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: 10548810
• 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 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10548810
• 关键词: Ablation; Address; Affinity; Antibodies; Anticoagulants; Antisense Oligonucleotides; Binding; Binding Proteins; Binding Sites; Biochemical; Biological; Blood Coagulation Disorders; Blood coagulation; Clinical; Coagulation Process; Collaborations; Complex; Disease; Dose; EGF gene; 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; Maps; Mediating; Mission; Modeling; Mus; National Heart, Lung, and Blood Institute; Peptide Hydrolases; Pharmacologic Substance; Phenotype; Physiological; Physiology; Plasma; Plasma Enhancement; Plasma Proteins; Positioning Attribute; Prevention; Protease Domain; Protein S; Protein S Deficiency; Proteins; Public Health; Recombinants; Recovery; Regulation; Research; Role; Structure; Testing; Therapeutic; Thrombin; Thrombophilia; Thrombosis; Variant; Woman; activated Protein C; cancer procoagulant; cofactor; defined contribution; hormonal contraception; in vivo; inhibitor; innovation; insight; man; novel; novel strategies; protein expression; 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S（PS）是一种非常重要的天然抗凝剂， 小鼠和人的纯合缺陷型凝血病。最初发现作为激活的凝血因子， 蛋白C（APC），随后的研究表明，大量的APC非依赖性抗凝剂 PS的机制没有明确证明其各自的生理相关性。最近，基因 在小鼠中进行的“再平衡”研究和直接的生化证据表明， PS和因子IXa（FIXa）本提案的目的是确定PS在体内调节FIXa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剂。 全身高凝状态。中心假设将通过追求三个具体目标进行测试：1）识别 调节人血浆中FIXa活性的主要机制，2）确定以下因素的贡献 蛋白S对重组FIX体内活性的亲和力（a），和3）确定蛋白S的贡献 缺乏FIX（a）活性的体内调节。为了实现这些目标，我们成立了一个小组， 对抗凝血酶（AT）、肝素和PS具有降低亲和力的重组人FIX变体。上 目的是检测PS、PS-TFPI抑制剂和PS-APC对内源性Xase复合物中FIXa活性的抑制作用 使用纯化的组分和FIXa变体。同样，AT-和PS-依赖性 将使用FIXa变体检查血浆凝血酶产生的调节机制， 免疫耗竭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的影响， 间隙其次，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： 在血友病B小鼠中建立模型，以评估AT和PS依赖性 机制等在第三个目标中，将使用Pros 1特异性反义寡核苷酸（阿索）来产生PS 在野生型和血友病B小鼠中缺乏。ASO介导的PS缺陷对出血和 将使用已建立的模型评价血栓形成表型，包括改善血栓形成的能力。 血友病B小鼠的基线出血表型。此外，ASO介导的PS缺陷对细胞凋亡的影响也不明显。 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缺乏效应的能力。这些目标的完成将阐明 PS-FIXa相互作用在内源性内FIXa促凝血活性的生理调节中的关键作用 Xase复合体。拟议的研究是创新的，因为它解决了一个新的体内意义 用于调节凝血反应中的限速步骤的机制。这一贡献将是巨大的 因为它将为操纵血友病和系统性血友病中的FIXa-PS相互作用提供概念基础。 与血浆FIXa活性水平升高相关的高凝状态。