Abstract
Factor Va is an essential cofactor for the activation of prothrombin catalyzed by factor Xa. The cofactor is a heterodimer composed of a light chain and a heavy chain that are associated noncovalently in the presence of divalent metal ions. The kinetics of the formation of factor Va from the isolated and separated subunits was examined by the time-dependent regain in cofactor activity using direct assays of prothrombin activation catalyzed by prothrombinase. The rate of reassociation at saturating concentrations of calcium ions was slow with a strong temperature dependence. The product of the association reaction was indistinguishable from native factor Va on the basis of activity. The second order rate constant for the process at 37 degrees C in the presence of 2 mM CaCl2 was 1.58 X 10(5) M-1.min-1. Manganese ion increased the rate of regain of activity without influencing the extent of the reaction. The previous identification of a single reactive sulfhydryl in each subunit of factor Va permitted the modification of the separated subunits with sulfhydryl-directed fluorophores. Subunit reassociation was directly measured by fluorescence energy transfer using light chain modified with 6-acryloyl-2-dimethylaminonaphthalene (fluorescence donor) and heavy chain modified with fluorescein 5-maleimide (fluorescence acceptor). Fluorescence measurements indicate that the heavy and light chains associate tightly (Kd = 5.9 x 10(-9) M) and reversibly with a stoichiometry of 1:1. The dissociation of the subunits from the cofactor is first order with a rate constant of 1.03 X 10(-3) min-1. These interpretations were confirmed by physical measurements of subunit reassociation by sedimentation velocity studies.
Highlights
Factor Va either directly or indirectly participates in the native factor Va on the basis of activity
Additional and reversibly witha stoichiometry of 1:l.The disso- evidence indicates that regions of the heavy chain may ciation of the subunits from the cofactoirs first order be involved in the interactions between factor Va and factor with a rate constant of 1.03 X
This enzyme complex catalyzes the proteolytic activation of the zymogen prothrombin to the serine protease a-thrombin which constitutes the penultimate reaction of the procoagulant cascade (1,2, 6-8)
Summary
The existing data indicate that the association process is slow with the possininic acid protein assay reagent, Pierce Chemical CO.)(34),by interpolating values from standard curves constructed using known amounts of unmodified heavy and light chains This procedure was bility of forming cofactor species of significantly greater spe- employed to minimize errors in the determination of protein concencific activity when the subunits are reassociated in the pres- tration due to spectral interference from the covalently incorporated ence of Mn2+ rather thaCn a2+(14,21).In this studyw, e have fluorophores. Initial rates of prothrombin activation were calculated from the initial, steady state portion of continuous progress curves and were converted to unitsdescribing molar IIa produced per unit time by assuming that fluorescence intensity at infinite time represented the quantitative conversion of substrate to product (28) In these measurements, the maximum concentration of factor Va present in the assay (based on the concentration of the limiting subunit in the reassociation mixture) was 1 nM.
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