Selective cleavage of heparin using aqueous 2-hydroxypyridine: Production of an aldose-terminating fragment with high anticoagulant activity

Unfractionated heparin (UFH) was partially depolymerized by heating at 115 °C with aqueous 2-hydroxypyridine. Compared to starting UFH, no significant loss of anticoagulant (anti-Xa) activity was observed. Products consisted of polysaccharide fragments and small quantities of ammonia, sulfate, and hexuronic acid. Fragments with aldose termini that reacted with [3H]NaBH4 (fragment A) were of relatively uniform size (6000 D) and increased as depolymerization time increased. Fragment A contained the anticoagulant activity, with 90–94% and 24–31% binding to Sepharose–thrombin and Sepharose–antithrombin, respectively. In contrast, a non-reducing fragment B that did not react with [3H]NaBH4 was more heterogeneous (6000–10,000 D) and did not have anticoagulant activity or Sepharose–antithrombin affinity. Given the polysaccharide 3H-incorporation, small release of monosaccharide products, and fragment A end-group analysis, thermolysis of UFH is likely limited to one site per molecule when protected by 2-hydroxypyridine. Thus, an anticoagulant fragment A is hydrolytically released from UFH leaving a variable-length fragment B complete with linkage region.