Binding of antithrombin III and thrombin to immobilized heparin under flow conditions.

Abstract

The chemical immobilization of heparin onto polymeric materials through hydrophilic spacer groups was performed to improve the hemocompatibility of blood-contacting devices. Significant data have been gathered attesting to the biological activity of immobilized heparin in static in vitro studies (clotting times) and dynamic in vivo studies (thrombus formation). However, few studies have been performed to investigate the binding kinetics of spacer-immobilized heparin under flow (shear stress) with antithrombin III (ATIII) and thrombin. To help elucidate this binding mechanism, a mathematical model was developed which parallels experiments to measure protein binding and dissociation at the heparin immobilized surface under flow conditions. Heparinized tubing was prepared by chemically immobilizing a high-ATIII-affinity fraction of heparin onto the surface of poly(ethylene)-oxide grafted, poly(styrene-co-p-aminostyrene)-coated polyethylene tubing. ATIII was first bound onto the immobilized heparin, followed by the introduction of thrombin to interact with ATIII. The concentration of thrombin-ATIII complex (TAT) flowing from the tubing was determined, and the dissociation rate constants (kD) of TAT from immobilized heparin were calculated as a function of flow rate. The results indicate that the dissociation rate constant of TAT varied with flow rate, especially low flow rates, high flow rates, and turbulent flow. As the TAT complex dissociates from immobilized heparin, this "recovered" heparin is available for subsequent binding of more ATIII and thrombin. These in vitro mathematical results may help support mechanisms and hypotheses generated for the biological activity of spacer-immobilized heparin observed during long-term in vivo and ex vivo experiments.