OPA1 an Estrogen-Mediated Modulator of Platelet Hyperactivation

强有力的流行病学证据将绝经前雌激素暴露与静脉炎风险增加联系起来 血栓形成的妇女，特别是在怀孕期间，在产后期间，并在妇女服用 含雌激素的口服避孕药（OCP）。本建议涉及一个重大的知识差距， 雌激素与血小板活化过去的研究集中在雌激素如何改变止血和 纤溶系统这一以血小板为重点的提案代表了以下方面富有成效的合作： 爱荷华州和犹他州的大学。我们在Fraurons队列和怀孕人类中的初步研究， 显示女性血小板OPA 1水平升高，可预测冠状动脉疾病的风险，或与 妊娠晚期血小板活化增加。在血小板缺陷型OPA 1敲除（KO）小鼠中 我们发现雌性OPA 1 KO小鼠与雄性相比免于血栓形成。的 血栓形成表型在卵巢切除术后逆转。基于这些初步发现，我们假设 OPA 1和OPA 1活性调节剂OMA 1和YMEL 1在血小板活化中调节血小板活化， 雌激素依赖方式和血小板OPA 1水平预测雌激素调节血小板 activation.为了解决R61阶段的这一假设，Aim 1将确定R61阶段的分子机制。 雌激素介导的和OPA-1依赖的血小板活化调节使用血小板特异性 OPA 1缺失并将产生OMA 1和YMEL 1血小板特异性KO小鼠，具有增加的OPA 1活性。 目的2检测血小板OPA 1、OMA 1和YMEL 1的表达水平，并将表达水平与血小板聚集相关。 在怀孕和使用OCP的情况下激活。我们还将开发人类iPSC模型系统， 产生MK以确定雌激素是否直接调节这些基因的表达。R33阶段将 剖析女性中OPA 1增加与血小板活化之间的分子机制。目标1将 对分离自OMA 1和YMEL 1的小鼠血小板进行详细的生理和分子表型分析 KO和OPA过表达转基因小鼠。此外，我们还将研究与增加的 OPA 1活性、线粒体能量学和血小板活化。Aim 2将通过以下方式补充动物研究： 检查孕妇和服用抗血小板药物的妇女的血小板中线粒体功能和信号传导的变化， OCP我们将从iPSC衍生的MK中基因切除OMA 1和YMEL 1，以直接测试雌激素 以OPA 1依赖性方式调节MK和血小板功能。通过整合体内和体外研究 在人类和小鼠模型中，我们的方法是转化的，创新的，对RFA高度敏感。 这些研究将首次确定是否增加雌激素暴露在怀孕和口服避孕药的使用 重新编程MK和血小板发育，以增加血栓并发症，并确定新的机制 对于这种增加的风险。这些研究的影响是，它们可以识别出可以预测的新型生物标志物 在怀孕期间或使用OCP之前，易感女性血栓形成的风险增加。

Strong epidemiological evidence links pre-menopausal estrogen exposure to increased risk of venous thrombosis in women, particularly during pregnancy, in the immediate postpartum period, and in women taking estrogen-containing oral contraceptives (OCP). This proposal addresses a significant knowledge gap linking estrogens with platelet activation. Studies in the past have focused on how estrogens alter the hemostatic and fibrinolytic system. This proposal, which focuses on platelets, represents a productive collaboration between the Universities of Iowa and Utah. Our preliminary studies in the Framingham cohort and in pregnant humans, reveal increased platelet OPA1 levels in females, that predict risk of coronary artery disease, or correlate with increased platelet activation in the third trimester of pregnancy. In platelet-deficient OPA1 knockout (KO) mice we discovered that female OPA1KO mice were protected from thrombosis compared to males. The thrombosis phenotype was reversed after oophorectomy. Based on these preliminary findings, we hypothesize that OPA1 and regulators of OPA1 activity, OMA1 and YMEL1, modulate platelet activation in an estrogen-dependent manner and OPA1 levels in platelets predict estrogen modulated platelet activation. To address this hypothesis in the R61 phase, Aim 1 will determine the molecular mechanisms for estrogen-mediated and OPA-1-dependent modulation of platelet activation using mice with platelet specific OPA1 deletion and will generate OMA1 and YMEL1 platelet specific KO mice, with increased OPA1 activity. Aim 2 will determine platelet OPA1, OMA1, and YMEL1 expression and correlate expression levels to platelet activation in the setting of pregnancy and OCP use. We will also develop a human iPSC model system to generate MKs to determine if estrogen directly regulates the expression of these genes. The R33 phase will dissect the molecular mechanisms linking increased OPA1 with platelet activation in females. Aim 1 will perform detailed physiological and molecular phenotyping of murine platelets isolated from OMA1 and YMEL1 KO and OPA overexpressing transgenic mice. In addition, we will examine the mechanisms linking increased OPA1 activity, mitochondrial energetics and platelet activation. Aim 2 will complement animal studies by examining changes in mitochondrial function and signaling in platelets from pregnant women and women on OCPs. We will genetically ablate OMA1 and YMEL1 from iPSC-derived MKs to directly test if estrogen modulates MK and platelet function in an OPA1 dependent manner. By integrating in vivo and in vitro studies in humans and mouse models, our approach is translational, innovative and highly responsive to the RFA. These studies will determine for the first time whether increased estrogen exposure in pregnancy and OCP use reprogram MKs and developing platelets to increase thrombotic complications and identify novel mechanisms for this increased risk. The impact of these studies, is that they may identify novel biomarkers that may predict increased risk of thrombosis in susceptible females during pregnancy or prior to OCP use.