| Objectives：Analyze clinical therapeutic dosage of arsenic trioxide (ATO) on platelet apoptoticcascades, whether ATO induces human platelet apoptosis. In the mean time, we alsodiscuss the signaling pathways involved in ATO induced platelet apoptosis.Methods：Fresh blood from health volunteers was collected and centrifugated, then washedplatelets and platelet rich plasma (PRP) were prepared. Washed platelets (3×108/ml) wereincubated with different concentrations of ATO or vehicle dimethyl sulfoxide (DMSO) at37°C for5hours. Then, mitochondrial inner transmembrane potential (ΔΨm),phosphatidylserine (PS) exposure, P-selection and platelet activated complex-1(PAC-1)binding were tested by flow cytometry. In the mean time, the treated platelets wereanalyzed by western blot for the expression levels of pro-apoptotic protein (Bax), andanti-apoptotic proteins (Bcl-2and Bcl-XL). Activation of caspase-3and c-junNH2-terminal kinase (JNK) were also examined by western blot. To test whetherinhibition of JNK can attenuate ATO-induced platelet apoptosis, JNK specific inhibitordicumarol was used and ΔΨm was analysis by flow cytometry. For platelet aggregationanalysis, PRP was incubated with ATO (2μM) or vehicle control (DMSO) at37℃for1hour, washed platelets were incubated with ATO (16μM) or vehicle control (DMSO) at37℃for2hours. Platelet aggregation assay were performed by addition of collagen (5μg/mL) or adenosine diphosphate (ADP,10μmol/L) into PRP, or thrombin (0.5U/mL)into washed platelets at37℃, and examined by a turbidometric platelet aggregometer (Chrono-log, PA, USA) at a stirring speed of1000rpm. To further investigate whetherATO incurs thrombocytopenia in vivo, clinical therapeutic dosage of ATO wasintraperitoneally injected into C57mice, and the numbers of circulating platelets werecounted by Sysmex KX-21N Blood Cell Analyser.Results：1. ATO induces platelet apoptosis.ATO dose-dependently induces depolarization of ΔΨm, up-regulation of Bax anddown-regulation of Bcl-2and Bcl-XL, caspase-3activation, and PS exposure in platelets.ATO did not induce surface expression of P-selectin and PAC-1binding. ATOdose-dependently induced JNK activation, and JNK specific inhibitor dicumarol obviouslyreduced ATO-induced ΔΨm depolarization in platelets. Taken together, the datademonstrate that ATO induces caspase-dependent apoptosis via mitochondria-mediatedintrinsic pathway in platelets. However, JNK inhibitor did not completely block ATOinduced platelet apoptosis suggesting other signaling pathway might be involved.2. ATO impairs platelet aggregation functions.It has been reported that ATO inhibited arachidonic acid, thrombin, U46619, andepinephrine induced human platelet aggregation. However, the effect of ATO oncollagen-induced platelet aggregation remains conflict. Our data clearly show that ATOreduced collagen, ADP, and thrombin induced platelet aggregation, thus further indicatingthatATO impairs platelet functions.3. In vivo results.Thrombocytopenia is one of the most frequent hematological side effects during thetreatment of ATO in patients with acute promyelocytic leukemia (APL) and other kinds ofmalignant diseases. However, the pathogenesis of ATO-related thrombocytopenia remainsunclear. The above data confirm that ATO incurs platelet apoptosis in vitro. To furtherinvestigate whether ATO reduces circulating platelets in vivo, clinical therapeutic dosage ofATO or0.9%normal saline (NS) control was intraperitoneally injected into C57mice, andthe numbers of circulating platelets were counted. Circulating platelets were moderate but significantly reduced after continuous injection of ATO for5days. The data directlydemonstrate that ATO reduces circulating platelets in C57mice.Conclusions：In conclusion, the data demonstrate that ATO induces caspase-dependent apoptosis viaJNK activation in platelets. ATO does not incur platelet activation, whereas, it not onlyimpairs platelet function but also decreases circulating platelets in vivo, suggesting thepossible pathogenesis of thrombocytopenia in patients treated withATO. |
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