Functional Domains of Coagulation Factor V

说明(申请人提供)：凝血酶原酶由蛋白质辅因子因子Va和因子Xa组成，在二价金属离子存在的情况下结合在细胞表面。将因子Va掺入凝血酶原酶并与因子Xa相互作用，与单独使用因子Xa催化反应相比，酶的催化效率提高了300,000倍。原辅因子因子V不参与凝血酶原酶。凝血酶激活因子V后，因子Va由重链和轻链通过二价金属离子结合而成。辅因子的两条链都与凝血因子Xa相互作用，而只有辅因子的重链与凝血酶原结合。只有在膜表面存在的情况下，活化蛋白C(APC)才能有效地下调辅因子Va重链的活性，导致辅因子不能与Xa因子结合。因此，与因子V激活和失活相关的正负调节过程直接与辅因子结合到凝血酶原酶和结合因子Xa的能力有关。负责凝血因子Va与凝血因子Xa和凝血酶原相互作用的氨基酸仍有待鉴定。我们有数据表明，辅因子的重链在氨基酸区域323-331内与因子Xa有结合区，而轻链的NH2末端部分(氨基酸残基1546-1558)也与因子Xa相互作用。此外，我们有数据表明，重链的COOH末端部分包含凝血酶原的相互作用部位，而先前的数据表明，凝血酶的结合部位位于原癌因子的B区。这项拨款计划的具体目的是：(1)鉴定和鉴定凝血因子Va轻链上的凝血因子Xa结合结构域(S)；(2)鉴定和鉴定凝血因子V分子上的凝血酶和凝血酶原结合结构域(S)；(3)通过研究凝血酶原酶在血小板上的组装和功能来检验我们发现的生理学相关性。为了实现这些目标，我们设计了一系列实验，这些实验被优先考虑，并与互补的分子和结构方法相结合。表征因子V的特定氨基酸区域对其功能至关重要，这将有助于深刻理解控制凝血酶原酶的大分子相互作用，以及其组装、功能和特异性所需的大分子相互作用。我们已经建立了一个研究磷脂驱动的大分子复合体形成的系统，这可能是一个形成细胞外和细胞内复合体的模型。

DESCRIPTION (provided by applicant): Prothrombinase is composed of the protein cofactor, factor Va, and the enzyme, factor Xa, associated on a cell surface in the presence of divalent metal ions. Incorporation of factor Va into prothrombinase and its interaction with factor Xa results in a 300,000-fold acceleration of the catalytic efficiency of the enzyme as compared to the catalysis of the reaction by factor Xa alone. The procofactor, factor V, does not participate in prothrombinase. Following activation of factor V by thrombin, factor Va is composed of heavy and light chains associated via divalent metal ions. Both chains of the cofactor interact with factor Xa while only the heavy chain of the cofactor binds prothrombin. The factor Va cofactor activity is efficiently down-regulated following proteolysis of the heavy chain by activated protein C (APC) only in the presence of a membrane surface and results in the inability of the cofactor to bind factor Xa. Thus, the positive and negative regulatory processes associated with factor V activation and its inactivation are directly associated with the capability of the cofactor to be incorporated into prothrombinase and to bind factor Xa. The amino acids responsible for the interaction of factor Va with factor Xa and prothrombin remain to be identified. We have data demonstrating that the heavy chain of the cofactor possesses a binding region for factor Xa within amino acid region 323-331, whereas the NH2-terminal portion of the light chain (amino acid residues 1546-1558) also interacts with factor Xa. In addition, we have data suggesting that the COOH-terminal portion of the heavy chain contain an interactive site for prothrombin while previous data have suggested that a binding site for thrombin is located on the B region of the procofactor. The specific aims of this grant proposal are: (1) to identify and characterize the factor Xa-binding domain(s) on factor Va light chain; (2) to identify and characterize the thrombin and prothrombin-binding domain(s) on the factor V molecule; (3) to test the physiological relevance of our findings by studying the assembly and function of prothrombinase on platelets. To achieve these goals we have designed a series of experiments that are prioritized and integrated with complementary molecular and structural approaches. Characterization of the specific amino acid regions of factor V that are critical for its function will allow for a profound understanding of the macromolecular interactions that control prothrombinase and are required for its assembly, function, and specificity. We have established a system to study phospholipids-driven macromolecular complex formation, which may be a model for the generation of complexes that form extra-and intra-cellularly.