Hormone therapy, oral contraceptives, and tamoxifen increase the risk of thrombotic disease. These compounds also reduce plasma content of tissue factor pathway inhibitor-1 (TFPI), which is the physiological inhibitor of the tissue factor pathway of coagulation. The current

E2, EE2, tamoxifen, raloxifene, and fulvestrant inhibited endothelial production of TFPI by a mechanism apparently independent of TFPI transcription.

Human endothelial cell cultures were treated with 17beta-estradiol (E2), 17alpha-ethinylestradiol (EE2), tamoxifen, raloxifene, or fulvestrant. Protein levels of TFPI in cell media and cell lysates were measured by an enzyme-linked immunosorbent assay, and TFPI mRNA levels were assessed by quantitative PCR. Expression of ERalpha was analysed by immunostaining.

All compounds (each in a concentration of 10 nM) reduced TFPI in cell medium, by 34% (E2), 21% (EE2), 16% (tamoxifen), and 28% (raloxifene), respectively, with identical inhibitory effects on cellular TFPI levels. Expression of TFPI mRNA was principally unchanged. Treatment with fulvestrant, which was also associated with down-regulation of secreted TFPI (9% with 10 nM and 26% with 1000 nM), abolished the TFPI-inhibiting effect of raloxifene, but not of the other compounds. Notably, the combination of 1000 nM fulvestrant and 10 nM raloxifene increased TFPI secretion, and, conversely, 10 nM of either tamoxifen or raloxifene seemed to partly (tamoxifen) or fully (raloxifene) counteract the inhibitory effect of 1000 nM fulvestrant. The cells did not express the regular nuclear 66 kDa ERalpha, but instead a 45 kDa ERalpha, which was not regulated by estrogens or ER modulators.