Inhibition of thrombin by antithrombin III in the presence of certain glycosaminoglycans found in the mammalian aorta

Chondroitin-4-sulphate, chondroitin-6-sulphate, dermatan sulphate, heparan sulphate and hyaluronic acid were compared with heparin in their abilities to influence the inactivation of bovine thrombin by rabbit antithrombin III. The effect of the glycosaminoglycans on the enzymic activity of thrombin was examined using the substrate α-N-benzoyl arginine ethyl ester. Heparin, dermatan sulphate, heparan sulphate and, to a smaller extent, chondroitin-6-sulphate increased the esterase activity, whereas chondroitin-4-sulphate and hyaluronic acid had a negligible effect. Heparan sulphate and dermatan sulphate markedly accelerated the inactivation of thrombin by antithrombin III, but chondroitin-4-sulphate, chondroitin-6-sulphate and hyaluronic acid did not significantly affect the reaction.The glycosaminoglycans were adsorbed on Sepharose-lysine or polyacrylamide-ethylenediamine to test their ability to bind thrombin or antithrombin III under conditions where neither protein could directly bind to the conjugates. The binding of thrombin to immobilised heparan sulphate or dermatan sulphate compared well with that to heparin. On immobilised chondroitin-6-sulphate, thrombin was retarded and on chondroitin-4-sulphate no thrombin binding was observed. In contrast to thrombin, antithrombin III only bound to heparin: antithrombin III did not bind to dermatan sulphate, heparan sulphate or the other glycosaminoglycans. Antithrombin III inactivation of 125I-thrombin adsorbed to either heparan sulphate or dermatan sulphate produced a thrombin-antithrombin III complex which was spontaneously released from the glycosaminoglycan. However, no complex was recovered from immobilised heparin unless the column was eluted by 1M NaCl. These results support the view that heparan sulphate and dermatan sulphate could play a role similar to that of heparin in the inactivation of thrombin by antithrombin III.