Competitive Inhibition of Heparinase by Persulfonated Glycosaminoglycans: A Tool to Detect Heparin Contamination

Abstract

Heparin and the low molecular weight heparins are extensively used as medicinal products to prevent and treat the formation of venous and arterial thrombi. In early 2008, administration of some heparin lots was associated with the advent of severe adverse effects, indicative of an anaphylactoid-like response. Application of orthogonal analytical tools enabled detection and identification of the contaminant as oversulfated chondroitin sulfate (OSCS) was reported in our earlier report. Herein, we investigate whether enzymatic depolymerization using the bacterially derived heparinases, given the structural understanding of their substrate specificity, can be used to identify the presence of OSCS in heparin. We also extend this analysis to examine the effect of other persulfonated glycosaminoglycans (GAGs) on the action of the heparinases. We find that all persulfonated GAGs examined were effective inhibitors of heparinase I, with IC50 values ranging from approximately 0.5–2 µg/mL. Finally, using this biochemical understanding, we develop a rapid, simple assay to assess the purity of heparin using heparinase digestion followed by size-exclusion HPLC analysis to identify and quantify digestion products. In the context of the assay, we demonstrate that less than 0.1% (w/w) of OSCS (and other persulfonated polysaccharides) can routinely be detected in heparin.

Crude Heparin Preparations Unveil the Presence of Structurally Diverse Oversulfated Contaminants

“…In previous work we have shown that the inhibition of heparinase by OSCS is irreversible, since addition of heparin does not dislodge the inhibitor [21]. Similar results were also found by other authors [22].…”

“…The differences observed in the figure are related to the fact that bovine heparin is a better substrate for heparitinase II compared to porcine heparin. The results from other authors, performing kinetics studies of heparitinase II in the presence of OSCS indicate that the enzyme is inhibited [22]. These apparent discrepancies may be related to the types of experiments performed by the groups.…”

Pharmacology of Heparin and Related Drugs

“…Other ingenious methods for detection and quantification of OSCS in heparin include a heparinase inhibition assay (Aich et al, 2011), a convenient disposable strip-type electrochemical polyanion sensor (Kang et al, 2011), pyrolysis MS (Nemes et al, 2013), and spectropolarimetry (Chmielewski et al, 2011;Stanley et al, 2011).…”

Preparation and Application of Nanosensor in Safeguarding Heparin Supply Chain

“…However, the C5 proton of 3-O-sulfonated uronic acid is still further away from the critical H151 amino acid residue of the enzyme, prohibiting the cleavage of OSCS, which leads to the enzyme inhibition. 18 In this technical report, we discuss the synthesis and application of the gold-heparin dye nanosensor in assessing OSCS levels in pharmaceutical heparin lots for safeguarding the complex global supply chain.…”