Daunorubicin induces procoagulant activity of cultured endothelial cells through phosphatidylserine exposure and microparticles release

Summary

Administration of various chemotherapeutic agents is associated with an increased risk of thrombotic events. Although vascular endothelium plays a predominant role in blood coagulation and fibrinolysis, the effect of cytotoxic drugs on the procoagulant activity (PCA) of endothelial cells has not been well evaluated. Our study aims to investigate the possibility that daunorubicin, a chemotherapeutic agent, exerts prothrombotic effect on endothelial cells. We tested the impact of daunorubicin on phosphatidylserine (PS) exposure, endothelial microparticles (EMPs) release and consequent PCA. Human umbilical vein endothelial cells (HUVECs) were treated with daunorubicin (0.1, 0.2, 0.5, 1, 2 μM) for 24 hours. PCA of HUVECs was measured using clotting time and purified coagulation complex assays. Counts and PCA of EMPs were evaluated by flow cytometry and clotting time assay, respectively. Lactadherin was used as a novel probe for detection of PS exposure and EMPs release. We found that daunorubicin dose-dependently increased the PS exposure and consequent PCA of HUVECs. Moreover, daunorubicin treatment also enhanced the release of EMPs which were highly procoagulant. This increment was especially significant at 0.2 μM of daunorubicin or more. Blockade of PS with lactadherin inhibited over 90% of HUVECs and EMPs PCA. However, anti-TF antibody had no significant inhibition effect. Our results demonstrate that daunorubicin treatment enhanced PCA of HUVECs through PS exposure and shedding of procoagulant EMPs. Lactadherin acts as an efficient anticoagulant in this process.

Keywords

Daunorubicin - endothelial cells - phosphatidylserine - microparticles - lactadherin

^* Theses authors contributed equally.

• References

• 1 Leone G, Sica S, Chiusolo P. Blood cells diseases and thrombosis. Haematologica 2001; 86: 1236-1244.
• 2 Negaard HF, Iversen PO, Østenstad B. et al. Hypercoagulability in patients with haematological neoplasia: no apparent initiation by tissue factor. Thromb Haemost 2008; 99: 1040-1048.
• 3 Uchiumi H, Matsushima T, Yamane A. et al. Prevalence and clinical characteristics of acute myeloid leukemia associated with disseminated intravascular coagulation. Int J Hematol 2007; 86: 137-142.
• 4 Haddad TC, Greeno EW. Chemotherapy-induced thrombosis. Thromb Res 2006; 118: 555-568.
• 5 Zhou J, Shi J, Hou J. et al. Phosphatidylserine exposure and procoagulant activity in acute promyelocytic leukemia. J Thromb Haemost 2010; 8: 773-782.
• 6 Zhou J, Zheng Y, Shi J. et al. Daunorubicin induces procoagulant response through phosphatidylserine exposure in red blood cells. Thromb Res 2010; 125: 178-183.
• 7 Schouten M, Wiersinga WJ, Levi M. et al. Inflammation, endothelium, and coagulation in sepsis. J Leukoc Biol 2008; 83: 536-545.
• 8 Gresele P, Momi S, Migliacci R. Endothelium, venous thromboembolism and ischaemic cardiovascular events. Thromb Haemost 2010; 103: 56-61.
• 9 Mallat Z, Benamer H, Hugel B. et al. Elevated levels of shed membrane microparticles with procoagulant potential in the peripheral circulating blood of patients with acute coronary syndromes. Circulation 2000; 101: 841-843.
• 10 Piccin A, Murphy W, Smith O. Circulating microparticles: pathophysiology and clinical implications. Blood 2006; 21: 157-171.
• 11 Chironi GN, Boulanger CM, Simon A. et al. Endothelial microparticles in diseases. Cell Tissue Res 2009; 335: 143-151.
• 12 Morrissey JH, Pureza V, Davis-Harrison RL. et al. Blood clotting reactions on nanoscale phospholipid bilayers. Thromb Res 2008; 122: S23-26.
• 13 Zwaal RF, Schroit AJ. Pathophysiologic implications of membrane phospholipid asymmetry in blood cells. Blood 1997; 89: 1121-1132.
• 14 Yeung T, Gilbert GE, Shi J. et al. Membrane phosphatidylserine regulates surface charge and protein localization. Science 2008; 319: 210-213.
• 15 Shi J, Shi Y, Waehrens LN. et al. Lactadherin detects early phosphatidylserine exposure on immortalized leukemia cells undergoing programmed cell death. Cytometry A 2006; 69: 1193-1201.
• 16 Shi J, Pipe SW, Rasmussen JT. et al. Lactadherin blocks thrombosis and hemostasis in vivo: correlation with platelet phosphatidylserine exposure. J Thromb Haemost 2008; 6: 1167-1174.
• 17 Burnier L, Fontana P, Kwak BR. et al. Cell-derived microparticles in haemostasis and vascular medicine. Thromb Haemost 2009; 101: 439-451.
• 18 Lechner D, Kollars M, Gleiss A. et al. Chemotherapy-induced thrombin generation via procoagulant endothelial microparticles is independent of tissue factor activity. J Thromb Haemost 2007; 5: 2445-2452.
• 19 Swystun LL, Shin LY, Beaudin S. et al. Chemotherapeutic agents doxorubicin and epirubicin induce a procoagulant phenotype on endothelial cells and blood monocytes. J Thromb Haemost 2009; 7: 619-626.
• 20 Stubbs J, Lekutis C, Singer K. et al. cDNA cloning of a mouse mammary epithelial cell surface protein reveals the existence of epidemal growth factor-like domains linked to factor VIII-like sequences. Proc Natl Acad Sci USA 1990; 87: 8417-8421.
• 21 Albanyan AM, Murphy MF, Rasmussen JT. et al. Measurement of phosphatidylserine exposure during storage of platelet concentrates using the novel probe lactadherin: a comparison study with annexin V. Transfusion 2009; 49: 99-107.
• 22 Shi J, Heegaard CW, Rasmussen JT. et al. Lactadherin binds selectively to membranes containing phosphatidyl-L-serine and increased curvature. Biochim Biophys Acta 2004; 1667: 82-90.
• 23 Shi J, Gilbert GE. Lactadherin inhibits enzyme complexes of blood coagulation by competing for phospholipid binding sites. Blood 2003; 101: 2628-2636.
• 24 Hvarregaard J, Andersen MH, Berglund L. et al. Characterization of glycoprotein PAS-6/7 from membranes of bovine milk fat globules. Eur J Biochem 1996; 240: 628-636.
• 25 Slapak CA, Lecerf JM, Daniel JC. et al. Energy-dependent accumulation of daunorubicin into subcellular compartments of human leukemia cells and cytoplasts. J Biol Chem 1992; 267: 10638-10644.
• 26 Zhou J, Liu S, Ma M. et al. Procoagulant activity and phosphatidylserine of amniotic fluid cells. Thromb Haemost 2009; 101: 845-851.
• 27 Andersen MH, Graversen H, Fedosov SN. et al. Functional analyses of two cellular binding domains of bovine lactadherin. Biochemistry 2000; 39: 6200-6206.
• 28 Shao C, Novakovic VA, Head JF. et al. Crystal structure of lactadherin C2 domain at 1.7A resolution with mutational and computational analyses of its membrane-binding motif. J Biol Chem 2008; 283: 7230-7241.
• 29 Nachman RL. Review: Stratton Lecture. Thrombosis and atherogenesis: molecular connections. Blood 1992; 79: 1897-1906.
• 30 Diehl P, Nagy F, Sossong V. et al. Increased levels of circulating microparticles in patients with severe aortic valve stenosis. Thromb Haemost 2008; 99: 711-719.
• 31 Mackman N. Role of tissue factor in hemostasis and thrombosis. Blood Cells Mol Dis 2006; 36: 104-107.
• 32 Bach RR. Tissue factor encryption. Arterioscler Thromb Vasc Biol 2006; 26: 456-461.
• 33 Kaushal V, Kaushal GP, Mehta P. Differential toxicity of anthracyclines on cultured endothelial cells. Endothelium 2004; 11: 253-258.
• 34 Wakabayashi I, Groschner K. Vascular actions of anthracycline antibiotics. Curr Med Chem 2003; 10: 427-436.
• 35 Lambers JW, Cammenga M, König BW. et al. Activation of human endothelial cell-type plasminogen activator inhibitor (PAI-1) by negatively charged phospholipids. J Biol Chem 1987; 262: 17492-17496.
• 36 Koopman G, Reutelingsperger CP, Kuijten GA. et al. Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood 1994; 84: 1415-1420.
• 37 Tait JF, Gibson D. Phospholipid binding of annexin V: effects of calcium and membrane phosphatidylserine content. Arch Biochem Biophys 1992; 298: 187-191.
• 38 Oling F, Bergsma-Schutter W, Brisson A. Trimers, dimers of trimers, and trimers of trimers are common building blocks of annexin a5 two-dimensional crystals. J Struct Biol 2001; 133: 55-63.
• 39 London F, Ahmad SS, Walsh PN. Annexin V inhibition of factor IXa-catalyzed factor X activation on human platelets and on negatively-charged phospholipid vesicles. Biochemistry 1996; 35: 16886-16897.