In Vivo Regulation of Factor IXa by Protein S in Hemophilia and Systemic Hypercoagulability

蛋白 S 在血友病和全身高凝状态中对因子 IXa 的体内调节

• 批准号: 10321939
• 负责人: JOHN Patrick SHEEHAN
• 金额: $ 38.88万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10321939
• 关键词: Ablation; Address; Affinity; Antibodies; Anticoagulants; Antisense Oligonucleotides; Antithrombins; Binding; Binding Sites; Biochemical; Biological; Blood Coagulation Disorders; Blood coagulation; Clinical; Coagulation Process; Collaborations; Complex; Disease; Dose; Embryo; Enzyme Precursors; Enzymes; Factor IXa; Factor VIIIa; Factor Va; Failure; Functional disorder; Generations; Genes; Genetic; Genotype; Goals; Half-Life; Hemophilia A; Hemophilia B; Hemorrhage; Hemostatic Agents; Hemostatic function; Heparin; Heparin Binding; Heparitin Sulfate; Human; Mediating; Mission; Modeling; Mus; National Heart, Lung, and Blood Institute; Peptide Hydrolases; Pharmacologic Substance; Phenotype; Physiological; Physiology; Plasma; Plasma Enhancement; Positioning Attribute; Prevention; Protease Domain; Protein S; Protein S Deficiency; Proteins; Public Health; Recombinants; Recovery; Regulation; Research; Role; Structure; TFPI; Testing; Therapeutic; Thrombin; Thrombophilia; Thrombosis; Variant; Woman; activated Protein C; base; cancer procoagulant; cofactor; defined contribution; hormonal contraception; in vivo; inhibitor; innovation; insight; man; novel; novel strategies; response; thrombogenesis; thrombotic

Protein S (PS) is a critically important natural anticoagulant as demonstrated by fatal consumptive coagulopathies in homozygous deficiency for mouse and man. Originally discovered as a cofactor for activated protein C (APC), subsequent studies have demonstrated a plethora of APC-independent anticoagulant mechanisms for PS without clear demonstration of their respective physiologic relevance. Recently, genetic “rebalancing” studies in the mouse and direct biochemical evidence suggest an important interaction between PS and factor IXa (FIXa) The objective of this proposal is to define the role of PS in the in vivo regulation of FIXa activity. The central hypothesis is that PS is an important physiologic regulator of FIXa in both hemostasis and systemic hypercoagulable states. The central hypothesis will be tested by pursuing three specific aims: 1) Identify the predominant mechanism(s) for regulation of FIXa activity in human plasma, 2) Determine the contribution of protein S affinity to the in vivo activity of recombinant FIX(a), and 3) Determine the contribution of protein S deficiency to in vivo regulation of FIX(a) activity. To pursue these aims, we have developed a panel of recombinant human FIX variants that possess reduced affinity for antithrombin (AT), heparin and PS. In the first aim, inhibition of FIXa activity in the intrinsic Xase complex by PS, PS-TFPI and PS-APC will be examined using purified components and the FIXa variants. Likewise, the contribution of AT- and PS-dependent mechanisms to the regulation of plasma thrombin generation will be examined with the FIXa variants and inhibitory antibodies in immunodepleted plasma. In the second aim, the human FIXa variants will first be employed to evaluate the impact of AT, heparin and PS affinity on zymogen and protease recovery and clearance. Secondly, dose-dependent in vivo hemostatic and thrombotic activity will be examined using established models in the hemophilia B mouse to evaluate the contribution of AT and PS-dependent mechanisms. In the third aim, Pros1 specific antisense oligonucleotides (ASO) will be employed to create PS deficiency in wild type and hemophilia B mice. The impact of the ASO-mediated PS deficiency on bleeding and thrombosis phenotypes will be evaluated using established models, including the ability to ameliorate the baseline bleeding phenotype in hemophilia B mice. Further, the effect of ASO-mediated PS deficiency on the in vivo hemostatic and thrombotic activity of injected FIXa variants will be examined in hemophilia B mice, including the ability of human PS to “rescue” the effects of PS deficiency. Completion of these aims will elucidate the critical role of the PS-FIXa interaction in the physiologic regulation of FIXa procoagulant activity within the intrinsic Xase complex. The proposed research is innovative because it addresses the in vivo significance of a novel mechanism for regulating the rate-limiting step in the coagulation response. This contribution will be significant because it will provide a conceptual basis for manipulating the FIXa-PS interaction in hemophilia and systemic hypercoagulable states associated with elevated levels of plasma FIXa activity.

致命的消耗性疾病证明蛋白质 S (PS) 是一种极其重要的天然抗凝剂 小鼠和人纯合缺陷的凝血病。最初被发现是作为激活的辅助因子 蛋白 C (APC)，随后的研究表明存在大量不依赖于 APC 的抗凝剂 PS 的机制尚未明确证明其各自的生理相关性。最近，遗传 小鼠“再平衡”研究和直接生化证据表明，两者之间存在重要的相互作用 PS 和因子 IXa (FIXa) 该提案的目的是确定 PS 在 FIXa 体内调节中的作用 活动。中心假设是 PS 是 FIXa 在止血和止血方面的重要生理调节因子。 全身高凝状态。将通过追求三个具体目标来检验中心假设：1）确定 调节人血浆中 FIXa 活性的主要机制，2) 确定 蛋白 S 对重组 FIX 体内活性的亲和力 (a)，以及 3) 确定蛋白 S 的贡献 FIX(a) 活性的体内调节缺陷。为了实现这些目标，我们开发了一个小组 重组人 FIX 变体对抗凝血酶 (AT)、肝素和 PS 的亲和力降低。在第一个 目的，将检查 PS、PS-TFPI 和 PS-APC 对内在 Xase 复合物中 FIXa 活性的抑制 使用纯化的组件和 FIXa 变体。同样，AT 和 PS 依赖的贡献 将使用 FIXa 变体检查血浆凝血酶生成的调节机制 免疫缺陷血浆中的抑制性抗体。在第二个目标中，人类 FIXa 变体将首先被 用于评估 AT、肝素和 PS 亲和力对酶原和蛋白酶回收率的影响 清除。其次，将使用以下方法检查剂量依赖性体内止血和血栓形成活性： 在 B 型血友病小鼠中建立模型以评估 AT 和 PS 依赖性的贡献 机制。第三个目标是利用Pros1特异性反义寡核苷酸（ASO）来创建PS 野生型和 B 型血友病小鼠中缺乏。 ASO 介导的 PS 缺乏对出血和出血的影响 将使用已建立的模型评估血栓形成表型，包括改善血栓形成的能力 B 型血友病小鼠的基线出血表型。此外，ASO 介导的 PS 缺乏对体内的影响 将在血友病 B 小鼠中检查注射的 FIXa 变体的体内止血和血栓形成活性，包括 人类PS“拯救”PS缺乏影响的能力。完成这些目标将阐明 PS-FIXa 相互作用在内在 FIXa 促凝血活性的生理调节中的关键作用 Xase复合物。拟议的研究具有创新性，因为它解决了小说的体内意义 调节凝血反应中限速步骤的机制。这一贡献将是巨大的 因为它将为操纵血友病和系统性 FIXa-PS 相互作用提供概念基础 高凝状态与血浆 FIXa 活性水平升高相关。