A Model Describing the Inactivation of Factor Va by APC:  Bond Cleavage, Fragment Dissociation, and Product Inhibition

Abstract

The inactivation of factor Va is a complex process which includes bond cleavage (at three sites) and dissociation of the A2N.A2C 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 VLEIDEN. APC cleavage of membrane-bound bovine factor Va (Arg306, Arg505, Arg662) leads to the dissociation of fragments of the A2 domain, residues 307-713 (A2N.A2C + A2C-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) (Kd = 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 VLEIDEN. The model accurately predicts the rates of inactivation of factor Va and factor VaLEIDEN, and the effect of product inhibition. Modeled reaction progress diagrams and activity profiles (from either factor Va or factor VaLEIDEN) 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 VLEIDEN.

Factor V: a combination of Dr Jekyll and Mr Hyde

“…98,99 APC binds to the light chain of factor Va in a competitive manner with factor Xa. [100][101][102][103] Conversely, factor Xa impairs APC cleavage and inactivation of the cofactor. 102,103 Thus, APC provides inhibition of coagulation by noncovalent competition with factor Xa and by cleavage and inactivation of factor Va. 95,96,[101][102][103] APC cleaves human factor Va heavy chain at Arg506, Arg306, and Arg679 with the kinetic order of these cleavages generally as specified ( Figure 1A,1C upper part).…”

“…102,103 Thus, APC provides inhibition of coagulation by noncovalent competition with factor Xa and by cleavage and inactivation of factor Va. 95,96,[101][102][103] APC cleaves human factor Va heavy chain at Arg506, Arg306, and Arg679 with the kinetic order of these cleavages generally as specified ( Figure 1A,1C upper part). 96,101 The cleavages at Arg506 and Arg306 are influenced by a membrane surface with the cleavage at Arg306 being virtually dependent on the presence of an anionic phospholipid-containing membrane. 96 Following cleavage at Arg506, the resulting cofactor (factor Va R506 ) displays lessened apparent affinity for factor Xa.…”

“…104 The inactivation rate of factor Va when explored as a function of APC concentration becomes independent of enzyme concentration, a clear signal that the final inactivation step involves not only cleavage but also dissociation of the A2 N and A2 C fragments from the rest of the molecule. 101,104 The human pro-cofactor, factor V, is also inactivated by APC but only when bound to a membranesurface. Human factor V is inactivated by cleavages at Arg306, Arg506, Arg679, and Lys994.…”

“…21 In empirical models conducted with purified reaction components and computer models of the blood coagulation system, ␣-thrombin cannot be generated in the absence of factor V when all inhibitors are present in the reaction system. 101,108,109 Conversely, humans with severe truncations in the factor V molecule that do not allow any synthesis of factor V by known conventional mechanisms have been observed to have either manageable or no pathology associated with bleeding. These observations suggest that there may be compensatory mechanisms present in human blood, which either bypass or reduce the need for factor V in generating levels of ␣-thrombin required for survival.…”

“…118 Arg506 is the initial cleavage site for APC on the heavy chain of human factor Va and facilitates the subsequent lipid-dependent inactivation cleavage at Arg306. 96,101,105 Because this abnormal molecule does not possess the APC-cleavage site at Arg506, factor Va LEIDEN is inactivated by APC at approximately one tenth the rate of normal factor Va. 119 However, inactivation still proceeds through cleavage at Arg306. 97,[119][120][121][122][123][124] Because elimination of the latent activity of membrane-bound factor V occurs because of an initial cleavage at Arg306, factor V LEIDEN is inactivated by APC at the same rate as normal factor V. 119 Platelet-factor Va and factor Va LEIDEN inactivation by APC proceed at similar rates and are delayed when compared with their plasma equivalents.…”