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

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