A model describing the inactivation of factor Va by APC: Bond cleavage, fragment dissociation, and product inhibition

The inactivation of factor Va is a complex process which includes bond clea vage (at three sites) and dissociation of the A2(N). A2(C) peptides, with i ntermediate activity in each species. Quantitation of the functional conseq uences of each step in the reaction has allowed for understanding of the pr esentation of disease in individuals possessing the factor V polymorphism f actor 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 membr ane-bound A1 . LC species. Evaluation of the dissociation process by light scattering yields invariant mass loss estimates as a function of APC concen tration. 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 f actor 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 a nd the reported rates of bond cleavage, binding constants, and product acti vity values for factor Va partial inactivation products, a model is develop ed which describes factor Va inactivation and accounts for the defect in fa ctor V-LEIDEN. The model accurately predicts the rates of inactivation of f actor Va and factor Va(LEIDEN), and the effect of product inhibition. Model ed reaction progress diagrams and activity profiles (from either factor Va or factor Va(LEIDEN)) are coincident with experimentally derived data, prov iding a mechanistic and kinetic explanation for all steps in the inactivati on of normal factor Va and the pathology associated with factor V-LEIDEN.