The effect of protein adsorption on the anticoagulant activity of surface immobilized heparin

The anticoagulant activity of albumin-heparin conjugates covalently immobilized on carboxylated polystyrene beads was determined before and after exposure to different plasma/PBS dilutions using a thrombin inhibition assay, a FXa inhibition assay, and a modified aPTT assay. Exposure of albumin-heparin modified surfaces (alb-hep surfaces) to plasma dilutions resulted in surfaces with a lower anticoagulant activity than surfaces which were not exposed to plasma dilutions. The reduction of the activity increased up to +/-80% when the surfaces were exposed to solutions containing more than 70% plasma. Alb-hep surfaces incubated in plasma which was preexposed to heparin-Sepharose retained 30% of their initial activity. These observations were attributed to non-specific adsorption of plasma proteins onto the surface and to interaction of heparin binding proteins with the immobilized heparin. Both processes result in a decreased accessibility of the immobilized heparin and thus in a reduced anticoagulant activity displayed by the heparinized surface. Identification of adsorbed proteins with SDS gel electrophoresis and immunoblotting revealed that many different proteins were present at the heparinized surface. Only small differences were observed between the gel electrophoresis pattern of adsorbed proteins obtained from heparinized surfaces and from a surface containing immobilized albumin.