Loss of PIKfyve in platelets causes a lysosomal disease leading to inflammation and thrombosis in mice - PubMed (original) (raw)

Aae Suzuki 1, Timothy J Stalker 1, Liang Zhao 1, Yuhuan Wang 2, Chris McKennan 3, Matthew J Riese 1, Jessica F Guzman 1, Suhong Zhang 4, Lurong Lian 1, Rohan Joshi 1, Ronghua Meng 5, Steven H Seeholzer 3, John K Choi 6, Gary Koretzky 1, Michael S Marks 5, Charles S Abrams 1

Affiliations

Loss of PIKfyve in platelets causes a lysosomal disease leading to inflammation and thrombosis in mice

Sang H Min et al. Nat Commun. 2014.

Abstract

PIKfyve is essential for the synthesis of phosphatidylinositol-3,5-bisphosphate [PtdIns(3,5)P2] and for the regulation of endolysosomal membrane dynamics in mammals. PtdIns(3,5)P2 deficiency causes neurodegeneration in mice and humans, but the role of PtdIns(3,5)P2 in non-neural tissues is poorly understood. Here we show that platelet-specific ablation of PIKfyve in mice leads to accelerated arterial thrombosis, and, unexpectedly, also to inappropriate inflammatory responses characterized by macrophage accumulation in multiple tissues. These multiorgan defects are attenuated by platelet depletion in vivo, confirming that they reflect a platelet-specific process. PIKfyve ablation in platelets induces defective maturation and excessive storage of lysosomal enzymes that are released upon platelet activation. Impairing lysosome secretion from PIKfyve-null platelets in vivo markedly attenuates the multiorgan defects, suggesting that platelet lysosome secretion contributes to pathogenesis. Our findings identify PIKfyve as an essential regulator for platelet lysosome homeostasis, and demonstrate the contributions of platelet lysosomes to inflammation, arterial thrombosis and macrophage biology.

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Figures

Figure 1

Figure 1. Platelet-specific ablation of PIKfyve causes multiorgan abnormalities in mice

(a) Schematic representation of genetic targeting of PIKfyve. The target exons 37and 38 (red bars) were targeted with lox P recombination sites (yellow arrows) to generate PIKfyve floxed alleles (PIKfyvefl). Mice expressing PIKfyvefl were then crossed with Pf4-Cre mice to induce homologous recombination of the PIKfyvefl (PIKfyve Post-Cre). (b) General appearance of control and PIKfyvefl/fl Pf4-Cre littermates at 24 weeks of age. Note the characteristic coarse facial features, body hair loss, and severely distended body morphology. (c) Body weight of control mice and PIKfyvefl/fl Pf4-Cre mice. The average numbers of mice were 8 per time point and per group. (d) Percent of total body fat in the control (n=3) and PIKfyvefl/fl Pf4-Cre (n=3) mice at 12–24 weeks of age. (e) Survival curves of control (n= 40) and PIKfyvefl/fl Pf4-Cre (n= 40) mice. *P<0.05, ** P<0.01, ***P<0.001. All error bars indicate mean +/− S.D.

Figure 2

Figure 2. Large vacuolated macrophages infiltrate multiple tissues in the PIKfyve fl/fl Pf4-Cre mice

(a) Representative histological sections of the lungs and livers stained with hematoxylin and eosin from control mice and PIKfyvefl/fl Pf4-Cre mice. Scale bar, 100µm. Insets show increased magnification of boxed areas in panel (a). Scale bar, 20µm. Arrowheads indicate the vacuolated cells. (b) Lung sections stained with Alcian blue. Scale bar, 100µm. Inset shows high magnification of the boxed area. (c) Liver structures of control mice and PIKfyvefl/fl Pf4-Cre mice were stained for macrophages with anti-F4/80 by immunohistochemistry. Scale bar, 100µm. Insets in the upper right corner of each image show higher magnification images of boxed areas. Scale bar, 20µm. (d) Cell diameters of F4/80+ cells in the livers of control mice (n=5) and PIKfyvefl/fl Pf4-Cre mice (n=5). ***P<0.001. All error bars indicate mean +/− S.D.

Figure 3

Figure 3. Platelets drive the multiorgan defects in the PIKfyvefl/fl Pf4-Cre mice

(a) The levels of PIKfyve protein were determined in the platelets and liver of the indicated mice by immunoblotting. Note the absence of the PIKfyve protein in the platelets, but its presence in the liver of PIKfyvefl/fl Pf4-Cre mice (n=3). Tissues from the wild-type mice, the PIKfyvefl/+ Pf4-Cre mice, and the PIKfyvefl/fl mice were used as positive controls. β-actin was used as a loading control. (b) X-gal staining of the lung tissues from PIKfyvefl/fl Rosa26-LacZ mice and PIKfyvefl/fl Pf4-Cre Rosa26-LacZ mice. Scale bar, 20µm. The arrow indicates the platelet clumps in the lung vessels expressing LacZ (blue stain). Arrowheads indicate the vacuolated macrophages that did not stain blue. (c) Representative general morphology of 15-weeks-old control mice and PIKfyvefl/fl Pf4-Cre mice after 6 weeks of either no intervention (untreated) or biweekly injections of anti-GP1bα antibodies (+GP1bα Ab). (d) Body weight change of the control mice and PIKfyvefl/fl Pf4-Cre mice after 6 weeks of no intervention or treatment with anti-GP1bα antibodies (n=3 for each group). *P<0.05. All error bars indicate mean +/− S.D.

Figure 4

Figure 4. PIKfyve is not necessary for the development of megakaryocytes nor for platelet production

(a) Electron micrographs of megakaryocytes grown in culture in the presence of thrombopoietin for 7 days. Note multiple enlarged vacuoles in the cytoplasm of PIKfyvefl/fl Pf4-Cre mice as compared to the control mice. Scale bar, 10µm. (b) CD41+ megakaryocyte counts of control PIKfyvefl/fl Pf4-Cre mice. (c) Megakaryocyte ploidy. (d–f) Counts of platelets (d), WBC (e), and Hb (f) of control mice and PIKfyvefl/fl Pf4-Cre mice.

Figure 5

Figure 5. PIKfyve is critical for the maturation, storage, and release of lysosomal enzymes in platelets

(a) Immunoblot of platelet lysates from the control mice and PIKfyvefl/fl Pf4-Cre mice showing the expression of EEA-1 and LAMP-1. β-actin was used as the loading control. (b) Quantification of protein bands shown in the panel a (n=3 per group). (c) β-hexosaminidase activity in the platelet lysates of control mice (n=3) and PIKfyvefl/fl Pf4-Cre mice (n=3). (d) β-hexosaminidase activity in the platelet releasate and plasma of control mice (n=3) and PIKfyvefl/fl Pf4-Cre mice (n=3). (e) Time to occlusion (TTO) of carotid arteries upon vascular injury with 7.5% of FeCl3 in the control mice (n=9) and the PIKfyvefl/fl Pf4-Cre mice (n=8 ). (f) Immunoblot of platelet lysates from control platelets and PIKfyvefl/fl Pf4-Cre platelets showing the Akt phosphorylation in resting condition or stimulated with 1U/mL of thrombin for 10 minutes. (g) SDS agarose electrophoresis showing the multimer pattern of plasma vWF from the control mice (n=3) and the PIKfyvefl/fl Pf4-Cre (n=3) mice. (h) Plasma levels of vWF from the control mice (n=3) and the PIKfyvefl/fl Pf4-Cre (n=3) mice were quantified by ELISA. *P<0.05. All error bars indicate mean +/− S.D.

Figure 6

Figure 6. Impaired secretion of platelet lysosomes attenuates the phenotypes of PIKfyvefl/fl Pf4-Cre mice

(a) General appearance of PIKfyvefl/fl Pf4-Cre mice and PIKfyvefl/fl Pf4-Cre/pallid mice on C57BL/6 background at 17 weeks of age. In the presence of the pallid mutation, the hair loss and weight gain are attenuated in the PIKfyvefl/fl Pf4-Cre/pallid mice. Note that the pallid mice have an underlying defect in melanosome biogenesis causing hypopigmented fur. (b) Body weight of the PIKfyvefl/fl Pf4-Cre mice and the aged-matched PIKfyve Pf4-Cre/pallid mice. The average numbers of mice were 5 per time point and per group. The dashed line indicates the mean ratio of body weight of the control mice. (c,d) Analysis of β-hexosaminidase activity in the platelet lysate (c) and plasma (d) of the PIKfyvefl/fl Pf4-Cre mice (n=3) and the PIKfyvefl/fl Pf4-Cre/pallid (n=3) mice. *P <0.05, ** P < 0.01. All error bars indicate mean +/− S.D.

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