The endogenous thrombin potential in patients with left ventricular assist device or heart transplant

BackgroundThe Heartmate 3 (HM 3) is a left ventricular assist device featuring less shear stress, milder acquired von Willebrand syndrome, and fewer bleeding incidences than its predecessor the Heartmate II (HM II). The novel surface coating of the HM 3 suggests less contact activation of plasmatic coagulation. We hypothesized that patients with HM 3 exhibit fewer aberrations in their thrombin potential than patients with HM II. We compared these results with the thrombin potential of patients with heart transplantation (HTX).MethodsThrombin generation in plasma samples of patients with HM II (n = 16), HM 3 (n = 20), and HTX (n = 13) was analyzed 3 days after implantation/transplantation and after long-term support (3–24 months) with HM II (n = 16) or HM 3 (n = 12) using calibrated automated thrombography. Heparin in postoperative samples was antagonized with polybrene.ResultsThree days postoperatively HM II patients exhibited a lower endogenous thrombin potential (ETP) than HM 3 and HTX patients (HM II: 947 ± 291 nM*min; HM 3: 1231 ± 176 nM*min; HTX: 1376 ± 162 nM*min, p < 0.001) and a lower velocity index of thrombin generation (HM II: 18.74 ± 10.90 nM/min; HM 3: 32.41 ± 9.51 nM/min; HTX: 37.65 ± 9.41 nM/min, p < 0.01). Subtle differences in the thrombin generation profiles remained in HM II and HM 3 patients under long-term support (Velocity Index: HM II: 38.70 ± 28.46 nM/min; HM 3: 73.32 ± 32.83 nM/min, p < 0.05). Prothrombin fragments 1 + 2 were higher in HM II than in HM 3 patients (HM II: 377.7 ± 208.4 pM; HM 3: 202.1 ± 87.7 pM, p < 0.05) and correlated inversely with the ETP (r = −0.584, p < 0.05).ConclusionWe observed a more aberrant thrombin generation in HM II than in HM 3 despite comparable anticoagulation and routine parameters. A trend toward lower values was still observable in HM 3 compared to HTX patients. Calibrated automated thrombography may be a good tool to monitor the coagulation state of these patients and guide anticoagulation in the future

Impact of infection on proteome-wide glycosylation revealed by distinct signatures for bacterial and viral pathogens

Mechanisms of infection and pathogenesis have predominantly been studied based on differential gene or protein expression. Less is known about posttranslational modifications, which are essential for protein functional diversity. We applied an innovative glycoproteomics method to study the systemic proteome-wide glycosylation in response to infection. The protein site-specific glycosylation was characterized in plasma derived from well-defined controls and patients. We found 3862 unique features, of which we identified 463 distinct intact glycopeptides, that could be mapped to more than 30 different proteins. Statistical analyses were used to derive a glycopeptide signature that enabled significant differentiation between patients with a bacterial or viral infection. Furthermore, supported by a machine learning algorithm, we demonstrated the ability to identify the causative pathogens based on the distinctive host blood plasma glycopeptide signatures. These results illustrate that glycoproteomics holds enormous potential as an innovative approach to improve the interpretation of relevant biological changes in response to infection

Relationship between molecular pathogen detection and clinical disease in febrile children across Europe: a multicentre, prospective observational study

BackgroundThe PERFORM study aimed to understand causes of febrile childhood illness by comparing molecular pathogen detection with current clinical practice.MethodsFebrile children and controls were recruited on presentation to hospital in 9 European countries 2016-2020. Each child was assigned a standardized diagnostic category based on retrospective review of local clinical and microbiological data. Subsequently, centralised molecular tests (CMTs) for 19 respiratory and 27 blood pathogens were performed.FindingsOf 4611 febrile children, 643 (14%) were classified as definite bacterial infection (DB), 491 (11%) as definite viral infection (DV), and 3477 (75%) had uncertain aetiology. 1061 controls without infection were recruited. CMTs detected blood bacteria more frequently in DB than DV cases for N. meningitidis (OR: 3.37, 95% CI: 1.92-5.99), S. pneumoniae (OR: 3.89, 95% CI: 2.07-7.59), Group A streptococcus (OR 2.73, 95% CI 1.13-6.09) and E. coli (OR 2.7, 95% CI 1.02-6.71). Respiratory viruses were more common in febrile children than controls, but only influenza A (OR 0.24, 95% CI 0.11-0.46), influenza B (OR 0.12, 95% CI 0.02-0.37) and RSV (OR 0.16, 95% CI: 0.06-0.36) were less common in DB than DV cases. Of 16 blood viruses, enterovirus (OR 0.43, 95% CI 0.23-0.72) and EBV (OR 0.71, 95% CI 0.56-0.90) were detected less often in DB than DV cases. Combined local diagnostics and CMTs respectively detected blood viruses and respiratory viruses in 360 (56%) and 161 (25%) of DB cases, and virus detection ruled-out bacterial infection poorly, with predictive values of 0.64 and 0.68 respectively.InterpretationMost febrile children cannot be conclusively defined as having bacterial or viral infection when molecular tests supplement conventional approaches. Viruses are detected in most patients with bacterial infections, and the clinical value of individual pathogen detection in determining treatment is low. New approaches are needed to help determine which febrile children require antibiotics.FundingEU Horizon 2020 grant 668303

Genomic investigations of unexplained acute hepatitis in children

Since its first identification in Scotland, over 1,000 cases of unexplained paediatric hepatitis in children have been reported worldwide, including 278 cases in the UK1. Here we report an investigation of 38 cases, 66 age-matched immunocompetent controls and 21 immunocompromised comparator participants, using a combination of genomic, transcriptomic, proteomic and immunohistochemical methods. We detected high levels of adeno-associated virus 2 (AAV2) DNA in the liver, blood, plasma or stool from 27 of 28 cases. We found low levels of adenovirus (HAdV) and human herpesvirus 6B (HHV-6B) in 23 of 31 and 16 of 23, respectively, of the cases tested. By contrast, AAV2 was infrequently detected and at low titre in the blood or the liver from control children with HAdV, even when profoundly immunosuppressed. AAV2, HAdV and HHV-6 phylogeny excluded the emergence of novel strains in cases. Histological analyses of explanted livers showed enrichment for T cells and B lineage cells. Proteomic comparison of liver tissue from cases and healthy controls identified increased expression of HLA class 2, immunoglobulin variable regions and complement proteins. HAdV and AAV2 proteins were not detected in the livers. Instead, we identified AAV2 DNA complexes reflecting both HAdV-mediated and HHV-6B-mediated replication. We hypothesize that high levels of abnormal AAV2 replication products aided by HAdV and, in severe cases, HHV-6B may have triggered immune-mediated hepatic disease in genetically and immunologically predisposed children

Neonatal Platelets: Lower G12/13 Expression Contributes to Reduced Secretion of Dense Granules

Despite fully functional primary hemostasis, platelets of healthy neonates exhibit hypoaggregability and secretion defects, which may be adaptations to specific requirements in this developmental stage. The etiologies for reduced signal transduction vary with the type of agonist. The discovered peculiarities are lower receptor densities, reduced calcium mobilization, and functional impairments of G proteins. Reduced secretion of dense granules has been attributed to lower numbers of granules. Signaling studies with adult platelets have shown a regulating effect of the G12/13 signaling pathway on dense granule secretion via RhoA. We comparatively analyzed secretion profiles using flow cytometry and expression levels of Gq, Gi, and G12/13 using Western blot analysis in platelets from cord blood and adults. Furthermore, we evaluated Rho activation after in vitro platelet stimulation with thrombin using a pulldown assay. We observed a markedly reduced expression of the dense granule marker CD63 on neonatal platelets after thrombin stimulation. Gα12/13 expression was significantly decreased in neonatal platelets and correlated with lower Rho activation after thrombin stimulation. We conclude that lower expression of G12/13 in neonatal platelets results in attenuated activation of Rho and may contribute to reduced secretion of dense granules after exposure to thrombin

Polyphosphate in Neonates: Less Shedding from Platelets and Divergent Prothrombotic Capacity Due to Lower TFPI Levels

Background: The neonatal hemostatic system exhibits a fragile balance featuring lower levels of clotting factors as well as inhibitors. Neonatal platelets show in-vitro hypoaggregability, but neonates exhibit well-functioning primary and secondary hemostasis despite this impairment. Recently, polyphosphate shed by activated platelets has been shown to induce a prothrombotic shift on the plasmatic coagulation system of adults. The impact of platelet derived polyphosphate might differ in neonates due to aforementioned peculiarities.Aims: We aimed to comparatively determine polyphosphate content and release from adult and neonatal platelets and to determine its impact on thrombin generation in plasma from adult and cord blood.Methods: Polyphosphate was extracted from adult and neonatal platelet lysates and releasates using silica spin-columns and quantified with a DAPI based fluorescence assay. The impact of exogenous polyphosphate in various concentrations (208–0.026 μg/ml) on thrombin generation was evaluated in plasma from adult and cord blood as well as in adult plasma with reduced tissue factor pathway inhibitor (TFPI) levels using calibrated automated thrombography.Results: Polyphosphate content was comparable in both groups, but the fraction of released polyphosphate upon stimulation with thrombin receptor activating peptide was lower in neonatal samples (adult: 84.1 ± 12.9%; cord: 58.8 ± 11.2%). Relative impact of polyphosphate on lag time of thrombin generation was higher in adult samples compared to samples from cord blood (adult: 41.0% [IQR: 35.2–71.8%] of vehicle; cord: 73.4% [IQR: 70.2–91.4%] of vehicle). However, in samples from cord blood, lower concentrations of polyphosphate were required to obtain maximal impact on thrombin generation (adult: 26 μg/ml; cord: 0.814 μg/ml). PolyP affected thrombin generation in adult plasma similarly to cord plasma, when the TFPI concentration was reduced to neonatal levels.Conclusion: Differences in the impact of polyphosphate on adult and neonatal coagulation are largely caused by differences in TFPI levels. Lower polyphosphate release from neonatal platelets, but lower optimum concentration to drive neonatal plasmatic hemostasis emphasizes the well-matched, but fragile interplay between platelets and coagulation in newborns. A potential developmental mismatch should be considered when transfusing adult platelets into neonates

The anticoagulant effects of ethyl pyruvate in whole blood samples.

BackgroundEthyl pyruvate (EP), the ethyl ester of pyruvate, has proven antiinflammatory and antioxidative properties. Additionally, anticoagulant properties have been suggested recently. EP, therefore, is a potentially antiatherosclerotic drug. We aimed to investigate whether EP possesses antiplatelet and anticoagulant properties particularly in the physiological environment of whole blood.MethodsWe investigated the effects of increasing concentrations of EP on platelet function, on the course of clot development, and on standard coagulation times. Additionally, clot ultrastructure using scanning electron microscopy was analysed.ResultsEP exerted significant antiplatelet actions: i) Impedance aggregometry amplitudes (11.7 ± 3.0 ohm, 0 μg/mL EP) dose dependently decreased (7.8 ± 3.1 ohm, 1000 μg/mL EP; -33.3%). ATP exocytosis (0.87 ± 0.24 nM, 0 μg/mL EP) measured by the luminiscent method dose-dependently decreased (0.56 ± 0.14 nM, 1000 μg/mL; -35.6%). ii) Closure times (104.4 ± 23.8 s, 0 μg/mL EP) using the Platelet function analyzer were dose-dependently prolonged (180.5 ± 82.5 s, 1000 μg/mL EP; +72.9%) using membranes coated with collagen/ADP. iii) Surface coverage (15.9 ± 5.1%, 0 μg/mL EP) dose-dependently decreased (9.0 ± 3.7%, 1000 μg/mL EP; -43.4%) using the Cone and Platelet analyzer. EP also exerted significant anticoagulant actions: Coagulation times (177.9 ± 37.8, 0 μg/mL EP) evaluated by means of thrombelastometry were dose-dependently prolonged (212.8 ± 57.7 s, 1000 μg/mL EP; +19.6%). Activated partial thromboplastin times (31.5 ± 1.8 s, 0 μg/mL EP) were dose-dependently prolonged (35.6 ± 2.3 s, 1000 μg/mL EP; +13.0%). Prothrombin times (0.94 ± 0.02 INR, 0 μg/mL EP) were dose-dependently prolonged (1.09 ± 0.04 INR, 1000 μg/mL EP; +16.0%).ConclusionWe found that EP possesses antiplatelet and anticoagulant properties in whole blood. Together with its proven anti-inflammatory and antioxidative properties, EP is a potentially antiatherogenic drug