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 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, ACE2, was not detected by mRNA or protein in platelets. Surprisingly, mRNA from the SARS-CoV-2 N1 gene was detected in platelets from 2/25 COVID-19 patients, suggesting 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 to 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.

Inbred mice are a useful tool for studying the in vivo functions of platelets. Nonetheless, the mRNA signature of mouse platelets is not known. Here, we use pairedend next-generation RNA sequencing (RNA-seq) to characterize the polyadenylated transcriptomes of human and mouse platelets. We report that RNA-seq provides unprecedented resolution of mRNAs that are expressed across the entire human and mouse genomes. Transcript expression and abundance are often conserved between the 2 species. Several mRNAs, however, are differentially expressed in human and mouse platelets. Moreover, previously described functional disparities between mouse and human platelets are reflected in differences at the transcript level, including protease activated receptor-1, protease activated receptor-3, platelet activating factor receptor, and factor V. This suggests that RNA-seq is a useful tool for predicting differences in platelet function between mice and humans. Our nextgeneration sequencing analysis provides new insights into the human and murine platelet transcriptomes. The sequencing dataset will be useful in the design of mouse models of hemostasis and a catalyst for discovery of new functions of platelets. Access to the dataset is found in the "Introduction." (Blood. 2011; 118(14):e101-e111)

Some familial platelet disorders are associated with predisposition to leukemia, myelodysplastic syndrome (MDS) or dyserythropoietic anemia.1,2 We identified a family with autosomal dominant thrombocytopenia, high erythrocyte mean corpuscular volume (MCV) and two occurrences of B-cell precursor acute lymphoblastic leukemia (ALL). Whole exome sequencing identified a heterozygous single nucleotide change in ETV6 (Ets Variant Gene 6), c.641C>T, encoding a p.Pro214Leu substitution in the central domain, segregating with thrombocytopenia and elevated MCV. A screen of 23 families with similar phenotype found two with ETV6 mutations. One family had the p.Pro214Leu mutation and one individual with ALL. The other family had a c.1252A>G transition producing a p.Arg418Gly substitution in the DNA binding domain, with alternative splicing and exon-skipping. Functional characterization of these mutations showed aberrant cellular localization of mutant and endogenous ETV6, decreased transcriptional repression and altered megakaryocyte maturation. Our findings underscore a key role for ETV6 in platelet formation and leukemia predisposition.

Neutrophils are highly specialized innate immune effector cells that evolved for antimicrobial host defense. In response to inflammatory stimuli and pathogens, they form neutrophil extracellular traps (NETs), which capture and kill extracellular microbes. Deficient NET formation predisposes humans to severe infection, but, paradoxically, dysregulated NET formation contributes to inflammatory vascular injury and tissue damage. The molecular pathways and signaling mechanisms that control NET formation remain largely uncharacterized. Using primary human neutrophils and genetically manipulated myeloid leukocytes differentiated to surrogate neutrophils, we found that mammalian target of rapamycin ( IntroductionNeutrophils (polymorphonuclear leukocytes, PMNs) are key effector cells in infection, inflammation, and tissue injury. 1 Formation of neutrophil extracellular traps (NETs), first identified with human PMNs, is a function of neutrophils. 2 NETs are complex lattices of decondensed chromatin that trap and kill bacteria, fungi, and some parasites by exposing them to high concentrations of NETassociated microbicidal factors. 3,4 Rapidly evolving studies indicate that NETs effect extracellular microbial killing while limiting the spread of pathogens in vivo. 3,5 The intracellular signaling pathways that regulate NET formation by PMNs remain largely unknown. There is evidence that generation of reactive oxygen species (ROS) is a key event. 4,6 Nevertheless, we showed in primary human PMNs that NET formation requires signaling events and regulatory pathways in addition to ROS generation. 4 Consistent with our results, subsequent studies in human HL-60 myeloid leukocytes and genetically altered mice indicate that activity of peptidylarginine deiminase 4, an enzyme responsible for chromatin decondensation, is also required. 5,7 Recent observations further suggest that NET formation requires enzymatic activity of neutrophil elastase (NE) and myeloperoxidase to initiate degradation of core histones that lead to chromatin decondensation before plasma membrane rupture. 8 Furthermore, ROS generation and NET formation can be dissociated under some conditions. 9,10 Thus, molecular regulation of NET formation is complex and may involve multiple signaling pathways and effector events, depending on the neutrophil agonists and inflammatory context.The mammalian target of rapamycin (mTOR) is a highly conserved PI3K-like serine/threonine kinase with functional homologs found in all studied eukaryotic organisms. 11 mTOR integrates nutrient, energy, oxygen sensing, and mitogenic input signals. 12 We found that mTOR also responds to inflammatory signals and mediates a previously unrecognized pathway of posttranscriptional gene regulation in human PMNs. 13 These results identified a new mechanism by which mTOR can regulate innate, as well as, adaptive, immune responses. Immunoregulatory activities of mTOR are now increasingly recognized. 14 Recent observations indicate that hypoxia inducible factor 1␣ (HIF-1␣), the regulated subunit of ...