Sympathetic stimulation facilitates thrombopoiesis by promoting megakaryocyte adhesion, migration, and proplatelet formation by Shilei Chen, Changhong Du, Mingqiang Shen, Gaomei Zhao, Yang Xu, Ke Yang, Xinmiao Wang, Fengju Li, Dongfeng Zeng, Fang Chen, Song Wang, Mo Chen, Cheng Wang, Ting He, Fengchao Wang, Aiping Wang, Tianmin Cheng, Yongping Su, Jinghong Zhao, and Junping Wang Blood Volume 127(8):1024-1035 February 25, 2016 ©2016 by American Society of Hematology

Continual stress exposure elevates the peripheral platelet level. Continual stress exposure elevates the peripheral platelet level. (A-B) Peripheral platelet counts in WT C57BL/6 mice subjected to noise stress or exhaustive exercise stress for 7 days. (C) High-pressure liquid chromatography (HPLC) analysis of plasma concentrations of NE and EPI in WT mice after stress. (D) Peripheral platelet counts in splenectomized mice exposed to continual noise or exhaustive exercise stress. (E) Peripheral platelet counts in Dbh−/− mice after exposure to continual noise or exhaustive exercise stress. (F-G) Peripheral platelet levels in WT mice injected intraperitoneally with saline, 15 mg/kg phentolamine, or 5 mg/kg propranolol 30 minutes prior to stress exposure. (H) Peripheral platelet counts in C57BL/6J-Mplhlb219/J mice after exposure to noise or exhaustive exercise stress. (I) Fold induction of platelet levels on day 7 in C57BL/6J-Mplhlb219/J and WT mice after exposure to noise or exhaustive exercise stress. *P < .05, **P < .01, vs control; #P < .05, ##P < .01, vs stress group; ▲P < .05, ▲▲P < .01, WT vs C57BL/6J-Mplhlb219/J mice. Each group contains 6 to 7 mice. Shilei Chen et al. Blood 2016;127:1024-1035 ©2016 by American Society of Hematology

NE and EPI administration elevate platelet levels in vivo. NE and EPI administration elevate platelet levels in vivo. (A-B) Changes in the peripheral platelet levels in normal C57BL/6 mice injected with saline, NE, or EPI once a day for 7 days. (C) Distributions of MKs in the BM (H&E staining) in the mice. Arrows point to the pseudopods of MKs (proplatelets) inserting into the vascular sinusoids. (D) Quantification of MKs in the BM in the mice. (E) Quantification of the distance between MKs and the vascular sinusoids in the BM. (F) Experimental schematic of NE and EPI administration in C57BL/6 mice subjected to irradiation. (G) Changes in the peripheral platelet counts in irradiated mice receiving NE or EPI treatment. (H) Time (days) needed for 50% recovery of platelet loss from the nadir in irradiated mice receiving NE or EPI treatment. (I) Experimental schematic of NE and EPI administration in C57BL/6 mice that received BMT after irradiation. (J) Changes in the peripheral platelet counts in grafted mice that received NE or EPI treatment. (K) Time (days) needed for 50% recovery of platelet loss from the nadir in grafted mice receiving NE or EPI treatment. *P < .05, **P < .01, vs control. Each group contains 6 to 7 mice. Shilei Chen et al. Blood 2016;127:1024-1035 ©2016 by American Society of Hematology

Long-term stress-induced thrombopoiesis contributes to the aggravation of atherosclerosis in ApoE−/− mice. Long-term stress-induced thrombopoiesis contributes to the aggravation of atherosclerosis in ApoE−/− mice. (A) Representative aortas from ApoE−/− mice subjected to noise or exhaustive exercise stress for 8 weeks. (B) Quantification of the lesion size in the whole aorta. Each data point represents a value from a single mouse; n = 6. (C) Immunofluorescence staining of platelets with the CD41 antibody in the atherosclerotic lesions in the right carotid artery. (D) Quantification of fluorescence intensity of CD41 in the atherosclerotic lesions in the right carotid artery. (E) Distributions of MKs in the BM in the mice. (F) Quantification of MKs in the BM. *P < .05, **P < .01, vs control. Shilei Chen et al. Blood 2016;127:1024-1035 ©2016 by American Society of Hematology

NE and EPI expand the population of CD34+ cells but have no significant effect on the commitment to MK lineage and the proliferation of MKs. Human cord blood–derived CD34+ cells were cultured with NE and EPI together with different cytokines for 7 days. NE and EPI expand the population of CD34+ cells but have no significant effect on the commitment to MK lineage and the proliferation of MKs. Human cord blood–derived CD34+ cells were cultured with NE and EPI together with different cytokines for 7 days. (A) Flow cytometric analysis of the numbers of CD34+ cells labeled with APC-conjugated anti-CD34 antibody after the cells being treated with NE and EPI at the indicated concentrations in the presence of 20 ng/mL rhSCF. (B) The expression of CD36+CD41+ on CD34+ cells analyzed by flow cytometric analysis. (C) Mean frequency of CD34+ cells expressing CD36+CD41+ after the cells being treated with different concentrations of NE or EPI in the presence of rhSCF. (D) Flow cytometric analysis of CD41+CD42b+ expression on the cells after treatment with NE (1 μM) or EPI (1 μM) in the presence of 20 ng/mL rhSCF. (E) Mean frequency of cells expressing CD41+CD42b+ after CD34+ cells being treated with different concentrations of NE or EPI in the presence of 20 ng/mL rhSCF. (F) Flow cytometric analysis of CD41+ cell counts after NE and EPI treatment at the indicated concentrations in the presence of 20 ng/mL rhTPO. (G) Histogram of DNA content in CD41+ cells after CD34+ cells being cultured with rhTPO and double-labeled with FITC-conjugated CD41 and PI. *P < .05, **P < .01 vs control. SCF, stem cell factor. Shilei Chen et al. Blood 2016;127:1024-1035 ©2016 by American Society of Hematology

NE and EPI promote the adherence and migration of MKs NE and EPI promote the adherence and migration of MKs. The progeny of CD34+ cells cultured with rhTPO for 13 days were purified first and then used for the experiments. NE and EPI promote the adherence and migration of MKs. The progeny of CD34+ cells cultured with rhTPO for 13 days were purified first and then used for the experiments. (A-B) Adherent and migrated MKs treated with different concentrations of NE or EPI. (C-D) Adherence and migration of MKs pretreated with phentolamine (10 μM) or propranolol (10 μM) for 20 minutes before incubation with 1 μM NE or EPI. (E) Western blot analysis for ERK1/2 phosphorylation in MKs after exposure to NE (1 μM) or EPI (1 μM) for the indicated times. (F) ERK1/2 phosphorylation in MKs pretreated with different adrenergic antagonists for 20 minutes followed by NE (1 μM) or EPI (1 μM) treatment of 30 minutes. (G-H) The inhibitory effect of yohimbine, prazosin, and U0126 on the adherence and migration of MKs induced by NE (1 μM) or EPI (1 μM). *P < .05, **P < .01, vs control; #P < .05, ##P < .01, vs NE or EPI treatment group. Shilei Chen et al. Blood 2016;127:1024-1035 ©2016 by American Society of Hematology

NE and EPI facilitate PPF NE and EPI facilitate PPF. (A) Representative photographs of PPF after NE (1 μM) or EPI (1 μM) stimulation. NE and EPI facilitate PPF. (A) Representative photographs of PPF after NE (1 μM) or EPI (1 μM) stimulation. Cytoskeleton actin (green) and the nucleus (blue) were stained. (B-C) Quantification of proplatelet-forming MKs treated with NE or EPI at different concentrations under an inverted microscope. (D-E) Western blot analysis of the expression of the phospho-RhoA in MKs prestimulated with yohimbine, prazosin, or U0126 for 20 minutes followed by NE or EPI treatment of 30 minutes. (F) Morphology exhibition of MKs treated with 10 μM of the ROCK inhibitor Y27632 for 20 minutes before stimulation with NE or EPI. Cytoskeleton actin (green), tubulin (red), and the nucleus (blue) were stained. (G-H) The counts of proplatelet-forming MKs exposed to 10 μM prazosin, 10 μM yohimbine, or 10 μM U0126 prior to NE or EPI stimulation. *P < .05, **P < .01, vs control; #P < .05, ##P < .01, vs NE or EPI treatment group. Shilei Chen et al. Blood 2016;127:1024-1035 ©2016 by American Society of Hematology

NE and EPI promote platelet production and activation. NE and EPI promote platelet production and activation. (A) Flow cytometric analysis of platelet production from MKs induced by NE or EPI. CD34+ cells were cultured with rhTPO for 13 days and then treated with NE (1 μM) or EPI (1 μM) for 24 hours. (B) Mean percentages of culture-derived platelets induced by NE or EPI. (C-D) Western blot analysis of ERK1/2 phosphorylation in isolated platelets from the health donors treated with NE or EPI for different times. (E-F) Flow cytometry analysis of the time-course changes of in CD62p expression in isolated platelets induced by NE (10 μM) or EPI (10 μM). (G-H) CD62p expression in isolated platelets treated with yohimbine (10 μM) and U0126 (10 μM) before NE and EPI stimulation. (I) Schematic illustration of the role of sympathetic stress in regulating thrombopoiesis and its pathophysiological implications. The elevated NE and EPI levels induced by sympathetic stress stimulate thrombopoiesis and platelet activation via α2-adrenoceptor–mediated ERK1/2 signaling. As a result, the increased platelet counts and activation promote 2 different outcomes (hemostasis facilitation or thrombosis aggravation) under different pathophysiological conditions. *P < .05, **P < .01, vs control; #P < .05, ##P < .01, vs NE or EPI treatment group. Shilei Chen et al. Blood 2016;127:1024-1035 ©2016 by American Society of Hematology