Influence of Selected Heparins on Human Neutrophil Functions In Vitro

Summary

The effects of four heparin derivatives [unfractionated heparin (UFH) at 5–50 IU/ml, low molecular weight heparin (LMWH) at 2–20 IU/ml, pentasaccharide (Penta) at 5 IU/ml and a synthetic heparinoid (PPS) at 10^–6–10^–5 M] on various polymorphonuclear (PMN) leukocyte end-functions (aggregation, chemotaxis, phagocytosis and burst) were examined. CR3 expression, actin polymerization and membrane surface charge were also studied to gain more insight on the mechanisms of the action of heparins on PMN. The different heparins were found to have rather different actions. PMN were found to be hyperreactive to PPS. Pentasaccharide hat no effect on PMN functions, while UFH and LMWH had intermediate reactivity, modulating responses in an adenosine-like manner. Interactions of heparins with PMN were attributed to biophysical properties of the molecules rather than to the presence of a specific sequence such as a pentasaccharide.

Our results show that certain heparin derivatives, apart their well-known anticoagulant action, modulate polymorphonuclear leukocyte functions that may be involved in vascular injury.

• REFERENCES

• 1 Jaques LB. Heparins - anionic polyelectrolyte drugs. Pharmacol Rev 1979; 31 (2): 099-166
• 2 Engler RL, Dahlgren MD, Morris DD, Peterson MA, Schmid-Schonbein GW. Role of leucocytes in the response to acute myocardial ischemia and reflow in dogs. Am J Physiol 1986; 251: 314-322
• 3 Engler RL, Dahlgren MD, Peterson MA, Dobbs A, Schmid-Schon-bein GW. Accumulation of polymorphonuclear leucocytes during 3-hours experimental myocardial ischemia. Am J Physiol 1986; 251: 093-100
• 4 Ernst E, Hammerschmidt DE, Bagge U, Matrai A, Dormandy JA. Leucocytes and the risk of ischemic disease. JAMA 1987; 257: 2318-2324
• 5 Schmid-Schonbein GW, Engler RL. Granulocytes as active participants in acute myocardial ischemia and infarction. J Cardiovasc Pathol 1986; 1: 15-30
• 6 Altieri DC, Morrissey JH, Edgington TS. Adhesive receptor Mac-1 coordinates the activation of factor X on stimulated cells of monocytic and myeloid differentiation: a alternative initiation of the coagulation protease cascade. Proc Natl Acad Sci 1988; 85: 7462-7466
• 7 Mahadoo J, Hiebert LM, Jacques LB. Vascular sequestration of heparin. Thromb Res 1977; 12: 79-80
• 8 Barzu T, van Rijn JLML, Petitou M, Molho P, Tobelem P. Endothelial binding sites for heparin. Specificity and role in heparin neutralization. Biochem J 1986; 238: 847-854
• 9 Van Rijn JLML, Trillou M, Mardiguian J, Tobelem G, Caen J. Selective binding of heparin to human endothelial cells. Implications of pharmacokinetics. Thromb Res 1987; 45: 211-222
• 10 Fareed JD. Heparin, its fractions, fragments and derivatives. Some newer perspectives. Semin Thromb Hemostas 1985; 11: 1-9
• 11 Vannuçchi S, Pasquali F, Chiarugi V, Ruggiero M. Internalization and metabolism of endogenous heparin by cultured endothelial cells. Biochem Biophys Res Commun 1986; 140: 249-301
• 12 Salzman E, Rosenberg R, Smith M, Lindon JN, Favreau L. Effect of heparin and heparin fractions on platelet aggregation. J Clin Invest 1980; 65: 64-73
• 13 Reches A, Eldor A, Salomon Y. Heparin inhibits PGE2-sensitive adenylate cyclase and antagonizes PGE1 antiaggregating effect in human platelets. J Lab Clin Med 1979; 93: 638
• 14 Eldor A, Weksler BB. Heparin and dextran sulfate antagonize PGI2 inhibition of platelet aggregation. Thromb Res 1979; 16: 617-618
• 15 Cairo MS, Allen J, Higgins C, Baehner RL, Boxer LA. Synergistic effect of heparin and chemotactic factor on polymorphonuclear leucocyte aggregation and degranulation. Am J Pathol 1983; 113: 67-74
• 16 Lazarowski ER, Santome JA, Behrens NH, Sanchez Avalos JC. Aggregation of human neutrophils by heparin. Thromb Res 1986; 41: 437-446
• 17 Berliner S, Fishelson Z, Wasserman L, Pinkhas J, Aronson M. Synergism between zymosan-activated serum and heparin in the induction of polymorphonuclear leucocyte aggregation. Biomed Phar-macother 1988; 42: 69-72
• 18 Laghi-Pasini F, Pasqusi AL, Ceccatelli L, Capecchi PL, Orrico A, Di Perri T. Heparin inhibition of polymorphonuclear leucocyte activation in vitro. A possible pharmacological approach to granulocyte-mediated vascular damage. Thromb Res 1984; 35: 527-537
• 19 Laghi-Pasini F, Pasqui AL, Ceccatelli L, Di Perri T. Heparin inhibits fMLP and CON-A dependent activation of human polymorphonuclear leucocytes in vitro. Int J Tiss Reac 1983; V (2): 144-151
• 20 Victor M, Weiss J, Elsbach P. Heparin inhibits phagocytosis by polymorphonuclear leucocytes. Infect Immun 1981; 32, 1: 295-299
• 21 Matzner Y, Marx G, Drexler R, Eldor A. The inhibitory effect of heparin and related glycosaminoglycans on neutrophil chemotaxis. Thromb Haemostas 1984; 52 2: 134-137
• 22 Nitzan DX, Pruzanski W, Saito S, Ranadive N. Modulation of locomotor activity of polymorphonuclear cells by cationic substances and cationic lysosomal fractions from human neutrophils. Inflammation 1985; 9 4: 375-387
• 23 Choay J, Petitou M, Lormeau JC, Sinay P, Casu B, Gatti G. Structure-activity relationship in heparin: a synthetic pentasaccharide with high affinity for antithrombin III and eliciting high anti-factor Xa activity. Biochem Biophys Commun 1983; 116: 492-499
• 24 Petitou M, Duchaussoy P, Choay J, Sinay P, Jacquinet JC, Torri G. Synthesis of heparin fragments. A chemical synthesis of the pentasaccharide 0-(2-deoxy-2sulfamido-6-0-sulfo-a-D-glucopyranosyl)-(1 > 4)-0-(β -D-glucopyranosyluronic acid)-(1 > 4)-0-(2-deoxy-2-sulfamido-3,6-di-0-sulfo-a-D-glucopyranosyl)-(1 > 4)-0-(2-0-sulfo-a-L-idopyranosyluronic acid)-(1 > 4)-2-deoxy-2-sulfamido-6-0-sulfo-D-glucopyranose decasodium salt, a heparin fragment having high affinity for antithrombin III. Carbohydr Res 1986; 147: 221-236
• 25 Duclos JP. L'héparine. Fabrication, structure propriétés, analyses. Ed Masson, Paris: 1984
• 26 Duque RE, Ward PA. Quantitative assessment of neutrophil function by flow cytometry. Anal Quant Cytol Histol 1987; 9: 42-48
• 27 Phillips WA, Hosking CS, Shelton MJ. Removal of contaminating erythrocytes from human polymorphonuclear leucocyte preparation. Scand J Clin Lab Invest 1983; 43: 621-627
• 28 Craddock PR, Hammerschmidt D, White JG, Dalmasso AP, Jacob HS. Complement (C5a)-induced granulocyte aggregation in vitro. A possible mechanism of complement-mediated leukostasis and leukopenia. J Clin Invest 1977; 60: 260-264
• 29 Nelson RD, Quie PG, Simmons RL. Chemotaxis under agarose: a new and simple method for measuring chemotaxis and spontaneous migration of PMNL and monocytes. J Immunol 1975; 115: 1650-1656
• 30 Phillips RD, Jennings LK, Edwards HH. Identification of membrane proteins mediating the interaction of human platelets. J Cell Biol 1980; 86: 77-86
• 31 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-685
• 32 Hasui M, Hirabayashi Y, Kobayashi Y. Simultaneous measurement by flow cytometry of phagocytosis and hydrogen peroxide production of neutrophils in whole blood. Immunol Meth 1989; 117: 53-58
• 33 Brandt R, Keston AS. Synthesis of diacetyldichlorofluoresin: a stable reagent for fluorometric analysis. Anal Biochem 1965; 11: 6-9
• 34 Weening RS, Wever R, Roos D. Quantitative aspects of the production of superoxide radicals by phagocytizing human granulocytes. J Lab Clin Med 1975; 85, 2: 245-252
• 35 Gallin JI, Duocher JR, Kaplan AP. Interaction of leucocyte chemotactic factors with the cell surface. I. Chemotactic factor induced changes in human granulocyte surface changes. J Clin Invest 1975; 55: 967-974
• 36 Fuhrmann GF, Ruhenstroth-Bauer G. Cell electrophoresis employing a rectangular measuring cuvette. In: Cell Electrophoresis. Ambrose EJ. (ed) Boston, MA: Little Brown,: 1965. p 22
• 37 Jichtman MA, Weed RI. Electrophoretic mobility and N-acetyl neuraminic acid content of human normal and leukemic lymphocytes and granulocytes, Blood. 1970; 35 1: 12-22
• 38 Philips MR, Buyon JP, Winchester R, Weissmann G, Abramson S. Upregulation of the iC3b receptor (CR3) is neither necessary nor sufficient to promote neutrophil aggregation. J Clin Invest 1988; 82: 495-501
• 39 Freyburger G, Belloc F, Boisseau MR. Pentoxifylline inhibits actin polymerization in human neutrophils after stimulation by chemoat-tractant factor. Agents Actions 1990; 31 (1/2): 72-78
• 40 Cronstein BN, Daguma L, Nichols D, Hutchison AJ, Williams M. The adenosine/neutrophil paradox resolved: human neutrophils possess both A1 and A2 receptors that promote chemotaxis and inhibit O₂-. generation, respectively. J Clin Invest. 1990; 85: 1150-1157