Crude Heparin Preparations Unveil the Presence of Structurally Diverse Oversulfated Contaminants.

Aline Mendes,Helena B Nader,Maria C Z Meneghetti,Marcelo A Lima,Jawed Fareed,Guilherme L Sassaki,Giselle Zenker Justo,Marcelly Valle Palladino

doi:10.3390/molecules24162988

Abstract

Nowadays, pharmaceutical heparin is purified from porcine and bovine intestinal mucosa. In the past decade there has been an ongoing concern about the safety of heparin, since in 2008, adverse effects associated with the presence of an oversulfated chondroitin sulfate (OSCS) were observed in preparations of pharmaceutical porcine heparin, which led to the death of patients, causing a global public health crisis. However, it has not been clarified whether OSCS has been added to the purified heparin preparation, or whether it has already been introduced during the production of the raw heparin. Using a combination of different analytical methods, we investigate both crude and final heparin products and we are able to demonstrate that the sulfated contaminants are intentionally introduced in the initial steps of heparin preparation. Furthermore, the results show that the oversulfated compounds are not structurally homogeneous. In addition, we show that these contaminants are able to bind to cells in using well known heparin binding sites. Together, the data highlights the importance of heparin quality control even at the initial stages of its production.

Highlights

Heparin is a linear, highly sulfated glycosaminoglycan (GAG) chain of various lengths with molecular weights varying from 2000 to 40,000 Da [1,2] and is composed of a repeating disaccharide units of 1,4 linked α-l-iduronic or β-d-glucuronic acid (d-GlcA), and α-d-glucosamine (d-GlcN).The predominant substitution pattern comprises 2-O-sulfation of the iduronate residues and N- and6-O-sulfation of the glucosamine residues [3].Heparin is an established anticoagulant drug for the prevention and control of thrombotic events owing to its interaction with a number of proteins of the blood clotting cascade, notably antithrombin increasing its inhibitory effect on thrombin and other coagulation serine proteases [4]
The degree of purity of the heparin preparations was initially evaluated by agarose gel electrophoresis in two different buffers (PDA and discontinuous Ba/PDA)
When comparing the results observed for standard heparin and the samples, the presence of other components which bind to the quaternary amine precipitating in the gel and exhibit metachromatic activity with toluidine blue becomes evident

Summary

Introduction

Highly sulfated glycosaminoglycan (GAG) chain of various lengths with molecular weights varying from 2000 to 40,000 Da [1,2] and is composed of a repeating disaccharide units of 1,4 linked α-l-iduronic or β-d-glucuronic acid (d-GlcA), and α-d-glucosamine (d-GlcN).The predominant substitution pattern comprises 2-O-sulfation of the iduronate residues and N- and6-O-sulfation of the glucosamine residues [3].Heparin is an established anticoagulant drug for the prevention and control of thrombotic events owing to its interaction with a number of proteins of the blood clotting cascade, notably antithrombin increasing its inhibitory effect on thrombin and other coagulation serine proteases [4]. The predominant substitution pattern comprises 2-O-sulfation of the iduronate residues and N- and. 6-O-sulfation of the glucosamine residues [3]. Heparin is an established anticoagulant drug for the prevention and control of thrombotic events owing to its interaction with a number of proteins of the blood clotting cascade, notably antithrombin increasing its inhibitory effect on thrombin and other coagulation serine proteases [4]. Antithrombin is the main regulator of coagulation proteases in vertebrates, acting upon thrombin and factors XIIa, XIa, IXa and Xa activities. The inhibitory effect is due to the fact that heparin induces conformational modifications in antithrombin, favoring its interaction with the serine proteases forming a ternary. Heparin is capable of potentiating cofactor II activity, which is another serpin of the coagulation cascade, specific for thrombin inhibition. Upon heparin treatment, endothelial cells enhance the synthesis and release of an antithrombotic heparan sulfate as well as tissue factor pathway inhibitor (TFPI) [7]

Methods

Results

Conclusion