Uptake and Inactivation of Thrombin on Rabbit Aortic Endothelium Studied with Two Different Substrates

Summary

The endothelium is an important compartment for uptake and inhibition of thrombin. The amount of enzymatically active bound thrombin can be detected with both small synthetic substrates and with aid of fibrinogen as substrate. The present study was designed to investigate the relation between endothelially bound thrombin with amidolytic activity towards a synthetic substrate (S-2238) and thrombin capable of converting fibrinogen by measuring generation of fibrinopeptide A (FPA). The luminal surfaces of rabbit aortae (2 cm²) were exposed in vitro to thrombin (0.625-5.0 NIH units/ml). Thrombin disappeared from the solution and a certain fraction was recovered on the surface. There was a linear relationship between the amount of thrombin on the surface and the concentration of thrombin in the incubation mixture. Approximately one third of the thrombin measured with S-2238 was also able to cleave fibrinogen. After incubation with defibrinogenated plasma almost total inhibition of fibrinogen splitting activity occurred within 30 sec. The inhibition of the amidolytic activity was less complete. When endothelially bound thrombin was exposed to plasma much less FPA was generated than in a fibrinogen solution. A minor fraction of endothelially bound thrombin was inhibited also upon incubation with Tyrode without recovery of any enzymatic activity in the solution. The results indicate that a fraction of thrombin bound to the endothelium has retained enzymatic activity and that a fraction of the enzymatically active thrombin is capable of converting fibrinogen. Inhibition of thrombin enzymatic activity occurs rapidly upon exposure to plasma. The endothelium itself has a minor inhibitory effect also in the absence of plasma. Thrombin with retained fibrinogen splitting activity may contribute to thrombotic events particularly if plasma inhibitors are insufficient. Thrombin with activity towards fibrinogen could be bound to GAG:s whereas thrombin active towards S-2238 could be bound to GAGrs TM or possibly other receptors.

Key words

Endothelium - Thrombin - Chromogenic substrate - Fibrinogen

• References

• 1 Lollar P, Owen WG. Clearance of Thrombin from Circulation in Rabbits by High-affinity Binding Sites on Endothelium. Possible role in the activation of thrombin by antithrombin III. J Clin Invest 1980; 66: 1222-1230
• 2 Dryjski M, Larsson R, Olsson P, Swedenborg J. Effect of gly-cosaminoglycans and antithrombin III on uptake and inhibition of thrombin by the vascular wall. Thromb Res 1983; 32: 355-363
• 3 Lollar P, Hoak JC, Owen WG. Binding of thrombin to cultured human endothelial cells. Nonequilibrium aspects. J Biol Chem 1980; 255: 10279-10283
• 4 Owen WG, Esmon CT. Functional properties of an endothelial cell cofactor for thrombin-catalyzed activation of Protein C. J Biol Chem 1981; 256: 5523-5535
• 5 Esmon CT, Esmon NL, Harris KW. Complex formation between thrombin and thrombomodulin inhibits both thrombin-catalyzed fibrin formation and factor V activation. J Biol Chem 1982; 257: 7944-7947
• 6 Hatton M WC, Dejana E, Casenave JP, Regoeczi E, Mustard JF. Heparin inhibits thrombin binding to rabbit thoracic aorta endothelium. J Lab Clin Med 1980; 96: 861-870
• 7 Olsson P, Larm O, Larsson R, Lins LE, Nilsson E, Swedenborg J. Requirements for thromboresistance of surface heparinized materials. Ann NY Acad Sci 1983; 146: 525-537
• 8 Busch C, Owen WG. Identification in vitro of an endothelial cell surface cofactor for antithrombin III. Parallel studies with isolated perfused rat hearts and microcarrier cultures of bovine endothelium. J Clin Invest 1981; 69: 726-729
• 9 Fenton JW, Fasco MJ, Stackrow AB, Aronson DL, Ljung AM, Finlayson JS. Human thrombins. Production, evaluation and properties of a-thrombin. J Biol Chem 1977; 252: 3587-3598
• 10 Nossel HL, Yudelman I, Canfield RE, Butler V PJr, Spanondis K, Wilner GD, Qureshi GD. Measurement of fibrinopeptide A in human blood. J Clin Invest 1974; 54: 43-53
• 11 Abildgaard U, Lie M, Ödegard OR. Antithrombin (heparin cofactor) assay with new chromogenic substrates (S-2238 and Chromozym TH). Thromb Res 1977; 11: 549-553
• 12 Owen WG. Interactions of thrombin, antithrombin III and protein C with endothelium. In: Biology of Endothelial Cells Jaffe EA. (ed) Martinus Nijhoff Publishers; 1984: 259-267
• 13 Dryjski M, Olsson P, Swedenborg J. Uptake and inhibition of thrombin by the vascular wall. Thromb Res 1982; 27: 467-475
• 14 Swedenborg J, Dryjski M, Olsson P. Inactivation of thrombin by the aortic endothelium. Thromb Haemostas 1983; 50: 635-638
• 15 Larsson R, Olsson P, Lindahl U. Inhibition of thrombin on surfaces coated with immobilized and heparin-like polysaccharides: A crucial non-thrombogenic principle. Thromb Res 1980; 19: 43-54
• 16 Barnhart MI, Chen ST. Vessel wall models for studying interaction capabilities with blood platelets. Semin Thromb Haemostas 1978; 5: 112-155
• 17 Goosen M FA, Sefton MV. Inactivation of thrombin by antithrombin III on a heparinized biomaterial. Thromb Res 1980; 20: 543-554
• 18 Becker CG, Harpel PC. Alpha₂-macroglobulin on human vascular endothelium. J Exp Med 1976; 144: 1-11
• 19 Blomback M, Blomback B, Olsson P, Svendsen L. The assay of antithrombin using a synthetic chromogenic substrate for thrombin. Thromb Res 1974; 5: 621-632