Interaction of Thrombin and Factor Xa with Bovine Vascular Endothelial Cells, Smooth Muscle Cells and Rat Hepatoma Cells

Maciej Dryjski; Be-Sheng Kuo; Thorir D Bjornsson

doi:10.1055/s-0038-1647020

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 1988; 60(02): 148-152
DOI: 10.1055/s-0038-1647020

Original Article

Schattauer GmbH Stuttgart

Interaction of Thrombin and Factor Xa with Bovine Vascular Endothelial Cells, Smooth Muscle Cells and Rat Hepatoma Cells

Authors

Maciej Dryjski

The Division of Clinical Pharmacology Departments of Medicine and Pharmacology, Duke University Medical Center, Durham, North Carolina, USA and the Division of Clinical Pharmacology, Department of Medicine and Department of Surgery, Jefferson Medical College, Philadelphia, Pennsylvania, USA
Be-Sheng Kuo

The Division of Clinical Pharmacology Departments of Medicine and Pharmacology, Duke University Medical Center, Durham, North Carolina, USA and the Division of Clinical Pharmacology, Department of Medicine and Department of Surgery, Jefferson Medical College, Philadelphia, Pennsylvania, USA
Thorir D Bjornsson

The Division of Clinical Pharmacology Departments of Medicine and Pharmacology, Duke University Medical Center, Durham, North Carolina, USA and the Division of Clinical Pharmacology, Department of Medicine and Department of Surgery, Jefferson Medical College, Philadelphia, Pennsylvania, USA

Abstract

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Summary

The aims of the present investigation were to characterize the binding and inhibition of thrombin and factor Xa to bovine vascular endothelial cells (EC), bovine smooth muscle cells (SMC), and rat hepatoma cells (RHC), and to evaluate the effects of plasma constituents on their inhibition. The enzymatic activities of bovine thrombin and factor Xa were assayed using chromogenic substrates. After 10 min incubation with the cells, thrombin activity in the solution had decreased by about 20% and was subsequently recovered on the cell surfaces. When the cells with the surface-bound thrombin were incubated with defibrinogenated plasma or antithrombin III (AT-III) for 30 sec only about 10% and 20-40%, respectively, of the initial activity could be recovered. In similar experiments with factor Xa, initial activity in the solution had decreased by 10% after 10 min incubation, and was subsequently recovered from the cell surfaces. After 30 sec incubation with AT-III, no cell surface-bound factor Xa activity was detected, whereas 10% of the bound factor Xa activity was recovered after incubation with defibrinogenated plasma. It is concluded that thrombin and factor Xa are taken up and inhibited by EC, SMC and RHC cell surfaces in similar ratios, suggesting that cell surface-mediated inhibition of clotting factors is not restricted to vascular wall cells. The inactivation of factor Xa was dependent on AT-III, however, the inactivation of thrombin was further promoted by an additional unidentified plasma constituent.

Keywords

Thrombin - Factor Xa - Endothelial cells - Smooth muscle cells

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References

References
1 Machovich R, Horvath I. Thrombin and haemostasis: Regulation of the biological functions of thrombin (Facts and perspectives). Haematologia 1981; 14: 339-359
2 Tollefsen DM, Feagler JR, Majerus PW. The binding of thrombin to the surface of human platelets. J Biol Chem 1974; 249: 2646-2651
3 Weksler BB, Lay CW, Jaffe EA. Stimulation of endothelial cell prostacyclin production by thrombin, trypsin and ionophore A23187. J Clin Invest 1978; 62: 923-930
4 Levin EG, Marzec U, Andersson J, Harker LA. Thrombin stimulates tissue plasminogen activator release from cultured human endothelial cell. J Clin Invest 1984; 74: 1988-1995
5 Owen WG, Esmon CT. Functional properties of an endothelial cell cofactor for thrombin-catalyzed activation of protein C. J Biol Chem 1981; 265: 5523-5535
6 Chen LB, Buchanan JM. Mitogenic activity of blood components. I. Thrombin and prothrombin. Proc Natl Acad Sci, USA 1975; 72: 131-135
7 Glenn KC, Cunningham DD. Thrombin-stimulated cell division involves proteolysis of its cell surface receptors. Nature 1979; 278: 711-714
8 Guyton JR, Rosenberg RD, Clowes AW, Karnowsky MJ. Inhibition of rat arterial smooth muscle cells proliferation by heparin. Circ Res 1980; 46: 625-634
9 Savion N, Farzame N. Comparative study of antithrombin III protease complex metabolism by fibroblasts and vascular endothelial cells. Thromb Res 1986; 41: 459-470
10 Swedenborg J, Dryjski M, Olsson P. Inactivation of thrombin by the aortic endothelium. Thromb Haemostas 1983; 50: 635-638
11 Shifman MA, Pizzo SV. The in vivo metabolism of antithrombin III and antithrombin III complexes. J Biol Chem 1982; 257: 3243-3248
12 Aubrey B, Hoak JC, Owen WG. Binding of human thrombin to cultured human endothelial cells. J Biol Chem 1979; 254: 4092-4095
13 Lollar P, Macintosh S, Owen WG. Reaction of antithrombin III with thrombin bound to the vascular endothelium. J Biol Chem 1984; 159: 4335-4338
14 Lollar P, Hoak JC, Owen WG. Binding of thrombin to cultured human endothelial cells. Nonequilibrium aspects. J Biol Chem 1980; 255: 10279-10283
15 Dryjski M, Olsson P, Swedenborg J. Uptake and inhibition of thrombin by the vascular wall. Thromb Res 1982; 27: 467-475
16 Dryjski M, Larsson R, Olsson P, Swedenborg J. Effect of glycosaminoglycans and antithrombin III on uptake and inhibition of thrombin by the vascular wall. Thromb Res 1983; 32: 355-363
17 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: 794-797
18 Swedenborg J, Dryjski M, Frebelius S, Olsson P. Uptake and inactivation of thrombin on rabbit aortic endothelium studies with two different substrates. Thromb Haemostas 1985; 54: 828-832
19 Hatton MW, Moar SL. A role of pericellular proteoglycan in the binding of thrombin or antithrombin III by the blood vessel endothelium? The effects of proteoglycan degrading enzymes and glycoaminoglycan binding proteins on ¹²⁵I-thrombin binding by the rabbit thoracic aorta in vitro. Thromb Haemostas 1985; 53: 228-234
20 Shimada K, Ozawa T. Evidence that cell surface heparin sulfate is involved in the high affinity thrombin binding to cultured porcine aortic endothelial cells. J Clin Invest 1985; 75: 1308-1316
21 Busch C, Owen WG. Identification in vitro of an endothelial cell surface cofactor for antithrombin III. J Clin Invest 1982; 69: 726-729
22 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
23 Fuchs HE, Pizzo SV. Regulation of factor Xa in vitro in human and mouse plasma and in vivo in mouse. J Clin Invest 1983; 72: 2041-2049
24 Heimark RL, Schwartz SM. Binding of coagulation factors Xa and X to the endothelial cell surface. Biochem Res Commun 1983; 111: 723-731
25 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
26 Nawroth PP, Mc Carthy D, Kisiel W, Handley D, Stern D. Cellular processing of bovine factor X and Xa by cultured bovine aortic endothelial cells. J Exp Med 1985; 162: 559-572
27 Gospodarowicz D, Moran J, Braun U, Birdwell CR. Clonal growth of bovine vascular endothelial cells: Fibroblast growth factor as a survival agent. Proc Natl Acad Sci, USA 1976; 73: 4120-4124
28 Jaffe EA, Hoyer LW, Nachman RL. Synthesis of antihemophilic factor antigen by cultured human endothelial cells. J Clin Invest 1973; 52: 2757-2764
29 Ross R. The smooth muscle cell. II. Growth of smooth muscle in culture and formation of elastic fibers. J Cell Biol 1971; 50: 172-186
30 Gimbrone MA. Vascular endothelium and atherosclerosis. In: Vascular Injury and Atherosclerosis. Moore S. (ed.) Marcel Dekker, Inc; New York: 1981: 25
31 Maruyama I, Majerus PW. The turnover of thrombin-thrombomodulin complex in cultured human umbilical vein endothelial cells and A 549 lung cancer cells. J Biol Chem 1985; 260: 15432-15438
32 Levin RI, Weksler BB, Marcus AJ, Jaffe EA. Prostacyclin production by endothelial cells. In: Biology of Endothelial Cells. Jaffe EA. (ed.) Martinus Nijhoff Publishers; Boston, MA: 1984: 228
33 Loskutoff DJ, Levin E. Properties of plasminogen activators produced by endothelial cells. In: Biology of Endothelial Cells. Jaffe EA. (ed.) Martinus Nijhoff Publishers; Boston, MA: 1984: 200
34 Tollefsen DM, Blank MK. Detection of a new heparin-dependent inhibitor of thrombin in human plasma. J Clin Invest 1981; 68: 589-596
35 Church F, Whinna HC. Rapid sulfopropyl-dish chromatographic purification of bovine and human thrombin. Analyt Biochem 1986; 157: 77-83