The antithrombin-dependent inhibition of prothrombinase, assembled at
a macroscopic surface, was studied under flow conditions utilizing a t
ubular flow reactor that consists of a phospholipid-coated glass capil
lary. Prothrombinase activity was determined from steady-state rates o
f thrombin production upon perfusion with prothrombin and from factor
Va-associated factor Xa activity present in the flow reactor. The prot
hrombinase density was maintained at a low level (0.03 fmol/cm(2)) to
assure that the rate of thrombin production reflected the amount of pr
othrombinase present in the capillary. Perfusion of the flow reactor w
ith antithrombin resulted in an exponential decrease of prothrombinase
activity in time. The second order rate constant (8.5 x 10(4) M(-1)mi
n(-1)) is comparable with the rate of inactivation of free factor Xa.
Inhibition was much faster when antithrombin was complexed with hepari
n. The second order rate constants of inhibition decreased with decrea
sing heparin chain length: 9.6 x 10(7), 4.5 x 10(7) and 0.39 x 10(7) M
(-1)min(-1) for unfractionated heparin, low molecular weight heparin a
nd synthetic pentasaccharide heparin, respectively. In the presence of
prothrombin (0.2 mu M), however, the heparin-dependent rate of inhibi
tion of prothrombinase was about 50-fold lower. The heparin-independen
t inhibition of prothrombinase by antithrombin (4 mu M) in the presenc
e of prothrombin (0.2 mu M) was virtually negligible. At a 70-fold hig
her surface density of prothrombinase (2 fmol/cm(2)) prothrombinase ac
tivity was much faster inactivated. The rate of thrombin production, h
owever, was not affected. In conclusion, at low prothrombinase densiti
es, prothrombin efficiently protects prothrombinase from inhibition. A
t high densities, prothrombinase is much less protected but the higher
rare of prothrombinase inactivation has no consequences for the throm
bin production because of the transport-limited regime.