key: cord-1036746-mwvryc6k
authors: Shrimali, Nishith M; Agarwal, Sakshi; Kaur, Simrandeep; Bhattacharya, Sulagna; Bhattacharyya, Sankar; Prchal, Josef T; Guchhait, Prasenjit
title: α-ketoglutarate augments prolyl hydroxylase-2 mediated inactivation of phosphorylated-Akt to inhibit induced-thrombosis and inflammation
date: 2021-08-06
journal: bioRxiv
DOI: 10.1101/2021.06.11.448037
sha: 1b41f32b9ab2c1b527d66e161dd23737ea8aa10b
doc_id: 1036746
cord_uid: mwvryc6k

Phosphorylation of Akt (pAkt) regulates multiple physiological and pathological processes including thrombosis and inflammation. In an approach to inhibit the pathological signalling of pAkt by prolyl-hydroxylase-2 (PHD2) we employed α-ketoglutarate (αKG), a cofactor of PHD2. Octyl-αKG supplementation to platelets promoted PHD2 activity through elevated intracellular αKG:succinate ratio and reduced aggregation in vitro by suppressing pAkt1(Thr308). Augmented PHD2 activity was confirmed by increased hydroxylated-proline alongside 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 platelet and leukocyte obtained from mice supplemented with dietary-αKG, unaccompanied by alteration in their counts. Dietary-αKG significantly reduced clot formation and leukocyte accumulation in various organs including lung of mice treated with thrombosis-inducing agent carrageenan. Importantly, we observed a significant rescue effect of dietary-αKG on inflamed lung of SARS-CoV-2 infected hamsters. αKG significantly reduced leukocyte accumulation, clot formation and viral load alongside downmodulation of pAkt in lung of the infected animals. Therefore, our study suggests a safe implementation of dietary-αKG in prevention of Akt-driven anomalies including thrombosis and inflammation, highlighting a better pulmonary management in COVID-19.

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) and therefore activated. Among the three isoforms, Akt1 is widely expressed in most of the cell types in both human and mice (1) (2) (3) (4) (5) . The poignant role of PI3K-Akt signalling is well investigated in platelet activation and functions including aggregation, adhesion and thrombus formation (1) (2) (3) (4) (5) (6) (7) . Platelets from Akt1−/− mice displayed an increased bleeding time and in ex vivo their platelets minimally responded to agonists (3) . Studies using inhibitors to Akt including SH-6, triciribine and Akti-X describe the important role of this signaling adaptor molecule in platelets functions including aggregation, clot formation and granule secretion in vitro and in vivo (4, (8) (9) .

Extensive studies have reported the crucial involvement of the PI3K-Akt pathway in the regulation of immune cell function in a broad range of inflammatory diseases such as rheumatoid arthritis, multiple sclerosis, asthma, chronic obstructive pulmonary disease, psoriasis, and atherosclerosis (10) (11) (12) (13) (14) (15) . Besides, the pathological signalling of Akt is well reported in the progression cancer or tumor cells (16) . Activation of PI3K-Akt pathway is highly relevant in infections like SARS-CoV-2 (17) (18) , SARS-CoV (19) , Dengue and Japanese Encephalitis (20) viruses. Akt signalling has been found to be pivotal for the virus entry and replication in host cells.

Therefore, extensive reports suggest Akt as a potential therapeutic target in various disease conditions (21) . The PI3K-Akt inhibitor wortmannin has been used to alleviate the severity of inflammation and improve the survival rate in rats with induced severe acute pancreatitis. Akt1−/− mice showed a markedly reduced carrageenan-induced paw edema and related inflammation alongside a significant decrease in neutrophil and monocyte infiltration (22) . Studies using inhibitors such as Akti-8 and Akt-siRNA describe the crucial regulatory role of Akt in inflammatory response of monocytes and macrophages in vitro and in vivo (23) (24) .

In the context of regulation of the Akt pathway, a study has 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 Hippel-Lindau protein (pVHL) binding to the hydroxylated site. pVHL then interacts with protein phosphatase 2A (PP2A), which dephosphorylates the Thr308 site, resulting in Akt1 inactivation (25) .

In this study, we describe a heretofore unreported role of PHD2 in the regulation of platelet and monocyte functions by inactivating pAkt1. Supplementation with dietary αKG, a metabolite of TCA cycle and a cofactor of PHD2, appears to be a potent suppressor of pAkt, significantly reducing clot formation and leukocyte accumulation and related thrombotic and inflammatory events in mice treated with thrombosis-inducing agent like carrageenan (26) . Further, we show a rescue effect of aKG on lung inflammation in SARS-CoV-2 infected hamsters. SARS-CoV-2 infection to golden hamsters induces significant inflammation of the bronchial epithelial cells and lung, in turn increases the disease severity between days 3-5 (27) . SARS-CoV-2 significantly increases phosphorylation of Akt1(Thr308) (17) , a known target of PHD2, in infected cells. We describe that dietary aKG significantly reduced clot formation, inflammation and viral load in conjunction with downmodulation of pAkt in the lungs of hamsters with SARS-CoV-2 infection.

Agonist mediated platelet activation is directly related to phosphorylation of Akt1 but inversely with prolyl-hydroxylase activity of PHD2

We checked the presence of all 3 isoforms PHD1, PHD2 and PHD3 in platelets ( Figure 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 in agonist-activated platelets and check if the PHD2 activity is functionally relevant. As expected, the phosphorylation of Pan-Akt or Akt1(Thr308) was increased with higher concentrations of agonists like collagen ( Figure 1B -D) and ADP (Supplemental Figure   1A ), indicating an insufficient activity of PHD2 enabling the hydroxylation of elevated level of pAkt after agonist stimulation. To confirm the prolyl-hydroxylation of elevated level of pAkt after agonist stimulation. To confirm prolyl-hydroxylation activity of PHD2, we measured expression of other known substrates of the enzyme, such as HIF1a and HIF2a in agonist-activated platelets.

We observed stabilization of both HIF1a and HIF2a in activated platelets under normal oxygen condition or normoxia ( Figure 1B ). It pertinently raised the speculation that the enzymatic activity of PHD2 remains below the threshold level to inactivate pAkt, which heightens in platelets after agonist stimulation. We then tested whether agonist or other chemical induced alteration in prolylhydroxylase 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) or pan-Akt, and also destabilized HIF1a and HIF2a in platelets under normoxia ( Figure 1E -G).

Interaction of pAkt1 with PHD2 was confirmed by immunoprecipitation of PHD2 followed by immunoblotting for pAkt1(Thr308). A significantly increased binding of PHD2 to pAkt1(Thr308) in presence of aKG, suggests a pre-existing condition of elevated prolyl-hydroxylation and inactivation of pAkt1 in collagen-activated platelets ( Figure 1H ). In order to check the enzymatic activity of PHD2 on pAkt, immunoprecipitation was performed using pan-pAkt antibody and immunoblotted for hydroxy proline. An increased level of pAkt-bound hydroxy proline ( Figure   1H ), suggests an elevated hydroxylation of proline on pAkt in platelets after aKG supplementation. On the other hand, the PHD2 inhibitor DKG promoted the Akt phosphorylation ( Figure 1E -G). Other known inhibitor of PHD2, such as ethyl-3-4-dihydroxybenzoic acid (DHB) also exhibited an elevated Akt phosphorylation (Supplemental Figure 1B) . 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 ( Figure 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) (Supplemental Figure 1C) , thus, confirming its specific target on pAkt.

Octyl aKG supplementation suppressed the expression of cell surface activation markers such as P-selectin, phosphatidylserine (PS) and PAC-1 binding to GPIIbIIIa integrin on collagen-activated platelets ( Figure 1J -L) and microparticle release from activated platelets (Supplemental Figure 3) in a concentration-dependent manner in vitro. In contrast, DKG enhanced the above parameters ( Figure 1J -L). Similarly, aKG suppressed platelet aggregation induced by collagen ( Figure 1M -N) or ADP (Supplemental Figure 4A -B) in a concentration-dependent manner, but DKG enhanced it. Another PHD2 inhibitor DHB also enhanced collagen-induced platelet aggregation (Supplemental Figure 4C -D). 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 DKGinduced thrombus formation ( Figure 1O -P). Collagen-activated platelets secreted large amount of sphingosine-1-phosphate (S1P), known stimulator of monocytes, which too was reduced by aKG supplementation ( Figure 1Q ).

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. Our data show that the aKG supplementation decreased pAkt1(Thr308) and increased degradation of HIF2a (Figure 2A -C). Simultaneous suppression in secretion of inflammatory cytokines including, IL1β, IL6, TNFa and IL10 was observed ( Figure 2D -G). Similar outcomes were observed in monocytes exposed to LPS after pre-treatment of aKG (Supplemental Figure 5 ).

We then confirmed that the above mechanism of aKG-induced suppression of monocyte activation is mediated primarily by pAkt, independent of HIFa. In HIF1a-depleted U937 monocytic cells (detailed protocol of shRNA-mediated depletion is mentioned in Supplemental Figure 6 ), aKG supplementation significantly reduced cytokine secretion ( Figure 2H -I) alongside downmodulated pAkt1(Thr308) ( Figure 2J -K).

To confirm that aKG-mediated suppression of pAkt in monocyte is mediated by PHD2, we performed the above experiment in PHD2-depleted U937 monocytic cells. Our data show that S1P-induced elevation of pAkt1 was not significantly suppressed by octyl aKG in PHD2-depleted cells ( Figure 2L -M), highlighting the role of PHD2 in the inactivation of pAkt1.

Elevated intracellular ratio of aKG to succinate correlates with augmented activity of PHD2 in platelet and monocyte after octyl aKG supplementation

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, although the level was unaltered in collagen-activated platelets compared to resting platelets in vitro ( Figure   3A ). Since, succinate, a product of aKG-dependent dioxygenase reaction in TCA cycle, inhibits PHD2 function; we measured its intracellular level and found no significant change in platelets after collagen activation as well as post aKG supplementation ( Figure 3B ). However, the intracellular aKG to succinate ratio was elevated in collagen-activated platelets after aKG supplementation, which might have played a role in augmentation of PHD2 activity ( Figure 3C) as suggested by others (28) as well as our recent work (29) . The intracellular level of other metabolites such as fumarate and pyruvate were found unaltered (Supplemental Figure 7A -B). We observed elevated level of lactate in the supernatant of activated platelets, which was reduced by aKG supplementation ( Figure 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 SIP-activated monocytes compared to untreated monocytes (Figure 3 E-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 detailed is mentioned in Figure 4A ) significantly inhibited platelet aggregation ex vivo in response to agonists such as collagen ( Figure 4B -C) and ADP (Supplemental Figure 9C ). The above aKG supplementation did not alter the counts of platelets and WBCs in mice (Supplemental Figure 8A- C), suggesting a safe implementation of the metabolite. In a recent work, we have mentioned the safe rescue effect of 1% dietary aKG in mice exposed to hypoxia treatment (29) .

We have investigated the enzymatic activity of PHD2 in mice from above experiment. Our data show that the dietary aKG supplementation elevated aKG level in plasma ( Figure 4D ) and also in platelets ( Figure 4E ). An increased intracellular ratio of aKG to succinate in platelets ( Figure 

A recent report suggested 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 along with decreased IL6 secretion in these infected cells after octyl-aKG supplementation ( Figure 6A -C), but viral load did not change significantly ( Figure 6D ). We then tested the rescue effect of dietary aKG (1%), administered via drinking water and oral gavage (protocol of treatment is mentioned in Figure 6E ), and observed significantly reduced clot injury spots on lung in SARS-CoV-2 infected hamsters ( Figure 6F ). The histopathology data showed a significantly reduced intravascular clot formation ( Figure 6G Figure 13A -B). The body weight decreased significantly in infected animals during day 3 -day 6, but no rescue effect of aKG on body weight was observed ( Figure 6M ). As reported by others (27), we did not observe death of SARS-CoV-2 infected hamsters. We also observed a gradual increase in body weight on day 8 onwards in another group of all infected hamsters. However, a decreased viral load in the lung was observed at day 6 in infected animals supplemented with aKG ( Figure 6N ). Supplementation with 1% dietary aKG for 6 days to control hamsters did not alter the count of blood cells including platelet and WBCs and granulocytes (Supplemental Figure 8D -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.

Also, we show heretofore unreported 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 unveils this pathway as another line of post translational modification for pAkt.

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 (30) platelets undergo Akt phosphorylation by PDK1. Akt phosphorylation is known to stimulate cell surface adhesion molecules like GPIIbIIIa and GPVI, and promote platelet aggregation and adhesion, and also secretion of granular contents (31) (32) . aKG supplementation significantly inhibits the above -mentioned functions of platelets by suppressing pAkt1. Contrastingly, 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. The above speculation was further supported by the aKG-mediated reduced expression of HIF1a and HIF2a, known substrates of PHD2, and DKG/DHB-driven stabilization of the same. Augmented PHD2 activity orchestrated these events was confirmed by increased hydroxylated proline alongside enhanced binding of PHD2 to pAkt in aKG-treated platelets. Besides, our study also describes an increased intracellular aKG: succinate ratio in platelets after aKG supplementation. PHD2 catalyses proline hydroxylation of its substrate by converting O2 and aKG to CO2 and succinate (33) , and succinate can inhibit PHD2 by competing with aKG (34). Therefore, an elevated intracellular ratio of aKG to succinate may serve as a stimulator of PHD2 activity as suggested (28) . It is notable that aKG: succinate ratio was unaltered in platelet after activation with agonist, but its elevated ratio in aKG-supplemented platelet significantly augmented PHD2 activity and in turn downmodulated pAkt1. Contrastingly, several studies have reported an increased pAkt in platelets and other cells in vitro (35) (36) and in vivo (37) after succinate supplementation. Thus, indicating that intracellular ratio of these two metabolites serves as a switch for the PHD2 activity.

We show that aKG supplementation significantly decreased pAkt in platelet to inhibit aggregation, thrombus formation and secretion of granular contents including inflammatory mediator such as sphingosin-1 phosphate (S1P) in vitro. S1P is one of the connecting between platelet activation and systemic inflammation as it can activate monocytes. Interestingly 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 primary through PHD2 in this context. We could also confirm that the outcomes of aKG usage were independent of HIF1a. Overall, our data upholds PHD2 as a potential target to abrogate Akt signalling. 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 highlights the implementation of dietary aKG to mice as one of the potential treatments to reduce the platelet aggregation and inflammatory response of monocyte by downmodulating pAkt1 without altering the count of these cell types. Therefore, it suggests a safe administration of this metabolite. In a recent work, we have described a safe rescue effect of this metabolite in mice from hypoxia-induced inflammation by downmodulating HIFa (29) . αKG has been used extensively for in vivo experimental therapies for manipulating multiple cellular processes related to organ development and viability of organisms (38) (39) , restriction of tumor growth and extending survival (40) , and preventing obesity (41) .

Another interesting part of our study describes the aKG-mediated rescue of clot formation and leukocyte accumulation alongside a reduction in cytokine secretion by these cells in lung and other organs in mice exposed to a thrombosis-inducing agent like carrageenan. Specifically, our study also reports a significant rescue effect of aKG on inflamed lung in SARS-CoV-2 infected hamsters. A significant reduction in intravascular clot formation and accumulation of leukocytes including macrophages and neutrophils in alveolar spaces of the lung of infected hamsters suggests a potential rescue effect of aKG. Thus, indicating that aKG usage can decelerate inflammation induced lung tissue damage reported in severe cases of SARS-CoV-2 infection and may eventually deter development of acute respiratory distress syndrome (42) (43) . However, this speculation needs further experimental evidences. Besides, aKG treatment also decreased viral load in the lung of the infected animals alongside diminished expression of pAkt. Although the exact role Akt in replication of SARS-CoV-2 remains to be delineated as reported for other viruses (44) . But its noteworthy that 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. Thus, suggesting that the augmentation of PHD2 activity by aKG would be a potential therapeutic strategy to inhibit pAkt-mediated anomalies like inflammation and thrombosis in host and also propagation of SARS-CoV-2. Therefore, our data together suggest a novel role of PHD2-pAkt axis in the regulation of platelet and leukocyte functions. Supplementation with αKG significantly increases the hydroxylase activity of PHD2 and therefore reduces phosphorylation of Akt and in turn supresses thrombotic and inflammatory functions of platelets and leukocytes respectively, depicted in schematic Figure 7 . Thus, suggesting a safe implementation of dietary aKG in prevention of Aktdriven thrombosis and inflammation in various disease conditions including inflamed lung in COVID-19. Study also highlights aKG-PHD2-pAkt axis as a potential target for better pulmonary management in these diseases.

Whole blood (16 ml) 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. 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 (45) .

Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll Hypaque (GE Healthcare, Freiburg, Germany) density gradient centrifugation as mentioned in our earlier work (46) . PBMCs were washed twice with PBS, pH 7.4 and seeded in cell culture-treated plates (Corning, NY, USA) in RPMI-1640 medium (Sigma Aldrich, USA) supplemented with 10% (v/v) fetal bovine serum (Gibco Invitrogen, San Diego, CA), 100 U/mL penicillin and 100 µg/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 mentioned hereafter.

Human PRP diluted (1:1) in Tyrod's buffer pH 7.2 was used for following assays. 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 mentioned (45) .

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

Platelet thrombus formation assay was performed by perfusing whole blood (collected in citrateanticoagulant from healthy individuals) over the petri plate immobilized with collagen. Whole blood was preincubated for 5 min with either αKG 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 mentioned in our previous study (47) .

PRP was pre-treated with/without αKG 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 (45) .

Primary monocytes and monocytic cell line were pre-treated with 1mM octyl α-ketoglutarate (Sigma Aldrich, USA) for 2 hrs and 4 hrs respectively followed by replacement with fresh media.

Cells were treated with either S1P (1 µM) 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-α, IL-1β, IL-6 and IL-10 were measured from human primary monocytes culture supernatant or mice plasma of different treatments as mentioned in results using CBA and analysed by FCAP array software (BD Biosciences, San Jose, CA,USA).

HIF-1α protein expression was depleted in human U937 cell line (ATCC, USA) using shRNA targeting HIF-1α (TCRN0000010819, Sigma Aldrich, USA) using a liposome mediated delivery (Life Technologies, Thermo Fisher Scientific, USA).

PHD2 was depleted in human U937 cell line using shRNA targeting EGLN1 as mentioned our publication (29) .

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

PRP was collected from control and αKG-treated mice 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 performed using PAP8 aggregometer. Collagen (7.5 µg/ml) or ADP (5 µM) was used as aggregation agonist.

BALB/c mice were used to develop carrageenan-induced thrombosis model (26) . Mice were injected with 100 µl of 10 mg/ml κ-carrageenan (Sigma Aldrich, USA) prepared in normal saline in intraperitoneal cavity. αKG 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 lung and liver of above mice. Lungs and liver samples were fixed in 4% formalin and paraffin embedded. 2.5 µm 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 40 X 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. Similarly, 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 color 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 (48) . 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 counts were determined by flow cytometry using CD45.2, CD11b, CD11c, Ly6G, Ly6C, and CD41 (46, 49) . 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 mentioned in our work (46) .

Male golden hamsters of 8 weeks old were given infection of 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 mentioned (27) .

1% dietary αKG was administered via drinking water and 400µl of 10% αKG was given through oral gavage on day 3 through day 5. During this phase they 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 mentioned in Figure 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 mentioned 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 (ATCC, USA) was seeded (6x10 4 cells/well) and pre-treated with 1mM octyl αKG 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.

Lung tissue sample from hamsters was homogenized in Trizol reagent (MRC, UK) using a handheld tissue-homogenizer and the total RNA extracted as per manufacturer's protocol. 1 µg 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 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, Akt, pAkt1(Thr 308), Akt1, HIF-1α, HIF-2α, β-Actin (Cell Signalling, USA) and α-tubulin (Thermo Fisher Scientific, USA) as described in detail in our previous work (45) . The detailed information of antibodies is mentioned in Supplemental Table 1 .

Steady-state level of α-ketoglutarate, Lactate, Fumarate, Pyruvate and Succinate was estimated in plasma, PBMC-granulocytes (10 5 ) and platelets (10 5 with αKG and collagen (5 µg/ml) and supernatant was used to estimate S1P level.

Data from at least three experiments are presented as Mean ± SEM (Standard Error of the Mean).

Statistical differences among experimental sets were analysed by Unpaired t test. Graph Pad Prism version 8.0 software was used for data analysis and P-values <0.05 were considered statistically significant.

Supplemental Supplemental Figure 14 : Densitometry of western blots.

Supplemental Table 1 : List of antibodies used in the study.

Human samples: Ethics approval was obtained from the Institutional Ethics Committee (IEC) for Schematic depicts that under normoxic environment supplementation with αKG (known cofactor of PHD2) increases PHD2 activity by elevating intracellular αKG: succinate ratio. Elevated PHD2 activity degrades pAkt1 and reduces platelet activation and aggregation. A similar mechanism also suppresses inflammatory function of monocyte. Thus, suggesting the involvement of αKG-PHD2-Akt1 axis in the regulation of events like thrombosis and inflammation. Importantly, dietary αKG supplementation significantly rescues mice from carrageenan-induced clot formation and leukocyte accumulation and inflammation in various organs including lung. Importantly, dietary αKG rescues significantly hamsters from SARS-CoV-2 induced clot formation and leukocyte accumulation in the lung, alongside downmodulation of pAkt.

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Improving frequency of thrombosis by altering blood flow in the carrageenan-induced rat tail thrombosis model

Pathogenesis and transmission of SARS-CoV-2 in golden hamsters

Cell-Permeating α-Ketoglutarate Derivatives Alleviate Pseudohypoxia in Succinate Dehydrogenase-Deficient Cells

Gain-of-function Tibetan PHD2D4E;C127S variant suppresses monocyte function: A lesson in inflammatory response to inspired hypoxia

Protease-activated receptors 1 and 4 do not stimulate Gi signaling pathways in the absence of secreted ADP and cause human platelet aggregation independently of Gi signaling

Integrins : dynamic scaffolds for adhesion and signaling in platelets Integrins : dynamic scaffolds for adhesion and signaling in platelets

Akt signaling in platelets and thrombosis

Oxygen Sensing by Metazoans: The Central Role of the HIF Hydroxylase Pathway

Prolyl 4-Hydroxylase

Succinate independently stimulates full platelet activation via cAMP and phosphoinositide 3-kinase-b signaling

Succinate induces skeletal muscle fiber remodeling via SUCNR1 signaling

The Succinate Receptor GPR91 Is Involved in Pressure Overload-Induced Ventricular Hypertrophy

The metabolite αketoglutarate extends lifespan by inhibiting ATP synthase and TOR

Yuan Alpha-ketoglutarate ameliorates age-related osteoporosis via regulating histone methylations

α-Ketoglutarate attenuates Wnt signaling and drives differentiation in colorectal cancer

Dietary alphaketoglutarate promotes beige adipogenesis and prevents obesity in middle-aged mice

Pulmonary vascular endothelialitis, thrombosis and angiogenesis in COVID-19

A Descriptive and Quantitative Immunohistochemical Study Demonstrating a Spectrum of Platelet Recruitment Patterns Across Pulmonary Infections Including COVID-19

Viral control of mitochondrial apoptosis

Hemoglobin interaction with GP1bα induces platelet activation and apoptosis: A novel mechanism associated with intravascular Hemolysis

Neutrophils develop rapid proinflammatory response after engulfing Hb-activated platelets under intravascular hemolysis

HbS Binding to GP1bα Activates platelets in sickle cell disease

Anti-inflammatory activity of aqueous extract and bioactive compounds identified from the fruits of Hancornia speciosa Gomes (Apocynaceae). B. M. C

Two physically, functionally, and developmentally distinct peritoneal macrophage subsets

Authors acknowledge generous help of Prof. Sudhanshu Vrati of Regional Centre for of India to PG.

The authors have declared that no conflict of interest exists.