Heparin: Pure and Simplified

In 1916, Jay McLean, a second-year medical school student walked into the office of William H. Howell, his research professor at Johns Hopkins University, placed a beaker of cat’s blood on a table and asked Howell to tell him when the blood clotted. McLean had added to the blood an extract prepared from liver that he called heparphosphatide. Instead of behaving like a thromboplastin and accelerating coagulation like most tissue-derived substances did, the heparphosphatide-treated blood “never did clot.”¹  This substance, subsequently named heparin by Howell and Luther E. Holt, immediately became the subject of an active investigation into its chemical, biosynthetic, physiologic, and pharmacologic properties that has continued to this day.

Crude preparations of heparin were first used clinically as an antithrombotic in 1935, followed by commercial preparation of unfractionated (UF) heparin from porcine intestine or bovine lung, and, much later, by lowmolecular-weight (LMW) and ultralow-molecular-weight (ULMW) heparins. Animal-derived heparin is a polysaccharide containing variable amounts of a disaccharide-repeating unit of either iduronic acid or glucuronic acid residues linked to a glucosamine residue. Each of these residues is variably sulfated within the polysaccharide chain. Both the variation in polysaccharide sequence and length and the variation in the degree of sulfation result in an extremely heterogeneous population of molecules that collectively is called heparin. UF heparin and LMW heparin, prepared through chemical or enzymatic degradation of UF heparin, contain an average of ~40 and ~14 monosaccharide units, respectively, leading to average molecular weights of 14,000 and 5,000 daltons, respectively. Fondaparinux sodium is a ULMW pentasaccharide that has a molecular weight of 1,508 daltons. In contrast to animal-derived UF and LMW heparins, fondaparinux is a synthetic heparin.

The identification of contaminated heparin formulations in 2007 that produced severe hypersensitivity-type reactions has increased the motivation to develop synthetic heparins to replace animal-derived heparins.²  However, the only commercial synthetic heparin, fondaparinux, is produced by a difficult manufacturing process involving approximately 50 steps. The lengthy chemical synthesis is necessitated by the introduction and removal of protecting groups and results in a yield of only ~0.1 percent. Partly as a result, fondaparinux is the most expensive heparin.

Now Xu et al., working in the laboratory of Jian Liu at the University of North Carolina at Chapel Hill, describe the scalable synthesis at ~40 percent yield of two ULMW heparins with molecular weights of 1,778 and 1,816 daltons, respectively (Figure). The method centers on the use of enzymes that are involved in the biosynthesis of heparan sulfate, a polysaccharide that is closely related to heparin. The chemoenzymatic synthesis includes the use of four sulfotransferases that add sulfo groups at specific sites in the polysaccharide chain. The size of polysaccharide starting material and intermediates, the sequence of sulfo group addition, and improved purification protocols were critical to the optimization process.

Heparin functions as an anticoagulant by binding to antithrombin and accelerating its capacity to inhibit factor Xa and thrombin. Xu et al. report that both of the new ULMW heparins bind antithrombin with affinities similar to that of fondaparinux. Additionally, the capacity of the new ULMW heparins to promote the inhibition of factor Xa by antithrombin is indistinguishable from that of fondaparinux as is the pharmacodynamic behavior of both constructs in rabbits as measured by anti-factor Xa activity.