TRANSGENICALLY PRODUCED HUMAN ANTITHROMBIN - STRUCTURAL AND FUNCTIONAL COMPARISON TO HUMAN PLASMA-DERIVED ANTITHROMBIN

Recombinant human antithrombin (rhAT) produced in transgenic goat milk was purified to greater than 99%. The specific activity of the rhAT w as identical to human plasma-derived AT (phAT) in an in vitro thrombin inhibition assay. However, rhAT had a fourfold higher affinity for he parin than phAT. The rhAT was analyzed and compared with phAT by rever se phase high-performance liquid chromatography, circular dichroism, f luorophore-assisted carbohydrate electrophoresis (FACE), amino acid se quence, and liquid chromatography/mass spectrography peptide mapping. Based on these analyses, rhAT was determined to be structurally identi cal to phAT except for differences in glycosylation. Oligomannose stru ctures were found on the Asn 155 site of the transgenic protein, where as only complex structures were observed on the plasma protein. RhAT c ontained a GalNAc for galactose substitution on some N-linked oligosac charides, as well as a high degree of fucosylation. RhAT was less sial ylated than phAT and contained both N-acetylneuraminic and N-glycolyl- neuraminic acid. We postulate that the increase in affinity for hepari n found with rhAT resulted from the presence of oligomannose-type stru ctures on the Asn 155 glycosylation site and differences in sialylatio n. (C) 1998 by The American Society of Hematology.