Prostacyclin protects basal and nitric oxide-induced megakaryocytes apoptosis

NEGROTTO S; POZNER RG; D'ATRI LP; LAZZARI MA; GÓMEZ RM; MAUGERI N; SCHATTNER M

Birmingham, Reino Unido

Congreso; XIX Congress of International Society for Thrombosis and Haemostasis; 2003

Birmingham, Reino Unido

Prostacyclin (PGI2) is a physiological endothelial derived regulatory molecule of platelet activation and vascular tone. Cytoprotection is another biological activity exerted by PGI2. We have previously demonstrated that nitric oxide (NO) induces apoptosis of megakaryocytes (Mks). In the present study we have investigated whether PGI2 had a regulatory effect on NO-induced apoptosis. Mks (CD34+ human cord blood cells incubated with 20 ng/ml thrombopoietin during 7-8 days, % of CD41+ cells >/= 80%) were treated with 100uM diethylenetriamine/NO (DETA/NO) and apoptosis was evaluated after 48 h by acridine orange/ethidium bromide staining. Results showed that treatment with DETA/NO augmented percentage of apoptotic cells compare to control values (49±9 vs 17± 2% respectively, p<.05, n=6). PGI2 (3 uM) protected Mks of the NO-proapoptotic effect (28±3, p<.05) in a concentration dependent manner (IC50=1uM, n=4), but did not modify basal apoptosis (16±3%). Since PGI2 and NO are constitutively synthesized in the bone marrow milieu, the effect of daily addition of both drugs during seven days was studied. DETA/NO (10 uM) increased percentage of apoptotic Mks compared to control values (23±2 vs 15±0.5% respectively, n=4, p<.05). PGI2 treatment (3uM) not only reverted NO-induced apoptosis to basal levels (16±1% p<.05) but also significatively inhibited basal Mks apoptosis (9±1.5% p<.05). Since cAMP is one of the major intracellular mediators involved in the PGI2 signaling pathway, we used a phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX). Like PGI2, treatment with IBMX (100 uM) for 48 hs significantly reduced DETA/NO pro-apoptotic effect (31±7 vs 51±6% respectively, p<.05, n=3). In attempt to clarify whether this PGI2 cytoprotective property could promote Mks development, DNA content was measured by propidium iodide incorporation and flow cytometry. The daily addition of PGI2 augmented the percentage of cells with a DNA content >2N compared to control cultures (25 vs 16% respectively, n=2). On the other hand apoptosis induced by DETA/NO treatment for 48 h was reflected by an increase in hypodiploid cells (39 vs control: 14%, n=2). The presence of PGI2 or IBMX reduced apoptotic cells induced by DETA/NO (21 and 15%) and promoted Mks polyploidization (26 and 25%). We conclude that PGI2 protects basal and NO-induced Mks apoptosis, promotes Mks maturation and cAMP seems to be at least one of the signaling pathways involved in this effect.     < 2N 2N > 2N         Control 14 70 16 DETA/NO 39 47 14 DETA/NO + PGI2 21 53 26 DETA/NO + IBMX 15 60 25