ANTITHROMBIN ACTIVITY OF SURFACE-BOUND HEPARIN STUDIED UNDER FLOW CONDITIONS

Polyacrylamide-grafted polyetherurethane sheets were modified by end-p oint and multipoint attachment of heparin. The surface-bound heparin w as firmly attached. No release of heparin activity could be detected w hen the surface was rinsed at a wall shear rate of 2000 s(-1). Uptake of antithrombin and thrombin inactivation were investigated under well -defined flow conditions by the use of a spinning device with an attac hed disk-shaped heparinized surface. It is demonstrated that the rate of thrombin inactivation at the antithrombin-heparin surface equals th e maximal rate of transport of thrombin toward the surface when the su rface coverage of antithrombin exceeds 10 pmol/cm(2). This result indi cates that a higher intrinsic catalytic efficiency of a surface does n ot necessarily result in a higher antithrombin activity. We varied the heparin content of the surfaces between 0 and 35 mu g/cm(2) by increa sing the number of functional groups to which heparin could be covalen tly attached. The uptake of antithrombin increased with the heparin co ntent of the surface, but the stoichiometry decreased from 2 to 0.5 pm ol antithrombin/mu g heparin. Apparently, antithrombin could not bind to heparins buried in the poly(acrylamide) layer. The rate of thrombin inactivation at surfaces with low heparin content (2 mu g/cm(2)) fell s below the transport limit of thrombin and became proportional with t he heparin content of the surface. Although the contribution of surfac e-bound heparin to the neutralization of fluid-phase thrombin was foun d to be negligible compared with the effect of fluid-phase antithrombi n at physiologic relevant concentrations, these heparinized surfaces m arkedly delayed the onset of thrombin generation in platelet-rich plas ma. It is concluded that the inhibition of locally produced thrombin m ight contribute to the thromboresistance of the heparinized surface. ( C) 1995 John Wiley & Sons, Inc.