Isolation and Characterization of the Glycosaminoglycan Component of Rabbit Thrombomodulin Proteoglycan

Previous studies on rabbit thrombomodulin (TM) re- vealed that certain anticoagulant activities expressed by TM depend on the presence of an acidic domain tentatively identified as a sulfated galactosaminogly- can (Bourin, M.-C., dhlin, A.-K., Lane, D., Stenflo, J., and Lindahl, U. (1988) J. Biol. Chem. 263, 8044- 8052). The glycan was released by alkaline &elimi- nation, isolated by ion-exchange chromatography, and radiolabeled by partial N-deacetylation (hydrazino- lysis) followed by re-N-[3H]acetylation. The labeled product behaved like standard chondroitin sulfate on ion-exchange chromatography, exhibited a M. of lo- 12 x lo3 on gel chromatography, and was susceptible to degradation by chondroitinase and testicular hya- luronidase. The major labeled degradation products following digestion of the glycosaminoglycan with chondroitinase were identified, depending on the in- cubation conditions, either as 4/6-mono-O-sulfated, 4,5-unsaturated disaccharides (AHexA-GalNAc(S)) and N-acetylgalactosamine 4,6-di-O-sulfate (GalcNAc (dis)), the latter component accounting for -25% of the total label, or as a major fraction of labeled trisac- charide, with the predominant structure GalNAc(diS)- GlcA-GalNAc(diS). The terminal GalNAc(diS) unit (not substituted at C3) was shown to be more suscep- tible to N-deacetylation during hydrazinolysis than were the internal GalNAc units (substituted at C3), and thus was more extensively labeled, resulting in over- representation of this unit. It is concluded that rabbit TM is a chondroitin sulfate proteoglycan, which car- ries a single glycan side chain characterized by an unusual accumulation of sulfate groups at the nonre- ducing terminus. Metabolically 35S-labeled TM was isolated from cultured rabbit heart endothelial cells and characterized as a chondroitin sulfate proteogly- can which accounted for l-2% of the total 35S-labeled cell-associated macromolecules. The isolated chondroi- tin sulfate showed weaker antithrombin-dependent anticoagulant activity, on a molar basis, than the intact TM proteoglycan. The anticoagulant action of TM thus depends on a unique form of functional collaboration between the different constituents of a glycoconjugate.