IMMOBILIZATION OF A LYSINE-TERMINATED HEPARIN TO POLYVINYL-ALCOHOL

Lysine terminated heparin, prepared by the nitrous acid partial depoly merization and reductive amination of heparin, failed to increase the active heparin content of a heparin-polyvinyl alcohol (heparin-PVA) hy drogel relative to the unmodified commercial heparin. The depolymeriza tion of heparin resulted in a loss of biological activity which outwei ghed the increase in the terminal amine groups (produced by reductive amination), that were used for glutaraldehyde immobilization to the PV A. The loss in anti-thrombin activity (thrombin time or chromogenic su bstrate) paralleled the increase in anhydromannose end groups due to d epolymerization making it necessary to optimize the loss of activity a gainst the increase in terminal amine groups after amination. For exam ple, 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 th e biological activity but achieved an anhydromannose content of 600 nm oles/mg (approximately 7 cleavage sites/molecule). After the anhydroma nnose groups were reductively aminated by lysine, the anhydromannose c ontent was reduced to 190 nmol/mg indicating a terminal lysine content of 410 nmol/mg. This resulted in an increase in heparin content of th e 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 hydr ogel with unmodified heparin in inactivating thrombin in a flow circui t. These results point out the need for care in interpreting heparin i mmobilization results and for new strategies to increase the active he parin content of this hydrogel.