The Factor V Activation Paradox

Thomas Orfeo,Nicole Brufatto,Michael E Nesheim,Hung Xu,Saulius Butenas,Kenneth G Mann

doi:10.1074/jbc.m400727200

Thomas Orfeo, Nicole Brufatto + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m400727200

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Abstract

The prothrombinase complex consists of the protease factor Xa, Ca2+, and factor Va assembled on an anionic membrane. Factor Va functions both as a receptor for factor Xa and a positive effector of factor Xa catalytic efficiency and thus is key to efficient conversion of prothrombin to thrombin. The activation of the procofactor, factor V, to factor Va is an essential reaction that occurs early in the process of tissue factor-initiated blood coagulation; however, the catalytic sequence leading to formation of factor Va is a subject of disagreement. We have used biophysical and biochemical approaches to establish the second order rate constants and reaction pathways for the activation of phospholipid-bound human factor V by native and recombinant thrombin and meizothrombin, by mixtures of prothrombin activation products, and by factor Xa. We have also reassessed the activation of phospholipid-bound human prothrombin by factor Xa. Numerical simulations were performed incorporating the various pathways of factor V activation including the presence or absence of the pathway of factor V-independent prothrombin activation by factor Xa. Reaction pathways for factor V activation are similar for all thrombin forms. Empirical rate constants and the simulations are consistent with the following mechanism for factor Va formation. alpha-Thrombin, derived from factor Xa cleavage of phospholipid-bound prothrombin via the prethrombin 2 pathway, catalyzes the initial activation of factor V; generation of factor Va in a milieu already containing factor Xa enables prothrombinase formation with consequent meizothrombin formation; and meizothrombin functions as an amplifier of the process of factor V activation and thus has an important procoagulant role. Direct activation of factor V by factor Xa at physiologically relevant concentrations does not appear to be a significant contributor to factor Va formation.

Highlights

Factor IXa and factor Xa require cofactors to function effectively; factor VIIIa complexes with factor IXa and functions to convert factor X to factor Xa, and factor Va complexes with factor Xa and catalyzes the conversion of prothrombin to ␣-thrombin
The formation of factor Va can be observed as a continuous process involving the dissociation of fragments of the B region from the factor V-vesicle complex. This offers some advantages both over activity-based assays, which involve sampling and quenching before assay and can be confounded by the variable affinity of factor V/Va activation intermediates for factor Xa, and over SDSPAGE analysis, which obscures all intermediates based on noncovalent interactions and, in the case of factor V, is complicated by the poor staining response of B region fragments
The data that we present for the activation of phospholipid-bound factor V using biophysical and biochemical techniques indicate the following order of catalytic efficacy: ␣-thrombin (k ϭ 6.4 ϫ 106 MϪ1 sϪ1) Ͼ meizothrombin (k ϭ 3.5 ϫ 106 MϪ1 sϪ1) ϾϾϾ factor Xa (k ϭ 0.1 ϫ 106 MϪ1 sϪ1)

Summary

Introduction

Factor IXa and factor Xa require cofactors to function effectively; factor VIIIa complexes with factor IXa (the intrinsic tenase) and functions to convert factor X to factor Xa, and factor Va complexes with factor Xa (prothrombinase) and catalyzes the conversion of prothrombin to ␣-thrombin. The propagation phase is driven by rapid factor Xa production derived from the emergence of the intrinsic tenase complex; the requirement for factor VIIIa has proven to be a relatively delayed event [8] Thrombin concentrations at this time are sufficient to account for both the formation of factor VIIIa and the increased rates of factor Va production needed to exploit the growing concentration of factor Xa. the prothrombinase complexes forming earlier during the initiation phase require a source of factor Va that precedes the thrombin formed by these same factor Xa-factor Va complexes. The initial studies into the potential of intact factor V to function as a cofactor showed that substitution of factor V for factor Va in a prothrombinase assay format resulted in prothrombinase activity Յ0.4% that of factor Va [9], a level of catalysis sufficient to function as the first source of ␣-thrombin in the initiating phase of clot generation [9]. Cleavage rates at Arg709 and Arg1018 are roughly equivalent and greater than that at Arg1545, resulting in a characteristic subset of high molecular weight intermediates and the delayed appearance of the light chain relative to the heavy chain when the process is visualized by SDS-PAGE [15, 17] Correlation with bioactivity measurements has indicated that the transition to fully active factor Va during ␣-thrombin catalysis is not complete until the cleavage at Arg1545 is complete (16, 18 –20)

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