Protein kinase A determines platelet life span and survival by regulating apoptosis
Lili Zhao, … , Changgeng Ruan, Kesheng Dai
Lili Zhao, … , Changgeng Ruan, Kesheng Dai
Published October 30, 2017
Citation Information: J Clin Invest. 2017;127(12):4338-4351. https://doi.org/10.1172/JCI95109.
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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 5

PKA regulates platelet apoptosis via mediation of phosphorylation of BAD at Ser155.

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PKA regulates platelet apoptosis via mediation of phosphorylation of BAD...
(A) Washed platelets were incubated with indicated concentrations of H89 or vehicle at 22°C for 160 minutes. Western blot analysis for the levels of indicated proteins with different antibodies. (B and C) Washed platelets were incubated with H89 (25 μM), forskolin (10 μM), or vehicle at 22°C for 120 minutes. The platelets were lysed and centrifugated. Western blot analysis for the levels of indicated proteins with different antibodies (B). The lysates were immunoprecipitated with anti-BAD antibody overnight. Western blot analysis for BAD, BCL-XL, and 14-3-3 with various antibodies (C). (D and E) Washed platelets were incubated with H89 (25 μM), forskolin (10 μM), or vehicle control at 22°C for 120 minutes. The mitochondria of the pretreated platelets were isolated and verified as described in Methods. Representative images of mitochondria stained for BAD and BCL-XL are shown. Scale bar: 5 μm (D). Western blot analysis for BAD and BCL-XL in the mitochondria, and quantification of the levels of BAD and BCL-XL from the Western blot data (E). Data are expressed as mean ± SD (n = 5). #P < 0.001, 1-way ANOVA. The blots and images shown in each panel are representative of at least 5 separate experiments with different donors. (F) Schematic representation of proposed mechanism for PKA-regulated platelet apoptosis. Thrombin generated in the circulation (or in the absence of endothelium-derived PGI2) reduces PKA activity. The decrease of PKA activity results in dephosphorylation of BAD at Ser155, which releases 14-3-3 and sequesters prosurvival BCL-XL on mitochondria, leading to apoptosis.