key: cord-0839106-z8zla3rz
authors: Shrimali, Nishith M; Agarwal, Sakshi; Kaur, Simrandeep; Bhattacharya, Sulagna; Bhattacharyya, Sankar; Prchal, Josef T; Guchhait, Prasenjit
title: α-Ketoglutarate Inhibits Thrombosis and Inflammation by Prolyl Hydroxylase-2 Mediated Inactivation of Phospho-Akt
date: 2021-11-02
journal: EBioMedicine
DOI: 10.1016/j.ebiom.2021.103672
sha: 801c790f1542250b506e688692ec70f2798344b8
doc_id: 839106
cord_uid: z8zla3rz

BACKGROUND: Phospho-Akt1 (pAkt1) undergoes prolyl hydroxylation at Pro125 and Pro313 by the prolyl hydroxylase-2 (PHD2) in a reaction decarboxylating α-ketoglutarate (αKG). We investigated whether the αKG supplementation could inhibit Akt-mediated activation of platelets and monocytes, in vitro as well as in vivo, by augmenting PHD2 activity. METHODS: We treated platelets or monocytes isolated from healthy individuals with αKG in presence of agonists in vitro and assessed the signalling molecules including pAkt1. We supplemented mice with dietary αKG and estimated the functional responses of platelets and monocytes ex vivo. Further, we investigated the impact of dietary αKG on inflammation and thrombosis in lungs of mice either treated with thrombosis-inducing agent carrageenan or infected with SARS-CoV-2. FINDINGS: Octyl αKG supplementation to platelets promoted PHD2 activity through elevated intracellular αKG to succinate ratio, and reduced aggregation in vitro by suppressing pAkt1(Thr308). Augmented PHD2 activity was confirmed by increased hydroxylated-proline and enhanced binding of PHD2 to pAkt in αKG-treated platelets. Contrastingly, inhibitors of PHD2 significantly increased pAkt1 in platelets. Octyl-αKG followed similar mechanism in monocytes to inhibit cytokine secretion in vitro. Our data also describe a suppressed pAkt1 and reduced activation of platelets and leukocytes ex vivo from mice supplemented with dietary αKG, unaccompanied by alteration in their number. Dietary αKG significantly reduced clot formation and leukocyte accumulation in various organs including lungs of mice treated with thrombosis-inducing agent carrageenan. Importantly, in SARS-CoV-2 infected hamsters, we observed a significant rescue effect of dietary αKG on inflamed lungs with significantly reduced leukocyte accumulation, clot formation and viral load alongside down-modulation of pAkt in the lung of the infected animals. INTERPRETATION: Our study suggests that dietary αKG supplementation prevents Akt-driven maladies such as thrombosis and inflammation and rescues pathology of COVID19-infected lungs. FUNDING: Study was funded by the Department of Biotechnology (DBT), Govt. of India (grants: BT/PR22881 and BT/PR22985); and the Science and Engineering Research Board, Govt. of India (CRG/000092).

The serine-threonine kinase Akt, also known as protein kinase B (PKB), contributes to a broad range of cellular functions including cell survival, proliferation, gene expression and migration of cells of most lineages. Akt plays a central role in both physiological and pathological signalling mechanisms. Upon exposure to stimuli, Akt is recruited to the cell membrane by phosphoinositide 3-kinase (PI3K), where it is phosphorylated by membrane associated 3-phosphoinositidedependent kinase-1 (PDK1), leading to its activation. Among the three identified isoforms, Akt1 is widely expressed in most human and mouse cells [1] [2] [3] [4] [5] . The crucial role of PI3K-Akt signalling has been elucidated in platelet activation and functional responses including aggregation, adhesion and thrombus formation [1] [2] [3] [4] [5] [6] [7] . Akt1 À/À mice displayed an increased bleeding time and their platelets showed decreased response to agonists ex vivo [3] . Both Akt1 and Akt2 are reported to facilitate GP2b3a binding to soluble fibrinogen and thus platelet aggregation at low concentration of agonist [2, 3] . Akt2 and Akt3 play important role in granule secretion and aggregation in response to thrombin and TxA2 in vitro and also in arterial thrombosis in vivo [3, 5] . Therefore, several studies have used Akt inhibitors like SH-6, triciribine and Akti-X in an attempt to abrogate platelet aggregation, clot formation and granule secretion in vitro as well as in vivo [4, [8] [9] .

The crucial involvement of PI3K-Akt pathway in regulation of immune cell functions in a broad range of inflammatory diseases such as rheumatoid arthritis, multiple sclerosis, asthma, chronic obstructive pulmonary disease, psoriasis and atherosclerosis has been documented [10] [11] [12] [13] [14] [15] . Pathological signalling of Akt is also well reported in progression of cancer [16] . Activation of PI3K-Akt pathway plays an important role in host immune response to infections including SARS-CoV-2, [17] [18] SARS-CoV, [19] Dengue and Japanese Encephalitis [20] viruses, wherein Akt signalling is pivotal for the virus entry and replication in host cells. Therefore, Akt is a potential therapeutic target in various disease conditions [21] . The PI3K-Akt inhibitor wortmannin has been used to alleviate severity of inflammation and improve survival rate in rats with induced severe acute pancreatitis. Akt1 À/À mice had reduced carrageenan-induced paw oedema and related inflammation alongside a significant decrease in neutrophil and monocyte infiltration [22] . Studies using inhibitors such as Akti-8 and Akt-siRNA support the crucial regulatory role of Akt in inflammatory response of monocytes and macrophages in vitro and in vivo [23] [24] .

It has been reported that phosphorylated Akt1 (pAkt1) is hydroxylated by an oxygen-dependent enzyme, prolyl hydroxylase 2 (PHD2). The pAkt1 undergoes prolyl hydroxylation at Pro125 and Pro313 by PHD2 in a reaction decarboxylating a-ketoglutarate (aKG) . This promotes von HippelÀLindau protein (pVHL) binding to the PHD2 hydroxylated site. pVHL then interacts with protein phosphatase 2A (PP2A), which dephosphorylates Thr308 and to a lesser extent Ser473, resulting in Akt1 inactivation [25] .

In this study, we describe a heretofore unreported role of PHD2 in regulation of platelet and monocyte functions by inactivating pAkt1.

Supplementation with dietary aKG, a metabolite of TCA cycle and a cofactor of PHD2, appears to be a potent suppressor of pAkt, significantly reducing thrombotic and inflammatory events in mice treated with thrombosis-inducing agent carrageenan [26] . Reports suggest that SARS-CoV-2 directly activates platelets [27, 28] and symptoms like thrombosis and inflammation are common in severe forms of COVID-19 [29] . We have tested the rescue effect of dietary aKG on lung inflammation in SARS-CoV-2 infected golden hamsters. The hamster model is well-studied for SARS-CoV-2 infection. Studies have reported that SARS-CoV-2 infection induces significant inflammation of the bronchial epithelial cells and lungs in hamsters [30] . Here, we show that SARS-CoV-2 increases phosphorylation of Akt1 (Thr308) [17] , a known target of PHD2, in infected cells. We describe that dietary aKG significantly reduces clot formation, inflammation and viral load in conjunction with down-modulation of pAkt in lungs of SARS-CoV-2 infected golden hamsters.

Human ethics approval was obtained from the Institutional Ethics Committee (IEC) for human research of Regional Centre for Biotechnology (RCB; ref no. RCB-IEC-H-08) to recruit healthy volunteers. Written informed consent was received from all participants.

Animal ethics approval was obtained from the Institutional Animal Ethics Committee (IAEC) of RCB (ref. no. RCB/ IAEC/2020/077) and experiments using BALB/c mouse strain (RRID: IMSR_JAX_000651) were conducted within the guidelines of IAEC in the Small Animal Facility (SAF) of our institute. Animal ethics approval for SARS-CoV-2 work was obtained from the IAEC (ref. no. RCB/IAEC/2020/069) and Institutional Biosafety Committee (IBSC; ref. no. RCB/IBSC/20-21/221) of RCB and experiments using Syrian golden hamster (available form ICMR-National Institute of Nutrition, Hyderabad, India) were conducted within the guidelines of IAEC in the BSL3 facility of our institute.

Whole blood was collected form healthy individuals in sodium citrate or ACD anticoagulant. Platelets and monocytes were isolated from whole blood and used for in vitro experiments. Volunteers were recruited on the basis of inclusion criteria: 1) healthy, 2) not taking any anti-platelet or anti-inflammatory drugs, 3) no major illness or chronic disease, and 4) no microbial infections within a month of recruitment. Number of healthy individuals recruited for each experiment has been mentioned in respective Figure legends. A written informed consent was received from all participants. 16 ml of whole blood was collected from healthy volunteers by venepuncture in vacutainers containing anti-coagulant sodium citrate or acid-citrate dextrose (ACD). Platelet rich plasma (PRP) was separated by centrifugation at 44 g for 15 min. Sodium citrate containing PRP was used for aggregation and activation studies. PRP in ACD was used for isolating washed platelets for further studies as described in our previous work [31] .

Evidence before this study It has been reported that prolyl hydroxylase-2 (PHD2) can inactivate phosphorylated Akt (pAkt). In von-Hippel-Lindau (VHL)deficient/suppressed cells and under hypoxic microenvironment, accumulation of pAkt is likely to promote tumor growth. The inhibition of pAkt partially reverses this tumorigenic effect. Studies, including the above work, have described that PHD2 catalyses proline hydroxylation of its substrates with concomitant conversion of O2 and a-ketoglutarate (aKG) to CO2 and succinate, respectively. Succinate can inhibit PHD2 by competing with aKG. Therefore, an elevated intracellular ratio of aKG to succinate may be employed as a marker of PHD2 activity.

Our study for the first time describes the regulatory role of the PHD2-pAkt axis in platelet and monocyte functional responses under normoxia. We report an inactivating impact of aKG mediated augmentation of PHD2 activity on phosphorylated Akt1 (pAkt1). An elevated ratio of aKG to succinate in aKG-supplemented platelets and monocytes significantly augmented PHD2 activity and in turn downmodulated pAkt1. Dietary aKG significantly reduced clot formation and leukocyte accumulation in various organs, including lungs, of mice treated with thrombosis-inducing agent carrageenan. Importantly, in SARS-CoV-2 infected hamsters, we observed a significant rescue effect of dietary aKG on inflamed lungs with significantly reduced leukocyte accumulation, clot formation, and viral load alongside downmodulation of pAkt in lungs of the infected animals.

This study proposes a safe supplement of dietary aKG to curtail Akt-driven thrombosis and inflammation in various pathologies, including the inflamed lungs of COVID-19 patients. The study also highlights aKG-PHD2-pAkt axis as a potential target for better pulmonary management in such diseases.

Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll Hypaque (GE Healthcare, Freiburg, Germany) density gradient centrifugation as described [32] . PBMCs were washed twice with phosphate buffered saline (PBS), pH 7.4 and seeded in cell culturetreated plates (Corning, NY, USA) in RPMI-1640 medium (Sigma Aldrich, USA) supplemented with 10% (v/v) foetal bovine serum (Gibco Invitrogen, San Diego, CA), 100 U/mL penicillin and 100 mg/ mL streptomycin for 2 hrs at 37°C in a humidified atmosphere with 5% CO 2 , to allow monocytes to adhere to the plate. After 2 hrs, supernatant containing non-adherent cells were removed and adhered monocytes were used for further treatments described hereafter.

Human PRP diluted (1:1) in Tyrod's buffer pH 7.2 was used for following assays. Diluted PRP was pre-treated with octyl a-ketoglutarate (Sigma Aldrich, USA) or inhibitors to PHD2 such as dimethyl ketoglutarate (DKG, Sigma Aldrich) or ethyl-3-4-dihydroxybenzoic acid (DHB, TCI America, Portland) and incubated with collagen (10 mg/ml) or ADP (2 mM, both from the Bio/Data Corporation, USA) 10 minutes. Platelets were labelled with P-selectin (PE Cy5), PAC-1 (FITC) and Annexin-V (FITC) antibodies (BD bioscience) for 15 min at 37°C and fixed in 1% paraformaldehyde. 20,000 events were acquired using flow cytometry (BD FACS Verse). The acquired data was analysed using the Flowjo software (Tree Star, USA), as described [31] . Supplementary Table S1 lists the details of antibodies that have been used in immunoblot, immunoprecipitation and flow cytometry based experiments.

Platelet aggregation was performed using PAP8 aggregometer (Bio/Data Corporation, USA). PRP was pre-treated with aKG or DKG and incubated with collagen (20 mg/ml) or ADP (5 mM) and aggregation percentage was measured.

Platelet thrombus formation assay was performed by perfusing whole blood (collected in citrate-anticoagulant from healthy individuals) in the Petri plate immobilized with collagen. Whole blood was preincubated for 5 min with either aKG or DKG or both before perfusion on collagen coated surface. A syringe pump (Harvard Apparatus Inc., USA) was connected to the outlet port that drew blood through the chamber at arterial shear stress of 25 dyne/cm 2 . The flow chamber was mounted onto a Nikon Eclipse Ti-E inverted stage microscope (Nikon, Japan) equipped with a high-speed digital camera. Movies were recorded at magnification 40X and analysed using NIS-Elements version 4.2 software as described in our previous study [33] .

PRP was pre-treated with/without aKG and incubated with collagen for 10 min. Platelet-free plasma was obtained by 2 sequential centrifugations: PRP at 1500 g for 7 min followed by platelet-poor plasma (PPP) at 1500 g for 15 min. Platelet-derived microparticles (MPs) were measured using flow cytometry after labelling with anti-CD41 PE antibody as mentioned [31] .

Primary monocytes and monocytic cell line were pre-treated with 1mM octyl a-ketoglutarate (Sigma Aldrich, USA) for 2 hrs and 4 hrs respectively followed by replacement with fresh media. Cells were treated with either S1P (1 mM) or LPS (500 ng/ml). Treated cell supernatant was used for assessing cytokines using the cytometric bead array (CBA). Protein lysate prepared from cell pellet was used for western blotting of signalling molecules.

Cytokines such as TNF-a, IL-1b, IL-6 and IL-10 were measured from human primary monocytes culture supernatant or mice plasma of different treatments as described in Results using CBA and analysed by FCAP array software (BD Biosciences, San Jose, CA,USA).

HIF-1a protein expression was depleted in human U937 cell line (RRID:CVCL_0007) using shRNA targeting HIF-1a (TCRN0000010819, Sigma Aldrich, USA) using a liposome mediated delivery (Life Technologies, Thermo Fisher Scientific, USA). The original cell line is validated and was also Mycoplasma contamination free.

2.9. Generation of PHD2 depleted monocyte cell line PHD2 was depleted in human U937 cell line using shRNA targeting EGLN1 as described [34] . The original cell line is validated and was also Mycoplasma contamination free.

Male BALB/c mice aged 5-7 weeks or Syrian golden hamsters of 8 weeks were supplemented with 1% of dietary a-ketoglutarate (SRL, Mumbai, India) in drinking water for 24 or 48 hrs (to mice), or for 6 days (to hamsters) as described in schematic Fig. 4 , 5 and 6. The blood cell counts and other assays were performed.

PRP was collected from control and aKG-treated mice. The sample size calculation was performed after doing a pilot study with 4 BALB/c mice per group. Based on the outcome of this pilot study, we calculated number of mice per group at a confidence interval of 95% and power of 80% to get a p-value less than 0.01 (http://www.lasec.cuhk. edu.hk/sample-size-calculation.html). The predicted number of mice to be kept in each group was calculated, n=6. PRP was and diluted with PBS (1:1 vol) and processed for centrifugation at 90 g using brake-free deceleration on a swinging bucket rotor for 10 min. Platelets counts were adjusted to 2.5£10 8 /ml and platelet aggregation was evaluated using PAP8 aggregometer. Collagen (7.5 mg/ml) or ADP (5 mM) was used as aggregation agonist.

2.12.Carrageenan. treatment to mice and measuring thrombosis and cellularity scores BALB/c mice were used to develop carrageenan-induced thrombosis model [35] . The sample size calculation was performed after doing a pilot study with 4 BALB/c mice per group. Based on the outcome of this pilot study, we calculated number of mice per group at a confidence interval of 95% and power of 80% to get a p-value less than 0.01 (http://www.lasec.cuhk.edu.hk/sample-size-calculation.html). The predicted number of mice to be kept in each group was calculated, n=7. Mice were injected with 100 ml of 10 mg/ml k-carrageenan (Sigma Aldrich, USA) prepared in normal saline in intraperitoneal cavity. aKG was supplemented via drinking water to these mice. After 48 hrs of carrageenan treatment, length of thrombus covered tail was measured and percentage tail thrombosis was calculated by length of thrombus covered tail/total length of tail£100.

Thrombosis score was measured in mice lungs and livers. Lungs and liver samples were fixed in 4% formalin and paraffin embedded.

2.5 mm thick sections were prepared and stained with Haematoxylin and Eosin (H&E) and Masson's trichrome (MT). Slides were observed under Nikon Eclipse Ti-E inverted stage microscope (Nikon, Japan) and images were acquired at 20 X to observe thrombi and at 40X to observe leukocyte accumulation. Thrombosis scoring was calculated using ImageJ software. Thrombi were selected using freehand selection tool in MT-stained slides. Percentage area covered was calculated as percentage of freehand selected area covering the total area. The lung sections were used for immunostaining using platelet marker CD42c antibody. The leukocyte accumulation was assessed as a marker of inflammation in the above lung section using percentage cellularity. Cellularity score was calculated using ImageJ software. Images were converted to RGB stack and from that all nuclei were selected based on the intensity of colour and size from H&E-stained slides. Percentage area covered by nucleated cells was calculated by measuring nuclear area as a percentage of the total tissue section area.

Carrageenan-induced peritoneal inflammation was measured in mice using a modified protocol as described [35, 36] . BALB/c mice received carrageenan (10 mg/ml) or saline intraperitoneally. At 3hrs and 6hrs, the animals were anaesthetized and peritoneal exudates were harvested in 3 ml of PBS. Different immune cell populations in peritoneal lavage were analysed and cell types were identified by flow cytometry using CD45.2, CD11b, CD11c, Ly6G, Ly6C, and CD41 expression [32, 33] . In another set of a similar experiment, BALB/c mice were injected with carrageenan. At 3 and 6 hrs, peritoneal inflammation was visualised using bioluminescence-based imaging of MPO activity by injecting luminol (i.p. 20 mg/100 g body weight) (Sigma Aldrich) 6 min prior to imaging using an in-vivo imaging system (IVIS; Perkin Elmer, Waltham, MA, USA) as described in our work [32] .

The hamster model of SARS-CoV-2 infection has been well established [30] . Male hamsters of 8 weeks old were infected with SARS-CoV-2 (isolate USA-WA-1/2020 from World Reference Center for Emerging Viruses and Arboviruses, from UTMB, Texas, USA), via nasal route inoculation using 1£10 6 plaque-forming units (PFU) as described [30] .

1% dietary aKG was administered via drinking water and 400ml of 10% aKG was given through oral gavage on day 3 through day 5. The sample size calculation was performed after doing a pilot study with 3 Male golden hamsters per group. Based on the outcome of this pilot study, we calculated number of hamsters per group at a confidence interval of 95% and power of 80% to get a p-value less than 0.01 (http://www.lasec.cuhk.edu.hk/sample-size-calculation.html).

The predicted number of hamsters to be kept in each group was calculated, n=5. During this phase, hamsters were symptomatic and were not drinking sufficient (male hamster of 100 gm B wt. drinks normally 5 ml per day) water. The schematic protocol of infection and therapy is described in Fig. 6 . At day-6, animals were sacrificed and lungs and liver samples were harvested, fixed in 4% formalin, paraffin embedded and processed for H&E and MT staining. The thrombosis and inflammation scores were measured as described above. The lung sections were used for immunohistochemistry staining for pAkt (Cell Signalling Tech, USA) Body weight was recorded on alternate days. The lung sections were used for measuring viral genome using RT-PCR.

Human liver cell line Huh7 (RRID:CVCL_0336) seeded (6x10 4 cells/well) and pre-treated with 1mM octyl aKG for 2 hrs and infected with 0.1 MOI of SARS-CoV-2 for 24 hrs in BSL3 facility. The cells pellet was lysed and fixed for using estimation of pAkt1 using Western blotting. The original cell line is validated and was also Mycoplasma contamination free.

Lung tissue sample from hamsters was homogenized in Trizol reagent (MRC, UK) using a hand-held tissue-homogenizer and the total RNA extracted as per manufacturer's protocol. 1 mg total RNA was reverse-transcribed using Superscript-III reverse-transcriptase (Invitrogen, USA) as per manufacturer's protocol, using random hexamers (Sigma Aldrich, USA). The cDNA was diluted in nuclease-free water (Promega, USA) and used for real-time PCR with either SARS-CoV-2 or GAPDH specific primers, using 2x SYBR-green mix (Takara Bio, Clontech, USA) in an Applied Biosystems Ò QuantStudio TM 6 Flex Real-Time PCR System. The oligonucleotides used were SARS-F (5 0 -CAATGGTTTAACAGGCACAGG-3 0 ) and SARS-R (5 0 -CTCAAGTGTCTGTGGATCACG-3 0 ) for SARS-CoV-2, and G3PDH-F (5 0 -GACATCAAGAAGGTGGTGAAGCA-3 0 ) and G3PDH-R (5 0 -CAT-CAAAGGTGGAAGAGTGGGA-3 0 ). The Ct value corresponding the viral RNA was normalised to that of G3PDH transcript. The relative level of SARS-CoV-2 RNA in mock-infected samples was arbitrarily taken as 1 and that of infected samples expressed as fold-enrichment (FE). The FE value for each infected sample was transformed to their logarithmic value to the base of 10 and plotted.

The viral genome was measured in Huh7 cell line treated with SARS-CoV-2 using RT-PCR as described above. The Ct value corresponding the viral RNA was normalised to that of RNAse P (RP) transcript.

The oligonucleotides used were RP-F (5 0 -AGATTTGGACCTGCGAGCG-3 0 ) and RP-R (5 0 -GAGCGGCTGTCTCCA-CAAGT-3 0 ).

PHD2 was immunoprecipitated from platelet lysate using protein G Sepharose beads and anti-PHD2 antibody (Cell Signalling Tech, USA). Similarly, phosphorylated Akt (pAkt) was immunoprecipitated using protein A Sepharose beads and anti-pAkt antibody (Cell Signalling Tech, USA). Briefly, washed platelets isolated from whole blood of healthy volunteer and lysed in lysis buffer (25 mM Tris, 150 mM sodium chloride, 1 mM EDTA, 1% NP-40, 50 mM sodium fluoride and 3% Glycerol) with protease and phosphatase inhibitor. Lysate was pre-cleared with protein A or G Sepharose beads and then added to antibody coated beds and incubated overnight. Beads were removed, washed with lysis buffer and collected protein sample was processed for western blotting.

The whole cell (platelets or primary monocyte or monocytic cell line) lysate was prepared using RIPA lysis buffer and protease-phosphatase inhibitor (Thermo Scientific Life Tech, USA). SDS-PAGE gel was followed by immunoblotting using primary antibodies against pAkt(Ser4730, Akt, pAkt1(Thr 308), Akt1, HIF-1a, HIF-2a, b-Actin (Cell Signalling, USA) and a-tubulin (Thermo Fisher Scientific, USA) as described in detail in our previous work [31] . The detailed information of antibodies is described in Table S1 .

Steady-state level of a-ketoglutarate, lactate, fumarate, pyruvate and succinate was estimated in plasma, PBMC-granulocytes (10 5 ) and platelets (10 5 ) of mice or human samples from different treatments as per manufacture protocol (Sigma Aldrich, USA catalogue no. MAK054, MAK064, MAK060, MAK071, MAK335 respectively).

aKG treated and control mice platelets were stimulated with collagen (10 mg/ml) in vitro and supernatant was collected and used to estimate Sphingosine-1-phosphate (S1P) level as per manufacturer's protocol [Cloud clone corp. (CEG031Ge)]. Similarly, human platelets were treated with aKG and collagen (5 mg/ml) and supernatant were used to estimate S1P level. The detailed information of reagents used in this study is described in Table S2 .

Data from at least three experiments are presented as mean § SEM (standard error of the mean). Statistical differences among experimental sets with normally distributed data were analysed by using either unpaired t test, or one-way or two-way ANOVA followed by Bonferroni's correction for multiple comparison. Kruskal Wallis test followed by Dunn's multiple comparison post-test was used for non-normally distributed data. D'Agostino-Pearson Test was used to check for normal distribution of data. Graph Pad Prism version 8.0 software was used for data analysis and P-values <0.05 were considered statistically significant.

This study is supported by BT/PR22881 and BT/PR22985 from the Department of Biotechnology (DBT), Govt. of India; and CRG/000092 from the Science and Engineering Research Board, Govt. of India to PG. The funder had no role in study design, data collection, data analysis, interpretation, or writing the manuscript.

We searched for the presence of all 3 isoforms PHD1, PHD2 and PHD3 in platelets (Fig. 1a) . A recent report has shown that PHD2 hydroxylates the proline residues of phosphorylated Akt1(Thr308) [pAkt1(Thr308)] eventually leading to inactivation [25] . Therefore, we measured phosphorylation status of Akt1 and PHD2 activity in agonist-activated platelets in normoxia. As expected, the phosphorylation of Akt(Ser473) and Akt1(Thr308) increased in tandem with higher concentrations of agonists, collagen (*P<0.05, **P<0.01, ****P<0.0001; Fig. 1b -d) and ADP (**P<0.01, ****P<0.0001; Fig. S1a ), indicating attenuated activity of PHD2. Also, we measured expression of other known substrates of PHD2 such as HIF1a and HIF2a in agonist-activated platelets. The increased expression of HIF1a and HIF2a (**P<0.01, ***P<0.001, ****P<0.0001; Fig. 1b ) suggested a lower activity of PHD2 in activated platelets. This suggested that the enzymatic activity of PHD2 remains below the threshold level to inactivate pAkt.. We then tested whether agonist or other chemical induced alteration in prolyl-hydroxylase activity of PHD2 can in turn alter the activation status of pAkt in platelet.

To alter the enzymatic activity of PHD2, we used a-ketoglutarate (aKG, a cofactor of PHD2) and dimethyl ketoglutarate (DKG, an inhibitor to PHD2). Octyl aKG (a membrane-permeating form) supplementation significantly decreased the collagen-induced phosphorylation of Akt1 (Thr308) and Akt(Ser473) (*P<0.05, ***P<0.001, ****P<0.0001; Fig. 1eg) , and also degraded HIF1a and HIF2a (*P<0.05, **P<0.01; Fig. 1e-g) in platelets under normoxia. Interaction of pAkt1 with PHD2 was confirmed by immunoprecipitation of PHD2 followed by immunoblotting for pAkt1(Thr308). In presence of aKG, enzymatically active PHD2 displayed increased binding to pAkt1(Thr308) (****P<0.0001; Fig. 1h ), resulting into the inactivation of pAkt1 in collagen-activated platelets. In order to evaluate the prolyl-hydroxylation activity of PHD2 on pAkt, immunoprecipitation was performed using Akt(Ser473) antibody and immunoblotted for hydroxylated proline. Increased levels of pAktbound hydroxy proline (*P<0.05; Fig. 1h ), suggested an elevated hydroxylation of proline on pAkt in platelets in presence of aKG. On the other hand, the PHD2 inhibitor DKG favoured the Akt phosphorylation ( Fig. 1e-g) . Other known inhibitor of PHD2, ethyl-3-4-dihydroxybenzoic acid (DHB), also elevated phosphorylation of Akt1(Thr308) and Akt (Ser473) (*P<0.05, **P<0.01; Fig. S1b ). Although, aKG and DKG altered the enzymatic function of the PHD2 to regulate pAkt, neither of the treatments altered the expression of PHD2 in platelets significantly (Fig. 1e) . We examined the effect of aKG on PI3K, activator of Akt. Our data show no significant effect of aKG on the expression of phosphorylated PI3K(p55) (Fig. S1c) , thus, confirming that its specific target is pAkt.

Octyl aKG supplementation suppressed the expression of cell surface activation markers P-selectin, PS, PAC-1 binding to GP2b3a integrin on collagen-activated platelets (*P<0.05; Fig. 1j -l) and microparticle release from activated platelets (Fig. S3 ) in a concentrationdependent manner in vitro. In contrast, DKG enhanced the above parameters (*P<0.05, **P<0.01,***P<0.001; Fig. 1j-l) . Similarly, aKG suppressed platelet aggregation induced by collagen (**P<0.01, ***P<0.001; Fig. 1m -n) or ADP (Fig. S4a-b) in a concentration-dependent manner, but DKG enhanced it (*P<0.05; Fig. 1m-n) . Another PHD2 inhibitor DHB also enhanced collagen-induced platelet aggregation (*P<0.05, **P<0.01; Fig. S4j-k) . Further, our data show that platelet thrombus formation was increased in a dose-dependent manner when whole blood was treated with DKG and perfused under flow shear condition on immobilized collagen surface. aKG significantly suppressed DKG-induced thrombus formation (****P<0.0001; Fig. 1o-p) . Collagen-activated platelets secreted large amount of sphingosine-1-phosphate (S1P), a known stimulator of monocytes, which too was reduced by aKG supplementation (*P<0.05; Fig. 1q ).

Furthermore, our data show that DKG can rescue the suppressive effect of aKG on pAkt1(Thr308) (*P<0.05), and P-selectin and PS expression, and PAC-1 binding on platelets (*P<0.05) and platelet aggregation (**P<0.01, Fig. S4c-i) .

We then investigated the PHD2-mediated inhibition of pAkt1 (Thr308) in monocytes, isolated from healthy individuals and activated with either S1P or LPS after pre-treatment with octyl aKG in vitro. We show that the aKG supplementation decreased both Akt (Ser473) and pAkt1(Thr308) (*P<0.05, ****P<0.0001; Fig. 2a-c) , and HIF1a and HIF2a (**P<0.01; Fig. 2a-c) . Simultaneous suppression in secretion of inflammatory cytokines including, IL1b, IL6, TNFa and IL10 was observed (*P<0.05, **P<0.01,***P<0.001, ****P<0.0001; Fig. 2d-g) . Similar outcomes were observed in monocytes exposed to LPS after pre-treatment of aKG (Fig. S5) . We then confirmed that the above mechanism of aKG-induced suppression of monocyte activation is mediated primarily by pAkt, independent of HIFa. In HIF1adepleted U937 monocytic cells (detailed protocol of shRNA-mediated depletion is described in Fig. S6 ), aKG supplementation significantly reduced cytokine secretion (*P<0.05; Fig. 2h -i) alongside down-modulated pAkt1(Thr308) (***P<0.001; Fig. 2j-k) .

To confirm that aKG-mediated suppression of pAkt in monocyte is mediated by PHD2, we performed the above experiment in PHD2- or DKG (10 and 100 mM) and the expression of above signalling molecules and PHD2 was measured using WB. (f-g) Densitometry data show suppression of collagen-induced elevation of pAkt and pAkt1 by aKG, but an elevation of these molecules in presence of DKG. Other densitometry data are described in Fig. S14d-f . (h-i) (h) Immunoprecipitation (IP) of PHD2 from lysate of washed-platelets from above experiment was performed and processed for WB of pAkt; further, IP of pAkt from same lysate and WB for hydroxy proline shows the interaction between the molecules. (i) Densitometry data of pAkt. Other densitometry data are described in Fig. S14g-h. (j-k) PRP from above experiment of Fig. 1c was processed for measuring surface (j) P-Selectin, (k) PS (Annexin-V binding) and (l) GP2b3a activation (PAC-1 binding) using flow cytometry. (m-n) (m) Platelet aggregation was performed using PRP pre-treated with aKG or DKG in response to collagen. (n) Percentage platelet aggregation was measured. Data from similar experiment in response to agonist ADP are described in Fig. S4. (o-p) (o) PRP from healthy individuals was incubated with aKG or DKG and perfused on immobilized collagen surface under arterial flow share condition 25 dyne/cm 2 and platelet thrombus formation was measured. Scale bar 50 mm. (p) Thrombus area was measured. (q) Secretion of Sphingosine-1-phosphate (S1P) was quantified from supernatant of aKGand collagen-treated washed platelets using ELISA. Data in above figure are mean § SEM from 3 independent experiments (one-way ANOVA, *P<0.05. **P<0.01, ***P<0.001, ****P<0.0001 and ns=non-significant). depleted U937 monocytic cells. Our data show that S1P-induced elevation of pAkt1 was not significantly suppressed by octyl aKG in PHD2-depleted cells (Fig. 2l-m) , highlighting the role of PHD2 in the inactivation of pAkt1.

To ascertain the mechanism of augmentation of PHD2 activity, we measured an elevated level of intracellular aKG in collagen-activated platelets after octyl aKG supplementation, even though the level was unaltered in collagen-activated platelets compared to resting platelets, in vitro (*P<0.05; Fig. 3a ). Since succinate, a product of aKG-dependent dioxygenase reaction in TCA cycle, inhibits PHD2 function, we measured its intracellular levels and found no significant change in platelets after collagen activation as well as post aKG supplementation (Fig. 3b) . However, the intracellular aKG to succinate ratio was elevated in collagenactivated platelets after aKG supplementation, which might have played a role in augmentation of PHD2 activity (*P<0.05, **P<0.01; Fig. 3c ) as suggested by others [37] as well as our recent work [34] . The intracellular level of other metabolites such as fumarate and pyruvate were found unaltered ( Fig. S7a-b) . We observed elevated lactate in supernatant of activated platelets, which levels of which were reduced by aKG supplementation (*P<0.05; Fig. 3d ).

We observed a similar elevation of intracellular aKG to succinate ratio in S1P-stimulated monocytes after octyl aKG supplementation, although the ratio was unaltered in S1P-activated monocytes compared to untreated monocytes (*P<0.05, **P<0.01, ***P<0.001; Fig. 3e-g) , which might have played a role in augmentation of PHD2 activity.

We investigated whether the supplementation of dietary aKG inhibits platelet aggregation in mice, and observed that 1% aKG via drinking water for 24 and 48 hrs (experimental details are described in Fig. 4a ) significantly inhibited platelet aggregation ex vivo, in response to agonists such as collagen (***P<0.001; Fig. 4b -c) and ADP (*P<0.05, **P<0.01; Fig. S9c ). The above aKG supplementation did not alter the number of platelets and WBCs counts of mice (Fig. S8ac) , suggesting a feasible administration of this metabolite. In a recent work, we have described the safe rescue effect of 1% dietary aKG in mice exposed to hypoxia treatment [34] .

We investigated the enzymatic activity of PHD2 in mice from above experiment. Our data show that the dietary aKG supplementation elevated aKG level in plasma (**P<0.01; Fig. 4d ) and also in platelets (**P<0.01; Fig. 4e ). An increased intracellular ratio of aKG to succinate in platelets (*P<0.05; Fig. 4f -g) might have augmented PHD2 activity. Indeed, the decreased expression of pAkt1 (**P<0.01) and HIF2a in platelets of aKG-supplemented mice has confirmed the augmented-activity of PHD2 (Fig. 4h-i) . The platelets from aKG-supplemented mice had a reduced secretion of inflammatory mediator S1P (**P<0.01; Fig. 4j ).

Similarly, PBMCs collected from aKG-supplemented mice had elevated level of intracellular aKG as well as aKG to succinate ratio (*P<0.05; Fig. 4k-m) . The PBMC of aKG-treated mice when exposed to stimulator like S1P in vitro, showed suppressed expression of pAkt and HIF2a (**P<0.01; Fig. 4n-o) . Based on these observations we further tested the effect of dietary aKG supplementation in animal models of induced thrombosis and inflammation.

Dietary supplementation of aKG (starting at 24 or 48 hrs before carrageenan treatment) significantly reduced tail thrombosis (**P<0.01; Fig. 5b-c) and clot formation in lungs and liver (**P<0.01, ***P<0.001, **** P<0.0001; Fig. 5d -g, and S10a-b), and platelet accumulation in lungs (***P<0.001; Fig. S10c-d) . We also found decrease in accumulation of leukocytes in lungs of carrageenan-treated mice supplemented with aKG (**P<0.01, **** P<0.0001; Fig. 5h-i, Fig. S11 ). aKG supplementation also suppressed the levels of inflammatory cytokines such as IL1ß, IL6 and TNFa in plasma of carrageenan treated mice (*P<0.05,**P<0.01, ***P<0.001, **** P<0.0001; Fig. 5jm) . Besides, aKG-supplemented mice when treated with carrageenan locally at abdomen and for a short period (3 and 6 hrs), displayed a decreased release of myeloperoxidase (MPO) (*P<0.05, **** P<0.0001; Fig. 5n-p) and less accumulation of monocytes, neutrophils and leukocyte-platelet aggregates (*P<0.05,**P<0.01, ***P<0.001, **** P<0.0001; Fig. 5q-t; Fig. S12b-c) in the peritoneum.

A recent report described the increased expression of pAkt1 (Thr308) in human alveolar epithelial type 2 cells after SARS-CoV-2 infection [17] . We also observed a similar elevated expression of pAkt1(Thr308) in human liver Huh7 cell line infected with this virus. As expected, we observed a significant reduction in pAkt1(Thr308) expression (***P<0.001, ****P<0.0001; Fig. 6a -b) along with decreased IL6 secretion (*P<0.05; Fig. 6c ) in these infected cells after octyl-aKG supplementation, but viral load did not change significantly (Fig. 6d) . No change in expression of PHD2 was observed in Huh7 cells after viral infection as well as after aKG treatment (Fig. 6a) . We then tested the rescue effect of dietary aKG (1%), administered via drinking water and oral gavage (protocol of treatment is described in Fig. 6e) . We observed significantly reduced evidence of lung thrombi in SARS-CoV-2 infected hamsters (Fig. 6f) . The histopathology data showed a significantly reduced intravascular clot formation (*P<0.05; Fig. 6g -h) and leukocyte accumulation in alveolar spaces (****P<0.0001; Fig. 6i-j) in the lung of SARS-CoV-2 infected hamsters after aKG administration. The elevated expression of pAkt in lung of SARS-CoV-2 infected animals was significantly reduced after aKG supplementation (**P<0.01; Fig. 6k-l) . The elevated expression of HIF2a in lungs of SARS-CoV-2 infected animals was also significantly reduced after aKG supplementation (**P<0.01; Fig. S13ab ). The body weight decreased significantly in infected animals during day 3 -6, but no rescue effect of aKG on body weight was observed (Fig. 6m) . As reported by others, [30] we did not observe death of SARS-CoV-2 infected hamsters. We also observed a gradual increase in body weight, day 8 onwards in another group of all infected hamsters. However, a decreased viral load in lungs was observed on day 6 in infected animals supplemented with aKG (*P<0.05; Fig. 6n ). Supplementation with 1% dietary aKG for 6 days to control hamsters did not alter the number of blood cells including Fig. 3 . Elevation in intracellular ratio of aKG to succinate promotes PHD2 activity in both platelet and monocyte. (a-d) PRP isolated from healthy individuals was pre-treated with aKG and activated in presence of collagen/ADP. Platelet pellet was washed and lysed, and used for measuring intracellular (a) aKG and (b) succinate using colorimetry-based assay. (c) Represents intracellular ratio of aKG to succinate. (d) Release of Lactate from supernatant of platelets from above experiment was quantified. Data of Pyruvate and Fumarate from platelet lysate is described in Fig. S7 . (e-f) Similarly, intracellular (e) aKG and (f) succinate were measured from cell lysate of primary monocytes pre-treated with aKG and stimulated with S1P as described in Fig 2a. (g) Represents ratio of aKG to succinate in monocytes. Data in above figure are mean § SEM from 3 independent experiments in duplicates (one-way ANOVA, *P<0.05 and **P<0.01, ***P<0.001 and ns=non-significant). platelet, WBCs and granulocytes ( Fig. S8d-f) , suggesting a safe implementation of the metabolite.

Our study for the first time describes the regulatory role of PHD2-pAkt axis in platelet function. We report an inactivating impact of a-ketoglutarate (aKG) mediated augmentation of prolyl hydroxylation activity of PHD2 on phosphorylated Akt1 (pAkt1). An earlier study has described that pAkt1 undergoes prolyl hydroxylation at Pro125 and Pro313, by PHD2, in a reaction decarboxylating aKG.

Hydroxylated pAkt1(Thr308) is dephosphorylated by Von Hippel-Lindau protein (pVHL) associated protein phosphatase 2A (PP2A), leading to Akt inactivation. Study thus unveiled this pathway as another mechanism of post translational modification for pAkt, and described that in VHL-deficient/suppressed setting and under hypoxic microenvironment, accumulation of pAkt is likely to promote tumour growth and its inhibition partially reverses the effect [25] .

We describe here that the supplementation with aKG, an intermediate of TCA cycle, significantly suppresses pAkt1 and reduces agonist-induced platelet activation under normoxia. Upon activation by agonists like collagen [3] and ADP [38] platelets undergo Akt phosphorylation by PDK1. Akt phosphorylation is known to stimulate cell surface adhesion molecules like GP2b3a and GPVI, and promote platelet aggregation, adhesion, and secretion of granular contents [39, 40] . aKG supplementation significantly inhibits these functions of platelets by suppressing pAkt1. In contrast, a marked amplification in platelet activity alongside increased pAkt1 was observed after treatment with PHD2 inhibitors like DKG or DHB. This indicates a crucial involvement of PHD2 in the regulation of platelet activation. This conclusion was further supported by the aKG-mediated attenuation of HIF1a and HIF2a expression, known substrates of PHD2, and there by DKG/DHB-driven stabilization. Augmented PHD2 activity orchestrated these events as confirmed by increased hydroxylated proline alongside enhanced binding of PHD2 to pAkt in aKG-treated platelets. Further, our study also describes an increased intracellular aKG: succinate ratio in platelets after aKG supplementation. PHD2 catalyses proline hydroxylation of its substrates by converting O 2 and aKG to CO 2 and succinate, [41] and succinate can inhibit PHD2 by competing with aKG [42] Therefore, an elevated intracellular ratio of aKG to succinate may serve as a stimulator of PHD2 activity [37] It is notable that aKG: succinate ratio was unaltered in platelets after activation with agonist, but its elevated ratio in aKG-supplemented platelets significantly augmented PHD2 activity and in turn downmodulated pAkt1. However, several studies have reported increased pAkt in platelets and other cells in vitro [43, 44] and in vivo [45] after succinate supplementation. This suggests that intracellular ratio of these two metabolites serves as a switch for the PHD2 activity. We show that aKG supplementation significantly decreased platelet's pAkt and inhibited aggregation, thrombus formation and secretion of granular contents including inflammatory mediator sphingosin-1 phosphate (S1P), in vitro. S1P is an intermediary between platelet activation and inflammation as it activates monocytes. We observed that aKG could also suppress S1P-mediated activation of monocytes in a pAkt1 dependent manner. When LPS was used as an activator to simulate a thrombo-inflammatory condition, aKG could deter the secretion of pro-inflammatory cytokines from monocytes. Importantly, our data showed no significant inactivation of pAkt1 by aKG in PHD2-deficient monocytic cell line, thus confirming that aKG imparts its effects primarily through PHD2. Overall, our data upholds PHD2 as a potential target to abrogate Akt signalling. Other studies have extensively used antagonists/inhibitors to target pathological signalling of Akt or PI3K-Akt to inhibit thrombosis and inflammation [8, 4, [23] [24] .

Our study underscores the implementation of dietary aKG supplementation to mice as a promising treatment to reduce the platelet aggregation and inflammatory response of monocytes by downmodulating pAkt1 without altering the number of these cell types, thus suggesting a safe administration of this metabolite. In a separate study, we have described a rescue effect of this metabolite in mice from hypoxia-induced inflammation by downmodulating HIF1/2a and NFkB [34] . Another recent study has mentioned the induction of IL10 by dietary aKG and suppression of chronic inflammation in mice [46] . aKG suppresses the NFkB mediated inflammation in piglets [47] . aKG has been used extensively for in vivo experimental therapies for manipulating multiple cellular processes related to organ development and viability of organisms, [48, 49] restriction of tumour growth and extending survival, [50] and preventing obesity [51] .

We also describe the aKG-mediated rescue of clot formation and leukocyte accumulation alongside a reduction in cytokine secretion by these cells in lungs and other organs in mice exposed to a thrombosis-inducing agent like carrageenan. Furthermore, our study also reports a significant rescue effect of aKG on inflamed lung in SARS-CoV-2 infected hamsters along with significant reduction in intravascular clot formation, prevention of accumulation of leukocytes including macrophages and neutrophils in alveolar spaces of the lung of infected hamsters. This indicates that aKG usage can decelerate inflammation induced lung tissue damage in severe cases of SARS-CoV-2 infection and may eventually delay or even abrogate the development of acute respiratory distress syndrome (ARDS), a known symptom of COVID-19 [52, 53] . However, more experimental evidence will be needed before the implementation of this metabolite in this clinical scenario. The aKG administration also decreased viral load in lungs of the infected animals along with concomitant decrease in pAkt. The exact role Akt in replication of SARS-CoV-2 remains to be delineated; but it has been reported in other viruses [54] However, a recent study has described an elevated pAkt1 (Thr308) in cells infected with SARS-CoV-2 [17] . Our in vitro data also show an elevated pAkt1(Thr308) alongside increased IL6 secretion by SARS-CoV-2 infected Huh7 cell line, which was further inhibited by aKG administration. All these observations underscore that the augmentation of PHD2 activity by aKG could be a potential therapeutic strategy to inhibit pAkt-mediated maladies like inflammation and thrombosis, as well as possible propagation of SARS-CoV-2.

We depict a novel role of PHD2-pAkt axis in the regulation of platelet and leukocyte functions. Supplementation with aKG significantly increases the hydroxylase activity of PHD2 and therefore reduces phosphorylation of Akt and in turn suppresses thrombotic and inflammatory functions of platelets and leukocytes respectively (schematic Figure 7) .

We propose a safe implementation of dietary aKG in prevention of Aktdriven thrombosis and inflammation in various disease scenarios including inflamed lung of COVID-19 patients.

In Fig 5, we used carrageenan-induced thrombosis mice model. We did not have access to more widely used thrombosis mice models such as FeCL 3 -induced mesenteric arteriole thrombosis model. . aKG mediated suppression of platelet and monocyte functions. Schematic depicts that under normoxic environment supplementation with aKG (known cofactor of PHD2) increases PHD2 activity by elevating intracellular aKG: succinate ratio. Elevated PHD2 activity degrades pAkt1 and reduces platelet activation and aggregation. A similar mechanism also suppresses inflammatory function of monocytes. Thus, suggesting the involvement of aKG-PHD2-Akt1 axis in the regulation of events like thrombosis and inflammation. Importantly, dietary aKG supplementation significantly rescues mice from carrageenan-induced clot formation, and leukocyte accumulation and inflammation in various organs including lung. Importantly, dietary aKG rescues significantly hamsters from SARS-CoV-2 induced clot formation and leukocyte accumulation in the lung, alongside downmodulation of pAkt.

We did not have access to use mice models of SARS-CoV-2 infection. We used hamster, which is also an established model for SARS-CoV-2 infection. The detailed inflammatory markers including specific immunes cells and cytokines were not assessed due to limitation in availability of these reagents.

NMS designed and performed all experiments, analysed data and wrote the manuscript. SA designed and performed mice experiment for immunophenotyping and histochemistry and analysed related data. SK and Sankar B designed and performed COVID-19 infection studies, and Sankar B has supervised the infection experiments. Sulagna B designed and performed metabolite estimation experiments, analysed and interpreted data, and edited the manuscript. JTP provided crucial conceptual inputs and edited the manuscript. PG designed and supervised the study, conceptualized the approach, designed the experiments, analyzed the data, and wrote the manuscript. All authors read, edited and approved the final manuscript.

The materials described in the manuscript, including all relevant raw data will be freely available from the corresponding author. Immunostaining of platelet marker in the lung sections from above experiment. Table S1 : List of antibodies used in the study. Supplementary Table S2 : List of reagents used in the study.

Authors declare that there is no conflict of interest.

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Authors acknowledge generous help of Prof. Sudhanshu Vrati of Regional Centre for Biotechnology, Faridabad, India for sharing SARS-CoV-2 virus strain. Authors acknowledge Dr. Arundhati Tiwari, Department of Biochemistry, Institute of Medical Sciences, Banaras Hindu University, India for editing the manuscript. Authors also acknowledge the funding by grants: BT/PR22881 and BT/PR22985 from the Department of Biotechnology (DBT), Govt. of India; and CRG/000092 from the Science and Engineering Research Board, Govt. of India to PG.

Supplementary material associated with this article can be found in the online version at doi:10.1016/j.ebiom.2021.103672.