Procoagulant activity of erythrocytes and platelets through phosphatidylserine exposure and microparticles release in patients with nephrotic syndrome

Summary

Recent studies showed that an imbalance of prothrombotic and antithrombotic factors and impaired thrombolytic activity contribute to the thrombophilia of the nephrotic syndrome (NS). However, it is not clear whether blood cell injury and/or activation is involved in hypercoagulability in NS patients. Our objectives were to study the increase in microparticle (MP) release and phosphatidylserine (PS) exposure on the outer membrane of MP-origin cells in NS patients, and to evaluate their procoagulant activity (PCA). The subjects were patients with membranous nephropathy (MN), minimal change nephrotic syndrome (MCNS) and healthy controls. Analyses of MPs and PS exposure were performed using a flow cytometer. PCA was determined by clotting time and purified coagulation complex assays. We found that lactadherin⁺ MPs, which derived from red blood cells (RBC), platelet and endothelial cell, increased in NS patients. Moreover, PS exposure on RBCs and platelets in each NS group, especially in MN, are higher than that in controls. MP shedding and PS exposure of RBCs/platelets were highly procoagulant in NS patients. However, blockade of PS with lactadherin inhibited over 90% of PCA while an anti-tissue factor antibody had no significant inhibition effect. Our results demonstrate that the thrombophilic susceptibility of NS may be partly ascribed to MP release and PS exposure of RBCs, platelets and endothelial cells. Lactadherin is a sensitive probe for PS that has high anticoagulant activity.

Keywords

Phosphatidylserine - microparticles - nephrotic syndrome - lactadherin

• References

• 1 Orth SR, Ritz E. The nephrotic syndrome. N Engl J Med 1998; 338: 1202-1211.
• 2 Barbour SJ, Greenwald A, Djurdjev O. et al. Disease-specific risk of venous thromboembolic events is increased in idiopathic glomerulonephritis. Kidney Int 2011; 81: 190-195.
• 3 Mahmoodi BK, ten Kate MK, Waanders F. et al. High absolute risks and predictors of venous and arterial thromboembolic events in patients with nephrotic syndrome, results from a large retrospective cohort study. Circulation 2008; 117: 224-230.
• 4 Kato S, Chernyavsky S, Tokita JE. et al. Relationship between proteinuria and venous thromboembolism. J Thromb Thrombolysis 2010; 30: 281-285.
• 5 Glassock RJ. Prophylactic anticoagulation in nephrotic syndrome, a clinical conundrum. J Am Soc Nephrol 2007; 18: 2221-2225.
• 6 Singhal R, Brimble KS. Thromboembolic complications in the nephrotic syndrome, Pathophysiology and clinical management. Thromb Res 2006; 118: 397-407.
• 7 Lionaki S, Derebail V, Hogan SL. et al. Venous Thromboembolism in Patients with Membranous Nephropathy. Clin J Am Soc Nephrol 2012; 07: 43-51.
• 8 Zaffanello M, Brugnara M, Fanos V. et al. Prophylaxis with AT III for thromboem-bolism in nephritic syndrome, why should it be done?. Int Urol Nephrol 2009; 41: 713-716.
• 9 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.
• 10 Burnier L, Fontana P, Kwak BR. et al. Cell-derived microparticles in haemostasis and vascular medicine. Thromb Haemost 2009; 101: 439-451.
• 11 Berckmans RJ, Nieuwland R, Böing AN. et al. Cell-derived microparticles circulate in healthy humans and support low grade thrombin generation. Thromb Haemost 2001; 85: 639-646.
• 12 Jung KH, Chu K, Lee ST. et al. Risk of macrovascular complications in type 2 diabetes mellitus: endothelial microparticle profiles. Cerebrovasc Dis 2011; 31: 485-493.
• 13 Yamaja Setty BN, Koneti Rao A, Stuart MJ. Thrombophilia in sickle cell disease, the red cell connection. Blood 2001; 98: 3228-3233.
• 14 Bonomini M, Dottori S, Amoroso L. et al. Increased platelet phosphatidylserine exposure and caspase activation in chronic uremia. J Thromb Haemost 2004; 02: 1275-1281.
• 15 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.
• 16 Zhou J, Shi J, Hou J. et al. Phosphatidylserine exposure and procoagulant activity in acute promyelocytic leukemia. J Thromb Haemost 2010; 08: 773-782.
• 17 Stubbs JD, Lekutis C, Singer KL. 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.
• 18 Shi J, Gilbert GE. Lactadherin inhibits enzyme complexes of blood coagulation by competing for phospholipid binding sites. Blood 2003; 101: 2628-2636.
• 19 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.
• 20 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.
• 21 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.
• 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 Yuana Y, Bertina RM, Osanto S. Pre-analytical and analytical issues in the analysis of blood microparticles. Thromb Haemost 2011; 105: 396-408.
• 24 Mause SF, Weber C. Microparticles: protagonists of a novel communication network for intercellular information exchange. Circ Res 2010; 107: 1047-1057.
• 25 van Ierssel SH, Van Craenenbroeck EM, Conraads VM. et al. Flow cytometric detection of endothelial microparticles (EMP): effects of centrifugation and storage alter with the phenotype studied. Thromb Res 2010; 125: 332-339.
• 26 Rubin O, Crettaz D, Tissot JD. et al. Pre-analytical and methodological challenges in red blood cell microparticle proteomics. Talanta 2010; 82: 1-8.
• 27 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.
• 28 Amabile N, Boulanger CM. Circulating microparticle levels in patients with coronary artery disease, a new indicator of vulnerability?. Eur Heart J 2011; 32: 1958-1960.
• 29 Pereira J, Alfaro G, Goycoolea M. et al. Circulating platelet-derived microparticles in systemic lupus erythematosus. Thromb Haemost 2006; 95: 94-99.
• 30 Machleidt C, Mettang T, Stärz E. et al. Multifactorial genesis of enhanced platelet aggregability in patients with nephrotic syndrome. Kidney Int 1989; 36: 1119-1124.
• 31 Ozanne P, Francis RB, Meiselman HJ. Red blood cell aggregation in nephrotic syndrome. Kidney Int 1983; 23: 519-525.
• 32 Chen CH, Jiang T, Yang JH. et al. Low-density lipoprotein in hypercholesterolemic human plasma induces vascular endothelial cell apoptosis by inhibiting fibroblast growth factor 2 transcription. Circulation 2003; 107: 2102-2108.
• 33 Caraba A, Romosan I. Endothelial dysfunction in the nephrotic syndrome. Med Pregl 2007; 60: 66-69.
• 34 Jackson CA, Greaves M, Patterson AD. et al. Relationship between platelet aggregation, thromboxane synthesis and albumin concentration in nephrotic syndrome. Br J Haematol 1982; 52: 69-77.
• 35 Couser WG. Mediation of immune glomerular injury. J Am Soc Nephrol 1990; 01: 13-29.
• 36 Bruschi M, Carnevali ML, Murtas C. et al. Direct characterization of target podocyte antigens and auto-antibodies in human membranous glomerulonephritis: Alfa-enolase and borderline antigens. J Proteomics 2011; 74: 2008-2017.
• 37 Zwaal RFA, Schroit AJ. Pathophysiologic implications of membrane phospholipid asymmetry in blood cells. Blood 1997; 89: 1121-1132.
• 38 Thompson C, Forbes CD, Prentice CRM. et al. Changes in blood coagulation and fibrinolysis in the nephritic syndrome. Q J Med 1974; 43: 399-407.
• 39 Østerud B. Tissue factor expression in blood cells. Thromb Res 2010; 125 (Suppl. 01) S31-34
• 40 Lu C, Shi J, Yu H. et al. Procoagulant activity of long-term stored red blood cells due to phosphatidylserine exposure. Transfus Med 2011; 21: 150-157.