ANTITHROMBIN-HEPARIN AFFINITY REDUCED BY FUCOSYLATION OF CARBOHYDRATEAT ASPARAGINE-155

The two human plasma antithrombin isoforms, alpha and beta, differ in glycosylation at asparagine 135. Only the alpha form carries carbohydr ate at this position and has lower affinity for heparin than the beta form. We previously found additional heterogeneity in a recombinant N1 35Q antithrombin variant, evidenced by two isoforms with a 2-fold diff erence in heparin affinity [Turko, I. V., Fan, B., & Gettins, P. G. W. (1993) FEES Lett. 335, 9-12]. To test whether this heterogeneity of h eparin affinity results from specific glycosylation differences, we ha ve determined the carbohydrate composition at the three remaining glyc osylation sites, asparagine residues 96, 155, and 192, in each of the two N135Q isoforms, by a combination of peptide fragmentation and elec trospray mass spectrometry. Patterns of glycosylation at residues 96 a nd 192 were similar for each isoform and showed the presence of mono-, bi-, and triantennary complex carbohydrate, as well as fucosylation o f all types of chains. At position 155, however, there was a marked di fference between the isoforms, with the form with lower heparin affini ty being 97% fucosylated at this position, whereas the form with highe r affinity for heparin was not fucosylated. Other differences in carbo hydrate type showed no strong correlation between the two isoforms. We conclude that formation of the two heparin-affinity isoforms of N135Q antithrombin results from the specific difference in fucosylation at residue 155, which may result in different structural properties of th e carbohydrate. Consistent with these findings was the elimination of heparin-affinity heterogeneity in a double N135Q-N155Q variant antithr ombin. It is possible that fucosylation of antithrombin may occur in v ivo as a means of modifying the physiological properties of the antith rombin through alteration of the amount of antithrombin bound to surfa ce heparin-like species.