Adenozin difosfat ile uyarılan trombositlerde Kaspaz-3 gen anlatımı [Caspase-3 gene expression in adenosine diphosphate stimulated platelets]

Abstract

Objectives: Platelets are known blood cells differentiated from megakaryocytes in bone marrow and they play a key role in coagulation mechanism. They live several days in blood circulation and they aggregate in respond to stimulants such as thrombin, collagen, ADP (adenosine diphosphate) and epinephrine. Although platelets are known anucleated cells, it has been reported in vivo and in vitro that they undergo to apoptosis. In this study apoptotic effects of ADP on the platelets was investigated. Materials and methods: P-selectin binding with flowcytometry, calcium levels with fluorometer, cytochrome-c levels with ELISA, caspase-3 gene expression with qRT-PCR, caspase-3 releasing with western blotting were determined in platelets. Results: Platelets were incubated for 1 hour at 37°C with 10µM ADP. P-selectin binding, calcium and cytochrome-c levels, caspase-3 releasing and gene expression are significantly increased compared to control in ADP stimulated platelets. Conclusions: Platelets can induce caspase-3 gene expression when stimulated with ADP which is a key enzyme plays an important role in apoptotic pathway.

Keywords

• Platelets, caspase-3, qRT-PCR, P-selectin, ADP

Adenozin difosfat ile uyarılan trombositlerde Kaspaz-3 gen anlatımı

Öz

Amaç: Trombositler kemik iliğinde megakaryositlerin farklılaşması sonucunda meydana gelen, pıhtılaşmada görev alan kan hücreleri olarak bilinirler. Dolaşımda birkaç günlük ömrü olan trombositler plazmada trombin, kollagen, adenozin difosfat (ADP) ve epinefrin gibi çeşitli uyarıcılarla karşı farklı sinyaller vererek agrege olurlar. Çekirdeksiz hücre olarak bilinmelerine karşın trombositlerin apoptoza uğradıkları yapılan in vivo ve in vitro çalışmalarla gösterilmiştir. Bu çalışmada ADP’nin trombositler üzerindeki apoptotik etkisi araştırılmıştır. Gereç ve yöntem: Trombositler 10µM ADP ile 37°C de 1 saat inkübe edilmiştir. Trombositlerde akım sitometride p-selektin bağlanması, fluorometre ile kalsiyum düzeyleri, ELISA ile sitokrom-c düzeyleri, qRT-PCR ile kaspaz-3 gen ekspresyonu, Western blot ile kaspaz-3 salınımı öl- çülmüştür. Bulgular: ADP ile uyarılan trombositlerde p-selektin, kalsiyum, sitokrom-c, kaspaz-3 salınımı ve gen ekspresyonu artmıştır. Sonuç: Trombositler ADP ile uyarıldıkları zaman apoptotik yolakta anahtar enzim olan kaspaz-3 gen anlatımını yapabilirler.

Anahtar Kelimeler

• Trombositler, kaspaz-3, qRT-PCR, P-selektin, ADP

Kaynakça

1. Deutsch VR, Tomer A. Megakaryocyte development and platelet production. Br J Haematol 2006;134(4):453-66.
2. Long MW. Megakaryocyte differentiation events. Semin Hematol 1998;35(2):192-9.
3. Avecilla ST, Hattori K, Heissig B, et al. Chemokine-me- diated interaction of hematopoietic progenitors with the bone marrow vascular niche is required for thrombopoi- esis. Nature Medicine 2004;10(1): 64-71.
4. Kaushansky K. The molecular mechanisms that control thrombopoiesis. J Clin Invest 2005; 115(12):3339-47.
5. Clemetson KJ, Clemetson JM. Michelson AD. Platelet receptors. In: Micheldon AD ed. Platelets, 2nd edn, San Diego, CA: Elsevier/Academic Press, 2007:117-43.
6. Yip J, Shen Y, Andrews KR. Primary platelet adhesion receptors. Australion Biochemist 2004;35(1):4-8.
7. Jin J, Daniel JL, Kunapuli SP. Molecular basis for ADP-induced platelet activation II. The P2Y1 recep- tor mediates ADP-induced intracellular calcium mo- bilization and shape change in platelets. J Biol Chem 1998;273(4):2030-4.
8. Tolhurst G, Vial C, Leon C, et al. Interplay between P2Y(1), P2Y(12), and P2X(1) receptors in the activa- tion of megakaryocyte cation influx currents by ADP: evidence that the primary megakaryocyte represents a fully functional model of platelet P2 receptor signaling. Blood 2005;106(5):1644-51.

9. Orrenius S, Zhivotovsky B, Nicotera P. Regulation of cell death: the calcium-apoptosis link. Nat Rev Mol Cell Biol 2003;4(7):552-65.
10. Zhang W, Liu J, Sun R, et al. Calpain activator di- bucaine induces platelet apoptosis. Int J Mol Sci 2011;12(4):2125-37.
11. Leytin V, Allen DJ, Mutlu A, et al. Platelet activation and apoptosis are different phenomena: Evidence from the sequential dynamics and the magnitude of responses during platelet storage. Br J Haematol 2008;142(4):480- 501.
12. Leytin V, Freedman J. Platelet apoptosis in stored plate- let concentrates and other models. Transfus Apher Sci 2003;28(2):285-95.
13. Vanags DM, Orrenius S, Aguilar-Santelises M. Altera- tions in Bcl-2/Bax protein levels in platelets form part of an ionomycin-induced process that resembles apopto- sis. Br J Haematol 1997;99(7):824-31.
14. Nicholson DW, Ali A, Thornberry NA, et al. Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis. Nature 1995;376(6535):37-43.
15. Chang YH, Yang X. Proteases for cell suicide: functions and regulation of caspases. Microbiol Mol Biol Rev 2000;64(4):821-46.
16. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utiliz- ing the principle of protein-dye binding. Anal Biochem 1976;72(2):248-54.
17. Cevik O, Oba R, Macit C, et al. Lycopene inhibits cas- pase-3 activity and reduces oxidative organ damage in a rat model of thermal injury. Burns 2012;38(6):861-71.
18. Mason DK, Carpinelli MR, Fletcher JI, et al. Pro- grammed anuclerar cell death delimits platelet life span. Cell 2007;128(9):1173-86.
19. Leytin V. Apoptosis in the anucleate platelet. Blood Rev 2012;26(2):51-63.
20. Shcherbina A, Remold-O’Donnell E. Role of caspase in a subset of human platelet activation responses. Blood 1999;93(12):4222-31.
21. Kroemer G, Galluzzi L, Brenner C. Mitochondrial membrane permeabilization in cell death. Physiol Rev 2007;87(1):99-163.
22. Leytin V, Allen DJ, Mutlu A, Gyulkhandanyan AV, Mykhaylov S, Freedman J. Mitochondrial control of platelet apoptosis: effect of cyclosporin A, an inhibitor of the mitochondrial permeability transition pore. Lab Invest 2009;89(4):374-84.
23. Gyulkhandanyan AV, Mutlu A, Freedman J, Leytin V. Markers of platelet apoptosis: methodology and appli- cations. J Thromb Thrombolysis 2012;33(4):397-411.
24. Michelson, A.D. Flow cytometry: A clinical test of platelet function. Blood 1996;87(12):4925-36.
25. Sener A, Ozsavci D, Oba R, et al. Do platelet apoptosis, activation, aggregation, lipid peroxidation and platelet- leukocyte aggregate formation occur simultaneously in hyperlipidemia? Clin Biochem 2005;38(12):1081-7.
26. Ozgen U, Ozerol E, Aminci M. Relationship between activation and apoptosis in platelets. Turk J Hematol 2007;24(2):171-6.
27. Rizzuto R, Pinton P, Ferrari D, et al. Calcium and apopto- sis: facts and hyotheses. Oncogene 2003;22(24):8619- 27.
28. Nicotera P, Orrenius S. The role of calcium in apoptosis. Cell Calcium 1998;23(2-3):173-80.
29. Juin P, Pelletier M, Oliver L, et al. Induction of a cas- pase-3-like activity by calcium in normal cytosolic ex- tracts triggers nuclear apoptosis in a cell-free system. J Biol Chem 1998;273(28):17559-64.
30. Affaticati P, Mignen O, Jambou F, et al. Sustained cal- cium signaling and caspase-3 activation involve NMDA receptors in thymocytes in contact with dendritic cells. Cell Death Differ 2011;18(1):99-108.
31. Cohen Z, Gonzales RF, Davis-Gorman GF, Copeland JG, McDonagh PF. Thrombin activity and platelet mi- croparticle formation are increased in type 2 diabetic platelets: a potential correlation with caspase activation. Thromb Res 2002;107(5):217-21.
32. Winkler J, Kroiss S, Rand ML, et al. Platelet apopto- sis in paediatric immune thrombocytopenia is amelio- rated by intravenous immunoglobulin. Br J Haematol 2012;156(4):508-15.
33. Amor NB, Pariente JA, Salido GM, Rosado JA, Bartegi A. Thrombin-induced caspases 3 and 9 translocation to the cytoskeleton is independent of changes in cyto- solic calcium in human platelets. Blood Cells Mol Dis 2006;36(3):392-401.
34. Cookson P, Sutherland J, Turner C,et al. Platelet apop- tosis and activation in platelet concentrates stored for up to 12 days in plasma or additive solution. Transfus Med 2010;20(6):392-402.
35. Reid S, Johnson L, Woodland N, Marks DC. Pathogen reduction treatment of buffy coat platelet concentrates in additive solution induces proapoptotic signaling. Trans- fusion 2012; doi: 10.1111/j.1537-2995.2011.03558.x.
36. Bertino AM, Qi XQ, Li J, Xia Y, Kuter DJ. Apoptotic mark- ers are increased in platelets stored at 37 degrees C. Transfusion 2003;43(7):857-66.