Protein kinase A determines platelet life span and survival by regulating apoptosis
Lili Zhao, Jun Liu, Chunyan He, Rong Yan, Kangxi Zhou, Qingya Cui, Xingjun Meng, Xiaodong Li, Yang Zhang, Yumei Nie, Yang Zhang, Renping Hu, Yancai Liu, Lian Zhao, Mengxing Chen, Weiling Xiao, Jingluan Tian, Yunxiao Zhao, Lijuan Cao, Ling Zhou, Anning Lin, Changgeng Ruan, Kesheng Dai
Lili Zhao, Jun Liu, Chunyan He, Rong Yan, Kangxi Zhou, Qingya Cui, Xingjun Meng, Xiaodong Li, Yang Zhang, Yumei Nie, Yang Zhang, Renping Hu, Yancai Liu, Lian Zhao, Mengxing Chen, Weiling Xiao, Jingluan Tian, Yunxiao Zhao, Lijuan Cao, Ling Zhou, Anning Lin, Changgeng Ruan, Kesheng Dai
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Research Article Hematology

Protein kinase A determines platelet life span and survival by regulating apoptosis

Abstract

Apoptosis delimits platelet life span in the circulation and leads to storage lesion, which severely limits the shelf life of stored platelets. Moreover, accumulating evidence indicates that platelet apoptosis provoked by various pathological stimuli results in thrombocytopenia in many common diseases. However, little is known about how platelet apoptosis is initiated or regulated. Here, we show that PKA activity is markedly reduced in platelets aged in vitro, stored platelets, and platelets from patients with immune thrombocytopenia (ITP), diabetes, and bacterial infections. Inhibition or genetic ablation of PKA provoked intrinsic programmed platelet apoptosis in vitro and rapid platelet clearance in vivo. PKA inhibition resulted in dephosphorylation of the proapoptotic protein BAD at Ser155, resulting in sequestration of prosurvival protein BCL-XL in mitochondria and subsequent apoptosis. Notably, PKA activation protected platelets from apoptosis induced by storage or pathological stimuli and elevated peripheral platelet levels in normal mice and in a murine model of ITP. Therefore, these findings identify PKA as a homeostatic regulator of platelet apoptosis that determines platelet life span and survival. Furthermore, these results suggest that regulation of PKA activity represents a promising strategy for extending platelet shelf life and has profound implications for the treatment of platelet number-related diseases and disorders.

Authors

Lili Zhao, Jun Liu, Chunyan He, Rong Yan, Kangxi Zhou, Qingya Cui, Xingjun Meng, Xiaodong Li, Yang Zhang, Yumei Nie, Yang Zhang, Renping Hu, Yancai Liu, Lian Zhao, Mengxing Chen, Weiling Xiao, Jingluan Tian, Yunxiao Zhao, Lijuan Cao, Ling Zhou, Anning Lin, Changgeng Ruan, Kesheng Dai

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Figure 7

Activation of PKA protects stored platelets from apoptosis, clearance, and loss of function.

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Activation of PKA protects stored platelets from apoptosis, clearance, a...
(A and B) Δψm depolarization and PS exposure of human platelets incubated with H89 (25 μM), forskolin (5 μM), or vehicle at 22°C for indicated times. Data are represented as mean ± SD from 4 independent experiments. *P < 0.05; **P < 0.01; #P < 0.001, 2-way ANOVA. (C) Mouse platelets were incubated with H89 (25 μM), forskolin (5 μM), or vehicle at 22°C for 72 hours. The platelets were labeled with calcein and injected into ICR mice (n = 5). The percentage of calcein-labeled platelets remaining in circulation was determined by flow cytometry. Data are represented as mean of normalized percentage of calcein-labeled platelets (time point 0 = 100%) ± SD of 3 independent experiments. **P < 0.01; #P < 0.001, 2-way ANOVA. (D and E) Human washed platelets were incubated with forskolin (5 μM) or vehicle (DMSO) at 22°C for 72 hours. The pretreated washed platelets were stimulated with ristocetin (1.25 mg/ml) plus von Willebrand factor (7.5 μg/ml) (D) and collagen (5 μg/ml) (E) at 37°C under constant stirring. Platelet aggregation was recorded in a CHRONO-LOG aggregometer. Histograms of maximal platelet aggregation under the indicated conditions are shown as mean ± SD of 5 independent experiments. **P < 0.01, Student’s t test. (F) Washed mouse platelets were incubated with forskolin (5 μM) or vehicle (DMSO) at 22°C for 72 hours and were labeled with calcein-AM (5 μg/ml). The recipient mice were injected intravenously with pretreated platelets (5 × 106/g). FeCl3-induced thrombosis in the mice was recorded by real-time microscopy at 3 minutes. Only forskolin-treated calcein-AM–labeled platelets were detected in the thrombus. No H89-treated or vehicle control–treated (DMSO) stored platelets were found in the thrombus. Each image is representative of 5 mice. Original magnification × 200 (× 10 eyepiece, × 20 objective).