Diverse human extracellular RNAs are widely detected in human plasma

There is growing appreciation for the importance of non-protein-coding genes in development and disease. Although much is known about microRNAs, limitations in bioinformatic analyses of RNA sequencing have precluded broad assessment of other forms of small-RNAs in humans. By analysing sequencing data from plasma-derived RNA from 40 individuals, here we identified over a thousand human extracellular RNAs including microRNAs, piwi-interacting RNA (piRNA), and small nucleolar RNAs. Using a targeted quantitative PCR with reverse transcription approach in an additional 2,763 individuals, we characterized almost 500 of the most abundant extracellular transcripts including microRNAs, piRNAs and small nucleolar RNAs. The presence in plasma of many non-microRNA small-RNAs was confirmed in an independent cohort. We present comprehensive data to demonstrate the broad and consistent detection of diverse classes of circulating non-cellular small-RNAs from a large population

Micro RNAs from DNA Viruses are Found Widely in Plasma in a Large Observational Human Population

Viral infections associate with disease risk and select families of viruses encode miRNAs that control an efficient viral cycle. The association of viral miRNA expression with disease in a large human population has not been previously explored. We sequenced plasma RNA from 40 participants of the Framingham Heart Study (FHS, Offspring Cohort, Visit 8) and identified 3 viral miRNAs from 3 different human Herpesviridae. These miRNAs were mostly related to viral latency and have not been previously detected in human plasma. Viral miRNA expression was then screened in the plasma of 2763 participants of the remaining cohort utilizing high-throughput RT-qPCR. All 3 viral miRNAs associated with combinations of inflammatory or prothrombotic circulating biomarkers (sTNFRII, IL-6, sICAM1, OPG, P-selectin) but did not associate with hypertension, coronary heart disease or cancer. Using a large observational population, we demonstrate that the presence of select viral miRNAs in the human circulation associate with inflammatory biomarkers and possibly immune response, but fail to associate with overt disease. This study greatly extends smaller singular observations of viral miRNAs in the human circulation and suggests that select viral miRNAs, such as those for latency, may not impact disease manifestation

Platelets and COVID-19: Inflammation, Hyperactivation and Additional Questions

The mechanisms underlining thrombosis in COVID-19 patients are not known and likely due to multiple processes including inflammation, oxygen demand injury, and plaque rupture triggered by the infection. Platelets mediate thrombotic vascular occlusion but are also increasingly recognized to have immunomodulatory activity. Several of our recent studies have characterized the role of viral infections in cardiac disease. Although robust data on the scope of acute myocardial infarction in COVID-19 are not yet available, myocardial infarction contributed to in-hospital mortality during previous severe acute respiratory syndrome/coronavirus epidemics. A recent study has also demonstrated that influenza and other respiratory viruses increase the incidence of acute myocardial infarction particularly in the first 7 days post-infection, suggesting that platelets may be directly involved in mediating an immune response but, when dysregulated, can also lead to thrombotic vascular occlusion

Platelets and Immunity: Going Viral

Platelets are the major blood component bridging immunity and thrombosis. Abundant in the circulation, platelets encounter pathogens at a higher rate than any circulating leukocyte. Viral particles of various blood-borne pathogens such as HIV,1 dengue,2 or even respiratory viruses such as influenza3 are found inside human platelets. Viral infections with these viruses,4 as well as the SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), can lead to thrombocytopenia5 along with thrombotic complications6–8 in patients. The presence of internalized viral particles, thrombocytopenia, and thrombosis implicates platelets as active participants in immunity during viral infections

SARS-CoV-2 Receptors are Expressed on Human Platelets and the Effect of Aspirin on Clinical Outcomes in COVID-19 Patients [preprint]

Coronavirus disease-2019 (COVID-19) caused by SARS-CoV-2 is an ongoing viral pandemic marked by increased risk of thrombotic events. However, the role of platelets in the elevated observed thrombotic risk in COVID-19 and utility of anti-platelet agents in attenuating thrombosis is unknown. We aimed to determine if human platelets express the known SARS-CoV-2 receptor-protease axis on their cell surface and assess whether the anti-platelet effect of aspirin may mitigate risk of myocardial infarction (MI), cerebrovascular accident (CVA), and venous thromboembolism (VTE) in COVID-19. Expression of ACE2 and TMPRSS2 on human platelets were detected by immunoblotting and confirmed by confocal microscopy. We evaluated 22,072 symptomatic patients tested for COVID-19. Propensity-matched analyses were performed to determine if treatment with aspirin or non-steroidal anti-inflammatory drugs (NSAIDs) affected thrombotic outcomes in COVID-19. Neither aspirin nor NSAIDs affected mortality in COVID-19. However, both aspirin and NSAID therapies were associated with increased risk of the combined thrombotic endpoint of (MI), (CVA), and (VTE). Thus, while platelets clearly express ACE2-TMPRSS2 receptor-protease axis for SARS-CoV-2 infection, aspirin does not prevent thrombosis and death in COVID-19. The mechanisms of thrombosis in COVID-19, therefore, appears distinct and the role of platelets as direct mediators of SARS-CoV-2-mediated thrombosis warrants further investigation

ACEing COVID-19: A Role For Angiotensin Axis Inhibition in SARS-CoV-2 infection

Cardiometabolic disease, especially hypertension, is a common risk factor for mortality among individuals with SARS-CoV-2 disease (COVID-19). The role of hypertension and vascular disease in COVID-19 has raised considerable debate around how to best manage anti-hypertensive therapy to alter disease trajectory. Given the role of the ACE2 receptor in SARS-CoV-2 viral entry6, angiotensin system modulation by ACE inhibitors (ACE-I) or angiotensin-II receptor blockers (ARBs) in clinical management have taken center stage in this controversy

Thrombosis and platelets: an update

Haemostasis and thrombosis are complex, multifactorial processes. There is an evolving understanding of the mechanisms influencing vascular occlusion and the role of inflammation and immunity. Despite major advances in elucidating the mechanistic pathways mediating platelet function and thrombosis, challenges in the treatment of vascular occlusive diseases persist. Pharmacological advances have greatly affected thrombotic outcomes, but this has led to the unwanted side effect of bleeding. Detailed assessment of the impact of non-thrombotic diseases on haemostasis and thrombosis is necessary to better evaluate thrombotic risk and establish optimal treatment. This review will focus on recent advances in understanding the contribution of evolving risk factors to thrombosis

A Translational Model for Venous Thromboembolism: MicroRNA Expression in Hibernating Black Bears

BACKGROUND: Hibernating American black bears have significantly different clotting parameters than their summer active counterparts, affording them protection against venous thromboembolism during prolonged periods of immobility. We sought to evaluate if significant differences exist between the expression of microRNAs in the plasma of hibernating black bears compared with their summer active counterparts, potentially contributing to differences in hemostasis during hibernation. MATERIALS AND METHODS: MicroRNA sequencing was assessed in plasma from 21 American black bears in summer active (n = 11) and hibernating states (n = 10), and microRNA signatures during hibernating and active state were established using both bear and human genome. MicroRNA targets were predicted using messenger RNA (mRNA) transcripts from black bear kidney cells. In vitro studies were performed to confirm the relationship between identified microRNAs and mRNA expression, using artificial microRNA and human liver cells. RESULTS: Using the bear genome, we identified 15 microRNAs differentially expressed in the plasma of hibernating black bears. Of these microRNAs, three were significantly downregulated (miR-141-3p, miR-200a-3p, and miR-200c-3p), were predicted to target SERPINC1, the gene for antithrombin, and demonstrated regulatory control of the gene mRNA expression in cell studies. CONCLUSIONS: Our findings suggest that the hibernating black bears\u27 ability to maintain hemostasis and achieve protection from venous thromboembolism during prolonged periods of immobility may be due to changes in microRNA signatures and possible upregulation of antithrombin expression

Pollen-derived RNAs Are Found in the Human Circulation

The presence of nonhuman RNAs in man has been questioned and it is unclear if food-derived miRNAs cross into the circulation. In a large population study, we found nonhuman miRNAs in plasma by RNA sequencing and validated a small number of pine-pollen miRNAs by RT-qPCR in 2,776 people. The presence of these pine-pollen miRNAs associated with hay fever and not with overt cardiovascular or pulmonary disease. Using in vivo and in vitro models, we found that transmission of pollen-miRNAs into the circulation occurs via pulmonary transfer and this transfer was mediated by platelet-pulmonary vascular cell interactions and platelet pollen-DNA uptake. These data demonstrate that pollen-derived plant miRNAs can be horizontally transferred into the circulation via the pulmonary system in humans. Although these data suggest mechanistic plausibility for pulmonary-mediated plant-derived miRNA transfer into the human circulation, our large observational cohort data do not implicate major disease or risk factor association

SARS-CoV-2 Initiates Programmed Cell Death in Platelets

Rationale: COVID-19 is characterized by increased incidence of microthrombosis with hyperactive platelets sporadically containing viral RNA. It is unclear if SARS-CoV-2 directly alters platelet activation or if these changes are a reaction to infection-mediated global inflammatory alterations. Importantly, the direct effect of SARS-CoV-2 on platelets has yet to be studied. Objective: To characterize the direct SARS-CoV-2-platelet interactions using in vitro studies with purified infectious virions and samples from infected patients. Methods and Results: Platelet RNA analyzed by ARTIC v3 sequencing for SARS-CoV-2 showed presence of fragmented viral genome in all COVID-19 patients. Immunofluorescent imaging of platelets from COVID-19 patients confirmed presence of SARS-CoV-2 proteins, while there was no detection of viral RNA by RT-qPCR. Transmission electron microscopy (TEM) of platelets incubated with purified SARS-CoV-2 virions demonstrated rapid internalization and digestion leading to distinct morphological changes, and resulted in a release of extracellular vesicles. Interactions between SARS-CoV-2 and platelets occurred with or without ACE2 presence as measured by immunofluorescence. TEM showed that SARS-CoV-2 virions became internalized when they were attached to microparticles, bypassing the need for ACE2. Enrichment analysis of platelet-transcriptome from patients with acute COVID-19, compared to those with clinical thrombosis, suggested upregulation of pathways related to virally mediated cell death, specifically necroptosis and apoptosis. Platelets incubated with infectious virus appeared to undergo cell death in 30 min post-incubation as assessed by TEM and platelets from COVID-19 patients showed evidence of increased markers of apoptosis and necroptosis by WB. Immunofluorescence confirmed colocalization of SARS-CoV-2 with phospho-MLKL and Caspase-3 on non-permeabilized platelets in vitro and in COVID-19 platelets. Conclusions:Platelets internalize SARS-CoV-2 virions, directly or attached to microparticles, and viral internalization leads to rapid digestion, programmed cell death and extracellular vesicle release. During COVID-19, platelets mediate a rapid response to SARS-CoV-2 and this response can contribute to dysregulated immunity and thrombosis