Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1996 Mar 15;314(Pt 3):1027–1033. doi: 10.1042/bj3141027

Oyxsterols induce membrane procoagulant activity in monocytic THP-1 cells.

K Aupeix 1, F Toti 1, N Satta 1, P Bischoff 1, J M Freyssinet 1
PMCID: PMC1217109  PMID: 8615754

Abstract

Oxidized cholesterol compounds or oxysterols are thought to be potent membrane-destabilizing agents. Anionic phospholipids, chiefly phosphatidylserine, have a procoagulant potential due to their ability to favour the membrane assembly of the characteristic clotting enzyme complexes including the tissue factor-dependent initiating complex. However, in resting cells, phosphatidylserine is sequestered in the inner leaflet of the plasma membrane. When THP-1 monocytic cells were cultured in the presence of 7beta-hydroxycholesterol (7beta-OH) or 25-hydroxycholesterol (25-OH), prothrombinase, which reflects anionic phospholipid exposure and tissue factor (TF) procoagulant activities, increased in a time- and dose-dependent manner. 7beta-OH appeared 1.5- to 2-fold more potent than 25-OH. Interestingly, no effect of cholesterol itself could be detected on procoagulant activities. Nevertheless, no difference in TF activity could be detected between oxysterol-treated and control cells after disruption. TF antigen expression was the same in oxysterol-treated and control cells as shown by flow cytometry. In contrast, the use of labelled annexin V, a protein probe of anionic phospholipids, revealed an elevated number of cells with exposed phosphatidylserine. Because the latter also constitutes a signal for phagocyte recognition of apoptotic cells and fragments, and a proportion of cells displayed altered morphology with condensed chromatin and membrane blebs, analysis of DNA was performed and indicated apoptosis in oxysterol-treated cells. Hence, oxysterol-induced phosphatidylserine exposure and enhanced TF activity may results from apoptosis. These results suggest relationships between oxysterol and the amplification of coagulation reactions by monocytic cells resulting from induced phosphatidylserine exposure.

Full Text

The Full Text of this article is available as a PDF (388.4 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Allen T. M., Williamson P., Schlegel R. A. Phosphatidylserine as a determinant of reticuloendothelial recognition of liposome models of the erythrocyte surface. Proc Natl Acad Sci U S A. 1988 Nov;85(21):8067–8071. doi: 10.1073/pnas.85.21.8067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Almus F. E., Rao L. V., Rapaport S. I. Regulation of factor VIIa/tissue factor functional activity in an umbilical vein model. Arterioscler Thromb. 1993 Jan;13(1):105–111. doi: 10.1161/01.atv.13.1.105. [DOI] [PubMed] [Google Scholar]
  3. Bach R., Rifkin D. B. Expression of tissue factor procoagulant activity: regulation by cytosolic calcium. Proc Natl Acad Sci U S A. 1990 Sep;87(18):6995–6999. doi: 10.1073/pnas.87.18.6995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Boissonneault G. A., Heiniger H. J. 25-Hydroxycholesterol-induced elevations in 45Ca uptake: permeability changes in P815 cells. J Cell Physiol. 1985 Dec;125(3):471–475. doi: 10.1002/jcp.1041250316. [DOI] [PubMed] [Google Scholar]
  5. Boissonneault G. A., Hennig B., Ouyang C. M. Oxysterols, cholesterol biosynthesis, and vascular endothelial cell monolayer barrier function. Proc Soc Exp Biol Med. 1991 Mar;196(3):338–343. doi: 10.3181/00379727-196-43198. [DOI] [PubMed] [Google Scholar]
  6. Brand K., Banka C. L., Mackman N., Terkeltaub R. A., Fan S. T., Curtiss L. K. Oxidized LDL enhances lipopolysaccharide-induced tissue factor expression in human adherent monocytes. Arterioscler Thromb. 1994 May;14(5):790–797. doi: 10.1161/01.atv.14.5.790. [DOI] [PubMed] [Google Scholar]
  7. Brand K., Fowler B. J., Edgington T. S., Mackman N. Tissue factor mRNA in THP-1 monocytic cells is regulated at both transcriptional and posttranscriptional levels in response to lipopolysaccharide. Mol Cell Biol. 1991 Sep;11(9):4732–4738. doi: 10.1128/mcb.11.9.4732. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Carpenter K. L., Wilkins G. M., Fussell B., Ballantine J. A., Taylor S. E., Mitchinson M. J., Leake D. S. Production of oxidized lipids during modification of low-density lipoprotein by macrophages or copper. Biochem J. 1994 Dec 1;304(Pt 2):625–633. doi: 10.1042/bj3040625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Carson S. D., Pirruccello S. J. Immunofluorescent studies of tissue factor on U87MG cells: evidence for non-uniform distribution. Blood Coagul Fibrinolysis. 1993 Dec;4(6):911–920. [PubMed] [Google Scholar]
  10. Carson S. D., Ross S. E., Bach R., Guha A. An inhibitory monoclonal antibody against human tissue factor. Blood. 1987 Aug;70(2):490–493. [PubMed] [Google Scholar]
  11. Chisolm G. M., Ma G., Irwin K. C., Martin L. L., Gunderson K. G., Linberg L. F., Morel D. W., DiCorleto P. E. 7 beta-hydroperoxycholest-5-en-3 beta-ol, a component of human atherosclerotic lesions, is the primary cytotoxin of oxidized human low density lipoprotein. Proc Natl Acad Sci U S A. 1994 Nov 22;91(24):11452–11456. doi: 10.1073/pnas.91.24.11452. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Christ M., Luu B., Mejia J. E., Moosbrugger I., Bischoff P. Apoptosis induced by oxysterols in murine lymphoma cells and in normal thymocytes. Immunology. 1993 Mar;78(3):455–460. [PMC free article] [PubMed] [Google Scholar]
  13. Connor J., Bucana C., Fidler I. J., Schroit A. J. Differentiation-dependent expression of phosphatidylserine in mammalian plasma membranes: quantitative assessment of outer-leaflet lipid by prothrombinase complex formation. Proc Natl Acad Sci U S A. 1989 May;86(9):3184–3188. doi: 10.1073/pnas.86.9.3184. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Dachary-Prigent J., Freyssinet J. M., Pasquet J. M., Carron J. C., Nurden A. T. Annexin V as a probe of aminophospholipid exposure and platelet membrane vesiculation: a flow cytometry study showing a role for free sulfhydryl groups. Blood. 1993 May 15;81(10):2554–2565. [PubMed] [Google Scholar]
  15. Donovan-Peluso M., George L. D., Hassett A. C. Lipopolysaccharide induction of tissue factor expression in THP-1 monocytic cells. Protein-DNA interactions with the promoter. J Biol Chem. 1994 Jan 14;269(2):1361–1369. [PubMed] [Google Scholar]
  16. Dvorak H. F., Quay S. C., Orenstein N. S., Dvorak A. M., Hahn P., Bitzer A. M., Carvalho A. C. Tumor shedding and coagulation. Science. 1981 May 22;212(4497):923–924. doi: 10.1126/science.7195067. [DOI] [PubMed] [Google Scholar]
  17. Edwards R. L., Rickles F. R. The role of leukocytes in the activation of blood coagulation. Semin Hematol. 1992 Jul;29(3):202–212. [PubMed] [Google Scholar]
  18. Faradji A., Bohbot A., Frost H., Schmitt-Goguel M., Siffert J. C., Dufour P., Eber M., Lallot C., Wiesel M. L., Bergerat J. P. Phase I study of liposomal MTP-PE-activated autologous monocytes administered intraperitoneally to patients with peritoneal carcinomatosis. J Clin Oncol. 1991 Jul;9(7):1251–1260. doi: 10.1200/JCO.1991.9.7.1251. [DOI] [PubMed] [Google Scholar]
  19. Freyssinet J. M., Beretz A., Klein-Soyer C., Gauchy J., Schuhler S., Cazenave J. P. Interference of blood-coagulation vitamin K-dependent proteins in the activation of human protein C. Involvement of the 4-carboxyglutamic acid domain in two distinct interactions with the thrombin-thrombomodulin complex and with phospholipids. Biochem J. 1988 Dec 1;256(2):501–507. doi: 10.1042/bj2560501. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Freyssinet J. M., Wiesel M. L., Grunebaum L., Pereillo J. M., Gauchy J., Schuhler S., Freund G., Cazenave J. P. Activation of human protein C by blood coagulation factor Xa in the presence of anionic phospholipids. Enhancement by sulphated polysaccharides. Biochem J. 1989 Jul 15;261(2):341–348. doi: 10.1042/bj2610341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Funakoshi T., Heimark R. L., Hendrickson L. E., McMullen B. A., Fujikawa K. Human placental anticoagulant protein: isolation and characterization. Biochemistry. 1987 Aug 25;26(17):5572–5578. doi: 10.1021/bi00391a053. [DOI] [PubMed] [Google Scholar]
  22. Goldstein J. L., Brown M. S. Regulation of the mevalonate pathway. Nature. 1990 Feb 1;343(6257):425–430. doi: 10.1038/343425a0. [DOI] [PubMed] [Google Scholar]
  23. Hughes H., Mathews B., Lenz M. L., Guyton J. R. Cytotoxicity of oxidized LDL to porcine aortic smooth muscle cells is associated with the oxysterols 7-ketocholesterol and 7-hydroxycholesterol. Arterioscler Thromb. 1994 Jul;14(7):1177–1185. doi: 10.1161/01.atv.14.7.1177. [DOI] [PubMed] [Google Scholar]
  24. Johnson K. A., Morrow C. J., Knight G. D., Scallen T. J. In vivo formation of 25-hydroxycholesterol from endogenous cholesterol after a single meal, dietary cholesterol challenge. J Lipid Res. 1994 Dec;35(12):2241–2253. [PubMed] [Google Scholar]
  25. Kalafatis M., Swords N. A., Rand M. D., Mann K. G. Membrane-dependent reactions in blood coagulation: role of the vitamin K-dependent enzyme complexes. Biochim Biophys Acta. 1994 Nov 29;1227(3):113–129. doi: 10.1016/0925-4439(94)90086-8. [DOI] [PubMed] [Google Scholar]
  26. Kappelmayer J., Kunapuli S. P., Wyshock E. G., Colman R. W. Characterization of monocyte-associated factor V. Thromb Haemost. 1993 Aug 2;70(2):273–280. [PubMed] [Google Scholar]
  27. Koopman G., Reutelingsperger C. P., Kuijten G. A., Keehnen R. M., Pals S. T., van Oers M. H. Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood. 1994 Sep 1;84(5):1415–1420. [PubMed] [Google Scholar]
  28. Kucuk O., Stoner-Picking J., Yachnin S., Gordon L. I., Williams R. M., Lis L. J., Westerman M. P. Inhibition of NK cell-mediated cytotoxicity by oxysterols. Cell Immunol. 1992 Feb;139(2):541–549. doi: 10.1016/0008-8749(92)90091-3. [DOI] [PubMed] [Google Scholar]
  29. Lau W. F., Das N. P. In vitro modulation of rat adipocyte ghost membrane fluidity by cholesterol oxysterols. Experientia. 1995 Jul 14;51(7):731–737. doi: 10.1007/BF01941271. [DOI] [PubMed] [Google Scholar]
  30. Lesnik P., Rouis M., Skarlatos S., Kruth H. S., Chapman M. J. Uptake of exogenous free cholesterol induces upregulation of tissue factor expression in human monocyte-derived macrophages. Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10370–10374. doi: 10.1073/pnas.89.21.10370. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Ottnad E., Parthasarathy S., Sambrano G. R., Ramprasad M. P., Quehenberger O., Kondratenko N., Green S., Steinberg D. A macrophage receptor for oxidized low density lipoprotein distinct from the receptor for acetyl low density lipoprotein: partial purification and role in recognition of oxidatively damaged cells. Proc Natl Acad Sci U S A. 1995 Feb 28;92(5):1391–1395. doi: 10.1073/pnas.92.5.1391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Ramasamy S., Boissonneault G. A., Hennig B. Oxysterol-induced endothelial cell dysfunction in culture. J Am Coll Nutr. 1992 Oct;11(5):532–538. doi: 10.1080/07315724.1992.10718258. [DOI] [PubMed] [Google Scholar]
  33. Rapaport S. I., Rao L. V. Initiation and regulation of tissue factor-dependent blood coagulation. Arterioscler Thromb. 1992 Oct;12(10):1111–1121. doi: 10.1161/01.atv.12.10.1111. [DOI] [PubMed] [Google Scholar]
  34. Ravanat C., Archipoff G., Beretz A., Freund G., Cazenave J. P., Freyssinet J. M. Use of annexin-V to demonstrate the role of phosphatidylserine exposure in the maintenance of haemostatic balance by endothelial cells. Biochem J. 1992 Feb 15;282(Pt 1):7–13. doi: 10.1042/bj2820007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Ravanat C., Torbet J., Freyssinet J. M. A neutron solution scattering study of the structure of annexin-V and its binding to lipid vesicles. J Mol Biol. 1992 Aug 20;226(4):1271–1278. doi: 10.1016/0022-2836(92)91066-x. [DOI] [PubMed] [Google Scholar]
  36. Reid V. C., Hardwick S. J., Mitchinson M. J. Fragmentation of DNA in P388D1 macrophages exposed to oxidised low-density lipoprotein. FEBS Lett. 1993 Oct 18;332(3):218–220. doi: 10.1016/0014-5793(93)80635-8. [DOI] [PubMed] [Google Scholar]
  37. Reisch J., Pera F., Cramer M. Detection and localization of autofluorescent substances in thymus tissue of sheep. Ann Anat. 1994 Dec;176(6):555–557. doi: 10.1016/s0940-9602(11)80395-x. [DOI] [PubMed] [Google Scholar]
  38. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993 Apr 29;362(6423):801–809. doi: 10.1038/362801a0. [DOI] [PubMed] [Google Scholar]
  39. Ruf W., Edgington T. S. Structural biology of tissue factor, the initiator of thrombogenesis in vivo. FASEB J. 1994 Apr 1;8(6):385–390. [PubMed] [Google Scholar]
  40. Sambrano G. R., Steinberg D. Recognition of oxidatively damaged and apoptotic cells by an oxidized low density lipoprotein receptor on mouse peritoneal macrophages: role of membrane phosphatidylserine. Proc Natl Acad Sci U S A. 1995 Feb 28;92(5):1396–1400. doi: 10.1073/pnas.92.5.1396. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Satta N., Toti F., Feugeas O., Bohbot A., Dachary-Prigent J., Eschwège V., Hedman H., Freyssinet J. M. Monocyte vesiculation is a possible mechanism for dissemination of membrane-associated procoagulant activities and adhesion molecules after stimulation by lipopolysaccharide. J Immunol. 1994 Oct 1;153(7):3245–3255. [PubMed] [Google Scholar]
  42. Savill J., Fadok V., Henson P., Haslett C. Phagocyte recognition of cells undergoing apoptosis. Immunol Today. 1993 Mar;14(3):131–136. doi: 10.1016/0167-5699(93)90215-7. [DOI] [PubMed] [Google Scholar]
  43. Sugimura M., Donato R., Kakkar V. V., Scully M. F. Annexin V as a probe of the contribution of anionic phospholipids to the procoagulant activity of tumour cell surfaces. Blood Coagul Fibrinolysis. 1994 Jun;5(3):365–373. [PubMed] [Google Scholar]
  44. Szostek R., Kucuk O., Lis L. J., Tracy D., Mata R., Dey T., Kauffman J. W., Yachnin S., Westerman M. P. Effect of inserted oxysterols on phospholipid packing in normal and sickle red blood cell membranes. Biochem Biophys Res Commun. 1991 Oct 31;180(2):730–734. doi: 10.1016/s0006-291x(05)81126-x. [DOI] [PubMed] [Google Scholar]
  45. Tipping P. G., Malliaros J., Holdsworth S. R. Procoagulant activity expression by macrophages from atheromatous vascular plaques. Atherosclerosis. 1989 Oct;79(2-3):237–243. doi: 10.1016/0021-9150(89)90129-9. [DOI] [PubMed] [Google Scholar]
  46. Zhou Q., Jimi S., Smith T. L., Kummerow F. A. The effect of 25-hydroxycholesterol on accumulation of intracellular calcium. Cell Calcium. 1991 Jul;12(7):467–476. doi: 10.1016/0143-4160(91)90029-e. [DOI] [PubMed] [Google Scholar]
  47. Zwijsen R. M., Oudenhoven I. M., de Haan L. H. Effects of cholesterol and oxysterols on gap junctional communication between human smooth muscle cells. Eur J Pharmacol. 1992 Sep 1;228(2-3):115–120. doi: 10.1016/0926-6917(92)90020-d. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES