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)：我们的主要目标是了解调节血液凝结和防止不需要的血液凝块(血栓)的抗凝机制。这些疾病会导致肺栓塞、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。我们将集中于S蛋白(PS-DIRECT)直接抗血栓活性的机制，包括与另一种抗凝蛋白-组织因子途径抑制物(TFP-I)相互依赖的新机制。洞察力可能会导致新的治疗方法，或者预防血栓形成的措施。基本原理：S蛋白在体内具有抗血栓作用，不依赖于碱性磷酸酶；证实了血浆中存在PS-DIRECT；活性蛋白S含有PS-DIRECT所必需的锌离子，这种锌离子在某些纯化方法中丢失；PS-DIRECT随着锌的丢失/恢复而丢失/恢复；蛋白质S与组织因子结合；在基于组织因子的凝血过程中与TFP I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是真实的，与健康相关；S蛋白缺乏会导致血栓形成的风险。具体目标：1.定义对PS-DIRECT敏感的凝血反应，阐明哪些是相互依赖的，哪些是不依赖TFPI的。扩展新的血浆和纯化组分活性分析，以发现PS-Direct的所有模式以及哪些模式是TFPI依赖的。找出对TFP I依赖的PS-Direct的要求，例如，对S蛋白、钙、磷脂、完整的凝血酶敏感环或S蛋白的任何作用的锌位点(S)的占据。2.确定S蛋白是直接与TFP I结合，还是与外源性FXase组分结合，并找到分子相互作用的部位。用物理方法测定蛋白质S与TFP I的结合：检验另一种假说，即蛋白质S直接与FXA、组织因子或FVIIa相互作用，引起有利于它们与TFP I结合的构象变化，或有助于将失活的缺锌蛋白S转化为亚稳定的活性形式。利用构建体、多肽、抗体和限制性定点突变等方法定位S参与的特异性蛋白结合位点。找到TFPI和FXA上的互补区。3.在蛋白质S上定位一个新的锌结合位点(S)，该位点是PS-Direct的关键位点，并确定该位点在蛋白质S与TFP I、FVA或FXA相互作用中是否起重要作用。通过使用现有的构建物来定位锌区域。使用3-D模型、已知的锌配位残基和有限数量的合理位点突变体来缩小该位点。测量锌含量，并以相互依赖或独立于TFPI的模式将其与PS-DIRECT相关联。检查构象变化。4.确定TFPI非依赖性PS-DIRECT的关键分子位点。利用现有的S结构蛋白、突变体、多肽和抗体，确定与抑制黄曲霉毒素A和黄曲霉毒素A有关的区域/残基。精确定位一个中和表位，找出它是否与FVA或FXA结合有关。使用3-D模型设计有限数量的新突变体，并建立一个连贯的发现图景。警惕增强或模仿PS-DIRECT或TFPI的潜在治疗剂。与公共卫生相关：我们的广泛目标是了解调节血液凝结和防止不需要的血液凝块(血栓)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500,000人住院和100,000人死亡。我们将集中于S蛋白(PS-DIRECT)直接抗血栓作用的机制，包括依赖于另一种抗凝蛋白-组织因子途径抑制物(TFP-I)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。