Platelet Aging In Vivo Is Associated with Activation of Apoptotic Pathways: Studies in a Model of Suppressed Thrombopoiesis in Dogs

Jaime Pereira; Mónica Soto; Iván Palomo; Mauricio Ocqueteau; Lindi-Marie Coetzee; Smiljan Astudillo; Eduardo Aranda; Diego Mezzano

doi:10.1055/s-0037-1613103

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2002; 87(05): 905-909
DOI: 10.1055/s-0037-1613103

Review Article

Schattauer GmbH

Platelet Aging In Vivo Is Associated with Activation of Apoptotic Pathways: Studies in a Model of Suppressed Thrombopoiesis in Dogs

Jaime Pereira

¹Department of Haematology-Oncology, School of Medicine, Catholic University of Chile, Santiago, Chile

,

Mónica Soto

¹Department of Haematology-Oncology, School of Medicine, Catholic University of Chile, Santiago, Chile

,

Iván Palomo

²Faculty of Health Sciences, University of Talca, Chile

,

Mauricio Ocqueteau

¹Department of Haematology-Oncology, School of Medicine, Catholic University of Chile, Santiago, Chile

,

Lindi-Marie Coetzee

³Department of Haematology and Cell Biology, University of the Orange Free State, Bloemfontein, South Africa

,

Smiljan Astudillo

¹Department of Haematology-Oncology, School of Medicine, Catholic University of Chile, Santiago, Chile

,

Eduardo Aranda

¹Department of Haematology-Oncology, School of Medicine, Catholic University of Chile, Santiago, Chile

,

Diego Mezzano

¹Department of Haematology-Oncology, School of Medicine, Catholic University of Chile, Santiago, Chile

› Author Affiliations

Abstract

Summary

The mechanism(s) involved in the clearance of senescent platelets are largely unknown. We have recently demonstrated that platelet aging in vivo is associated with loss of membrane phospholipid asymmetry, a universal phenomenon in cells undergoing apoptosis. Thus, we postulated that senescent platelets may exhibit programmed cell death changes, which may trigger their removal from circulation. Since platelets contain the apoptosis machinery as well as mitochondria, a key organelle in the regulation of apoptosis, we studied the appearance of apoptotic-like changes during platelet aging in vivo. To investigate this, we assessed changes in mitochondrial membrane potential (Δψ) in circulating canine platelets during decline in platelet count after suppression of thrombopoiesis by estradiol injection, a validated model to obtain circulating platelets of increasing mean age. Phosphatidylserine (PS) exposure was determined by flow cytometry by binding of FITC-labeled annexin V. Mitochondrial Δψ was studied with the cationic lipophilic dye DIOC₆ (3) and the J-aggregate-forming cation JC-1 and analysis by flow cytometry. The proportion of platelets with exposed PS rose significantly with age, from 2.88% before to 6.7%, 8 days after estradiol injection. By flow cytometry it was demonstrated a significant decreased in DIOC₆ (3) fluorescence (median fluorescence intensity 791 ± 98 vs 567 ± 102 day 0 vs day 8 post injection of estradiol, respectively; n: 11; p <0.01), consistent with mitochondrial Δψ collapse. JC-1 has the unique property of forming J-aggregates under high mitochondrial Δψ (red fluorescence, FL2) whereas the monomeric form fluoresces in green (FL1). Aged platelets in vivo, loaded with JC-1, exhibited a significant increase in FL1/FL2 ratio (2.5 ± 1.7 vs 4.7 ± 1.6, day 0 vs day 8 post injection of estradiol, respectively; n: 13; p <0.05), confirming the mitochondrial Δψ alteration.

The results show that platelet aging in vivo is associated with a decrease in mitochondrial Δψ and PS exposure. In conclusion, our data provide for the first time, evidence that platelet senescence is associated with changes characteristics of apoptosis, which may promote their removal from circulation.

Keywords

Platelet aging - apoptosis - mitochondria - platelet removal

Full Text

References

References
1 George J, Dale G. Platelet kinetics. In: Beutler E, Lichtman M, Coller B, Kipps T. eds. Hematology. New York: McGraw-Hill Inc; 1995: 1202-5.
2 Savill J, Fadok V, Henson P, Haslett C. Phagocyte recognition of cells undergoing apoptosis. Immunol Today 1993; 14: 131-6.
3 Zwaal RF, Schroit AJ. Pathophysiologic implications of membrane phospholipid asymmetry in blood cells. Blood 1997; 89: 1121-32.
4 Bevers EM, Comfurius P, Zwaal RF. Changes in membrane phospholipid distribution during platelet activation. Biochim Biophys Acta 1983; 736: 57-66.
5 Blumenfeld N, Zachowski A, Galacteros F, Beuzard Y, Devaux PF. Transmembrane mobility of phospholipids in sickle erythrocytes: effect of deoxygenation on diffusion and asymmetry. Blood 1991; 77: 849-54.
6 Connor J, Pak CC, Schroit AJ. Exposure of phosphatidylserine in the outer leaflet of human red blood cells. Relationship to cell density, cell age, and clearance by mononuclear cells. J Biol Chem 1994; 269: 2399-404.
7 Utsugi T, Schroit AJ, Connor J, Bucana CD, Fidler IJ. Elevated expression of phosphatidylserine in the outer membrane leaflet of human tumor cells and recognition by activated human blood monocytes. Cancer Res 1991; 51: 3062-6.
8 Fadok VA, Savill JS, Haslett C. et al. Different populations of macrophages use either the vitronectin receptor or the phosphatidylserine receptor to recognize and remove apoptotic cells. J Immunol 1992; 149: 4029-35.
9 Martin SJ, Reutelingsperger CP, McGahon AJ. et al. Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl. J Exp Med 1995; 182: 1545-56.
10 Pereira J, Palomo I, Ocqueteau M, Soto M, Aranda E, Mezzano D. Platelet aging in vivo is associated with loss of membrane phospholipid asymmetry. Thromb Haemost 1999; 82: 1318-21.
11 Jacobson MD, Burne JF, Raff MC. Programmed cell death and Bcl-2 protection in the absence of a nucleus. Embo J 1994; 13: 1899-910.
12 Vanags DM, Orrenius S, Aguilar-Santelises M. Alterations in Bcl-2/Bax protein levels in platelets form part of an ionomycin-induced process that resembles apoptosis. Br J Haematol 1997; 99: 824-31.
13 Wolf BB, Goldstein JC, Stennicke HR. et al. Calpain functions in a caspase-independent manner to promote apoptosis- like events during platelet activation. Blood 1999; 94: 1683-92.
14 Brown SB, Clarke MC, Magowan L, Sanderson H, Savill J. Constitutive death of platelets leading to scavenger receptor-mediated phagocytosis. A caspase-independent cell clearance program. J Biol Chem 2000; 275: 5987-96.
15 Li J, Xia Y, Bertino AM, Coburn JP, Kuter DJ. The mechanism of apoptosis in human platelets during storage. Transfusion 2000; 40: 1320-9.
16 Kroemer G, Dallaporta B, Resche-Rigon M. The mitochondrial death/life regulator in apoptosis and necrosis. Annu Rev Physiol 1998; 60: 619-42.
17 Castedo M, Hirsch T, Susin SA. et al. Sequential acquisition of mitochondrial and plasma membrane alterations during early lymphocyte apoptosis. J Immunol 1996; 157: 512-21.
18 From AH, Fong JS, Chiu T, Good RA. Role of platelets in the pathogenesis of canine endotoxin shock. Infect Immun 1976; 13: 1591-4.
19 Aranda E, Pizarro M, Pereira J, Mezzano D. Accumulation of 5-hydroxytryptamine by aging platelets: studies in a model of suppressed thrombopoiesis in dogs. Thromb Haemost 1994; 71: 488-92.
20 Salvioli S, Ardizzoni A, Franceschi C, Cossarizza A. JC-1, but not DiOC₆ (3) or rhodamine 123, is a reliable fluorescent probe to assess delta psi changes in intact cells: implications for studies on mitochondrial functionality during apoptosis. FEBS Lett 1997; 411: 77-82.
21 Bortner CD, Cidlowski JA. Caspase independent/dependent regulation of K(+), cell shrinkage, and mitochondrial membrane potential during lymphocyte apoptosis. J Biol Chem 1999; 274: 21953-62.
22 Chiu T. Studies on estrogen-induced proliferative disorders of hemopoietic tissue in dogs. St. Paul: University of Minnesota; 1974: 1-270.
23 Berger G, Hartwell DW, Wagner DD. Platelet death, an apoptotic-like process?. Blood 1998; 92: 347a.
24 Salvioli S, Barbi C, Dobrucki J. et al. Opposite role of changes in mitochondrial membrane potential in different apoptotic processes. FEBS Lett 2000; 469: 186-90.
25 Susin SA, Zamzami N, Kroemer G. Mitochondria as regulators of apoptosis: doubt no more. Biochim Biophys Acta 1998; 1366: 151-65.
26 Bernardi P, Scorrano L, Colonna R, Petronilli V, Di Lisa F. Mitochondria and cell death. Mechanistic aspects and methodological issues. Eur J Biochem 1999; 264: 687-701.
27 Kroemer G, Reed JC. Mitochondrial control of cell death. Nat Med 2000; 06: 513-9.
28 Denecker G, Dooms H, Van Loo G. et al. Phosphatidyl serine exposure during apoptosis precedes release of cytochrome c and decrease in mitochondrial transmembrane potential. FEBS Lett 2000; 465: 47-52.
29 van Engeland M, Nieland LJ, Ramaekers FC, Schutte B, Reutelingsperger CP. Annexin V-affinity assay: a review on an apoptosis detection system based on phosphatidylserine exposure. Cytometry 1998; 31: 1-9.
30 Reers M, Smith TW, Chen LB. J-aggregate formation of a carbocyanine as a quantitative fluorescent indicator of membrane potential. Biochemistry 1991; 30: 4480-6.
31 Smiley ST, Reers M, Mottola-Hartshorn C. et al. Intracellular heterogeneity in mitochondrial membrane potentials revealed by a J-aggregate-forming lipophilic cation JC-1. Proc Natl Acad Sci USA 1991; 88: 3671-5.
32 Cossarizza A, Baccarani-Contri M, Kalashnikova G, Franceschi C. A new method for the cytofluorimetric analysis of mitochondrial membrane potential using the J-aggregate forming lipophilic cation 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolcarbocyanine iodide (JC-1). Biochem Biophys Res Commun 1993; 197: 40-5.