Comparison of endothelial biomarkers according to reversibility of pulmonary hypertension secondary to congenital heart disease.

The reversibility of pulmonary arterial hypertension (PAH) in children with congenital heart disease (CHD) is strongly associated with the degree of intimal proliferation, vessel narrowing, and number of circulating endothelial cells (CECs). Circulating endothelial cells may arise from either endothelial damage or accelerated turnover during vessel remodeling, but nothing is known about endothelial microparticles (EMPs) and other biomarkers reflecting endothelial alterations. This study aimed to document endothelial markers further according to the irreversibility of PAH secondary to CHD. The study investigated soluble markers of endothelial damage or activation (thrombomodulin, soluble endothelial protein C receptor, and soluble E-selectin), inflammation (interleukin-6), and angiogenic cytokine levels [vascular endothelial growth factor (VEGF) and placental growth factor (PlGF)] in 26 patients with CHD, 16 with reversible PAH (median age, 2 years) and 10 with irreversible PAH (median age, 9 years). Endothelial activation/apoptosis was evaluated by measuring EMP levels. Plasma procoagulant activity also was measured. The results show that the levels of soluble markers indicating endothelial activation were not predictors of PAH irreversibility. Lower levels of PlGF were observed in reversible compared with irreversible PAH but were not associated with the CEC level, the mean pulmonary artery pressure (mPAP), or age. No significant difference in procoagulant activity or EMP level was found between irreversible and reversible PAH. Among a large panel of biomarkers reflecting endothelial activation, regeneration, and injury, the high CEC levels previously described proved to be the only marker allowing discrimination between reversible and irreversible PAH secondary to CHD.

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.

The TF-603A/G gene promoter polymorphism and circulating monocyte tissue factor gene expression in healthy volunteers.

Three single nucleotide polymorphisms (-603A/G, -1322C/T, -1812C/T) and one deletion/insertion polymorphism (-1208D/I) are present in the tissue factor (TF) gene promoter sequence. These polymorphisms are in complete linkage disequilibrium, determining two haplotypes with almost equal frequency. The -603A/-1208D/-1322C/-1812C haplotype, presently defined as TF-603A, has been linked to venous thromboembolic disease, with a potentially protecting effect. The effects of the TF-603A/G gene polymorphism on monocyte gene expression and on a whole-blood clotting time (WBCT) are not known. We determined the WBCT in basal conditions (H0) and after 4 hours of LPS stimulation ex vivo (H4LPS) on blood samples from 100 young healthy caucasian male subjects on 2 visits, 7 days apart. Monocyte TF mRNA was quantified at H0 and H4LPS by real-time quantitative reverse-transcription PCR. The monocyte TF mRNA values determined at the first and second visits were concordant. In H4LPS samples, TF mRNA levels were increased 70-fold. The TF-603A haplotype was associated with 40%-lower TF mRNA levels at H0 (P=0.0002) and this association followed the same trend but was no longer significant at H4LPS. At H4LPS, TF mRNA levels were associated with WBCT shortening (P=0.0003). WBCT at H0 was not concordant over time, precluding any genotype-phenotype analysis. WBCT at H4LPS was concordant over time but was not related to the TF-603A/G polymorphism. The TF-603A/G gene promoter polymorphism thus significantly influences constitutive TF gene expression in human monocytes but has no major effect on TF gene expression or on WBCT in LPS stimulated conditions.

Impact of aspirin and clopidogrel interruption on platelet function in patients undergoing major vascular surgery.

AIMS: To investigate functional platelet recovery after preoperative withdrawal of aspirin and clopidogrel and platelet function 5 days after treatment resumption. METHODS/RESULTS: We conducted an observational study, which prospectively included consecutive patients taking aspirin, taking clopidogrel, and untreated controls (15 patients in each group). The antiplatelet drugs were withdrawn five days before surgery (baseline) and were reintroduced two days after surgery. Platelet function was evaluated by optical aggregation in the presence of collagen, arachidonic acid (aspirin) and ADP (clopidogrel) and by VASP assay (clopidogrel). Platelet-leukocyte complex (PLC) level was quantified at each time-point. At baseline, platelet function was efficiently inhibited by aspirin and had recovered fully in most patients 5 days after drug withdrawal. PLC levels five days after aspirin reintroduction were similar to baseline (+4±10%; p = 0.16), in line with an effective platelet inhibition. Chronic clopidogrel treatment was associated with variable platelet inhibition and its withdrawal led to variable functional recovery. PLC levels were significantly increased five days after clopidogrel reintroduction (+10±15%; p = 0.02), compared to baseline. CONCLUSIONS: Aspirin withdrawal 5 days before high-bleeding-risk procedures was associated with functional platelet recovery, and its reintroduction two days after surgery restored antiplaletet efficacy five days later. This was not the case of clopidogrel, and further work is therefore needed to define its optimal perioperative management.

Effects of rabeprazole on the antiplatelet effects and pharmacokinetics of clopidogrel in healthy volunteers.

BACKGROUND: Several studies have suggested that proton-pump inhibitors (PPIs), mostly omeprazole, interact with clopidogrel efficacy by inhibiting the formation of its active metabolite via CYP2C19 inhibition. Whether this occurs with all PPIs is a matter of debate. As rabeprazole is a less potent CYP2C19 inhibitor than other PPIs, we studied the interaction between rabeprazole and the antiplatelet actions and pharmacokinetics of clopidogrel. AIM: To demonstrate the non-inferiority of rabeprazole over placebo using change in platelet reactivity index (PRI; vasodilator-stimulated phosphoprotein [VASP] assay) in a predefined population of good clopidogrel responders. Omeprazole was used as the positive control. METHODS: In this randomized three-period crossover study in healthy volunteers, 36 healthy men received clopidogrel (75 mg/day for 7 days) with placebo, omeprazole (20mg/day) or rabeprazole (20mg/day). Clopidogrel antiplatelet effects and disposition kinetics were assessed on day 7 of combination therapy. Non-inferiority threshold was predefined as an upper limit of the 90% confidence interval for the difference in change in PRI between placebo and rabeprazole of<10% in good clopidogrel responders. RESULTS: In good clopidogrel responders (inhibition of VASP index>30%), the clopidogrel antiplatelet effect remained non-inferior to placebo during rabeprazole (difference 3.4% [-1.7; 8.5]) but not omeprazole (difference 7.5% [2.5; 12.6]) co-administration. The AUC0-24 and Cmax of active clopidogrel metabolite decreased with both omeprazole and rabeprazole, and conditions of bioequivalence were not met, except for AUC0-24 with rabeprazole. CONCLUSIONS: Rabeprazole does not interact with clopidogrel to the same extent as omeprazole. However, under our experimental conditions and proton-pump inhibitor doses, there was no significant pharmacodynamic interaction between rabeprazole or omeprazole and clopidogrel, despite a significant decrease in the formation of clopidogrel active metabolite.

Effect of clopidogrel on circulating biomarkers of angiogenesis and endothelial activation.

Angiogenic cytokines have been shown to influence vessel injury, and platelets represent a disposable circulating pool of angiogenic molecules. In the present study, objectives were to determine whether clopidogrel could have a potential effect on levels of circulating biomarkers of angiogenesis and endothelial activation. We explored 28 healthy white male volunteers treated for 7 days with clopidogrel 75 mg/day. We quantified angiogenic growth factors that have been shown to be correlated to cardiovascular risk or endothelial progenitor cell mobilization such as vascular endothelial growth factor (VEGF)-A and its soluble receptor forms VEGFR1 and VEGFR2, placenta growth factor, and stromal cell-derived factor-1. We also quantified soluble E-selectin and von Willebrand factor to evaluate endothelial activation. Blood samples were drawn just before the first clopidogrel intake on day 1, and after the last dosing (day 7). As expected, we observed a decrease in platelet reactivity in response to clopidogrel, confirmed by vasodilator-stimulated phosphoprotein phosphorylation assay. However, the 7-day intake of clopidogrel did not significantly modify the levels of the selected angiogenic factors or biomarkers of endothelial activation. These results show that circulating angiogenic factor level in healthy subjects is not driven by P2Y12 platelet receptor-induced activation and clopidogrel does not modify in a significant way the endothelial activation level.

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.

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.

Elevated growth-arrest-specific protein 6 plasma levels in patients with severe sepsis.

OBJECTIVE: Growth-arrest-specific protein 6 (Gas6), an intracellular protein released by apoptotic cells, has been detected in normal plasma. As the Gas6 system has been implicated in mouse susceptibility to sepsis, and as leukocyte apoptosis is thought to play a major role in the physiopathology of human severe sepsis, we studied Gas6 plasma levels and possibly related variables in patients with severe sepsis. DESIGN: Matched case-control study. SETTING: Adult intensive care unit in a university hospital. PATIENTS: Thirty patients with severe sepsis, 30 patients with organ failure not related to infection, and 30 healthy subjects matched for age and gender. INTERVENTIONS: Blood draw. MEASUREMENTS AND MAIN RESULTS: Gas6 plasma levels were quantified using enzyme-linked immunosorbent assay. Whole-blood gas6 messenger RNA levels were measured by quantitative real-time polymerase chain reaction. Gas6 plasma levels were elevated (110 ng/mL [75, 139]; median values [interquartile range]) in severe sepsis patients compared with organ failure patients (85 ng/mL [56, 101]) and healthy subjects (54 ng/mL [49, 68]). In patients with severe sepsis, this increase correlated with the Acute Physiology and Chronic Health Evaluation II severity score, the organ failure Organ Dysfunction and Infection (ODIN) score, and the existence of a septic shock. Gas6 messenger RNA levels were increased in patients with severe sepsis and correlated specifically with the monocyte count. CONCLUSIONS: In severe sepsis, the recently described anti-apoptotic protein Gas6 was found at high levels in plasma and correlated well with the degree of organ dysfunction.