New Aspects of Protein S Anticoagulant Activity

描述（由申请人提供）：我们的主要目标是了解调节血液凝固和防止不必要的血栓形成的抗凝机制。这些导致肺栓塞、中风和静脉血栓形成，在美国每年导致50万人住院，10万人死亡。我们将重点关注蛋白S （PS-direct）直接抗血栓活性的机制，包括与另一种抗凝蛋白组织因子途径抑制剂（TFPI）相互依赖的新机制。洞察可能会导致新的治疗方法，或预防血栓形成的措施。理由：蛋白S在体内具有抗血栓作用，独立于APC；验证了PS-direct在血浆中的作用；活性蛋白S含有对PS-direct至关重要的Zn2+，在某些纯化方法中会丢失；PS-direct损失/恢复，Zn损失/恢复；与TFPI结合的蛋白S；在基于组织因子的凝血过程中显示与TFPI的相互作用；血浆蛋白S单体和多聚体抑制FXa/FVa的能力相似。因此，PS-direct是真实的和健康相关的；蛋白S缺乏会导致血栓形成风险。具体目标：1；定义对PS-direct敏感的凝血反应，并澄清哪些是相互依赖的，哪些是独立于TFPI的。扩展新的血浆和纯化组分活性测定，以发现PS-direct的所有模式以及哪些模式是tpi共依赖的。查找对tpi共依赖PS-direct的要求，例如，占据蛋白s、Ca、磷脂、完整的凝血酶敏感环或蛋白s多聚体的任何作用。2. 确定蛋白S是直接与TFPI结合，还是与外部FXase成分结合，并找到分子相互作用的位点。通过物理方法测量蛋白质S与TFPI的结合：验证另一种假设，即蛋白质S直接与FXa、组织因子或FVIIa相互作用，并导致有利于其与TFPI结合的构象改变，或有利于将无活性缺锌蛋白S转化为亚稳态活性形式。利用构建体、多肽、抗体和有限的位点特异性诱变来定位特定的蛋白S结合位点。求TFPI和FXa上的互补区域。3. 在蛋白s上找到一个对PS-direct至关重要的新的Zn结合位点，并确定该位点是否对蛋白s与TFPI、FVa或FXa的相互作用很重要。通过使用现有构造来定位Zn区域。使用三维模型，已知的锌配位残基和有限数量的合理位点突变来缩小位点。测量Zn含量，并将其与PS-direct在相互依赖或独立于TFPI的模式下进行关联。检查构象的变化。4. 确定与tpi无关的PS-direct的关键分子位点。利用现有的蛋白S构建体、突变体、多肽、抗体，确定与FVa和FXa抑制相关的区域/残基。确定一个中和表位，并发现它是否参与FVa或FXa的结合。使用三维模型来设计有限数量的新突变体，并建立一个有凝聚力的发现图。警惕潜在的增强或模拟PS-direct或TFPI的治疗药物。公共卫生相关性：我们的广泛目标是了解抗凝血机制，以调节血液凝固和防止不必要的血栓（血栓形成）。这些血块导致肺栓塞、中风和静脉血栓形成，在美国每年导致50万人住院，10万人死亡。我们将重点关注蛋白S （PS-direct）的直接抗血栓活性机制，包括依赖于另一种抗凝蛋白组织因子途径抑制剂（TFPI）的新机制。洞察可能导致新的治疗方法，或预防血栓形成的措施。

DESCRIPTION (provided by applicant): Our broad goal is to understand anticoagulant mechanisms that regulate blood clotting and prevent unwanted blood clots (thrombosis). These cause pulmonary embolism, stroke, and venous thrombosis, leading to 500,000 hospitalizations and 100,000 deaths a year in the USA. We will focus on mechanisms for the direct antithrombotic activity of protein S (PS-direct), including new mechanisms that are codependent with another anticoagulant protein, tissue factor pathway inhibitor (TFPI). Insights may lead to new treatments, or to measures that prevent thrombosis. Rationale: Protein S was antithrombotic in vivo, independent of APC; PS-direct in plasma was verified; active protein S contained Zn2+ that was crucial for PS-direct, and that was lost during some purification methods; PS-direct was lost/regained with Zn loss/regain; protein S bound to TFPI; interplay with TFPI was shown during tissue factor-based clotting; plasma protein S monomers and multimers had similar ability to inhibit FXa/FVa. Thus, PS-direct is real and health-relevant; protein S deficiency leads to thrombotic risk. Specific Aims: 1. Define coagulation reactions that are sensitive to PS-direct and clarify which are codependent or independent of TFPI. Expand novel plasma and purified component activity assays to find all modes of PS-direct and which ones are TFPI-codependent. Find the requirements for TFPI-codependent PS-direct, e.g., occupation of a Zn site(s) on protein S, Ca, phospholipids, intact thrombin-sensitive loop, or any role of protein S multimers. 2. Determine if protein S binds directly to TFPI, or to an extrinsic FXase component, and find the sites of molecular interaction. Measure binding of protein S to TFPI by physical methods: Test the alternate hypothesis that protein S interacts directly with FXa, tissue factor, or FVIIa and causes a conformation change conducive to their binding to TFPI, or conducive to conversion of inactive Zn-deficient protein S to a metastable active form. Locate the specific protein S binding site involved by use of constructs, peptides, antibodies and limited site-specific mutagenesis. Find the complementary region on TFPI and FXa. 3. Locate a new Zn binding site(s) on protein S that is crucial for PS-direct and find if this site is important for protein S interaction with TFPI, FVa, or FXa. Locate the Zn region through use of existing constructs. Narrow the site using a 3-D model, known Zn-coordinating residues and a limited number of rational site mutants. Measure Zn content and correlate it with PS-direct in modes that are codependent or independent of TFPI. Examine conformation changes. 4. Identify molecular sites crucial for TFPI-independent PS-direct. Define regions/residues implicated in inhibition of FVa and FXa, using existing protein S constructs, mutants, peptides, antibodies. Pinpoint a neutralizing epitope and find if it is involved in FVa or FXa binding. Use a 3-D model to design a limited number of new mutants and to build a cohesive picture of findings. Be alert for potential therapeutic agents that enhance or mimic PS-direct or TFPI. PUBLIC HEALTH RELEVANCE: Our broad goal is to understand anticoagulant mechanisms that regulate blood clotting and prevent unwanted blood clots (thrombosis). These clots cause pulmonary embolism, stroke, and venous thrombosis, leading to 500,000 hospitalizations and 100,000 deaths a year in the USA. We will focus on mechanisms for the direct antithrombotic activity of protein S (PS-direct), including new mechanisms that depend on another anticoagulant protein, tissue factor pathway inhibitor (TFPI). Insights may lead to new treatments, or measures that prevent thrombosis.