Summary
A well established technique to improve blood compatibility of artificial materials
for use in the circulation is to coat the surface with heparin. The present report
describes the antithrombin mediated inhibition of thrombin and factor Xa by surfaces
modified with end point immobilized heparin. The reaction was followed by conventional
chromogenic substrate based enzyme assays as well as by immunological measurement
of the enzyme inhibitor (thrombin-antithrombin) complex formation. Both enzymes were
rapidly inactivated by heparin surfaces after selective presaturation with antithrombin
on the immobilized high affinity heparin molecules. The thrombin inhibitory capacity
was enhanced when both high and low affinity heparin were preadsorbed with inhibitor.
The main part of the thrombin-antithrombin complex formed remained bound to the surface,
however, without functionally blocking the activity of the high affinity sequence
of the immobilized heparin.
Aliquots of recalcified plasma were slowly rotated in loops of heparinized tubing
to investigate whether the main thromboresistant function of the surface was exerted
at the level of thrombin or by inactivation of preceding enzymes. After 1 h no visible
clotting occurred and only trace amounts of thrombin (0.07 IU/ml), measured as thrombin-antithrombin
complexes, had been formed. In non-heparinized loops and in the test tube plasma clotted
after 20 min. The thrombin generation when clotting occurred was in the order of 10
IU/ml. It is concluded that the immobilized heparin mediates inhibition of the coagulation
cascade prior to prothrombin activation.
