FACTOR IX COMPLEXES ON MEMBRANE SURFACES

膜表面上的因子 IX 复合物

• 批准号: 6657095
• 负责人: Bruce Furie
• 金额: $ 18.67万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-01 至 2003-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6657095
• 关键词: X ray crystallography; affinity labeling; binding sites; chemical structure function; clotting factor; coagulation factor IX; coagulation factor VIII; gamma carboxyglutamate; human subject; intermolecular interaction; membrane activity; membrane proteins; membrane structure; nuclear magnetic resonance spectroscopy; phosphoserine; protein binding; protein structure function; prothrombin; site directed mutagenesis

Factor IX, a plasma protein involved in blood coagulation, is defective in patients with hemophilia B. This application examines the g- carboxyglutamic acid-rich domain of Factor IX to understand its role in membrane binding and in complex formation with Factor VIIIa. Two themes are the focus of this application: the nature of Factor IX- membrane interaction in chemical terms and of the Factor IXa-Factor VIIIa complex on membrane studies. We will explore the membrane binding properties of Factor IX, particularly the contribution of phosphatidylethanolamine and soluble phospholipids. Based on our X-ray crystal structure of pro-thrombin bound to phosphatidylserine, we will examine the homologues residues in Factor IX that form contact sites with phospholipid by site-specific mutagenesis. P-Benzoylphenylalanine-FIX (1-47) will be used as photoaffinity labels of platelet membranes to identify the protein or lipids to which Factor IXa binds. Based on our successful application of 2D NMR spectroscopy to the determination of the soluble structure of the Gla domain of Factor IX, we will determine the solution structure of the ternary Factor IX (1-47) phospholipid: calcium complex. The focus will be on defining the membrane finding site in Factor IX and the contact residues that determine specificity for acidic phospholipids. The metal generate Factor IX crystals in the presence of calcium phosphatidylserine suitable for X-ray crystallography. The solution structure of the Gla domains of other vitamin K-dependent proteins will be determined in the presence of phosphatidylserine in order to compare their interaction with phospholipid to that of Factor IX. Finally, Factor IXa-Factor VIIIA complex formation will be studied to understand formation of the Xase complex on membrane surfaces. Based upon the preliminary observation that a membrane finding peptide based upon Factor VIII greatly accelerates Factor X activation by Factor IXa in the absence of Factor VIIIa and phospholipid, we will determine the sites on Factor IXa and Factor X to which this peptide binds using a p-Bpa analog of FVIII (2303-2323) as photoaffinity label. The interaction of FVIII (2303-2323) with Factor IXa and Factor X as well as with FIX (1-47) will be characterized by UV difference and NMR spectroscopy. A high affinity peptide inhibitor of Factor IXa-Factor VIIIa interaction will be identified by screening a combinatorial phase library. Inhibitory peptides will be characterized for their mechanism of inhibition of binding of Factor IXa with Factor VIIIa. Understanding the details of Factor IX-membrane interaction and Factor IXa-Factor VIIIa complex formation will contribute to our knowledge of hemophilia and the treatment of thrombotic disease.

因子IX是一种参与血液凝结的血浆蛋白，在血友病B的患者中有缺陷。该应用研究了因子IX的G-羧谷氨酸富含含量的G-羧谷氨酸结构域，以了解其在膜结合和与因子VIIIA的复杂形成中的作用。两个主题是该应用的重点：在化学术语和膜研究中，IX-膜相互作用的性质和IXA因子VIIIA复合物的性质。我们将探索IX因子的膜结合特性，尤其是磷脂酰乙醇胺和可溶性磷脂的贡献。基于我们与磷脂酰丝氨酸结合的凝血酶蛋白的X射线晶体结构，我们将检查IX因子IX中的同源物残基，该残基由位点特异性诱变形成与磷脂的接触位点。 P-苯甲酰苯胺固定（1-47）将用作血小板膜的光亲和力标签，以识别IXA结合的因子与之结合的蛋白质或脂质。 基于我们成功地应用了2D NMR光谱法在因子IX因子GLA结构域的可溶性结构中的确定，我们将确定三元因子IX（1-47）磷脂的溶液结构：钙复合体。重点将放在定义因子IX中的膜查找位点以及确定酸性磷脂特异性的接触残基。金属在适用于X射线晶体学的钙磷脂酰丝氨酸的情况下产生IX晶体。其他维生素K依赖性蛋白的GLA结构域的溶液结构将在磷脂酰丝氨酸的存在下确定，以将其与磷脂的相互作用与IX因子IX的相互作用进行比较。最后，将研究因子IXA因子VIIIA复合物的形成，以了解膜表面上的Xase复合物的形成。 Based upon the preliminary observation that a membrane finding peptide based upon Factor VIII greatly accelerates Factor X activation by Factor IXa in the absence of Factor VIIIa and phospholipid, we will determine the sites on Factor IXa and Factor X to which this peptide binds using a p-Bpa analog of FVIII (2303-2323) as photoaffinity label. FVIII（2303-2323）与因子IXA和因子X以及固定（1-47）的相互作用将以UV差异和NMR光谱法来表征。 IXA因子VIIIA相互作用的高亲和肽抑制剂将通过筛选组合相文库来识别。抑制性肽的特征是其抑制因子与因子VIIIA结合的机理。 了解因子IX膜相互作用和IXA因子VIIIA复合物的细节将有助于我们对血友病的了解和血栓性疾病的治疗。