Bovine Heparin Demonstrates the Same Interaction with HIT Antibodies As Porcine Heparin

Heparin, an anticoagulant widely used in numerous medical applications, is considered an essential medicine by the WHO. Due to its high volume use and that it is the parent material for low molecular weight heparins, there is potential for the raw material to be in short supply. The African swine fever epidemic in China, ongoing since August 2018, has added further restraints on heparin source material supply. At present medical grade heparin in the US is only derived from porcine intestinal mucosa; however, there are explorations into using bovine, ovine, and other sources. Bovine heparin, once common place in the US pharmaceutical sector, is again under consideration by the US FDA. This study focused on the primary immunogenic activity associated with heparin, that is heparin-induced thrombocytopenia (HIT), and how the interaction of bovine heparin with functional HIT antibodies compares to that of porcine heparin.

Materials and Methods

Bovine unfractionated heparin from multiple manufacturers was compared to commercial medical grade porcine heparin obtained from US pharmacies. The US Pharmacopeia porcine heparin standard was used to determine potency equivalence. Antibodies to the complex of platelet factor 4/heparin (PF4/heparin) from banked clinically confirmed HIT patient apheresis fluids were combined with heparin and donor platelet rich plasma (PRP; blood collected in citrate from volunteers after signing a consent document). Heparins were tested at final concentrations of 0.1, 0.4, 0.8, 1, and 100 U/mL. The platelet activation response was determined on the BioData PAP-8 Platelet Aggregometer and quantitated in terms of primary slope (PS), area under the curve (AUC), maximum aggregation (MA), and final aggregation (FA).

Characterization of the biophysical interaction between varying molar ratios of human PF4 and heparin was performed using photon correlation spectroscopy (PCS) and zeta potential (Zp) measurements of PF4/heparin complexes using Zetasizer Nano ZS instrumentation and software.

Differences between bovine and porcine heparin were assessed by t-test or Mann-Whitney test. Concentration-response curves were analyzed by two-way ANOVA followed by the Holm-Sidak multiple comparison test using SigmaPlot software.

Results

Platelet activation to PF4/heparin antibodies at bovine and porcine heparin concentrations of 0.1 U/mL (56 ± 9 % vs. 54 ± 11 % MA) and 0.4 U/mL (59 ± 10 % vs. 65 ± 8 % MA) were the same with the expected inhibition (9 ± 4% MA) at the supra-therapeutic concentration of 100 U/mL. Consistent responses were obtained across 21 lots of bovine heparin, 30 lots of porcine heparin, and 38 platelet-HIT antibody combinations. The HIT potential of bovine heparin and porcine heparin was not statistically different (p>0.05). At higher medical use doses, the platelet aggregation response in the presence of HIT antibodies was actually lower for bovine heparin than porcine heparin (0.8 U/mL, 49 ± 10 % vs. 64 ± 9 % MA, p<0.05; and 1.0 U/mL, 45 ± 11 % vs. 62 ± 9 % MA, p<0.05).

By PCS, it was observed that the maximal aggregation between PF4 and either porcine or bovine heparin occurred at comparable molar ratios (7.3 ± 1.5 vs. 6.4 ± 0). Although the porcine and bovine heparins exhibited comparable molecular weights (16,333 ± 153 vs. 16,790 ± 230 Da) and polydispersities (1.19 ± 0.02 vs. 1.15 ± 0.01), porcine heparin formed somewhat larger complexes with PF4 (1113 ± 65 nm) than did bovine heparin (863 ± 68 nm). The molar ratios of PF4 to heparin at which the charge of the complex was fully neutralized (Zp = 0) was comparable for porcine and bovine heparin (9.04 ± 0.19 vs. 9.97 ± 0.65). Consistent responses were obtained across 4 lots of bovine heparin and 3 lots of porcine heparin.

Conclusions

Bovine heparin and porcine heparin had the same in vitro functional platelet activation response in the presence of HIT antibodies, the same potential to form complexes with human PF4, and the same associated features that make PF4 immunogenic. This investigation demonstrates that bovine heparins should have a similar immunogenic response as porcine heparin at equi-unit dosing. Current refinements in the manufacturing process for bovine and porcine heparins have led to well-characterized and purified products which is reflected in their comparable biological behavior.