Heparanase procoagulant activity is elevated in women using oral contraceptives

Human Reproduction, Vol.28, No.9 pp. 2372 –2380, 2013 Advanced Access publication on June 25, 2013 doi:10.1093/humrep/det257 ORIGINAL ARTICLE Fertility control Heparanase procoagulant activity is elevated in women using oral contraceptives Moshe Matan 1, Elena Axelman 2, Benjamin Brenner 2, and Yona Nadir 2,* 1 Faculty of Medicine, The Bruce Rappaport Faculty of Medicine, Technion, Haifa, Israel 2Thrombosis and Hemostasis Unit, Department of Hematology, Rambam Health Care Campus, Haifa, Israel *Correspondence address. Tel: +972-4-8453520; Fax: +972-4-8543886; E-mail: study question: What is the effect of estrogen on heparanase procogulant activity? summary answer: Estrogen increases heparanase procoagulant activity. what is known already: Estrogen therapy increases the risk of thrombosis and was previously found to up-regulate heparanase expression. Heparanase is involved in angiogenesis and metastasis, and has been shown to form a complex with tissue factor (TF) and also shown to enhance the generation of factor Xa. study design, size, duration: A case –control study. Thirty-four healthy women using oral contraceptives (OC) and 41 women not using hormonal therapy and not pregnant per history were enrolled, over a 5-month period, at the Rambam Medical Center, Haifa, Israel. In vitro, estrogen receptor-positive (MCF-7) and -negative (MDA-231) cell lines were incubated with estrogen, tamoxifen and ICI-182.780 a pure estrogen receptor antagonist. The cell medium was evaluated for TF/heparanase complex activity, TF activity and heparanase procoagulant activity by chromogenic substrate. participants/materials, setting, methods: Exclusion criteria included age ,18 years, post-menopausal women, concomitant medications other than supplement minerals and vitamins, acute or chronic illness. main results and the role of chance: The study demonstrates increased risk of high heparanase procoagulant activity in OC users. When a cutoff level of 0.25 (absorbance 405 –490 nm) was set, the odds ratio was 131 (P , 0.0001). When all results were studied by quartiles, in quartiles 3 and 4 the results were almost exclusively of the OC users (P , 0.0001). In cell cultures, estrogen and tamoxifen increased heparanase procoagulant activity in the medium of estrogen receptor-positive (MCF-7) cells. limitations, reasons for caution: The main limitation of the current study is that the two estrogens given to the women and cell cultures, ethinyl estradiol (EE) and 17-b-estradiol (E2), respectively, may have different effects on the coagulation system, although an increase in heparanase procoagulant activity was demonstrated in both of them. Although the sample size of the study group was limited, significant differences in the activation of the extrinsic coagulation pathway were demonstrated. wider implications of the findings: The clinical relevance of the heparanase procoagulant activity assay as a screening tool in thrombophilia work-up should further be elucidated. study funding/competing interest(s): No external funding was sought for this study. Authors Nadir and Brenner are named in a US Provisional Patent Application No. 29509/WO/12 filed on 18.01.2012. The other authors have no conflict of interest to declare. trial registration number: N/A. Key words: heparanase / coagulation / oral contraceptives / estrogen / tamoxifen Introduction The use of oral contraceptives (OC) is a well-established risk factor for venous thrombosis. Evidence on hormonal contraceptive is derived almost exclusively from observational studies and points to a 2–6-fold increased relative risk of venous thromboembolism (Deitcher and Gomes, 2004). Acquired protein C resistance resulting from reduced levels of protein C, protein S and elevated factor VIII is the main & The Author 2013. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: Submitted on September 12, 2012; resubmitted on March 31, 2013; accepted on May 23, 2013 2373 Heparanase activity in oral contraceptives users Materials and Methods Study group The study was approved by the institutional Ethic Committee on human research at the Rambam Medical Center. Thirty-four healthy women using OC and 41 women not using hormonal therapy and not pregnant per history were enrolled, over a 5-month period, at the Rambam Medical Center. Exclusion criteria included age ,18 years, post-menopausal women, concomitant medications other than supplement minerals and vitamins, acute or chronic illness. After obtaining informed consent, a total of 6 ml of peripheral blood was collected, with 3.2% sodium citrate as an anticoagulant. Plasma was obtained by centrifugation (1500g for 15 min at 48C) and all plasma samples were frozen and thawed once. Cell culture MCF-7 and MDA-MB-231 human breast carcinoma cells were obtained from the American Type Culture Collection. Cells were routinely maintained in Dulbecco’s modified Eagle’s medium (MDA-MB-231 cells) or RPMI 1640 (MCF-7 cells) medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Invitrogen), at 378C. In order to avoid estrogen preinduction or addition, before estrogen treatment, cells were maintained for 96 h in phenol red-free medium supplemented with charcoal-stripped fetal bovine serum (HyClone, Logan, UT, USA). Then, medium was changed to phenol red-free serum-free medium and estrogen with or without tamoxifen (a partial estrogen receptor antagonist) or ICI-182.780 (a pure estrogen receptor antagonist) were added. Estrogen (17-b-estradiol, E2) and 4-hydroxy-tamoxifen were obtained from Sigma (St. Louis, MO) and dissolved in absolute ethanol. ICI-182.780 was obtained from Sigma (St. Louis, MO) and dissolved in dimethylsulfoxide (DMSO). Control cultures were treated with the corresponding vehicle (0.1% ethanol or DMSO). Medium was collected after 16 h and refrigerated at 2868C. Reagents and antibodies Single-chain GS3 heparanase gene construct, comprising the 8 and 50 kDa heparanase subunits (8 + 50) was purified from the conditioned medium of baculovirus-infected cells. GS3 heparanase was assayed for the presence of bacterial endotoxin by Biological Industries (Beit Haemek, Israel), using the gel – clot technique (Limulus amebocyte lysate—LAL test) and was found to contain ,10 pg/ml endotoxin (Nadir et al., 2006). Monoclonal anti-heparanase antibody 1E1 was generated by immunizing Balb/C mice with the entire 65 kDa heparanase protein (Shafat et al., 2006). Polyclonal antibody 1453 was raised in rabbits against the entire 65 kDa heparanase precursor isolated from the conditioned medium of heparanase-transfected HEK-293 cells. The antibody was affinity-purified on immobilized bacterially expressed 50 kDa heparanase GST fusion protein (Zetser et al., 2004). Recombinant human factor VIIa and plasma-derived human factor X were purchased from American Diagnostica (Stanford, CT, USA) (...truncated)