Phospholipid surfaces regulate the delivery of substrate to tissue factor:VIIa and the removal of product.
For many years, the essential role of tissue factor (TF) in coagulation and thrombogenesis has been recognized. The catalytic complex of TF and VIIa (TF:VIIa) is membrane-bound whereas its substrate, factor X (FX), is distributed between a phospholipid-bound fraction and one that is in true solution in 3-dimensional space. This complicates analytical solutions for the kinetic mechanisms describing this reaction because dimensionality must be preserved. We believe that, at the time of activation, FX is simultaneously bound to TF:VIIa and the phospholipid surface. The hydrolysis of a peptide bond activates FX and the product, Xa, is yet bound to the catalytic complex in a manner such that it must leave before a new molecule of X encounters the complex. This means that, in principle, the classically defined Vmax does not apply because on a surface, infinite substrate and its attendant infinite collision frequency do not apply. We show that increasing the lipid surface area available to each TF:VIIa increases the apparent k(cat) and that it approaches a maximum asymptotically, exhibiting a K(1/2) at a 40 nm lipid radius. Notably, this is of the same order as transient confinement zones that have been identified on the surface of living cells. We believe the increased lipid surface area allows the Xa to easily diffuse away from the enzyme complex along the 2D lipid surface, thereby allowing new substrate to approach the enzyme and minimizing collisions between the product and the enzyme complex (product inhibition). Thus, after Xa leaves the vicinity of the enzyme, a new FX molecule is able to bind TF:VIIa and the rate at which this complex forms cannot exceed the leaving rate of Xa from the TF:VIIa and phospholipid sites. Thus, this parameter is of critical interest. Starting with the off-rate of Xa from appropriate phospholipid surfaces, we note that the literature values differ by a factor of approximately 500. Using energy transfer techniques between 30% phosphatidylserine/70% phosphatidylcholine vesicles and human F.Xa, we measured this off rate and found it agrees closely with the Biacore generated data. We have determined the binding parameters of Xa to vesicles and a continuous supported bilayer. Our data are in excellent agreement with the data derived using a lipid coated Biacore chip.
Hathcock
James J
JJ
Department of Medicine, Mt. Sinai School of Medicine, New York, NY 10029, USA. James.Hathcock@mssm.edu
Rusinova
Elena
E
Andree
Harry
H
Nemerson
Yale
Y
D002384:Catalysis; D004058:Diffusion; D015942:Factor VIIa / Q000378:metabolism*; D005170:Factor X / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008954:Models, Biological; D010743:Phospholipids / Q000378:metabolism*; D021381:Protein Transport; D013499:Surface Properties; D013925:Thromboplastin / Q000378:metabolism*
D010743:Phospholipids; D005170:Factor X; D013925:Thromboplastin; D015942:Factor VIIa