Diffusion control in blood coagulation. Activation of factor X by factors IXa/VIIIa assembled on human monocyte membranes.
This study examines mechanisms that regulate the activation of blood coagulation proteases on intact cell membranes. The activation of factor X by factors IXa and VIIIa assembled on viable monocytes is presented as a biologically relevant model for membrane-dependent proteolysis of coagulation zymogens. The hypothesis that this reaction is limited by diffusion was tested by comparing predicted with observed concentration dependence, temperature dependence, and effective rate coefficient. Rates of factor X catalysis were measured using a chromogenic substrate specific for the product, factor Xa. The value of KR and of K1/2, i.e. concentrations giving half-maximal rates in reciprocal functional titrations with substrate and enzyme, respectively, were directly correlated with the concentration of the titrated component. Arrhenius plots constructed over temperatures encompassing 10-35 degrees C were biphasic with downward concavity. Apparent activation energies were 6.01 +/- 0.93 and 35.84 +/- 8.9 kcal/mol for the interval above and below the inflection point, respectively. The effective rate coefficient calculated from apparent kinetic parameters was 3.58 +/- 0.1 x 10(12) M-1 s-1. This rate is similar to the maximal rate of collision between factor X molecules and the monocyte, i.e. 2.9 x 10(12) M-1 s-1 estimated from the steady-state von Smoluchowski equation for uniformly reacting spherical particles. The observed agreement between predicted and experimental results indicates that under biologically relevant conditions, the rate of factor X activation by the intrinsic protease is controlled by diffusion of factor X toward the catalytic site.
McGee
M P
MP
Department of Medicine, Bowman Gray School of Medicine, Winston-Salem, North Carolina 27157-1058.
Li
L C
LC
Xiong
H
H
D001777:Blood Coagulation; D002462:Cell Membrane / Q000378:metabolism; D004058:Diffusion; D015949:Factor IXa / Q000378:metabolism*; D015944:Factor VIIIa / Q000378:metabolism*; D005170:Factor X / Q000378:metabolism*; D015951:Factor Xa / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008433:Mathematics; D008962:Models, Theoretical; D009000:Monocytes / Q000378:metabolism*; D013816:Thermodynamics
D015944:Factor VIIIa; D005170:Factor X; D015949:Factor IXa; D015951:Factor Xa