Age-Related Decrease in Mitofusin 1 Results in Platelet Dysfunction and Thrombosis

项目摘要/摘要 衰老是血栓性疾病的最大危险因素之一，如深静脉血栓形成、心肌梗死 脑梗塞和中风。通过激活和聚集，血小板是血栓形成的中心。之前 研究表明，与年龄相关的血小板活化增加，但其分子机制尚不清楚。 尽管血小板是无核的，但它们包含有功能的线粒体和活跃的泛素蛋白小体系统 (UPS)。UPS可以去除陈旧和受损的蛋白质，以维持细胞的动态平衡。我们的初步数据 提示从健康老年人(75岁)分离的血小板显示UPS活性增加和 与分离的血小板相比，关键线粒体融合蛋白Mfn1的表达降低 来自健康青年受试者(18-35岁)。老年人的血小板也表现出增加的趋势 线粒体氧化剂的产生和血小板的激活。基于这些观察，我们将测试 UPS随年龄变化导致线粒体功能障碍加重的假说 导致线粒体活性氧簇(MtROS)的产生以刺激血小板激活 以及随后的血栓形成。在目标1中，我们将描述年轻人和老年人分离的血小板中UPS的特征 健康的人体受试者。同时进行Mfn1水平和血小板活化测量，以 确定这些系统如何因UPS中与年龄相关的变化而改变。建立一种小鼠模型 对于分子机制，我们将在青年(8-10周)和老年(&gt；1.5岁)进行相同的测量 野生型(WT)小鼠。这些研究将得到人类来源的体外细胞培养系统的补充。 CD34+祖细胞，可分化为血小板，用于机制研究，利用 慢病毒专门针对UPS组件。在目标2中，我们产生了血小板特异性的Mfn1基因敲除 (KO)小鼠确定血小板中Mfn1减少是否会导致mtROS增加和易感性增加 血栓形成。我们将测量血小板形态、mtROS产生和线粒体的变化。 在年轻和老年血小板特异性Mfn1 KO小鼠中进行生物能量学研究，并将其与年轻和老年WT小鼠进行比较。 将使用腺病毒在老年WT小鼠和血小板特异性Mfn1 KO小鼠中过表达Mfn1，以确定它是否 拯救老年人的表型。此外，年轻和老年WT和血小板特异性Mfn1(KO)小鼠将被 受制于激光诱导的血管损伤，以确定血小板活化和血栓形成易感性是否增加 当Mfn1不存在时。慢病毒敲除和过表达Mfn1在我们的CD34+细胞培养系统中将 补充这些机械论研究。最后，目标3将确定临床使用的药物MitoQ (mtROS清道夫)或Bortezomib(蛋白酶体抑制剂)可以减弱年龄依赖性血小板的激活和 WT和血小板特异性Mfn1 KO小鼠模型中的血栓形成。这个项目的成功完成将揭开面纱 健康衰老过程中血小板活化和血栓形成的新机制及新策略 调整临床可用药物的用途，以减轻与年龄相关的血栓形成。

PROJECT SUMMARY/ABSTRACT Aging is one of the greatest risk factors for thrombotic diseases such as deep vein thrombosis, myocardial infarction and stroke. Platelets are central to thrombus formation through their activation and aggregation. Prior studies show age-dependent increases in platelet activation, but the molecular mechanisms remain unknown. Though platelets are anucleate they contain functional mitochondria and an active ubiquitin-proteosome system (UPS). The UPS removes old and damaged proteins to maintain cellular homeostasis. Our preliminary data suggests that platelets isolated from healthy aged (>75 years) human subjects show increased UPS activity and decreased expression of the key mitochondrial fusion protein mitofusin 1 (MFN1) compared to platelets isolated from healthy young subjects (18-35 years). Platelets from aged individuals also exhibited increased mitochondrial oxidant production and platelet activation. Based on these observations, we will test the hypothesis that age-dependent changes in the UPS lead to increased mitochondrial dysfunction resulting in mitochondrial reactive oxygen species (mtROS) production to stimulate platelet activation and subsequent thrombosis. In Aim 1, we will characterize the UPS in isolated platelets from young and aged healthy human subjects. Concomitant MFN1 levels and platelet activation measurements will be made to determine how these systems are altered by the age-related changes in the UPS. To establish a murine model for molecular mechanism, we will perform identical measurements in young (8-10 week) and aged (>1.5 year) wildtype (WT) mice. These studies will be complemented by an in vitro cell culture system of human derived CD34+ progenitor cells which can be differentiated into platelets and used for mechanistic studies that utilize lentivirus to specifically target the UPS components. In Aim 2, we generated platelet-specific MFN1 knockout (KO) mice to determine whether decreased MFN1 in platelets results in increased mtROS and susceptibility to thrombosis. We will measure changes in platelet morphology, mtROS production, and mitochondrial bioenergetics in young and aged platelet-specific MFN1 KO mice and compare it to young and aged WT mice. Adenovirus will be used to overexpress MFN1 in old WT and platelet-specific MFN1 KO mice to determine if it rescues the aged phenotype. Additionally, young and aged WT and platelet-specific MFN1 (KO) mice will be subject to laser-induced vascular injury to determine if platelet activation and thrombosis susceptibility increases when MFN1 is absent. Lentivirus knockdown and overexpression of MFN1 in our CD34+ cell culture system will complement these mechanistic studies. Finally, Aim 3 will determine whether the clinically used drugs MitoQ (mtROS scavenger) or Bortezomib (proteasome inhibitor) can attenuate age-dependent platelet activation and thrombosis in WT and platelet-specific MFN1 KO murine models. Successful completion of this project will unveil a novel mechanism by which platelet activation and thrombosis occur during healthy aging and new strategies to repurpose clinically available drugs to mitigate age-related thrombosis.