Flow and the inhibition of prothrombinase by antithrombin III and heparin
P Schoen and T Lindhout
Department of Biochemistry, University of Limburg, Maastricht, The
Netherlands.
Inhibition of prothrombinase by antithrombin III (ATIII) and heparin was
investigated in a continuous-flow system. Phospholipid-coated capillaries,
containing phospholipid-bound factor Xa and factor Va, were perfused with
1.0 mumol/L prothrombin and 0.5 nmol/L factor Va. At 25 degrees C and a
flow rate of 32 microL/min (shear rate 28 seconds-1) the steady-state rates
of prothrombin conversion depended linearly on the surface concentration of
prothrombinase up to 2 fmol/cm2. The rate of thrombin generation was 952
+/- 43 (SE) mol/min/mol prothrombinase. When ATIII was included in the
perfusate for 10 minutes, the free thrombin concentration at the outlet of
the capillary was markedly reduced: a 50% neutralization was obtained at
0.7 mumol/L ATIII. However, the prothrombinase activity was not inhibited,
as could be established after a subsequent perfusion with prothrombin and
factor Va. At an ATIII concentration typical of normal plasma (2 mumol/L) a
slight neutralization of prothrombinase was observed: 10% neutralization
following a 10-minute perfusion. During a perfusion with ATIII in the
absence of prothrombin, or in its presence with hirudin (2 mumol/L) also
included in the perfusate, a more pronounced neutralization of
prothrombinase was observed: 40% residual activity was obtained after a
10-minute perfusion. From this observation the suggestion comes forward
that thrombin, continuously produced at the surface, consumes ATIII in the
boundary layer. In this case the true ATIII concentration in the vicinity
of surface-bound prothrombinase will be but a small fraction of the initial
ATIII concentration in the bulk fluid. Unfractionated heparin and an
ultra-low molecular weight heparin (pentasaccharide) did enhance the
ATIII-dependent neutralization of prothrombinase, but to a much lesser
extent than observed with small unilaminar phospholipid vesicles as the
catalytic sites for prothrombinase assembly. The findings reported here
support the notion that regulation of prothrombinase by heparin under in
vivo conditions occurs at the stage of its formation, ie, through
inhibition of free factor Xa and/or the generation of factor Va, rather
than by direct inhibition of the prothrombinase activity.
Volume 78,
Issue 1,
pp. 118-124,
07/01/1991
Copyright © 1991 by The American Society of Hematology
