Understanding and Enhancing the Therapeutic Efficacy of Factor VIIa for Emergency Hemostasis

颅内出血(ICH)是导致中风的第二大常见原因，也是发病率最高的原因 与中风相关的发病率和死亡率。虽然脑出血可以是自发的，但它也是众所周知的 重型颅脑损伤(TBI)。每年有170万美国人患有脑外伤，其中近一半的人将 导致长期残疾。此外，颅脑损伤已成为最近军事行动的标志性伤口。 在伊拉克和阿富汗服役期间，17.3%的退伍军人符合创伤性脑损伤标准。 尽管在重症监护方面做出了密集的研究努力并取得了进展，但ICH的30天死亡率并没有 在过去的30年里发生了巨大的变化。目前的管理在很大程度上仍然局限于优化 并提供支持性护理。而使用重组因子VIIa(RFVIIa) 作为一种显著减少出血性中风和脑外伤患者血肿生长的止血剂， 死亡率方面的好处仍不清楚。这在一定程度上是由于弥漫性并发症的重大风险。 炎症和微血管功能障碍可加剧出血并导致继发性神经元 通过不适合手术或止血干预的机制造成的伤害。此外，还使用了 RFVIIa受到血栓并发症的严重风险的限制，特别是在较高剂量时。因此，a 同时具有止血和抗炎活性的分子可能比现有的分子具有明显的优势 通过减少出血的原发和继发并发症进行治疗。 RFVIIa的疗效依赖于与激活的血小板的结合；然而，以前的尝试改善 这种药物之所以失败，部分原因是人们对血小板与rFVIIa结合的机制还不太清楚。 因此，拟议研究的总体目标是阐明血小板-rFVIIa的机制。 相互作用作为设计rFVIIa变体的一种手段，具有增强的止血效果，降低血栓形成的风险， 以及潜在的抗炎和细胞保护作用。 目前提案中的研究将集中在促凝血剂血小板如何储存和调节表面。 表达一种有助于rFVIIa结合和止血活性的新蛋白。这些研究 将使用共聚焦显微镜和免疫金染色相结合的方法，然后再使用透射电子 显微镜来表征这种蛋白质的亚细胞定位，并进一步确定它的来源，运输， 以及对其血小板表面表达的潜在调节。这一数据将通过进一步的研究得到加强 这将利用化学交联和蛋白质组学方法来确定组成其他合作伙伴(S) 血小板与rFVIIa结合所需的复杂的同时相互作用。最后，这项工作还将 确定一种新的蛋白C-FVIIa嵌合体的治疗潜力 相对于rFVIIa，提高了止血效果并降低了血栓风险，同时保留了 蛋白C的炎症和神经保护活性流式细胞仪和酶活性分析 将在体外决定这个嵌合体的血小板结合和活性。止血效果和血栓形成 将使用小鼠隐静脉出血和静脉瘀血模型在体内确定潜伏期， 分别进行了分析。潜在的细胞保护和抗炎作用也将通过细胞来确定。 培养和小鼠血管通透性测定。 完成后，这项工作将通过阐明必要的相互作用而产生积极的翻译影响 为血小板-rFVIIa结合提供了对血小板如何调节表面表达的洞察力 促凝血蛋白。此外，PC-FVIIa嵌合体不仅代表了一种可能是 开发用于临床，它也是一个工具，以解决基本的止血机制和指导 设计其他潜在的治疗方法，以解决重大的未得到满足的需求，需要更好的战略来改进 退伍军人卒中或严重脑外伤后脑出血的转归。

Intracranial hemorrhage (ICH) is the second most common cause of stroke and results in the highest rates of stroke-related morbidity and mortality. While ICH can be spontaneous, it is also a well-known feature of severe traumatic brain injury (TBI). Each year 1.7 million Americans sustain a TBI and nearly half of these will result in long-term disability. In addition, TBI has become the signature wound of recent military operations and training with 17.3% of veterans meeting criteria for TBI during military service in Iraq and Afghanistan. Despite intensive research efforts and advances in critical care, the 30-day mortality rate from ICH has not changed significantly in the last 30 years. Current management remains largely restricted to optimizing cerebral perfusion pressure and providing supportive care. While the use of recombinant Factor VIIa (rFVIIa) as a hemostatic agent significantly reduces hematoma growth for patients with hemorrhagic stroke and TBI, the mortality benefit remains unclear. This is, in part, due to the significant risk of complications from diffuse inflammation and microvascular dysfunction that can exacerbate hemorrhage and cause secondary neuronal injury via mechanisms that are not amenable to surgical or hemostatic intervention. In addition, the use of rFVIIa is limited by a significant risk of thrombotic complications, particularly at higher doses. Therefore, a molecule with both hemostatic and anti-inflammatory activities could have distinct advantages over existing therapies by reducing both the primary and secondary complications of bleeding. The efficacy of rFVIIa is dependent on binding to activated platelets; however, previous attempts to improve this drug have failed, in part, because the mechanism of platelet-rFVIIa binding is not well understood. Therefore, the overall objective of the proposed studies is to elucidate the mechanisms of platelet-rFVIIa interaction as a means to design rFVIIa variants with enhanced hemostatic efficacy, reduced thrombotic risk, and potential anti-inflammatory and cytoprotective properties. Studies in the current proposal will focus on how procoagulant platelets store and regulate the surface expression of a novel protein that contributes to the binding and hemostatic activity of rFVIIa. These studies will use a combination of confocal microscopy and immunogold staining followed by transmission electron microscopy to characterize the subcellular localization of this protein and further define its origin, trafficking, and the potential regulation of its platelet-surface expression. This data will be enhanced by further studies that will utilize a chemical cross-linking and proteomics approach to identify additional partner(s) that make up the complex simultaneous interactions required for platelet-rFVIIa binding. Finally, this work will also determine the therapeutic potential of a novel Protein C-FVIIa chimera designed with the potential for increased hemostatic efficacy and reduced thrombotic risk relative to rFVIIa while retaining the anti- inflammatory and neuroprotective activities of Protein C. Flow cytometric analyses and enzyme activity assays will determine the platelet binding and activity of this chimera in vitro. Hemostatic efficacy and thrombogenic potential will be determined in vivo using murine saphenous vein bleeding and venous stasis models, respectively. The potential cytoprotective and anti-inflammatory effects will also be determined using cell culture and murine vascular permeability assays. When completed, this work will have a positive translational impact by elucidating the interactions necessary for platelet-rFVIIa binding and providing insights into how platelets regulate the surface expression of procoagulant proteins. In addition, the PC-FVIIa chimera not only represents a hemostatic agent that could be developed for clinical use, it also serves as a tool to address basic mechanisms of hemostasis and guide the design of other potential therapies to address the significant unmet need for better strategies to improve outcomes in veterans with ICH due to stroke or severe TBI.