Non-Genomic Effects of Estradiol on Human Platelets: Regulation of Mitogen Activated Protein Kinase Activation and Enhanced Spreading and Aggregation.

17β-estradiol or estrogen (E2) is a sex hormone that modulates platelet function and is widely used in hormone replacement therapy (HRT). We and others have previously demonstrated that human megakaryocytes and platelets posses estrogen receptors ERα and ERβ. HRT treatment augmented the bone marrow megakaryocytes without increasing other bone marrow cells, suggesting that E2 can modulate proliferation of megakaryocytes. Since mitogen activated protein kinases (MAPKs) are critical for 1) cell proliferation, 2) megakarytocyte differentiation and proplatelet formation and estrogen activates MAPK in other cell types, we hypothesized that estrogen regulates the activation of MAPK in human platelets. Signaling was studied using washed platelets from male and female subjects in response to varying concentrations of estrogen. Compared to the ethanol (vehicle) treated platelets, 1 nM E2 treated platelets for 60 seconds resulted in an enhanced activation of extracellular signal-regulated kinase 2 (ERK 2) and P38 but not Jun N-kinase (JNK). These results suggest that E2 can cause a non-genomic signaling in human platelets. The MEK inhibitors PD98059 and U0216 blocked the E2 effect, suggesting that the activation of ERK 2 was mediated through the upstream mitogen activated protein kinase kinase (MAPKK). Because E2 can modulate actin reorganization in other cell types and cell spreading is promoted by ERK 2 activation, we examined the effect of E2 on platelet spreading - a process not dependent on agonist stimulation. Compared to ethanol treated platelets, platelets preincubated with 100 nM E2 for 60 seconds and stained with rhodamine phallodine exhibited a ~60 % greater spreading at 5 and 15 minutes. This observation suggests that that E2 can cause rapid actin cytoskeletal reorganization in platelets. Since inhibition of ERK 2 activation blocks aggregation to low doses of thrombin and collagen, we examined a role for E2 in platelet aggregation. E2 alone did not induce platelet aggregation. However, E2 potentiated aggregation with low but not high doses of thrombin and collagen related peptide (CRP) (P=0.05 for 0.02 μg/ml thrombin and P<0.001 for 0.2 μg/ml CRP). Our data demonstrates that E2 can activate MAPK through a non-genomic mechanism and this activation correlates with greater platelet functions like spreading and aggregation. Our findings support a mechanism whereby a consistent non-genomic enhancement of platelet signaling and reactivity by E2 may underlie the increased cardiovascular events observed in recent randomized clinical trials with HRT.