Comparative Studies on the Interaction of Heparins from Various Origins with Heparin Cofactor II and Antithrombin

Abstract

IntroductionUnfractionated heparins (UFH) of mucosal origin are currently obtained from bovine, ovine, and porcine sources. In the USP potency assays, bovine mucosal heparin (BMH) exhibit much lower activities (140 U/mg) in comparison to porcine mucosal heparin (PMH; 195 U/mg) and ovine mucosal heparin (OMH; 200 U/mg). Besides molecular and structural differences, these heparins have differential interactions with serpins such as antithrombin (AT) and heparin cofactor II (HCII). The purpose of this study is to compare the inhibitory profiles of various heparins in systems enriched with AT and HCII.Materials andMethodsThe active pharmaceutical ingredients (API) of BMH, OMH, and PMH were obtained from Kin Master (Brazil), Ronssi (China), and Medefil (USA). Bovine (B.LMWH), ovine (O.LMWH), and porcine (P.LMWH) derived low‐molecular‐weight heparins (LMWH, enoxaparin) were obtained from Ronssi (China). Purified human AT, HCII, and thrombin (IIa) were obtained from Enzyme Research (USA). All heparins (UFH’s and LMWH’s) were supplemented to AT and HCII enriched buffer systems at various gravimetric and potency equated concentrations. The relative inhibition of IIa was measured in terms of percent inhibition using an amidolytic method employing IIa specific substrates. Percent inhibition was calculated for each concentration in both systems for each of the three heparins. Inhibitory concentrations required to inhibit 50% of thrombin (IC‐50) was calculated for each UFH and LMWH in terms of µg/mL and U/mL.ResultsIn the AT supplemented system using gravimetric concentrations of various UFH’s, PMH and OMH showed comparable values, whereas BMH exhibited much higher IC‐50’s. However, the LMWH’s of various origins showed comparable IC‐50 values. In this same system, the USP potency adjusted heparins exhibited comparable results for all UFH’s (Figure 1). In the HCII supplemented system, UFH’s of various origins exhibited similar IC‐50 values in both gravimetric and USP potency adjusted studies. Similarly, the LMWH’s when compared in gravimetric amounts demonstrated similar IC‐50 values (Figure 2). The relative IC‐50 values of the LMWH’s in both the AT and HCII enriched systems were higher than the UFH’s.ConclusionThese results show that potency adjusted UFH’s exhibit comparable inhibition of IIa in AT and HCII enriched systems. The differing BMH results observed in these two systems at gravimetric concentrations may partly be due to differences in charge density and AT‐binding consensus sequences. The depolymerized enoxaparin of different origins did not reveal any significant differences in the IC‐50 values. This may be due to the chemical modifications that occur during the manufacturing of these agents. Future studies may be warranted to further investigate these cofactors and their impacts on the function of various heparins.