View metadata, citation and similar papers at core.ac.uk brought to you by CORE Journal of the American College of Cardiology providedVol. by 40,Elsevier No. 11,- Publisher 2002 Connector © 2002 by the American College of Cardiology Foundation ISSN 0735-1097/02/$22.00 Published by Elsevier Science Inc. PII S0735-1097(02)02565-2 Oral Conjugated Equine Estrogen Increases Plasma von Willebrand Factor in Postmenopausal Women LeRoy E. Rabbani, MD, FACC, Nicole A. Seminario, Robert R. Sciacca, ENGSCD, Hong Jun Chen, MD, Elsa-Grace V. Giardina, MD, FACC New York, New York

OBJECTIVES We sought to test whether one month of daily oral conjugated equine estrogen (CEE) or transdermal estradiol alters hemostatic factors in postmenopausal subjects. BACKGROUND Estrogen replacement therapy and hormonal replacement therapy (HRT) effect an early increase in cardiovascular events in postmenopausal women. Circulating plasma von Wille- brand factor (vWF) antigen is a marker of generalized endothelial dysfunction and atherothrombosis. METHODS Thirty-eight healthy postmenopausal women (average 59 Ϯ 7 years) were randomized to receive daily oral CEE, 0.625 mg (n ϭ 21); transdermal estradiol, 0.1 mg/day (n ϭ 7); or oral placebo (n ϭ 10) for one month. Blood samples were collected at baseline and after two weeks and four weeks of therapy for measurement of circulating plasma hormones, lipid concen- trations, and hemostatic factors. RESULTS Oral CEE decreased total cholesterol (p Ͻ 0.01) and low-density lipoprotein cholesterol (p Ͻ 0.01), although it increased both triglycerides (p Ͻ 0.05) and high-density lipoprotein cholesterol (p Ͻ 0.01). Transdermal estradiol had no significant effect on lipids. Plasminogen activator inhibitor-1 antigen declined in both oral CEE and transdermal estradiol users, but did not achieve statistical significance. Fibrin D-dimer antigen did not vary significantly in any group. However, oral CEE users had a significant increase in vWF from baseline to four weeks (p Ͻ 0.03) and a decrease in tissue-type plasminogen activator antigen from baseline to four weeks (p Ͻ 0.004), which was significantly different from the change observed in the transdermal estradiol group (p Ͻ 0.05). CONCLUSIONS These data suggest that the oral CEE-mediated increase in plasma vWF may have clinical relevance given the early atherothrombotic effects of HRT in postmenopausal women. (J Am Coll Cardiol 2002;40:1991–9) © 2002 by the American College of Cardiology Foundation

Cardiovascular disease is the leading cause of mortality for protective effects of HRT. The secondary prevention trial— women in the U.S. (1,2). The role of hormone replacement the Heart and Estrogen/progestin Replacement Study therapy (HRT) with estrogen and progesterone for primary (HERS)—examined 2,763 subjects with proven coronary and secondary prevention of coronary artery disease in artery disease and randomized them to conjugated equine- women remains controversial. Observational studies have estrogen (CEE) with medroxyprogesterone acetate (MPA) demonstrated that women who had used estrogen- or placebo (16). Over a four-year period, there was no replacement therapy (ERT) with or without progestins (i.e., difference in the number of secondary cardiovascular events HRT) during the menopausal years had a lower incidence of and adverse events from peripheral vascular disease between cardiovascular events, as compared with untreated women the HRT-treated subjects and the placebo-treated subjects (3,4). Indeed, several potential mechanisms attributed to (16). The group receiving HRT experienced a 50% higher ERT or HRT had been considered to account for a cardiac event rate during the first year (absolute excess of beneficial effect. For example, oral exogenous estrogen 1.4%) and a lower rate from the third year onward. The increases the level of high-density lipoprotein (HDL) cho- investigators considered that the results might be attribut- lesterol and decreases the concentration of low-density able to dual effects, such as an immediate prothrombotic, lipoprotein (LDL). Moreover, several studies have sug- proarrhythmic, or proischemic effect that was later out- gested that ERT or HRT exerts a profibrinolytic effect by weighed by slower progression of atherosclerosis. In further reducing plasminogen activator inhibitor-1 (PAI-1), the support of the lack of a benefit from HERS, the Estrogen endogenous inhibitor of tissue-type plasminogen activator Replacement and Atherosclerosis (ERA) trial demonstrated (t-PA) and urokinase plasminogen activator (5–15). that three years of treatment with either CEE or CEE with However, recent studies have cast doubt on the cardio- MPA did not slow the rate of angiographic coronary artery disease progression, compared with placebo, in 309 post- From the Cardiology Division and Center for Women’s Health, Department of Medicine, Columbia University College of Physicians and Surgeons, New York, New menopausal women with established coronary artery disease York. This study was supported in part by Grant HL-07406 from the Department of (17). Health and Human Services; Grant RR-00645 from the Research Resources The von Willebrand factor (vWF) has recently been Administration, Bethesda, Maryland; and a grant from Linda and Peter Nisselson. Manuscript received May 2, 2002; revised manuscript received July 1, 2002, proposed as a marker of generalized endothelial dysfunc- accepted August 19, 2002. tion, and as such, may contribute to the development of 1992 Rabbani et al. JACC Vol. 40, No. 11, 2002 Oral Estrogen Increases vWF December 4, 2002:1991–9

four weeks of therapy. At enrollment, each had a history and Abbreviations and Acronyms physical examination. Venous blood samples were collected CEE ϭ conjugated equine estrogen during each visit. At baseline and after four weeks, blood CRP ϭ C-reactive protein was collected for a complete blood count, chemical- ϭ ERT estrogen replacement therapy metabolic profile—including electrolytes, hepatic and renal HDL ϭ high-density lipoprotein cholesterol HRT ϭ hormone replacement therapy function, total cholesterol, LDL cholesterol, HDL choles- LDL ϭ low-density lipoprotein cholesterol terol, triglycerides, estradiol, follicle-stimulating hormone, MPA ϭ medroxyprogesterone acetate progesterone, luteinizing hormone—and urinalysis, as well ϭ PAI-1 plasminogen activator inhibitor-1 as t-PA antigen, PAI-1 antigen, fibrin D-dimer, vWF ϭ t-PA tissue-type plasminogen activator antigen, and fibrinogen. After two weeks, blood was also vWF ϭ von Willebrand factor collected for hormonal and hemostatic factors to assess patient compliance. Subjects were entered into a randomized, double-blind, atherothrombotic disease (18,19). vWF is synthesized by placebo-controlled trial using one of two forms of hormonal the endothelium and megakaryocytes, and increased con- replacement: oral CEE (Premarin), 0.625 mg/day, or trans- centrations of vWF have been found in patients with dermal estrogen (Climara), 0.1 mg/day. Because concurrent peripheral vascular disease, cerebral vascular disease, and progestins given in conjunction with CEE may offset coronary artery disease (20–22). In addition, patients with estrogenic effects, we elected to treat subjects with estrogen known disease are at greater risk of cardiovascular mortality monotherapy. Subjects were randomized by means of a if they have high concentrations of vWF (21–25). The random table to three groups in a ratio of 2:1:1, as to Hoorn study of 631 patients demonstrated that increased whether they received oral CEE, transdermal estrogen, or vWF is independently associated with cardiovascular and oral placebo, respectively. The rationale for the randomiza- all-cause mortality in both diabetic and nondiabetic patients tion scheme, which favored oral administration, was the (26). consideration that the majority of women prescribed post- We hypothesized that the early effects of treatment with menopausal ERT use CEE. oral CEE in postmenopausal women might increase mark- There were 55 subjects enrolled: 48 were randomized, ers associated with endothelial dysfunction, such as vWF. and 38 completed the trial. Three subjects randomized to Therefore, we conducted a short-term study and measured oral CEE did not complete the study because of stomach the concentrations of vWF, lipids, and other hemostatic upset (n ϭ 1) or noncompliance (n ϭ 2). Three subjects factors in postmenopausal subjects receiving daily oral CEE randomized to transdermal estrogen did not complete the versus transdermal estradiol or placebo for one month. In study because of vaginal bleeding (n ϭ 1), noncompliance this study, we demonstrate that one-month treatment with (n ϭ 1), and elective withdrawal (n ϭ 1). Four randomized oral CEE, but not transdermal estradiol, significantly in- to placebo did not complete the study because of migraine creases vWF, thereby potentially increasing the likelihood headache (n ϭ 1), noncompliance (n ϭ 1), a hormone of cardiovascular events. concentration that did not meet entry criteria for menopause ϭ METHODS (n 1), and generalized discomfort following medication (n ϭ 1). Thirty-eight postmenopausal subjects completed The Institutional Review Board of the Columbia– the study. The subjects (average age 59 Ϯ 7 years) received Presbyterian Medical Center approved the protocol, and daily oral CEE, 0.625 mg (n ϭ 21); transdermal estradiol, written, informed consent was obtained from each subject, 0.1 mg/day (n ϭ 7); or oral placebo (n ϭ 10) for one month. in accordance with institutional guidelines. Subjects who Plasma samples were collected before and after treatment were postmenopausal for at least one year participated. in the morning, at the same time of the day for each visit. Menopausal status was confirmed by measures of the Blood samples were drawn at rest from a large antecubital estradiol concentration (Ͻ50 pg/ml) and follicle- vein into 0.1 mol/liter trisodium citrate tubes for analysis of stimulating hormone (Ͼ20 mU/ml), consistent with meno- t-PA antigen, PAI-1 antigen, fibrin D-dimer antigen, and pause. No subject was currently taking or had previously vWF. The tubes were immediately centrifuged and spun at taken ERT within the past year. Exclusion criteria included 3,000g for 20 min at 4°C. This centrifugation was per- a personal or immediate family history of breast or endo- formed within 1 h. The plasma was removed into aliquots metrial cancer, previous myocardial infarction, coronary and stored at Ϫ80°C for up to four months before analysis. artery disease, thrombophlebitis or a history of thromboem- The assays for PAI-1, t-PA, and vWF were determined bolic disease, and a previous adverse reaction to estrogen. using the respective enzyme-linked immunosorbent assay Only subjects with a normal mammogram within the year (ELISA) kits purchased from American Diagnostics, Inc. were enrolled. Subjects with an abnormal metabolic-renal (Greenwich, Connecticut). The t-PA antigen measured by profile, including hyperglycemia and hypercalcemia, were ELISA was the total amount of t-PA present (free and not enrolled. complexed t-PA, single-chain and double-chain t-PA) (27). Subjects underwent testing at baseline and after two and The PAI-1 antigen measured by ELISA was the total JACC Vol. 40, No. 11, 2002 Rabbani et al. 1993 December 4, 2002:1991–9 Oral Estrogen Increases vWF

Table 1. Baseline Characteristics of Subjects concentrations were assessed using a log-transform of the Transdermal various factors. Variable Placebo Oral CEE Estradiol Age (yrs) 60 Ϯ 458Ϯ 760Ϯ 10 Time since menopause (yrs) 11 Ϯ 68Ϯ 99Ϯ 8 RESULTS Weight (kg) 70 Ϯ 14 68 Ϯ 11 70 Ϯ 10 Body mass index (kg/m2) 28 Ϯ 627Ϯ 528Ϯ 4 Table 1 shows the baseline characteristics of the assigned Estradiol (pg/ml) 15 Ϯ 918Ϯ 516Ϯ 10 subjects. There were no significant differences among the Follicle-stimulating 63 Ϯ 18 69 Ϯ 20 63 Ϯ 28 placebo, oral CEE, and transdermal estradiol groups at hormone (mU/ml) Ϯ Ϯ Ϯ baseline. Progesterone (ng/ml) 0.2 0.1 0.3 0.2 0.3 0.1 Table 2 shows the effect of oral CEE and transdermal Luteinizing hormone 24 Ϯ 12 24 Ϯ 927Ϯ 17 (mU/ml) estradiol therapy on hormones and lipids after one month. Total cholesterol (mg/dl) 221 Ϯ 39 229 Ϯ 32 218 Ϯ 34 Subjects assigned to oral CEE experienced a significant Triglycerides (mg/dl) 139 Ϯ 54 121 Ϯ 75 134 Ϯ 46 increase in estradiol (p Ͻ 0.01) and a decrease in follicle- Ϯ Ϯ Ϯ HDL cholesterol (mg/dl) 53 16 61 15 43 8 stimulating hormone from baseline to four weeks (p Ͻ LDL cholesterol (mg/dl) 140 Ϯ 31 144 Ϯ 29 148 Ϯ 32 0.01). Transdermal estradiol users experienced a nonsignif- Data are presented as the mean value Ϯ SD. icant increase in estradiol (p ϭ NS) and a significant CEE ϭ conjugated equine estrogen; HDL ϭ high-density lipoprotein; LDL ϭ low-density lipoprotein. decrease in follicle-stimulating hormone at four weeks (p Ͻ 0.01). Subjects assigned to oral CEE experienced a signif- quantity of PAI-1 present (free or complexed with t-PA; icant decrease in total cholesterol (p Ͻ 0.01) and LDL Ͻ Ͻ active or inactive form) (27). Fibrin D-dimer was analyzed cholesterol (p 0.01) and an increase in triglycerides (p using the Asserachrom D-Di ELISA from Diagnostica 0.05) and HDL cholesterol (p Ͻ 0.01). Neither transdermal Stago (Asnieres-Sur-Seine, France). Estradiol, progester- estradiol nor placebo had a significant effect on lipids. one, follicle-stimulating hormone, and luteinizing hormone Table 3 and Figure 1 show the effect of oral CEE and concentrations were determined by their specific immuno- transdermal estradiol on hemostatic factors; there was no assays from Diagnostic Products Corp. (Los Angeles, Cal- effect of placebo on hemostatic factors. For the subjects ifornia), according to the manufacturer’s instructions. Inter- randomized to oral CEE and transdermal estradiol, PAI-1 assay coefficient of variation for estradiol at high, middle, (ng/ml) declined, but it achieved only borderline signifi- and low control concentrations are 3.5%, 4.6%, and 9.6%, cance (p Ͻ 0.08), and only in the oral CEE subjects. There respectively. Total cholesterol, LDL cholesterol, HDL cho- was a significant decrease in t-PA antigen (p Ͻ 0.004) after lesterol, triglyceride, and fibrinogen concentrations were oral CEE. Fibrin D-dimer did not vary significantly in any determined by conventional enzymatic methods. group. However, oral CEE users experienced a significant Statistical analysis. Data on baseline characteristics were increase (p Ͻ 0.03) in vWF from baseline to four weeks, analyzed by ANOVA, with post-hoc differences in mean which was significantly different from the change observed values among groups assessed using Tukey’s procedure. in the transdermal estradiol group. Neither oral CEE nor Statistical significance was defined as a two-sided p value transdermal estradiol had significant effects on fibrinogen. Ͻ0.05. The effect of estrogen within each group on hor- The change observed for t-PA in the oral CEE group was mones and lipid concentrations was determined using a significantly different from that observed in the transdermal two-tailed, paired Student t test. In addition, to correct for group (p Ͻ 0.05) and that in the placebo group (p Ͻ 0.05). individual differences in baseline values among subjects, Subjects using placebo experienced no significant change in relative changes in the effect of estrogen treatment within any hormone, lipid, or hemostatic factor values from base- each group on hormones, lipids, and hemostatic factor line to two and four weeks.

Table 2. Effect of Oral CEE or Transdermal Estradiol on Hormones and Lipids After One Month Oral CEE Transdermal Estradiol Before After Before After Variable Treatment Treatment p Value Treatment Treatment p Value Estradiol (pg/ml) 18 Ϯ 576Ϯ 28 Ͻ 0.01 16 Ϯ 10 59 Ϯ 66 NS Follicle-stimulating hormone (ng/ml) 69 Ϯ 20 39 Ϯ 13 Ͻ 0.01 63 Ϯ 28 33 Ϯ 11 Ͻ 0.01 Progesterone (ng/ml) 0.3 Ϯ 0.2 0.2 Ϯ 0.1 NS 0.3 Ϯ 0.1 0.3 Ϯ 0.2 NS Luteinizing hormone (mU/ml) 24 Ϯ 921Ϯ 11 NS 27 Ϯ 17 20 Ϯ 10 NS Total cholesterol (mg/dl) 229 Ϯ 32 209 Ϯ 24 Ͻ 0.01 218 Ϯ 34 214 Ϯ 35 NS Triglycerides (mg/dl) 121 Ϯ 75 144 Ϯ 75 Ͻ 0.05 134 Ϯ 46 120 Ϯ 37 NS HDL cholesterol (mg/dl) 61 Ϯ 15 65 Ϯ 14 Ͻ 0.01 43 Ϯ 843Ϯ 7NS LDL cholesterol (mg/dl) 144 Ϯ 29 115 Ϯ 27 Ͻ 0.01 148 Ϯ 32 147 Ϯ 32 NS

Data are presented as the mean value Ϯ SD. NS ϭ not significant; other abbreviations as in Table 1. 1994 Rabbani et al. JACC Vol. 40, No. 11, 2002 Oral Estrogen Increases vWF December 4, 2002:1991–9

Table 3. Effect of Oral Conjugated Equine Estrogen or Transdermal Estradiol on Hemostatic Factors After One Month Oral CEE Transdermal Estradiol Before After Before After Variable Treatment Treatment % Change p Value* Treatment Treatment % Change p Value* PAI-1 (mg/ml) 42 Ϯ 21 36 Ϯ 19 Ϫ8.2 0.08 34 Ϯ 10 30 Ϯ 12 Ϫ9.2 0.34 t-PA (mg/ml) 11.2 Ϯ 8.0 9.7 Ϯ 8.1 Ϫ17.8 0.004 7.8 Ϯ 4.0 9.0 Ϯ 4.0 19.5 0.23 vWF (mU/ml) 835 Ϯ 365 963 Ϯ 372 22.7 0.03 1,151 Ϯ 159 1,025 Ϯ 238 Ϫ10.5 0.24 Fibrin D-dimer (mU/ml) 339 Ϯ 141 467 Ϯ 354 67.7 0.06 452 Ϯ 116 446 Ϯ 222 Ϫ4.7 0.72 Fibrinogen (mg/dl) 354 Ϯ 67 368 Ϯ 65 6.9 0.33 363 Ϯ 61 348 Ϯ 45 Ϫ2.7 0.54

*Relates to the log difference reflecting the percent change. Data are presented as the mean value Ϯ SD. CEE ϭ conjugated equine estrogen; PAI-1 ϭ plasminogen activator inhibitor-1; t-PA ϭ tissue-type plasminogen activator; vWF ϭ von Willebrand factor.

DISCUSSION CEE had no significant effect on fibrin D-dimer or fibrin- ogen. In contrast, one month of transdermal estradiol In this study, we report that oral CEE but not transdermal therapy or placebo had no effect on PAI-1, t-PA, fibrin estradiol therapy for one month significantly increases the D-dimer, vWF antigen, fibrinogen, or lipids. The observa- plasma concentration of vWF antigen in postmenopausal women. Our findings with CEE and vWF in postmeno- tion that oral, but not transdermal, administration of estro- pausal women are intriguing when viewed in the context of gen alters several markers, including lipoproteins and PAI-1 several recent trials such as HERS and ERA, which have levels, suggests that the hepatic effects of estrogen regulate cast doubt on the cardioprotective effect of HRT in post- their synthesis and/or clearance (5,10,15), accounting for menopausal women (16,17). Although we found that treat- the difference in response. ment for one month with oral CEE decreased PAI-1, t-PA Elevated serum t-PA levels are present in cardiovascular antigen, total cholesterol, and LDL cholesterol, it signifi- conditions involving endothelial cell damage (28). Most of cantly increased vWF antigen. Oral CEE also increased the t-PA in the plasma travels while bound to PAI-1 HDL cholesterol and triglyceride concentrations. Oral molecules to form circulating, inactive t-PA/PAI-1 com-

Figure 1. The percent change after oral conjugated equine estrogen (CEE) or transdermal estrogen on plasminogen activator inhibitor-1 (PAI-1), tissue-type plasminogen activator (t-PA), and von Willebrand factor (vWF), compared with baseline. For oral CEE, the test result of the percent change (p value by analysis of variance [ANOVA]) was p ϭ 0.08 for PAI-1, p Ͻ 0.004 for t-PA, and p ϭ 0.03 for vWF. For transdermal estrogen, the test result of the percent change (p value by ANOVA) was not significant: p ϭ 0.34 for PAI-1; p ϭ 0.23 for t-PA; and p ϭ 0.24 for vWF. Solid bars ϭ PAI-1; hatched bars ϭ t-PA; and open bars ϭ vWF. JACC Vol. 40, No. 11, 2002 Rabbani et al. 1995 December 4, 2002:1991–9 Oral Estrogen Increases vWF plexes (29,30). Therefore, oral CEE-induced decreased mary, we conclude that variability in the findings could be t-PA and PAI-1 antigen levels may reflect diminished related to study design: estrogen formulations (e.g., CEE, t-PA/PAI-1 complexes and, hence, augmented fibrinolytic estradiol valerate, micronized estradiol, 17-beta-estradiol), activity. the size of dose, the method of estrogen replacement (e.g., Recent studies have suggested that increased circulating cyclical, continuous), monotherapy or combination with vWF antigen denotes a poor prognosis in unstable angina progestin, the age of subjects, the time since the start of and non–ST-segment elevation myocardial infarction (31). menopause, the duration of treatment, and the time of Moreover, patients undergoing percutaneous coronary in- sampling. Although there are several reports relating the terventions who have unstable angina have higher vWF effect of ERT and HRT on PAI-1, few measured vWF. No antigen levels than do patients with stable angina, and the other study measured vWF at one month, but more likely decrease in vWF antigen after a successful percutaneous measured it at 3 to 4, 6, 12, and 36 months. Two studies intervention may reflect plaque re-endothelialization and collected samples as early as three months; however, one stabilization (32). Indeed, vWF facilitates platelet aggrega- used micronized estradiol plus 1 or 2 mg dydrogesterone tion at sites of vascular injury under high shear stress, and (35) and found that vWF decreased, and the other used elevated plasma vWF antigen concentrations correlate with CEE plus 5 mg medrogestone (12) and found no effect on enhanced platelet adhesion to collagen types I, III, and vWF. IV—all extremely potent physiologic inducers of platelet Thus, it is possible that oral CEE exerts very early effects, aggregation at the site of plaque rupture and vascular injury increasing vWF antigen after one month, which may then (33). Studies with vWF-deficient mice indicate that the dissipate over time with no effects found on vWF levels by absence of vWF predominantly affects aortic regions with three months to one year. We found that one month of disturbed flow that are prone to atherosclerosis (34). vWF transdermal estradiol had no effect on vWF antigen levels, a may recruit platelets and leukocytes to the lesion in a finding consistent with the results of a study of transdermal flow-dependent fashion or may be part of the mechano- estradiol in 28 postmenopausal hysterectomized subjects transduction pathway that regulates the endothelium’s re- after four months of therapy (42). sponse to shear stress (34). Our finding that oral, but not transdermal, estrogen Therefore, the oral CEE-mediated increase of vWF increases plasma vWF antigen after one month of therapy is antigen at one month in our study may partially explain the noteworthy in that vWF is not only a marker of endothelial results of HERS, which found an early increase in cardio- dysfunction and enhanced atherothrombotic activity, but vascular events after oral CEE and MPA. It should be noted also is a marker of inflammation (36,41). In this regard, that van Baal et al. (35) found a 14% decrease in vWF in 14 recent reports have discovered that HRT initially increases healthy postmenopausal subjects after three months of oral levels of C-reactive protein (CRP), an inflammatory marker CEE. Cushman et al. (36) found no effect of four different that is a potent prognosticator of an increased risk of estrogen/progestin hormone preparations on vWF antigen cardiovascular events in both women and men (36,37, levels in 365 patients after 12 months. One study measured 42,50). Indeed, HRT appears to increase high-sensitivity the effect of oral CEE and transdermal estrogen after one CRP within four to eight months of the initiation of month; however, in that study, vWF was not measured (5). therapy. Therefore, oral CEE may cause an early increase in In a study by Lip et al. (37), 27 premenopausal women both inflammation and hemostasis, as evidenced by early (average age 43.6 years) received CEE, 0.625 mg, following elevations in both vWF and CRP, which result in an early a hysterectomy and bilateral salpingo-oophorectomy. They increase in cardiovascular events. Furthermore, our findings found a decrease in plasma levels of both vWF and t-PA that oral CEE, but not transdermal estradiol, induced the after six weeks of CEE (37). In contrast to the subjects aforementioned effects is intriguing considering that the described here, no subjects were naturally postmenopausal proinflammatory cytokine, interleukin-6, which is the pri- for at least one year; all had surgically induced menopause; mary stimulant of hepatic CRP (51), is also a potent inducer all were considerably younger (as expected of a premeno- of vWF (52). pausal population); and all had vasomotor symptoms. Fur- Oral CEE increased HDL cholesterol and decreased thermore, the investigators acknowledge that stress and LDL cholesterol and total cholesterol, even in subjects who blood loss, a consequence of surgery versus naturally occur- were overweight (Table 2), which corresponds to previous ring menopause, might have accounted for the findings. studies demonstrating that oral CEE has beneficial effects The opportunity to evaluate the effects of CEE and on the lipid profile (53,54). However, we also found that transdermal estradiol after one month may provide insight oral CEE concomitantly increases triglycerides, as evi- into the time course of variables affected by estrogen. denced by previous studies (53,54). In addition, it should be Although there are a considerable number of reports docu- noted that, despite decreasing total cholesterol and LDL menting estrogen’s effect on markers of fibrinolysis and cholesterol, oral CEE actually decreases the LDL particle hemostasis, the results are not uniform. Tables 4 and 5 size, increases the very low density lipoprotein size, and only summarize the existing, sometimes contradictory, findings minimally reduces the concentration of apolipoprotein B of 20 studies (5,10–13,15,35,36,38–49). From this sum- (53,54). Furthermore, our study demonstrates that trans- 1996 Rabbani et al. JACC Vol. 40, No. 11, 2002 Oral Estrogen Increases vWF December 4, 2002:1991–9

Table 4. Reports Evaluating Hemostatic and Fibrinolytic Markers in Postmenopausal Women After Estrogen Design; Number of Subjects (n); Time of Study (Ref.) and Age (years) Sampling Oral Estrogen Transdermal Estrogen Andersen et al. (38) Active treatment and reference group; Baseline and 3, 6, Estradiol valerate, 2 mg/day Not administered n ϭ 60; age 40–60 years and 12 months (cont or cyc) ϩ CA, 1 mg/day Boschetti et al. (39) Random allocation; n ϭ 40 Baseline and 2, 4, CEE, 0.625 mg (3 one-week Transdermal estradiol, and 12 months cycles) ϩ MPA, 10 mg/day 0.05 mg/day (3 one-week (third week) cycles) ϩ MPA, 10 mg/ day (third week) Chen et al. (40) n ϭ 41 Baseline and 3, 6, Estradiol valerate, 2 mg/day Estradiol, 1.5 mg ϩ MPA, 5 and 12 months (cont or cyc) ϩ CA,1mg mg Cushman et al. (36) PEPI: randomized double-blind, Baseline and 12 CEE, 0.625 mg; CEE ϩ MPA, Not administered placebo-controlled; n ϭ 383; and 36 months 10 mg/day (cyc); CEE ϩ mean age 56 years MPA, 2.5 mg/day (cont); CEE ϩ MP, 200 mg/day (cyc) Cushman et al. (41) Cardiovascular health study: Annually CEE (92%); CEE ϩ MPA Not administered population-based, longitudinal; (cont or cyc) n ϭ 290; mean age 73 years DeMitrio et al. (42) Hysterectomized women; n ϭ 28; Baseline and 4 Not administered 17-beta-estradiol, 50 ␮g/24 h age range 44–65 years (mean 47) months Gilabert et al. (11) n ϭ 12 oral, n ϭ 12 transdermal Baseline and 3–4 Estradiol valerate, 2 mg/day Estradiol, 0.5 mg ϩ MPA, and 12 months ϩ MPA, 2.5 mg/day 2.5 mg/day Gottsater et al. (43) Part 1: double-blind, randomized trial Baseline and 3 Estradiol valerate, 2 mg ϩ MPA, Not administered for 3 months; part 2: open study and 12 months 10 mg for 10 days every for 9 months; postmenopausal 3 months women; n ϭ 51; age range 53–54 years Hoibraaten et al. (44) Estrogen in women with Baseline and 3 Not administered 17-beta-estradiol, 50 ␮g/24 h atherosclerosis study: open and 12 months ϩ MPA, 5 mg (cyc) randomized trial; n ϭ 118; age Ͻ71 years Koh et al. (5) Randomized, crossover; n ϭ 50 (30 Baseline and CEE, 0.625 mg/day or CEE, Estradiol, 0.1 mg/day or oral, 20 transdermal); mean age 1 month 0.625 mg/day ϩ MPA, 2.5 mg estradiol, 0.1 mg/day ϩ for oral 55 years, mean age for MPA, 2.5 mg transdermal 56 years Kroon et al. (10) Crossover; n ϭ 23; age Յ65 years Baseline and CEE, 0.625 mg/day 17-beta-estradiol, 0.05 mg/ 6 weeks day Lindoff et al. (45) Open, prospective trial with active 2 years Not administered 17-beta-estradiol, 50 ␮g/24 h treatment vs. control; n ϭ 42 ϩ MPA, 5 mg (cyc) treated, n ϭ 18 untreated Luyer et al. (46) Placebo-controlled; n ϭ 26; Baseline and CEE, 0.625 mg/day Not administered age range 55–80 years 3 months Scarabin et al. (15) Open, randomized trial with control Baseline and 17-beta-estradiol, 2 mg/day 17-beta-estradiol gel, group; n ϭ 45; age 45–64 years 6 months ϩ MP, 200 mg/day (cyc) 2.5 mg/day ϩ MP, 200 mg/day (cyc) Seljeflot et al. (47) Postmenopausal women with Baseline and 3 Not administered 17-beta-estradiol, 50 ␮g/day coronary artery disease; n ϭ 98; and 12 months ϩ sequential MPA, 5 mg age range 59–71 years for 14 days every third month Shahar et al. (13) ARIC study cohort with case control; Baseline Estrogen or estrogen progestin Not specified n ϭ 59; age 45–64 years combination; type not specified Teede et al. (48) Double-blind, placebo-controlled; Baseline and 6 Estradiol, 2 mg ϩ continuous Not administered n ϭ 42; age 50–75 years weeks norethisterone acetate, 1 mg van Baal et al. (35) Prospective, randomized, open; Baseline and 3, 12, Micronized estradiol, 1 mg ϩ Not administered n ϭ 27; mean age 52 years and 15 months dydrogesterone, 5–10 mg or micronized estradiol, 2 mg ϩ dydrogesterone Vehkavaara et al. (49) Randomized, placebo-controlled; Baseline and 2 Estradiol, 2 mg Estradiol, 50 ␮g/day n ϭ 27; age Ն52 years; and 12 weeks mean age 55 years Winkler et al. (12) n ϭ 42 Baseline and CEE, 0.6 mg ϩ medrogestone, 17-beta-estradiol 50 ␮g/day 3 months 5 mg (cyc)

ARIC ϭ Atherosclerosis Risk In Communities study; CA ϭ cyprotereone acetate, anti-androgen, progestational compound with androgen receptor blocking capacity; CEE ϭ conjugated equine estrogen; cont ϭ continuous; cyc ϭ cyclical; MP ϭ micronized progesterone; MPA ϭ medroxyprogesterone acetate; PEPI ϭ Postmenopausal Estrogen/Progestin Interventions study. JACC Vol. 40, No. 11, 2002 Rabbani et al. 1997 December 4, 2002:1991–9 Oral Estrogen Increases vWF

Table 5. Reports Evaluating Hemostatic and Fibrinolytic Markers in Postmenopausal Women After Estrogen Oral CEE Transdermal Estrogen

t-PA D- t-PA D- Study (Ref.) PAI-1 Antigen vWF Dimer Fibrinogen PAI-1 Antigen vWF Dimer Fibrinogen Andersen et al. (38) 2p Ͻ 0.05 2p Ͻ 0.05 ——2p Ͻ 0.05 ————— Boschetti et al. (39) — p ϭ NS ——p ϭ NS — p ϭ NS ——1p Ͻ 0.05 Chen et al. (40) p ϭ NS p ϭ NS ——p ϭ NS p ϭ NS p ϭ NS ——p ϭ NS Cushman et al. (36) ——p ϭ NS — 1p Ͻ 0.02 ————— Cushman et al. (41) 2p Ͻ 0.001 ——p ϭ NS 2p Ͻ 0.001 ————— DeMitrio et al. (42) ———————p ϭ NS —— Gilabert et al. (11) 2p Ͻ 0.05 ————p ϭ NS ———— Gottsater et al. (43) p ϭ NS 2p Ͻ 0.01 p ϭ NS — 2p Ͻ 0.01 ————— Hoibraaten et al. (44) —————2p Ͻ 0.047 p ϭ NS — p ϭ NS p ϭ NS Koh et al. (5) 2p Ͻ 0.001 ——p ϭ NS — p ϭ NS ———— Kroon et al. (10) 2p Ͻ 0.05 ————p ϭ NS ———— Lindoff et al. (45) —————2p ϭ NS 1p ϭ 0.01 ——2p Ͻ 0.01 Luyer et al. (46) 2p ϭ NS ———p ϭ NS ————— Seljeflot et al. (47) ——————p ϭ NS 2p Ͻ 0.001 —— Scarabin et al. (15) 2p Ͻ 0.02 2p Ͻ 0.001 p ϭ NS p ϭ NS p ϭ NS p ϭ NS p ϭ NS p ϭ NS p ϭ NS p ϭ NS Shahar et al. (13) 2p ϭ NS 2p Ͻ 0.01 — p ϭ NS — ————— Teede et al. (48) 2p Ͻ 0.0005 ——1p Ͻ 0.03 — ————— Van Baal et al. (35) ——2p Ͻ 0.05 — 2p ϭ NS ————— Vehkavaara et al. (49) 2p Ͻ 0.05 2p Ͻ 0.05 — 1p Ͻ 0.05 — p ϭ NS p ϭ NS — p ϭ NS — Winkler et al. (12) 2p Ͻ 0.05 — p ϭ NS — p ϭ NS p ϭ NS — p ϭ NS — p ϭ NS

2 ϭ decrease after estrogen; 1 ϭ increase after estrogen; — ϭ level not reported; NS ϭ not significant; other abbreviations as in Table 3. dermal estradiol has no significant effect on lipids. We CEE may have very early deleterious effects promoting found a positive correlation between the oral CEE-induced atherothrombosis by increasing a hemostatic factor, which increase in vWF and HDL cholesterol. The mechanism encompasses the interaction of endothelial dysfunction, underlying this interesting observation merits further inves- platelet aggregation, and inflammation. tigation. Study limitations. Of the 48 subjects randomized, 38 Reprint requests and correspondence: Dr. Elsa-Grace V. Giar- (79%) completed the trial and 10 (21%) discontinued dina, Cardiology Division, Columbia University College of Phy- treatment because of adverse events or because they were sicians and Surgeons, 630 West 168th Street, PH 3-346, New deemed no longer eligible or noncompliant. In subjects York, New York 10032. E-mail: [email protected]. taking ERT for the first time, continuance of ERT is sporadic, and the current subjects parallel this experience. In REFERENCES the Massachusetts Women’s Health Survey, among patients 1. U.S. Department of Health and Human Services, Public Health taking ERT for the first time, 20% stopped taking the drug Service Centers for Disease Central, National Center for Health within nine months and 10% used it on an intermittent Statistic, Monthly Vital statistics Report 1995;43:1–76. basis (55). In a survey from The North American Meno- 2. Shaw L, Miller D, Rorers J, et al. Patterns in coronary heart disease—morbidity and mortality in the sexes: a 26-year following of pause Society, among 833 women, 8% who had used ERT the Framingham population. Ann Intern Med 1994;111:383–90. or HRT in the past stopped taking it, and among 58% who 3. Stampfer MJ, Colditz GA, Willet WC, et al. Postmenopausal estro- had never used it, 14% never tried it, even though it was gen therapy and cardiovascular disease: ten-year follow-up from the Nurses’ Health Study. N Engl J Med 1991;325:756–62. prescribed (56). A second limitation is that the number of 4. Stampfer MJ, Colditz GA. Estrogen replacement therapy and coro- subjects assigned to transdermal estradiol and placebo is nary heart disease: a quantitative assessment of the epidemiologic relatively small. The rationale for the randomization evidence. Prev Med 1991;20:47–63. 5. Koh KK, Mincemoyer R, Bui MN, et al. Effects of hormone- scheme, which favored oral administration, was the consid- replacement therapy on fibrinolysis in postmenopausal women. N Engl eration that the majority of postmenopausal women treated J Med 1997;336:683–90. with hormones use CEE, and furthermore, oral CEE was 6. Gebara OCE, Mittleman MA, Sutherland P, et al. Association between increased estrogen status and increased fibrinolytic potential used in the HERS and ERA study, as well as the Women’s in the Framingham Offspring Study. Circulation 1995;91:1952–8. Health Initiative. Thus, the findings reported here may 7. Sporrong T, Mattson LA, Samsive G, Stigendal L, Hellgren M. provide insight into the mechanisms of benefit and risk, as Hemostatic changes during continuous estradiol progestogen treat- ment of postmenopausal women. Br J Obstet Gynaecol 1990;97:939– well as the time course of markers, following administration 44. of CEE in the postmenopausal years. 8. Scarabin PY, Plu-Bureau G, Bara L, et al. Haemostatic variables and Conclusions. One month of oral CEE, but not transder- menopausal status: influence of hormone replacement therapy. Thromb Haemost 1993;70:584–7. mal estradiol, significantly increases the plasma levels of 9. van Wersch JWJ, Ubachs JMH, van den Ende A, van Enk A. The vWF antigen. Taken together, these data suggest that oral effect of two regimens of hormone replacement therapy on the 1998 Rabbani et al. JACC Vol. 40, No. 11, 2002 Oral Estrogen Increases vWF December 4, 2002:1991–9

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