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.

蛋白质S（PS）是一种至关重要的天然抗凝剂 小鼠和人纯合缺乏的凝血病。最初被发现为激活的辅助因子 蛋白C（APC），随后的研究表明，大量与APC无关的抗凝剂 PS的机制，没有明确证明其各自的生理相关性。最近，通用 小鼠和直接生化证据的“重新平衡”研究表明 PS和因子IXA（FIXA）该提案的目的是定义PS在FIXA的体内调节中的作用 活动。中心假设是PS是止血和止血和 全身性高凝状态。中心假设将通过追求三个具体目标来检验：1）确定 调节人血浆中固定活性的主要机制，2）确定的贡献 蛋白质对重组固定体内活性（a）的亲和力（3）确定蛋白质的贡献 固定活性（a）活性的体内调节不足。为了追求这些目标，我们已经开发了一个小组 重组人固定变体具有降低对抗凝血酶（AT），肝素和PS的亲和力。在第一个 目的，将检查PS，PS-TFPI和PS-APC对固有Xase复合物中固定活性的抑制作用 使用纯化的组件和固定变体。同样，依赖AT和PS的贡献 将使用FIXA变体检查血浆凝血酶生成调节的机制 免疫部血浆中的抑制性抗体。在第二个目标中，人类fixa变种将首先是 用于评估AT，肝素和PS亲和力对酶原和蛋白酶恢复的影响以及 清除。其次，将使用剂量依赖性体内止血和血栓性活性。 在血友病B小鼠中建立的模型，以评估AT和PS依赖性的贡献 机制。在第三个目标中，将雇用Pros1特定的反义寡核苷酸（ASO）来创建PS 野生型和血友病小鼠的缺乏。 ASO介导的PS缺乏对出血和 血栓形成表型将使用既定模型进行评估，包括改善的能力 血友病小鼠的基线出血表型。此外，ASO介导的PS缺乏对IN的影响 注射固定剂变体的体内止血和血小板活性将在血友病小鼠中检查，包括 人类PS“拯救” PS缺乏症的影响的能力。这些目标的完成将阐明 PS-FIXA相互作用在固有的固定凝胶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，因为它解决了新颖的体内意义 控制凝结响应中限速步骤的机制。这项贡献将是重要的 因为它将为操纵血友病和全身性的FIXA-PS相互作用提供概念基础 与血浆固定活性水平升高有关的高凝状态。