Research ArticleHematology Open Access | 10.1172/JCI189801

Chengjie Gao,1 Yitian Dai,2 Paul A. Spezza,2 Paul Boasiako,2 Alice Tang,2 Giselle Rasquinha,2 Hui Zhong,3 Bojing Shao,4 Yunfeng Liu,5 Patricia A. Shi,6 Cheryl A. Lobo,7 Xiuli An,1 Anqi Guo,5 William B. Mitchell,8 Deepa Manwani,8 Karina Yazdanbakhsh,5 and Avital Mendelson2

1Laboratory of Membrane Biology,

2Laboratory of Stem Cell Biology and Engineering Research,

3Laboratory of Immune Regulation,

4Laboratory of Vascular Inflammation and Thrombosis Research,

5Laboratory of Complement Biology,

6Sickle Cell Clinical Research Program, and

7Laboratory of Blood Borne Parasites, New York Blood Center, New York, New York, USA.

8Department of Pediatrics, Montefiore Health Center, Albert Einstein College of Medicine, Children’s Hospital at Montefiore, Bronx, New York, USA.

Address correspondence to: Avital Mendelson, 310 E67 Street, New York, New York, 10065, USA. Phone: 212.570.3463; Email: amendelson@nybc.org.

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1Laboratory of Membrane Biology,

2Laboratory of Stem Cell Biology and Engineering Research,

3Laboratory of Immune Regulation,

4Laboratory of Vascular Inflammation and Thrombosis Research,

5Laboratory of Complement Biology,

6Sickle Cell Clinical Research Program, and

7Laboratory of Blood Borne Parasites, New York Blood Center, New York, New York, USA.

8Department of Pediatrics, Montefiore Health Center, Albert Einstein College of Medicine, Children’s Hospital at Montefiore, Bronx, New York, USA.

Address correspondence to: Avital Mendelson, 310 E67 Street, New York, New York, 10065, USA. Phone: 212.570.3463; Email: amendelson@nybc.org.

Find articles by Dai, Y. in: JCI | PubMed | Google Scholar

1Laboratory of Membrane Biology,

2Laboratory of Stem Cell Biology and Engineering Research,

3Laboratory of Immune Regulation,

4Laboratory of Vascular Inflammation and Thrombosis Research,

5Laboratory of Complement Biology,

6Sickle Cell Clinical Research Program, and

7Laboratory of Blood Borne Parasites, New York Blood Center, New York, New York, USA.

8Department of Pediatrics, Montefiore Health Center, Albert Einstein College of Medicine, Children’s Hospital at Montefiore, Bronx, New York, USA.

Address correspondence to: Avital Mendelson, 310 E67 Street, New York, New York, 10065, USA. Phone: 212.570.3463; Email: amendelson@nybc.org.

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1Laboratory of Membrane Biology,

2Laboratory of Stem Cell Biology and Engineering Research,

3Laboratory of Immune Regulation,

4Laboratory of Vascular Inflammation and Thrombosis Research,

5Laboratory of Complement Biology,

6Sickle Cell Clinical Research Program, and

7Laboratory of Blood Borne Parasites, New York Blood Center, New York, New York, USA.

8Department of Pediatrics, Montefiore Health Center, Albert Einstein College of Medicine, Children’s Hospital at Montefiore, Bronx, New York, USA.

Address correspondence to: Avital Mendelson, 310 E67 Street, New York, New York, 10065, USA. Phone: 212.570.3463; Email: amendelson@nybc.org.

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1Laboratory of Membrane Biology,

2Laboratory of Stem Cell Biology and Engineering Research,

3Laboratory of Immune Regulation,

4Laboratory of Vascular Inflammation and Thrombosis Research,

5Laboratory of Complement Biology,

6Sickle Cell Clinical Research Program, and

7Laboratory of Blood Borne Parasites, New York Blood Center, New York, New York, USA.

8Department of Pediatrics, Montefiore Health Center, Albert Einstein College of Medicine, Children’s Hospital at Montefiore, Bronx, New York, USA.

Address correspondence to: Avital Mendelson, 310 E67 Street, New York, New York, 10065, USA. Phone: 212.570.3463; Email: amendelson@nybc.org.

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1Laboratory of Membrane Biology,

2Laboratory of Stem Cell Biology and Engineering Research,

3Laboratory of Immune Regulation,

4Laboratory of Vascular Inflammation and Thrombosis Research,

5Laboratory of Complement Biology,

6Sickle Cell Clinical Research Program, and

7Laboratory of Blood Borne Parasites, New York Blood Center, New York, New York, USA.

8Department of Pediatrics, Montefiore Health Center, Albert Einstein College of Medicine, Children’s Hospital at Montefiore, Bronx, New York, USA.

Address correspondence to: Avital Mendelson, 310 E67 Street, New York, New York, 10065, USA. Phone: 212.570.3463; Email: amendelson@nybc.org.

Find articles by Rasquinha, G. in: JCI | PubMed | Google Scholar

1Laboratory of Membrane Biology,

2Laboratory of Stem Cell Biology and Engineering Research,

3Laboratory of Immune Regulation,

4Laboratory of Vascular Inflammation and Thrombosis Research,

5Laboratory of Complement Biology,

6Sickle Cell Clinical Research Program, and

7Laboratory of Blood Borne Parasites, New York Blood Center, New York, New York, USA.

8Department of Pediatrics, Montefiore Health Center, Albert Einstein College of Medicine, Children’s Hospital at Montefiore, Bronx, New York, USA.

Address correspondence to: Avital Mendelson, 310 E67 Street, New York, New York, 10065, USA. Phone: 212.570.3463; Email: amendelson@nybc.org.

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1Laboratory of Membrane Biology,

2Laboratory of Stem Cell Biology and Engineering Research,

3Laboratory of Immune Regulation,

4Laboratory of Vascular Inflammation and Thrombosis Research,

5Laboratory of Complement Biology,

6Sickle Cell Clinical Research Program, and

7Laboratory of Blood Borne Parasites, New York Blood Center, New York, New York, USA.

8Department of Pediatrics, Montefiore Health Center, Albert Einstein College of Medicine, Children’s Hospital at Montefiore, Bronx, New York, USA.

Address correspondence to: Avital Mendelson, 310 E67 Street, New York, New York, 10065, USA. Phone: 212.570.3463; Email: amendelson@nybc.org.

Find articles by Shao, B. in: JCI | PubMed | Google Scholar

1Laboratory of Membrane Biology,

2Laboratory of Stem Cell Biology and Engineering Research,

3Laboratory of Immune Regulation,

4Laboratory of Vascular Inflammation and Thrombosis Research,

5Laboratory of Complement Biology,

6Sickle Cell Clinical Research Program, and

7Laboratory of Blood Borne Parasites, New York Blood Center, New York, New York, USA.

8Department of Pediatrics, Montefiore Health Center, Albert Einstein College of Medicine, Children’s Hospital at Montefiore, Bronx, New York, USA.

Address correspondence to: Avital Mendelson, 310 E67 Street, New York, New York, 10065, USA. Phone: 212.570.3463; Email: amendelson@nybc.org.

Find articles by Liu, Y. in: JCI | PubMed | Google Scholar |

1Laboratory of Membrane Biology,

2Laboratory of Stem Cell Biology and Engineering Research,

3Laboratory of Immune Regulation,

4Laboratory of Vascular Inflammation and Thrombosis Research,

5Laboratory of Complement Biology,

6Sickle Cell Clinical Research Program, and

7Laboratory of Blood Borne Parasites, New York Blood Center, New York, New York, USA.

8Department of Pediatrics, Montefiore Health Center, Albert Einstein College of Medicine, Children’s Hospital at Montefiore, Bronx, New York, USA.

Address correspondence to: Avital Mendelson, 310 E67 Street, New York, New York, 10065, USA. Phone: 212.570.3463; Email: amendelson@nybc.org.

Find articles by Shi, P. in: JCI | PubMed | Google Scholar |

1Laboratory of Membrane Biology,

2Laboratory of Stem Cell Biology and Engineering Research,

3Laboratory of Immune Regulation,

4Laboratory of Vascular Inflammation and Thrombosis Research,

5Laboratory of Complement Biology,

6Sickle Cell Clinical Research Program, and

7Laboratory of Blood Borne Parasites, New York Blood Center, New York, New York, USA.

8Department of Pediatrics, Montefiore Health Center, Albert Einstein College of Medicine, Children’s Hospital at Montefiore, Bronx, New York, USA.

Address correspondence to: Avital Mendelson, 310 E67 Street, New York, New York, 10065, USA. Phone: 212.570.3463; Email: amendelson@nybc.org.

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1Laboratory of Membrane Biology,

2Laboratory of Stem Cell Biology and Engineering Research,

3Laboratory of Immune Regulation,

4Laboratory of Vascular Inflammation and Thrombosis Research,

5Laboratory of Complement Biology,

6Sickle Cell Clinical Research Program, and

7Laboratory of Blood Borne Parasites, New York Blood Center, New York, New York, USA.

8Department of Pediatrics, Montefiore Health Center, Albert Einstein College of Medicine, Children’s Hospital at Montefiore, Bronx, New York, USA.

Address correspondence to: Avital Mendelson, 310 E67 Street, New York, New York, 10065, USA. Phone: 212.570.3463; Email: amendelson@nybc.org.

Find articles by An, X. in: JCI | PubMed | Google Scholar

1Laboratory of Membrane Biology,

2Laboratory of Stem Cell Biology and Engineering Research,

3Laboratory of Immune Regulation,

4Laboratory of Vascular Inflammation and Thrombosis Research,

5Laboratory of Complement Biology,

6Sickle Cell Clinical Research Program, and

7Laboratory of Blood Borne Parasites, New York Blood Center, New York, New York, USA.

8Department of Pediatrics, Montefiore Health Center, Albert Einstein College of Medicine, Children’s Hospital at Montefiore, Bronx, New York, USA.

Address correspondence to: Avital Mendelson, 310 E67 Street, New York, New York, 10065, USA. Phone: 212.570.3463; Email: amendelson@nybc.org.

Find articles by Guo, A. in: JCI | PubMed | Google Scholar

1Laboratory of Membrane Biology,

2Laboratory of Stem Cell Biology and Engineering Research,

3Laboratory of Immune Regulation,

4Laboratory of Vascular Inflammation and Thrombosis Research,

5Laboratory of Complement Biology,

6Sickle Cell Clinical Research Program, and

7Laboratory of Blood Borne Parasites, New York Blood Center, New York, New York, USA.

8Department of Pediatrics, Montefiore Health Center, Albert Einstein College of Medicine, Children’s Hospital at Montefiore, Bronx, New York, USA.

Address correspondence to: Avital Mendelson, 310 E67 Street, New York, New York, 10065, USA. Phone: 212.570.3463; Email: amendelson@nybc.org.

Find articles by Mitchell, W. in: JCI | PubMed | Google Scholar

1Laboratory of Membrane Biology,

2Laboratory of Stem Cell Biology and Engineering Research,

3Laboratory of Immune Regulation,

4Laboratory of Vascular Inflammation and Thrombosis Research,

5Laboratory of Complement Biology,

6Sickle Cell Clinical Research Program, and

7Laboratory of Blood Borne Parasites, New York Blood Center, New York, New York, USA.

8Department of Pediatrics, Montefiore Health Center, Albert Einstein College of Medicine, Children’s Hospital at Montefiore, Bronx, New York, USA.

Address correspondence to: Avital Mendelson, 310 E67 Street, New York, New York, 10065, USA. Phone: 212.570.3463; Email: amendelson@nybc.org.

Find articles by Manwani, D. in: JCI | PubMed | Google Scholar

1Laboratory of Membrane Biology,

2Laboratory of Stem Cell Biology and Engineering Research,

3Laboratory of Immune Regulation,

4Laboratory of Vascular Inflammation and Thrombosis Research,

5Laboratory of Complement Biology,

6Sickle Cell Clinical Research Program, and

7Laboratory of Blood Borne Parasites, New York Blood Center, New York, New York, USA.

8Department of Pediatrics, Montefiore Health Center, Albert Einstein College of Medicine, Children’s Hospital at Montefiore, Bronx, New York, USA.

Address correspondence to: Avital Mendelson, 310 E67 Street, New York, New York, 10065, USA. Phone: 212.570.3463; Email: amendelson@nybc.org.

Find articles by Yazdanbakhsh, K. in: JCI | PubMed | Google Scholar

1Laboratory of Membrane Biology,

2Laboratory of Stem Cell Biology and Engineering Research,

3Laboratory of Immune Regulation,

4Laboratory of Vascular Inflammation and Thrombosis Research,

5Laboratory of Complement Biology,

6Sickle Cell Clinical Research Program, and

7Laboratory of Blood Borne Parasites, New York Blood Center, New York, New York, USA.

8Department of Pediatrics, Montefiore Health Center, Albert Einstein College of Medicine, Children’s Hospital at Montefiore, Bronx, New York, USA.

Address correspondence to: Avital Mendelson, 310 E67 Street, New York, New York, 10065, USA. Phone: 212.570.3463; Email: amendelson@nybc.org.

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Published February 27, 2025 - More info

Published in Volume 135, Issue 8 on April 15, 2025
J Clin Invest. 2025;135(8):e189801. https://doi.org/10.1172/JCI189801.
© 2025 Gao et al. This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Published February 27, 2025 - Version history
Received: December 3, 2024; Accepted: February 19, 2025 View PDF Abstract

Newly produced platelets acquire a low activation state, but whether the megakaryocyte plays a role in this outcome has not been fully uncovered. Mesenchymal stem cells (MSCs) were previously shown to promote platelet production and lower platelet activation. We found that healthy megakaryocytes transfer mitochondria to MSCs, which is mediated by connexin 43 (Cx43) gap junctions on MSCs and leads to platelets at a low energetic state with increased LYN activation, characteristic of resting platelets with increased LYN activation, characteristic of resting platelets. On the contrary, MSCs have a limited ability to transfer mitochondria to megakaryocytes. Sickle cell disease (SCD) is characterized by hemolytic anemia and results in heightened platelet activation, contributing to numerous disease complications. Platelets in SCD mice and human samples had a heightened energetic state with increased glycolysis. MSC exposure to heme in SCD led to decreased Cx43 expression and a reduced ability to uptake mitochondria from megakaryocytes. This prevented LYN activation in platelets and contributed to increased platelet activation at steady state. Altogether, our findings demonstrate an effect of hemolysis in the microenvironment leading to increased platelet activation in SCD. These findings have the potential to inspire new therapeutic targets to relieve thrombosis-related complications of SCD and other hemolytic conditions.

Graphical Abstract Introduction

The bone marrow niche for regulating megakaryocyte (MK) function and preventing platelet activation remains poorly understood. We recently identified a role for mesenchymal stem cells (MSCs) from both bone marrow and umbilical cord tissue, which can lower baseline platelet activation (1), though the mechanism remains unclear. Platelets in a skin wound healing model can transfer mitochondria to MSCs leading to a proangiogenic phenotype and enhanced tissue repair (2). While intercellular mitochondria transfer has been demonstrated between MSCs and various cell types, such as hematopoietic stem and progenitor cells, immune cells, or alveolar epithelia (3–7), whether mature MKs can also transfer mitochondria to MSCs, leading to the production of platelets with low baseline activation, has not been explored.

Sickle cell disease (SCD) is an inherited blood disorder, which results in the polymerization of hemoglobin S and sickling of red blood cells, leading to hemolysis along with thrombotic and inflammatory alterations (8–11). Heightened platelet activation in SCD is believed to contribute to numerous complications, including stroke, thromboembolism, and cardiovascular complications, among others (8, 12, 13). Platelet inhibition with aspirin treatment was investigated in preventing vaso-occlusion in SCD, but clinical trial results were mainly negative (14–16). A recent study identified reduced platelet aggregation following treatment with crizanlizumab (17), which targets P selectin on the surface of platelets and endothelial cells to prevent pain crises, though further investigation is ongoing to confirm its therapeutic benefits. A trial of P2Y12 inhibition with prasugrel also did not identify sufficient benefits for reducing vaso-occlusive crisis events (18). While primary clinical trials of ticagrelor P2Y12 inhibitor displayed promising results, phase III studies were halted due to lack of therapeutic benefit (19, 20). Thus, alternative antiplatelet and antithrombotic strategies for treating SCD are needed. Previously we found that SCD MSCs have severely impaired functionality, including decreased expression of hematopoietic maintenance genes and impaired self-renewal capacity (21). As a consequence, the ability of SCD MSCs to maintain hematopoietic stem cells in the bone marrow was also lowered (21). Whether SCD MSCs also have a reduced ability to communicate with MKs leading to increased platelet activation remains unknown.

Here, we examined the mechanism by which bone marrow–derived MSCs can promote low baseline platelet activation at steady state. We assessed mitochondrial transfer between MKs and MSCs, mediated by connexin 43 (Cx43) gap junctions, leading to metabolic changes among MKs and MSCs. This in turn translated to activation of LYN signaling and low baseline activation levels in platelets. MKs in SCD were also examined for their metabolic changes due to dysfunctional MSCs leading to increased platelet activation. Reducing baseline platelet activation in SCD could have cascading effects, resulting in lower inflammation and reduced complications of the disease.

Results

MKs transfer mitochondria to MSCs. Given that platelet mitochondria serve functions in platelet activation, metabolism, and ATP production (22), we were interested to investigate whether changes to the mitochondria may occur at the MK level due to MSC-MK interactions. Primary murine wild-type MKs cultured together with wild-type murine MSCs were found to have reduced mitochondria content by mitotracker green analysis (Figure 1A). Human cord blood CD34+ cell–derived MKs cultured together with human bone marrow MSCs also showed reduced mitochondrial content following coculture (Figure 1B). MKs from murine MSC-MK cocultures were further found to have a lower Nd1/Hk2 ratio by RT-PCR (Figure 1C) compared with MKs cultured alone, further confirming a reduced mitochondrial content. We examined the possibility of mitochondrial transfer occurring between MKs and MSCs using cells from a PhAM-floxed;E2a-cre mouse model in which mitochondria are fluorescently labeled with GFP (23). Coculture of wild-type MKs with PhAM-floxed MSCs was conducted to examine mitochondrial transfer from MSCs to MKs. This resulted in only a slight increase in GFP signal within cocultured MKs (Figure 1D). In a second coculture, we assessed whether there may be mitochondrial transfer occurring from MKs to MSCs using MKs from PhAM-floxed;E2a-cre mice with MSCs from wild-type mice. Surprisingly, we found a significant increase in GFP signal among wild-type MSCs from the coculture (Figure 1E and Supplemental Videos 1 and 2). Cultures were analyzed at 18 hours because a time-course study revealed mitochondrial transfer levels from MKs at this time point were among maximal levels (Supplemental Figure 1A). To further verify mitochondrial transfer, murine wild-type MKs were prelabeled with PKmito deep red mitochondria dye followed by coculture with wild-type MSCs. MKs and platelets from cocultures displayed a reduction in mitochondrial signal compared with MKs cultured alone (Supplemental Figure 1, B and C), while MSCs acquired mitochondrial signal from cocultured MKs (Supplemental Figure 1D). Importantly, isolated MKs and MSCs from cocultures remained pure and devoid of cross-contamination (Supplemental Figure 1, E–G). When MKs were pretreated with Oligomycin A to inhibit mitochondrial ATP synthetase, mitochondrial transfer to MSCs was abrogated (Supplemental Figure 1H). Overall, this suggests that MKs are transferring mitochondria to MSCs while MKs receive a minimal amount of mitochondria from MSCs. To examine mitochondrial uptake in vivo, we transplanted wild-type mice with bone marrow from PhAM-floxed;E2a-cre mice and transplanted PhAM-floxed;E2a-cre mice with bone marrow from wild-type mice (Figure 1F). Bone marrow MSC and MK GFP intensity in PhAM-floxed mice were comparable at baseline (Supplemental Figure 2A), and PhAM GFP+ signal could be clearly distinguished in comparison with wild-type mice (Supplemental Figure 2B). At 12 weeks after transplant, approximately 30% of wild-type host MSCs were found to contain GFP signal from transplanted PhAM-floxed bone marrow (Figure 1, G and H, and Supplemental Figure 2C), whereas only 4% of MKs from transplanted wild-type bone marrow contained host mitochondrial GFP signal (Figure 1, I and J, and Supplemental Figure 2D), further suggesting a biased directionality of mitochondrial transfer.

Figure 1

Megakaryocytes transfer mitochondria to mesenchymal stem cells. C57BL/6 murine MKs were cultured together with C57BL/6 MSCs or cultured alone. By flow cytometry, MKs from cocultures were analyzed for (A) mean fluorescence intensity (MFI) of mitotracker green (MTG) (n = 5) and (B) MFI of MTG from human cord blood CD34+ cell-derived MKs cocultured with human bone marrow MSCs (n = 4). (C) RT-PCR expression of Nd1/Hk2 in murine MKs from cocultures (n = 5). (D) C57BL/6 murine MKs were cultured together with MSCs from PhAM-floxed;E2a-cre mice or cultured alone. MFI of mitochondria PhAM signal in MKs by flow cytometry (n = 5). (E) Wild-type murine MSCs were cultured together with murine MKs from PhAM-floxed;E2a-cre mice or cultured alone. MFI of PhAM mitochondrial signal in MSCs by flow cytometry (n = 4). (F) Schematic of transplant design in which C57BL/6 mice were transplanted with PhAM-floxed;E2a-cre bone marrow cells or PhAM-floxed;E2a-cre mice were transplanted with C57BL/6 cells. (G) Bone marrow MSCs analyzed from transplanted mice for percentage PhAM+ MSCs and (H) numbers of PhAM+ MSCs per femur. (I) Bone marrow MKs analyzed from transplanted mice for percentage PhAM+ MKs and (J) numbers of PhAM+ MKs per femur. n = 3–4 mice for G–J. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Data were analyzed with 2-tailed, unpaired Student’s t test. Data are presented as mean ± SEM.

MKs undergo metabolic changes after transferring mitochondria. To examine if MKs cocultured with MSCs led to alterations in the metabolic properties of both cell types, we conducted a Seahorse Glycolytic Rate Assay (Agilent). Following coculture with MKs, MSCs were found to be more glycolytic compared with MSCs cultured alone (Figure 2A), including significantly increased basal glycolysis (Figure 2B) and compensatory glycolysis (Figure 2C). Interestingly, MKs cocultured with MSCs had a reduction in glycolysis compared with MKs cultured alone (Figure 2D), leading to both lower basal glycolysis (Figure 2E) and compensatory glycolysis (Figure 2F). MKs had a reduced ability to uptake glucose following MSC coculture compared with MKs cultured alone (Supplemental Figure 1I) and MKs pretreated with oligomycin following by coculture with MSCs (Supplemental Figure 1I). Given that MKs enhance their mitochondrial biogenesis with increased maturation (24) and the presence of MSCs leads to greater platelet formation by MKs (1), we found an increased MK ratio of ATP/ADP following MSC-MK coculture compared with MKs cultured alone (Supplemental Figure 1J). A high ATP/ADP ratio in cocultured MKs led to inhibition of glycolysis, whereas a decreased ATP/ADP ratio in MKs cultured alone and in MKs pretreated with oligomycin followed by coculture led to a glycolytic phenotype (Supplemental Figure 1, I and J). Thus, MSCs following MK coculture have an enhanced energetic state, whereas MKs following MSC coculture have lowered metabolic activity.

Figure 2

Metabolic changes to megakaryocytes and MSCs following coculture. C57BL/6 murine MKs were cultured together with C57BL/6 MSCs or cultured alone. (A) A representative Seahorse glycolytic rate assay conducted on MSCs following coculture. (B) Basal glycolysis levels of MSCs from coculture. (C) Compensatory glycolysis of MSCs following coculture. (D) Representative Seahorse glycolytic rate assay conducted on MKs following coculture. (E) Basal glycolysis levels of MKs from coculture. (F) Compensatory glycolysis of MKs following coculture. n = 3. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. Data were analyzed with 2-tailed, unpaired Student’s t test. Data are presented as mean ± SEM.

Cx43 gap junctions on MSCs mediate mitochondrial acceptance. Cx43 gap junctions have been demonstrated to mediate the transfer of intracellular content between cells (4, 7, 25, 26). Using a mouse model in which Cx43 is selectively depleted from Lepr+ MSCs, we examined whether mitochondrial transfer between MKs and MSCs occurs through Cx43 gap junctions on MSCs. MSCs isolated from conditional Cx43-KO (Cx43-floxed) mice were cocultured with MKs from PhAM-floxed;E2a-cre mice, which resulted in mitochondrial retention in MKs (Figure 3A) compared with coculture with wild-type MSCs. We also observed increased mitochondrial content in platelets produced within cocultures containing Cx43-floxed MSCs compared with cocultures with wild-type MSCs (Figure 3B). Cx43-floxed MSCs had less mitochondrial uptake ability compared with wild-type MSCs (Figure 3C). Furthermore, platelets produced in culture with Cx43-floxed MSCs had increased expression of platelet activation markers CD62P (Figure 3D) and JonA (Figure 3E) compared with cultures containing wild-type MSCs. Accordingly, we also observed a reduced ability for platelets from Cx43-floxed mice to activate and aggregate in response to thrombin (Supplemental Figure 3, A–D) and CRP compared with platelets from wild-type mice (Supplemental Figure 3, E–H). Platelets from Cx43-floxed mice subjected to the T-TAS atheroma chip coagulation assay led to a reduced channel occlusion time compared with control platelets (Supplemental Figure 3I). Platelets from cocultures with Cx43 inhibited MSCs, and healthy MKs were subjected to a flow cytometry–based aggregation assay and displayed a reduced aggregation (27) ability compared with healthy MSC-MK cocultures (Supplemental Figure 3J). Evaluation of the MK glycolytic profile by Seahorse revealed that MKs cocultured with Gap19-treated MSCs to inhibit Cx43 led to increased glycolysis compared with coculture with wild-type MSCs (Figure 3, F–H). Compared with MKs cocultured with control MSCs, coculturing MKs with Gap19-treated MSCs also resulted in increased glucose uptake ability (Supplemental Figure 1I) and reduced intracellular ATP/ADP ratios (Supplemental Figure 1J), which was confirmed by increased expression of glycolysis genes pyruvate kinase M2 (Pkm2), phosphofructokinase (Pkfm), hexokinase 1 (Hk1), and solute carrier family 3 member 1 (Slc3a3) by RT-PCR (Figure 3I). Therefore, our data suggest that MSC Cx43 gap junctions may mediate mitochondrial transfer from MKs to MSCs, which in turn leads to downstream alterations in platelet activation.

Figure 3

MSC CX43 gap junctions mediate mitochondrial transfer from megakaryocytes. PhAM-floxed;E2a-cre murine MKs cultured together with C57BL/6 MSCs or MSCs from CX43-floxed;Lepr-cre mice. Following coculture, flow cytometry assessment of (A) mean fluorescence intensity (MFI) of PhAM signal in MKs (n = 5), (B) MFI of PhAM signal in platelets (n = 5), (C) MFI of PhAM signal in MSCs (n = 5), (D) CD62P expression on platelets (n = 4), and (E) JonA (αIIbβ3) expression on platelets (n = 4). (F) Representative Seahorse glycolytic rate assay conducted on MKs following coculture with wild-type or Gap19-treated MSCs (n = 3). (G) Basal glycolysis levels of MKs from coculture (n = 3). (H) Compensatory glycolysis of MKs following coculture (n = 3). (I) RT-PCR analysis of glycolytic genes (Pkm2, Pfkm, Hk1, and Slc3a3) in MKs from cocultures with either C57BL/6 MSCs or C57BL/6 MSCs pretreated with Gap19 to inhibit CX43 expression (n = 3–4). *P ≤ 0.05, **P ≤ 0.01. Data were analyzed with 2-tailed, unpaired Student’s t test. Data are presented as mean ± SEM.

Given that gap junction–mediated intracellular trafficking requires close proximity (28), we examined the possibility that Cx43 deletion in MSCs leads to altered MK localization away from MSCs compared with wild-type MSC-MK localization. Through whole-mount bone marrow imaging, we found MKs in Cx43-floxed mice localized at an average distance of 22.9 μm away from Lepr+ MSCs, whereas MKs in wild-type mice localized at an average distance of 20.8 μm (Supplemental Figure 4) from Lepr+ MSCs. This raises the possibility that increased distance between MKs and MSCs may further prevent mitochondrial transfer between MKs and MSCs due to inhibited Cx43 expression.

MSCs alter the transcriptome signature of platelets. Following coculture of wild-type murine MKs with wild-type MSCs or MKs cultured alone, a transcriptomics analysis was conducted on platelets generated in cultures. A principal component analysis revealed the distribution and clustering between samples within each group (Figure 4A). The differentially expressed genes among platelets from MSC cocultures compared with platelets from MKs cultured alone displayed stark differences between groups (Figure 4B). Differentially expressed genes are listed in Supplemental Table 1. Among the top pathways of differentially expressed genes was mitochondrial organization, ATP metabolic process, mitochondrial transport and cellular respiration (Figure 4C). Additionally, platelets from MSC-MK cocultures were downregulated in numerous genes involved in platelet activation (Figure 4D).

Figure 4

MSCs alter platelet transcriptomics signature. C57BL/6 murine MKs were cultured together with C57BL/6 MSCs or cultured alone. Platelets from cultures were pooled to conduct bulk RNA-Seq analysis (n = 4 mice per group). (A) Principal component analysis. (B) Top differentially expressed genes among platelets from cultures. (C) Top 10 pathways of differentially expressed genes in platelets from MK-MSC cocultures compared with MKs cultured alone. (D) Gene expression related to platelet aggregation in platelets from MK-MSC cocultures compared with MKs cultured alone.

Reduced levels of mitochondria lead to activation of LYN signaling in platelets. Previously it was shown that LYN activation in platelets negatively regulates integrin αIIβ3 signaling (29) and prevents their activation (30). Platelets from MSC-MK cocultures were found to have increased total LYN expression by flow cytometry (Figure 5A) compared with control MK-only cultures. Examination of phosphorylation of LYN (p-LYN) revealed that coculture of MK and MSC led to increased p-LYN signaling compared with MKs cultured alone (Figure 5, B and C, and Supplemental Figure 5, A and B). Treatment of MSCs with Gap19 to inhibit Cx43 prevented LYN signaling activation (Figure 5, B and C, and Supplemental Figure 5, A and B). Tunneling nanotubes are often formed between cells to aid in intracellular organelle trafficking (31). Furthermore, tunneling nanotubes can form by actin polymerization and connect gap junctions between cells (32, 33). MSCs treated with cytochalasin D actin polymerization inhibitor prevented LYN signaling from occurring (Figure 5, B and C, and Supplemental Figure 5, A and B), suggesting that tunneling nanotubes formed between MSCs and MKs are mediating mitochondrial transfer between cells.

Figure 5

MSCs upregulate LYN signaling in platelets. C57BL/6 murine MKs were cultured together with C57BL/6 MSCs or cultured alone. Platelets from cultures were analyzed by flow cytometry for (A) total LYN mean fluorescence intensity (MFI). C57BL/6 murine MKs were cultured together with C57BL/6 MSCs, with Gap19-treated MSCs, cytochalasin D–treated MSCs, or cultured alone. (B) Histogram of p-LYN signal among platelets from cocultures. (C) MFI p-LYN signal among platelets from cocultures. *P ≤ 0.05. n = 3–4 mice. Data in A were analyzed with 2-tailed, unpaired Student’s t test. Data in C were analyzed by 1-way ANOVA with Tukey’s multiple comparison test. Data are presented as mean ± SEM.

Platelets from both SCD mice and patients with SCD are more glycolytic. Platelets from the Townes mouse model of SCD were found to have increased CD62P expression (Figure 6A) and JonA expression (Figure 6B) compared with control SA heterozygous mice, confirming previous findings (34). Platelets from SCD mice had reduced activation of p-LYN (Figure 6C and Supplemental Figure 5, C and D) signaling compared with SA mice platelets. Seahorse analysis of platelets from SCD mice revealed increased glycolysis (Figure 6D), including enhanced basal glycolysis (Figure 6E) as well as compensatory glycolysis (Figure 6F), compared with SA control mice. MKs from SCD mice displayed significantly increased expression of numerous genes related to glycolysis compared with SA MKs, including Slc2a1, Slc3a3, Hk1, Gpi1, Pkm2, Gapdh, and Ldha (Figure 6G). Platelets from patients with SCD also displayed similarly glycolytic profiles (Figure 6, H–J). We examined the localization of SCD or SA control MKs to Lepr+ MSCs using whole-mount imaging of bone marrow and found that SCD MKs are located at an average distance of 26.1 μm away from the nearest MSC whereas SA MKs are located 21.0 μm away from the nearest MSC (Supplemental Figure 6).

Figure 6

Activation and metabolic profile of platelets from SCD mice. Peripheral blood platelets from the Townes model of SCD or SA control mice were analyzed by flow cytometry for (A) CD62P expression (n = 3 mice) and (B) JonA expression (N = 4 mic