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 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) with 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<0.0001) and death as outcome (P<0.0005). Illness severity correlated directly with plasma MPO-DNA complexes (P=0.0360), while PaO2/FiO2 correlated inversely(P=0.0340). 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.

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.

Tissue factor (TF) is an essential cofactor for the activation of blood coagulation in vivo. We now report that quiescent human platelets express TF pre-mRNA and, in response to activation, splice this intronic-rich message into mature mRNA. Splicing of TF pre-mRNA is associated with increased TF protein expression, procoagulant activity, and accelerated formation of clots. Pre-mRNA splicing is controlled by Cdc2-like kinase (Clk)1, and interruption of Clk1 signaling prevents TF from accumulating in activated platelets. Elevated intravascular TF has been reported in a variety of prothrombotic diseases, but there is debate as to whether anucleate platelets—the key cellular effector of thrombosis—express TF. Our studies demonstrate that human platelets use Clk1-dependent splicing pathways to generate TF protein in response to cellular activation. We propose that platelet-derived TF contributes to the propagation and stabilization of a thrombus.

Platelets are chief effector cells in hemostasis. In addition, they are multifaceted inflammatory cells with functions that span the continuum from innate immune responses to adaptive immunity. Activated platelets have key “thromboinflammatory” activities in a variety of vascular disorders and vasculopathies. Recently-identified inflammatory and immune activities provide insights into the biology of these versatile blood cells that are directly relevant to human vascular diseases.

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 ...

BACKGROUND: Prospective studies on the incidence of VTE during severe sepsis and septic shock remain absent, hindering effi cacy assessments regarding VTE prevention strategies in sepsis.

Key Points Developed a targeted sequencing platform covering 63 genes linked to heritable bleeding, thrombotic, and platelet disorders. The ThromboGenomics platform provides a sensitive genetic test to obtain molecular diagnoses in patients with a suspected etiology.

Platelets are anucleate cells produced by megakaryocytes. In recent years, a robust body of literature supports the evolving role of platelets as key sentinel and effector cells in infectious diseases, especially critical in bridging hemostatic, inflammatory, and immune continuums. Upon intravascular pathogen invasion, platelets can directly sense viral, parasitic, and bacterial infections through pattern recognition receptors and integrin receptors or pathogen: immunoglobulin complexes through Fc and complement receptors—although our understanding of these interactions remains incomplete. Constantly scanning for areas of injury or inflammation as they circulate in the vasculature, platelets also indirectly respond to pathogen invasion through interactions with leukocytes and the endothelium. Following antigen recognition, platelets often become activated. Through a diverse repertoire of mechanisms, activated platelets can directly sequester or kill pathogens, or facilitate pathogen clearance by activating macrophages and neutrophils, promoting neutrophil extracellular traps (NETs) formation, forming platelet aggregates and microthrombi. At times, however, platelet activation may also be injurious to the host, exacerbating inflammation and promoting endothelial damage and thrombosis. There are many gaps in our understandings of the role of platelets in infectious diseases. However, with the emergence of advanced technologies, our knowledge is increasing. In the current review, we mainly discuss these evolving roles of platelets under four different infectious pathogen infections, of which are dengue, malaria, Esterichia coli (E. coli) and staphylococcus aureus S. aureus, highlighting the complex interplay of these processes with hemostatic and thrombotic pathways.