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Thromb Haemost 2006; 96(01): 29-37
DOI: 10.1160/TH06-01-0052
Platelets and Blood Cells
Schattauer GmbH

Flow cytometry-sorted non-viable endotoxin-treated human monocytes are strongly procoagulant

Carola E. Henriksson
1   The R&D Group, Department of Clinical Chemistry, Ullevaal University Hospital, Oslo, Norwa
,
Marit Hellum   *
1   The R&D Group, Department of Clinical Chemistry, Ullevaal University Hospital, Oslo, Norwa
,
Kirsti S. Landsverk   *
1   The R&D Group, Department of Clinical Chemistry, Ullevaal University Hospital, Oslo, Norwa
,
Olav Klingenberg
1   The R&D Group, Department of Clinical Chemistry, Ullevaal University Hospital, Oslo, Norwa
2   Department of Medical Biochemistry, Rikshospitalet University Hospital, Oslo, Norway
,
Gun-Britt Joø
1   The R&D Group, Department of Clinical Chemistry, Ullevaal University Hospital, Oslo, Norwa
,
Peter Kierulf
1   The R&D Group, Department of Clinical Chemistry, Ullevaal University Hospital, Oslo, Norwa
› Author Affiliations
Further Information

Publication History

Received 26 January 2006

Accepted after resubmission 08 June 2006

Publication Date:
29 November 2017 (online)

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Summary

Monocytes/macrophages are important in disease states such as gram-negative sepsis and coronary artery disease. Following exposure to lipopolysaccharide (LPS), monocytes express tissue factor (TF), the main initiator of blood coagulation. We previously demonstrated that human monocytes treated with high concentrations of LPS, or with LPS and calcium ionophore, displayed higher TF activity than monocytes treated with only low concentrations of LPS, even though the monocytes under all conditions expressed similar amounts of cell surface TF antigen. Such restrained TF activity is often referred to as encryption and its release as de-encryption. We also observed that the increase in TF activity, de-encryption, coincided with an increase in cell surface phosphatidylserine (PS) representing apoptosis and necrosis. In the present work, we separated LPS and LPS and calcium ionophore-treated human monocytes into two populations, one of mainly viable, PS negative cells, and one of mainly non-viable, PS positive cells, by sorting flow-cytometry. We observed that non-viable cells expressed considerably less TF antigen than viable cells. Despite this, non-viable cells were clearly more procoagulant than viable cells in two different coagulation assays. Procoagulant activity was dependent on both TF and PS. We consider the higher content of externalized PS in non-viable monocytes as the major reason for the stronger procoagulant activity of these cells. Thus, TF de-encryption appears largely to occur on PS positive, non-viable cells under these conditions. This supports the important role of PS in coagulation, and it suggests that PS expression signifying cell death, may be clinically relevant.

Keywords

Tissue factor - coagulation - phosphatidylserine - monocyte - sorting flow cytometry

* These authors contributed equally.


 

  • References

  • 1 Brandtzaeg P, Kierulf P, Gaustad P. et al. Plasma endotoxin as a predictor of multiple organ failure and death in systemic meningococcal disease. J Infect Dis 1989; 159: 195-204.
    CrossrefPubMedSearch in Google Scholar
  • 2 Gregory SA, Morrissey JH, Edgington TS. Regulation of tissue factor gene expression in the monocyte procoagulant response to endotoxin. Mol Cell Bio 1989; 09: 2752-5.
    CrossrefPubMedSearch in Google Scholar
  • 3 Nemerson Y. Tissue factor and hemostasis. Blood 1988; 71: 1-8.
    PubMedSearch in Google Scholar
  • 4 Henriksson CE, Klingenberg O, Øvstebø R. et al. Discrepancy between tissue factor activity and tissue factor expression in endotoxin-induced monocytes is associated with apoptosis and necrosis. Thromb Haemost 2005; 94: 1236-44.
    Thieme ConnectPubMedSearch in Google Scholar
  • 5 Robinson RA, Worfolk L, Tracy PB. Endotoxin enhances the expression of monocyte prothrombinase activity. Blood 1992; 79: 406-16.
    PubMedSearch in Google Scholar
  • 6 Bruckheimer EM, Schroit AJ. Membrane phospholipid asymmetry: host response to the externalization of phosphatidylserine. J Leukoc Biol 1996; 59: 784-8.
    CrossrefPubMedSearch in Google Scholar
  • 7 Hanshaw RG, Smith BD. New reagents for phos-phatidylserine recognition and detection of apoptosis. Bioorg Med Chem 2005; 13: 5035-42.
    CrossrefPubMedSearch in Google Scholar
  • 8 Lund PK, Namork E, Brorson SH. et al. The fate of monocytes during 24 h of culture as revealed by flow cytometry and electron microscopy. J Immunol Methods 2002; 270: 63-76.
    CrossrefPubMedSearch in Google Scholar
  • 9 Krishnaswamy S, Field KA, Edgington TS. et al. Role of the membrane surface in the activation of human coagulation factor X. J Biol Chem 1992; 267: 26110-20.
    PubMedSearch in Google Scholar
  • 10 Rapaport SI, Rao LV. Initiation and regulation of tissue factor-dependent blood coagulation. Arterioscle Thromb 1992; 12: 1111-21.
    PubMedSearch in Google Scholar
  • 11 Bach R, Rifkin DB. Expression of tissue factor pro-coagulant activity: regulation by cytosolic calcium. Proc Natl Acad Sci USA 1990; 87: 6995-9.
    CrossrefPubMedSearch in Google Scholar
  • 12 Osterud B, Flaegstad T. Increased tissue thrombo plastin activity in monocytes of patients with meningococcal infection: related to an unfavourable prognosis. Thromb Haemost 1983; 49: 5-7.
    Thieme ConnectPubMedSearch in Google Scholar
  • 13 Hatakeyama K, Asada Y, Marutsuka K. et al. Localization and activity of tissue factor in human aortic atherosclerotic lesions. Atherosclerosis 1997; 133: 213-9.
    CrossrefPubMedSearch in Google Scholar
  • 14 Libby P, Theroux P. Pathophysiology of coronary artery disease. Circulation 2005; 111: 3481-8.
    CrossrefPubMedSearch in Google Scholar
  • 15 Taubman MB, Fallon JT, Schecter AD. et al. Tissue factor in the pathogenesis of atherosclerosis. Thromb Haemost 1997; 78: 200-4.
    Thieme ConnectPubMedSearch in Google Scholar
  • 16 Huang DR, Wang J, Kivisakk P. et al. Absence of monocyte chemoattractant protein 1 in mice leads to decreased local macrophage recruitment and antigen-specific T helper cell type 1 immune response in experimental autoimmune encephalomyelitis. J Exp Med 2001; 193: 713-26.
    CrossrefPubMedSearch in Google Scholar
  • 17 Bach RR. Tissue factor encryption. Arterioscler Thromb Vasc Biol. 2006 doi:10.1161/01.
    PubMedSearch in Google Scholar
  • 18 Bouchard BA, Shatos MA, Tracy PB. Human brain pericytes differentially regulate expression of procoagulant enzyme complexes comprising the extrinsic pathway of blood coagulation. Arterioscler Thromb Vasc Biol 1997; 17: 1-9.
    CrossrefPubMedSearch in Google Scholar
  • 19 Le DT, Rapaport SI, Rao LV. Relations between factor VIIa binding and expression of factor VIIa/tissue factor catalytic activity on cell surfaces. J Biol Chem 1992; 267: 15447-54.
    PubMedSearch in Google Scholar
  • 20 Wolberg AS, Monroe DM, Roberts HR. et al. Tissue factor de-encryption: ionophore-treatment induces changes in tissue factor activity by phosphatidylserine-dependent and -independent mechanisms. Blood Coagul Fibrinolysis 1999; 10: 201-10.
    CrossrefPubMedSearch in Google Scholar
  • 21 Greeno EW, Bach RR, Moldow CF. Apoptosis is associated with increased cell surface tissue factor procoagulant activity. Lab Invest 1996; 75: 281-9.
    PubMedSearch in Google Scholar
  • 22 Ruf W, Rehemtulla A, Morrissey JH. et al. Phospholipid-independent and -dependent interactions required for tissue factor receptor and cofactor function. J Biol Chem 1991; 266: 16256.
    PubMedSearch in Google Scholar
  • 23 Ruf W, Edgington TS. Structural biology of tissue factor, the initiator of thrombogenesis in vivo . FASEB J 1994; 08: 385-90.
    CrossrefPubMedSearch in Google Scholar
  • 24 Lecoeur H, Oliveira-Pinto LM, Gougeon ML. Multiparametric flow cytometric analysis of biochemical and functional events associated with apoptosis and oncosis using the 7-aminoactinomycin D assay. J Immunol Methods 2002; 265: 81-96.
    CrossrefPubMedSearch in Google Scholar
  • 25 Henriksson CE, Klingenberg O, Hellum M. et al. Calcium ionophore-induced de-encryption of tissue factor in monocytes is associated with extensive cell death. Thromb Res. in press.
    PubMed
  • 26 Wharton SA, Riley PA. The suppression of endogenous protein degradation by fractions of foetal calf serum: dialysed serum is less able to suppress degradation in aged cells. Cell Biochem Funct 1986; 04: 189-95.
    CrossrefPubMedSearch in Google Scholar
  • 27 Lund PK, Joo GB, Westvik AB. et al. Isolation of monocytes from whole blood by density gradient centrifugation and counter-current elutriation followed by cryopreservation: six years’ experience. Scand J Clin Lab Invest 2000; 60: 357-65.
    CrossrefPubMedSearch in Google Scholar
  • 28 Hansen JB, Halvorsen DS, Haldorsen BC. et al. Retention of phagocytic functions in cryopreserved human monocytes. J Leukoc Biol 1995; 57: 235-41.
    CrossrefPubMedSearch in Google Scholar
  • 29 Hori S, Heike Y, Takei M. et al. Freeze-thawing procedures have no influence on the phenotypic and functional development of dendritic cells generated from peripheral blood CD14+ monocytes. J Immunother 2004; 27: 27-35.
    CrossrefPubMedSearch in Google Scholar
  • 30 Lund PK, Westvik AB, Joo GB. et al. Flow cytometric evaluation of apoptosis, necrosis and recovery when culturing monocytes. J Immunol Methods 2001; 252: 45-55.
    CrossrefPubMedSearch in Google Scholar
  • 31 Osnes LT, Westvik AB, Kierulf P. Procoagulant and profibrinolytic activities of cryopreserved human monocytes. Thromb Res 1994; 76: 373-83.
    CrossrefPubMedSearch in Google Scholar
  • 32 Ollivier V, Houssaye S, Ternisien C. et al. Endotoxin-induced tissue factor messenger RNA in human monocytes is negatively regulated by a cyclic AMP-dependent mechanism. Blood 1993; 81: 973-9.
    PubMedSearch in Google Scholar
  • 33 Jungi TW. A turbidimetric assay in an ELISA reader for the determination of mononuclear phagocyte procoagulant activity. J Immunol Methods 1990; 133: 21-9.
    CrossrefPubMedSearch in Google Scholar
  • 34 Satta N, Toti F, Feugeas O. et al. Monocyte vesiculation is a possible mechanism for dissemination of membrane-associated procoagulant activities and adhesion molecules after stimulation by lipopolysaccharide. J Immunol 1994; 153: 3245-55.
    PubMedSearch in Google Scholar
  • 35 Darzynkiewicz Z, Juan G, Li X. et al. Cytometry in cell necrobiology: analysis of apoptosis and accidental cell death (necrosis). Cytometry 1997; 27: 1-20.
    CrossrefPubMedSearch in Google Scholar
  • 36 Bach RR, Moldow CF. Mechanism of tissue factor activation on HL-60 cells. Blood 1997; 89: 3270-6.
    PubMedSearch in Google Scholar
  • 37 Hansen CB, van Deurs B, Petersen LC. et al. Discordant expression of tissue factor and its activity in polarized epithelial cells. Asymmetry in anionic phospholipid availability as a possible explanation. Blood 1999; 94: 1657-64.
    PubMedSearch in Google Scholar
  • 38 Ramstrom S, Ranby M, Lindahl TL. Platelet phosphatidylserine exposure and procoagulant activity in clotting whole blood--different effects of collagen, TRAP and calcium ionophore A23187. Thromb Haemost 2003; 89: 132-41.
    Thieme ConnectPubMedSearch in Google Scholar
  • 39 Ashcroft RG, Lopez PA. Commercial high speed machines open new opportunities in high throughput flow cytometry (HTFC). J Immunol Methods 2000; 243: 13-24.
    CrossrefPubMedSearch in Google Scholar
  • 40 Szaniszlo P, Wang N, Sinha M. et al. Getting the right cells to the array: Gene expression microarray analysis of cell mixtures and sorted cells. Cytometry 2004; 59: 191-202.
    PubMedSearch in Google Scholar