Uremic Medium Disturbs the Hemostatic Balance of Cultured Human Endothelial Cells

Mireia Serradell; Maribel Díaz-Ricart; Aleix Cases; María J. Zurbano; José Aznar-Salatti; José López-Pedret; Antonio Ordinas; Ginés Escolar

doi:10.1055/s-0037-1616539

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2001; 86(04): 1099-1105
DOI: 10.1055/s-0037-1616539

Special Article

Schattauer GmbH

Uremic Medium Disturbs the Hemostatic Balance of Cultured Human Endothelial Cells

Mireia Serradell

¹Servicios de Hemoterapia-Hemostasia y Nefrología, Hospital Clínic, IDIBAPS, Barcelona, Spain

,

Maribel Díaz-Ricart

¹Servicios de Hemoterapia-Hemostasia y Nefrología, Hospital Clínic, IDIBAPS, Barcelona, Spain

,

Aleix Cases

¹Servicios de Hemoterapia-Hemostasia y Nefrología, Hospital Clínic, IDIBAPS, Barcelona, Spain

,

María J. Zurbano

¹Servicios de Hemoterapia-Hemostasia y Nefrología, Hospital Clínic, IDIBAPS, Barcelona, Spain

,

José Aznar-Salatti

²Aventis Behring, Barcelona, Spain

,

José López-Pedret

¹Servicios de Hemoterapia-Hemostasia y Nefrología, Hospital Clínic, IDIBAPS, Barcelona, Spain

,

Antonio Ordinas

¹Servicios de Hemoterapia-Hemostasia y Nefrología, Hospital Clínic, IDIBAPS, Barcelona, Spain

,

Ginés Escolar

¹Servicios de Hemoterapia-Hemostasia y Nefrología, Hospital Clínic, IDIBAPS, Barcelona, Spain

› Author Affiliations

Abstract

Summary

We have investigated the ability of serum from uremic patients to modify the thrombogenic properties of the endothelium. The effects of uremic medium on the morphology of endothelial cells (ECs), and their resistance to flow was analyzed. The influence of uremic media on the reactivity of the extracellular matrix (ECM) generated by ECs towards normal platelets was evaluated in a parallel-plate perfusion chamber. Exposure of ECs to uremic medium resulted in abnormal cell morphology and signs of an accelerated growth. Detachment of ECs exposed to circulating blood was increased when cells had been grown with media supplemented with uremic serum (21% vs. 14% non exposed). Platelet deposition was significantly elevated on ECMs generated in the presence of uremic media (uremicECMs) (p<0.01 vs. control studies). Effects of uremic serum were not observed at short incubation periods (5 h) but were evident after 24 or 72 h of incubation. Northern blot analysis revealed increased expression of tissue factor (TF) mRNA in ECs exposed to uremic conditions. Immunocytochemical methods detected an augmented expression of TF antigen on uremic ECMs. Incubation of ECMs with an antibody to human tissue factor prevented the increase in platelet deposition observed in uremic ECMs, suggesting that the presence of TF in ECM could be responsible for the enhanced platelet deposition. Results from our study indicate that uremic medium impairs the antithrombotic functions of cultured endothelial cells.

Keywords

Uremia - endothelial cells - tissue factor - hemostatic balance - atherothrombosis

Full Text

References

References
1 Lewis J, Zucker M, Ferguson J. Bleeding tendency in uremia. Blood 1956; 11: 1073-76.
2 Lindner A, Charra B, Sherrard DJ, Scribner BH. Accelerated atherosclerosis in prolonged maintenance hemodialysis. N Engl J Med 1974; 290: 697-701.
3 Huysmans K, Lins RL, Daelemans R, Zachee P, De Broe ME. Hypertension and accelerated atherosclerosis in end stage renal disease. J Nephrol 1998; 11: 185-95.
4 Lindsay RM, Friesen M, Aronstam A, Andrus F, Clark WF, Linton AL. Improvement of platelet function by increased frequency of hemodialysis. Clin Nephrol 1978; 10: 67-70.
5 Remuzzi G, Livio M, Marchiaro G, Mecca G, Degaetano G. Bleeding in renal failure: altered platelet function in chronic uraemia only partially corrected by haemodialysis. Nephron 1978; 22: 347-53.
6 Diminno G, Martinez J, Mckean ML, Delarosa J, Burke JF, Murphy S. Platelet dysfunction in uremia. Multifaceted defect partially corrected by dialysis. Am J Med 1985; 79: 552-59.
7 Wardle EN, Piercy DA. Studies of contact activation of blood in haemodialysis. J Clin Pathol 1972; 25: 1045-49.
8 Hernandez MR, Galan AM, Lozano M, Bozzo J, Cases A, Escolar G, Calls J, Ordinas A. Platelet-leukocyte activaton during hemodialysis detected with a monoclonal antibody to leukocyte integrin CD11b. Nephron 1998; 80: 197-203.
9 Reverter JC, Escolar G, Sanz C, Cases A, Villamor N, Nieuwenhuis HK, Lopez J, Ordinas A. Platelet activation during hemodialysis measured through exposure of p-selectin: analysis by flow cytometric and ultrastructural techniques. J Lab Clin Med 1994; 124: 79-85.
10 Pearson JD. Endothelial cell function and thrombosis. Bailliere Clin Haem 1999; 12: 329-41.
11 Eberst ME, Berkowitz LR. Hemostasis in renal disease: pathophysiology and management. Am J Med 1994; 96: 168-79.
12 Van Guldener C, Lambert J, Janssen MJ, Donker AJ, Stehouwer CD. Endothelium-dependent vasodilatation and distensibility of large arteries in chronic haemodialysis patients. Nephrol Dial Transplant 1997; 12 (Suppl. 02) 14-8.
13 Aznar-Salatti J, Escolar G, Cases A, Gómez-Ortiz G, Antón P, Castillo R, Revert L, Ordinas A. Uraemic medium causes endothelial cell dysfunction characterized by an alteration of the properties of its subendothelial matrix. Nephrol Dial Transplant 1995; 10: 2199-204.
14 Gordge MP, Neild GH. Platelet function in uraemia. Platelets 1991; 2: 115-23.
15 Jaffe EA, Nachman RL, Becker CG. Culture of human endothelial cells derived from umbilical veins. J Clin Invest 1973; 52: 2745-56.
16 Aznar-Salatti J, Diaz-Ricart M, Anton P, Bastida E, Escolar G, Ordinas A. Variations in the thrombogenic properties of the extracellular matrix produced by endothelial cells in consecutive passages. Sangre 1990; 35: 173-7.
17 Fiore MM, Neuenschwander PF, Morrissey JH. An unusual antibody that blocks tissue factor/factor VIIa function by inhibiting cleavage only of macromolecular substrates. Blood 1992; 80: 3127-34.
18 Sakariassen KS, Aarts PA, de Groot PG, Houdijk WP, Sixma JJ. A perfusion chamber developed to investigate platelet interaction in flowing blood with human vessel wall cells, their extracellular matrix, and purified components. J Lab Clin Med 1983; 102: 522-35.
19 Diaz-Ricart M, Carretero M, Castillo R, Ordinas A, Escolar G. Digital image analysis of platelet-extracellular matrix interactions: studies in von willebrand disease patients and aspirin-treated donors. Haemostasis 1994; 24: 219-29.
20 De Mey J, Moeremans M, Geuens G, Nuydens R, De Brabander M. High resolution light and electron microscopic localization of tubulin with the IGS (immuno gold staining) method. Cell Biol Int Rep 1981; 5: 889-99.
21 Scopsi L, Larsson LI, Bastholm L, Nielsen MH. Silver-enhanced colloidal gold probes as markers for scanning electron microscopy. Histochemistry 1986; 86: 35-41.
22 Hu Y, Oscarson M, Johansson I, Yue QY, Dahl ML, Tabone M, Arinco S, Albano E, Ingelman-Sundberg M. Genetic polymorphism of human CYP2E1: characterization of two variant alleles. Mol Pharmacol 1997; 51: 370-76.
23 Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987; 162: 156-59.
24 Gálvez A, Gómez Ortiz G, Diaz-Ricart M, Escolar G, Gonzalez Sarmiento R, Zurbano MJ, Ordinas A, Castillo R. Desmopressin (DDAVP) enhances platelet adhesion to the extracellular matrix of cultured human endothelial cells through increased expression of tissue factor. Thromb Haemost 1997; 77: 975-80.
25 Ross R. The pathogenesis of atherosclerosis-an update. N Engl J Med 1986; 314: 488-500.
26 Harker LA. Platelets and vascular thrombosis. N Engl J Med 1994; 330: 1006-7.
27 Escolar G, Krumwiede M, White JG. Organization of the actin cytoskeleton of resting and activated platelets in suspension. Am J Pathol 1986; 123: 86-94.
28 Zwaginga JJ, de Boer HC, IJsseldijk MJ, Kerkhof A, Muller-Berghaus G, Gruhlichhenn J, Sixma JJ, de Groot PG. Thrombogenicity of vascular cells. Comparison between endothelial cells isolated from different sources and smooth muscle cells and fibroblasts. Arteriosclerosis 1990; 10: 437-48.
29 Zwaginga JJ, IJsseldijk MJ, Beeser-Visser N, de Groot PG, Vos J, Sixma JJ. High von Willebrand factor concentration compensates a relative adhesion defect in uremic blood. Blood 1990; 75: 1498-508.
30 Lozano M, Bos A, Degroot PG, Vanwilligen G, Meuleman DG, Ordinas A, Sixma JJ. Suitability of low-molecular-weight heparin(oid)s and a pentasaccharide for an in vitro human thrombosis model. Arterioscl Thromb Vas 1994; 14: 1215-22.
31 Rao LVM, Rapaport SI, Bajaj SP. Activation of human factor VII in the initiation of tissue factor-dependent coagulation. Blood 1986; 65: 685-91.
32 Weiss HJ, Lages B. Evidence for tissue factor-dependent activation of the classic extrinsic coagulation mechanism in blood obtained from bleeding time wounds. Blood 1988; 71: 629-35.
33 Weiss HJ, Hawiger J, Ruggeri ZM, Turitto VT, Thiagarajan P, Hoffmann T. Fibrinogen-independent platelet adhesion and thrombus formation on subendothelium mediated by glycoprotein IIb-IIIa complex at high shear rate. J Clin Invest 1989; 83: 288-97.
34 Nemerson Y. Tissue factor and hemostasis. Blood 1988; 71: 1-8.
35 Orvim U, Roald HE, Stephens RW, Roos N, Sakariassen KS. Tissue factor-induced coagulation triggers platelet thrombus formation as efficiently as fibrillar collagen at arterial blood flow conditions. Arterioscl Thromb Vas 1994; 14: 1976-83.
36 Salatti JA, Fenton J, Anton P, Sakariassen KS. alpha-thrombin bound to extracellular endothelial matrix induces pronounced fibrin deposition and platelet thrombus growth in flowing non-anticoagulated human blood. Blood Coagul Fibrin 1994; 5: 561-66.
37 Witko-Sarsat V, Friedlander M, Capeillere-Blandin C, Nguyen-Khoa T, Nguyen AT, Zingraff J, Jungers P, Descamps-Latscha B. Advanced oxidation protein products as a novel marker of oxidative stress in uremia. Kidney Int 1996; 49: 1304-13.
38 Miyata T, Wada Y, Cai Z, Iida Y, Horie K, Yasuda Y, Maeda K, Kurokawa K, Van Yperseled. Implication of an increased oxidative stress in the formation of advanced glycation end products in patients with end-stage renal failure. Kidney Int 1997; 51: 1170-81.
39 Krebs M, Kaun C, Lorenz M, Haag-Weber M, Geiger M, Binder BR. Pro-tease dependent activation of endothelial cells by peritoneal dialysis effluents. Thromb Haemost 1999; 82: 1334-41.
40 Hawrylowicz CM, Howells GL, Feldmann M. Platelet-derived interleukin 1 induces human endothelial adhesion molecule expression and cytokine production. J Exp Med 1991; 174: 785-90.
41 Zimmerman J, Herlinger S, Pruy A, Metzger T, Wanner C. Inflammation enhances cardiovascular risk and mortality in hemodialysis patients. Kidney Int 1999; 55: 648-58.
42 Estebanell E, Rendu F, Cases A, Serradell M, Aznar-Salatti J, Escolar G, Ordinas A, Diaz-Ricart M. Uremic media alters the hemostatic balance of cultured human endothelial cells [Abstract]. Thromb Haemost 1999; (Supplement) 148.