Role of the glycosaminoglycan component of thrombomodulin in its acceleration of the inactivation of single-chain urokinase-type plasminogen activator by thrombin

Thrombomodulin (TM), a membrane proteoglycan on endothelial cells, binds thrombin in a 1:1 complex, accelerates the protein C activation by thrombin, promotes the thrombin inactivation by antithrombin III and inhibits the procoagulant properties of thrombin. The inactivation of single-chain urokinase-type plasminogen activator (scu-PA) by thrombin is accelerated about 70-fold by TM [De Munk, Groeneveld and Rijken (1991) J. Clin. Invest. 88, 1680-1684]. The present study investigates the role of the O-linked glycosaminoglycan moiety of TM in the latter reaction. In the presence of an excess of a fully-glycosylated soluble recombinant human TM mutant (high-M(r) rec-TM), 0.11 nM thrombin inactivated 50% of 4.4nM scu-PA in 45min at 37°C. In the presence of a soluble recombinant TM mutant lacking the glycosaminoglycans (low-M(r) rec-TM), 1.9nM thrombin was needed to inactivate 50% scu-PA, as compared with 4.7nM thrombin in the absence of TM. Using the scu-PA inactivation assay the dissociation constant for the thrombin-TM interaction was found to be 0.4nM for high-M(r) rec-TM and 14nM for low-M(r) rec-TM. Treatment of high-M(r) rec-TM with chondroitinase ABC to digest the glycosaminoglycans decreased the accelerating effect to the level of low-M(r) rec-TM. A similar decrease was observed after treatment of solubilized rabbit TM with chondroitinase ABC. As expected, chondroitinase ABC had no influence on the accelerating effect of low-M(r) rec-TM. The free glycosarninoglycans obtained by alkaline treatment of TM or chondroitin sulphate A also accelerated the inactivation of scu-PA by thrombin, but about 1000-fold higher concentrations than with TM were needed to obtain the same acceleration. It is concluded that the major glycosaminoglycan of TM plays a pivotal role in the inactivation of scu-PA by the TM-thrombin complex, both in the formation and in the activity of the complex. Chemicals/CAS: chondroitin 4 sulfate, 24967-93-9; chondroitin ABC lyase, 9024-13-9; thrombin, 9002-04-4; thrombomodulin, 112049-68-0; urokinase, 139639-24-0; Chondroitin Lyases, EC 4.2.2.-; Glycosaminoglycans; Receptors, Cell Surface; Receptors, Thrombin; Recombinant Proteins; Thrombin, EC 3.4.21.5; Urinary Plasminogen Activator, EC 3.4.21.73