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MOLECULAR BIOLOGY OF BLOOD COAGULATION FACTORS VII AND X

凝血因子 VII 和 X 的分子生物学

基本信息

批准号：
2224388
负责人：
HAROLD L JAMES
金额：
$ 17.02万
依托单位：
UNIVERSITY OF TEXAS HLTH CTR AT TYLER
依托单位国家：
美国
项目类别：
财政年份：
1993
资助国家：
美国
起止时间：
1993-07-01 至 1999-06-30
项目状态：
已结题

项目摘要

Factors VII and X are the two serine coagulation proteases which engage in the initial interactions leading to prothrombin activation and clot formation via the extrinsic blood coagulation pathway. Factor VII in its activated form, VIIa, is the component of the initial extrinsic pathway macromolecular complex, which includes tissue factor (non- proteolytic cofactor) and calcium, that converts factor X to Xa. Factor VII can be activated by factors IXa, Xa or XIIa, and additionally activates factor IX. Factor VII thus undergoes multiple specific protein-protein interactions. Factor X is pivotal in the blood coagulation mechanisms, being activated at the point of convergence of the extrinsic and intrinsic pathways. In the intrinsic pathway factor Xa is formed through the interaction of factor IXa and factor VIIIa (non-proteolytic cofactor) on phospholipid surface in the presence of calcium. Factor Xa associates with factor Va (non- proteolytic cofactor) and calcium in another membrane surface oriented complex responsible for activation of prothrombin. Thus, factor X participates in three unique macromolecular complexes, suggesting, as for factor VII, the existence of several regions on the surface of the molecule that control its intermolecular associations. Enzyme kinetic and immunologic studies in this laboratory using inhibitory peptides derived from factor VII and factor X primary sequences and antipeptide antibodies strongly suggested specific surface regions on factors VII and X for their multiple types of specific interactions. Interactive sites have been proposed as existing largely in so-called variable regions of factors VII and X which can be identified on the basis of primary sequence homology among serine proteases. Such regions would be expected to contain structural properties conferring specificity for protein-protein interactions. These findings, and observations on dysfunctional inherited variants of actors VII and X for which point mutations have been identified form the background and rationale for this proposal. Site-directed mutagenesis and expression in eukaryotic cells will be used to confirm the association of point/other mutations with dysfunctional forms of factors VII and X. Secondly, site-directed and domain mutagenesis will be used to produce expressed variant species of factor VII and factor X molecules modified within specific sequences thought to participate in their respective activator, cofactor or substrate binding sites. Finally, new generations of maximally effective factor VII- and X-derived inhibitory peptides will be designed. Concepts of molecular modeling and protein folding will be used as tools to further understand how structural perturbations in mutated factors VII and X may affect their various functions. It is anticipated that the findings will contribute to understanding of the structure/function correlates of factors VII and X and lead to design of management of thrombosis.

因子VII和因子X是两种丝氨酸凝固蛋白酶，在最初的相互作用中导致凝血酶原激活和凝血通过外源性凝血途径形成。因子vii 其活化形式VIIa是初始外源性途径大分子复合物，其中包括组织因子（非蛋白水解辅因子）和钙，其将因子X转化为Xa。因子VII可以被因子IXa、Xa或XIIa激活，并且另外激活因子IX。因此，因子VII经历多个特异性蛋白质间相互作用。 X因子在血液凝固机制，被激活的点，外在途径和内在途径的融合。在本征途径因子XA是通过因子IXA和因子VIIIa（非蛋白水解辅因子）在磷脂表面上，钙的存在。因子Xa与因子Va（非- 蛋白水解辅因子）和钙在另一个膜表面取向负责激活凝血酶原的复合物。因此，因子X 参与了三种独特的大分子复合物，这表明，对于因子VII，在膜表面上存在几个区域，控制其分子间缔合的分子。酶动力学在这个实验室里用抑制肽进行免疫学研究衍生自因子VII和因子X一级序列和抗肽抗体强烈提示因子VII的特异性表面区域和X，因为它们有多种特定的相互作用。互动网站已被提议为存在很大程度上在所谓的变量因子VII和因子X的区域，可根据丝氨酸蛋白酶之间的一级序列同源性。这些区域将预期含有赋予特异性的结构性质，蛋白质相互作用这些发现和观察功能失调的遗传性变体的演员VII和X 突变已经从背景和基本原理中确定，这个提议。定点突变和真核表达细胞将用于确认点/其他突变的关联 VII因子和X因子功能失调第二，现场指导和结构域诱变将用于产生表达的变体因子VII和因子X分子的种类在特定的被认为参与其各自激活剂的序列，辅因子或底物结合位点。最后，新一代最大有效的因子VII-和X-衍生的抑制肽将被设计。分子建模和蛋白质折叠的概念将作为工具，以进一步了解结构扰动如何在突变的因子VII和X可能影响它们的各种功能。是预计调查结果将有助于了解因子VII和X的结构/功能相关性，并导致设计血栓形成的管理。