Determinants of platelet conjugate formation with polymorphonuclear leukocytes or monocytes in whole blood

Summary

Following preliminary in-vitro experiments, platelet-leukocyte conjugates and their determinants were evaluated in citrated whole blood from 349 subjects (209 women, age 16–92 years) randomly recruited from the general population. Platelet activation by ADP/collagen but not leukocyte stimulation by fMLP or LTB4 resulted in formation of platelet conjugates with PMN or monocytes. In the population study, mixed cell conjugates, platelet P-selectin and leukocyte CD11b were measured by flow cytometry both at baseline and after in-vitro stimulation with ADP/collagen. The latter significantly increased platelet conjugates with either PMN or monocytes, platelet P-selectin and leukocyte CD11b expression. Platelet count significantly correlated with platelet-PMN, platelet-monocyte conjugates and P-selectin both at baseline and upon stimulation. In all conditions, both conjugate levels correlated with each other, when adjusted for gender, age and platelet count. Age correlated with platelet-PMN conjugate numbers in basal and stimulated conditions and with basal P-selectin. ADP/collagen stimulation resulted in higher P-selectin and conjugates values in women. Among risk factors, a significant correlation was found between conjugate and glucose levels. In conclusion, the presence and formation in whole blood from a large population of plateletleukocyte conjugates reflects primary platelet – but not leukocyte – activation and varies with gender, age, platelet count and blood glucose.

Keywords

Platelet-leukocyte conjugates - platelet P-selectin - cell activation markers - flow cytometry - general population

^* Both authors contributed equally to this work.

^# On leave of absence from Department of Allergology and Internal Medicine, Medical University of Bialystok, Poland.

• References

• 1 Hagberg IA, Lyberg T. Evaluation of circulating platelet-leukocyte conjugates: a sensitive flow cytometric assay well suited for clinical studies. Platelets 2000; 11: 151-160.
• 2 Michelson AD, Barnard MR, Krueger LA. et al. Circulating monocyte-platelet aggregates are a more sensitive marker of in vivo platelet activation than platelet surface P-selectin: studies in baboons, human coronary intervention, and human acute myocardial infarction. Circulation 2001; 104: 1533-1537.
• 3 Joseph JE, Harrison P, Mackie IJ. et al. Increased circulating platelet-leucocyte complexes and platelet activation in patients with antiphospholipid syndrome, systemic lupus erythematosus and rheumatoid arthritis. Br J Haematol 2001; 115: 451-459.
• 4 Wun T, Cordoba M, Rangaswami A. et al. Activated monocytes and platelet-monocyte aggregates in patients with sickle cell disease. Clin Lab Haematol 2002; 24: 81-88.
• 5 Falanga A, Marchetti M, Vignoli A. et al. Leukocyte- platelet interaction in patients with essential thrombocytemia a nd polycythemia vera. Exp Hematol 2005; 33: 523-530.
• 6 Palabrica T, Lobb R, Furie BC. et al. Leukocyte accumulation promoting fibrin deposition is mediated in vivo by P-selectin on adherent platelets. Nature 1992; 359: 848-851.
• 7 Cerletti C, Evangelista V, Molino M. et al. Platelet activation by polymorphonuclear leukocytes: role of cathepsin G and P-selectin. Thromb Haemost 1995; 74: 218-223.
• 8 Diacovo TG, Roth SJ, Buccola JM. et al. Neutrophil rolling, arrest, and transmigration across activated, surface- adherent platelets via sequential action of P-selectin and the beta 2-integrin CD11b/CD18. Blood 1996; 88: 146-157.
• 9 Ma YQ, Plow EF, Geng JG. P-selectin binding to P-selectin glycoprotein ligand-1 induces an intermediate state of alphaMbeta2 activation and acts cooperatively with extracellular stimuli to support maximal adhesion of human neutrophils. Blood 2004; 104: 2549-2556.
• 10 Weber C, Springer TA. Neutrophil accumulation on activated, surface-adherent platelets in flow is mediated by interaction of Mac-1 with fibrinogen bound to IIbIIIa and stimulated by platelet-activating factor. J Clin Invest 1997; 100: 2085-2093.
• 11 McEver RP. Adhesive interactions of leukocytes, platelets, and the vessel wall during hemostasis and inflammation. Thromb Haemost 2001; 86: 746-756.
• 12 Ruggeri ZM. Platelets in atherothrombosis. Nat Med 2002; 8: 1227-1234.
• 13 Cerletti C, Maugeri N, Evangelista V. et al. Plateletleukocyte interaction and atherothrombosis. In: Thrombosis. Fundamental and clinical aspects. Leuven University Press; 2003: 305-326.
• 14 Kirchhofer D, Riederer MA, Baumgartner HR. Specific accumulation of circulating monocytes and polymorphonuclear leukocytes on platelet thrombi in a vascular injury model. Blood 1997; 89: 1270-278.
• 15 Yang J, Furie BC, Furie B. The biology of P-selectin glycoprotein ligand-1: its role as a selectin counterreceptor in leukocyte-endothelial and leukocyte-platelet interaction. Thromb Haemost 1999; 81: 1-7.
• 16 Cerletti C, Evangelista V, de Gaetano G. P-selectinbeta 2-integrin cross-talk: a molecular mechanism for polymorphonuclear leukocyte recruitment at the site of vascular damage. Thromb Haemost 1999; 82: 787-793.
• 17 Martins Pda C, Zwaginga JJ. Leukocyte-platelet aggregates: new particles reflecting and effecting cardiovascular disease. Thromb Haemost 2005; 94: 1120-1121.
• 18 Chignard M, Selak MA, Smith JB. Direct evidence for the existence of a neutrophil-derived platelet activator (neutrophilin). Proc Natl Acad Sci USA 1986; 83: 8609-8613.
• 19 Evangelista V, Manarini S, Rotondo S. et al. Platelet/ polymorphonuclear leukocyte interaction in dynamic conditions: evidence of adhesion cascade and cross talk between P-selectin and the ß2 integrin CD11b/CD18. Blood 1996; 88: 4183-4194.
• 20 Russo P, Lauria F, Loguercio M. et al.; European Collaborative Group of the IMMIDIET. –344C/T Variant in the promoter of the aldosterone synthase gene (CYP11B2) is associated with metabolic syndrome in men. Am J Hypertens 2007; 20: 218-222.
• 21 Maugeri N, Giordano G, Petrilli MP. et al. Inhibition of tissue factor expression by hydroxyurea in polymorphonuclear leukocytes from patients with myeloproliferative disorders: a new effect for an old drug?. J Thromb Haemost 2006; 4: 2593-2598.
• 22 Maugeri N, Di Fabio G, Barbanti M. et al. Parnaparin, a low molecular weight heparin, prevents P-selectin dependent formation of platelet-leukocyte aggregates in human whole blood. Thromb Haemost 2007; 97: 156-157.
• 23 Harding SA, Din JN, Sarma J. et al. Flow cytometric analysis of circulating platelet-mpnocyte aggregates in whole blood: methodological considerations. Thromb Haemost 2007; 98: 451-456.
• 24 Barnard MR, Linden MD, Frelinger 3rd AL. et al. Effects of platelet binding on whole blood flow cytometry assays of monocyte and neutrophil procoagulant activity. J Thromb Haemost 2005; 3: 2563-2570.
• 25 Rinder HM, Bonan JL, Rinder CS. et al. Dynamics of leukocyte-platelet adhesion in whole blood. Blood 1991; 78: 1730-1737.
• 26 Ernst E, Hammerschmidt DE, Bagge U. et al. Leukocytes and the risk of ischemic diseases. J Am Med Assoc 1987; 257: 2318-2324.
• 27 Li N, Goodall AH, Hjemdahl P. A sensitive flow cytometric assay for circulating platelet-leukocyte aggregates. Br J Haematol 1997; 99: 808-816.
• 28 Ludbrook J. Multiple comparison procedures updated. Clin Exp Pharmacol Physiol 1998; 25: 1032-1037.
• 29 Li N, Hu H, Lindqvist M. et al. Platelet-leukocyte cross talk in whole blood. Arterioscler Thromb Vasc Biol 2000; 20: 2702-2708.
• 30 Vermylen J, Hoylaerts M, Arnout J. Increased mortality with long-term platelet glycoprotein IIb/IIIa antagonists: an explanation?. Circulation 2001; 104: E109.
• 31 Straub A, Wendel HP, Azevedo R. et al. The GP IIb/ IIIa inhibitor abciximab (ReoPro) decreases activation and interaction of platelets and leukocytes during in vitro cardiopulmonary bypass simulation. Eur J Cardiothorac Surg 2005; 27: 617-621.
• 32 Evangelista V, Manarini S, Sideri R. et al. Platelet/ polymorphonuclear leukocyte interaction: P-selectin triggers protein-tyrosine phosphorylation-dependent CD11b/CD18 adhesion: role of PSGL-1 as a signalling molecule. Blood 1999; 93: 876-885.
• 33 Piccardoni P, Sideri R, Manarini S. et al. Platelet/ polymorphonuclear leukocyte adhesion: a new role for SRC kinases in Mac-1 adhesive function triggered by P-selectin. Blood 2001; 98: 108-116.
• 34 Polgar J, Matuskova J, Wagner DD. The P-selectin, tissue factor, coagulation triad. J Thromb Haemost 2005; 3: 1590-1596.
• 35 Ahn KC, Jun AJ, Pawar P. et al. Preferential binding of platelets to monocytes over neutrophils under flow. Biochem Biophys Res Commun 2005; 329: 345-355.
• 36 Kappelmayer J, Kiss A, Karaszi E. et al. Identification of P-selectin glycoprotein ligand-1 as a useful marker in acute myeloid leukaemias. Br J Haematol 2001; 115: 903-909.
• 37 Evangelista V, Piccardoni P, White JG. et al. Cathepsin G-dependent platelet stimulation by activated polymorphonuclear leukocytes and its inhibition by antiproteinases: role of P-selectin-mediated cell-cell adhesion. Blood 1993; 81: 2947-2957.
• 38 Neelamegham S, Taylor AD, Shankaran H. et al. Shear and time-dependent changes in Mac-1, LFA-1, and ICAM-3 binding regulate neutrophil homotypic adhesion. J Immunol 2000; 164: 3798-3805.
• 39 Maugeri N, Santarelli MT, Lazzari MA. Circulating platelet/polymorphonuclear leukocyte mixed-cell aggregates in patients with mechanical heart valve replacement. Am J Hematol 2000; 65: 93-98.
• 40 Villmow T, Kemkes-Matthes B, Matzdorff AC. Markers of platelet activation and platelet-leukocyte interaction in patients with myeloproliferative syndromes. Thromb Res 2003; 108: 139-145.
• 41 Arellano-Rodrigo E, Alvarez-Larran A, Reverter JC. et al. Increased platelet and leukocyte activation as contributing mechanisms for thrombosis in essential thrombocytemia and correlation with the JAK2 mutational status. Haematologica 2006; 91: 169-175.
• 42 Johnson M, Ramey E, Ramwell PW. Sex and age differences in human platelet aggregation. Nature 1975; 253: 355-357.
• 43 Faraday N, Goldschmidt-Clermont PJ, Bray PF. Gender differences in platelet GPIIb-IIIa activation. Thromb Haemost 1997; 77: 748-754.
• 44 Sudic D, Razmara M, Forslund M. et al. High glucose levels enhance platelet activation: involvement of multiple mechanisms. Br J Haematol 2006; 133: 315-322.
• 45 Vaidyula VR, Boden G, Rao AK. Platelet and monocyte activation by hyperglycemia and hyperinsulinemia in healthy subjects. Platelets 2006; 17: 577-585.