| Description |
Heparin is a heterogeneous polysaccharide the prolongs the clotting time of blood by preventing the fibrin formation. Previous research has demonstrated that heparin immobilized onto a polymer substrate using grafted hydrophilic spacer improved the blood compatibility in in vitro, ex vivo, and in vivo experiments. In this current research, the previous observations were confirmed and explained by studying the binding kinetics of immobilized heparin with antithrombin III (ATIII), thrombin, and fibronectin, both under static and flow conditions. Heparin, with its high affinity for ATIII and monodispersed molecular weight (mw 6,000), was bound to polyethylene oxide (PEO, mw 3,400) which was grafted onto poly(styrene-co-p-aminostyrene). Heparin was immobilized by 1:1 binding with the PEO spacer, resulting in 16.2 % bioactivity, compared to that of free heparin. For spacer-immobilized heparin, the dominant pathway in the anticoagulant processes involved spacer-immobilized heparin initially binding to ATIII, thereby accelerating the binding of thrombin to ATIII. Directly-immobilized heparin, however, bound only to thrombin. So, the mechanism of immobilized heparin was changed by spacer. The TAT complex was then dissociated from the spacer-immobilized heparin quickly, thereby recovering the bioactivity of heparin. The effect of fibronectin on the binding of immobilized heparin with ATIII depended upon the concentration of fibronectin in bulk. Fibronectin could be displaced by ATIII on the heparin binding site at the plasma concentration. To study flow effect on the reactions of immobilized heparin, the dissociation rate of TAT complex and the mechanism of protein binding to immobilized heparin were determined by comparing the theoretical data in numerical simulation with the parallel experimental data. The dissociation rate constant of the TAT complex from spacer-immobilized heparin was gradually increased with increasing flow rate, and the binding of thrombin to heparin-ATIII complex at the surface was controlled by diffusion. Spacer-immobilized heparin reacts with ATIII and thrombin via the main pathway in low flow rates as mentioned above. Therefore, spacer-immobilized heparin surfaces can work at low flow rates as well as high flow rates. |
