New Aspects of Protein S Anticoagulant Activity

Protein S 抗凝活性的新方面

• 批准号: 7524692
• 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/7524692
• 关键词: 3-Dimensional; Affect; Affinity; Amino Acids; Anions; Anticoagulants; Binding; Binding Proteins; Binding Sites; Biological Assay; Blocking Antibodies; 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; Numbers; Occupations; Pathway interactions; Peptide antibodies; Peptides; Phase; Phospholipids; Plasma; Plasma Proteins; Play; Process; Protein Binding; Protein Deficiency; Proteins; Public Health; Pulmonary Embolism; Reaction; Reporting; Research; Risk; Role; SHBG gene; Site; Site-Directed Mutagenesis; Stroke; Structure; Surface Plasmon Resonance; TFPI; Techniques; Testing; Therapeutic Agents; Thrombin; Thromboplastin; Thrombosis; Venous Thrombosis; activated Protein C; base; cancer procoagulant; design; in vivo; insight; molecular site; monomer; mutant; novel; prevent; 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）：我们的主要目标是了解调节血液凝固和防止不必要的血液凝块（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500，000人住院和100，000人死亡。我们将集中在蛋白S（PS直接）的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）共依赖的新机制。洞察力可能会导致新的治疗方法，或预防血栓形成的措施。基本原理：蛋白S在体内是抗血栓形成的，不依赖于APC;血浆中的PS-直接被验证;活性蛋白S含有对PS-直接至关重要的Zn 2+，并且其在一些纯化方法期间丢失; PS-直接随着Zn损失/恢复而损失/恢复;蛋白S与TFPI结合;在基于组织因子的凝血期间显示与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-直接是真实的和健康相关的;蛋白S缺乏导致血栓形成的风险。具体目标：1。定义对PS直接敏感的凝血反应，并阐明哪些是共同依赖或独立于TFPI的。扩展新型血浆和纯化组分活性测定，以发现PS直接的所有模式以及哪些模式是TFPI共依赖性的。找到TFPI相关PS直接的要求，例如，蛋白S、Ca、磷脂、完整凝血酶敏感环上的Zn位点的占据，或蛋白S多聚体的任何作用。2.确定蛋白S是否直接与TFPI或外部FXase组分结合，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：检验另一种假设，即蛋白S直接与FXa、组织因子或FVIIa相互作用，并导致有利于其与TFPI结合的构象变化，或有利于无活性的锌缺陷蛋白S转化为亚稳态活性形式。通过构建体、肽、抗体和有限的位点特异性突变定位所涉及的特异性蛋白S结合位点。找到TFPI和FXa上的互补区域。3.定位蛋白S上对PS直接至关重要的新Zn结合位点，并确定该位点对蛋白S与TFPI、FVa或FXa的相互作用是否重要。通过使用现有构造定位Zn区域。使用3-D模型、已知的锌配位残基和有限数量的理性位点突变体缩小位点。测量锌含量，并将其与PS直接相关的模式是相互依赖或独立的TFPI。检查构象变化。4.鉴定对TFPI-独立PS-直接至关重要的分子位点。使用现有的蛋白S构建体、突变体、肽、抗体来定义涉及FVa和FXa抑制的区域/残基。精确定位中和表位，并发现它是否参与FVa或FXa结合。使用三维模型设计有限数量的新突变体，并建立一个有凝聚力的图片的发现。警惕增强或模拟PS直接或TFPI的潜在治疗药物。公共卫生相关性：我们的广泛目标是了解调节血液凝固和防止不必要的血液凝块（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50万人住院治疗和10万人死亡。我们将集中在蛋白S（PS直接）的直接抗血栓活性的机制，包括新的机制，依赖于另一种抗凝蛋白，组织因子途径抑制剂（TFPI）。洞察力可能会导致新的治疗方法，或预防血栓形成的措施。