17β-Estradiol (E2) regulates estrogen receptor-α (ERα) target gene transcription through the two independent activation functions (AFs), AF1 and AF2, located in the N-terminal and ligand binding domain of ERα, respectively. We previously reported that ERα is required for the E2 atheroprotective action as well as for its accelerative action on endothelial healing, but its AF1 function is dispensable. Here, we investigated the role of ERαAF2 in these two major beneficial actions of E2 by electively targeting ERαAF2 (named ERαAF2 0 ). Our results prove four points. (i) Compared with WT ERα, the ability of ERαAF2 0 to stimulate the C3 complement or the estrogen response element-thymidine kinase promoter in two cell lines was dramatically decreased, confirming the importance of AF2 in the E2-induced transcriptional activity of ERα. (ii) The uterotrophic action of E2 was totally absent in ERαAF2 0 mice, showing the crucial role of ERαAF2 in E2-induced uterus hyperplasia. (iii) ERαAF2 was dispensable for the accelerative action of E2 on endothelial healing, underlining the functionality of ERαAF2 0 in vivo. (iv) Finally, the atheroprotective effect of E2 was abrogated in ERαAF2 0 LDL-r −/− mice. Thus, whereas ERαAF1 and ERαAF2 are both required for the uterotrophic action of E2, we show that only ERαAF2 is necessary for its atheroprotective effect.atherosclerosis | nuclear receptor | transactivating function E strogens, and particularly, 17β-estradiol (E2), play a pivotal role in sexual development and reproduction and are also implicated in a large number of physiological processes, particularly in the cardiovascular system. Although epidemiological studies (1, 2) and the Nurses' Health Study (3) suggested and all animal models of early atheroma (4, 5) clearly showed a vasculoprotective action of both endogenous and exogenous estrogens, the Women's Health Initiative did not confirm the preventive action of estrogens against coronary heart disease (CHD) (6, 7). However, women who initiate hormone therapy closer to menopause have reduced CHD risk compared with the increase in CHD risk among women more distant from menopause (8). It is clear that these hormones have important effects on vascular physiology and pathophysiology, with potential therapeutic implications such as acceleration of endothelial healing (9) and atheroprotection (10). Interestingly, a delay between ovariectomy and E2 replacement abrogates the prevention of atheroma in the monkey (4) as well as the mouse (11), suggesting that these animal models of atheroma could mimic the problem of timing observed in women in terms of coronary artery risk (2, 4, 12).The action of E2 is mediated by two nuclear receptors, estrogen receptor-α (ERα) and ERβ (13), encoded by two distinct genes, Esr1 and Esr2, respectively. Both ERs belong to the nuclear receptor subfamily of ligand-inducible transcription factors whose members, based on structural and functional similarities, can be subdivided into six distinct regions termed A to F (13). Ligand-induced transcription of...

Although estrogens are known to have a deleterious effect on the venous thrombosis risk and a preventive action on the development of arterial atheroma, their effect on platelet function in vivo remains unclear. Here, we demonstrate that a chronic high physiologic level of estradiol (E2) in mice leads to a marked decrease in platelet responsiveness ex vivo and in vivo compared with ovariectomized controls. E2 treatment led to increased bleeding time and a resistance to thromboembolism. Hematopoietic chimera mice harboring a selective deletion of estrogen receptors (ERs) ␣ or ␤ were used to demonstrate that the effects of E2 were exclusively because of hematopoietic ER␣. Within ER␣ the activation function-1 domain was not required for resistance to thromboembolism, as was previously shown for atheroprotection. This domain is mandatory for E2-mediated reproductive function and suggests that this role is controlled independently. Differential proteomics indicated that E2 treatment modulated the expression of platelet proteins including ␤1 tubulin and a few other proteins that may impact platelet production and activation. Overall, these data demonstrate a previously unrecognized role for E2 in regulating the platelet proteome and platelet function, and point to new potential antithrombotic and vasculoprotective therapeutic strategies. (Blood. 2012;120(8):1703-1712) IntroductionEstradiol-17␤ (E2) and estrogen receptors (ERs) are well known for their pivotal role in sexual development and reproduction. However, they can also modulate cardiovascular and metabolic risk, autoimmune disease progression, and cancer growth. These pleiotropic effects are a consequence of both the widespread expression of ER in many cell populations within the body as well as possibly reflecting the ancestral status of ER in the steroid receptor family. 1 Epidemiologic and experimental studies now support an atheroprotective effect of both endogenous and exogenous estrogens. The Women's Health Initiative study did not show a coronary protective effect of estrogen in postmenopausal women, 2 but subsequent studies have shown that this was because of both inappropriate timing (ie, administering the hormone therapy too late) and the identity of the associated progestin. 3,4 The route of administration of estrogens (oral versus transdermal) has also been shown to have a major impact on the risk of venous thromboembolism. 5 Together these studies indicate that the beneficial or deleterious action of estrogens are strongly influenced by the dose, route, and timing of the hormonal treatment alongside age, genetic, and environmental factors.Although the impact of estrogens on coagulation and venous thromboembolism risk has received much attention, their effect on platelet function remains poorly characterized in vivo. After estrogen treatment, one study reported reduced platelet activation 6 whereas 2 others found increased activation. 7,8 Platelet activation markers were also found at higher levels in postmenopausal compared with premenopausal women...