MOLECULAR BIOLOGY OF BLOOD COAGULATION FACTORS VII AND X

凝血因子 VII 和 X 的分子生物学

• 批准号: 3367479
• 负责人: HAROLD L JAMES
• 金额: $ 11.01万
• 依托单位: UNIVERSITY OF TEXAS HLTH CTR AT TYLER
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-01-01 至 1996-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3367479
• 关键词: active sites; blood chemistry; blood coagulation; chemical models; coagulation factor VII; coagulation factor X; cofactor; complementary DNA; computer program /software; computer simulation; enzyme complex; enzyme mechanism; gene expression; human genetic material tag; human tissue; intermolecular interaction; molecular biology; molecular cloning; nucleic acid sequence; peptide chemical synthesis; point mutation; polymerase chain reaction; protein folding; protein purification; protein structure function; radioimmunoassay; site directed mutagenesis; synthetic peptide; tissue /cell culture; western blottings

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 X 是两种丝氨酸凝固蛋白酶，它们参与 在导致凝血酶原激活和凝块的初始相互作用中 通过外源性凝血途径形成。 因子VII 其激活形式 VIIa 是初始外源性的组成部分 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，将因子 X 转化为 Xa。 因子VII可以被因子IXa、Xa或XIIa激活，并且 另外还激活因子 IX。 因此，因子VII经历了多重 特定的蛋白质-蛋白质相互作用。 因素 X 至关重要 血液凝固机制，在以下时刻被激活 外在途径和内在途径的融合。 在内在的 途径因子 Xa 是通过因子 IXa 和因子 IXa 相互作用形成的。 磷脂表面的因子 VIIIa（非蛋白水解辅助因子） 钙的存在。 因子 Xa 与因子 Va 相关（非 蛋白水解辅因子）和钙在另一个膜表面定向 负责激活凝血酶原的复合物。 因此，因子 X 参与三种独特的大分子复合物，表明， 对于因子 VII，表面存在多个区域 控制其分子间缔合的分子。 酶动力学 本实验室使用抑制肽进行免疫学研究 源自因子 VII 和因子 X 一级序列和抗肽 抗体强烈表明因子 VII 上的特定表面区域 X 代表其多种类型的特定相互作用。 交互的 据建议，这些地点主要存在于所谓的可变区域中。 因子 VII 和 X 的区域可以根据 丝氨酸蛋白酶之间的一级序列同源性。 这些地区将 预计包含赋予特异性的结构特性 蛋白质-蛋白质相互作用。 这些发现和观察 演员 VII 和 X 的功能失调的遗传变体，对于这一点 突变已从背景和原理中确定 这个建议。 真核生物中的定点突变和表达 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。 其次，位点定向 域诱变将用于产生表达变体 在特定范围内修饰的因子 VII 和因子 X 分子种类 被认为参与各自激活剂的序列， 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 用作进一步了解结构扰动如何进行的工具 突变的因子 VII 和 X 可能会影响它们的各种功能。 这是 预计研究结果将有助于理解 因子 VII 和 X 的结构/功能相关并导致设计 血栓形成的管理。