Neutralization of Unfractionated Heparin (UFH) and a Bioengineered Heparin Dodecasccharide (12‐mer) by Protamine Sulfate
Authors: Kouta Ahmed , Duff Richard , Hoppensteadt Debra , Jeske Walter , Abro Schuharazad , Fareed Jawed 
Published: November 2023

Introduction

Heparin, a highly sulfated glycosaminoglycan (GAG), is used extensively as an anticoagulant. It consists of repeating disaccharide units. Heparin, and its analogues, are used during surgery and dialysis, and are often used to coat indwelling catheters and other devices where the vascular system is exposed. Heparin is obtained from mucosal tissue of porcine intestine, and to a lesser extent bovine lung. Chemoenzymatic methods provide an alternate approach to synthesize heparin‐related oligosaccharides. The 12mer represents a homogeneous oligosaccharide with high affinity to antithrombin and exhibits anti‐Xa activity along with very weak anti‐IIa actions which may be comparable to LMWHs. In this study we investigated the neutralization of UFH and 12‐mer by protamine sulfate following intravenous administration to non‐human primates.

Materials & Methods

Porcine UFH and Protamine sulfate were commercially obtained in powder form. 12‐mer was synthesized by a chemo‐enzymatic method and obtained in a powder form. Normal monkey plasma (NMP) was obtained from ten healthy male and female monkeys for in vitro neutralization and calibration studies. In vivo neutralization studies were carried out in individual groups of male and female primate (n=4). Baseline blood samples were drawn and either 12‐mer or UFH treated groups were administered at 500 ug/kg IV to primates (n=4). A second blood samples was drawn at 5 minutes' post drug administration and the protamine sulfate was immediately given at 1.0 mg/kg IV. Additional blood samples were drawn at 5 minutes' post protamine and 30 minutes' post protamine administration. Blood samples were centrifuged to obtain platelet poor plasma (PPP). Samples were analyzed in the two activated partial thrombin time (aPTT) assays using clot based methods. In addition, anti‐IIa activity and anti‐Xa activity were measured using chromogenic methods. Additionally, tissue factor pathway inhibitor (TFPI) antigen level was measured using ELISA method.

Results

In the clot based assays, administration of UFH resulted in a marked increase in the aPTT by both methods (aPTT >300 sec). In contrast the aPTT with the 12‐mer was only marginally increased in both assays (aPTT<50 sec). In the anti‐IIa assay, UFH produced a strong inhibition at 5 mins whereas 12‐mer showed a marginal activity (<10%). In the anti‐Xa assay, both UFH and 12‐mer produced strong inhibitory effect (>90%) 5 mins after administration. Administration of protamine sulfate resulted in complete neutralization of the anticoagulant effect of UFH as measured by aPTT assays and partial neutralization in terms of anti‐IIa and anti‐Xa activities. The administration of protamine had minimal effect on the activity of 12‐mer. In the TFPI assay protamine sulfate administration significantly decreased the circulating TFPI level in heparin‐treated primates, but did not significantly decrease the TFPI release by 12‐mer over the period of the study.

Conclusion

12mer exhibits minimum anti‐coagulant and anti‐IIa activities, yet markedly higher anti‐Xa activity after I.V. administration. Protamine sulfate almost completely neutralized the anticoagulant effects as measured by APTTs and significantly reduced the anti‐Xa effects for UFH. In contrast, protamine sulfate was not effective in neutralizing the effects of 12‐mer in all assays.