HHS Public Access Author manuscript Author Manuscript
Cell Rep. Author manuscript; available in PMC 2017 September 15. Published in final edited form as: Cell Rep. 2017 July 25; 20(4): 881–894. doi:10.1016/j.celrep.2017.06.083.
Deletion of GLUT1 and GLUT3 Reveal Multiple Roles for Glucose Metabolism in Platelet and Megakaryocyte Function Trevor P. Fidler1,2,3, Robert A. Campbell2,4, Trevor Funari3, Nicholas Dunne3, Enrique Balderas Angeles5, Elizabeth Middleton2,4, Dipayan Chaudhuri5, Andrew S. Weyrch2,4, and E. Dale Abel2,3,*,# 1Department
Author Manuscript
2Program
of Pharmacology and Toxicology University of Utah, Salt Lake City, UT, 84112, USA
in Molecular Medicine University of Utah, Salt Lake City, UT, 84112, USA
3Fraternal
Order of Eagles Diabetes Research Center and Division of Endocrinology and Metabolism, Carver College of Medicine, University of Iowa, Iowa City, IA, 52242, USA
4Department
of Internal Medicine, University of Utah, Salt Lake City, UT, 84112, USA
5Nora
Eccles Harrison Cardiovascular Research and Training Institute, University of Utah, Salt Lake City, UT, 84112, USA
Summary
Author Manuscript
Anucleate platelets circulate in the blood to facilitate thrombosis and diverse immune functions. Platelet activation leading to clot formation correlates with increased glycogenolysis, glucose uptake, glucose oxidation, and lactic acid production. Simultaneous deletion of glucose transporter (GLUT) 1 and GLUT3 (double knockout (DKO)) specifically in platelets completely abolished glucose uptake. In DKO platelets mitochondrial oxidative metabolism of non-glycolytic substrates such as glutamate, increased. Thrombosis and platelet activation were decreased through impairment at multiple activation nodes including Ca2+ signaling, degranulation, and integrin activation. DKO mice developed thrombocytopenia, secondary to impaired pro-platelet formation from megakaryocytes, and increased platelet clearance resulting from cytosolic calcium overload and calpain activation. Systemic treatment with oligomycin, inhibiting mitochondrial metabolism, induced rapid clearance of platelets with circulating counts dropping to zero in DKO but not wildtype mice, demonstrating an essential role for energy metabolism in platelet viability. Thus substrate metabolism is essential for platelet production, activation and survival.
Author Manuscript
eTOC blurb
*
Corresponding author, [email protected]. #Lead Contact
Author Contribution Conceptualization, EDA, ASW, and TPF; Methodology, EDA, ASW, TPF, and RC; Investigation, TPF, RC, TF, EBA, and ND; Writing-original draft, TPF; Writing-Review and Editing, TPF, EDA, ASW, and RC; Funding Acquisition, EDA and ASW; Resources, EDA and ASW; Supervision DC, EDA, and ASW Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Fidler et al.
Page 2
Author Manuscript
Fidler et al. show that glucose metabolism is essential for platelet production, activation, and clearance. Their findings reveal complementary roles for glycolysis versus mitochondrial metabolism in platelet viability. Blocking both metabolic pathways leads to complete clearance of platelets from the circulation, due to calcium overload and calpain activation.
Author Manuscript Introduction
Author Manuscript
Glucose enters cells via glucose transporters, of which platelets express the facilitative glucose transporter 1 (GLUT1) (Craik, 1995) and glucose transporter 3 (GLUT3) (Heijnen et al., 1997). ~85% of GLUT3 is located in α-granule membranes with ~15% localized in the plasma membrane. Upon degranulation, GLUT3 translocates to the plasma membrane and is believed to account for the increased glucose uptake following activation (Heijnen et al., 1997). Platelet activation also increases glycolysis and lactic acid production (Karpatkin, 1967), glycogenolysis (Scott and Cooper, 1967) and glucose oxidation (Warshaw et al., 1966). In vitro inhibition of these processes blunts platelet activation (Akkerman, 1981; Yamagishi, 2001), while incubation under hyperglycemic conditions potentiates activation in response to ADP, arachidonic acid, thrombin and collagen (Yamagishi, 2001). However, these associations have not been evaluated in vivo.
Author Manuscript
Although the correlation between activation and increased metabolism is well established, the mechanisms underling these relationships are unknown. Studies of permeabilized platelets, lacking cytosol indicate that ATP is an essential cofactor for Ca2+-induced degranulation (Flaumenhaft, 1999). Administration of the competitive glucose uptake inhibitor 2-deoxyglucose (2-DOG) to platelets suggested that glucose uptake and metabolism plays an important role in platelet aggregation and degranulation. Although these changes correlate with ATP content, a direct relationship between platelet glucose uptake and ATP content remains to be demonstrated (Akkerman, 1979). Conversely, platelets isolated from diabetic patients demonstrate dysfunctional Ca2+ signaling (Li, 2001), although it is unknown if platelet activation in this context arises from altered metabolism.
Cell Rep. Author manuscript; available in PMC 2017 September 15.
Fidler et al.
Page 3
Author Manuscript
Mitochondrial respiration has also been linked to platelet activation. Inhibitors of mitochondrial respiration blunt platelet activation (Garcia-Souza and Oliveira, 2014). Upon activation, a subpopulation of platelets demonstrate mitochondrial depolarization and generation of reactive oxygen species (ROS) which facilitates phosphatidylserine (PS) exposure to the outer leaflet of the plasma membrane that promotes platelet procoagulant activity (Garcia-Souza and Oliveira, 2014). In addition to thrombotic functions, platelet mitochondria occupy a central role in the intrinsic apoptosis pathway (Mason et al., 2007). The balance between BCL-XL activity and formation of mitochondrial Bak/Bax pores regulates platelet apoptosis and platelet viability. However, the relationship between mitochondrial metabolism and apoptotic cell death pathways in platelets remain to be clarified. Moreover, mice infected with dengue demonstrate an additional mechanism of platelet clearance that correlated with PS exposure in vivo (Alonzo et al., 2012). Given the diverse mechanisms leading to platelet clearance, the integration of these mechanisms in the regulation of platelet viability and the contribution of platelet metabolism to these processes remains to be clarified.
Author Manuscript Author Manuscript
Given the dearth of information regarding the role of energy metabolism in regulating platelet biology in vivo, we generated mice lacking GLUT1 and GLUT3 specifically in platelets. Analyses of these mice reveal a dynamic interaction between glucose utilization and mitochondrial energy metabolism to maintain platelet energy requirements. Importantly, glucose metabolism is essential for pro-platelet formation from megakaryocytes. Glucose metabolism is essential for agonist mediated Ca2+ signaling, and downstream signaling events leading to platelet activation. We demonstrate a critical role for glycolytic and mitochondrial metabolism in regulating platelet clearance establishing a paradigm whereby impaired platelet energy metabolism undermines calcium homeostasis, leading to increased cytoplasmic Ca2+ that activates a calpain-mediated cell death and clearance pathway.
Results Glucose metabolism is abolished in DKO platelets
Author Manuscript
To investigate the contribution of glucose metabolism to platelet function, GLUT1 and GLUT3 were individually or simultaneously deleted from platelets by crossing mice expressing a Pf4 promoter-driven Cre recombinase to mice harboring homozygous floxed GLUT1 and GLUT3 alleles individually or in combination. Immunoblot analysis of platelet protein lysates isolated from GLUT1 single knockout (GLUT1-KO), GLUT3 single knockout (GLUT3-KO) and GLUT1/GLUT3 double knockout (DKO) and their respective littermate controls (Figure 1A) confirmed the absence of the respective proteins. Glucose uptake was measured in control quiescent platelets at ~10 pmoles/1×106 platelets/30 minutes, which after thrombin stimulation increased two-fold. Deletion of GLUT3 reduced basal glucose uptake by ~22% and abolished thrombin stimulated glucose uptake (Figure 1B) illustrating that GLUT3 mediates post-activation glucose uptake. Surprisingly, GLUT1KO platelets revealed no changes in basal or thrombin-mediated glucose uptake relative to controls. However, deletion of both GLUT1 and GLUT3 completely abolished glucose uptake under basal and thrombin stimulated conditions indicating that GLUT1 and GLUT3 are the biologically relevant glucose transporters in platelets. These data also demonstrate
Cell Rep. Author manuscript; available in PMC 2017 September 15.
Fidler et al.
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that GLUT1 and GLUT3 have unique, but also overlapping roles in the regulation of platelet glucose uptake.
Author Manuscript
DKO platelets do not undergo glycolysis. Quantitative analysis of 13C-1,6-glucose flux demonstrated DKO platelets fail to convert exogenous glucose to lactic acid (Figure 1C), whereas controls generated 0.0085pg lactic acid/minute/1×106 platelets. In addition, with 13C-glucose as the exclusive exogenous glucose source, 12C-lactic acid release, which represents the contribution of glycogen to glycolysis, was also abolished (Figure 1D). Qualitative analysis of glycolysis determined by the extracellular acidification rate (ECAR) was markedly repressed in DKO platelets (Figure 1E). Of note, ECAR was not zero in DKO platelets potentially reflecting acidification of the media, by acids other than lactate (TeSlaa and Teitell, 2014). Consistent with studies in human platelets (Karpatkin, 1968), thrombin increased glycolysis by ~2-fold in control murine platelets. In contrast, DKO platelets did not increase glycolysis following thrombin stimulation, or in the presence of inhibitors of mitochondrial metabolism (Figure 1E). Platelets contain large glycogen stores, which can be mobilized upon activation (Scott and Cooper, 1967). Glycogen content in DKO platelets was reduced ~7-fold to levels near the limit of detection (Figure 1F). Thus glucose uptake, and metabolism are largely abolished in DKO platelets. DKO platelets increase utilization of alternative mitochondrial substrates
Author Manuscript Author Manuscript
In the absence of glucose uptake, alternative metabolic pathways may sustain cellular ATP to maintain cellular viability. DKO platelets displayed a significantly increased ratio of [AMP+ADP]/[ATP] (Figure 2A), suggesting metabolic stress. Consistent with this, freshly isolated DKO platelets demonstrated increased phosphorylated AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) (Figure 2B). Interestingly, mitochondrial membrane potential was significantly increased in freshly isolated DKO platelets independent of exogenous substrate present in the media (Figure 2C). Seahorse analysis of mitochondrial oxygen consumption rates (OCR) in the presence of glucose, glutamate, and pyruvate indicated that at baseline and under maximal respiration conditions, OCR in DKO platelets were ~3-fold higher than controls (Figure 2D). Increased mitochondrial respiration in DKO reflects increased capacity to utilize mitochondrial substrates. OCR were equivalent in control and DKO platelets with glucose as the sole metabolic substrate, however following addition of glutamate and pyruvate, mitochondrial OCR increased by ~70% in DKO, but was unchanged in controls (Figure 2E). DKO platelets exhibited decreased intracellular lactic acid and relatively increased pyruvate leading to a significantly reduced lactate/pyruvate ratio (Figure 2F), suggesting induction of alternative pathways of pyruvate generation such as the transamination of alanine that requires mitochondrial α-ketoglutarate (Gray et al., 2014). Mitochondrial content and gross structure of DKO platelets were unchanged (Figure 3A and 3B), suggesting a qualitative rather than a quantitative change in mitochondrial function. Thus, absence of glucose metabolism in DKO platelets increased metabolism of alternative mitochondrial substrates. Platelet activation is decreased in DKO platelets DKO platelets exhibited blunted platelet activation. Non-stimulated DKO platelets displayed similar organelle distribution, with the exception of decreased α-granule number (Figure 3A
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and 3B), which may suggest a biogenesis defect. In addition, following stimulation with 250μM PAR4 peptide, degranulation was impaired in DKO platelets as evidenced by retention of α-granules in platelets (Figure 3A and 3C). In vitro analysis of platelets incubated in 5mM glucose for 1-hour, revealed decreased activation of GPIIbIIIa measured by JonA geo. MFI (Figure 3D), and α-granule degranulation marked by CD62p surface translocation (CD62p geo. MFI) (Figure 3E) in response to PAR4 peptide, the thromboxane receptor agonist U46619, ADP, and convulxin. However, this loss of activation was only partially rescued (ADP and U46619 – JonA) when glutamate and pyruvate was added to the media (Figure 3D and 3E), suggesting a direct link between glucose metabolism and platelet activation, that is not rescued in the presence of alternate mitochondrial substrates in DKO platelets. Agonist mediated calcium signaling is impaired in DKO platelets
Author Manuscript Author Manuscript Author Manuscript
In response to activating stimuli, platelets increase cytoplasmic Ca2+ that activates the Ca2+ sensitive scramblase TMEM16f, to facilitate the translocation of PS to the outer leaflet of the plasma membrane leading to a procoagulant response (Jobe et al., 2008; Van Kruchten, 2012). Although PS is exposed, this procoagulant process is distinct from apoptosismediated PS exposure and is not thought to lead to an “eat me” signal that leads to platelet clearance (Van Kruchten, 2012). In response to thrombin plus convulxin, DKO platelets demonstrate impaired exposure of PS to the outer leaflet of the plasma membrane measured by annexin V binding (Figure 4A and S1A–F). Restoring Ca2+ flux by administration of the Ca2+ ionophore ionomycin, completely restored annexin V binding (Figure 4A). In response to thrombin, cytoplasmic Ca2+ flux was blunted in DKO platelets (Figure 4B). Importantly, following the rise in agonist-mediated cytoplasmic Ca2+, control platelets restored cytoplasmic Ca2+ to near basal levels (Figure 4B), whereas levels remained increased in DKO platelets for >10 minutes following stimulation. This inability to export/sequester Ca2+ following stimulation suggests a defect in sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) and/or plasma membrane Ca2+-ATPase (PMCA)-mediated Ca2+ transport. Using an independent methodology, we confirmed impairment of platelet Ca2+ mobilization following combined stimulation by thrombin and convulxin of DKO platelets (Figure 4C and 4D). Following thapsigargin treatment to deplete SERCA, DKO platelets failed to restore Ca2+ concentrations to control levels (Figure 4E and 4F), revealing a defect in store operated Ca2+ entry. DKO platelets exhibit impaired GPIIbIIIa activation and CD62p surface translocation following treatment with thrombin plus convulxin, the Ca2+ ionophore ionomycin, or the SERCA inhibitor thapsigargin (Figure 4G and 4H). These data indicate that glucose metabolism regulates PS exposure to the outer leaflet of the plasma membrane via its effect on calcium mobilization, but even when calcium flux is rescued, glucose metabolism mediates additional steps required for integrin activation and α-granule degranulation. Platelet glucose metabolism is essential for in vivo thrombosis The contribution of platelet glucose metabolism to thrombosis and hemostasis in vivo is unknown. In a tail-bleeding assay, DKO mice exhibited significantly longer time to bleeding cessation, with many mice failing to stop prior to assay completion (Figure 4I). Although spontaneous bleeding was not observed, hematocrit was modestly yet significantly reduced Cell Rep. Author manuscript; available in PMC 2017 September 15.
Fidler et al.
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in untreated DKO mice (Table S1). Arterial thrombosis was also evaluated using 7.5% ferric chloride. DKO mice had significantly increased time to arterial occlusion relative to littermate controls; with many failing to occlude after 20 minutes of observation (Figure 4J). In a collagen/epinephrine-induced pulmonary embolism model, which is dependent on in vivo platelet aggregation, DKO mice demonstrated increased survival (Figure 4K). Thus platelet glucose metabolism is essential for in vivo thrombosis. DKO mice are thrombocytopenic
Author Manuscript
Platelet counts were reduced in DKO mice (Figure 5A, Table S1); however, platelet counts were unchanged in mice with GLUT1 or GLUT3-deficient platelets respectively. To determine if thrombocytopenia resulted from decreased platelet production from megakaryocytes, we administered antibodies to GPIbα that depletes platelets within 18 hours (Figure 5B). In control mice platelet counts completely recovered after 96 hours, whereas DKO mice required 168 hours for platelet recovery, suggesting that megakaryocytemediated platelet biogenesis may be impaired under conditions associated with increased platelet consumption. GLUT1 and GLUT3 single knockout mice exhibited no changes in platelet recovery (Figure S2A and S2B). Cross-sectional analysis of megakaryocytes in femurs and spleens of mice under basal conditions revealed no significant change in megakaryocyte number in DKO mice (Figure 5C and 5D). Megakaryocytes cultured from DKO bone marrow displayed significantly reduced GLUT1 and GLUT3 protein content (Figure S2C and S2D), and demonstrated virtually no glucose uptake (Figure 5E). Importantly, the ability of bone marrow derived megakaryocytes, to produce platelets was significantly impaired in DKO cultures (Figure 5F and 5G), even in the presence of glutamate and pyruvate. These observations define an obligate role for glucose metabolism in platelet generation from megakaryocytes.
Author Manuscript
Phosphatidylserine exposure is increased in DKO platelets in response to metabolic stress
Author Manuscript
Circulating platelet counts reflect the balance between platelet production and clearance. Therefore we investigated circulating half-life of platelets by administration of a Dylight 488 labeled anti-GP1bβ antibody. GLUT1 and GLUT3 single knockout mice demonstrated no reduction in circulating half-life (Figure S2E and S2F), However, DKO platelets demonstrated significantly reduced circulating half-life compared to controls (Figure 6A), suggesting that increased clearance also contributes to the thrombocytopenia observed. An important mechanism of platelet clearance occurs via protein desialylation, which can be monitored by Ricinus communis aggulutinin I (RCA-1) binding (Li et al., 2015). However, DKO platelets demonstrated no change RCA-1 binding (Figure S3A), rendering this mechanism unlikely. We also ruled out increased autophagy or oxidative stress as mechanisms for increased clearance (Figure S3B, S3C). Platelet clearance can also be potentiated by increasing extracellular exposure of PS on plasma membranes (Alonzo et al., 2012). Therefore we monitored annexin V binding on platelets in diluted whole blood, 72 hours post administration of Dylight 488 labeled anti-GP1bβ antibody. Interestingly, GP1bβ positive platelets, which represent older platelets, displayed increased relative annexin V binding marked by the geo. MFI (Figure 6B), but no significant increase was observed in GP1bβ negative platelets or in the population as a whole, suggesting that as DKO platelets Cell Rep. Author manuscript; available in PMC 2017 September 15.
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age they increase PS exposure to the outer leaflet of the plasma membrane. In vitro, control platelets incubated in DMEM with 5mM glucose ± 2mM glutamate and 1mM pyruvate had minimal annexin V positive platelets after 22 hours of incubation (Figure 6C). In contrast, after 22 hours, DKO platelets incubated in glucose alone exhibited ~70% annexin V positivity (Figure 6C). Supplementation of the media with the mitochondrial substrates glutamate and pyruvate significantly reduced but did not normalize annexin V binding in DKO platelets (Figure 6C). Due to optimal signal resolution, a 6-hour in vitro incubation time was chosen for subsequent signaling studies. After 6-hours incubation, DKO platelets incubated in glucose alone exhibited ~25% annexin V positivity, while addition of glutamate and pyruvate decreased annexin V binding by half to ~12%. Control platelets regardless of exogenous substrates demonstrated
Cell Rep. Author manuscript; available in PMC 2017 September 15. Published in final edited form as: Cell Rep. 2017 July 25; 20(4): 881–894. doi:10.1016/j.celrep.2017.06.083.
Deletion of GLUT1 and GLUT3 Reveal Multiple Roles for Glucose Metabolism in Platelet and Megakaryocyte Function Trevor P. Fidler1,2,3, Robert A. Campbell2,4, Trevor Funari3, Nicholas Dunne3, Enrique Balderas Angeles5, Elizabeth Middleton2,4, Dipayan Chaudhuri5, Andrew S. Weyrch2,4, and E. Dale Abel2,3,*,# 1Department
Author Manuscript
2Program
of Pharmacology and Toxicology University of Utah, Salt Lake City, UT, 84112, USA
in Molecular Medicine University of Utah, Salt Lake City, UT, 84112, USA
3Fraternal
Order of Eagles Diabetes Research Center and Division of Endocrinology and Metabolism, Carver College of Medicine, University of Iowa, Iowa City, IA, 52242, USA
4Department
of Internal Medicine, University of Utah, Salt Lake City, UT, 84112, USA
5Nora
Eccles Harrison Cardiovascular Research and Training Institute, University of Utah, Salt Lake City, UT, 84112, USA
Summary
Author Manuscript
Anucleate platelets circulate in the blood to facilitate thrombosis and diverse immune functions. Platelet activation leading to clot formation correlates with increased glycogenolysis, glucose uptake, glucose oxidation, and lactic acid production. Simultaneous deletion of glucose transporter (GLUT) 1 and GLUT3 (double knockout (DKO)) specifically in platelets completely abolished glucose uptake. In DKO platelets mitochondrial oxidative metabolism of non-glycolytic substrates such as glutamate, increased. Thrombosis and platelet activation were decreased through impairment at multiple activation nodes including Ca2+ signaling, degranulation, and integrin activation. DKO mice developed thrombocytopenia, secondary to impaired pro-platelet formation from megakaryocytes, and increased platelet clearance resulting from cytosolic calcium overload and calpain activation. Systemic treatment with oligomycin, inhibiting mitochondrial metabolism, induced rapid clearance of platelets with circulating counts dropping to zero in DKO but not wildtype mice, demonstrating an essential role for energy metabolism in platelet viability. Thus substrate metabolism is essential for platelet production, activation and survival.
Author Manuscript
eTOC blurb
*
Corresponding author, [email protected]. #Lead Contact
Author Contribution Conceptualization, EDA, ASW, and TPF; Methodology, EDA, ASW, TPF, and RC; Investigation, TPF, RC, TF, EBA, and ND; Writing-original draft, TPF; Writing-Review and Editing, TPF, EDA, ASW, and RC; Funding Acquisition, EDA and ASW; Resources, EDA and ASW; Supervision DC, EDA, and ASW Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Fidler et al.
Page 2
Author Manuscript
Fidler et al. show that glucose metabolism is essential for platelet production, activation, and clearance. Their findings reveal complementary roles for glycolysis versus mitochondrial metabolism in platelet viability. Blocking both metabolic pathways leads to complete clearance of platelets from the circulation, due to calcium overload and calpain activation.
Author Manuscript Introduction
Author Manuscript
Glucose enters cells via glucose transporters, of which platelets express the facilitative glucose transporter 1 (GLUT1) (Craik, 1995) and glucose transporter 3 (GLUT3) (Heijnen et al., 1997). ~85% of GLUT3 is located in α-granule membranes with ~15% localized in the plasma membrane. Upon degranulation, GLUT3 translocates to the plasma membrane and is believed to account for the increased glucose uptake following activation (Heijnen et al., 1997). Platelet activation also increases glycolysis and lactic acid production (Karpatkin, 1967), glycogenolysis (Scott and Cooper, 1967) and glucose oxidation (Warshaw et al., 1966). In vitro inhibition of these processes blunts platelet activation (Akkerman, 1981; Yamagishi, 2001), while incubation under hyperglycemic conditions potentiates activation in response to ADP, arachidonic acid, thrombin and collagen (Yamagishi, 2001). However, these associations have not been evaluated in vivo.
Author Manuscript
Although the correlation between activation and increased metabolism is well established, the mechanisms underling these relationships are unknown. Studies of permeabilized platelets, lacking cytosol indicate that ATP is an essential cofactor for Ca2+-induced degranulation (Flaumenhaft, 1999). Administration of the competitive glucose uptake inhibitor 2-deoxyglucose (2-DOG) to platelets suggested that glucose uptake and metabolism plays an important role in platelet aggregation and degranulation. Although these changes correlate with ATP content, a direct relationship between platelet glucose uptake and ATP content remains to be demonstrated (Akkerman, 1979). Conversely, platelets isolated from diabetic patients demonstrate dysfunctional Ca2+ signaling (Li, 2001), although it is unknown if platelet activation in this context arises from altered metabolism.
Cell Rep. Author manuscript; available in PMC 2017 September 15.
Fidler et al.
Page 3
Author Manuscript
Mitochondrial respiration has also been linked to platelet activation. Inhibitors of mitochondrial respiration blunt platelet activation (Garcia-Souza and Oliveira, 2014). Upon activation, a subpopulation of platelets demonstrate mitochondrial depolarization and generation of reactive oxygen species (ROS) which facilitates phosphatidylserine (PS) exposure to the outer leaflet of the plasma membrane that promotes platelet procoagulant activity (Garcia-Souza and Oliveira, 2014). In addition to thrombotic functions, platelet mitochondria occupy a central role in the intrinsic apoptosis pathway (Mason et al., 2007). The balance between BCL-XL activity and formation of mitochondrial Bak/Bax pores regulates platelet apoptosis and platelet viability. However, the relationship between mitochondrial metabolism and apoptotic cell death pathways in platelets remain to be clarified. Moreover, mice infected with dengue demonstrate an additional mechanism of platelet clearance that correlated with PS exposure in vivo (Alonzo et al., 2012). Given the diverse mechanisms leading to platelet clearance, the integration of these mechanisms in the regulation of platelet viability and the contribution of platelet metabolism to these processes remains to be clarified.
Author Manuscript Author Manuscript
Given the dearth of information regarding the role of energy metabolism in regulating platelet biology in vivo, we generated mice lacking GLUT1 and GLUT3 specifically in platelets. Analyses of these mice reveal a dynamic interaction between glucose utilization and mitochondrial energy metabolism to maintain platelet energy requirements. Importantly, glucose metabolism is essential for pro-platelet formation from megakaryocytes. Glucose metabolism is essential for agonist mediated Ca2+ signaling, and downstream signaling events leading to platelet activation. We demonstrate a critical role for glycolytic and mitochondrial metabolism in regulating platelet clearance establishing a paradigm whereby impaired platelet energy metabolism undermines calcium homeostasis, leading to increased cytoplasmic Ca2+ that activates a calpain-mediated cell death and clearance pathway.
Results Glucose metabolism is abolished in DKO platelets
Author Manuscript
To investigate the contribution of glucose metabolism to platelet function, GLUT1 and GLUT3 were individually or simultaneously deleted from platelets by crossing mice expressing a Pf4 promoter-driven Cre recombinase to mice harboring homozygous floxed GLUT1 and GLUT3 alleles individually or in combination. Immunoblot analysis of platelet protein lysates isolated from GLUT1 single knockout (GLUT1-KO), GLUT3 single knockout (GLUT3-KO) and GLUT1/GLUT3 double knockout (DKO) and their respective littermate controls (Figure 1A) confirmed the absence of the respective proteins. Glucose uptake was measured in control quiescent platelets at ~10 pmoles/1×106 platelets/30 minutes, which after thrombin stimulation increased two-fold. Deletion of GLUT3 reduced basal glucose uptake by ~22% and abolished thrombin stimulated glucose uptake (Figure 1B) illustrating that GLUT3 mediates post-activation glucose uptake. Surprisingly, GLUT1KO platelets revealed no changes in basal or thrombin-mediated glucose uptake relative to controls. However, deletion of both GLUT1 and GLUT3 completely abolished glucose uptake under basal and thrombin stimulated conditions indicating that GLUT1 and GLUT3 are the biologically relevant glucose transporters in platelets. These data also demonstrate
Cell Rep. Author manuscript; available in PMC 2017 September 15.
Fidler et al.
Page 4
Author Manuscript
that GLUT1 and GLUT3 have unique, but also overlapping roles in the regulation of platelet glucose uptake.
Author Manuscript
DKO platelets do not undergo glycolysis. Quantitative analysis of 13C-1,6-glucose flux demonstrated DKO platelets fail to convert exogenous glucose to lactic acid (Figure 1C), whereas controls generated 0.0085pg lactic acid/minute/1×106 platelets. In addition, with 13C-glucose as the exclusive exogenous glucose source, 12C-lactic acid release, which represents the contribution of glycogen to glycolysis, was also abolished (Figure 1D). Qualitative analysis of glycolysis determined by the extracellular acidification rate (ECAR) was markedly repressed in DKO platelets (Figure 1E). Of note, ECAR was not zero in DKO platelets potentially reflecting acidification of the media, by acids other than lactate (TeSlaa and Teitell, 2014). Consistent with studies in human platelets (Karpatkin, 1968), thrombin increased glycolysis by ~2-fold in control murine platelets. In contrast, DKO platelets did not increase glycolysis following thrombin stimulation, or in the presence of inhibitors of mitochondrial metabolism (Figure 1E). Platelets contain large glycogen stores, which can be mobilized upon activation (Scott and Cooper, 1967). Glycogen content in DKO platelets was reduced ~7-fold to levels near the limit of detection (Figure 1F). Thus glucose uptake, and metabolism are largely abolished in DKO platelets. DKO platelets increase utilization of alternative mitochondrial substrates
Author Manuscript Author Manuscript
In the absence of glucose uptake, alternative metabolic pathways may sustain cellular ATP to maintain cellular viability. DKO platelets displayed a significantly increased ratio of [AMP+ADP]/[ATP] (Figure 2A), suggesting metabolic stress. Consistent with this, freshly isolated DKO platelets demonstrated increased phosphorylated AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) (Figure 2B). Interestingly, mitochondrial membrane potential was significantly increased in freshly isolated DKO platelets independent of exogenous substrate present in the media (Figure 2C). Seahorse analysis of mitochondrial oxygen consumption rates (OCR) in the presence of glucose, glutamate, and pyruvate indicated that at baseline and under maximal respiration conditions, OCR in DKO platelets were ~3-fold higher than controls (Figure 2D). Increased mitochondrial respiration in DKO reflects increased capacity to utilize mitochondrial substrates. OCR were equivalent in control and DKO platelets with glucose as the sole metabolic substrate, however following addition of glutamate and pyruvate, mitochondrial OCR increased by ~70% in DKO, but was unchanged in controls (Figure 2E). DKO platelets exhibited decreased intracellular lactic acid and relatively increased pyruvate leading to a significantly reduced lactate/pyruvate ratio (Figure 2F), suggesting induction of alternative pathways of pyruvate generation such as the transamination of alanine that requires mitochondrial α-ketoglutarate (Gray et al., 2014). Mitochondrial content and gross structure of DKO platelets were unchanged (Figure 3A and 3B), suggesting a qualitative rather than a quantitative change in mitochondrial function. Thus, absence of glucose metabolism in DKO platelets increased metabolism of alternative mitochondrial substrates. Platelet activation is decreased in DKO platelets DKO platelets exhibited blunted platelet activation. Non-stimulated DKO platelets displayed similar organelle distribution, with the exception of decreased α-granule number (Figure 3A
Cell Rep. Author manuscript; available in PMC 2017 September 15.
Fidler et al.
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and 3B), which may suggest a biogenesis defect. In addition, following stimulation with 250μM PAR4 peptide, degranulation was impaired in DKO platelets as evidenced by retention of α-granules in platelets (Figure 3A and 3C). In vitro analysis of platelets incubated in 5mM glucose for 1-hour, revealed decreased activation of GPIIbIIIa measured by JonA geo. MFI (Figure 3D), and α-granule degranulation marked by CD62p surface translocation (CD62p geo. MFI) (Figure 3E) in response to PAR4 peptide, the thromboxane receptor agonist U46619, ADP, and convulxin. However, this loss of activation was only partially rescued (ADP and U46619 – JonA) when glutamate and pyruvate was added to the media (Figure 3D and 3E), suggesting a direct link between glucose metabolism and platelet activation, that is not rescued in the presence of alternate mitochondrial substrates in DKO platelets. Agonist mediated calcium signaling is impaired in DKO platelets
Author Manuscript Author Manuscript Author Manuscript
In response to activating stimuli, platelets increase cytoplasmic Ca2+ that activates the Ca2+ sensitive scramblase TMEM16f, to facilitate the translocation of PS to the outer leaflet of the plasma membrane leading to a procoagulant response (Jobe et al., 2008; Van Kruchten, 2012). Although PS is exposed, this procoagulant process is distinct from apoptosismediated PS exposure and is not thought to lead to an “eat me” signal that leads to platelet clearance (Van Kruchten, 2012). In response to thrombin plus convulxin, DKO platelets demonstrate impaired exposure of PS to the outer leaflet of the plasma membrane measured by annexin V binding (Figure 4A and S1A–F). Restoring Ca2+ flux by administration of the Ca2+ ionophore ionomycin, completely restored annexin V binding (Figure 4A). In response to thrombin, cytoplasmic Ca2+ flux was blunted in DKO platelets (Figure 4B). Importantly, following the rise in agonist-mediated cytoplasmic Ca2+, control platelets restored cytoplasmic Ca2+ to near basal levels (Figure 4B), whereas levels remained increased in DKO platelets for >10 minutes following stimulation. This inability to export/sequester Ca2+ following stimulation suggests a defect in sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) and/or plasma membrane Ca2+-ATPase (PMCA)-mediated Ca2+ transport. Using an independent methodology, we confirmed impairment of platelet Ca2+ mobilization following combined stimulation by thrombin and convulxin of DKO platelets (Figure 4C and 4D). Following thapsigargin treatment to deplete SERCA, DKO platelets failed to restore Ca2+ concentrations to control levels (Figure 4E and 4F), revealing a defect in store operated Ca2+ entry. DKO platelets exhibit impaired GPIIbIIIa activation and CD62p surface translocation following treatment with thrombin plus convulxin, the Ca2+ ionophore ionomycin, or the SERCA inhibitor thapsigargin (Figure 4G and 4H). These data indicate that glucose metabolism regulates PS exposure to the outer leaflet of the plasma membrane via its effect on calcium mobilization, but even when calcium flux is rescued, glucose metabolism mediates additional steps required for integrin activation and α-granule degranulation. Platelet glucose metabolism is essential for in vivo thrombosis The contribution of platelet glucose metabolism to thrombosis and hemostasis in vivo is unknown. In a tail-bleeding assay, DKO mice exhibited significantly longer time to bleeding cessation, with many mice failing to stop prior to assay completion (Figure 4I). Although spontaneous bleeding was not observed, hematocrit was modestly yet significantly reduced Cell Rep. Author manuscript; available in PMC 2017 September 15.
Fidler et al.
Page 6
Author Manuscript
in untreated DKO mice (Table S1). Arterial thrombosis was also evaluated using 7.5% ferric chloride. DKO mice had significantly increased time to arterial occlusion relative to littermate controls; with many failing to occlude after 20 minutes of observation (Figure 4J). In a collagen/epinephrine-induced pulmonary embolism model, which is dependent on in vivo platelet aggregation, DKO mice demonstrated increased survival (Figure 4K). Thus platelet glucose metabolism is essential for in vivo thrombosis. DKO mice are thrombocytopenic
Author Manuscript
Platelet counts were reduced in DKO mice (Figure 5A, Table S1); however, platelet counts were unchanged in mice with GLUT1 or GLUT3-deficient platelets respectively. To determine if thrombocytopenia resulted from decreased platelet production from megakaryocytes, we administered antibodies to GPIbα that depletes platelets within 18 hours (Figure 5B). In control mice platelet counts completely recovered after 96 hours, whereas DKO mice required 168 hours for platelet recovery, suggesting that megakaryocytemediated platelet biogenesis may be impaired under conditions associated with increased platelet consumption. GLUT1 and GLUT3 single knockout mice exhibited no changes in platelet recovery (Figure S2A and S2B). Cross-sectional analysis of megakaryocytes in femurs and spleens of mice under basal conditions revealed no significant change in megakaryocyte number in DKO mice (Figure 5C and 5D). Megakaryocytes cultured from DKO bone marrow displayed significantly reduced GLUT1 and GLUT3 protein content (Figure S2C and S2D), and demonstrated virtually no glucose uptake (Figure 5E). Importantly, the ability of bone marrow derived megakaryocytes, to produce platelets was significantly impaired in DKO cultures (Figure 5F and 5G), even in the presence of glutamate and pyruvate. These observations define an obligate role for glucose metabolism in platelet generation from megakaryocytes.
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Phosphatidylserine exposure is increased in DKO platelets in response to metabolic stress
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Circulating platelet counts reflect the balance between platelet production and clearance. Therefore we investigated circulating half-life of platelets by administration of a Dylight 488 labeled anti-GP1bβ antibody. GLUT1 and GLUT3 single knockout mice demonstrated no reduction in circulating half-life (Figure S2E and S2F), However, DKO platelets demonstrated significantly reduced circulating half-life compared to controls (Figure 6A), suggesting that increased clearance also contributes to the thrombocytopenia observed. An important mechanism of platelet clearance occurs via protein desialylation, which can be monitored by Ricinus communis aggulutinin I (RCA-1) binding (Li et al., 2015). However, DKO platelets demonstrated no change RCA-1 binding (Figure S3A), rendering this mechanism unlikely. We also ruled out increased autophagy or oxidative stress as mechanisms for increased clearance (Figure S3B, S3C). Platelet clearance can also be potentiated by increasing extracellular exposure of PS on plasma membranes (Alonzo et al., 2012). Therefore we monitored annexin V binding on platelets in diluted whole blood, 72 hours post administration of Dylight 488 labeled anti-GP1bβ antibody. Interestingly, GP1bβ positive platelets, which represent older platelets, displayed increased relative annexin V binding marked by the geo. MFI (Figure 6B), but no significant increase was observed in GP1bβ negative platelets or in the population as a whole, suggesting that as DKO platelets Cell Rep. Author manuscript; available in PMC 2017 September 15.
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age they increase PS exposure to the outer leaflet of the plasma membrane. In vitro, control platelets incubated in DMEM with 5mM glucose ± 2mM glutamate and 1mM pyruvate had minimal annexin V positive platelets after 22 hours of incubation (Figure 6C). In contrast, after 22 hours, DKO platelets incubated in glucose alone exhibited ~70% annexin V positivity (Figure 6C). Supplementation of the media with the mitochondrial substrates glutamate and pyruvate significantly reduced but did not normalize annexin V binding in DKO platelets (Figure 6C). Due to optimal signal resolution, a 6-hour in vitro incubation time was chosen for subsequent signaling studies. After 6-hours incubation, DKO platelets incubated in glucose alone exhibited ~25% annexin V positivity, while addition of glutamate and pyruvate decreased annexin V binding by half to ~12%. Control platelets regardless of exogenous substrates demonstrated
