TY - JOUR
T1 - Inhibition of prothrombinase by antithrombin-heparin at a macroscopic surface
AU - Speijer, Han
AU - Billy, Didier
AU - Willems, George
AU - Hemker, H. Coenraad
AU - Lindhout, Theo
PY - 1995
Y1 - 1995
N2 - The antithrombin-dependent inhibition of prothrombinase, assembled at a macroscopic surface, was studied under flow conditions utilizing a tubular flow reactor that consists of a phospholipid-coated glass capillary. Prothrombinase activity was determined from steady-state rates of thrombin production upon perfusion with prothrombin and from factor Va-associated factor Xa activity present in the flow reactor. The prothrombinase density was maintained at a low level (0.03 fmol/cm(2)) to assure that the rate of thrombin production reflected the amount of prothrombinase present in the capillary. Perfusion of the flow reactor with antithrombin resulted in an exponential decrease of prothrombinase activity in time. The second order rate constant (8.5 x 10(4) M(-1)min(-1)) is comparable with the rate of inactivation of free factor Xa. Inhibition was much faster when antithrombin was complexed with heparin. The second order rate constants of inhibition decreased with decreasing 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 and synthetic pentasaccharide heparin, respectively. In the presence of prothrombin (0.2 mu M), however, the heparin-dependent rate of inhibition of prothrombinase was about 50-fold lower. The heparin-independent inhibition of prothrombinase by antithrombin (4 mu M) in the presence of prothrombin (0.2 mu M) was virtually negligible. At a 70-fold higher surface density of prothrombinase (2 fmol/cm(2)) prothrombinase activity was much faster inactivated. The rate of thrombin production, however, was not affected. In conclusion, at low prothrombinase densities, prothrombin efficiently protects prothrombinase from inhibition. At high densities, prothrombinase is much less protected but the higher rare of prothrombinase inactivation has no consequences for the thrombin production because of the transport-limited regime.
AB - The antithrombin-dependent inhibition of prothrombinase, assembled at a macroscopic surface, was studied under flow conditions utilizing a tubular flow reactor that consists of a phospholipid-coated glass capillary. Prothrombinase activity was determined from steady-state rates of thrombin production upon perfusion with prothrombin and from factor Va-associated factor Xa activity present in the flow reactor. The prothrombinase density was maintained at a low level (0.03 fmol/cm(2)) to assure that the rate of thrombin production reflected the amount of prothrombinase present in the capillary. Perfusion of the flow reactor with antithrombin resulted in an exponential decrease of prothrombinase activity in time. The second order rate constant (8.5 x 10(4) M(-1)min(-1)) is comparable with the rate of inactivation of free factor Xa. Inhibition was much faster when antithrombin was complexed with heparin. The second order rate constants of inhibition decreased with decreasing 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 and synthetic pentasaccharide heparin, respectively. In the presence of prothrombin (0.2 mu M), however, the heparin-dependent rate of inhibition of prothrombinase was about 50-fold lower. The heparin-independent inhibition of prothrombinase by antithrombin (4 mu M) in the presence of prothrombin (0.2 mu M) was virtually negligible. At a 70-fold higher surface density of prothrombinase (2 fmol/cm(2)) prothrombinase activity was much faster inactivated. The rate of thrombin production, however, was not affected. In conclusion, at low prothrombinase densities, prothrombin efficiently protects prothrombinase from inhibition. At high densities, prothrombinase is much less protected but the higher rare of prothrombinase inactivation has no consequences for the thrombin production because of the transport-limited regime.
U2 - 10.1055/s-0038-1653835
DO - 10.1055/s-0038-1653835
M3 - Article
SN - 0340-6245
VL - 73
SP - 648
EP - 653
JO - Thrombosis and Haemostasis
JF - Thrombosis and Haemostasis
IS - 4
ER -