New Aspects of Protein S Anticoagulant Activity

Protein S 抗凝活性的新方面

• 批准号: 7895735
• 负责人: MARY J HEEB
• 金额: $ 42.64万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7895735
• 关键词: 3-Dimensional; Affect; Affinity; Amino Acids; Anions; Anticoagulants; Binding; Binding Proteins; Binding Sites; Biological Assay; Blood Clot; Blood Coagulation Factor; Blood coagulation; Cessation of life; Coagulation Process; Data; Epitopes; Goals; Homologous Protein; Hospitalization; Ions; Kringles; Lead; Light; Liquid substance; Measures; Methods; Minor; Modeling; Molecular Conformation; Monoclonal Antibodies; Occupations; Pathway interactions; Peptide antibodies; Peptides; Phase; Phospholipids; Plasma; Plasma Proteins; Play; Process; Protein Binding; Protein Deficiency; Proteins; Pulmonary Embolism; Reaction; Reporting; Research; Risk; Role; SHBG gene; Site; Site-Directed Mutagenesis; Stroke; Structure; Surface Plasmon Resonance; TFPI; Testing; Therapeutic Agents; Thrombin; Thromboplastin; Thrombosis; Venous Thrombosis; activated Protein C; base; cancer procoagulant; design; in vivo; insight; molecular site; monomer; mutant; novel; prevent; public health relevance; three dimensional structure

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.

描述（由申请人提供）：我们的总体目标是了解调节血液凝固并防止不必要的血栓（血栓形成）的抗凝机制。这些会导致肺栓塞、中风和静脉血栓形成，在美国每年导致 50 万人住院和 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。见解可能会带来新的治疗方法或预防血栓形成的措施。理由：Protein S 在体内具有抗血栓作用，与 APC 无关； PS-direct在血浆中得到验证；活性蛋白 S 含有 Zn2+，这对于 PS-direct 至关重要，但在某些纯化方法中会丢失； PS-direct 丢失/恢复，Zn 丢失/恢复；蛋白S与TFPI结合；在基于组织因子的凝血过程中显示了与 TFPI 的相互作用；血浆蛋白S单体和多聚体具有相似的抑制FXa/FVa的能力。因此，PS-direct 是真实且与健康相关的；蛋白质 S 缺乏会导致血栓风险。具体目标： 1. 定义对 PS-direct 敏感的凝血反应，并阐明哪些反应与 TFPI 相互依赖或独立。扩展新型血浆和纯化成分活性测定，以找到 PS 直接的所有模式以及哪些模式是 TFPI 共同依赖性的。查找 TFPI 共依赖性 PS-direct 的要求，例如，蛋白质 S、Ca、磷脂、完整凝血酶敏感环或蛋白质 S 多聚体上的 Zn 位点的占据。 2. 确定蛋白 S 是否直接与 TFPI 或外在 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 S 与 TFPI 的结合：测试另一种假设，即蛋白 S 直接与 FXa、组织因子或 FVIIa 相互作用，并引起有利于它们与 TFPI 结合的构象变化，或有利于无活性缺锌蛋白的转化S 至亚稳态活性形式。通过使用构建体、肽、抗体和有限的位点特异性诱变来定位所涉及的特定蛋白质 S 结合位点。找到 TFPI 和 FXa 上的互补区域。 3. 找到蛋白质 S 上对于 PS-direct 至关重要的新 Zn 结合位点，并确定该位点对于蛋白质 S 与 TFPI、FVa 或 FXa 的相互作用是否重要。通过使用现有构造来定位 Zn 区域。使用 3-D 模型、已知的 Zn 配位残基和有限数量的合理位点突变体缩小位点。测量 Zn 含量，并将其与 PS-direct 以相互依赖或独立于 TFPI 的模式关联起来。检查构象变化。 4. 确定对于不依赖 TFPI 的 PS-direct 至关重要的分子位点。使用现有的蛋白 S 构建体、突变体、肽、抗体，定义与 FVa 和 FXa 抑制相关的区域/残基。查明中和表位并确定它是否参与 FVa 或 FXa 结合。使用 3-D 模型设计有限数量的新突变体，并构建一个连贯的研究结果图。警惕增强或模仿 PS-direct 或 TFPI 的潜在治疗药物。公共健康相关性：我们的总体目标是了解调节血液凝固并防止不必要的血栓（血栓形成）的抗凝机制。这些血栓会导致肺栓塞、中风和静脉血栓形成，在美国每年导致 50 万人住院和 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。见解可能会带来新的治疗方法或预防血栓形成的措施。