Véronique Remones

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.

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.

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.

LETTER TO THE EDITOR: P2Y1 gene polymorphism and ADP-induced platelet response

1 Mannucci PM, Duga S, Peyvandi F. Recessively inherited coagulation disorders. Blood 2004; 104: 1243–52. 2 Maghzal GJ, Brennan SO, Homer VM, George PM. The molecular mechanisms of congenital hypofibrinogenaemia. Cell Mol Life Sci 2004; 61: 1427–38. 3 Neerman-Arbez M. The molecular basis of inherited afibrinogenaemia. Thromb Haemost 2001; 86: 154–63. 4 Hanss MM, Ffrench PO, Mornex JF, Chabuet M, Biot F, De Mazancourt P, Dechavanne M. Two novel fibrinogen variants found in patients with pulmonary embolism and their families. J Thromb Haemost 2003; 1: 1251–7. 5 Homer VM, Brennan SO, Ockelford P, George PM. Novel fibrinogen truncation with deletion of Bbeta chain residues 440–461 causes hypofibrinogenaemia. Thromb Haemost 2002; 88: 427–31. 6 Zhang JZ, Redman CM. Identification of Bbeta chain domains involved in human fibrinogen assembly. J Biol Chem 1992; 267: 21727– 32. 7 Brennan SO, Maghzal G, Shneider BL, Gordon R, Magid MS, George PM. Novel fibrinogen gamma 375 Arg fi Trp mutation (fibrinogen Aguadilla) causes hepatic endoplasmic reticulum storage and hypofibrinogenemia. Hepatology 2002; 36: 652–8. 8 Huang S, Mulvihill ER, Farrell DH, Chung DW, Davie EW. Biosynthesis of human fibrinogen. Subunit interactions and potential intermediates in the assembly. J Biol Chem 1993; 268: 8919–26. 9 Okumura N, Terasawa F, Yonekawa O, Hamada E, Kaneko H. Hypofibrinogenemia associated with a heterozygous C>T nucleotide substitution at position )1138 bp of the 5¢flanking region of the fibrinogen Aa-chain gene. Ann NY Acad Sci 2001; 936: 526–30. 10 Brennan SO, Hammonds B, George PM. Aberrant hepatic processing causes removal of activation peptide and primary polymerisation site from fibrinogen Canterbury (A alpha 20 Val fi Asp). J Clin Invest 1995; 96: 2854–8. 11 Farrell DH, Huang S, Davie EW. Processing of the carboxyl 15-aminoacid extension in the alpha-chain of fibrinogen. J Biol Chem 1993; 268: 10351–5.

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.

P2Y1 gene polymorphism and ADP-induced platelet response: Letter to the Editor

1 Mannucci PM, Duga S, Peyvandi F. Recessively inherited coagulation disorders. Blood 2004; 104: 1243–52. 2 Maghzal GJ, Brennan SO, Homer VM, George PM. The molecular mechanisms of congenital hypofibrinogenaemia. Cell Mol Life Sci 2004; 61: 1427–38. 3 Neerman-Arbez M. The molecular basis of inherited afibrinogenaemia. Thromb Haemost 2001; 86: 154–63. 4 Hanss MM, Ffrench PO, Mornex JF, Chabuet M, Biot F, De Mazancourt P, Dechavanne M. Two novel fibrinogen variants found in patients with pulmonary embolism and their families. J Thromb Haemost 2003; 1: 1251–7. 5 Homer VM, Brennan SO, Ockelford P, George PM. Novel fibrinogen truncation with deletion of Bbeta chain residues 440–461 causes hypofibrinogenaemia. Thromb Haemost 2002; 88: 427–31. 6 Zhang JZ, Redman CM. Identification of Bbeta chain domains involved in human fibrinogen assembly. J Biol Chem 1992; 267: 21727– 32. 7 Brennan SO, Maghzal G, Shneider BL, Gordon R, Magid MS, George PM. Novel fibrinogen gamma 375 Arg fi Trp mutation (fibrinogen Aguadilla) causes hepatic endoplasmic reticulum storage and hypofibrinogenemia. Hepatology 2002; 36: 652–8. 8 Huang S, Mulvihill ER, Farrell DH, Chung DW, Davie EW. Biosynthesis of human fibrinogen. Subunit interactions and potential intermediates in the assembly. J Biol Chem 1993; 268: 8919–26. 9 Okumura N, Terasawa F, Yonekawa O, Hamada E, Kaneko H. Hypofibrinogenemia associated with a heterozygous C>T nucleotide substitution at position )1138 bp of the 5¢flanking region of the fibrinogen Aa-chain gene. Ann NY Acad Sci 2001; 936: 526–30. 10 Brennan SO, Hammonds B, George PM. Aberrant hepatic processing causes removal of activation peptide and primary polymerisation site from fibrinogen Canterbury (A alpha 20 Val fi Asp). J Clin Invest 1995; 96: 2854–8. 11 Farrell DH, Huang S, Davie EW. Processing of the carboxyl 15-aminoacid extension in the alpha-chain of fibrinogen. J Biol Chem 1993; 268: 10351–5.