MAPK-interacting kinase 1 regulates platelet production, activation, and thrombosis.

The MAPK-interacting kinase (Mnk) family includes Mnk1 and Mnk2, which are phosphorylated and activated in response to extracellular stimuli. Mnk1 contributes to cellular responses by regulating messenger RNA (mRNA) translation, and mRNA translation influences platelet production and function. However, the role of Mnk1 in megakaryocytes and platelets has not previously been studied. The present study investigated Mnk1 in megakaryocytes and platelets using both pharmacological and genetic approaches. We demonstrate that Mnk1, but not Mnk2, is expressed and active in human and murine megakaryocytes and platelets. Stimulating human and murine megakaryocytes and platelets induced Mnk1 activation and phosphorylation of eIF4E, a downstream target of activated Mnk1 that triggers mRNA translation. Mnk1 inhibition or deletion significantly diminished protein synthesis in megakaryocytes as measured by polysome profiling and [35S]-methionine incorporation assays. Depletion of Mnk1 also reduced megakaryocyte ploidy and proplatelet forming megakaryocytes in vitro and resulted in thrombocytopenia. However, Mnk1 deletion did not affect the half-life of circulating platelets. Platelets from Mnk1 knockout mice exhibited reduced platelet aggregation, α granule secretion, and integrin αIIbß3 activation. Ribosomal footprint sequencing indicated that Mnk1 regulates the translation of Pla2g4a mRNA (which encodes cPLA2) in megakaryocytes. Consistent with this, Mnk1 ablation reduced cPLA2 activity and thromboxane generation in platelets and megakaryocytes. In vivo, Mnk1 ablation protected against platelet-dependent thromboembolism. These results provide previously unrecognized evidence that Mnk1 regulates mRNA translation and cellular activation in platelets and megakaryocytes, endomitosis and thrombopoiesis, and thrombosis.

Platelets in Pulmonary Immune Responses and Inflammatory Lung Diseases.

Platelets are essential for physiological hemostasis and are central in pathological thrombosis. These are their traditional and best known activities in health and disease. In addition, however, platelets have specializations that broaden their functional repertoire considerably. These functional capabilities, some of which are recently discovered, include the ability to sense and respond to infectious and immune signals and to act as inflammatory effector cells. Human platelets and platelets from mice and other experimental animals can link the innate and adaptive limbs of the immune system and act across the immune continuum, often also linking immune and hemostatic functions. Traditional and newly recognized facets of the biology of platelets are relevant to defensive, physiological immune responses of the lungs and to inflammatory lung diseases. The emerging view of platelets as blood cells that are much more diverse and versatile than previously thought further predicts that additional features of the biology of platelets and of megakaryocytes, the precursors of platelets, will be discovered and that some of these will also influence pulmonary immune defenses and inflammatory injury.

Platelet MHC class I mediates CD8+ T-cell suppression during sepsis.

Circulating platelets interact with leukocytes to modulate host immune and thrombotic responses. In sepsis, platelet-leukocyte interactions are increased and have been associated with adverse clinical events, including increased platelet-T-cell interactions. Sepsis is associated with reduced CD8+ T-cell numbers and functional responses, but whether platelets regulate CD8+ T-cell responses during sepsis remains unknown. In our current study, we systemically evaluated platelet antigen internalization and presentation through major histocompatibility complex class I (MHC-I) and their effects on antigen-specific CD8+ T cells in sepsis in vivo and ex vivo. We discovered that both human and murine platelets internalize and proteolyze exogenous antigens, generating peptides that are loaded onto MHC-I. The expression of platelet MHC-I, but not platelet MHC-II, is significantly increased in human and murine platelets during sepsis and in human megakaryocytes stimulated with agonists generated systemically during sepsis (eg, interferon-γ and lipopolysaccharide). Upregulation of platelet MHC-I during sepsis increases antigen cross-presentation and interactions with CD8+ T cells in an antigen-specific manner. Using a platelet lineage-specific MHC-I-deficient mouse strain (B2Mf/f-Pf4Cre), we demonstrate that platelet MHC-I regulates antigen-specific CD8+ T-cell proliferation in vitro, as well as the number and functional responses of CD8+ T cells in vivo, during sepsis. Loss of platelet MHC-I reduces sepsis-associated mortality in mice in an antigen-specific setting. These data identify a new mechanism by which platelets, through MHC-I, process and cross-present antigens, engage antigen-specific CD8+ T cells, and regulate CD8+ T-cell numbers, functional responses, and outcomes during sepsis.

Platelet gene expression and function in patients with COVID-19.

There is an urgent need to understand the pathogenesis of coronavirus disease 2019 (COVID-19). In particular, thrombotic complications in patients with COVID-19 are common and contribute to organ failure and mortality. Patients with severe COVID-19 present with hemostatic abnormalities that mimic disseminated intravascular coagulopathy associated with sepsis, with the major difference being increased risk of thrombosis rather than bleeding. However, whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection alters platelet function to contribute to the pathophysiology of COVID-19 remains unknown. In this study, we report altered platelet gene expression and functional responses in patients infected with SARS-CoV-2. RNA sequencing demonstrated distinct changes in the gene-expression profile of circulating platelets of COVID-19 patients. Pathway analysis revealed differential gene-expression changes in pathways associated with protein ubiquitination, antigen presentation, and mitochondrial dysfunction. The receptor for SARS-CoV-2 binding, angiotensin-converting enzyme 2 (ACE2), was not detected by messenger RNA (mRNA) or protein in platelets. Surprisingly, mRNA from the SARS-CoV-2 N1 gene was detected in platelets from 2 of 25 COVID-19 patients, suggesting that platelets may take-up SARS-COV-2 mRNA independent of ACE2. Resting platelets from COVID-19 patients had increased P-selectin expression basally and upon activation. Circulating platelet-neutrophil, -monocyte, and -T-cell aggregates were all significantly elevated in COVID-19 patients compared with healthy donors. Furthermore, platelets from COVID-19 patients aggregated faster and showed increased spreading on both fibrinogen and collagen. The increase in platelet activation and aggregation could partially be attributed to increased MAPK pathway activation and thromboxane generation. These findings demonstrate that SARS-CoV-2 infection is associated with platelet hyperreactivity, which may contribute to COVID-19 pathophysiology.

Neutrophil extracellular traps contribute to immunothrombosis in COVID-19 acute respiratory distress syndrome.

COVID-19 affects millions of patients worldwide, with clinical presentation ranging from isolated thrombosis to acute respiratory distress syndrome (ARDS) requiring ventilator support. Neutrophil extracellular traps (NETs) originate from decondensed chromatin released to immobilize pathogens, and they can trigger immunothrombosis. We studied the connection between NETs and COVID-19 severity and progression. We conducted a prospective cohort study of COVID-19 patients (n = 33) and age- and sex-matched controls (n = 17). We measured plasma myeloperoxidase (MPO)-DNA complexes (NETs), platelet factor 4, RANTES, and selected cytokines. Three COVID-19 lung autopsies were examined for NETs and platelet involvement. We assessed NET formation ex vivo in COVID-19 neutrophils and in healthy neutrophils incubated with COVID-19 plasma. We also tested the ability of neonatal NET-inhibitory factor (nNIF) to block NET formation induced by COVID-19 plasma. Plasma MPO-DNA complexes increased in COVID-19, with intubation (P < .0001) and death (P < .0005) as outcome. Illness severity correlated directly with plasma MPO-DNA complexes (P = .0360), whereas Pao2/fraction of inspired oxygen correlated inversely (P = .0340). Soluble and cellular factors triggering NETs were significantly increased in COVID-19, and pulmonary autopsies confirmed NET-containing microthrombi with neutrophil-platelet infiltration. Finally, COVID-19 neutrophils ex vivo displayed excessive NETs at baseline, and COVID-19 plasma triggered NET formation, which was blocked by nNIF. Thus, NETs triggering immunothrombosis may, in part, explain the prothrombotic clinical presentations in COVID-19, and NETs may represent targets for therapeutic intervention.

Sepsis alters the transcriptional and translational landscape of human and murine platelets.

There is increasing recognition that platelets have a functional role in the pathophysiology of sepsis, though this role has not been precisely defined. Whether sepsis alters the human platelet transcriptome and translational landscape has never been established. We used parallel techniques of RNA sequencing and ribosome footprint profiling to interrogate the platelet transcriptome and translatome in septic patients and healthy donors. We identified 1806 significantly differentially expressed (false discovery rate <0.05) transcripts in platelets from septic patients. Platelet translational events during sepsis were also upregulated. To explore the relevance of a murine model of sepsis, cecal ligation and puncture (CLP), we compared sepsis-induced changes in platelet gene expression between septic patients and mice subjected to CLP. Platelet transcriptional (ρ = 0.42, P = 3.2 × 10-285) and translational (ρ = 0.65, P = 1.09 × 10-56) changes were significantly correlated between septic patients and mice. We focused on ITGA2B, tracking and validating the expression, regulation, and functional impact of changes in ITGA2B during sepsis. Increased ITGA2B was identified in bone marrow megakaryocytes within 24 hours of sepsis onset. Subsequent increases in ITGA2B were seen in circulating platelets, suggesting dynamic trafficking of the messenger RNA. Transcriptional changes in ITGA2B were accompanied by de novo protein synthesis of αIIb and integrin αIIbß3 activation. Increased αIIb was associated with mortality in humans and mice. These findings provide previously unrecognized evidence that human and murine sepsis similarly alters the platelet transcriptional and translational landscape. Moreover, ITGA2B is upregulated and functional in sepsis due to trafficking from megakaryocytes and de novo synthesis in platelets and is associated with increased mortality.

Amicus or Adversary Revisited: Platelets in Acute Lung Injury and Acute Respiratory Distress Syndrome.

Platelets are essential cellular effectors of hemostasis and contribute to disease as circulating effectors of pathologic thrombosis. These are their most widely known biologic activities. Nevertheless, recent observations demonstrate that platelets have a much more intricate repertoire beyond these traditional functions and that they are specialized for contributions to vascular barrier integrity, organ repair, antimicrobial host defense, inflammation, and activities across the immune continuum. Paradoxically, on the basis of clinical investigations and animal models of disease, some of these newly discovered activities of platelets appear to contribute to tissue injury. Studies in the last decade indicate unique interactions of platelets and their precursor, the megakaryocyte, in the lung and implicate platelets as essential effectors in experimental acute lung injury and clinical acute respiratory distress syndrome. Additional discoveries derived from evolving work will be required to precisely define the contributions of platelets to complex subphenotypes of acute lung injury and to determine if these remarkable and versatile blood cells are therapeutic targets in acute respiratory distress syndrome.

Glucose Transporter 3 Potentiates Degranulation and Is Required for Platelet Activation.

OBJECTIVE: On activation, platelets increase glucose uptake, glycolysis, and glucose oxidation and consume stored glycogen. This correlation between glucose metabolism and platelet function is not well understood and even less is known about the role of glucose metabolism on platelet function in vivo. For glucose to enter a cell, it must be transported through glucose transporters. Here we evaluate the contribution of GLUT3 (glucose transporter 3) to platelet function to better understand glucose metabolism in platelets. APPROACH AND RESULTS: Platelet-specific knockout of GLUT3 was generated by crossing mice harboring GLUT3 floxed allele to a PF4 (platelet factor 4)-driven Cre recombinase. In platelets, GLUT3 is localized primarily on α-granule membranes and under basal conditions facilitates glucose uptake into α-granules to be used for glycolysis. After activation, platelets degranulate and GLUT3 translocates to the plasma membrane, which is responsible for activation-mediated increased glucose uptake. In vivo, loss of GLUT3 in platelets increased survival in a collagen/epinephrine model of pulmonary embolism, and in a K/BxN model of autoimmune inflammatory disease, platelet-specific GLUT3 knockout mice display decreased disease progression. Mechanistically, loss of GLUT3 decreased platelet degranulation, spreading, and clot retraction. Decreased α-granule degranulation is due in part to an impaired ability of GLUT3 to potentiate exocytosis. CONCLUSIONS: GLUT3-mediated glucose utilization and glycogenolysis in platelets promotes α-granule release, platelet activation, and postactivation functions.

Autophagy and its consequences for platelet biology.

Autophagy, the continuous recycling of intracellular building blocks, molecules, and organelles is necessary to preserve cellular function and homeostasis. In this context, it was demonstrated that autophagy plays an important role in megakaryopoiesis, the development and differentiation of hematopoietic progenitor cells into megakaryocytes. Furthermore, in recent years, autophagic proteins were detected in platelets, anucleate cells generated by megakaryocytes, responsible for hemostasis, thrombosis, and a key cell in inflammation and host immune responses. In the last decade studies have indicated the occurrence of autophagy in platelets. Moreover, autophagy in platelets was subsequently demonstrated to be involved in platelet aggregation, adhesion, and thrombus formation. Here, we review the current knowledge about autophagy in platelets, its function, and clinical implications. However, at the advent of platelet autophagy research, additional discoveries derived from evolving work will be required to precisely define the contributions of autophagy in platelets, and to expand the ever increasing physiologic and pathologic roles these remarkable and versatile blood cells play.

Alterations in the megakaryocyte transcriptome impacts platelet function in sepsis and COVID-19 infection.

Platelets and their parent cell, the megakaryocyte (MK), are increasingly recognized for their roles during infection and inflammation. The MK residing in the bone marrow or arising from precursors trafficked to other organs for development go on to form platelets through thrombopoiesis. Infection, by direct and indirect mechanisms, can alter the transcriptional profile of MKs. The altered environment, whether mediated by inflammatory cytokines or other signaling mechanisms results in an altered platelet transcriptome. Platelets released into the circulation, in turn, interact with each other, circulating leukocytes and endothelial cells and contribute to the clearance of pathogens or the potentiation of pathophysiology through such mechanisms as immunothrombosis. In this article we hope to identify key contributions that explore the impact of an altered transcriptomic landscape during severe, systemic response to infection broadly defined as sepsis, and viral infections, including SARS-CoV2. We include current publications that outline the role of MKs from bone-marrow and extra-medullary sites as well as the circulating platelet. The underlying diseases result in thrombotic complications that exacerbate organ dysfunction and mortality. Understanding the impact of platelets on the pathophysiology of disease may drive therapeutic advances to improve the morbidity and mortality of these deadly afflictions.

Characterization of all small RNAs in and comparisons across cultured megakaryocytes and platelets of healthy individuals and COVID-19 patients.

BACKGROUND: The small noncoding RNAs (sncRNAs) in megakaryocytes (MKs) and platelets are not well characterized. Neither is the impact of SARS-CoV-2 infection on the sncRNAs of platelets. OBJECTIVES: To investigate the sorting of MK sncRNAs into platelets, and the differences in the platelet sncRNAomes of healthy donors (HDs) and COVID-19 patients. METHODS: We comprehensively profiled sncRNAs from MKs cultured from cord blood-derived CD34+ cells, platelets from HDs, and platelets from patients with moderate and severe SARS-CoV-2 infection. We also comprehensively profiled Argonaute (AGO)-bound sncRNAs from the cultured MKs. RESULTS: We characterized the sncRNAs in MKs and platelets and can account for ∼95% of all sequenced reads. We found that MKs primarily comprise microRNA isoforms (isomiRs), tRNA-derived fragments (tRFs), rRNA-derived fragments (rRFs), and Y RNA-derived fragments (yRFs) in comparable abundances. The platelets of HDs showed a skewed distribution by comparison: 56.7% of all sncRNAs are yRFs, 34.4% are isomiRs, and <2.0% are tRFs and rRFs. Most isomiRs in MKs and platelets are either noncanonical, nontemplated, or both. When comparing MKs and platelets from HDs, we found numerous isomiRs, tRFs, rRFs, and yRFs showing opposite enrichments or depletions, including molecules from the same parental miRNA arm, tRNA, rRNA, or Y RNA. The sncRNAome of platelets from patients with COVID-19 is skewed compared to that of HDs with only 19.8% of all sncRNAs now being yRFs, isomiRs increasing to 63.6%, and tRFs and rRFs more than tripling their presence to 6.1%. CONCLUSION: The sncRNAomes of MKs and platelets are very rich and more complex than it has been believed. The evidence suggests complex mechanisms that sort MK sncRNAs into platelets. SARS-CoV-2 infection acutely alters the contents of platelets by changing the relative proportions of their sncRNAs.

Tissue factor activity of small and large extracellular vesicles in different diseases.

Background: Circulating procoagulant extracellular vesicles (EVs) are increased in diseases, such as cancer, sepsis, and COVID-19. EV tissue factor (TF) activity is associated with disseminated intravascular coagulation in sepsis and venous thrombosis in patients with pancreatic cancer and COVID-19. EVs are commonly isolated by centrifugation at ∼20,000 g. Objectives: In this study, we analyzed the TF activity of 2 EV populations enriched for large and small EVs in patients with either sepsis, pancreatic cancer, or COVID-19. Methods: EVs were isolated from plasma by sequential centrifugation at 20,000 g (large EVs, LEVs) and then 100,000 g (small EVs, SEVs). We analyzed EVs from plasma prepared from whole blood samples from healthy individuals with or without lipopolysaccharide (LPS) stimulation as well as EVs from plasma samples from patients with either sepsis, pancreatic cancer, or COVID-19. TF-dependent (EV-TF activity) and TF-independent factor Xa (FXa) generation of the EVs was measured. Results: LPS increased EV-TF activity in LEVs but not SEVs. Similarly, in 2 patients with sepsis who had EV-TF activity above the background of the assay we observed EV-TF activity in LEVs but not SEVs. Patients with pancreatic cancer or COVID-19 had circulating EV-TF activity in both LEVs and SEVs. Conclusion: We recommend that EVs are isolated from plasma from patients by centrifugation at 100,000 g rather than 20,000 g to obtain a more accurate measure of levels of circulating EV-TF activity.

Intravenous aviptadil and remdesivir for treatment of COVID-19-associated hypoxaemic respiratory failure in the USA (TESICO): a randomised, placebo-controlled trial.

BACKGROUND: There is a clinical need for therapeutics for COVID-19 patients with acute hypoxemic respiratory failure whose 60-day mortality remains at 30-50%. Aviptadil, a lung-protective neuropeptide, and remdesivir, a nucleotide prodrug of an adenosine analog, were compared with placebo among patients with COVID-19 acute hypoxaemic respiratory failure. METHODS: TESICO was a randomised trial of aviptadil and remdesivir versus placebo at 28 sites in the USA. Hospitalised adult patients were eligible for the study if they had acute hypoxaemic respiratory failure due to confirmed SARS-CoV-2 infection and were within 4 days of the onset of respiratory failure. Participants could be randomly assigned to both study treatments in a 2 × 2 factorial design or to just one of the agents. Participants were randomly assigned with a web-based application. For each site, randomisation was stratified by disease severity (high-flow nasal oxygen or non-invasive ventilation vs invasive mechanical ventilation or extracorporeal membrane oxygenation [ECMO]), and four strata were defined by remdesivir and aviptadil eligibility, as follows: (1) eligible for randomisation to aviptadil and remdesivir in the 2 × 2 factorial design; participants were equally randomly assigned (1:1:1:1) to intravenous aviptadil plus remdesivir, aviptadil plus remdesivir matched placebo, aviptadil matched placebo plus remdesvir, or aviptadil placebo plus remdesivir placebo; (2) eligible for randomisation to aviptadil only because remdesivir was started before randomisation; (3) eligible for randomisation to aviptadil only because remdesivir was contraindicated; and (4) eligible for randomisation to remdesivir only because aviptadil was contraindicated. For participants in strata 2-4, randomisation was 1:1 to the active agent or matched placebo. Aviptadil was administered as a daily 12-h infusion for 3 days, targeting 600 pmol/kg on infusion day 1, 1200 pmol/kg on day 2, and 1800 pmol/kg on day 3. Remdesivir was administered as a 200 mg loading dose, followed by 100 mg daily maintenance doses for up to a 10-day total course. For participants assigned to placebo for either agent, matched saline placebo was administered in identical volumes. For both treatment comparisons, the primary outcome, assessed at day 90, was a six-category ordinal outcome: (1) at home (defined as the type of residence before hospitalisation) and off oxygen (recovered) for at least 77 days, (2) at home and off oxygen for 49-76 days, (3) at home and off oxygen for 1-48 days, (4) not hospitalised but either on supplemental oxygen or not at home, (5) hospitalised or in hospice care, or (6) dead. Mortality up to day 90 was a key secondary outcome. The independent data and safety monitoring board recommended stopping the aviptadil trial on May 25, 2022, for futility. On June 9, 2022, the sponsor stopped the trial of remdesivir due to slow enrolment. The trial is registered with ClinicalTrials.gov, NCT04843761. FINDINGS: Between April 21, 2021, and May 24, 2022, we enrolled 473 participants in the study. For the aviptadil comparison, 471 participants were randomly assigned to aviptadil or matched placebo. The modified intention-to-treat population comprised 461 participants who received at least a partial infusion of aviptadil (231 participants) or aviptadil matched placebo (230 participants). For the remdesivir comparison, 87 participants were randomly assigned to remdesivir or matched placebo and all received some infusion of remdesivir (44 participants) or remdesivir matched placebo (43 participants). 85 participants were included in the modified intention-to-treat analyses for both agents (ie, those enrolled in the 2 x 2 factorial). For the aviptadil versus placebo comparison, the median age was 57 years (IQR 46-66), 178 (39%) of 461 participants were female, and 246 (53%) were Black, Hispanic, Asian or other (vs 215 [47%] White participants). 431 (94%) of 461 participants were in an intensive care unit at baseline, with 271 (59%) receiving high-flow nasal oxygen or non-invasive ventiliation, 185 (40%) receiving invasive mechanical ventilation, and five (1%) receiving ECMO. The odds ratio (OR) for being in a better category of the primary efficacy endpoint for aviptadil versus placebo at day 90, from a model stratified by baseline disease severity, was 1·11 (95% CI 0·80-1·55; p=0·54). Up to day 90, 86 participants in the aviptadil group and 83 in the placebo group died. The cumulative percentage who died up to day 90 was 38% in the aviptadil group and 36% in the placebo group (hazard ratio 1·04, 95% CI 0·77-1·41; p=0·78). The primary safety outcome of death, serious adverse events, organ failure, serious infection, or grade 3 or 4 adverse events up to day 5 occurred in 146 (63%) of 231 patients in the aviptadil group compared with 129 (56%) of 230 participants in the placebo group (OR 1·40, 95% CI 0·94-2·08; p=0·10). INTERPRETATION: Among patients with COVID-19-associated acute hypoxaemic respiratory failure, aviptadil did not significantly improve clinical outcomes up to day 90 when compared with placebo. The smaller than planned sample size for the remdesivir trial did not permit definitive conclusions regarding safety or efficacy. FUNDING: National Institutes of Health.

Human platelets display dysregulated sepsis-associated autophagy, induced by altered LC3 protein-protein interaction of the Vici-protein EPG5.

Platelets mediate central aspects of host responses during sepsis, an acute profoundly systemic inflammatory response due to infection. Macroautophagy/autophagy, which mediates critical aspects of cellular responses during inflammatory conditions, is known to be a functional cellular process in anucleate platelets, and is essential for normal platelet functions. Nevertheless, how sepsis may alter autophagy in platelets has never been established. Using platelets isolated from septic patients and matched healthy controls, we show that during clinical sepsis, the number of autophagosomes is increased in platelets, most likely due to an accumulation of autophagosomes, some containing mitochondria and indicative of mitophagy. Therefore, autophagy induction or early-stage autophagosome formation (as compared to decreased later-stage autophagosome maturation or autophagosome-late endosome/lysosome fusion) is normal or increased. This was consistent with decreased fusion of autophagosomes with lysosomes in platelets. EPG5 (ectopic P-granules autophagy protein 5 homolog), a protein essential for normal autophagy, expression did increase, while protein-protein interactions between EPG5 and MAP1LC3/LC3 (which orchestrate the fusion of autophagosomes and lysosomes) were significantly reduced in platelets during sepsis. Furthermore, data from a megakaryocyte model demonstrate the importance of TLR4 (toll like receptor 4), LPS-dependent signaling for regulating this mechanism. Similar phenotypes were also observed in platelets isolated from a patient with Vici syndrome: an inherited condition caused by a naturally occurring, loss-of-function mutation in EPG5. Together, we provide evidence that autophagic functions are aberrant in platelets during sepsis, due in part to reduced EPG5-LC3 interactions, regulated by TLR4 engagement, and the resultant accumulation of autophagosomes.Abbreviations: ACTB: beta actin; CLP: cecal ligation and puncture; Co-IP: co-immunoprecipitation; DAP: death associated protein; DMSO: dimethyl sulfoxide; EPG5: ectopic P-granules autophagy protein 5 homolog; ECL: enhanced chemiluminescence; HBSS: Hanks' balanced salt solution; HRP: horseradish peroxidase; ICU: intensive care unit; LPS: lipopolysaccharide; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; MKs: megakaryocytes; PFA: paraformaldehyde; PBS: phosphate-buffered saline; PLA: proximity ligation assay; pRT-PCR: quantitative real-time polymerase chain reaction; RT: room temperature; SQSTM1/p62: sequestosome 1; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TLR4: toll like receptor 4; TEM: transmission electron microscopy; WGA: wheat germ agglutinin.

Eligibility for Posthospitalization Venous Thromboembolism Prophylaxis in Hospitalized Patients With COVID-19: A Retrospective Cohort Study.

Background A recent randomized trial, the MICHELLE trial, demonstrated improved posthospital outcomes with a 35-day course of prophylactic rivaroxaban for patients hospitalized with COVID-19 at high risk of venous thromboembolism. We explored how often these findings may apply to an unselected clinical population of patients hospitalized with COVID-19. Methods and Results Using a 35-hospital retrospective cohort of patients hospitalized between March 7, 2020, and January 23, 2021, with COVID-19 (MI-COVID19 database), we quantified the percentage of hospitalized patients with COVID-19 who would be eligible for rivaroxaban at discharge per MICHELLE trial criteria and report clinical event rates. The main clinical outcome was derived from the MICHELLE trial and included a composite of symptomatic venous thromboembolism, pulmonary embolus-related death, nonhemorrhagic stroke, and cardiovascular death at 35 days. Multiple sensitivity analyses tested different eligibility and exclusion criteria definitions to determine the effect on eligibility for postdischarge anticoagulation prophylaxis. Of 2016 patients hospitalized with COVID-19 who survived to discharge and did not have another indication for anticoagulation, 25.9% (n=523) would be eligible for postdischarge thromboprophylaxis per the MICHELLE trial criteria (range, 2.9%-39.4% on sensitivity analysis). Of the 416 who had discharge anticoagulant data collected, only 13.2% (55/416) were actually prescribed a new anticoagulant at discharge. Of patients eligible for rivaroxaban per the MICHELLE trial, the composite clinical outcome occurred in 1.2% (6/519); similar outcome rates were 5.7% and 0.63% in the MICHELLE trial's control (no anticoagulation) and intervention (rivaroxaban) groups, respectively. Symptomatic venous thromboembolism events and all-cause mortality were 6.2% (32/519) and 5.66% in the MI-COVID19 and MICHELLE trial control cohorts, respectively. Conclusions Across 35 hospitals in Michigan, ≈1 in 4 patients hospitalized with COVID-19 would qualify for posthospital thromboprophylaxis. With only 13% of patients actually receiving postdischarge prophylaxis, there is a potential opportunity for improvement in care.

Homogeneous surrogate virus neutralization assay to rapidly assess neutralization activity of anti-SARS-CoV-2 antibodies.

The COVID-19 pandemic triggered the development of numerous diagnostic tools to monitor infection and to determine immune response. Although assays to measure binding antibodies against SARS-CoV-2 are widely available, more specific tests measuring neutralization activities of antibodies are immediately needed to quantify the extent and duration of protection that results from infection or vaccination. We previously developed a 'Serological Assay based on a Tri-part split-NanoLuc® (SATiN)' to detect antibodies that bind to the spike (S) protein of SARS-CoV-2. Here, we expand on our previous work and describe a reconfigured version of the SATiN assay, called Neutralization SATiN (Neu-SATiN), which measures neutralization activity of antibodies directly from convalescent or vaccinated sera. The results obtained with our assay and other neutralization assays are comparable but with significantly shorter preparation and run time for Neu-SATiN. As the assay is modular, we further demonstrate that Neu-SATiN enables rapid assessment of the effectiveness of vaccines and level of protection against existing SARS-CoV-2 variants of concern and can therefore be readily adapted for emerging variants.

IFITM3 regulates fibrinogen endocytosis and platelet reactivity in nonviral sepsis.

Platelets and megakaryocytes are critical players in immune responses. Recent reports suggest infection and inflammation alter the megakaryocyte and platelet transcriptome to induce altered platelet reactivity. We determined whether nonviral sepsis induces differential platelet gene expression and reactivity. Nonviral sepsis upregulated IFN-induced transmembrane protein 3 (IFITM3), an IFN-responsive gene that restricts viral replication. As IFITM3 has been linked to clathrin-mediated endocytosis, we determined whether IFITM3 promoted endocytosis of α-granule proteins. IFN stimulation enhanced fibrinogen endocytosis in megakaryocytes and platelets from Ifitm+/+ mice, but not Ifitm-/- mice. IFITM3 overexpression or deletion in megakaryocytes demonstrated IFITM3 was necessary and sufficient to regulate fibrinogen endocytosis. Mechanistically, IFITM3 interacted with clathrin and αIIb and altered their plasma membrane localization into lipid rafts. In vivo IFN administration increased fibrinogen endocytosis, platelet reactivity, and thrombosis in an IFITM-dependent manner. In contrast, Ifitm-/- mice were completely rescued from IFN-induced platelet hyperreactivity and thrombosis. During murine sepsis, platelets from Ifitm+/+ mice demonstrated increased fibrinogen content and platelet reactivity, which was dependent on IFN-α and IFITMs. Platelets from patients with nonviral sepsis had increases in platelet IFITM3 expression, fibrinogen content, and hyperreactivity. These data identify IFITM3 as a regulator of platelet endocytosis, hyperreactivity, and thrombosis during inflammatory stress.