Martine Aiach

Effects of Oral and Transdermal Estrogen/Progesterone Regimens on Blood Coagulation and Fibrinolysis in Postmenopausal Women A Randomized Controlled Trial

Postmenopausal hormone replacement therapy is associated with a reduction in the incidence of coronary heart disease. However, inconclusive results have been reported with respect to the risk of stroke, and recent studies consistently showed an increased risk of venous thromboembolism in postmenopausal women using oral estrogen. There are surprisingly few interventional studies to assess the true effects of estrogen-progestin regimens on blood coagulation and fibrinolysis, and the impact of the route of estrogen administration on hemostasis has not been well documented. Therefore, we investigated the effects of oral and transdermal estradiol/progesterone replacement therapy on hemostatic variables. Forty-five healthy postmenopausal women, aged 45 to 64 years, were assigned randomly to one of the three following groups: cyclic oral or transdermal estradiol, both combined with progesterone, or no hormonal treatment. Hemostatic variables were assayed at baseline and after a 6-month period. Pairwise differences in the mean change between the three groups were compared using nonparametric tests. Oral but not transdermal estradiol regimen significantly increased the mean value of prothrombin activation peptide (F1 + 2) and decreased mean antithrombin activity compared with no treatment. Differences in fragment F1 + 2 levels between active treatments were significant. The oral estrogen group was associated with a significant decrease in both mean tissue-type plasminogen (t-PA) concentration and plasminogen activator inhibitor (PAI-1) activity and a significant rise in global fibrinolytic capacity (GFC) compared with the two other groups. A transdermal estrogen regimen had no significant effect on PAI-1, t-PA, and GFC levels. There were no significant changes in mean values of fibrinogen, factor VII, von Willebrand factor, protein C, fibrin D-dimer, and plasminogen between and within the three groups. We conclude that oral estrogen/progesterone replacement therapy may result in coagulation activation and increased fibrinolytic potential, whereas opposed transdermal estrogen appears without any substantial effects on hemostasis. Whereas these results may account for an increased risk of venous thromboembolism in users of oral postmenopausal estrogen, they emphasize the potential importance of the route of estrogen administration in prescribing hormone replacement therapy to postmenopausal women, especially to those at high risk of thrombotic disease.

Interdonor variability of platelet response to thrombin receptor activation : influence of PlA2 polymorphism

Considering that platelet response to thrombin receptor activation might be critical for the development of arterial thrombosis, we measured the dense granule release under stimulation by the thrombin receptor activating peptide (TRAP) in a series of 102 healthy volunteers. The threshold TRAP concentration which initiated a secretion ranged from 3 to 20u2003μm. A good concordance (79%, ku2003=u20030.677) between two tests performed at a 1 month interval indicated that platelet response to thrombin receptor activation was characteristic of each individual donor. Since the threshold concentration required to initiate secretion corresponded to the threshold concentration which induced a biphasic aggregation, all volunteers were genotyped for the PlA2 polymorphism, the Pro33 variant of GPIIIa. Platelets from subjects with the PlA2 polymorphism required higher TRAP concentrations to aggregate than those from subjects with no PlA2 allele (Pu2003=u20030.0012). However, they also required a higher ADP concentration to aggregate. In order to exclude any influence of GPIIIa polymorphism on TRAP‐induced secretion, we studied the variability of platelet response to TRAP among the 77 individuals with no PlA2 allele, and found the same interdonor variability with the same distribution of threshold TRAP concentrations as for the 102 individuals. The results suggest that (i) platelet secretion in response to thrombin receptor activation could be a genetically controlled phenotype independent of the GPIIIa polymorphism; (ii) the PlA2 polymorphism is associated with platelet hypoaggregability.

Impact of progestagens on activated protein C (APC) resistance among users of oral contraceptives

Summary.u2002 Oral contraceptive (OC) use is associated with an increased risk of venous thromboembolism. Previous data reported higher thrombotic risk in women using third‐generation combined OC than in those using second generation OC. The difference could be explained by differential effects of progestagens on plasma sensitivity to activated protein C (APC). The main purpose of this cross‐sectional study was to assess the influence of a progestagen‐only OC (chlormadinone acetate) as well as the effect of several combined OC with different progestagen components on APC resistance. The effect of APC on endogenous thrombin potential (ETP) was investigated in the plasma of healthy women using either combined OC (nu2003=u200382) or progestagen‐only OC (nu2003=u200328), and in non‐users (nu2003=u200364). Carriers of factor V Leiden were excluded. Compared with non‐users, there was no significant change in APC resistance in women using progestagen‐only OC. Women who used combined OC were less sensitive to APC than non‐users (Pu2003<u20030.001) and the difference was significantly more pronounced in women using third‐generation OC (nu2003=u200341) than in those who used second‐generation OC containing levonorgestrel (nu2003=u200322) (Pu2003<u20030.05). Compared with OC containing levonorgestrel, use of norethisterone‐containing OC (nu2003=u20039) was associated with an increased resistance to APC (Pu2003<u20030.05). Women who used cyproterone‐containing OC (nu2003=u200310) were less sensitive to APC than those using third‐generation OC (Pu2003<u20030.05) or second‐generation OC containing levonorgestrel (Pu2003<u20030.05). Protein S, factor II and FVIII levels explained in part the OC‐related changes in APC sensitivity variations. ETP‐based APC resistance may contribute to explain why different brands of OC can be associated with different levels of thrombogenicity.

Endothelial progenitor cells: Characterization, in vitro expansion, and prospects for autologous cell therapy

Injection of hematopoietic stem cells or endothelial progenitor cells (EPCs) expanded ex vivo has been shown to augment neovascularization in adult patients, but the precise origin and identity of the cell population responsible for these clinical benefits are controversial. The limited quantity of EPCs in the circulation has been the main obstacle to clinical trials. Several authors have therefore attempted to expand these cells ex vivo in order to obtain a homogeneous cell therapy product. One possible means of expanding EPCs ex vivo is to activate the thrombin receptor PAR-1 with the specific peptide SFLLRN. Indeed, PAR-1 activation promotes cell proliferation and C-X-C chemokine receptor type 4 (CXCR4) dependent migration and differentiation, with an overall angiogenic effect. This review summarizes the results and rationale of clinical trials of angiogenic therapy, the nature of EPCs, the different methods of ex vivo expansion, and current methods of quantification.

Mutations in Promoter Region of Thrombomodulin and Venous Thromboembolic Disease

The present study was designed to analyze the thrombomodulin proximal promoter region spanning nucleotides -293 to -12 to search for polymorphisms that could modify thrombomodulin gene expression in patients with venous thromboembolic disease. The study population comprised 205 patients and 394 healthy subjects of similar age and sex distribution. No polymorphisms and only 1 point mutation (G-33A) were found. The G-33A mutation was present at the heterozygous state in 2 patients and in 1 control. Being more frequent in the patients (0.97%) than in the controls (0.25%), the G-33A mutation might be a risk factor for venous thrombosis. To investigate the effect of this mutation on the thrombomodulin promoter activity, the proximal promoter region of the gene (bearing or not bearing the G-33A mutation) was inserted into a promotorless expression vector, upstream of the firefly luciferase gene, and transiently transfected into EA.hy926 endothelial cells. Under the conditions of the assay, the G-33A mutation mildly decreased the promoter activity. This study confirms that abnormalities of the thrombomodulin proximal promoter are not frequent in patients with venous thromboembolism.

Thrombomodulin Promoter Mutations, Venous Thrombosis, and Varicose Veins

Abstract —We analyzed the distal promoter region of the thrombomodulin (TM) gene (nucleotides −300 to −2052) in subjects from the Paris Thrombosis Study (PATHROS), a French case-control study of venous thrombosis, to identify polymorphisms that might modify TM gene expression. Eight novel mutations were found in the 40 DNA samples initially screened. Two of these mutations (−1748G/C and −1208/−1209 del TT) were frequent. One rare transition (−1166G/A) might have functional consequences owing to its position. These 3 mutations were screened for in the entire study population of 327 patients and 398 controls. None of the 3 was significantly associated with thrombosis. Interestingly, the −1208/−1209 TT deletion was associated with varicose veins in the patients. This mutation was in tight linkage disequilibrium with the +1418 C/T change in the coding sequence, a known polymorphism that predicts an Ala 455 Val substitution in the sixth epidermal growth factor–like TM module, a domain previously implicated in the proliferative functions of TM. This linkage suggests that the Ala 455 Val mutation may promote changes in these functions and thus be involved in varicose vein formation.

Haemostatic effects of a new combined oral contraceptive, nomegestrol acetate/17β-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 17β-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.

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 CaCl2 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.

Repeated Fetal Losses and the Lupus Anticoagulant

Excerpt To the Editor:Combined therapy with prednisone and low doses of aspirin has been reported to reduce the risk for fetal loss in women with the lupus anticoagulant (1, 2). We report a case in...

Role of the P2Y12 gene polymorphism in platelet responsiveness to clopidogrel in healthy subjects

Clopidogrel is an inhibitor of platelet function. Its active metabolite irreversibly blocks the platelet ADP receptor P2Y12 [1] and prevents ischemic events in patients with atherosclerosis [2]. The antiplatelet effect of clopidogrel shows wide interindividual variability and in 5–30% of patients the recommended dose of 75 mg day fails to inhibit platelet aggregation ex vivo [3–5]. We examined the role of the gain-of-function polymorphism (H2 allele) of the P2Y12 gene [6], which encodes the clopidogrel target receptor, in the extent of clopidogrel pharmacodynamic response. To investigate the possible influence of the H2 allele free from confounding factors, such as medications or cardiovascular diseases, we evaluated clopidogrel responsiveness in young healthy subjects during a 1-week oral course of clopidogrel 75 mg day. Among 225 Caucasian men aged 18–35 years screened for the H1H2 genotype [7], we consecutively included 29 healthy volunteers (14 H1H1, 11 H1H2, and 4 H2H2). The study was approved by the Paris-Cochin ethics committee and all the subjects gave their written informed consent. The volunteers were asked to come to the Clinical Investigations Center each day at 09:00 hours where they were given a standard dose of clopidogrel 75 mg once daily for 7 days in the presence of medical staff. Blood samples for pharmacodynamic studies were drawn just before the first clopidogrel intake on days 1, 2, 3, 4, 5, and 7, and on days 9, 10, 11, and 14. Platelet response to 10 lmol L ADP was evaluated with platelet aggregation and vasodilator-stimulated phosphoprotein (VASP) phosphorylation, which is specific for P2Y12 inhibition. Aggregation studies were performed at baseline, on treatment, and after treatment completion at 37 C using a photometric method on a four-channel aggregometer (Regulest, Amneville, France). ADP-induced aggregation (10 lmol L final concentration, Sigma-Aldrich, St-Quentin Fallavier, France) was reported as maximal aggregation or as a percentage of baseline aggregation. VASP was measured in whole blood at baseline, on day 4 and on day 7 of clopidogrel intake using a flow cytometric assay (Platelet VASP , Diagnostica Stago, Biocytex, Marseille, France) adapted to a Cy-Flow apparatus (Partec GmbH, SteGeneviève-des-Bois, France). Results were expressed as a platelet reactivity index (PRI, %) [8]. All experiments were performed blindly to the P2Y12 genotype. The effect of the genotype on pharmacodynamic parameters was assessed at each time point by the Kruskal–Wallis test. When this test was significant (P < 0.05), paired comparisons of the three genotype groups were made usingWilcoxon’s test. Clopidogrel responsiveness was also evaluated in each genotype group across time using Friedman’s test. At baseline, ADP-induced platelet aggregation did not vary with the genotype: median values were 79.5% [interquartile range (IQR) 75.0; 81.0] in H1H1 subjects; 76.0% (IQR 72.0; 78.0) in H1H2 subjects; and 79.0% (IQR 76.5; 81.0) in H2H2 subjects (P 1⁄4 0.25). The aggregation fell significantly between days 1 and 7 of clopidogrel intake in the whole population [median at day 7 (IQR): 54% (45.5; 65.5), P < 0.0001], but the response did not differ between H1H1 subjects and H1H2 + H2H2 subjects (P 1⁄4 0.58). However, while median platelet aggregation significantly fell in H1H1 [54% (46; 68)] and H1H2 [49% (29; 59)] groups on day 7 of clopidogrel intake, no significant fall occurred in the H2H2 subjects [76% (65; 82), P < 0.05 vs. H1H1 and H1H2 groups] (Fig. 1). The inhibition of platelet aggregation relative to baseline was 7% (3; 9) in H2H2 subjects, 28% (15; 38) in H1H2 subjects, and 23% (11; 32) in H1H1 subjects (P 1⁄4 0.01 vs. H2H2 subjects). Interestingly, when individual responses to the 7-day oral course of clopidogrel were classified in quartiles of relative reduction in ADP-induced platelet aggregation from baseline (first quartile 0–13.8%; second quartile 13.9–30.8%; third quartile 30.9–42.2%; fourth quartile> 42.3%), the fourH2H2 homozygotes were situated in the first quartile, which may define poor responders although this was also the case in three of the H1H1 subjects. At day 7, the PRI (VASP phosphorylation assay) fell significantly after clopidogrel intake in the H1H1 group [PRI baseline: 76.9% (IQR 63.3; 89.4); PRI day 7: 46.3% (5.6; 67.5), P 1⁄4 0.0015] and in the H1H2 group [PRI baseline: 69.7% (58.1; 75.8); PRI day 7: 42.9% (16; 66.1), P 1⁄4 0.0051] with no significant difference between the H1H1 and H1H2 + H2H2 groups. However, the median fall in the PRI did not reach statistical significance in the H2H2 Correspondence: Pascale Gaussem, INSERM U 765, Service d’Hématologie Biologique A, Hôpital Européen Georges Pompidou, 20 rue Leblanc, 75908 Paris Cedex 15, France. Tel.: +33 1 56 09 39 36; fax: +33 1 56 09 39 13; e-mail: pascale. gaussem@egp.aphp.fr