MOLECULAR BIOLOGY OF BLOOD COAGULATION FACTORS VII AND X

凝血因子VII和X是两种相互作用的丝氨酸凝固酶 在导致凝血酶原激活和凝血的最初相互作用中 通过外源性凝血途径形成。第VII因子in 它的激活形式，VIIa，是初始外在的组成部分 途径大分子复合体，其中包括组织因子(非 蛋白水解性辅因子)和钙，将X因子转化为Xa。 因子VII可被因子IXa、Xa或XIIa激活，以及 此外还能激活因子IX。因此，第七因子经历了多个 特定的蛋白质-蛋白质相互作用。X因子在全球范围内 凝血机制，在以下点被激活 外在路径和内在路径的汇聚。在内在的 途径因子Xa是通过因子IXa和Xa相互作用形成的 磷脂表面VIIIa因子(非蛋白水解性辅因子) 钙的存在。因子Xa与因子Va(非 蛋白水解性辅因子)和钙在另一种膜表面定向 负责激活凝血酶原的复合体。因此，因子X 参与了三个独特的大分子络合物，表明 对于因子VII，在表面上存在几个区域 控制其分子间联系的分子。酶动力学 在这个实验室中使用抑制性多肽进行免疫学研究 来源于凝血因子VII和凝血因子X的一级序列和反肽 抗体强烈提示凝血因子VII的特异性表面区 X代表他们多种类型的特定交互。互动 已经提出的站点主要存在于所谓的变量中 因子VII和X的区域，可根据以下公式确定 丝氨酸蛋白酶之间的初级序列同源性。这些地区将 应包含赋予以下特性的结构属性 蛋白质之间的相互作用。这些发现，以及对 演员VII和X的功能失调的遗传变体 已经从背景和理由中确定了突变 这项提议。定点突变及其在真核细胞中的表达 细胞将被用来确认点/其他突变的关联 与因子VII和X的功能失调形式。第二，部位定向 而结构域突变将被用于产生表达的变体 在特定条件下修饰的因子VII和因子X分子的种类 被认为参与了各自激活剂的序列， 辅因子或底物结合部位。最后，新一代的 最有效的第VII和第X因子衍生的抑制性多肽 被设计好。分子建模和蛋白质折叠的概念 被用作工具来进一步理解结构扰动是如何 突变的因子VII和X可能会影响它们的各种功能。它是 预计这些发现将有助于理解 因子VII和因子X的结构/功能相关并导致设计 血栓形成的管理。

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.