genetic polymorphisms of the coding sequence of the TP gene and platelet function tests. We investigated 100 healthy volunteers twice, one week apart by performing platelet aggregation and secretion tests. We sequenced the coding region of the TP gene and confronted the genetic variants with the phenotypic results. We identified five single nucleotide polymorphisms (SNP); one of them, T1712C, replaces Leu by Pro at position 133 of the isoform beta of the TP. Homozygosity for the minor allele of the C795T, C924T or the G1686A SNP was associated with a decreased expression of CD62P when platelets were stimulated with the TP agonist U46619. As C795T and C924T have been linked to clinical disorders in which TxA2 plays a key role, the possible role of the G1686A and T1712C SNP should also be examined in selected diseases.

Cytochrome P450 2C19 loss-of-function polymorphism is a major determinant of clopidogrel responsiveness in healthy subjects.

The capacity of clopidogrel to inhibit ADP-induced platelet aggregation shows wide intersubject variability. To determine whether frequent functional variants of genes coding for candidate cytochrome P450 (CYP) isoenzymes involved in clopidogrel metabolic activation (CYP2C19*2, CYP2B6*5, CYP1A2*1F, and CYP3A5*3 variants) influence the platelet responsiveness to clopidogrel, we conducted a prospective pharmacogenetic study in 28 healthy white male volunteers treated for 7 days with clopidogrel 75 mg/d. We observed that pharmacodynamic response to clopidogrel was significantly associated with the CYP2C19 genotype. Twenty of the subjects were wild-type CYP2C19 (*1/*1) homozygotes, while the other 8 subjects were heterozygous for the loss-of-function polymorphism CYP2C19*2 (*1/*2). Baseline platelet activity was not influenced by the CYP2C19 genotype. In contrast, platelet aggregation in the presence of 10 muM ADP decreased gradually during treatment with clopidogrel 75 mg once daily in *1/*1 subjects, reaching 48.9% +/- 14.9% on day 7 (P < .001 vs baseline), whereas it did not change in *1/*2 subjects (71.8% +/- 14.6% on day 7, P = .22 vs baseline, and P < .003 vs *1/*1 subjects). Similar results were found with VASP phosphorylation. The CYP2C19*2 loss-of-function allele is associated with a marked decrease in platelet responsiveness to clopidogrel in young healthy male volunteers and may therefore be an important genetic contributor to clopidogrel resistance in the clinical setting.