Biphasic Effect of Thyroid Hormone on Megakaryopoiesis and Platelet Production

Abstract

Background:

Abnormal platelet counts are frequently observed in patients with thyroid dysfunction; however, the direct impact of thyroid hormones on thrombopoiesis remains largely undefined.

Methods:

This study elucidates the dose–response effect of the thyroid hormone triiodothyronine (T₃) on megakaryocyte (MK) development and thrombopoiesis using both a murine model of hyperthyroidism/hypothyroidism and in vitro cultures of human cord blood CD34⁺ cell-derived MKs. After the application of inhibitors to MKs, the examination of total and phosphorylated protein levels of the phosphoinositide 3-kinase (PI3K)/AKT pathway was utilized to assess the specific mechanisms of T₃ action. The use of autophagy dual-staining lentivirus and transmission electron microscopy was employed to evaluate the impact of T₃ on the autophagy flux in MKs. Mouse whole-body irradiation and bone marrow transplantation models are applied to assess the influence of T₃ on the recovery of MKs/platelets in vivo.

Results:

We found that physiological or slightly elevated thyroid hormone levels are essential for sustaining MK development and thrombopoiesis, primarily through the TRα-PI3K/AKT signaling pathway. In contrast, supraphysiological thyroid hormone concentrations induce MK apoptosis via excessive autophagy, thereby reducing platelet production.

Conclusions:

Here, we present evidence that the thyroid hormone influences MK development and platelet production in a concentration-dependent manner, exhibiting a dualistic role. Our discoveries shed new light on the intricate relationship between thyroid hormones and platelet formation, offering novel perspectives on the pathophysiological consequences of thyroid disorders.

Introduction

Platelets, released from megakaryocyte (MK) lysis, play essential roles in hemostasis and inflammation.^1
–3 The interplay between the endocrine system and megakaryopoiesis can effectively assist platelet production. The main cytokine promoting platelet production is thrombopoietin (TPO), which binds to receptors to activate downstream intracellular pathways. Meanwhile, several endocrine hormones can also support platelet homeostasis through complementary pathways,^4,5 which provides a protective mechanism of the organism and may also account for the fluctuations in platelet counts often associated with endocrine disorders. Thyroid hormone imbalance is a prevalent condition, encompassing hyperthyroidism and hypothyroidism.⁶ Thyroid hormone abnormalities may have a particular impact in patients with concomitant abnormalities in platelet counts or function by causing delayed development of hematopoietic cells and aberrant signal transduction.^7
–9

Thyroid hormone receptors TRα and TRβ are widely distributed, and their activation can trigger a series of signaling pathways that promote cell growth, proliferation, and energy metabolism regulation.¹⁰ The non-genomic effects of triiodothyronine (T₃) are highly regarded for their extensive influence, engaging with TRα and p85α, a regulatory subunit of phosphoinositide 3-kinase (PI3K).^11,12 This activation leads to increased cell proliferation. In contrast, thyroid hormone activation enhances lysosomal activity and induces autophagy under metabolic stress.^13,14 Therefore, excessive thyroid hormones can induce cell apoptosis or excessive autophagy, indicating their broad and dose-dependent effects. While the involvement of thyroid hormones in erythropoiesis is well-established,^15,16 their effects on MK development and thrombopoiesis are not fully known.

This study revealed that the thyroid hormone exerts a bidirectional effect on MK development and thrombopoiesis, directly related to its concentration. These findings elucidate the effects of thyroid hormone on thrombopoiesis and provide new insights into the physiopathological implications of thyroid dysfunction.

Methods

Mice and treatment

Mature (8–14 weeks) male wild-type C57BL/6J mice were used. The mice were maintained at a controlled temperature (22 ± 2°C) and a 12-h light/dark period, with ad libitum access to water and food. Animal experiments were approved by the Animal Care and Use Committee of the Army Medical University (No. AMUWEC20230409) and conducted according to the institutional guidelines.

MK induction and culture

MKs from mice and humans were both induced and cultured as previously reported.¹⁷ Briefly, human cord blood was used to separate CD34⁺ cells, followed by culturing in serum-free medium (StemSpan SFEM; STEMCELL) supplemented with 20 ng/mL recombinant human TPO (PeproTech) to induce human MKs. Mouse c-kit⁺ cells were sorted and seeded in the same medium in the presence of 50 ng/mL recombinant mouse TPO (PeproTech), 30 ng/mL recombinant mouse stem cell factor (PeproTech), and 10 ng/mL recombinant mouse interleukin 3 (PeproTech). A one-step BSA gradient was used to enrich mature MKs, which were then cultured for future use.

Flow cytometry assay

Bone marrow cells from femora were flushed by certification at 12,000 rpm for 30 seconds and homogenized. After lysis of red blood cells, cells were washed twice with PBS and then resuspended in PBS for further use. For analysis of MK differentiation, the MKs were stained with CD41-APC and CD42b-PE antibodies. For quantification of ploidy, the MKs were stained with CD41-APC antibody and then treated with PI/RNase Staining Buffer (BD Pharmingen) for fixation and staining. For apoptosis analysis, cells were labeled with annexin V and 7-AAD. Analysis was performed using flow cytometry (ID7000; Sony Biotechnology Inc., Japan) and data were analyzed by FlowJo software (Tree Star Inc., USA).

ELISA

Mouse pericardial blood was collected and left at room temperature for 30 minutes, followed by centrifugation at 400 g for 15 minutes to take the supernatant serum. The serum total T₃ (DNOV053, NovaTec Immundiagnostica GmbH, Germany), total T₄ (thyroxine; EIA-1781, DRG Diagnostics, Germany), thyrotropin (KE1295, Immunoway, USA), and TPO (EMTHPO, ThermoFisher Scientific Inc., USA) were quantified following the manufacturer’s instructions.

Statistical analysis

The results were presented as means ± standard deviation, and p < 0.05 indicates statistical significance. GraphPad Prism was used for analyses, applying Student’s t-test for two-group comparisons or one-way ANOVA with Tukey’s test for multiple groups.

Result

Thyroid hormone disorders result in low platelet counts in vivo

To investigate the T₃ effect on thrombopoiesis, mice were treated with T₃ (10 μg/100 g body weight) or propylthiouracil (PTU)/low iodine diet (LID) for 10 days at room temperature to induce models of hyperthyroidism and hypothyroidism (Fig. 1A, B).¹⁸ Lower platelet counts in the peripheral blood were observed in both hyperthyroidism and hypothyroidism mice compared with the control group (Fig. 1C, D). Platelet volume transiently increased 7 days after T₃ and PTU/LID treatment (Supplementary Fig. S1A, B).

FIG. 1.

Thyroid disorders are reflected as abnormal platelet counts. (A) A schematic shows the establishment of hyperthyroidism and hypothyroidism mouse models. (B) Serum levels of total T₃, total T₄, and TSH in PTU/LID or T₃-treated and control mice at day 10. ***p < 0.001, Student’s t-test, n = 6. (C–D) Peripheral platelets in C57BL/6J mice. Significance according to ***p < 0.001, **p < 0.01, *p < 0.05, two-tailed unpaired t-test, n = 8. (E–F) H&E staining for MK quantification in the BM from PTU/LID or T₃-treated and control mice. Eight high-power fields per sample were counted. The black arrow indicates MKs. Scale bar: 10 μm. ***p < 0.001, Student’s t-test, n = 8. (G) The CD41⁺CD42b⁺ MK percentage in the BM from PTU/LID or T₃-treated mice was compared with the control group using flow cytometry. *p < 0.05, ***p < 0.001, one-way ANOVA, n = 6. (H) Detection of plasma TPO level by using enzyme-linked immunosorbent assay. ns, not significant, Student’s t-test, n = 12. T₃, triiodothyronine; T₄, thyroxine; H&E, hematoxylin and eosin; MK, megakaryocyte; TSH, thyrotropin; PTU, propylthiouracil; LID, low iodine diet; TPO, thrombopoietin.

Further study of bone marrow sections from these model mice revealed a significant reduction in mature MKs (Fig. 1E, F). Flow cytometry analysis was utilized to determine the growth of bone marrow MK. The percentage and quantity of bone marrow MK progenitors (MKPs) in the hypothyroidism model mice declined slightly. However, hyperthyroidism did not affect MKP levels (Supplementary Fig. S1C, D). Without effect of plasma TPO levels, mature MK proportions and quantities in the bone marrow were lower in hyperthyroidism and hypothyroidism model mice compared with the control group (Fig. 1G, H). Polyploid MKs (with DNA content >4 N) are recognized as mature forms, exhibiting an enhanced capacity for platelet production. The hypothyroidism state decreased the proportion and quantity of >4 N polyploids (Supplementary Fig. S1E). These results suggest that thyroid hormone imbalances suppress MK in the bone marrow, reducing platelet counts in peripheral blood.

Biphasic dose-effect of thyroid hormone on thrombopoiesis

To assess the T₃ effect on MKs, CD34⁺ cells sourced from human peripheral blood were cultured across a range of T₃ concentrations (0–1000 nM). The expression of megakaryocytic lineage markers CD41, CD42b, and CD61 was quantified using flow cytometry. T₃ at low concentrations (≤1 nM) enhanced MK proliferation, whereas higher concentrations (10–100 nM) suppressed MK generation (Fig. 2A). The proportions of MKs expressing CD41, CD42b, and CD61 were measured on day 12 for groups treated with 0.1 and 10 nM T₃. The CD42b⁺ and CD61⁺ MK percentages were significantly higher in the 0.1 nM T₃ group compared with the control group, but were reduced in the 10 nM T₃ group (Figs. 2B–D). Moreover, mature MKs, characterized by CD41 and CD42b co-expression, exhibited semiresponses to 0.5 and 50 nM T₃ treatments (Fig. 2E). The polyploidy distribution analysis revealed that 0.5 nM T₃ increased the proportion of MKs with a ploidy of >4 N, whereas 50 nM T₃ reduced the proportion of >4 N MKs (Fig. 2F). Note that 0.5 nM T₃ significantly increased the production of cultured platelets compared with both the control group and the 50 nM T₃ group (Fig. 2G). There were no variations in MKP percentages between the control group, the group treated with 0.5 nM T₃, and the group treated with 50 nM T₃ (Supplementary Fig. S2A). The data suggest that T₃ has a dual influence on MK differentiation and maturation.

FIG. 2.

The effect of thyroid hormone on MK differentiation and maturation in vitro. (A) Human peripheral blood-derived CD34⁺ cells were cultured with different concentrations of T₃ (0, 0.1, 1, 5, 10, 100, and 1000 nM) for 12 days. The expression of CD41, CD42b, and CD61 was analyzed by flow cytometry. (B–D) The CD41⁺, CD42b⁺, and CD61⁺ MK proportions of the T₃ 0.1 nM, T₃ 10 nM, and control groups at day 12. *p < 0.05, **p < 0.01, ns, not significant, Student’ t-test, n = 3. (E) The CD41⁺CD42b⁺ MK percentage in the T₃ 0.5 nM and T₃ 50 nM group compared with the control group using flow cytometry. *p < 0.05, ***p < 0.001, Student’s t-test, n = 5. (F) Polyploid MK distribution at day 10 in the T₃ 0.5 nM, T₃ 50 nM, and control groups according to flow cytometric analysis. **p < 0.01, ***p < 0.001, Student’s t-test, n = 6. (G) Flow cytometric analysis of platelet production from MKs induced by T₃ 0.5 nM, T₃ 50 nM, and the control group. ***p < 0.001, Student’ t-test, n = 5.

To evaluate the T₃ toxicity, the MK cell viability was observed at different time points in all treatment groups. After 12-day incubation, the number of MK cells in the control group increased from 1 × 10⁵ to 1.98 × 10⁶. MK counts were comparable among all groups during the initial 3–7 days of culture (Supplementary Fig. S2B). By day 12, the MK count in the 0.5 nM T₃ group significantly exceeded that of the control group, while the 50 nM T₃ group showed similar levels to the control group (Supplementary Fig. S2B). The percentage of CD61⁺ Ki-67⁺ MKs showed no significant variation between the control, 0.5 nM T₃, and 50 nM T₃ groups on both day 7 and day 10 (Supplementary Fig. S2C). These findings suggest that the suppressive effect of 50 nM T₃ on MKs is not due to toxicity.

Thyroid hormone promotes THRA-PI3K axis connection in MKs

To determine whether genes associated with thyroid hormones change expression at critical phases during MK development, the expression of major thyroid hormone transporters¹⁹ (LAT1, LAT2, MCT8, MCT10, and SLCO1C1) and thyroid hormone metabolism enzymes²⁰ iodothyronine deiodinase type 1 (DIO1), DIO2, and DIO3 in MKs at 7 days (development phase),²¹ 10 days (maturation phase), and 14 days (platelet-producting phase), were analyzed (Fig. 3A, B; Supplementary Fig. S3A, B). Results indicate a notable upregulation in the expression of transporter genes, particularly during the maturation of MKs. Meanwhile, there is an elevation in the expression and activity of DIOs at maturation and terminal platelet-producting phase (Fig. 3C–E).

FIG. 3.

MKs have a basis for responding to thyroid hormone stimulation. (A, B) Quantitative polymerase chain reaction (qPCR) analysis of thyroid hormone transporters gene (LAT1, LAT2, MCT10, MCT8, and SLCO1C1) and metabolism enzymes (DIO1, DIO2, and DIO3) expression during MKs development. *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA, n = 5. (C–E) Activity assay of DIO1, DIO2, and DIO3 during MKs development. *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA, n = 5. (F) qPCR analysis of thyroid hormone receptor genes in MKs from human CD34⁺ cell and mouse c-kit⁺ cell differentiation. ***p < 0.001, Student’s t-test, n = 5. (G) DNA agarose gel electrophoresis analysis of thyroid hormone receptor genes in MKs from human CD34⁺ cell and mouse c-kit⁺ cell differentiation. ***p < 0.001, Student’s t-test, n = 5. (H) Representative confocal images of MKs in different groups. MKs were stained with TRα (red), phosphoinositide 3-kinase (PI3K)-p85α (green), and DAPI (blue). TRα (red) mean pixel intensity (MPI) in PI3K-p85α (green) mask of T₃-stimulated MKs. ***p < 0.001, one-way ANOVA, n = 10.

In humans, the genes THRA and THRB encode three distinct thyroid hormone-binding receptor isoforms: α1, β1, and β2.²² PCR data show that THRA (Thra) expression is significantly higher than THRB (Thrb), consistent with predictions from the GEXC database (Fig. 3F, G; Supplementary Fig. S3C, D). These indicated the necessity and significance of T₃ in MK development.

Thyroid hormone can activate the PI3K/AKT pathway,²³ crucial for MK terminal differentiation and platelet production.^24,25 TRα, when bound to T₃, physically interacts with PI3K-p85α, promoting its phosphorylation and activating downstream pathways.¹¹ MKs were stimulated with 0.5 and 50 nM T₃ for 24 hours, and the co-localization of TRα and PI3K-p85α was observed through immunofluorescence staining. Results showed that 0.5 nM T₃ stimulation increased the co-localization of TRα and PI3K-p85α compared with the control and 50 nM groups (Fig. 3H). The results indicate that TRα is the primary thyroid hormone receptor in MK/platelet and that optimal T₃ levels improve TRα-PI3K interactions in MKs.

A low concentration of thyroid hormone facilitates thrombopoiesis through the PI3K/AKT-CDC42-cofilin axis

We then focused on how T₃ activates the PI3K/AKT signaling pathway, crucial for MK growth and development. MKs were exposed to several doses (0–1000 nM) of T₃ to evaluate the activation of the PI3K/AKT pathway. Results indicated that T₃ within the concentration range of 0.1–1 nM significantly increased the expression of phosphorylated PI3K (pPI3K) and phosphorylated AKT (pAKT). In contrast, concentrations above 10 nM inhibited the activation of the PI3K/AKT pathway (Fig. 4A–C, Supplementary Fig. S4A). Furthermore, MKs were treated with various combinations of TRα inhibitors, PI3K inhibitors, and AKT inhibitors. The findings revealed that the TRα inhibitor (SR35021) and the PI3K inhibitor (LY294002) blocked the expression of pPI3K and pAKT induced by 0.5 nM T₃. The AKT inhibitor (MK2206) only blocked the expression of pAKT (Fig. 4D–F). This indicates that T₃ promotes MK development through the TRα-PI3K/AKT pathway.

FIG. 4.

The role of PI3K/AKT in thyroid hormone promotion of thrombopoiesis. (A–C) Western blot analysis of PI3K, Akt, and ACTB levels in lysates from MKs cultured with different concentrations of T₃ (0, 0.1, 1, 10, 100, and 1000 nM). The values of p-PI3K/PI3K and p-Akt/Akt were reflected in the bar graph. p, phosphorylated; AU, arbitrary units. (D–F) Western blot analysis of PI3K, Akt, and ACTB levels in lysates from MKs cultured with 0.5 nM T₃ and inhibitors (SR35021, LY294002, and MK2206). The values of p-PI3K/PI3K and p-Akt/Akt were reflected in the bar graph. (G–I) Western blot analysis of Rac1/Cdc42, myosin light chain 2 (MLC2), cofilin, and ACTB levels in lysates from MKs cultured with 0.5 nM T₃ and inhibitors (SR35021, LY294002, and EHOP-016). The values of p-MLC2 and p-cofilin were reflected in the bar graph. (J) Typical PPF from MKs captured by confocal microscope in T₃ 0.5 nM, T₃ 50 nM, and the control group. MKs were stained with actin (red), β-tubulin (green), and DAPI (blue). Scale bar, 15 μm. MKs of proplatelet formation in the unit field were quantified. *p < 0.05, ***p < 0.001, one-way ANOVA, n = 10.

Proplatelet formation and shedding depend on the coordinated cytoskeletal rearrangement of tubulin and actin for mechanical force.²⁶ The small GTPases of the Rho family, such as Cdc42 and Rac1, along with their effector proteins, control the dynamics of the cytoskeleton.²⁷ This signaling pathway regulates key proteins such as cofilin, an actin-depolymerizing factor, and myosin light chain 2, which modulate the actin cytoskeleton.^28,29 Western blot analysis showed that the TRα inhibitor (SR35021) and the PI3K inhibitor (LY294002) both effectively blocked the activation of the Cdc42/Rac1-cofilin signaling pathway induced by 0.5 nM T₃, similar to the inhibition of Rac1 (EHOP-016) (Fig. 4G–1). Confocal pictures of MKs generating proplatelets showed that, regarding platelet precursor formation, MKs treated with 0.5 nM T₃ exceeded those treated with 50 nM and the control group (Fig. 4J). These results suggest that T₃ when bound to TRα, promotes MK development and platelet precursor formation through the PI3K/AKT-CDC42-cofilin signaling axis.

A high concentration of thyroid hormone induces autophagy through the AMPK-ULK1-mTOR pathway

Studies suggest that thyroid hormones can stimulate autophagy in cells, and excessive autophagy could impede cell growth and trigger cell death.^13,30 To determine the effect of T₃ on autophagy in MKs, MKs from hyperthyroid mice treated with T₃ at 10 μg/100 g body weight for 10 days were analyzed. Elevated levels of lipidated MAP1LC3B/LC3B (MAP1LC3B-II) and decreased SQSTM1/p62 were detected, indicating enhanced autophagic flux (Fig. 5A, B). Consistently, electron microscopy revealed an increase in autophagic vesicles in these MKs (Fig. 5C).

FIG. 5.

Excessive thyroid hormones cause MK autophagy. (A, B) Increased autophagic flux in MKs from hyperthyroid mice. Immunoblots and densitometry showing MAP1LC3B-II and SQSTM1/p62 levels in MKs of hyperthyroid mice. **p < 0.01, ***p < 0.001, Student’s t-test, n = 3. (C) Electron microscopy images showing increased autophagic vesicles in hyperthyroid MKs. Scale bar: 10 μm. (D) Western blot analysis of AMPK, mTOR, and ULK1 levels in lysates from MKs cultured with 50 nM T₃ and control MKs. The values of p-AMPK/AMPK, p-mTOR/mTOR, and p-ULK1/ULK1 were reflected in the bar graph. p, phosphorylated; AU, arbitrary units. **p < 0.01, one-way ANOVA, n = 3. (E) MKs were pretreated with SBI (10 µM) for 1 hour, followed by exposure to T₃ (50 nM) for an additional 24 hours. MKs were collected and lysed, and then Western blot analysis was performed. The bar graph shows the quantification of the indicated proteins. (F) MKs were pretreated with rapamycin (RAPA) (20 nM) for 1 hour, followed by exposure to T₃ (50 nM) for an additional 24 hours. MKs were collected and lysed, and then Western blot analysis was performed. The bar graph shows the quantification of the indicated proteins.

Previous studies have established AMPK as a pivotal regulator of autophagy.^31
–33 High T₃ concentrations induced AMPK and ULK1 phosphorylation while reducing mTOR phosphorylation in MKs (Fig. 5D). AMPK inhibition reversed the T₃-triggered increase in pULK1 levels; however, it did not significantly alter T₃-induced pmTOR expression in MKs (Supplementary Fig. S4B). Furthermore, ULK1 inhibition by SBI-0206965 (SBI) significantly controlled T₃-induced autophagy (Fig. 5E). Similarly, T₃ reduced the levels of pmTOR, and the mTOR inhibitor rapamycin significantly influenced T₃-induced LC3B conversion and p62 degradation, suggesting that T₃-induced autophagy activation is mTOR-dependent in MKs (Fig. 5F). This effect likely originates from the high concentration of T₃ inhibiting the PI3K/AKT pathway. Thus, the AMPK-ULK1-mTOR pathway is essential for T₃-induced autophagy and MKs death.

Excessive autophagy caused by high concentrations of thyroid hormone resulting in MK apoptosis

T₃ elevated MAP1LC3B-II levels in MKs in a time-dependent manner (Fig. 6A–C). Bafilomycin A1 (Baf), an inhibitor of vacuolar H⁺ATPase/lysosomal acidification, caused MAP1LC3B-II accumulation in MKs. Adding Baf to T₃ treatment increased MAP1LC3B-II levels more than T₃ treatment alone, indicating that T₃ enhances autophagosome synthesis and improves autophagic flux (Fig. 6D–F).

FIG. 6.

Thyroid hormone-mediated MK death in an autophagy-dependent manner. (A–C) Time course of MAP1LC3B-II and p62 induction in MKs treated with 50 nM T₃ for 0, 3, 6, and 24 hours. The bar graph shows the quantification of the indicated proteins. **p < 0.01, ***p < 0.001, one-way ANOVA, n = 3. (D–F) Autophagic flux analysis showing accumulation of MAP1LC3B-II following autophagy inhibition. MKs were treated with 50 nM T₃ for 24 hours. MKs were treated with 50 nM Baf to block autophagosome clearance 6 hours before harvest. The bar graph shows the quantification of the indicated proteins. **p < 0.01, ***p < 0.001, one-way ANOVA, n = 3. (G) Confocal images showing MK cell line Meg-01 transfected with mCherry-EGFP-MAP1LC3B lentivirus and treated with 50 nM T₃ for 24 hours. The yellow color represents MAP1LC3B-II on autophagosomes. The red color represents MAP1LC3B-II on lysosomes. Scale bar: 20 μm. Quantitative analysis of the mCherry (red) fluorescence relative to total (yellow) signal. ImageJ software was used to quantify images (at least 10 transfected cells per sample in 3 different fields). (H) Representative flow cytometry of MKs treated with 50 nM T₃ and chloroquine (CQ). CD41⁺ MKs were gated and analyzed for ANXA5/annexin-V surface expression and 7-AAD staining. *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA, n = 6. (I, J) MKs were pretreated with 3-methyladenine (3-MA) (5 mM) and CQ (5 µM) for 1 hour, followed by treatment with T₃ (50 nM) for another 24 hours. (I) Cell viability was measured by a CCK-8 detection kit and (J) cell death was detected with PI staining by flow cytometry. ***p < 0.001, one-way ANOVA, n = 3. (K) Western blot analysis of BAX, BCL2, and BCL2L1 protein levels in MKs from wild-type (WT), hyperthyroid, and hyperthyroid + CQ mice. ACTB was used as a control.

In order to construct an autophagy-monitoring MKs, we transfected the mature MK cell line derived from humans, Meg-01 (human megakaryoblastic leukemia cells), with an adenovirus carrying tandem fluorescent MAP1LC3B (mCherry-eGFP-MAP1LC3B) plasmid. This construct encodes MAP1LC3B with both green and red fluorescent tags. The strength of the red signal corresponds to the rate at which autophagy occurs. Confocal microscopy analysis of T₃-treated MKs showed an augmentation in the number of red puncta and a greater red-to-yellow signal ratio (Fig. 6G), indicating an enhanced autophagic flux.

The crosstalk between autophagy triggered by higher T₃ concentrations and MK apoptosis was investigated. MKs were treated with 50 nM T₃, and a control group with autophagy inhibited by chloroquine (CQ) was established. The 50 nM T₃ treatment caused a significant increase in the number of early and late apoptotic MKs compared with the control group. However, inhibition of autophagy by CQ effectively alleviated T₃-induced MK apoptosis (Fig. 6H). To investigate the role of autophagy in MK cell death caused by 50 nM T₃, 3-methyladenine (3-MA) and CQ were employed, which block the initial and final phases of autophagy. Figure 6I demonstrates that administering 3-MA and CQ improved the survival of MK cells exposed to 50 nM T₃. Moreover, 3-MA and CQ had no significant effects on MK viability (Fig. 6J). In vivo experiments also confirmed the protective effect of autophagy inhibition on MK apoptosis in hyperthyroidism (Fig. 6K). These results indicate that autophagy contributes to the cytotoxicity of high concentrations of T₃ in MKs.

Influence of thyroid hormone on MK reconstitution and platelet counts in mice radiation thrombocytopenia

The decrease in platelet count and the difficulty in MK reconstitution are significant characteristics of radiation-induced bone marrow injury.^17,34 The radiation model also examines the parameters that influence the development of MK and thrombopoiesis. The mice were subjected to whole-body γ-ray irradiation at a dose of 6.5 Gy and then underwent bone marrow transplantation. The mice were administered different T₃ concentrations daily from 5 to 10 days after the transplant (Fig. 7A). The low-concentration group exhibited improved restoration of white blood cells, hemoglobin, and platelet counts compared with the high-concentration and control groups (Fig. 7B–D).

FIG. 7.

Thyroid hormone significantly influences platelet reconstitution in mice after BMT in vivo. (A) Schematic of T₃ administration and peripheral blood monitoring after BMT in irradiated mice. (B–D) WBC, HGB, and PLT reconstruction after syngeneic BMT from mice treated with different doses of T₃ (control: 0 mg/mouse; lo-T₃: 0.5 mg/mouse; hi-T₃: 5 mg/mouse) (3–5 mice per group). (E) Representative images of H&E staining of bone marrow sections from the control, lo-T₃, and hi-T₃ group mice are shown. The black arrows indicate MKs. Scale bar: 10 μm. (F) The CD41⁺ cell percentage in the BM from the lo-T₃ and hi-T₃ group mice compared with the control group using flow cytometry. *p < 0.05, ***p < 0.001, one-way ANOVA, n = 5. (G) Percentage of reticulated platelets (thiazole orange [TO]⁺) in the control, lo-T₃, and hi-T₃ group mice. **p < 0.01, ***p < 0.001, ns, not significant, one-way ANOVA, n = 10. (H) A schematic shows the biphasic effect of thyroid hormone on megakaryopoiesis and platelet production.

Further analysis focused on MKs and platelet recovery. In bone marrow sections, the number and proportion of mature MKs per unit area in the lo-T₃ group were higher than in the hi-T₃ and control groups (Fig. 7E). Flow cytometry results also confirmed the pro-megakaryopoiesis effect of low thyroid hormone concentrations (Fig. 7F). Furthermore, peripheral platelets in the lo-T₃ group exhibited a higher thiazole orange staining rate, indicating a higher number of newly formed platelets (Fig. 7G). Low thyroid hormone concentrations have been shown to help bone marrow MKs recover and may even be utilized to treat radiation-induced thrombocytopenia.

Discussion

Investigating thyroid hormone effects on platelet production is important for understanding endocrine failures and their effects on hematological diseases and body system interactions.³⁵ In this study, a commonly used mouse model of hyperthyroidism and hypothyroidism was established through in vitro administration of thyroid hormone or PTU. Hemogram and bone marrow analyses revealed that abnormal thyroid hormone levels in vivo significantly reduce platelet production and counts. This indicates that the effects of thyroid hormone on MK formation and thrombopoiesis are dose-dependent, with high and low thyroid hormone levels being harmful. This finding is consistent with the aberrant platelet counts found in hypothyroidism and hyperthyroidism patients, as well as research on hematopoietic suppression in hyperthyroidism patients.^7,36

Studies have shown that TRα can interact with PI3K-p85α and promote the phosphorylation of PI3K and the activation of subsequent signaling pathways.²³ The activation of the PI3K/AKT pathway occurred following stimulation with an appropriate dose of thyroid hormone, consistent with previous reports. The PI3K/AKT pathway is involved in MK proliferation and pre-platelet formation.²⁵ Multiple pathway inhibitors further established the role of the TRα-PI3K/AKT-CDC42/cofilin axis in promoting thrombopoiesis (Fig. 7H). Thyroid hormones affect cellular energy regulation and promote autophagy.^14,37 The AMPK/ULK1/mTOR pathway was examined to know how thyroid hormones trigger autophagy. This pathway is consistent with the thyroid hormones’ previously documented role in regulating cellular energy. These findings showed that stimulated cells had significant upregulation of the AMPK/ULK1/mTOR pathway, with reduced mTOR phosphorylation due to high thyroid hormone suppressing the PI3K-AKT pathway (Fig. 7H).³⁸

The dose–response effect of thyroid hormones deserves further attention. According to our clinical data, the normal total T₃ (tT₃) levels are in the range of 0.52–3.40 nM, while hyperthyroid patients have an average serum tT₃ of 10.31 ± 3.312 nM, with the range in hypothyroid patients being 0.37 ± 0.947 nM. Published clinical studies show that patients with thyrotoxicosis have an average serum tT₃ level of 8.20 ± 3.415 nM, while the level in those with hypothyroidism is 0.42 ± 0.984 nM.^39

–42 In vitro studies on mammalian cells typically use T₃ doses in the 0–100 nM range.^43

–46 Nevertheless, we acknowledge that the 50 nM T₃ concentration used in some of our in vitro experiments is much higher than the physiological levels, and the inhibitory effects observed at such high concentrations may also have questionable clinical relevance, as such high T₃ concentrations are rare in clinical practice.

This study highlights thyroid hormone’s important role in platelet production and hematopoietic system maintenance, providing further evidence of endocrine–hematopoietic system crosstalk. This study provides insights in understanding blood abnormalities in thyroid patients and may have potential future treatment implications.

Footnotes

Acknowledgment

The authors would like to thank MJEditor () for its linguistic assistance during the preparation of this article.

Authors’ Contributions

J.W., B.X., and X.Y. designed and performed the experiments. B.X. and X.Y. performed electron microscopy on megakaryocytes. S.C. provided guidance on the CD34⁺ cell differentiation. J.W. and B.X. formulated the research idea and designed the study. J.W. supervised the study. B.X., X.Y., Z.W., J.C., Y.X., M.C., and M.S. analyzed the data and drafted the first version of the article, which was read and commented on by all authors. There were no AI tools used during the writing of the article.

Data Sharing Statement

Reasonable requests to view the original data will be considered on receipt of an email by Junping Wang (wangjunping@tmmu.edu.cn) or Shilei Chen (chen.shilei@foxmail.com).

Author Disclosure Statement

The authors declare no competing interests.

Funding Information

This work was supported by the National Natural Science Foundation of China (No. 82222060, 82430103, 82473572, 82073487, 81930090), Science Foundation of State Key Laboratory of Trauma and Chemical Poisoning (2024K004), and High-level Talent Program of Third Military Medical University (2022XRC02).

Supplementary Material

Supplementary Data

Supplementary Figure S1

Supplementary Figure S2

Supplementary Figure S3

Supplementary Figure S4

Supplementary Figure S5

References

J-J

, Liu

, Li

, et al. Differentiation route determines the functional outputs of adult megakaryopoiesis. Immunity, 2024; 57(3):478–494.e6; doi: 10.1016/j.immuni.2024.02.006

Koupenova

, Clancy

, Corkrey

, et al. Circulating platelets as mediators of immunity, inflammation, and thrombosis. Circ Res, 2018; 122(2):337–351; doi: 10.1161/CIRCRESAHA.117.310795

Poscablo

, Worthington

, Smith-Berdan

, et al. An age-progressive platelet differentiation path from hematopoietic stem cells causes exacerbated thrombosis. Cell, 2024; 187(12):3090–3107.e21; doi: 10.1016/j.cell.2024.04.018

Chen

, Du

, Shen

, et al. Sympathetic stimulation facilitates thrombopoiesis by promoting megakaryocyte adhesion, migration, and proplatelet formation. Blood, 2016; 127(8):1024–1035; doi: 10.1182/blood-2015-07-660746

, Xu

, Yang

, et al. Estrogen promotes megakaryocyte polyploidization via estrogen receptor beta-mediated transcription of GATA1. Leukemia, 2017; 31(4):945–956; doi: 10.1038/leu.2016.285

Taylor

, Albrecht

, Scholz

, et al. Global epidemiology of hyperthyroidism and hypothyroidism. Nat Rev Endocrinol, 2018; 14(5):301–316; doi: 10.1038/nrendo.2018.18

Cao

Y-T

, Zhang

K-Y

, Sun

, et al. Platelet abnormalities in autoimmune thyroid diseases: A systematic review and meta-analysis. Front Immunol, 2022; 13:1089469; doi: 10.3389/fimmu.2022.1089469

Sullivan

, McDonald

. Thyroxine suppresses thrombocytopoiesis and stimulates erythropoiesis in mice. Proc Soc Exp Biol Med, 1992; 201(3):271–277; doi: 10.3181/00379727-201-43507

Ioachimescu

, Makdissi

, Lichtin

, et al. Thyroid disease in patients with idiopathic thrombocytopenia: A Cohort Study. Thyroid, 2007; 17(11):1137–1142; doi: 10.1089/thy.2007.0066

10.

Mullur

, Liu

Y-Y

, Brent

. Thyroid hormone regulation of metabolism. Physiol Rev, 2014; 94(2):355–382; doi: 10.1152/physrev.00030.2013

11.

Hiroi

, Kim

H-H

, Ying

, et al. Rapid nongenomic actions of thyroid hormone. Proc Natl Acad Sci U S A, 2006; 103(38):14104–14109; doi: 10.1073/pnas.0601600103

12.

Cao

, Kambe

, Yamauchi

, et al. Thyroid-hormone-dependent activation of the phosphoinositide 3-kinase/Akt cascade requires Src and enhances neuronal survival. Biochem J, 2009; 424(2):201–209; doi: 10.1042/BJ20090643

13.

Liu

, Shoji-Kawata

, Sumpter

, et al. Autosis is a Na+,K+-ATPase-regulated form of cell death triggered by autophagy-inducing peptides, starvation, and hypoxia-ischemia. Proc Natl Acad Sci U S A, 2013; 110(51):20364–20371; doi: 10.1073/pnas.1319661110

14.

Yau

, Singh

, Lesmana

, et al. Thyroid hormone (T ₃) stimulates brown adipose tissue activation via mitochondrial biogenesis and MTOR-mediated mitophagy. Autophagy, 2019; 15(1):131–150; doi: 10.1080/15548627.2018.1511263

15.

Park

, Han

, Park

, et al. Defective erythropoiesis caused by mutations of the thyroid hormone receptor α gene. Grimes HL. ed. PLoS Genet, 2017; 13(9):e1006991; doi: 10.1371/journal.pgen.1006991

16.

Colonnello

, Criniti

, Lorusso

, et al. Thyroid hormones and platelet activation in COVID-19 patients. J Endocrinol Invest, 2023; 46(2):261–269; doi: 10.1007/s40618-022-01896-2

17.

, Wang

, Shen

, et al. hGH promotes megakaryocyte differentiation and exerts a complementary effect with c-Mpl ligands on thrombopoiesis. Blood, 2014; 123(14):2250–2260; doi: 10.1182/blood-2013-09-525402

18.

Park

, Zhu

, Kim

, et al. Thyroid hormone receptor α1 mutants impair B Lymphocyte development in a mouse model. Thyroid, 2021; 31(6):994–1002; doi: 10.1089/thy.2019.0782

19.

Groeneweg

, van Geest

, Peeters

, et al. Thyroid hormone transporters. Endocr Rev, 2020; 41(2):bnz008; doi: 10.1210/endrev/bnz008

20.

Luongo

, Dentice

, Salvatore

. Deiodinases and their intricate role in thyroid hormone homeostasis. Nat Rev Endocrinol, 2019; 15(8):479–488; doi: 10.1038/s41574-019-0218-2

21.

Chen

, Qi

, Wang

, et al. Melatonin enhances thrombopoiesis through ERK1/2 and Akt activation orchestrated by dual adaptor for phosphotyrosine and 3-phosphoinositides. J Pineal Res, 2020; 68(3):e12637; doi: 10.1111/jpi.12637

22.

Ortiga-Carvalho

, Sidhaye

, Wondisford

. Thyroid hormone receptors and resistance to thyroid hormone disorders. Nat Rev Endocrinol, 2014; 10(10):582–591; doi: 10.1038/nrendo.2014.143

23.

Carrillo-Sepúlveda

, Ceravolo

, Fortes

, et al. Thyroid hormone stimulates NO production via activation of the PI3K/Akt pathway in vascular myocytes. Cardiovasc Res, 2010; 85(3):560–570; doi: 10.1093/cvr/cvp304

24.

Chen

, Hu

, Shen

, et al. IGF-1 facilitates thrombopoiesis primarily through Akt activation. Blood, 2018; 132(2):210–222; doi: 10.1182/blood-2018-01-825927

25.

Chen

, Sun

, Xu

, et al. Akt-mediated mitochondrial metabolism regulates proplatelet formation and platelet shedding post vasopressin exposure. J Thromb Haemost, 2023; 21(2):344–358; doi: 10.1016/j.jtha.2022.11.018

26.

Bornert

, Boscher

, Pertuy

, et al. Cytoskeletal-based mechanisms differently regulate in vivo and in vitro proplatelet formation. Haematologica, 2021; 106(5):1368–1380; doi: 10.3324/haematol.2019.239111

27.

Chang

, Auradé

, Larbret

, et al. Proplatelet formation is regulated by the Rho/ROCK pathway. Blood, 2007; 109(10):4229–4236; doi: 10.1182/blood-2006-04-020024

28.

Gilles

, Bluteau

, Boukour

, et al. MAL/SRF complex is involved in platelet formation and megakaryocyte migration by regulating MYL9 (MLC2) and MMP9. Blood, 2009; 114(19):4221–4232; doi: 10.1182/blood-2009-03-209932

29.

Bender

, Eckly

, Hartwig

, et al. ADF/n-cofilin-dependent actin turnover determines platelet formation and sizing. Blood, 2010; 116(10):1767–1775; doi: 10.1182/blood-2010-03-274340

30.

Sinha

, You

S-H

, Zhou

, et al. Thyroid hormone stimulates hepatic lipid catabolism via activation of autophagy. J Clin Invest, 2012; 122(7):2428–2438; doi: 10.1172/JCI60580

31.

Zhang

, Xie

, Yan

, et al. Negative pressure wound therapy improves bone regeneration by promoting osteogenic differentiation via the AMPK-ULK1-autophagy axis. Autophagy, 2022; 18(9):2229–2245; doi: 10.1080/15548627.2021.2016231

32.

, Chen

. AMPK and autophagy. Adv Exp Med Biol, 2019; 1206:85–108; doi: 10.1007/978-981-15-0602-4_4

33.

Hardie

. Hot stuff: Thyroid hormones and AMPK. Cell Res, 2010; 20(12):1282–1284; doi: 10.1038/cr.2010.153

34.

C-H

, Wu

Y-D

, Yang

, et al. Apoptosis-resistant megakaryocytes produce large and hyperreactive platelets in response to radiation injury. Mil Med Res, 2023; 10(1):66; doi: 10.1186/s40779-023-00499-z

35.

Gancz

, Gilboa

. Hormonal control of stem cell systems. Annu Rev Cell Dev Biol, 2013; 29:137–162; doi: 10.1146/annurev-cellbio-101512-122331

36.

Weetman

. Immune reconstitution syndrome and the thyroid. Best Pract Res Clin Endocrinol Metab, 2009; 23(6):693–702; doi: 10.1016/j.beem.2009.07.003

37.

Zhou

, Gauthier

, Ho

, et al. Thyroid hormone receptor α regulates autophagy, mitochondrial biogenesis, and fatty acid use in skeletal muscle. Endocrinology, 2021; 162(8):bqab112; doi: 10.1210/endocr/bqab112

38.

, Wei

, Liu

. Attacking the PI3K/Akt/mTOR signaling pathway for targeted therapeutic treatment in human cancer. Semin Cancer Biol, 2022; 85:69–94; doi: 10.1016/j.semcancer.2021.06.019

39.

Balkrishna

, Paliwal

, Maity

, et al. Thyrogrit, supplemented with a sub-optimal dose of levothyroxine, restores thyroid function in rat model of propylthiouracil-induced hypothyroidism. Clin Phytosci, 2024; 10(1):8; doi: 10.1186/s40816-024-00371-0

40.

, Yuan

, Liu

, et al. Clinical evaluation of various thyroid hormones on thyroid function. Int J Endocrinol, 2014; 2014:618572; doi: 10.1155/2014/618572

41.

Mehran

, Amouzegar

, Foroutan

, et al. Pharmacodynamic and pharmacokinetic properties of the combined preparation of levothyroxine plus sustained- release liothyronine; a randomized controlled clinical trial. BMC Endocr Disord, 2023; 23(1):182; doi: 10.1186/s12902-023-01434-y

42.

Kim

, Joung

, Kang

, et al. A modest protective effect of thyrotropin against bone loss is associated with plasma triiodothyronine levels. PLoS One, 2015; 10(12):e0145292; doi: 10.1371/journal.pone.0145292

43.

Gnoni

, Rochira

, Leone

, et al. 3,5,3′triiodo-L-thyronine induces SREBP-1 expression by non-genomic actions in human HEP G2 cells. J Cell Physiol, 2012; 227(6):2388–2397; doi: 10.1002/jcp.22974

44.

Vicinanza

, Coppotelli

, Malacrino

, et al. Oxidized low-density lipoproteins impair endothelial function by inhibiting non-genomic action of thyroid hormone-mediated nitric oxide production in human endothelial cells. Thyroid, 2013; 23(2):231–238; doi: 10.1089/thy.2011.0524

45.

de Oliveira

, Rodrigues

, Olimpio

RMC

, et al. Adiponectin and Serine/Threonine Kinase Akt Modulation by Triiodothyronine and/or LY294002 in 3T3-L1 Adipocytes. Lipids, 2019; 54(2–3):133–140; doi: 10.1002/lipd.12135

46.

Martinez de Mena

, Scanlan

, Obregon

M-J

. The T3 receptor beta1 isoform regulates UCP1 and D2 deiodinase in rat brown adipocytes. Endocrinology, 2010; 151(10):5074–5083; doi: 10.1210/en.2010-0533