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; 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）是中风的第二大原因，导致率最高 与中风相关的发病率和死亡率。虽然ICH可能是自发的，但它也是一个众所周知的功能 严重的脑损伤（TBI）。每年有170万美国人维持TBI，其中近一半将 导致长期残疾。此外，TBI已成为最近军事行动的签名伤口 在伊拉克和阿富汗服兵役期间，有17.3％的退伍军人符合TBI标准的退伍军人培训。 尽管进行了深入的研究工作和重症监护方面的进步，但ICH的30天死亡率尚未 在过去30年中发生了重大变化。当前的管理在很大程度上仅限于优化 脑灌注压力并提供支持性护理。而重组因子VIIA（RFVIIA）的使用 作为止血剂可显着降低出血性中风和TBI患者的血肿生长， 死亡率益处尚不清楚。这部分是由于弥漫性出现并发症的重大风险 炎症和微血管功能障碍会加剧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受伤。另外，使用 RFVIIA受到血栓形成并发症的重大风险的限制，尤其是在较高剂量下。因此， 具有止血和抗炎活性的分子可能比现有的优势有明显的优势 通过减少出血的主要和继发并发症来疗法。 RFVIIA的功效取决于与活化血小板的结合。但是，以前的尝试改进 该药物部分失败了，因为血小板-RFVIIA结合的机理尚不清楚。 因此，拟议的研究的总体目的是阐明血小板-RFVIIA的机制 相互作用作为设计RFVIIA变体具有增强止血功效，降低血小板风险的一种手段， 以及潜在的抗炎和细胞保护特性。 当前提案中的研究将重点介绍procagulant血小板如何存储和调节表面 新型蛋白质的表达有助于RFVIIA的结合和止血活性。这些研究 将结合共聚焦显微镜和免疫染色，然后是透射电子 显微镜以表征该蛋白质的亚细胞定位，并进一步定义其起源，运输， 以及其血小板表达的潜在调节。这些数据将通过进一步的研究来增强 这将利用化学交联和蛋白质组学方法来识别构成的其他合作伙伴 血小板-RFVIIA结合所需的复杂同时相互作用。最后，这项工作也将 确定新型蛋白C-FVIIA嵌合体的治疗潜力 相对于RFVIIA的止血疗效和血小板风险降低，同时保留了抗 蛋白质C的炎症和神经保护活性。流式细胞仪分析和酶活性测定 将在体外确定该嵌合体的血小板结合和活性。止血和血栓形成 使用鼠类隐静脉出血和静脉暂停模型，将在体内确定电势， 分别。潜在的细胞保护和抗炎作用也将使用细胞确定 培养和鼠血管通透性测定法。 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结合并提供有关血小板如何调节表面表达的见解 Procagulant蛋白。此外，PC-FVIIA嵌合体不仅代表了可能是的止血剂 供临床使用开发，它也可以作为解决止血的基本机制的工具，并指导 设计其他潜在疗法，以满足对改进的更好策略的重要需求 由于中风或严重的TBI，具有ICH的退伍军人的结果。