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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
› Author Affiliations
Further Information

Publication History

Received 23 August 1996

Accepted after resubmission 29 November 1996

Publication Date:
11 July 2018 (online)

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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
    PubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar
  • 3 Glusa E, Markwardt F. Platelet factions in recombinant hirudin-anticoagulated blood. Haemostasis 1990; 20: 112-118
    PubMedGoogle Scholar
  • 4 Glusa E. Hirudin and platelets. Semin Thromb Hemost 1991; 17: 122-125
    Article in Thieme ConnectPubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 6 Fejgin MD, Lourwood DL. Low molecular weight heparins and their use in obstetrics and gynecology. Obstet Gynecol Surv 1994; 49: 424-431
    CrossrefPubMedGoogle Scholar
  • 7 Witt DP, Lander AD. Differential binding of chemokines to glycosaminoglycan subpopulations. Curr Biol 1994; 04: 394-400
    CrossrefPubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar
  • 11 Horne MK, Chao ES. Heparin binding to resting and activated platelets. Blood 1989; 74: 238-243
    PubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 13 Willuweit B, Aktories K. Heparin uncouples a2-adrenoceptors from the Gi-protein in membranes of human platelets. Biochem J 1988; 249: 857-863
    CrossrefPubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 15 Markwardt F. Hirudin and derivatives as anticoagulant agents. Thromb Haemost 1991; 66: 141-152
    Article in Thieme ConnectPubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar
  • 20 Yam LT, Li CY, Crosby WH. Cytochemical identification of monocytes and granulocytes. Am J Clin Pathol 1971; 55: 283-290
    CrossrefPubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 23 Glaser KG, Mobilio D, Chang JY, Senko N. Phospholipase A2 enzymes: Regulation and inhibition. TIPS 1993; 14: 92-98
    PubMedGoogle Scholar
  • 24 Wright SD, Kolesnick RN. Does endotoxin stimulate cells by mimicking ceramide? Immunol Today. 1995; 16: 297-302
    PubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 26 Dekker LV, Parker PJ. Protein kinase C – a question of specificity. TIBS 1994; 19: 73-77
    PubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    Article in Thieme ConnectPubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 34 Høgåsen AKM, Abrahamsen TG. Heparin suppresses lipopolysaccharideinduced monocyte production of several cytokines, but simultaneously stimulates C3 production. Thromb Res 1995; 80: 179-184
    CrossrefPubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar
  • 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
    Google Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar