PB0582 - Differential neutralization of bovine heparins as measured by functional assays and absolute quantification of glycosaminoglycan context

Background: Bovine heparin has been used as anti-coagulant in South East Asian countries and in the Middle East. On a mass basis, bovine heparins exhibit lower anti-coagulant activity in comparison to porcine heparins.

Aims: This study compares the neutralization profile of bovine heparin by protamine sulfate using functional assays.

Methods: The anti-coagulant effects and the anti-Factor Xa/ IIa activities of these Heparins were measured at 2.5 ug/mL upon supplementation in the blood bank plasma prior to and after protamine sulfate neutralization at equi-gravimetric amounts. Absolute glycosaminoglycan (GAG) levels were determined by a fluorescence probe assay (Red Probes, Germany).

Results: The absolute quantification of glycosaminoglycans (GAG) using Heparin Red revealed variable results with average of 87 ± 11% quenching of the fluorescence. Upon protamine sulfate supplementation, the quenching was markedly reduced to an average of 31± 15%.
Bovine Heparins exhibited variable inhibitory effects in the Anti-Factor Xa Assay. The averaged Xa inhibition was 44± 6% at 2.5 ug/mL and range of inhibition was 37- 58% in these samples. The anti-IIa effects of the bovine heparins showed a mean of 19 ± 7% and range of inhibition was 8 – 30% at 2.5 ug/mL. Upon protamine sulfate supplementation, both the anti-factor Xa and IIa effects were completely neutralized. In the aPTT assay, all of the bovine heparin batches at a concentration of 2.5ug/mL produced an increased clotting time with a mean of 62 ± 5 sec. After supplementation of protamine sulfate, all of the bovine heparin batches at 2.5ug/mL produced a reduction in clotting time to a mean of 51 ± 9 sec.

Conclusion(s): In these studies, protamine sulfate did not completely neutralize the glycosaminoglycan (GAG) as measured by Heparin Red method at equi-gravimetric amounts. This suggests that the antithrombin binding consensus sites are more susceptible to protamine neutralization in comparison to other components.