Establishment of an in vitro assay to detect antibody-mediated procoagulant platelet formation on a single cell level in real time

Introduction Procoagulant platelets (PLTs), a subpopulation of PLTs that is characterized by increased externalization of phosphatidylserine (PS), are increasingly identified to promote a prothrombotic environment in different diseases. Recently we observed that procoagulant PLT formation can be induced via engagement of immune receptor Fc-gamma-RIIA by COVID-19, VITT and HIT patient immunoglobulin subclass G (IgG) antibodies (Abs). Here, Fc-gamma- RIIA engagement by patient Abs resulted in significant formation of procoagulant PLTs and loss of mitochondrial potential that was associated with high thrombin formation as well as increased thrombus formation. In the cur- rent study, we aim to establish a PLT adhesion assay that allows investigation of PLT mitochondria during procoagulant PLT formation. Method PLTs were spread on human serum albumin, fibrinogen or collagen precoated glass slides. Adhesion and subsequent shape change of PLTs as well as procoagulant PLT formation were investigated in real time using immune fluorescence microscopy. For the detection of PLT shape change, mitochondrial dynamics and PS externalization, PLTs were double stained with MitoTracker green, a mitochondrial dye that binds to free thiol groups of cysteine residues in the mitochondrial membrane, and Annexin-V, respectively. For the visualization of mitochondrial release from PLTs intracellular compartment, a monoclonal Ab that binds to a subunit of the translocase of the outer membrane (TOM) complex on the mitochondrial membrane, namely TOM22, was used. Results During the observation period, a subgroup of PLTs that was spread on collagen became procoagulant as determined by an increased binding of Annexin- V on the PLT surface. Contrary, these changes were nearly absent in PLTs that adhered to fibrinogen (percentage [ %] of Annexin-V positive cells: 19.80 +/- 3.42 % vs. 1.92 +/- 0.62 %, p value 0.0357). Interestingly, procoagulant PLT formation was associated with a significant loss of MitoTracker green signal in PLTs while it remained constant in non-procoagulant PLTs attached on both extracellular matrix coatings. Loss of MitoTracker green signal was associated with translocation of mitochondrial proteins from intracellular to extracellular, as a higher count of TOM22 Ab-positive labelled structures, most likely extracellular mitochondria were detected on collagen but not on fibrinogen coated glass slides. Conclusion Our findings indicate, that the formation of procoagulant PLTs is associated with dramatic changes of the mitochondrial integrity in PLTs. Further attempts, that investigate the potential pathophysiological role of PLT mitochondrial release in Ab-mediated prothrombotic disorders may contribute to a further understanding of the role of PLT mitochondria in these complex diseases.

Procoagulant platelet activity in VITT induced increased thrombus formation

Background: Vaccines against SARS-CoV-2 virus significantly reduce morbidity and mortality of the pandemic. But with millions of people vaccinated in a short period of time, even very rare side effects like the clotting disorder vaccine-induced thrombotic thrombocytopenia (VITT) became apparent. We recently identified an increase in procoagulant platelets in these patients, which is even higher than in a previously reported cohort of COVID-19 patients. Method(s): 8 patients (4 female and 4 male) who were hospitalized with suspected thrombotic complications 5 to 16 days after ChAdOx1 nCoV-19 vaccination were included in this study. The median age was 38 years. All patients had thrombocytopenia at admission. Three had a fatal outcome and five were successfully treated. The blood samples were analyzed by using enzyme immune assays, flow cytometry, ex vivo thrombus formation assay and heparin-induced platelet aggregation assay. Result(s): All sera from VITT patients contained antibodies against PF4 [OD 3.0+/-0.68] with the ability to activate platelets (8/8). Sera induced significant increase in procoagulant markers (CD62P and phosphatidylserine externalization) [CD62P/PS positive PLTs: 40.82+/-7.02%] compared to COVID-19 patients [FI CD62P/PS positive PLTs:15.71+/-7.70];p=0.8977. The formation of procoagulant platelets could be significantly reduced by use of the monoclonal IV.3 antibody as well as IVIG [FI CD62P/PS positive PLTs:1.01+/-0.36];p=0.0001. In thrombus formation model, IgGs from VITT patients induced increase platelet surface area (8.64+/-0.53, SAC+/-SEM) compared to control (0.72+/-0.0.07, SAC+/-SEM);p=0.001), which was inhibited by IVIG (4.07+/-0.51, p= 0.001). Conclusion(s): Our ex vivo microfluidic thrombus formation model supports the significance of procoagulant platelet in the pathogenesis of VITT. It may offer significant clinical implications and therapeutic options like evaluation of IVIG as a recommended therapy or other drugs for treatment of clinical picture of VITT.

Cardiac Neurobiology of Arrhythmia in Human Cell Models: Novel Therapeutic Targets

Heightened sympathetic drive (dysautonomia) is a hallmark of several cardiovascular diseases including SARS-CoV-2. It is also a powerful prognostic predictor for arrhythmia and sudden cardiac death, especially in patients with channelopathies (long QT syndrome-LQTS, and catecholaminergic polymorphic ventricular tachycardia-CPVT). However, little is known about the molecular targets underlying this dysautonomia. We have identified a novel pathway using a combination of single cell and bulk RNAseq, neurochemistry, FRET imaging and single cell electrophysiology. This pathway involves impairment of cyclic nucleotide coupled phosphodiesterases (PDE) linked to enhanced intracellular calcium transients and exocytosis from rat sympathetic neurons. In particular, the adaptor protein Nos1-ap, Pde2A, and Ace2 are associated with sympathetic hyperexcitability. These proteins are also conserved in human stellates from patients with LQTS and CPVT, although their role in neuronal-myocyte cellular function is unknown. We have developed a unique human iPSC sympathetic-cardiac co-culture model for target discovery in LQTS and CPVT. The lecture will highlight the use of gene manipulation of these proteins to determine their role in driving abnormal transmission and arrhythmia.

Platelets' morphology, metabolic profile, exocytosis, and heterotypic aggregation with leukocytes in relation to severity and mortality of COVID-19-patients.

Roles of platelets during infections surpass the classical thrombus function and are now known to modulate innate immune cells. Leukocyte-platelet aggregations and activation-induced secretome are among factors recently gaining interest but little is known about their interplay with severity and mortality during the course of SARS-Cov-2 infection. The aim of the present work is to follow platelets' bioenergetics, redox balance, and calcium homeostasis as regulators of leukocyte-platelet interactions in a cohort of COVID-19 patients with variable clinical severity and mortality outcomes. We investigated COVID-19 infection-related changes in platelet counts, activation, morphology (by flow cytometry and electron microscopy), bioenergetics (by Seahorse analyzer), mitochondria function (by high resolution respirometry), intracellular calcium (by flow cytometry), reactive oxygen species (ROS, by flow cytometry), and leukocyte-platelet aggregates (by flow cytometry) in non-intensive care unit (ICU) hospitalized COVID-19 patients (Non-ICU, n=15), ICU-survivors of severe COVID-19 (ICU-S, n=35), non-survivors of severe COVID-19 (ICU-NS, n=60) relative to control subjects (n=31). Additionally, molecular studies were carried out to follow gene and protein expressions of mitochondrial electron transport chain complexes (ETC) in representative samples of isolated platelets from the studied groups. Our results revealed that COVID-19 infection leads to global metabolic depression especially in severe patients despite the lack of significant impacts on levels of mitochondrial ETC genes and proteins. We also report that severe patients' platelets exhibit hyperpolarized mitochondria and significantly lowered intracellular calcium, concomitantly with increased aggregations with neutrophil. These changes were associated with increased populations of giant platelets and morphological transformations usually correlated with platelets activation and inflammatory signatures, but with impaired exocytosis. Our data suggest that hyperactive platelets with impaired exocytosis may be integral parts in the pathophysiology dictating severity and mortality in COVID-19 patients.

Anti-Biotics And Anti-Viral Drugs In Covid Management: Systemic Review Based On Current Evidence

This review focuses on the management of Novel Corona Virus with antiviral drugs and antibiotics and therefore the worldwide dissemination of COVID-19 has been accompanied by increased use of antibiotics, according to this review, which focuses on the therapy of Novel Corona Virus with antiviral medicines and antiviral. This is linked to COVID-19 patients' priority of viral infections. In low-and middle-income countries, identifying viruses is difficult because to a lack of medical or cheap infrastructure that is easily accessible and inexpensive among diseases and pathogens. The possibility of COVID-19 spreading has increased public awareness of the need of antibiotic management systems, as well as infection control and control measures that can minimize microbial load. In underdeveloped nations, these measures are commonly employed. During the COVID-19 pandemic, studies were conducted as a test for worldwide antibiotic resistance. Respiratory problems are being blamed on the Novel Corona Virus that Include pneumonia, colds, sneezing and coughing, and other respiratory diseases. Humans are infected with the Coronavirus by airborne droplets. The World Health Organization has warned against visiting public areas and avoiding close contact with an infected individual. First, on December 31, 2019, the Coronavirus (2019-nCoV) was separated from the Wuhan market in China, resulting in the COVID-19 pandemic of extremely complicated viral illnesses. Patients with risk factors are more prone to develop secondary infections, which necessitate the use of antibiotics. Attempts to duplicate the medication, on the other hand, raised knowledge of the antibiotics' significance beyond infection management. Antiviral, immunomodulatory action, and unique pharmacokinetic profile of antibiotics play a significant part in the therapy of pneumonia;other benefits include cardiac safety, improved lung tissue access, and possible antiviral, and immunomodulation, but some adverse effects by usage. SARS-CoV-2 has generated an epidemic of the highly infectious new coronavirus 2019 (COVID-19), which poses a severe public health concern. Given the potential for a COVID-19 outbreak, a better knowledge of the virus is critical in the event of therapeutic alternatives. We offer a thorough analysis of antimicrobials and antiviral COVID-19 in this review. We also go about COVID-19's current treatments. Copyright © 2022, Anka Publishers. All rights reserved.

Sera from donors of COVID-19 convalescent plasma do not induce procoagulant phenotype in platelets

Background: COVID-19 convalescent plasma (CCP) has been suggested to be beneficial to prevent disease progression in COVID-19. However, concerns have been expressed whether plasma components in CCP can shift the already imbalanced coagulation system to a more hypercoagulable state. Aim(s): To investigate the effect of CCP on platelet phenotype and activation. Method(s): We investigated platelets from CCP donors who had a history of mild COVID-19 infection. Donors who did not have COVID-19 were used as controls (non-CCP donors). We analyzed phosphatidylserine (PS) externalization, CD62p expression, and GPVI shedding in healthy platelets after incubation with sera from CCP and non-CCP donors using flow cytometry. The study protocol was approved by the ethics committee of the University Hospital of Tubingen. (Figure Presented) Results: Forty-seven CCP donors [22 Male, 25 Female;and mean age (+/-SD) 41.4 +/- 13.7 years] with a history of mild COVID-19 infection were included. Median duration after acute COVID-19 infection was 97 days (range, 34-401). Compared to sera from non-CCP donors, sera from CCP donors did not induce higher PS externalization (Fold increase [FI] of PS positive platelets: 1.16% +/- 0.66 vs. 1.51% +/- 0.74, respectively, p = 0.11) or increased the rate of CD62p/PS double positive procoagulant phenotype (FI in CD62p/PS positive platelets: 1.86 +/- 0.87 vs. 1.37 +/- 0.63, respectively, p = 0.10) in platelets from healthy persons. Of note, CD62p expression in healthy platelets after incubation with sera from CCP plasma donors was significantly lower compared to sera from non-CCP donors (FI in CD62p: 2.09 +/- 1.36 vs. 1.16 +/- 0.45, p< 0.01). Sera-mediated GPVI shedding was similar between non-CCP and CCP donors (1.07 +/- 0.16 vs. 1.27 +/- 0.91, p = 0.52). Conclusion(s): Our findings support data from clinical studies, which indicate that transfusion of CCP to treat or prevent severe COVID-19 is not associated with increased risk of exacerbation of the coagulopathy in COVID-19.

Increased thrombus formation via procoagulant platelets in vaccine induced thrombotic thrombocytopenia (VITT)

Background: Vaccines against SARS-CoV- 2 virus reduce morbidity and mortality of the pandemic. But with millions of people vaccinated in a short period of time, even very rare side effects like the clotting disorder vaccine-induced thrombotic thrombocytopenia (VITT) became apparent. We recently identified an increase in procoagulant platelets in these patients. Aim(s): Investigation of the impact of procoagulant platelets in thrombus formation. Method(s): 8 patients (4 female, 4 male) who were hospitalized with suspected thrombotic complications 5 to 16 days after ChAdOx1 nCoV-19 vaccination were included in this study. The blood samples were analyzed by using enzyme immune assays, flow cytometry, ex vivo thrombus formation assay and heparin-induced platelet aggregation assay. Result(s): The median age was 38 years. All patients had thrombocytopenia at admission. Three had a fatal outcome and five were successfully treated. All sera from VITT patients contained high titer antibodies against platelet factor 4 (PF4) [OD: 3.0 +/- 0.68] with the ability to activate platelets in the HIPA assay (8/8). Sera from VITT patients induced significant increase in procoagulant markers (P-selectin [CD62P] and phosphatidylserine externalization) [% CD62P/ PS positive PLTs: 40.82 +/- 7.02] compared to COVID-19 patients [% CD62P/PS positive PLTs: 15.71 +/- 7.70]. The generation of procoagulant platelets was PF4 dependent. The formation of procoagulant platelets could be significantly reduced by use of the monoclonal IV.3 [% CD62P/PS positive PLTs: 1.05 +/- 0.21];p = 0.0001 antibody as well as IVIG [% CD62P/PS positive PLTs: 1.01 +/- 0.36];p = 0.0001. In thrombus formation model, IgGs from VITT patients induced increase platelet surface area (Mean % SAC +/- SEM: 10.38 +/- 1.30) compared to control IgG, which was inhibited by IVIG (4.08 +/- 0.96), p = 0.001. Conclusion(s): Our ex vivo microfluidic thrombus formation model supports the significance of procoagulant platelet in the pathogenesis of VITT. It may offer significant clinical implications and therapeutic options like evaluation of IVIG as a recommended therapy or other drugs for treatment of clinical picture of VITT. (Table Presented).

Endothelial cell activation, Weibel-Palade body secretion and enhanced angiogenesis in severe COVID-19

Background: Severe COVID-19 is associated with marked endothelial cell (EC) activation that plays a key role in immunothrombosis and pulmonary microvascular occlusion. However, the biological mechanisms through which SARS-CoV-2 causes EC activation and damage remain poorly defined. Aim(s): We investigated EC activation in patients with acute COVID-19, and in particular focused on how proteins stored within Weibel-Palade bodies (WPBs) may impact key aspects of disease pathogenesis. Method(s): 39 patients with confirmed COVID-19 were recruited. Weibel-Palade body biomarkers [von Willebrand factor (VWF), angiopoietin-2 (Ang-2) and osteoprotegerin (OPG)] and soluble thrombomodulin (sTM) levels were determined. In addition, EC activation and angiogenesis were assessed in the presence or absence of COVID-19 plasma incubation. Result(s): Markedly elevated plasma VWF:Ag, Ang-2, OPG and sTM levels were observed in acute COVID-19 patients. The increased levels of both sTM and WPB components (VWF, OPG and Ang-2) correlated with COVID-19 severity. Incubation of COVID-19 plasma with ECs triggered enhanced VWF secretion and increased Ang-2 expression (Figure 1). In keeping with the autopsy reports of intussusceptive angiogenesis, treatment with COVID-19 plasma also caused significantly increased EC angiogenesis (Figure 1). Conclusion(s): We propose that as COVID-19 develops, progressive loss of TM and increased sTM, as well as increased Ang-2 expression result in loss of EC quiescence, WPB exocytosis, and a local pro-angiogenic state.

Erythema multiforme related to AstraZeneca SARS-CoV-2 vaccine

A 44-year-old man of Pakistani origin presented to emergency 6 days following his first dose of the AstraZeneca (AZ) SARSCoV- 2 vaccine. He developed flu-like symptoms followed by erythematous pruritic rash. Physical examination showed a maculopapular rash associated with purpura and targetoid lesions affecting his distal extremities, trunk and mucous membranes. He also had crusting and ulceration of his oral and genital mucosal areas. He had no other significant past medical history. A biopsy was taken from his right arm and sent for urgent histology and direct immunofluorescence. Histology revealed parakeratotic scale with interface dermatitis comprising basal layer vacuolation and lymphocyte exocytosis. The epidermis showed prominent dyskeratotic keratinocytes scattered throughout the epidermis. The papillary dermis showed a mild perivascular lymphocytic infiltrate including eosinophils and melanophages. Other investigations showed leucocytosis (12 × 109 L-1), high eosinophils (0.9 × 109 L-1), raised liver enzymes with alkaline phosphatase 159 U L-1 and alanine aminotransferase 172 U L-1. A full infection screen, including herpes simplex virus, SARS-CoV-2 and atypical viral infection, was negative. Immunology was also reported as negative. Based on the findings, a diagnosis of erythema multiforme (EM) secondary to AZ vaccine was made. He was treated with topical steroids and emollients, leading to resolution of his skin and mucosal areas in 4-6 weeks. Recently, there have been a few reported cases of EM in patients with COVID-19 (Jimenez-Cauhe J, Ortega-Quijano D, Carretero- Barrio I et al. Erythema multiforme-like eruption in patients with COVID-19 infection: clinical and histological findings. Clin Exp Dermatol 2020;45: 892-5) and two patients who have had the Pfizer-BioNTech vaccine [Kim M, Kim J, Kim M et al. Generalized erythema multiforme-like skin rash following the first dose of COVID-19 vaccine (Pfizer-BioNTech). J Eur Acad Dermatol Venereol 2021], but the information is limited. Our case emphasizes the need for further studies into the cutaneous adverse effects related to COVID-19vaccines.

Differential Functional Responses of Neutrophil Subsets in Severe COVID-19 Patients.

Neutrophils play a significant role in determining disease severity following SARS-CoV-2 infection. Gene and protein expression defines several neutrophil clusters in COVID-19, including the emergence of low density neutrophils (LDN) that are associated with severe disease. The functional capabilities of these neutrophil clusters and correlation with gene and protein expression are unknown. To define host defense and immunosuppressive functions of normal density neutrophils (NDN) and LDN from COVID-19 patients, we recruited 64 patients with severe COVID-19 and 26 healthy donors (HD). Phagocytosis, respiratory burst activity, degranulation, neutrophil extracellular trap (NET) formation, and T-cell suppression in those neutrophil subsets were measured. NDN from severe/critical COVID-19 patients showed evidence of priming with enhanced phagocytosis, respiratory burst activity, and degranulation of secretory vesicles and gelatinase and specific granules, while NET formation was similar to HD NDN. COVID LDN response was impaired except for enhanced NET formation. A subset of COVID LDN with intermediate CD16 expression (CD16Int LDN) promoted T cell proliferation to a level similar to HD NDN, while COVID NDN and the CD16Hi LDN failed to stimulate T-cell activation. All 3 COVID-19 neutrophil populations suppressed stimulation of IFN-γ production, compared to HD NDN. We conclude that NDN and LDN from COVID-19 patients possess complementary functional capabilities that may act cooperatively to determine disease severity. We predict that global neutrophil responses that induce COVID-19 ARDS will vary depending on the proportion of neutrophil subsets.

Amphiphilic anti-SARS-CoV-2 drug remdesivir incorporates into the lipid bilayer and nerve terminal membranes influencing excitatory and inhibitory neurotransmission.

Remdesivir is a novel antiviral drug, which is active against the SARS-CoV-2 virus. Remdesivir is known to accumulate in the brain but it is not clear whether it influences the neurotransmission. Here we report diverse and pronounced effects of remdesivir on transportation and release of excitatory and inhibitory neurotransmitters in rat cortex nerve terminals (synaptosomes) in vitro. Direct incorporation of remdesivir molecules into the cellular membranes was shown by FTIR spectroscopy, planar phospholipid bilayer membranes and computational techniques. Remdesivir decreases depolarization-induced exocytotic release of L-[14C] glutamate and [3H] GABA, and also [3H] GABA uptake and extracellular level in synaptosomes in a dose-dependent manner. Fluorimetric studies confirmed remdesivir-induced impairment of exocytosis in nerve terminals and revealed a decrease in synaptic vesicle acidification. Our data suggest that remdesivir dosing during antiviral therapy should be precisely controlled to prevent possible neuromodulatory action at the presynaptic level. Further studies of neurotropic and membranotropic effects of remdesivir are necessary.

Respiratory symptoms of the 2019 novel coronavirus when the source of the symptoms is intra-cardiac thrombus not the pneumonia

Case Report The 2019 Novel Coronavirus (COVID-19) is currently causing a global pandemic. Common symptoms are fever, cough, myalgia, fatigue, headache, dyspnea, sore throat, vomiting, and diarrhea. Patients may present with end-organ failure, ARDS, shock, acute kidney injury, or even death. We present a case of COVID-19 with shortness of breath caused by an intra-cardiac thrombus. Case presentation An 84-year-old woman with COPD and diastolic heart failure presented with shortness of breath. She had hypoxemia on room air upon presentation. Lungs were clear on physical examination. COVID-19 PCR was positive. Her chest radiograph demonstrated no pulmonary infiltrates. Transthoracic echocardiography (TTE) demonstrated a large, irregularly shaped echogenic mass in both the right atrium and right ventricle consistent with a large thrombus. The mass in the right atrium was 3.9∗3.6 cm;the portion in the ventricle was 3.2∗2.2 cm. A previous TTE study in this patient did not reveal an intra-cardiac thrombus. No deep venous thrombosis was found. She was begun on anticoagulation and refused catheter-directed therapy. She improved and was discharged to her home. Discussion Thromboembolic complications of COVID-19 have been described in the literature. The most common are deep venous thrombosis and pulmonary embolism in critically ill patients despite the use of prophylactic anticoagulation. Several studies have reported post-mortem biopsies with widespread microthrombi. Arterial thrombosis with stroke and limb ischemia has also been described. Our case had an unusual presentation since the cause of her shortness of breath was the intra-cardiac thrombus. The pathogenesis beyond the hypercoagulability in COVID is not well understood. Some studies propose direct endothelial injury by the COVID-19 virus, causing microvascular inflammation, endothelial exocytosis, and endothelitis. Some experts propose a hypercoagulable state in COVID-19 patients based on elevated factor VIII, elevated fibrinogen, circulating prothrombotic microparticles, and neutrophil extracellular traps (NETs). Yet, no definitive mechanism has been identified. (Figure Presented).

SARS CoV-2;a closer look at the agent of the pandemic of modern times

Due to the COVID-19 pandemic, as of September 2021, a total of 222,309,456 people were infected in the world and a total of 4,592,685 patients were lost. The pandemic, which has a fatality rate of around 2%, has made and continues to make us live thhrough all experiences of epidemics that we have only read about in Annals of Medicine and Microbiology and that deeply affected the World at their times. The virus causing the pandemic has a positive polarity RNA genome of 30,000 bases and produces a total of 29 proteins. Of these proteins, 4 are structural, 16 are nonstructural, and 9 are accessory proteins. SARS-CoV-2 is an enveloped RNA virus with a diameter of 150-200 nm, has an S (spike-spike-tassel) glycoprotein on its surface, which, like other coronaviruses, creates the crown appearance unique to these viruses. After the S protein is synthesized as a polyprotein, it is cleaved into S1 and S2 subunits. The S1 subunit binds to the target cell, and the S2 subunit performs fusion with the cell membrane to be infected. Since these functions are critical features of a successful viral infection, the S protein is the main target of all interventions to prevent virus infection. In this context, the main target of neutralizing antibodies and drugs to stop virus infection before it starts is the S protein. The S protein has a trimer structure similar to hemagglutinin in influenza virus and contains the fusion peptide that becomes exposed during transition from the prefusion configuration to the fusion configuration and facilitates the fusion function with the cellular/endosomal membranes. Apart from the S protein, SARS-CoV-2 has structural proteins known as E (envelope), M (membrane), and N (nucleocapsid) proteins;The N protein binds to the RNA genome and together with the S, E and M proteins and the RNA genome form the virion. While SARS-CoV-2 S protein attaches to cells using Cellular Angiotensin Converting Enzyme 2 (HCoV- NL63, SARS-CoV and SARS-CoV-2), other coronaviruses use different receptors (Aminopeptidase N-HCoV-229E;dipeptidyl peptidase 4- MERS-CoV). Unlike viruses in this group, the SARS CoV-2 S1 protein with receptor binding domain (RBD) has a cleavage site made up of polybasic amino acids at the S1-S2 border and used by the cellular furin protease, which is believed to provide advantages to the virus in proteolytic cleavage, cell tropism, virulence and pathogenicity. ACE-2 is important in the renin-angiotensin-aldosterone system and although it is rarely found in the circulation, it is widely expressed in organs and is an enzyme involved in the regulation of blood pressure and fluid balance. Following intracellular entry and fusion of membranes, the SARS-CoV-2 genome is released into the cytoplasm and gene expression proceeds as a temporally and spatially well-regulated process. Non-structural proteins, which are produced from direct translation of ORF1a and ORF1b regions of positive sense genomic RNA, form the replication and transcription complex. These complexes establish the infrastructure for the next steps. The common features of coronaviruses such as cytoplasmic replication, viral gene expression through sub-genomic nested set messages, exocytosis of mature virions within vesicles occur in SARS-CoV-2 as well. One of the most important problems in the COVID-19 pandemic has been the emergence of variant viruses. These viruses adversely affecting the transmission rate, virulence, clinical course, and the effectiveness of the diagnostic or therapeutic methods carry mutations that lead to amino acid changes, especially in the RBD region. The World Health Organization and other authorities refer to these viruses as variants of concern or variants of interest. As of September 2021, WHO lists Alpha (UK, September 2020), Beta (South Africa, May 2020), Gamma (Brazil, November 2020), and Delta (India, October 2020) viruses as variants of concern. Also, Eta (December 2020), Iota (USA, November 2020), Kappa (India, October 2020), Lambda (Peru December, 2020) and Mu (Colombia, January 2021) mutant viruses are on he list variants of interest. In conclusion, less than 2 years of time has passed since the emergence of the COVID-19 agent SARS CoV-2 virus. However, this virus has been the most extensively studied viral agent in the history of medicine and the most detailed information has been gathered about the infection. Despite all these, it is difficult to indicate that the fight against this pathogen has been successful nor are we any closer to declare that the enormous danger the virus poses to humanity is reduced.

Channels and Transporters of the Pulmonary Lamellar Body in Health and Disease.

The lamellar body (LB) of the alveolar type II (ATII) cell is a lysosome-related organelle (LRO) that contains surfactant, a complex mix of mainly lipids and specific surfactant proteins. The major function of surfactant in the lung is the reduction of surface tension and stabilization of alveoli during respiration. Its lack or deficiency may cause various forms of respiratory distress syndrome (RDS). Surfactant is also part of the innate immune system in the lung, defending the organism against air-borne pathogens. The limiting (organelle) membrane that encloses the LB contains various transporters that are in part responsible for translocating lipids and other organic material into the LB. On the other hand, this membrane contains ion transporters and channels that maintain a specific internal ion composition including the acidic pH of about 5. Furthermore, P2X4 receptors, ligand gated ion channels of the danger signal ATP, are expressed in the limiting LB membrane. They play a role in boosting surfactant secretion and fluid clearance. In this review, we discuss the functions of these transporting pathways of the LB, including possible roles in disease and as therapeutic targets, including viral infections such as SARS-CoV-2.

SARS-CoV-2 Ion Channel ORF3a Enables TMEM16F-Dependent Phosphatidylserine Externalization to Augment Procoagulant Activity of the Tenase and Prothrombinase Complexes

Severe SARS-CoV-2 infection is complicated by dysregulation of the blood coagulation system and high rates of thrombosis, but virus-intrinsic mechanisms underlying this phenomenon are poorly understood. Increased intracellular calcium concentrations promote externalization of phosphatidylserine (PS), the membrane anionic phospholipid required for assembly and activation of the tenase and prothrombinase complexes to drive blood coagulation. TMEM16F is a ubiquitous phospholipid scramblase that mediates externalization of PS in a calcium-dependent manner. As SARS-CoV-2 ORF3a encodes a presumed cation channel with the ability to transport calcium, we hypothesized that ORF3a expression by infected host cells perturbs the cellular calcium rheostat, driving TMEM16F-dependent externalization of PS and enhancing procoagulant activity. Using a doxycycline-inducible system, synchronized expression of ORF3a in A549 pulmonary epithelial cells resulted in a time-dependent augmentation of tissue factor (TF) procoagulant activity exceeding 9-fold by 48 hours (p < 0.0001), with no change in TF cell-surface expression. This enhancement was dependent upon PS as determined by inhibition with the PS-binding protein lactadherin. Over 2-fold enhancement of prothrombinase activity (p < 0.0001) was also observed by 48 hours. ORF3a increased intracellular calcium levels by 18-fold at 48 hours (p < 0.0001), as determined by the intracellular calcium indicator fluo-4. After 16 hours of ORF3a expression, more than 60% of cells had externalized PS (p < 0.001) without increased cell death, as quantified by flow cytometry following annexin V binding. Immunofluorescence microscopy staining for ORF3a, annexin V, and nuclei confirmed ORF3a expression within internal and cell surface membranes and increased PS externalization. PS externalization was insensitive to the pan-caspase inhibitor z-VAD-FMK, and there was no evidence of apoptotic activation as determined by caspase-3 cleavage. By contrast, ORF3a expression did not augment coagulation in cells deficient in the calcium-dependent phospholipid scramblase TMEM16F. Similarly, ORF3a-enhanced TF procoagulant activity (p < 0.01) and prothrombinase activity (p<0.05) was completely abrogated using TMEM16 inhibitors, including the uricosuric agent benzbromarone that has been registered for human use in over 20 countries. Live SARS-CoV-2 infection of A549-ACE2 cells increased cell surface factor Xa generation at MOI 0.1 (p < 0.01) but not MOI 0.01 or following heat inactivation of the virus, and RNA sequencing confirmed ORF3a induction without increased F3 expression. RNA sequencing of human SARS-CoV-2 infected lung autopsy and control tissue (n= 53) confirmed these findings in vivo. Immunofluorescence staining for ORF3a and KRT8/18 and CD31 in SARS-CoV-2 infected human lung autopsy specimens demonstrated ORF3a expression in pulmonary epithelium and endothelial cells, highlighting the potential pathologic relevance of this mechanism. Here we demonstrate that expression of the SARS-CoV-2 accessory protein ORF3a increases the intracellular calcium concentration and TMEM16F-dependent PS scrambling to augment procoagulant activity of the tenase and prothrombinase complexes. Our studies of human cells and tissues infected with SARS-CoV-2 support the pathologic relevance of this mechanism. We highlight the therapeutic potential to target the ORF3a-TMEM16F axis as with benzbromarone to mitigate dysregulation of coagulation and thrombosis during severe SARS-CoV-2 infection. Disclosures: Schwartz: Miromatrix Inc: Membership on an entity's Board of Directors or advisory committees;Alnylam Inc.: Consultancy, Speakers Bureau. Schulman: CSL Behring: Consultancy, Research Funding.

Effect of Crizanlizumab, a P-Selectin Inhibitor, in COVID-19: A Placebo-Controlled, Randomized Trial.

COVID-19 is characterized by vascular inflammation and thrombosis, including elevations in P-selectin, a mediator of inflammation released by endothelial cells. We tested the effect of P-selectin inhibition on biomarkers of thrombosis and inflammation in patients with COVID-19. Hospitalized patients with moderate COVID-19 were randomly assigned to receive either placebo or crizanlizumab, a P-selectin inhibitor, in a double-blind fashion. Crizanlizumab reduced P-selectin levels by 89%. Crizanlizumab increased D-dimer levels by 77% and decreased prothrombin fragment. There were no significant differences between crizanlizumab and placebo for clinical endpoints. Crizanlizumab was well tolerated. Crizanlizumab may induce thrombolysis in the setting of COVID-19. (Crizanlizumab for Treating COVID-19 Vasculopathy [CRITICAL]; NCT04435184).

Autophagy and treatment of patients with COVID-19;which drugs target the autophagy pathway?

Autophagy is a way to create new cellular structures, clear cells invaded by microbes, and block accumulating proteins that can cause disease. Moreover, it can destroy all cellular organs and pathogens, including fungi, parasites, bacteria, and viruses, either randomly or selectively. Many research groups are examining a strategy to combat COVID-19. In particular, research is underway to identify drugs that can target autophagy in COVID-19 virus infection. Several known drugs are currently under clinical evaluation for the autophagy process, given that regulating autophagy is a way to combat COVID-19. This study introduces drugs that target the autophagy pathway.

ORF3a of SARS-CoV-2 promotes lysosomal exocytosis-mediated viral egress.

Viral entry and egress are important determinants of virus infectivity and pathogenicity. ß-coronaviruses, including the COVID-19 virus SARS-CoV-2 and mouse hepatitis virus (MHV), exploit the lysosomal exocytosis pathway for egress. Here, we show that SARS-CoV-2 ORF3a, but not SARS-CoV ORF3a, promotes lysosomal exocytosis. SARS-CoV-2 ORF3a facilitates lysosomal targeting of the BORC-ARL8b complex, which mediates trafficking of lysosomes to the vicinity of the plasma membrane, and exocytosis-related SNARE proteins. The Ca2+ channel TRPML3 is required for SARS-CoV-2 ORF3a-mediated lysosomal exocytosis. Expression of SARS-CoV-2 ORF3a greatly elevates extracellular viral release in cells infected with the coronavirus MHV-A59, which itself lacks ORF3a. In SARS-CoV-2 ORF3a, Ser171 and Trp193 are critical for promoting lysosomal exocytosis and blocking autophagy. When these residues are introduced into SARS-CoV ORF3a, it acquires the ability to promote lysosomal exocytosis and inhibit autophagy. Our results reveal a mechanism by which SARS-CoV-2 interacts with host factors to promote its extracellular egress.

PTP-MEG2 regulates quantal size and fusion pore opening through two distinct structural bases and substrates.

Tyrosine phosphorylation of secretion machinery proteins is a crucial regulatory mechanism for exocytosis. However, the participation of protein tyrosine phosphatases (PTPs) in different exocytosis stages has not been defined. Here we demonstrate that PTP-MEG2 controls multiple steps of catecholamine secretion. Biochemical and crystallographic analyses reveal key residues that govern the interaction between PTP-MEG2 and its substrate, a peptide containing the phosphorylated NSF-pY83 site, specify PTP-MEG2 substrate selectivity, and modulate the fusion of catecholamine-containing vesicles. Unexpectedly, delineation of PTP-MEG2 mutants along with the NSF binding interface reveals that PTP-MEG2 controls the fusion pore opening through NSF independent mechanisms. Utilizing bioinformatics search and biochemical and electrochemical screening approaches, we uncover that PTP-MEG2 regulates the opening and extension of the fusion pore by dephosphorylating the DYNAMIN2-pY125 and MUNC18-1-pY145 sites. Further structural and biochemical analyses confirmed the interaction of PTP-MEG2 with MUNC18-1-pY145 or DYNAMIN2-pY125 through a distinct structural basis compared with that of the NSF-pY83 site. Our studies thus provide mechanistic insights in complex exocytosis processes.