RESUMO
Extracellular vesicles (EVs) represent nanometer-sized, subcellular spheres, that are released from almost any cell type and carry a wide variety of biologically relevant cargo. In severe cases of coronavirus disease 2019 (COVID-19) and other states of systemic pro-inflammatory activation, EVs, and their cargo can serve as conveyors and indicators for disease severity and progression. This information may help distinguish individuals with a less severe manifestation of the disease from patients who exhibit severe acute respiratory distress syndrome (ARDS) and require intensive care measures. Here, we investigated the potential of EVs and associated miRNAs to distinguish normal ward patients from intensive care unit (ICU) patients (N = 10/group), with 10 healthy donors serving as the control group. Blood samples from which plasma and subsequently EVs were harvested by differential ultracentrifugation (UC) were obtained at several points in time throughout treatment. EV-enriched fractions were characterized by flow cytometry (FC), nanoparticle tracking analysis (NTA), and qPCR to determine the presence of selected miRNAs. Circulating EVs showed specific protein signatures associated with endothelial and platelet origin over the course of the treatment. Additionally, significantly higher overall EV quantities corresponded with increased COVID-19 severity. MiR-223-3p, miR-191-5p, and miR-126-3p exhibited higher relative expression in the ICU group. Furthermore, EVs presenting endothelial-like protein signatures and the associated miR-126-3p showed the highest area under the curve in terms of receiver operating characteristics regarding the requirement for ICU treatment. In this exploratory investigation, we report that specific circulating EVs and miRNAs appear at higher levels in COVID-19 patients, especially when critical care measures are indicated. Our data suggest that endothelial-like EVs and associated miRNAs likely represent targets for future laboratory assays and may aid in clinical decision-making in COVID-19.
RESUMO
BACKGROUND: Viscoelastically guided coagulation factor concentrate-based algorithms for the treatment of trauma-induced coagulopathy include the administration of prothrombin complex concentrates (PCCs). However, the exact role of PCC preparations in this context is a matter of debate. Particularly, the ideal diagnostic trigger for their administration and potential differences between heparin-containing and heparin-free preparations remain unclear. We investigated the hypothesis that 2 different PCCs might have distinct influences on in vitro blood coagulation. METHODS: We conducted a direct comparison of 2 commercially available PCC preparations (the heparin-containing Beriplex P/N and the heparin-free Cofact) in an in vitro hemodilution model. Sole fibrinogen substitution served as the control group. To characterize the hemostatic changes, we utilized conventional coagulation tests, a thrombin generation assay (TGA), and 2 different viscoelastic hemostatic assays (VHAs; ROTEM delta and ClotPro). RESULTS: Irrespective of the diagnostic assay used, no significant differences between the 2 PCC groups were observed. Fibrinogen levels remained stable from the baseline throughout every dilution level. The control group already showed an increased endogenous thrombin potential (ETP; nM·L -1 ·min -1 ) at all dilution levels compared to baseline (baseline, 2829.4 (432.8); 40% dilution, 4211.7 (391.6); 60% dilution, 4290.9 (300.8); 80% dilution, 3861.4 (303.5); all P < .001). Spiking with both PCC preparations led to a further-pronounced thrombin elevation in comparison to the control group (ETP at 40% dilution, PCC1: 4913.3 [370.2], PCC2: 4988.1 [265.7]; 60% dilution, PCC1: 5174.5 [234.7], PCC2: 5390.4 [334.9]; 80% dilution, PCC1: 5253.8 [357.9], PCC2: 5392.6 [313.4]; all P < .001). Conventional coagulation tests did not mirror the TGA results. Despite increased thrombin generation, prothrombin time was significantly prolonged at all dilution levels for the control group, and both PCC groups exhibited significant prolongations at the 60% and 80% dilution levels (all P < .001) compared to baseline. Similarly, VHA did not depict the thrombin elevation. Furthermore, descriptive analyses revealed relevant differences between the 2 VHA devices, particularly at baseline. CONCLUSIONS: Both PCC preparations (ie, irrespective of heparin content) induced significant elevation of thrombin generation, which was not depicted by conventional coagulation tests or VHA. Our in vitro results suggest that diagnostic assays routinely used to guide PCC administration might not adequately reflect thrombin generation in bleeding patients.
