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

蛋白质S(PS)是一种重要的天然抗凝剂，其致命性消耗证明 小鼠和人纯合子缺乏症的凝固性疾病。最初被发现为激活的辅因子 蛋白C(APC)，随后的研究证明了过多的APC非依赖性抗凝剂 PS的机制没有清楚地证明它们各自的生理相关性。最近，基因 小鼠的“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的作用 活动。中心假设是PS是FixA的一个重要的生理调节因子，在止血和 全身高凝状态。核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FixA活性的主要机制(S)，2)决定了 蛋白S亲和力对体内重组FIX活性的影响(A)，以及3)确定S蛋白的贡献 缺乏体内对FIX(A)活性的调节。为了实现这些目标，我们开发了一个小组 重组人FIX变异体对抗凝血酶(AT)、肝素和PS的亲和力降低。在第一个 目的：研究PS、PS-TFP I和PS-APC对Xase内源复合体固定酶活性的抑制作用。 使用纯化的成分和Fixa变种。同样，AT依赖和PS依赖的贡献 调节血浆凝血酶生成的机制将用FixA变体和 免疫耗竭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 用来评估AT、肝素和PS亲和力对酶原和蛋白酶回收的影响以及 通行证。其次，体内止血和血栓形成活性的剂量依赖关系将使用 在血友病B小鼠中建立模型以评估AT和PS依赖的贡献 机械装置。在第三个目的中，将使用Pros1特异性反义寡核苷酸(ASO)来创建PS 野生型和血友病B小鼠的缺乏症。ASO介导的PS缺陷对出血和血管紧张素转换酶的影响 血栓表型将使用已建立的模型进行评估，包括改善血栓形成表型的能力 血友病B小鼠的基线出血表型。此外，ASO介导的PS缺乏对IN的影响 将在血友病B小鼠身上检测注射的FixA变体的活体止血和血栓活性，包括 人类PS“拯救”PS缺乏症的能力。完成这些目标将阐明 PS-FixA相互作用在内源性FixA促凝活性生理调节中的关键作用 Xase复合体。这项拟议的研究具有创新性，因为它解决了一部小说的活体意义 调节凝血反应中限速步骤的机制。这一贡献将是巨大的。 因为它将为控制血友病和系统性红斑狼疮的FixA-PS相互作用提供一个概念基础。 高凝状态与血浆FixA活性升高有关。

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.