Heparanase Procoagulant Activity in Women Using Oral Contraceptives
Heparanase Procoagulant Activity in Women Using Oral Contraceptives
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-β-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.
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 mechanism known today to explain the increased risk of venous thromboembolism among users of OC (Rosing et al., 1999).
Heparanase is an endo-β-d-glucuronidase capable of cleaving heparan sulfate (HS) side chains at a low pH (5.8–6.2), yielding HS fragments of still appreciable size (~5–7 kDa) (Freeman and Parish, 1998; Pikas et al., 1998). Heparanase activity is implicated in tumor growth, neovascularization and inflammation (Dempsey et al., 2000; Parish et al., 2001; Vlodavsky and Friedmann, 2001). Applying active site-mutated heparanase devoid of enzymatic activity, it was noted that heparanase also exerts non-enzymatic activities, at physiological pH, associated with tissue remodeling, angiogenesis and cell invasion (Goldshmidt et al., 2003; Gingis-Velitski et al., 2004; Zetser et al., 2006). Recently, we have shown that heparanase is directly involved in the activation of the coagulation system. Heparanase was demonstrated to interact with tissue factor (TF) and enhance the generation of factor Xa (Nadir et al., 2011a). In addition, we lately developed an assay to evaluate heparanase procoagulant activity showing significant increased heparanase procoagulant activity in women at the end of pregnancy and in patients following orthopedic surgery (Nadir et al., 2011b; Peled et al., 2012). Elkin et al. identified four putative estrogen response elements in the heparanase promoter region and found that heparanase promoter genes were significantly up-regulated in estrogen receptor-positive MCF-7 human breast carcinoma cells after estrogen treatment. In vivo, exposure to estrogen augmented levels of heparanase protein in MCF-7 cells embedded in Matrigel plugs and correlated with increased plug vascularization (Elkin et al., 2003).
In the present work, estrogen effect on the procoagulant activity of heparanase was evaluated in breast cancer cell lines and women using OC.
Abstract and Introduction
Abstract
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-β-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.
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 mechanism known today to explain the increased risk of venous thromboembolism among users of OC (Rosing et al., 1999).
Heparanase is an endo-β-d-glucuronidase capable of cleaving heparan sulfate (HS) side chains at a low pH (5.8–6.2), yielding HS fragments of still appreciable size (~5–7 kDa) (Freeman and Parish, 1998; Pikas et al., 1998). Heparanase activity is implicated in tumor growth, neovascularization and inflammation (Dempsey et al., 2000; Parish et al., 2001; Vlodavsky and Friedmann, 2001). Applying active site-mutated heparanase devoid of enzymatic activity, it was noted that heparanase also exerts non-enzymatic activities, at physiological pH, associated with tissue remodeling, angiogenesis and cell invasion (Goldshmidt et al., 2003; Gingis-Velitski et al., 2004; Zetser et al., 2006). Recently, we have shown that heparanase is directly involved in the activation of the coagulation system. Heparanase was demonstrated to interact with tissue factor (TF) and enhance the generation of factor Xa (Nadir et al., 2011a). In addition, we lately developed an assay to evaluate heparanase procoagulant activity showing significant increased heparanase procoagulant activity in women at the end of pregnancy and in patients following orthopedic surgery (Nadir et al., 2011b; Peled et al., 2012). Elkin et al. identified four putative estrogen response elements in the heparanase promoter region and found that heparanase promoter genes were significantly up-regulated in estrogen receptor-positive MCF-7 human breast carcinoma cells after estrogen treatment. In vivo, exposure to estrogen augmented levels of heparanase protein in MCF-7 cells embedded in Matrigel plugs and correlated with increased plug vascularization (Elkin et al., 2003).
In the present work, estrogen effect on the procoagulant activity of heparanase was evaluated in breast cancer cell lines and women using OC.
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