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

了解和增强因子 VIIa 紧急止血的治疗效果

• 批准号: 10394710
• 负责人: AMMON M FAGER
• 金额: --
• 依托单位: DURHAM VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10394710
• 关键词: Address; Affect; Afghanistan; Alpha Granule; American; Anti-Inflammatory Agents; Antiinflammatory Effect; Area; Binding; Binding Sites; Biological Assay; Biology; Blood Platelets; Brain hemorrhage; Cell Culture Techniques; Cerebral perfusion pressure; Cessation of life; Chemicals; Chimera organism; Clinical; Complex; Confocal Microscopy; Critical Care; Data; Development; Diffuse; Dose; Emergency Situation; Exhibits; Factor VIIa; Goals; Growth; Health Care Costs; Hematoma; Hemorrhage; Hemostatic Agents; Hemostatic function; Human; In Vitro; Inflammation; Inflammatory; Intervention; Intracranial Hemorrhages; Iraq; Knowledge; Life; Medical; Membrane Proteins; Microvascular Dysfunction; Modeling; Molecular; Morbidity - disease rate; Mus; Nature; Neuronal Injury; Operative Surgical Procedures; Outcome; Patient-Focused Outcomes; Patients; Persons; Pharmaceutical Preparations; Property; Protein C; Proteins; Proteomics; Recombinants; Regulation; Relative Risks; Research; Risk; Saphenous Vein; Stains; Stroke; Supportive care; Surface; TBI treatment; Testing; Therapeutic; Thromboplastin; Time; Training; Translating; Transmission Electron Microscopy; Traumatic Brain Injury; Treatment Efficacy; Variant; Vascular Permeabilities; Venous; Veterans; Work; activated protein C receptor; base; cost; crosslink; design; disability; enzyme activity; falls; improved; improved outcome; in vivo; insight; meetings; military operation; military service; mortality; novel; receptor; receptor expression; recombinant FVIIa; thrombogenesis; thrombotic; thrombotic complications; tool; trafficking; translational impact; treatment strategy; wound

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.

颅内出血（ICH）是中风的第二大常见原因，其发病率最高 中风相关的发病率和死亡率。虽然ICH可以是自发的，但它也是一个众所周知的特征， 严重创伤性脑损伤（TBI）。每年有170万美国人遭受TBI，其中近一半将 导致长期残疾。此外，TBI已成为近期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TBI标准。 尽管在重症监护方面进行了深入的研究和取得了进展，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。目前的管理仍然主要局限于优化 脑灌注压和提供支持性护理。虽然使用重组因子VIIa（rFVIIa） 作为止血剂显著减少出血性中风和TBI患者的血肿生长， 死亡率效益仍不清楚。这部分是由于弥漫性的并发症的重大风险， 炎症和微血管功能障碍可加重出血并引起继发性神经元 通过不适合手术或止血干预的机制造成的损伤。此外，使用 rFVIIa受到血栓性并发症的显著风险的限制，特别是在较高剂量下。因此 同时具有止血和抗炎活性的分子可能比现有的 通过减少出血的原发性和继发性并发症来治疗。 rFVIIa的疗效依赖于与活化血小板的结合;然而，先前的改善rFVIIa的效果的尝试并不成功。 这种药物已经失败，部分原因是血小板-rFVIIa结合的机制还不清楚。 因此，拟议研究的总体目标是阐明血小板-rFVIIa的机制 相互作用作为设计具有增强的止血功效、降低的血栓形成风险 以及潜在的抗炎和细胞保护特性。 目前建议的研究将集中在促凝血血小板如何储存和调节表面 表达有助于rFVIIa的结合和止血活性的新蛋白质。这些研究 将使用共聚焦显微镜和免疫金染色的组合，然后进行透射电子显微镜检查。 显微镜来表征这种蛋白质的亚细胞定位，并进一步确定其来源，运输， 和其血小板表面表达的潜在调节。这一数据将通过进一步研究得到充实 它将利用化学交联和蛋白质组学方法来鉴定组成 血小板-rFVIIa结合所需的复杂的同时相互作用。最后，这项工作还将 确定新型蛋白质C-FVIIa嵌合体的治疗潜力，所述嵌合体被设计为具有以下潜力： 相对于rFVIIa，止血功效增加，血栓形成风险降低，同时保留抗- 蛋白C的炎症和神经保护活性。流式细胞术分析和酶活性测定 将决定这种嵌合体在体外的血小板结合和活性。止血效果和血栓形成 将使用鼠隐静脉出血和静脉停滞模型在体内测定潜力， 分别潜在的细胞保护和抗炎作用也将使用细胞免疫学方法来确定。 培养和鼠血管渗透性测定。 完成后，这项工作将通过阐明必要的相互作用产生积极的翻译影响 用于血小板-rFVIIa结合，并提供血小板如何调节 促凝血蛋白此外，PC-FVIIa嵌合体不仅代表了一种止血剂， 为临床使用而开发，它也可作为一种工具，用于解决止血的基本机制，并指导 设计其他潜在的治疗方法，以解决对更好的策略的重大未满足需求， 因中风或严重TBI而患有ICH的退伍军人的结局。