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

Carola E. Henriksson; Marit Hellum; Kirsti S. Landsverk; Olav Klingenberg; Gun-Britt Joø; Peter Kierulf

doi:10.1160/TH06-01-0052

Thrombosis and Haemostasis, Table of Contents

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

Authors

Carola E. Henriksson

¹The R&D Group, Department of Clinical Chemistry, Ullevaal University Hospital, Oslo, Norwa
Marit Hellum^*

¹The R&D Group, Department of Clinical Chemistry, Ullevaal University Hospital, Oslo, Norwa
Kirsti S. Landsverk^*

¹The R&D Group, Department of Clinical Chemistry, Ullevaal University Hospital, Oslo, Norwa
Olav Klingenberg

¹The R&D Group, Department of Clinical Chemistry, Ullevaal University Hospital, Oslo, Norwa

²Department of Medical Biochemistry, Rikshospitalet University Hospital, Oslo, Norway
Gun-Britt Joø

¹The R&D Group, Department of Clinical Chemistry, Ullevaal University Hospital, Oslo, Norwa
Peter Kierulf

¹The R&D Group, Department of Clinical Chemistry, Ullevaal University Hospital, Oslo, Norwa

Abstract

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

Full Text

References

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.
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.
3 Nemerson Y. Tissue factor and hemostasis. Blood 1988; 71: 1-8.
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.
5 Robinson RA, Worfolk L, Tracy PB. Endotoxin enhances the expression of monocyte prothrombinase activity. Blood 1992; 79: 406-16.
6 Bruckheimer EM, Schroit AJ. Membrane phospholipid asymmetry: host response to the externalization of phosphatidylserine. J Leukoc Biol 1996; 59: 784-8.
7 Hanshaw RG, Smith BD. New reagents for phos-phatidylserine recognition and detection of apoptosis. Bioorg Med Chem 2005; 13: 5035-42.
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.
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.
10 Rapaport SI, Rao LV. Initiation and regulation of tissue factor-dependent blood coagulation. Arterioscle Thromb 1992; 12: 1111-21.
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.
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.
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.
14 Libby P, Theroux P. Pathophysiology of coronary artery disease. Circulation 2005; 111: 3481-8.
15 Taubman MB, Fallon JT, Schecter AD. et al. Tissue factor in the pathogenesis of atherosclerosis. Thromb Haemost 1997; 78: 200-4.
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.
17 Bach RR. Tissue factor encryption. Arterioscler Thromb Vasc Biol. 2006 doi:10.1161/01.
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.
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.
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.
21 Greeno EW, Bach RR, Moldow CF. Apoptosis is associated with increased cell surface tissue factor procoagulant activity. Lab Invest 1996; 75: 281-9.
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.
23 Ruf W, Edgington TS. Structural biology of tissue factor, the initiator of thrombogenesis in vivo . FASEB J 1994; 08: 385-90.
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.
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.
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.
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.
28 Hansen JB, Halvorsen DS, Haldorsen BC. et al. Retention of phagocytic functions in cryopreserved human monocytes. J Leukoc Biol 1995; 57: 235-41.
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.
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.
31 Osnes LT, Westvik AB, Kierulf P. Procoagulant and profibrinolytic activities of cryopreserved human monocytes. Thromb Res 1994; 76: 373-83.
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.
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.
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.
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.
36 Bach RR, Moldow CF. Mechanism of tissue factor activation on HL-60 cells. Blood 1997; 89: 3270-6.
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.
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.
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.
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.