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 4

Platelets lacking PKA Cα exhibit shortened life span and apoptosis.

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Platelets lacking PKA Cα exhibit shortened life span and apoptosis. (A) ...
(A) A PKA Cα conditional KO mouse was generated by introducing a loxP site on either side of exons 6, 7, and 8 of the gene (Pkacafl/fl). Mice with KO specific to megakaryocytes and platelets were produced by crossing the PKA Cα floxed mice with transgenic mice expressing PF4 promoter–driven Cre recombinase (Pf4-Cre+). (B) Western blot analysis of PKA Cα, BAD, GPIbβ, phosphorylated BAD at Ser155, and phosphorylated GPIbβ at Ser166 in platelets from PKA–/–, PKA–/+, and PKA+/+ mice. The immunoblots shown are representative of at least 5 mice of each genotype. (C–E) Platelets from PKA–/–, PKA+/–, and PKA+/+ mice were analyzed for Δψm depolarization (red population) (C), shrinkage (red population) (D), and morphology by scanning electron microscopy (E). Plots or images shown in each panel are representative of at least 3 mice. Scale bars: 2 μm. (F) Automated analysis of platelet counts. Data are represented as mean ± SD of 7 PKA+/+, 7 PKA+/–, and 5 PKA–/– male mice. *P < 0.05, compared with PKA+/+ mice, Student’s t test. (G) Anti-mouse platelet antibody R300 (0.15 mg/kg) was intraperitoneally injected into PKA+/+ or PKA+/– mice. Platelet counting was performed at indicated time points. Data are represented as mean ± SD of 6 PKA+/+ and 6 PKA+/– mice. *P < 0.05; **P < 0.01, compared with PKA+/+ mice, 2-way ANOVA. (H) The life span of PKA–/– platelets is obviously decreased. Mice were intravenously injected with NHS-biotin. Peripheral blood samples were taken from PKA+/+ and PKA–/– mice 1, 2, 3, 4, 5, and 6 days after injection. The percentage of biotinylated platelets was determined by flow cytometry. Data are represented as mean ± SD of 5 mice at each time point. #P < 0.001, 2-way ANOVA.