INTRINSIC SYSTEM CLOTTING: STRUCTURE AND FUNCTION

内在系统凝血：结构和功能

• 批准号: 3342238
• 负责人: Arthur Rumford Thompson
• 金额: $ 13.6万
• 依托单位: BLOODWORKS
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-12-01 至 1993-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3342238
• 关键词: RNA splicing; activation product; biological polymorphism; calcium binding protein; chemical binding; clotting factor; coagulation factor IX; coagulation factor VIII; congenital blood protein disorder; epidermal growth factor; hemophilia B; hemostasis; human population genetics; human tissue; immunochemistry; immunologic techniques; laboratory mouse; laboratory rabbit; monoclonal antibody; nucleic acid probes; protein sequence; protein structure function; site directed mutagenesis; synthetic peptide; tissue /cell culture; vascular endothelium; von Willebrand's disease

Functions domains of factor IX will be defined by four overlapping approaches. Congenital defects provide dozens of distinct mutants, the structural bases of which can be correlated to clinical severity and specific levels of functional, in vitro interactions. Specific antisera,including monoclonal antibodies and polycolonal fractions, assist in localizing defects or functions. Synthetic peptides can be used to prepare specific antisera and to directly inhibit functional interactions. Finally, for regions in which synthetic peptides inadequately reflect surface structure, synthetic fragments can be produced by expression of mutated cDNAs in an appropriate vector which has been transfected into a cultured cell line. This proposal focuses upon distinct functional domains of factor IX. These are presented from the N to the C terminus. First, we will define the defect in a hemophilic IX which as abnormal calcium-binding properties. A synthetic leader peptide will be tested further for both stimulation of Gla formation in cell expression systems and with Dr. Suttie, for its structural requirements for interaction with the carboxylase system in vitro. Secondly, hemophilic defects involving the fourth exon will be characterized by analyses of their DNA and isolated proteins. A major effort will mounted to produce recombinant factor IX fragments containing the growth factor-like regions(s) in order to probe its role in hemostasis. Both locally with Dr. Heimark and with Dr. Stern in New York, peptides, antibodies and isolated abnormal IXs will be studied for IX binding to endothelial cells. Other potential roles of this region will be assessed by in vitro clotting and binding studies and animal survival measurements of the recombinant fragments. We will confirm that a specific antibody fraction to an exon IV eptiope recognizes a non-Gla, calcium binding sire. The use of a monoclonal a-IX recognizing an exonic polymorphism will be extended from IX to VIII and von Willebrand factor to develop rapid immunoassays for carrier testing in hemophilia A and von Willebrand disease. Attempts will be made to prepare antibodies specific for factor IXa. Peptides to sequences in the variable portion of IXa's heavy chain will be studied and new reagents developed to probe the function of the active enzyme. Binding antibodies in patients with severe defects will be characterized to develop strategies for localization of their defects. A heavy chain defect due to a point mutation in a Taq1 cleavage site will be defined by oligonucleotide probes, to compare with functional data of the hemophilic mutation.

因子IX的功能域将由四个重叠的 接近。 先天性缺陷提供了几十种不同的突变体， 其结构基础可以与临床 功能性体外相互作用的严重程度和特定水平。 特异性抗血清，包括单克隆抗体和多克隆抗体 分数，协助定位缺陷或功能。 合成 肽可用于制备特异性抗血清 抑制功能性相互作用。 最后，对于那些 合成肽不能充分反映表面结构， 合成片段可以通过表达突变的cDNA 在已经转染到培养的 细胞系 这一建议侧重于不同的功能域的因素 九. 这些从N端到C端呈现。 一是 将血友病IX的缺陷定义为异常 钙结合性能。 合成前导肽将是 进一步测试刺激细胞中Gla形成 表达系统，并与Suttie博士，其结构 在体外与羧化酶系统相互作用的要求。 第二，涉及第四外显子的血友病缺陷将被排除。 通过分析它们的DNA和分离的蛋白质来表征。 一 将大力生产重组因子IX 含有生长因子样区域的片段， 探讨其止血作用。 在当地和海马克医生一起， 在纽约的斯特恩博士那里， 将研究异常IX与内皮细胞的IX结合。 该区域的其他潜在作用将通过体外试验进行评估。 凝血和结合研究以及动物存活率测量 重组片段。 我们将确认一个具体的 针对外显子IV表位的抗体部分识别非Gla， 钙离子结合型 使用单克隆α-IX识别一种 外显子多态性将从IX扩展到VIII，并且von Willebrand因子开发用于携带者检测的快速免疫测定法 血友病A和血管性血友病 将尝试 来制备针对因子IXa的抗体肽与 IXa重链可变部分中的序列将是 研究和新的试剂开发，以探测的功能， 活性酶 严重缺陷患者的结合抗体 将以制定本地化战略为特点， 他们的缺点。 由于一个基因的点突变导致的重链缺陷， Taq 1切割位点将由寡核苷酸探针确定， 与血友病突变的功能数据进行比较。