α-Ketoglutarate Inhibits Thrombosis and Inflammation by Prolyl Hydroxylase-2 Mediated Inactivation of Phospho-Akt.

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).

Neutrophil-to-lymphocyte ratio, platelets-to-lymphocyte ratio, and eosinophils correlation with high-resolution computer tomography severity score in COVID-19 patients.

Inflammation has an important role in the progression of various viral pneumonia, including COVID-19. Circulating biomarkers that can evaluate inflammation and immune status are potentially useful in diagnosing and prognosis of COVID-19 patients. Even more so when they are a part of the routine evaluation, chest CT could have even higher diagnostic accuracy than RT-PCT alone in a suggestive clinical context. This study aims to evaluate the correlation between inflammatory markers such as neutrophil-to-lymphocyte ratio (NLR), platelets-to-lymphocytes ratio (PLR), and eosinophils with the severity of CT lesions in patients with COVID-19. The second objective was to seek a statically significant cut-off value for NLR and PLR that could suggest COVID-19. Correlation of both NLR and PLR with already established inflammatory markers such as CRP, ESR, and those specific for COVID-19 (ferritin, D-dimers, and eosinophils) were also evaluated. One hundred forty-nine patients with confirmed COVID-19 disease and 149 age-matched control were evaluated through blood tests, and COVID-19 patients had thorax CT performed. Both NLR and PLR correlated positive chest CT scan severity. Both NLR and PLR correlated positive chest CT scan severity. When NLR value is below 5.04, CT score is lower than 3 with a probability of 94%, while when NLR is higher than 5.04, the probability of severe CT changes is only 50%. For eosinophils, a value of 0.35% corresponds to chest CT severity of 2 (Se = 0.88, Sp = 0.43, AUC = 0.661, 95% CI (0.544; 0.779), p = 0.021. NLR and PLR had significantly higher values in COVID-19 patients. In our study a NLR = 2.90 and PLR = 186 have a good specificity (0.89, p = 0.001, respectively 0.92, p<0.001). Higher levels in NLR, PLR should prompt the clinician to prescribe a thorax CT as it could reveal important lesions that could influence the patient's future management.

Immature platelets as a biomarker for disease severity and mortality in COVID-19 patients.

COVID-19, caused by SARS-CoV-2, is a contagious life-threatening viral disease that has killed more than three million people worldwide to date. Attempts have been made to identify biomarker(s) to stratify disease severity and improve treatment and resource allocation. Patients with SARS-COV-2 infection manifest with a higher inflammatory response and platelet hyperreactivity; this raises the question of the role of thrombopoiesis in COVID-19 infection. Immature platelet fraction (IPF, %) and immature platelet counts (IPC, ×109 /l) can be used to assess thrombopoiesis. This study investigates whether the level of thrombopoiesis correlates with COVID-19 severity. A large cohort of 678 well-characterized COVID-19 patients was analyzed, including 658 (97%) hospitalized and 139 (21%) admitted to the intensive care unit (ICU). Elevated percentage IPF at presentation was predictive of length of hospitalization (P < 0·01) and ICU admission (P < 0·05). Additionally, percentage IPF at the peak was significantly higher among ICU patients than non-ICU patients (6·9 ± 5·1 vs 5·3 ± 8·4, P < 0·01) and among deceased patients than recovered patients (7·9 ± 6·3 vs 5·4 ± 7·8, P < 0·01). Furthermore, IPC at the peak was significantly higher among ICU patients than non-ICU patients (18·5 ± 16·2 vs. 13·2 ± 8·3, P < 0·05) and among patients on a ventilator than those not (22·1 ± 20·1 vs.13·4 ± 8·4, P < 0·05). Our study demonstrated that elevated initial and peak values of percentage IPF and IPC might serve as prognostic biomarkers for COVID-19 progression to severe conditions.

Neutrophil-to-lymphocyte ratio and platelet-to-lymphocyte ratio as mortality predictor of advanced stage non-small cell lung cancer (NSCLC) with COVID-19 in Indonesia.

OBJECTIVE: This study aimed to compare the mortality rate between advanced-stage non-small cell lung cancer patients (NSCLC) with and without COVID-19. This study also explores the possible laboratory characteristics used for prognostication in patients with NSCLC and COVID-19. Additionally, this study evaluated potential differences in laboratory values between the case and control groups. PATIENTS AND METHODS: This is a single-center retrospective cohort study conducted in Dharmais National Cancer Hospital, Indonesia, enrolling patients with NSCLC undergoing chemotherapy or targeted therapy between May 2020 and January 2021. All patients with NSCLC and COVID-19 in these periods were enrolled into the case group. The control group was age-matched NSCLC patients without COVID-19 that was derived from the NSCLC cohort through randomization. RESULTS: There were 342 patients with NSCLC between May 2020 and January 2021. Twenty-seven (7.9%) of the patients were infected by COVID-19. To facilitate comparison, thirty-five age-matched controls with NSCLC were selected from the cohort. The mortality rate in patients with COVID-19 was 46.2%. Eleven patients (40.7%) had severe COVID-19, of which none survived. NLR >8.35 has a sensitivity of 83.3%, specificity of 92.9%, LR+ of 12, and LR- of 0.18. The AUC was 0.946 (95% CI 0.867-1.000), p<0.001. PLR >29.14 has a sensitivity of 75.0%, specificity of 71.4%, LR+ 2.62, LR- 0.35, and AUC 0.851 (95% CI 0.706-0.996), p=0.002. Both NLR and PLR were associated with shorter time-to-mortality in the unadjusted and adjusted model CONCLUSIONS: NLR and PLR are independent predictors of mortality in COVID-19 patients with NSCLC.

Proteomics: A Tool to Study Platelet Function.

Platelets are components of the blood that are highly reactive, and they quickly respond to multiple physiological and pathophysiological processes. In the last decade, it became clear that platelets are the key components of circulation, linking hemostasis, innate, and acquired immunity. Protein composition, localization, and activity are crucial for platelet function and regulation. The current state of mass spectrometry-based proteomics has tremendous potential to identify and quantify thousands of proteins from a minimal amount of material, unravel multiple post-translational modifications, and monitor platelet activity during drug treatments. This review focuses on the role of proteomics in understanding the molecular basics of the classical and newly emerging functions of platelets. including the recently described role of platelets in immunology and the development of COVID-19.The state-of-the-art proteomic technologies and their application in studying platelet biogenesis, signaling, and storage are described, and the potential of newly appeared trapped ion mobility spectrometry (TIMS) is highlighted. Additionally, implementing proteomic methods in platelet transfusion medicine, and as a diagnostic and prognostic tool, is discussed.

COVID-19-related coagulopathy: A review of pathophysiology and pharmaceutical management.

December 2019 will never be forgotten in the history of medicine when an outbreak of pneumonia of unknown etiology in Wuhan, China sooner or later prompted the World Health Organization to issue a public health warning emergency. This is not the first nor will it be the last time that a member of ß-coronaviruses (CoVs) is waging a full-scale war against human health. Notwithstanding the fact that pneumonia is the primary symptom of the novel coronavirus (2019nCoV; designated as SARS-CoV-2), the emergence of severe disease mainly due to the injury of nonpulmonary organs at the shadow of coagulopathy leaves no choice, in some cases, rather than a dreadful death. Multiple casual factors such as inflammation, endothelial dysfunction, platelet and complement activation, renin-angiotensin-aldosterone system derangement, and hypoxemia play a major role in the pathogenesis of coagulopathy in coronavirus disease 2019 (COVID-19) patients. Due to the undeniable role of coagulation dysfunction in the initiation of several complications, assessment of coagulation parameters and the platelet count would be beneficial in early diagnosis and also timely prediction of disease severity. Although low-molecular-weight heparin is considered as the first-line of treatment in COVID-19-associated coagulopathy, several possible therapeutic options have also been proposed for better management of the disease. In conclusion, this review would help us to gain insight into the pathogenesis, clinical manifestation, and laboratory findings associated with COVID-19 coagulopathy and would summarize management strategies to alleviate coagulopathy-related complications.

Platelet hyperactivity in COVID-19: Can the tomato extract Fruitflow® be used as an antiplatelet regime?

The ongoing coronavirus disease 2019 (COVID-19) pandemic caused by the SARS-CoV-2 virus is now considered a global public health threat. The primary focus has been on reducing the viral spread and treating respiratory symptoms; as time goes on, the impact of COVID-19 on neurological and haemostatic systems becomes more evident. The clinical data suggest that platelet hyperactivity plays a role in the pathology of COVID-19 from its onset and that platelets may serve critical functions during COVID-19 progression. Hyperactivation of blood platelets and the coagulation system are emerging as important drivers of inflammation and may be linked to the severity of the 'cytokine storm' induced in severe cases of COVID-19, in which disseminated intravascular coagulation, and platelet hyperactivity are associated with poor prognosis and increased risk of mortality. We propose that targeting platelet hyperactivity in the early stages of COVID-19 infection may reduce the immunothrombotic complications of COVID-19 and subdue the systemic inflammatory response. Lowering baseline platelet activity may be of particular importance for higher-risk groups. As an alternative to antiplatelet drugs, an inappropriate intervention in public health, we propose that the dietary antiplatelet agent Fruitflow®, derived from tomatoes, may be considered a suitable therapy. Fruitflow® contains antiplatelet and anti-inflammatory compounds that target the mechanisms of platelet activation specific to COVID-19 and can be considered a safe and natural antiplatelet regime.

COVID-19 induces a hyperactive phenotype in circulating platelets.

Coronavirus Disease 2019 (COVID-19), caused by the novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), has affected over 30 million globally to date. Although high rates of venous thromboembolism and evidence of COVID-19-induced endothelial dysfunction have been reported, the precise aetiology of the increased thrombotic risk associated with COVID-19 infection remains to be fully elucidated. Therefore, we assessed clinical platelet parameters and circulating platelet activity in patients with severe and nonsevere COVID-19. An assessment of clinical blood parameters in patients with severe COVID-19 disease (requiring intensive care), patients with nonsevere disease (not requiring intensive care), general medical in-patients without COVID-19, and healthy donors was undertaken. Platelet function and activity were also assessed by secretion and specific marker analysis. We demonstrated that routine clinical blood parameters including increased mean platelet volume (MPV) and decreased platelet:neutrophil ratio are associated with disease severity in COVID-19 upon hospitalisation and intensive care unit (ICU) admission. Strikingly, agonist-induced ADP release was 30- to 90-fold higher in COVID-19 patients compared with hospitalised controls and circulating levels of platelet factor 4 (PF4), soluble P-selectin (sP-selectin), and thrombopoietin (TPO) were also significantly elevated in COVID-19. This study shows that distinct differences exist in routine full blood count and other clinical laboratory parameters between patients with severe and nonsevere COVID-19. Moreover, we have determined all COVID-19 patients possess hyperactive circulating platelets. These data suggest abnormal platelet reactivity may contribute to hypercoagulability in COVID-19 and confirms the role that platelets/clotting has in determining the severity of the disease and the complexity of the recovery path.

Immunothrombotic Dysregulation in COVID-19 Pneumonia Is Associated With Respiratory Failure and Coagulopathy.

BACKGROUND: Severe acute respiratory syndrome corona virus 2 infection causes severe pneumonia (coronavirus disease 2019 [COVID-19]), but the mechanisms of subsequent respiratory failure and complicating renal and myocardial involvement are poorly understood. In addition, a systemic prothrombotic phenotype has been reported in patients with COVID-19. METHODS: A total of 62 subjects were included in our study (n=38 patients with reverse transcriptase polymerase chain reaction-confirmed COVID-19 and n=24 non-COVID-19 controls). We performed histopathologic assessment of autopsy cases, surface marker-based phenotyping of neutrophils and platelets, and functional assays for platelet, neutrophil functions, and coagulation tests, as well. RESULTS: We provide evidence that organ involvement and prothrombotic features in COVID-19 are linked by immunothrombosis. We show that, in COVID-19, inflammatory microvascular thrombi are present in the lung, kidney, and heart, containing neutrophil extracellular traps associated with platelets and fibrin. Patients with COVID-19 also present with neutrophil-platelet aggregates and a distinct neutrophil and platelet activation pattern in blood, which changes with disease severity. Whereas cases of intermediate severity show an exhausted platelet and hyporeactive neutrophil phenotype, patients severely affected with COVID-19 are characterized by excessive platelet and neutrophil activation in comparison with healthy controls and non-COVID-19 pneumonia. Dysregulated immunothrombosis in severe acute respiratory syndrome corona virus 2 pneumonia is linked to both acute respiratory distress syndrome and systemic hypercoagulability. CONCLUSIONS: Taken together, our data point to immunothrombotic dysregulation as a key marker of disease severity in COVID-19. Further work is necessary to determine the role of immunothrombosis in COVID-19.

Nanofiltration of growth media supplemented with human platelet lysates for pathogen-safe xeno-free expansion of mesenchymal stromal cells.

BACKGROUND AIMS: Human platelet lysate can replace fetal bovine serum (FBS) for xeno-free ex vivo expansion of mesenchymal stromal cells (MSCs), but pooling of platelet concentrates (PCs) increases risks of pathogen transmission. We evaluated the feasibility of performing nanofiltration of platelet lysates and determined the impact on expansion of bone marrow-derived MSCs. METHODS: Platelet lysates were prepared by freeze-thawing of pathogen-reduced (Intercept) PCs suspended in 65% storage solution (SPP+) and 35% plasma, and by serum-conversion of PCs suspended in 100% plasma. Lysates were added to the MSC growth media at 10% (v/v), filtered and subjected to cascade nanofiltration on 35- and 19-nm Planova filters. Media supplemented with 10% starting platelet lysates or FBS were used as the controls. Impacts of nanofiltration on the growth media composition, removal of platelet extracellular vesicles (PEVs) and MSC expansion were evaluated. RESULTS: Nanofiltration did not detrimentally affect contents of total protein and growth factors or the biochemical composition. The clearance factor of PEVs was >3 log values. Expansion, proliferation, membrane markers, differentiation potential and immunosuppressive properties of cells in nanofiltered media were consistently better than those expanded in FBS-supplemented media. Compared with FBS, chondrogenesis and osteogenesis genes were expressed more in nanofiltered media, and there were fewer senescent cells over six passages. CONCLUSIONS: Nanofiltration of growth media supplemented with two types of platelet lysates, including one prepared from pathogen-reduced PCs, is technically feasible. These data support the possibility of developing pathogen-reduced xeno-free growth media for clinical-grade propagation of human cells.