Objective-The purpose of this study was to investigate whether the effect of transdermal estrogen therapy in postmenopausal women differs from that of oral therapy with regard to resistance to activated protein C (APC), an important risk factor for venous thrombosis, and levels of related proteins, such as protein S, protein C, and prothrombin. Methods and Results-In a randomized, double-blind, placebo-controlled study, 152 healthy hysterectomized postmenopausal women received daily either placebo (nϭ49), transdermal 17␤-estradiol (E 2 ) 50 g (tE 2 group, nϭ33), oral E 2 1 mg (oE 2 group, nϭ37), or oral E 2 1 mg combined with gestodene 25 g (oE 2 ϩG group, nϭ33) for 13 28-day treatment cycles, followed by 4 cycles of placebo for each group. Plasma samples were collected at baseline and in cycles 4, 13, and 17. In cycle 13, significant increases versus baseline and placebo were found in normalized APC sensitivity ratios (nAPCsr) in all treated groups (tE 2 , ϩ26.9%; oE 2 , ϩ102.7%; oE 2 ϩG, ϩ69.9%). Increases in nAPCsr were significantly higher in the oral treatment groups than in the tE 2 group. In addition, compared with baseline and placebo, after 13 cycles, decreases were observed in total protein S (tE 2 , Ϫ4.1%; oE 2 , Ϫ7.9%; oE 2 ϩG, Ϫ5.8%), free protein S (tE 2 , Ϫ7.1%; oE 2 , Ϫ8.4%; oE 2 ϩG, Ϫ5.2%), and protein C in the oE 2 ϩG group (Ϫ6.4%), but these changes did not explain the increase in nAPCsr. Changes in prothrombin were small and also did not affect the nAPCsr. Conclusions-Increases were observed in resistance to APC, which were more pronounced in the oral treatment groups than in the transdermal group. The increase in resistance to APC was not explained by changes in protein S, protein C, or prothrombin and may contribute to the increased incidence of venous thrombosis in users of hormone replacement therapy. Key Words: resistance to activated protein C Ⅲ protein S Ⅲ protein C Ⅲ estrogen replacement therapy Ⅲ venous thrombosis W omen using oral estrogen containing hormone replacement therapy 1-3 or oral contraceptives 4 have an increased risk of developing venous thrombosis. The risk for oral contraceptive users is higher in women who use ethynylestradiol in combination with so-called third-generation progestogens (desogestrel or gestodene) than among women using ethynylestradiol in combination with the secondgeneration progestogen levonorgestrel. 4 This difference might be explained by a differential effect of the progestogens on resistance to activated protein C (APC), 5,6 a risk factor for venous thrombosis. 7,8 It is plausible that the observed increased risk of venous thrombosis in hormone replacement users can also, at least in part, be explained by an increase in resistance to APC.The effect of hormone replacement therapy on resistance to APC, on protein S, and on protein C has been investigated in several studies. However, only few had a randomized, placebo-controlled design. 9 -16 Only uncontrolled 17,18 and cross-sectional 19 studies on the effect of transdermal estradiol on resistance...

Our findings support the hypothesis that the effect of an oral contraceptive on SHBG levels might be a marker for the thrombotic risk.

Summary. In 56 women with a lymph-node-positive breast carcinoma and 28 matched healthy control subjects, the sensitivity to activated protein C (APC-sr) was determined with an APC resistance test that quantifies the effect of APC on thrombin generation initiated via the extrinsic coagulation pathway. Carriers of the Factor V Leiden mutation were excluded from the study. Significant resistance to APC was found in the breast cancer patients: median APC-sr 2AE02 vs 1AE03 in the healthy control subjects (P < 0AE001). No difference in APC-sr was found between patients with metastases and without metastases. In patients with metastases, protein S levels were significantly elevated compared with patients without metastases and healthy control subjects: 108AE0% vs 96AE0% and 94AE5% (P ¼ 0AE008 and P ¼ 0AE007). The APC-sr correlated with protein S in the control subjects and in patients without metastases but not in patients with metastases. The disturbance of the haemostatic balance probed by the tissue-factor-based APC resistance test might contribute to the cancer-related hypercoagulability.Keywords: activated protein C, APC resistance, thrombin generation, breast cancer, protein S.Activation of the coagulation system in cancer patients is a long known but still poorly understood phenomenon (Goad & Gralnick, 1996;Green & Silverstein, 1996;Francis et al, 1998). It is becoming clear that activation of the haemostatic system is involved in tumour cell growth and angiogenesis (Zhang et al, 1994;Shoji et al, 1998;Zacharski & Ornstein, 1998), and symptomatic and recurrent episodes of (particularly) venous thromboembolism in cancer patients are common (Baron et al, 1998;Sörensen et al, 1998). However, in many cancer patients, the coagulation system is activated without clinical signs of thromboembolism.The hypercoagulability that occurs with cancer has been described to associate with increased levels of coagulation factors, e.g. tissue factor and factor VIIa (Kakkar et al, 1995). The possibility of increased thrombin formation as a result of decreased levels of anticoagulant factors, e.g. antithrombin or the proteins of the protein C pathway has gained much less attention.Hereditary resistance to activated protein C (APC), due to a mutation in coagulation factor V at an APC cleavage site, is associated with hypercoagulability and an increased risk of venous thromboembolism (Dahlback et al, 1993;Griffin et al, 1993;Koster et al, 1993). Recently, it was reported that APC resistance in the absence of factor V Leiden (e.g. acquired APC resistance) is an independent risk factor for venous thrombosis (De Visser et al, 1999;Rodeghiero & Tosetto, 1999). Moreover, acquired APC resistance has been proposed to explain the increased risk of venous thromboembolism associated with the use of oral contraceptives (Rosing et al, 1997;Curvers et al, 1999;Rosing et al, 1999). Although hereditary APC resistance as such is not more common in cancer patients than in the normal population, there are indications that cancer patients without the f...

SummaryThe normalized activated protein C sensitivity ratio (nAPC-sr) determined with an assay that quantifies the effect of APC on thrombin formation initiated via the extrinsic coagulation pathway identifies hereditary and acquired defects of the protein C system. We investigated the influence of assay conditions (analytical variables) and plasma handling (pre-analytical variables) on nAPC-sr obtained with this APC resistance test. The effect of the analytical variables (CaCl2, phospholipid and APC concentrations and the concentration and source of tissue factor) was determined in pooled normal plasma. Inhibition of thrombin formation by APC was dependent on the APC concentration and was also affected by the tissue factor, Ca2+ and phospholipid concentrations. Thus, strict standardization of reactant concentrations is required to obtain reproducible nAPC-sr. Three different tissue factor preparations were compared by determining nAPCsr in plasma samples obtained from 90 healthy individuals. nAPC-sr were similar for all three tissue factor preparations although, compared with the noncommercially available tissue factor used in earlier studies, values determined with commercial tissue factor preparations showed larger variation. Pre-analytical variables, investigated in plasma of nine volunteers (3 normal individuals and 6 individuals with an APCresistant phenotype) were: concentration of anticoagulant (3.2% vs. 3.8% trisodiumcitrate), time before processing of blood (0, 4 and 24 h), centrifugation speed, storage temperature of plasma (–20° C vs. –80° C) and sample thawing. Multiple linear regression analysis showed that only the citrate concentration affected the nAPC-sr, which was higher in samples collected in 3.2% trisodiumcitrate than in samples collected in 3.8% trisodiumcitrate.