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DOI: 10.1055/s-0038-1651068
Formation of Activated Protein C and Inactivation of Cell-Bound Thrombin by Antithrombin III at the Surface of Cultured Vascular Endothelial Cells - A Comparative Study of Two Anticoagulant Mechanisms[*]
Publication History
Received 01 September 1986
Accepted after revision 26 November 1986
Publication Date:
06 July 2018 (online)
![](https://www.thieme-connect.de/media/10.1055-s-00035024/198701/lookinside/thumbnails/10-1055-s-0038-1651068-1.jpg)
Summary
Intact vascular endothelium provides several anticoagulant mechanisms for the maintenance of blood fluidity and the prevention of thrombosis. High-affinity binding of proteolytic active thrombin to thrombomodulin at the cell surface effectively facilitates the activation of the potent anticoagulant protein C (PC). Rapid inactivation of cell-bound thrombin by antithrombin III (AT III) accelerated by heparin-like structures represents another anticoagulant mechanism. In the present investigation the interference of these two events has been studied. Inhibition of thrombin bound to cultured bovine aortic endothelial cells (BAEC) by AT III and the effect of the inhibitor on the activation of PC has been studied using purified components of bovine origin. Exposure of thrombin (45 nM) with prewashed confluent BAEC-monolayers for 10 min resulted in the binding of 12% thrombin. The subsequent incubation with various concentrations (0.3-2.4 μM) of ATIII revealed no acceleration of the inhibition of thrombin by ATIII at the endothelial cell surface when compared with the uncatalyzed fluid phase reaction. However, heparin added to the reaction mixture substantially increased the inactivation of cell-bound thrombin. Modified ATIII that did not possess heparin cofactor activity presented a comparable inactivation pattern for endothelial cell bound-thrombin as native ATIII indicating that heparin-like structures did not accelerate the interaction. When PC (32 nM) and ATIII (1.8 μM) competed for thrombin bound to BAEC, activation of PC was demonstrated within the initial 6 min of the incubation amounting to 62% of the activated PC formation in the absence of ATIII. Preincubation of BAEC with blocking antibodies against ATIII excluded a possible influence of BAEC-bound ATIII on the capacity of cell-bound thrombin to activate PC. The results suggest that an enhancing mechanism for the inactivation of thrombin by ATIII was not operative at the surfaces of cultured bovine aortic endothelial cells, but instead, appreciable amounts of activated PC become generated in the presence of an excess of ATIII.
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References
- 1 Awbrey BJ, Hoak JC, Owen WG. Binding of human thrombin to cultured human endothelial cells. J Biol Chem 1979; 254: 4092-4095
- 2 Lollar P, Hoak JC, Owen WG. Binding of thrombin to cultured human endothelial cells: nonequilibrium aspects. J Biol Chem 1980; 225: 10279-10283
- 3 Bauer PI, Machovich R, Aranyi R, Büki G, Csonka E, Horvath I. Mechanism of thrombin binding to endothelial cells. Blood 1983; 61: 368-372
- 4 Levine JD, Harlan JM, Harker LA, Joseph ML, Counts RB. Thrombin-mediated release of factor VIII antigen from human umbilical vein endothelial cells in culture. Blood 1982; 60: 531-534
- 5 Pearson JD, Gordon JL. Vascular endothelial and smooth muscle cells in culture selectively release adenine nucleotides. Nature 1979; 281: 384-386
- 6 Weksler BB, Ley CW, Jaffe EA. Stimulation of endothelial cell prostacyclin production by thrombin, trypsin, and the ionophore A 23187. J Clin Invest 1978; 62: 923-930
- 7 Levin EG, Marzek U, Anderson J, Harker LA. Thrombin stimulates tissue plasminogen-activator release from cultured human endothelial cells. J Clin Invest 1984; 74: 1988-1995
- 8 Esmon CT, Owen WG. Identification of an endothelial cell cofactor for thrombin catalyzed activation of protein C. Proc Natl Acad Sci USA 1981; 78: 2249-2252
- 9 Esmon NL, Owen WG, Esmon CT. Isolation of a membrane-bound cofactor for thrombin-catalyzed activation of protein C. J Biol Chem 1982; 257: 859-864
- 10 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
- 11 Esmon NL, Caroll RC, Esmon CT. Thrombomodulin blocks the ability of thrombin to activate platelets. J Biol Chem 1983; 258: 12238-12242
- 12 Fulcher CA, Gardiner JE, Griffin JH, Zimmerman TS. Proteolytic inactivation of human factor VIII procoagulant protein by activated human protein C and its analogy with factor V. Blood 1984; 63: 486-489
- 13 Walker FJ, Sexton PW, Esmon CT. The inhibition of blood coagulation by activated protein C through the selective inactivation of activated factor V. Biochim Biophys Acta 1979; 571: 333-342
- 14 Marcum JA, McKenney JB, Rosenberg RD. Acceleration of thrombin-antithrombin complex-formation in rat hindquarters via heparin like molecules bound to the endothelium. J Clin Invest 1984; 74: 341-350
- 15 Marcum JA, Rosenberg RD. Heparin-like molecules with anticoagulant activity are synthesized by cultured endothelial cells. Biochem Biophys Res Commun 1985; 126: 365-372
- 16 Delvos U, Gajdusek C, Sage H, Harker LA, Schwartz SM. Interactions of vascular wall cells with collagen gels. Lab Invest 1982; 46: 61-72
- 17 Jaffe EA, Hoyer LW, Nachman RL. Synthesis of antihemophilic factor antigen by cultured human endothelial cells. J Clin Invest 1973; 52: 2757-2764
- 18 Ross R. The smooth muscle cell II. Growth of smooth muscle cells in culture and formation of elastic fibers. J Cell Biol 1971; 50: 172-186
- 19 Bertina RM, Broekmans AW, van der Linden IK, Mertens K. Protein C deficiency in a Dutch family with thrombotic disease. Thromb Haemostas 1982; 48: 1-5
- 20 Miletich JP, Broze GJ, Majerus PW. Purification of human coagulation factors II, IX and X using sulfated dextran beads. Meth Enzymol 1981; 80: 221-228
- 21 Miller-Andersson M, Borg H, Andersson LO. Purification of antithrombin III by affinity chromatography. Thromb Res 1974; 5: 439-452
- 22 Thaler E, Schmer G. A simple two-step isolation procedure for human and bovine antithrombin Ililll (Heparin cofactor): a comparison of two methods. Br J Haematol 1975; 31: 233-243
- 23 Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72: 248-254
- 24 Thiel W, Preissner KT, Delvos U, Muller-Berghaus G. A simplified functional assay for protein C in plasma samples. Blut 1986; 52: 169-177
- 25 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4 . Nature 1970; 227: 680-685
- 26 Morita T, Iwanaga S. Prothrombin activator from Echis carinatus venom. Meth Enzymol 1981; 80: 303-311
- 27 Fenton JW, Fasco MJ. Polyethylene glycol 6,000 enhancement of the clotting of fibrinogen solutions in visual and mechanical assays. Thromb Res 1974; 4: 809-813
- 28 Blackbum MN, Smith RL, Carson J, Sibley CC. The heparin binding site of antithrombin III. J Biol Chem 1983; 259: 939-941
- 29 Lollar P, Owen WG. Clearance of thrombin from circulation of rabbits by high-affinity binding sites on endothelium: possible role in the inactivation of thrombin by antithrombin III. J Clin Invest 1980; 66: 1222-1230
- 30 Busch C, Owen WG. Identification in vitro of an endothelial cell surface cofactor for antithrombin III. J Clin Invest 1982; 69: 726-729
- 31 Marcum JA, Atha DH, Fritze L MS, Nawroth P, Stem D, Rosenberg RD. Cloned bovine aortic endothelial cells synthesize anticoagulantly active heparan sulfate proteoglycan. J Biol Chem 1986; 261: 7507-7517
- 32 Shimada K, Ozawa T. Evidence that cell surface heparan sulfate is involved in the high affinity thrombin binding to cultured porcine aortic endothelial cells. J Clin Invest 1985; 75: 1308-1316
- 33 Hatton M WC, Moar SL. A role for pericellular proteoglycan in the binding of thrombin or antithrombin III by the blood vessel endothelium? The effects of proteoglycan-degrading enzymes and glycosaminoglycan-binding proteins on 125-1-thrombin binding by the rabbit thoracic aorta in vitro. Thromb Haemostas 1985; 53: 228-234
- 34 Lollar P, Macintosh SC, Owen WG. Reaction of antithrombin III with thrombin bound to the vascular endothelium: analysis in a recirculating perfused rabbit heart preparation. J Biol Chem 1984; 259: 4335-4338
- 35 Jakubowski HV, Kline MD, Owen WG. The effect of bovine thrombomodulin on the specificity of bovine thrombin. J Biol Chem 1986; 261: 3876-3882
- 36 Chan TK, Chan V. Antithrombin III, the major modulator of intravascular coagulation, is synthesized by human endothelial cells. Thromb Haemostas 1981; 46: 504-506
- 37 Stern DM, Nawroth PP, Kisiel W, Handley D, Drillings M, Bartos J. A coagulation pathway on bovine aortic segments leading to generation of factor Xa and thrombin. J Clin Invest 1984; 74: 1910-1921
- 38 Stern D, Nawroth P, Marcum J, Handley D, Kisiel W, Rosenberg R, Stem K. Interaction of antithrombin III with bovine aortic segments: role of heparin in binding and enhanced anticoagulant activity. J Clin Invest 1985; 75: 272-279