Role of erythrocytes and platelets in the hypercoagulable status in polycythemia vera through phosphatidylserine exposure and microparticle generation

Summary

The development of thrombosis in polycythaemia vera (PV) involves multifactorial processes including pathological activation of blood cells. Release of microparticles (MPs) by activated cells in diseases is associated with thrombotic risk, but relatively few data are available in PV. The aim of the present study was to investigate the increase in MP release and exposure of phosphatidylserine (PS) on the outer membrane of MP-origin cells in patients with PV, and to analyse their procoagulant activity (PCA). PS-positive MPs and cells were detected by flow cytometry, while PCA was assessed with clotting time and purified coagulation complex assays. We found that PV patients had elevated circulating lactadherin⁺ MPs, which mostly originating from erythrocytes, platelets, granulocytes, and endothelial cells, as well as increased PS exposing erythrocytes/platelets as compared to secondary polycythaemia patients or healthy controls. These PS-bearing MPs and cells were highly procoagulant. Moreover, lactadherin competed factor V and VIII to PS and inhibited about 90% of the detected PCA in a dose-response manner while anti-TF antibody did no significant inhibition. Treatment with hydroxyurea is associated with a decrease in PS exposure and lactadherin⁺ MP release of erythrocytes/platelets. Our data demonstrate that PV patients are characterised by increased circulating procoagulant MPs and PS exposing erythrocytes/platelets, which could contribute to the hypercoagulable state in these patients.

Keywords

Phosphatidylserine - microparticles - polycythaemia vera - lactadherin

• References

• 1 Spivak JL. Polycythemia vera: myths, mechanisms, and management. Blood 2002; 100: 4272-4290.
• 2 Landolfi R, Di Gennaro L, Falanga A. Thrombosis in myeloproliferative disorders: pathogenetic facts and speculation. Leukemia 2008; 22: 2020-2028.
• 3 Sonmez M, Saglam F, Karahan SC. et al. Treatment related changes in antifibrinolytic activity in patients with polycythemia vera. Haematology 2010; 15: 391-396.
• 4 Landolfi R, Di Gennaro L. Thrombosis in myeloproliferative and myelodys-plastic syndromes. Haematology 2012; 17 (Suppl. 01) S174-S176.
• 5 Di Nisio M, Barbui T, Di Gennaro L. et al. The haematocrit and platelet target in polycythemia vera. Br J Haematol 2007; 136: 249-259.
• 6 Michiels JJ, Berneman Z, Schroyens W. et al. The paradox of platelet activation and impaired function: platelet-von Willebrand factor interactions, and the etiology of thrombotic and hemorrhagic manifestations in essential thrombo-cythemia and polycythemia vera. Semin Thromb Hemost 2006; 32: 589-604.
• 7 Leventis PA, Grinstein S. The distribution and function of phosphatidylserine in cellular membranes. Annu Rev Biophys 2010; 39: 407-427.
• 8 Yeung T, Gilbert GE, Shi J. et al. Membrane phosphatidylserine regulates surface charge and protein localisation. Science 2008; 319: 210-213.
• 9 Shi J, Shi Y, Waehrens LN. et al. Lactadherin detects early phosphatidylserine exposure on immortalised leukemia cells undergoing programmed cell death. Cytometry A 2006; 69: 1193-1201.
• 10 Morel O, Jesel L, Freyssinet JM. et al. Cellular mechanisms underlying the formation of circulating microparticles. Arterioscler Thromb Vasc Biol 2011; 31: 15-26.
• 11 Montoro-Garcia S, Shantsila E, Marin F. et al. Circulating microparticles: new insights into the biochemical basis of microparticle release and activity. Basic Res Cardiol 2011; 106: 911-923.
• 12 Shet AS, Aras O, Gupta K. et al. Sickle blood contains tissue factor-positive microparticles derived from endothelial cells and monocytes. Blood 2003; 102: 2678-2683.
• 13 Gao C, Xie R, Yu C. et al. Procoagulant activity of erythrocytes and platelets through phosphatidylserine exposure and microparticles release in patients with nephrotic syndrome. Thromb Haemost 2012; 107: 681-689.
• 14 Weiss E, Rees DC, Gibson JS. Role of calcium in phosphatidylserine externalisation in red blood cells from sickle cell patients. Anemia 2011; 2011: 379894.
• 15 Tsimerman G, Roguin A, Bachar A. et al. Involvement of microparticles in diabetic vascular complications. Thromb Haemost 2011; 106: 310-321.
• 16 Presseizen K, Friedman Z, Shapiro H. et al. Phosphatidylserine Expression on the Platelet Membrane of Patients with Myeloproliferative Disorders and its Effect on Platelet-Dependent Thrombin Formation. Clin Appl Thromb Hemost 2002; 08: 33-39.
• 17 Fujita H, Sakuma R, Tomiyama J. et al. Increased phosphatidylserine exposure on the erythrocyte membrane in patients with polycythaemia vera. Br J Haema-tol 2011; 152: 238-240.
• 18 Shi J, Heegaard CW, Rasmussen JT. et al. Lactadherin binds selectively to membranes containing phosphatidyl-L-serine and increased curvature. Biochim Bio-phys Acta 2004; 1667: 82-90.
• 19 Hou J, Fu Y, Zhou J. et al. Lactadherin functions as a probe for phosphatidylserine exposure and as an anticoagulant in the study of stored platelets. Vox Sang 2011; 100: 187-195.
• 20 Shi J, Gilbert GE. Lactadherin inhibits enzyme complexes of blood coagulation by competing for phospholipid-binding sites. Blood 2003; 101: 2628-2636.
• 21 Fu Y, Zhou J, Li H. et al. Daunorubicin induces procoagulant activity of cultured endothelial cells through phosphatidylserine exposure and microparticles release. Thromb Haemost 2010; 104: 1235-1241.
• 22 Vardiman JW, Thiele J, Arber DA. et al. The 2008 revision of the World Health Organisation (WHO) classification of myeloid neoplasms and acute leukemia: rationale and important changes. Blood 2009; 114: 937-951.
• 23 Hvarregaard J, Andersen MH, Berglund L. et al. Characterisation of glycoprotein PAS-6/7 from membranes of bovine milk fat globules. Eur J Biochem 1996; 240: 628-636.
• 24 Xie R, Gao C, Li W. et al. Phagocytosis by macrophages and endothelial cells inhibits procoagulant and fibrinolytic activity of acute promyelocytic leukemia cells. Blood 2012; 119: 2325-2334.
• 25 Mause SF, Weber C. Microparticles: Protagonists of a Novel Communication Network for Intercellular Information Exchange. Circ Res 2010; 107: 1047-1057.
• 26 Dignat-George F, Freyssinet JM, Key NS. Centrifugation is a crucial step impacting microparticle measurement. Platelets 2009; 20: 225-226.
• 27 Lacroix R, Robert S, Poncelet P. et al. Standardisation of platelet-derived micro-particle enumeration by flow cytometry with calibrated beads: results of the International Society on Thrombosis and Haemostasis SSC Collaborative workshop. J Thromb Haemost 2010; 08: 2571-2574.
• 28 Zhou J, Liu S, Ma M. et al. Procoagulant activity and phosphatidylserine of amniotic fluid cells. Thromb Haemost 2009; 101: 845-851.
• 29 Duchemin J, Ugo V, Ianotto JC. et al. Increased circulating procoagulant activity and thrombin generation in patients with myeloproliferative neoplasms. Thromb Res 2010; 126: 238-242.
• 30 Falanga A, Marchetti M, Evangelista V. et al. Polymorphonuclear leukocyte activation and hemostasis in patients with essential thrombocythemia and polycythemia vera. Blood 2000; 96: 4261-4266.
• 31 Presti RL, Caracciolo C, Montana M. et al. Erythrocyte deformability evaluated by laser diffractometry in polycythemia vera. Clin Hemorheol Microcirc 2012; 50: 189-192.
• 32 Haynes Jr J, Obiako B, Hester RB. et al. Hydroxyurea attenuates activated neut-rophil-mediated sickle erythrocyte membrane phosphatidylserine exposure and adhesion to pulmonary vascular endothelium. Am J Physiol Heart Circ Physiol 2008; 294: H379-H385.
• 33 Gerotziafas GT, Van Dreden P, Chaari M. et al. The acceleration of the propagation phase of thrombin generation in patients with steady-state sickle cell disease is associated with circulating erythrocyte-derived microparticles. Thromb Haemost 2012; 107: 1044-1052.
• 34 Trappenburg MC, van Schilfgaarde M, Marchetti M. et al. Elevated procoagulant microparticles expressing endothelial and platelet markers in essential thrombocythemia. Haematologica 2009; 94: 911-918.
• 35 Yuana Y, Bertina RM, Osanto S. Pre-analytical and analytical issues in the analysis of blood microparticles. Thromb Haemost 2010; 105: 396-408.
• 36 Suades R, Padró T, Vilahur G. et al. Circulating and platelet-derived micropar-ticles in human blood enhance thrombosis on atherosclerotic plaques. Thromb Haemost 2012; 108: 1208-1219.
• 37 van Doormaal F, Kleinjan A, Berckmans RJ. et al. Coagulation activation and microparticle-associated coagulant activity in cancer patients. An exploratory prospective study. Thromb Haemost 2012; 108: 160-165.
• 38 Auwerda JJ, Yuana Y, Osanto S. et al. Microparticle-associated tissue factor activity and venous thrombosis in multiple myeloma. Thromb Haemost 2011; 105: 14-20.
• 39 Osterud B. Tissue factor expression in blood cells. Thromb Res 2010; 125 (Suppl. 01) S31-S34.