KRINGLE DOMAINS IN BLOOD COAGULATION PROTEINS

凝血蛋白中的 KRINGLE 结构域

• 批准号: 3087811
• 负责人: David A Roth
• 金额: $ 8.68万
• 依托单位: TUFTS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-07-01 至 1996-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3087811
• 关键词: CHO cells; SDS polyacrylamide gel electrophoresis; aminoacid; binding proteins; blood proteins; calcium; chimeric proteins; coagulation factor V; coagulation factor X; enzyme complex; fibrinolysis; homeostasis; human tissue; immunoaffinity chromatography; laboratory rabbit; membrane activity; nucleic acid sequence; phospholipids; plasminogen; polymerase chain reaction; protein biosynthesis; protein purification; protein structure function; prothrombin; radioimmunoassay; site directed mutagenesis; spectrometry; tissue /cell culture; CHO cells; SDS polyacrylamide gel electrophoresis; aminoacid; binding proteins; blood proteins; calcium; chimeric proteins; coagulation factor V; coagulation factor X; enzyme complex; fibrinolysis; homeostasis; human tissue; immunoaffinity chromatography; laboratory rabbit; membrane activity; nucleic acid sequence; phospholipids; plasminogen; polymerase chain reaction; protein biosynthesis; protein purification; protein structure function; prothrombin; radioimmunoassay; site directed mutagenesis; spectrometry; tissue /cell culture

The kringle domain is a highly conserved structure found in five proteins involved in hemostasis: prothrombin, plasminogen, tissue plasminogen activator, urokinase and Factor XII. The exact function of the kringle domain remains uncertain, however, the two kringle domains in prothrombin likely play a role in the recognition of prothrombin as a substrate for the enzyme complex, Factor Xa and Factor Va (prothrombinase). The major goals of this project are to elucidate the role of prothrombin kringle domains in prothrombinase complex assembly on membrane surfaces, and to identify specific regions and amino acids within the kringle domain that define the interaction of prothrombin with Factor Xa and Factor Va. Prothrombin kringle deletion mutants have been constructed by oligonucleotide-directed loop-out mutagenesis. Prothrombin mutants in which the kringle domains are substituted for one another, and prothrombin chimera in which kringle domains are replaced by kringle domains from plasminogen or tissue plasminogen activator will be constructed as follows. The kringle domains of prothrombin will be selectively removed by loop-out mutagenesis and replaced by PCR generated kringle domains from prothrombin, plasminogen or tissue plasminogen activator. In addition, prothrombin mutants will be constructed with regional and point mutations in the kringle domains. The cDNA encoding these prothrombin mutants will be transfected into CHO cells, expressed, and the mutant proteins purified by immunoaffinity chromatography. To determine if the activity and functional specificity of these mutants has been altered, they will be evaluated for specific coagulant activity as well as for binding to, and activation by the Factor Xa/Factor Va (prothrombinase) complex on membrane surfaces. Mutants will be evaluated for direct binding to Factor Va in the presence and absence of Factor Xa, phospholipid and calcium ions, by measurement of fluorescence energy transfer. Their ability to serve as substrates for Factor Xa in the presence and absence of the cofactor, Factor Va, will be evaluated by fluorimetric kinetic analysis of DAPA binding to thrombin. Competitive inhibition of prothrombin activation will be assessed using thrombin- active-site-inhibited prothrombin kringle mutants. The results of these experiments will identify critical amino acids in the prothrombin kringle domain(s) which are involved in prothrombin binding to and activation by prothrombinase, and they will determine the functional role of kringle domains in conferring biological activity and specificity to blood coagulation proteins.

kringle结构域是一种在五种蛋白质中发现的高度保守的结构 参与止血：凝血酶原，纤溶酶原，组织纤溶酶原 激活剂，尿激酶和因子XII。 Kringle的确切功能 但是，域仍然不确定，但是，凝结凝结物中的两个kringle域 可能在识别凝血酶原作为底物的底物中发挥作用 酶复合物，因子Xa和因子VA（黑凝结酶）。 主要目标 这个项目的旨在阐明凝血酶原kringle域在 凝结凝结酶复合物组件在膜表面上，并识别 克林勒域内的特定区域和氨基酸定义 凝血酶原与因子XA和VA因子的相互作用。 克林勒缺失突变体是由寡核苷酸定向构建的 循环诱变。 凝血酶蛋白突变体，其中kringle域是 彼此代替，凝凝结蛋白嵌合体中有kringle 域被纤溶酶原或组织的kringle结构域取代 纤溶酶原激活剂将被构造如下。 kringle域 凝血酶原的凝血酶素将通过循环诱变和 由凝血酶原，纤溶酶原或 组织纤溶酶原活化剂。 另外，凝血酶原突变体将是 用克林格域中的区域和点突变构建。 这 编码这些凝血酶蛋白突变体的cDNA将被转染到CHO细胞中， 表达，通过免疫亲和力纯化的突变蛋白 色谱法。 确定活动和功能特异性是否 这些突变体已被改变，将评估它们的特定 凝结剂活性以及与因子结合和激活 膜表面上的XA/因子VA（凝血酶原酶）复合物。 突变者会 在存在和不存在的情况下评估与因子VA的直接结合 Xa因子，磷脂和钙离子，通过测量荧光 能量转移。 它们作为因子Xa的基材的能力 辅因子因子VA的存在和不存在将通过 DAPA与凝血酶结合的氟化动力学分析。 竞争的 将使用凝血酶来评估凝血酶蛋白激活的抑制作用 活动位置抑制的凝血酶原kringle突变体。 这些结果 实验将鉴定凝血酶原kringle中的关键氨基酸 与凝血酶原结合并激活凝血酶原结合的结构域 凝血酶聚集酶，他们将确定克林格的功能作用 赋予生物学活性和对血液特异性的领域 凝血蛋白。