A model describing the inactivation of factor Va by APC: Bond cleavage, fragment dissociation, and product inhibition
A model describing the inactivation of factor Va by APC: Bond cleavage, fragment dissociation, and product inhibition
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DOI：
10.1021/bi981966e
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发表时间：
1999-05-25
期刊：
影响因子：
2.9
通讯作者：
Mann, KG
中科院分区：
文献类型：
--
作者：
Hockin, MF;Cawthern, KM;Mann, KG
Va 因子的失活是一个复杂的过程，包括键断裂（在三个位点）和 A2(N) 的解离。 A2(C) 肽，在每个物种中具有中等活性。对反应中每个步骤的功能后果进行定量可以了解具有因子 V 多态性因子 V-LEIDEN 的个体的疾病表现。膜结合牛因子 Va (Arg(306)、Arg(505)、Arg(662)) 的 ApC 裂解导致 A2 结构域片段、残基 307-713 (A2(N).A2(C) + A2(C-肽)) 解离，留下膜结合 A1。 LC 种。通过光散射评估解离过程可得出作为 APC 浓度函数的不变质量损失估计值。 A2 片段解离的速率常数随 [APC] 变化，达到最大值 k = 0.028 s(-1)，即 A2 结构域片段解离的单分子速率常数。 APC 结合位点位于因子 Va 轻链 (LC) (K-d = 7 nM)，表明膜结合的 LC 。 A1 产品将起到隔离 APC 的作用。这种抑制相互作用（LC.A1.APC）被证明与纯化的因子Va LC 或因子Va 失活的产物存在。利用这些实验数据和报道的因子Va部分失活产物的键断裂速率、结合常数和产物活性值，开发了一个模型，该模型描述因子Va失活并解释因子V-LEIDEN的缺陷。该模型准确预测了因子Va和因子Va(LEIDEN)的失活率以及产物抑制的效果。建模的反应进程图和活性曲线（来自因子 Va 或因子 Va(LEIDEN)）与实验得出的数据一致，为正常因子 Va 失活的所有步骤以及与因子 V-LEIDEN 相关的病理学提供了机械和动力学解释。
The inactivation of factor Va is a complex process which includes bond cleavage (at three sites) and dissociation of the A2(N). A2(C) peptides, with intermediate activity in each species. Quantitation of the functional consequences of each step in the reaction has allowed for understanding of the presentation of disease in individuals possessing the factor V polymorphism factor V-LEIDEN. ApC cleavage of membrane-bound bovine factor Va (Arg(306), Arg(505), Arg(662)) leads to the dissociation of fragments of the A2 domain, residues 307-713 (A2(N). A2(C) + A2(C-peptide)), leaving behind the membrane-bound A1 . LC species. Evaluation of the dissociation process by light scattering yields invariant mass loss estimates as a function of APC concentration. The rate constant for A2 fragment dissociation varies with [APC], reaching a maximal value of k = 0.028 s(-1), the unimolecular rate constant for A2 domain fragment dissociation. The APC binding site resides in the factor Va Light chain (LC) (K-d = 7 nM), suggesting that the membrane-bound LC . A1 product would act to sequester APC. This inhibitory interaction (LC . A1 . APC) is demonstrated to exist with either purified factor Va LC or the products of factor Va inactivation. Utilizing these experimental data and the reported rates of bond cleavage, binding constants, and product activity values for factor Va partial inactivation products, a model is developed which describes factor Va inactivation and accounts for the defect in factor V-LEIDEN. The model accurately predicts the rates of inactivation of factor Va and factor Va(LEIDEN), and the effect of product inhibition. Modeled reaction progress diagrams and activity profiles (from either factor Va or factor Va(LEIDEN)) are coincident with experimentally derived data, providing a mechanistic and kinetic explanation for all steps in the inactivation of normal factor Va and the pathology associated with factor V-LEIDEN.