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风险增加联系在一起 妇女中的血栓形成，特别是在怀孕期间、产后即刻和服用 含雌激素口服避孕药(OCP)。这项提议解决了一个重大的知识鸿沟 雌激素与血小板活化有关。过去的研究主要集中在雌激素如何改变止血和 纤溶系统。这项专注于血小板的提案代表了两国之间富有成效的合作 爱荷华州大学和犹他州大学。我们在弗雷明翰队列和孕妇身上进行的初步研究， 发现女性血小板OPA1水平升高，预测冠状动脉疾病的风险，或与 妊娠晚期的血小板活化增加。血小板缺陷OPA1基因敲除(KO)小鼠 我们发现，与雄性相比，雌性OPA1KO小鼠免受血栓形成的保护。这个 卵巢切除后血栓表型逆转。基于这些初步发现，我们假设 OPA1和OPA1活性调节剂OMA1和YMEL1调节血小板活化 雌激素依赖方式和血小板中OPA1水平预测雌激素调节的血小板 激活。为了在R61阶段解决这一假设，目标1将确定 雌激素介导和OPA-1依赖的血小板特异性小鼠对血小板活化的调节 OPA1缺失，并将产生OMA1和YMEL1血小板特异性KO小鼠，OPA1活性增加。 AIM 2将检测血小板OPA1、OMA1和YMEL1的表达并将其与血小板的表达水平相关联 在妊娠和OCP使用的设置中激活。我们还将开发一个人类IPSC模型系统来 产生巨噬细胞集落刺激因子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OPA1升高与血小板活化相关的分子机制。目标1将 对从OMA1和YMEL1分离的小鼠血小板进行详细的生理和分子表型鉴定 KO和OPA过表达转基因小鼠。此外，我们将研究增加联系的机制 OPA1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 检测孕妇和女性血小板线粒体功能和信号的变化 OCPS。我们将从基因上去除IPSC来源的MKs中的OMA1和YMEL1，以直接测试雌激素是否 以OPA1依赖的方式调节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，我们的方法是翻译的、创新的，并且对RFA高度响应。 这些研究将首次确定怀孕期间雌激素暴露增加和OCP使用 重新编程MKs和开发血小板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，他们可能识别出新的生物标记物，可以预测 在怀孕期间或使用口服避孕药之前，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.