Factor Xa-factor Va complex assembles in two dimensions with unexpectedly high affinity: an experimental and theoretical approach.
The influence of phospholipid vesicle concentration and size on the affinity and the kinetics of assembly of the prothrombin activation complex are examined. Activation of prethrombin 1 was used to monitor complex formation between factors Va and Xa. When activation rates were measured immediately after the addition of the reactants, the rate of activation increased, and subsequently decreased, as a function of increasing vesicle concentration. Larger vesicles did not inhibit the reaction to a comparable extent until much higher phospholipid concentrations were present. The inhibition by high vesicle concentrations was significantly reduced by a prolonged incubation period. These results are interpreted as an initial step of factors Va and Xa binding independently to separate phospholipid vesicles, followed by a slow redistribution between vesicles to maximize complex formation. These experiments indicated that the Kd < or = 25 pM, much tighter than previously reported. Two-dimensional binding on the membrane surface was investigated under conditions where all of the proteins were membrane bound. The complex formation was independent of the surface density of the reactions, indicating a near complete complex formation at the lowest surface density of the reactants. Thus, we conclude that (i) the overall affinity of factor Va-factor Xa interaction in the presence of vesicles is higher than previously appreciated, and (ii) factor Va and factor Xa complex once they bind to the same vesicle.
Ye
J
J
Oklahoma Medical Research Foundation Department of Pathology, University of Oklahoma Health Sciences Center, Oklahoma City, USA.
Esmon
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CT
D000818:Animals; D002417:Cattle; D004789:Enzyme Activation; D015943:Factor Va / Q000737:chemistry* / Q000378:metabolism; D015951:Factor Xa / Q000737:chemistry* / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D046911:Macromolecular Substances; D008567:Membranes, Artificial; D010713:Phosphatidylcholines / Q000737:chemistry; D010718:Phosphatidylserines / Q000737:chemistry; D011485:Protein Binding; D011516:Prothrombin / Q000378:metabolism*
D046911:Macromolecular Substances; D008567:Membranes, Artificial; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D015943:Factor Va; D011516:Prothrombin; D015951:Factor Xa