Transport rate limited catalysis on macroscopic surfaces: the activation of factor X in a continuous flow enzyme reactor.
Blood coagulation is initiated on cells which present a macroscopic surface to the flowing blood stream. We have used a continuous flow enzyme reactor to model this system and to investigate the effects of shear rate and mass transport on the activation of factor X by the complex of the transmembrane protein, tissue factor, and the serine protease, factor VIIa. This initial step of blood coagulation was found to be half-maximal at very low enzyme densities (0.03-0.06%) on the wall of the capillaries. In agreement with hydrodynamic theory, the apparent Km in the flow reactor was correlated with the cube root of the wall shear rate. These data indicate that at high tissue factor densities (> 0.6%) the activation of 150 nM factor X is controlled by the flux of X toward the surface, which is controlled by wall shear rate and substrate concentration. The appearance of the product, Xa, in the effluent was delayed to 8-12 min, which was caused by high-affinity binding of Xa to the phospholipid. This delay was considerably shortened by embedding tissue factor into PC or by coating the PS/PC surface with the phospholipid binding protein, annexin V. At low tissue factor densities, annexin V inhibited X activation by 45%, while no inhibition was observed at high densities. We demonstrate that when the reaction is limited by substrate flux, addition of further enzyme does not increase reaction rates. This contrasts with classical three-dimensional catalysis in which the initial velocity is ordinarily linear with the enzyme concentration.
Andree
H A
HA
Department of Biochemistry, Mt. Sinai School of Medicine, City University of New York, New York 10029.
Contino
P B
PB
Repke
D
D
Gentry
R
R
Nemerson
Y
Y
D000595:Amino Acid Sequence; D017304:Annexin A5 / Q000378:metabolism; D001692:Biological Transport; D002384:Catalysis; D004800:Enzymes, Immobilized; D015942:Factor VIIa / Q000378:metabolism; D005170:Factor X / Q000096:biosynthesis / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010713:Phosphatidylcholines / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism; D012212:Rheology / Q000295:instrumentation; D013925:Thromboplastin / Q000378:metabolism
D017304:Annexin A5; D004800:Enzymes, Immobilized; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; C042511:1,2-dioleoylphosphatidylserine; D005170:Factor X; D013925:Thromboplastin; D015942:Factor VIIa; C017251:1,2-oleoylphosphatidylcholine