Immobilization of a lysine-terminated heparin to polyvinyl alcohol

Lysine terminated heparin, prepared by the nitrous acid partial depolymerization and reductive amination of heparin, failed to increase the active heparin content of a heparin-polyvinyl alcohol (heparin-PVA) hydrogel relative to the unmodified commercial heparin. The depolymerization of heparin resulted in a loss of biological activity which outweighed the increase in the terminal amine groups (produced by reductive amination), that were used for glutaraldehyde immobilization to the PVA. The loss in anti-thrombin activity (thrombin time or chromogenic substrate) paralleled the increase in anhydromannose end groups due to depolymerization making it necessary to optimize the loss of activity against the increase in terminal amine groups after amination. For example, depolymerization at a high sodium nitrite concentration (81 g/l) at pH4 and 25 degrees C for 20 min, resulted in a loss of 22-40% of the biological activity but achieved an anhydromannose content of 600 nmoles/mg (approximately 7 cleavage sites/molecule). After the anhydromannose groups were reductively aminated by lysine, the anhydromannose content was reduced to 190 nmol/mg indicating a terminal lysine content of 410 nmol/mg. This resulted in an increase in heparin content of the final hydrogel by 53% on mass terms. However, given the reduction in biological activity, it was not surprising that the modified heparin-PVA hydrogel coated on a polyethylene tube was no better than the hydrogel with unmodified heparin in inactivating thrombin in a flow circuit. These results point out the need for care in interpreting heparin immobilization results and for new strategies to increase the active heparin content of this hydrogel.