Platelets are circulating anucleated cell fragments that play a major role in thrombosis and hemostasis in response to a number of stimuli. Platelet activation is a multi-step processes that can be modulated via several known mechanisms. It has long been suspected that sex hormones, like estrogen, have an effect on platelet aggregation and thrombosis; the exact role which estrogen plays in these processes remains unclear. Pre-menopausal women have a lower risk for coronary thrombosis than men– presumably due to higher estrogen levels. However, in postmenopausal women, hormone replacement paradoxically increases the risk for thrombotic events such as coronary and venous thrombosis. Current literature on the subject of estrogen effect on platelets varies considerably.
Previous and current research indicates potential estrogen mechanisms for thrombosis; yet the complete biochemical pathway has not been elucidated. Current literature describes the aggregatory effects of estrogen, but little is said about the fact that premenopausal women still have a lower risk for coronary thrombosis in relation to their male counterparts despite having comparatively higher estrogen levels.
The goal of our study was to better delineate estrogen mediated biochemical pathways involved in platelet aggregation. Blood was collected from healthy male donors and fractionated into platelet rich plasma (PRP). PRP was centrifuged to prepare platelet poor plasma (PPP) to be used as a control during platelet aggregation assays. PRP samples were incubated with varying physiologic concentrations of 17-b-estradiol for 1-10 minutes and then activated with agonist, and compared to activated control samples in the absence of estrogen. Agonists tested include TRAP-1 (activates PAR-1), gamma thrombin (activates PAR-4), collagen, and ADP. Our results show that estrogen at physiologic levels (60-80 nM) has a synergistic effect on platelet aggregation when combined with sub-threshold doses of agonist. We also showed that this synergy is at least in part mediated by lipid rafts, a fact demonstrated in other studies. A role for lipid rafts in the estrogen effect was demonstrated by incubating PRP with the lipid raft disruptor, methyl β cyclodextrin (MβCD), which resulted in the inhibition of estrogen-induced synergy. Incubation with MβCD alone had no effect on platelet aggregation.
We demonstrated that estrogen-induced synergy was absent when platelets, derived from male donors, were washed free of plasma components. Upon the addition of PPP (10-25 uL), synergy was restored. We postulated the necessity of some plasma protein in presenting estrogen to its receptor (ERα and ERβ) for there to be any synergistic effect. Using immunoflourescent microscopy we were able to identify that platelets contain both ERα and ERβ and that the expression of each is altered upon binding of estrogen and TRAP-1. Using filtration techniques we were able to determine that the molecular weight of the necessary protein to restore synergy is greater than 90-100,000 kDa. The most likely candidate was the dimerized plasma sex hormone binding globulin (SHBG), due to its high affinity for estrogens. Support for this comes from the ability of anti-SHBG antibodies to block the estrogen-induced synergy of agonist-induced platelet aggregations.
Platelets derived from premenopausal females showed no estrogen-induced synergy of platelet aggregations under the identical conditions used with platelets derived from males. However, platelets derived from postmenopausal women did show synergistic effects of estrogen mirroring effects observed with male platelets samples. In all cases where estrogen-induced synergy of aggregation was observed, incubation of the same platelet samples with estrogen for 2-24hrs totally reversed the synergistic effect. This would be comparable to the premenopausal woman whose platelets in vivo are constantly exposed to high levels of estrogen. Our studies demonstrate that the SHBG must present estrogen to the platelet membrane estrogen receptors and that these receptors are down-regulated upon prolonged exposure to estrogen resulting in reduced levels of platelet aggregation in the presence of sub-optimal concentrations of agonists. Results of our studies would account for the varied effects of estrogen on platelet activation reported in the literature. Studies are in progress with testosterone and second messengers.
No relevant conflicts of interest to declare.
