Thromb Haemost 1997; 77(04): 690-696
DOI: 10.1055/s-0038-1656035
Coagulation
Schattauer GmbH Stuttgart

Modulation of Blood Cell Activation by Four Commonly Used Anticoagulants

Charlotte Sissener Engstad
1   The Department of Biochemistry, Institute of Medical Biology, University of Tromsø
,
Tore Jarl Gutteberg
2   The Microbiology Department, University Hospital of Tromsø, Tromsø, Norway
,
Bjarne Østerud
1   The Department of Biochemistry, Institute of Medical Biology, University of Tromsø
› Institutsangaben
Weitere Informationen

Publikationsverlauf

Received 23. August 1996

Accepted after resubmission 29. November 1996

Publikationsdatum:
11. Juli 2018 (online)

Summary

In the past years, our group has made several observations suggesting that blood cells behave differently and when stimulated, release different levels of cytokines, depending on which anticoagulant the blood has been drawn into. The aim of this study was therefore to compare the effect of the four anticoagulants EDTA, citrate, heparin and hirudin on monocyte, neutrophil (PMN), and platelet function in human whole blood. Human whole blood was employed as an ex vivo model of cytokine production and protein secretion, and lipopolysaccharide (LPS) induced tissue factor (TF) activity in monocytes and LPS induced tumor necrosis factor α (TNFα) release were chosen as parameters of monocyte activation. Platelet factor 4 (PF4) secretion and LPS induced lacto ferrin release were chosen as parameters of platelet and PMN activation, respectively. When human whole blood was stimulated with 5 ng/ml LPS for 2 h, TF activity in monocytes isolated from EDTA blood was found to be 2.9 mU/106 cells, whereas TF activity in monocytes isolated from citrated, heparinized and hirudinized blood was 14.7, 24.7 and 28.5 mU/106 cells, respectively. TNFα concentrations in platelet poor plasma (PPP) isolated from whole blood stimulated with 5 ng/ml LPS for 2 h was increased with 200,400 and 350% in citrated, heparinized and hirudinized blood respectively, as compared to EDTA blood. Next, the effect of the anticoagulant on PMN secretion was measured. PPP isolated from whole blood incubated with 5 ng/ml LPS for 90 min contained 1170 ng/ml (EDTA blood), 2880 ng/ml (citrated blood), 4220 ng/ml (heparinized blood), and 5520 ng/ml lactoferrin (hirudinized blood). When studying the platelet parameter PF4, whole blood was incubated without any stimuli for 60 min, and we found that heparin PPP contained 1180 ng/ml PF4, while hirudin PPP contained 469 ng/ml, citrate PPP 440 ng/ml, and EDTA PPP 217 ng/ml PF4, respectively. Finally, the low molecular weight heparin compound Fragmin had no enhancing effect on PF4 levels in whole blood. It is concluded that the anticoagulant used in in vitro experiments has a large influence on the parameters measured.

 
  • References

  • 1 Petersen F, Flad HD, Brandt E. Neutrophil-activating peptides NAP-2 and IL-8 bind to the same sites on neutrophils but interact in different ways. J Immunol 1994; 152: 2467-2478
  • 2 Harvath L, Robbins JD, Russell AA, Seamon KB. cAMP and human neutrophil chemotaxis. Elevation of cAMP differentially affects chemotactic responsiveness. J Immunol 1991; 146: 224-232
  • 3 Glusa E, Markwardt F. Platelet factions in recombinant hirudin-anticoagulated blood. Haemostasis 1990; 20: 112-118
  • 4 Glusa E. Hirudin and platelets. Semin Thromb Hemost 1991; 17: 122-125
  • 5 Pratt CW, Church FC. General features of the heparin-binding serpins antithrombin, heparin cofactor II and protein C inhibitor. Blood Coagul Fibrinol 1993; 04: 479-490
  • 6 Fejgin MD, Lourwood DL. Low molecular weight heparins and their use in obstetrics and gynecology. Obstet Gynecol Surv 1994; 49: 424-431
  • 7 Witt DP, Lander AD. Differential binding of chemokines to glycosaminoglycan subpopulations. Curr Biol 1994; 04: 394-400
  • 8 Nelson RM, Cecconi O, Roberts WG, Aruffo A, Linhardt RJ, Bevilacqua MP. Heparin oligosaccharides bind Land P-selectin and inhibit acute inflammation. Blood 1993; 82: 3253-3258
  • 9 Skinner MP, Lucas CM, Bums GF, Chesterman CN, Berndt MC. GMP-140 binding to neutrophils is inhibited by sulfated glycans. J Biol Chem 1991; 266: 5371-5374
  • 10 Leung L, Saigo K, Grant D. Heparin binds to human monocytes and modulates their procoagulant activities and secretory phenotypes. Effect of histidine-rich glycoprotein. Blood 1989; 73: 177-184
  • 11 Horne MK, Chao ES. Heparin binding to resting and activated platelets. Blood 1989; 74: 238-243
  • 12 Chen J, Karlberg KE, Sylven C. Heparin enhances platelet aggregation irrespective of anticoagulation with citrate or with hirudin. Thromb Res 1992; 67: 253-262
  • 13 Willuweit B, Aktories K. Heparin uncouples a2-adrenoceptors from the Gi-protein in membranes of human platelets. Biochem J 1988; 249: 857-863
  • 14 Rohrer MJ, Kestin AS, Ellis PA, Barnard MR, Rodino L, Breckwoldt WL, Li JM, Michelson AD. High-dose heparin suppresses platelet alpha granule secretion. J Vase Surg 1992; 15: 1000-1008
  • 15 Markwardt F. Hirudin and derivatives as anticoagulant agents. Thromb Haemost 1991; 66: 141-152
  • 16 van GinkelCJW, van AkenWG, Oh JIH, Vreeken J. Stimulation of monocyte procoagulant activity by adherence to different surfaces. Br J Hematol 1977; 35: 35-45
  • 17 Haskill S, Johnson C, Eierman D, Becker S, Warren K. Adherence induces selectively mRNA expression of monocyte mediators and proto-oncogenes. J Immunol 1988; 140: 1690-1694
  • 18 Hofsli E, Lamvik J, Nissen-Meyer J. Evidence that tumour necrosis factor (TNF) is not constitutively present in vivo. Scand J Immunol 1988; 28: 435-441
  • 19 Sporn SA, Eierman DF, Johnson CE, Morris J, Martin G, Ladner M, Haskill S. Monocyte adherence results in selective induction of novel genes sharing homology with mediators of inflammation and tissue repair. J Immunol 1990; 144: 4434-4441
  • 20 Yam LT, Li CY, Crosby WH. Cytochemical identification of monocytes and granulocytes. Am J Clin Pathol 1971; 55: 283-290
  • 21 Engstad CS, Lia K, Rekdal Ø, Olsen JO, østerud B. A novel biological effect of platelet factor 4 (PF4): Enhancement of LPS-induced tissue factor activity in monocytes. J Leuk Biol 1995; 58: 575-581
  • 22 Gutteberg TJ, Røkke O, Jørgensen T, Andersen O. Lactoferrin as an indicator of septicemia and endotoxemia in pigs. Scand J Infect Dis 1988; 20: 659-666
  • 23 Glaser KG, Mobilio D, Chang JY, Senko N. Phospholipase A2 enzymes: Regulation and inhibition. TIPS 1993; 14: 92-98
  • 24 Wright SD, Kolesnick RN. Does endotoxin stimulate cells by mimicking ceramide? Immunol Today. 1995; 16: 297-302
  • 25 Østerud B, Olsen JO, Wilsgard L. The role of arachidonic acid release and lipoxygenase pathway in lipopolysaccharide-induced thromboplastin activity in monocytes. Blood Coagul Fibrinol 1990; 01: 41-46
  • 26 Dekker LV, Parker PJ. Protein kinase C – a question of specificity. TIBS 1994; 19: 73-77
  • 27 van derLogt CPE, Dirven RJ, Reitsma PH, Bertina RM. Expression of tissue factor and tissue factor pathway inhibitor in monocytes in response to bacterial lipopolysaccharide and phorbol ester. Blood Coagul Fibrinol 1994; 05: 211-220
  • 28 Temisien C, Ramani M, Ollivier V, Vu T, Hakim J, de Prost D. Endotoxininduced tissue factor in human monocytes is dependent upon protein kinase C activation. Thromb Haemost 1993; 70: 800-806
  • 29 de BoerJP, Wolbink GJ, Thijs LG, Baars JW, Wagstaff J, Hac CE. Interplay of complement and cytokines in the pathogenesis of septic shock. Immunopharmacol 1992; 24: 135-148
  • 30 Østerud B, Eskeland T. The mandatory role of complement in the endotoxin-induced synthesis of tissue thromboplastin in blood monocytes. FEBS Lett 1982; 149: 75-79
  • 31 Jørgensen L, Nilsen GJ, Perry DW, Mustard JF, Kinlough-Rathbone RL. Rabbit lung macrophages stimulate platelets in vitro as observed by density gradient centrifugation and transmission electron microscopy. Scand J Clin Lab Invest 1993; 53: 711-724
  • 32 Supattapone S, Worley PF, Baraban JM, Snyder SH. Solubilization, purification, and characterization of an inositol trisphosphate receptor. J Biol Chem 1988; 263: 1530-1534
  • 33 Ogwan’g R, Mwangi J, Gachihi G, Nwachuku A, Roberts CR, Martin SK. Use of pharmacological agents to implicate a role for phosphoinositide hydrolysis products in malaria gamete formation. Biochem Pharmacol 1993; 46: 1601-1606
  • 34 Høgåsen AKM, Abrahamsen TG. Heparin suppresses lipopolysaccharideinduced monocyte production of several cytokines, but simultaneously stimulates C3 production. Thromb Res 1995; 80: 179-184
  • 35 Dedrick RJ, Conlon PJ. Prolonged expression of lipopolysaccharide (LPS)induced inflammatory genes in whole blood requires continual exposure to LPS. Infect Immun 1995; 63: 1362-1368
  • 36 Taylor FB. Studies on the inflammatory-coagulant axis in the baboon response to E. coli: Regulatory roles of proteins C, S, C4bBP and of inhibitors of tissue factor. In: Bacterial Endotoxins: Basic science to anti-sepsis strategies. Wiley-Liss, Inc. 1994. pp 175-194
  • 37 Ramani M, Ollivier V, Khechai F, Vu T, Temisien C, Bridey F, Prost D. Interleukin-10 inhibits endotoxin-induced tissue factor mRNA production by human monocytes. FEBS Lett 1993; 334: 114-116
  • 38 Meszaros K, Aberle S, Dedrick R, Machovich R, Horwitz A, Birr C, Theofan G, Parent JB. Monocyte tissue factor induction by lipopolysaccharide (LPS): Dependence on LPS-binding protein and CD 14, and inhibition by a recombinant fragment of bactericidal/permeability-increasing protein. Blood 1994; 83: 2516-2525