Biologic Potency Variation Among Bovine, Ovine and Porcine Heparins May be Due to their Differential Affinity to Antithrombin
Authors: Yao Yiming , Bouchard Olivia , Abro Schuharazad , Kahn Daniel , Hoppensteadt Debra , Fareed Jawed 
Published: November 2023

IntroductionCurrently, the resourcing of heparin includes bovine (BH), ovine (OH) and porcine mucosal (PH) tissues. The biologic activities including the anti‐coagulant and anti‐protease actions of these agents vary. While the data on the OH is limited, it is reported that BH are relatively less potent than PH. This study, was designed to compare the AT affinity in a biochemically defined system utilizing the functional Anti‐Xa and Anti‐IIa assays.Materials and MethodsSix PH were obtained commercially from the US market. The six OH were obtained from Ronnsi Pharmaceuticals, JiangSu, China. The six BH were obtained from Kin Master, Passo Fundo, Brazil. Amidolytic methods were used to determine the Anti‐Xa and Anti‐IIa activities in an AT supplemented systems. Briefly, 250 uL of AT supplemented buffer containing varying concentrations of each of the individual heparins in the range of 0–10 ug/mL was mixed with a known amount of thrombin or Factor Xa. The relative inhibition of these enzymes was measured using a kinetic analyzer, monitoring the amidolysis of chromogenic substrates. The IC‐50 was calculated as the amount of heparin which produced 50% of the inhibition of the enzymatic activity.ResultsThe IC‐50 for the OH ranged from 0.91–2.58 ug/mL for the Anti‐Xa effects compared to the PH which ranged from 2.18–2.99 ug/mL. The BH ranged from 3.03–4.16 ug/mL. The IC‐50 for the OH ranged from 0.23–0.40 ug/mL for the Anti‐IIa effects. The PH ranged from 0.25–0.83 ug/mL. The BH ranged from 0.37–0.85 ug/mL.The averaged Anti‐Xa IC‐50 value for the OH was 1.74 ug/mL. The averaged value for the PH was 2.43 ug/mL. The averaged value for the BH was 3.45 ug/mL. In the Anti‐IIa assay, the average IC‐50 value for the OH was 0.29 ug/mL. The average for the PH was 0.46 ug/mL. The average for the BH was 0.59 ug/mL.DiscussionThe structural characteristics of AT binding profiles of BH and PH have been recently reported (Naggi, et al. J Pharm and Biomed Analysis 118 (2016) 52–63 ). There were structural variations in the AT binding region (ATBR) and there was higher content of N‐sulfated components in the BH in comparison to the PH. Interestingly, increased N‐sulfation has also been reported for the OH in comparison to PH. However, there is no definitive information on the ATBR for the OH. In the reported studies, a comparison of six individual batches of BH, OH and PH was carried out. In the functional assay in which AT supplementation was used, the relative inhibition of Factor Xa and Factor IIa was quantified in terms of IC‐50 values. The results of these studies confirmed the reported data on the ATBR in the PH revealing that it is a stronger inhibitory agent in both the Anti‐Xa and Anti‐IIa assay than the BH. In this study, the results on OH show that this heparin has even stronger affinity to AT and exhibits even a lower IC‐50 profile for both the Anti‐Xa and Anti‐IIa assays.ConclusionAlthough the NMR data on the ATBR of OH is not available, the data reported in this study indicated that OH may even have a stronger affinity to AT and thus produces stronger anti‐protease effects. To validate these observations, NMR data may provide additional supportive data for OH.