Cleavage of the antithrombin Ⅲ binding site in heparin by heparinases and its implication in the generation of low molecular weight heparin

摘要

Heparin has been used as a clinical anticoagulant for more than S0 years, making it one of the most effective pharmacological agents known. Much of heparin's activity can be traced to its ability to bind antithrombin Ⅲ (AT-Ⅲ). Low molecular weight heparin (LMWH), derived from heparin by its controlld breakdown, main- tains much of the antithrombotic activity of heparin without many of the serious side effects. The clinical significance of LMWH has highlighted the need to understand and develop chemical or enzymatic means to generate it. The primary enzymatic tools used for the production of LMWH are the heparinases from Flavobac- terium heparinum, specifically heparinases Ⅰ and Ⅱ. Using pentasac- charide and hexasaccharide model compounds, we show that heparinases Ⅰ and Ⅱ, but not heparinase Ⅲ, cleave the AT-Ⅲ binding site, leaving only a partially intact site. Furthermore, we show herein that glucosamine 3-O sulfation at the reducing end of a glycosidic linkage imparts resistance to heparinase Ⅰ. Ⅱ, and Ⅲ cleavage. Finally, we examine the biological and pharmacological consequences of a heparin oligosaccharide that contains only a partial AT-Ⅲ binding site. We show that such an oligosaccharide lacks some of the functional attributes of heparin- and heparan sulfate-like glycosaminoglycans containing an intact AT-Ⅲ site.