KRINGLE DOMAINS IN BLOOD COAGULATION PROTEINS

Kringle结构域是一种高度保守的结构，存在于五种蛋白质中 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。 圣诞老人的确切功能 结构域仍然不确定，然而，凝血酶原中的两个kringle结构域 可能在识别凝血酶原作为底物的过程中发挥作用， 酶复合物，因子Xa和因子Va（凝血酶原酶）。 主要目标 本项目的目的是阐明凝血酶原kringle结构域在 凝血酶原酶复合物在膜表面的组装，并鉴定 Kringle结构域内的特定区域和氨基酸，其定义了 凝血酶原与因子Xa和因子Va相互作用。 凝血酶原 Kringle缺失突变体已经通过寡核苷酸指导的 环出突变。 其中kringle结构域是 和凝血酶原嵌合体，其中Kringle 结构域被来自纤溶酶原或组织的Kringle结构域取代 纤溶酶原激活剂将如下构建。 Kringle域 将通过环出诱变选择性地去除凝血酶原， 由PCR产生的来自凝血酶原、纤溶酶原或 组织型纤溶酶原激活剂 此外，凝血酶原突变体将是 在Kringle结构域中具有区域和点突变。 的 将编码这些凝血酶原突变体的cDNA转染到CHO细胞中， 表达，并通过免疫亲和纯化突变蛋白 层析 确定的活性和功能特异性是否 这些突变体已经被改变，它们将被评估为特定的 凝血活性以及与因子的结合和激活 Xa/因子Va（凝血酶原酶）复合物。 突变株 在存在和不存在以下物质的情况下，评价与因子Va的直接结合： 因子Xa、磷脂和钙离子，通过测量荧光 能量转移 它们作为凝血因子Xa底物的能力， 辅因子因子Va的存在和不存在将通过 达帕与凝血酶结合的荧光动力学分析。 竞争 凝血酶原激活的抑制将使用凝血酶- 活性位点抑制的凝血酶原Kringle突变体。 的结果予以 实验将确定凝血酶原kringle中的关键氨基酸 参与凝血酶原结合和激活的结构域 凝血酶原酶，它们将决定Kringle的功能作用 赋予血液生物活性和特异性的结构域 凝血蛋白

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.