Background Thrombosis with thrombocytopenia syndrome is a rare condition known to occur spontaneously or after heparin use. With the advent of COVID-19 vaccines during the pandemic, thrombosis with thrombocytopenia syndrome cases emerged post administration of adenoviral vaccines, termed vaccine-induced immune thrombosis and thrombocytopenia. In response, the thrombosis with thrombocytopenia syndrome consortium was formed to deepen our understanding of this syndrome post vaccination. Methods The consortium employed a comprehensive approach across five work packages. This included designing cohort studies covering the entire English population and analysing local linked regional data sets to detect thrombosis with thrombocytopenia syndrome occurrences in real time. Various patient and healthy control specimens, including those from vaccinated individuals, underwent testing for antiplatelet factor 4 antibodies using three different assays. Patients who developed vaccine-induced immune thrombosis and thrombocytopenia after the AstraZeneca (AZD1222) COVID-19 vaccine underwent whole-genome and ribonucleic acid sequencing to identify genetic susceptibility factors. Multiple studies were conducted to investigate the mechanism of antiplatelet factor 4 antibody formation, including assessments of adenoviral vector structure and binding to platelet factor 4. Detailed studies were also conducted to understand the immune response to vaccines, the role of immune complexes involving platelet factor 4 and their effects on proinflammatory cytokines, neutrophil extracellular traps and platelets in the pathogenesis of the syndrome. Results Cohort studies revealed a higher risk of arterial and venous thromboses after COVID-19 infection compared to vaccination. Specifically, regarding vaccines, the risk of thrombosis and/or thrombocytopenia was higher after the first dose of the AZD1222 vaccine but not with subsequent doses of. Regional linked data indicated that real-time ascertainment of diseases across multiple acute hospital sites’ secure data environments is not yet feasible at scale. The overall background seroprevalence of antiplatelet factor 4 antibodies was low in healthy individuals, vaccinated individuals and those infected with COVID-19. Whole-genome sequencing did not identify significant variants predisposing to vaccine-induced immune thrombosis and thrombocytopenia, with ongoing work on ribonucleic acid sequencing. An electrostatic interaction between the hexon hypervariable regions of the ChAdOx1 capsid and platelet factor 4 was suggested as a possible mechanism for antiplatelet factor 4 antibody development. Strong immune response drove the formation of neutrophil extracellular traps, significant inflammatory responses and clot formation in distant organs. Platelet activation post immune complex formation against platelet factor 4 was dependent on FcγRIIa but independent of complement, also occurring through binding with c-Mpl. T-cell reactivity against the AZD1222 vaccine indicates potential cross-reactivity with prevalent human adenoviruses. Conclusions The consortium’s comprehensive work has uncovered new potential mechanisms of vaccine-induced immune thrombosis and thrombocytopenia and identified novel biomarkers and therapeutic strategies for further development and validation. This is crucial, as the combination of thrombosis and thrombocytopenia, alongside antiplatelet factor 4 antibodies, can occur without exposure to heparin or adenovirus vaccines. Future considerations Recommendations include the development of a national reference laboratory and registry for diagnosis and further study of thrombosis with thrombocytopenia syndrome; future vaccine development using the adenoviral vector platform to focus on the reduction of the electrostatic interaction between viral hexons and platelet factor 4; international genomics collaboration; and studies focused on understanding the symptoms suffered by patients as well as strategies to ameliorate them. Limitations Direct identification of vaccine-induced immune thrombosis and thrombocytopenia patients was hindered by poor recording. The rarity of vaccine-induced immune thrombosis and thrombocytopenia limited the number of patients recruited for genomic and mechanistic studies. Funding This synopsis presents independent research funded by the National Institute for Health and Care Research (NIHR) Efficacy and Mechanism Evaluation (EME) programme as award number NIHR135073. Plain language summary Thrombosis with thrombocytopenia syndrome is rare: it is characterised by thrombosis and lowered platelet counts together with the development of an antibody against a protein called platelet factor 4. This syndrome has been linked to heparin use or can occur spontaneously. With COVID-19 vaccines, a new form called vaccine-induced immune thrombosis and thrombocytopenia appeared. The thrombosis with thrombocytopenia syndrome consortium formed to better understand this syndrome. The consortium used various methods, like studying the data of the entire English population and analysing local data in real time. They tested patient and healthy control samples for antiplatelet factor 4 antibodies and sequenced genes from patients who got vaccine-induced immune thrombosis and thrombocytopenia after the AZD1222 COVID-19 vaccine. They also studied how these antibodies form and their effects, including changes in cytokines and platelet involvement. Our studies showed a higher thrombosis risk after COVID-19 infection compared to vaccination. The first dose of the AZD1222 vaccine had higher risks of thrombosis and lowered platelets (occurring separately), but subsequent doses or mRNA vaccines were safer. Identifying vaccine-induced immune thrombosis and thrombocytopenia patients directly was difficult due to poor records. Real-time tracking of diseases across hospitals was not yet possible at scale. The prevalence of antiplatelet factor 4 antibodies was low in healthy, vaccinated and COVID-19-infected individuals. Genetic sequencing didn’t find significant variants causing vaccine-induced immune thrombosis and thrombocytopenia, but there are ongoing ribonucleic acid studies. Our studies found a possible mechanism for antiplatelet factor 4 antibody development involving the AZD1222 vaccine. The immune response caused generalised inflammation and clotting in distant organs. Platelet activation was influenced by certain factors. T-cell reactivity against the AZD1222 vaccine hinted at potential cross-reactivity with common human viruses. The consortium’s work has uncovered new insights into vaccine-induced immune thrombosis and thrombocytopenia, suggesting potential new diagnostic and treatment strategies. This is crucial, as thrombosis with thrombocytopenia syndrome can occur without exposure to heparin or adenovirus vaccines.

Severe deficiency of ADAMTS13 (<10 iu/dL) is diagnostic of thrombotic thrombocytopenic purpura (TTP) and leads to accumulation of ultra-large vWF multimers, platelet aggregation, and widespread microthrombi, which can be life-threatening. However, the clinical implications of a low ADAMTS13 activity level are not only important in an acute episode of TTP. In this article, we discuss the effects of low ADAMTS13 activity in congenital and immune-mediated TTP patients not only at presentation but once in a clinical remission. Evidence is emerging of the clinical effects of low ADAMTS13 activity in other disease areas outside of TTP, and here, we explore the wider impact of low ADAMTS13 activity on the vascular endothelium and the potential for recombinant ADAMTS13 therapy in other thrombotic disease states.

BACKGROUND:Circulating histones are released by extensive tissue injury or cell death and play important pathogenic roles in critical illnesses. Their interaction with circulating plasma components and the potential roles in the clinical setting are not fully understood. OBJECTIVES:We aimed to characterize the interaction of histones with fibrinogen and explore its roles in vitro, in vivo, and in patient samples. METHODS:Histone-fibrinogen binding was assessed by electrophoresis and enzyme-linked immunosorbent assay-based affinity assay. Functional significance was explored using washed platelets and endothelial cells in vitro and histone-infusion mouse models in vivo. To determine clinical translatability, a retrospective single-center cohort study was conducted on patients requiring intensive care admission (n = 199) and validated in a cohort of hospitalized patients with COVID-19 (n = 69). RESULTS:Fibrinogen binds histones through its D-domain with high affinity (calf thymus histones, KD = 18.0 ± 5.6 nM; histone 3, KD = 2.7 ± 0.8 nM; and histone 4, KD = 2.0 ± 0.7 nM) and significantly reduces histone-induced endothelial damage and platelet aggregation in vitro and in vivo in a histone-infusion mouse model. Physiologic concentrations of fibrinogen can neutralize low levels of circulating histones and increase the cytotoxicity threshold of histones to 50 μg/mL. In a cohort of patients requiring intensive care, a histone:fibrinogen ratio of ≥6 on admission was associated with moderate-severe thrombocytopenia and independently predicted mortality. This finding was validated in a cohort of hospitalized patients with COVID-19. CONCLUSION:Fibrinogen buffers the cytotoxic properties of circulating histones. Detection and monitoring of circulating histones and histone:fibrinogen ratios will help identify critically ill patients at highest risk of adverse outcomes who might benefit from antihistone therapy.

Atrial fibrillation (AF) is the most common cardiac arrhythmia and is a potentially life-threatening condition that can contribute to stroke and thromboembolism. While anticoagulation has reduced the risk of thromboembolism in patients with AF, morbidity and mortality remain unacceptably high, hence the recent move in guidelines toward a more holistic or integrated care approach to improve clinical outcomes.[1] [2]

Introduction Non-ventilator-associated hospital-acquired pneumonia (nv-HAP) is the most common healthcare-associated infection (HCAI), is associated with high mortality and morbidity and places a major burden on healthcare systems. Diagnosis currently relies on chest x-rays to confirm pneumonia and sputum cultures to determine the microbiological cause. This approach leads to over-diagnosis of pneumonia, rarely identifies a causative pathogen and perpetuates unnecessary and imprecise antibiotic use. The HAP-FAST study aims to evaluate the feasibility of a randomised trial to evaluate the clinical impact of low-dose, non-contrast-enhanced thoracic CT scans and rapid molecular sputum analysis using the BIOFIRE® FILMARRAY® pneumonia plus panel (FAPP) for patients suspected with nv-HAP.Methods and analysis The HAP-FAST feasibility study consists of a pilot randomised trial, a qualitative study, a costing analysis and exploratory analyses of clinical samples to investigate the immune-pathophysiology of HAP. Participants are identified and recruited from four acute hospitals in the Northwest of the UK. Using a Research Without Prior Consent model, the pilot trial will recruit 220 adult participants, with or without mental capacity, and with suspected HAP. HAP-FAST is a non-blinded, sequential, multiple assignment, randomised trial with two possible stages of randomisation: first, chest x-ray (CXR) or CT; second, if treated as nv-HAP, FAPP or standard microbiological processing alone (no FAPP). Pathogen-specific antibiotic guidance will be provided for FAPP results. Randomisation uses a web-based platform and followed up for 90 days. The feasibility of a future trial will be determined by assessing trial processes, outcome measures and patient and staff experiences.Ethics and dissemination This study has undergone combined review by the UK NHS Research Ethics Committee and Health Research Authority. Results will be disseminated via peer-reviewed journals, via the funders’ website and through a range of media to engage the public.Trial registration number NCT05483309.

BACKGROUND: By causing inflammation and tissue damage, neutrophil extracellular traps (NETs) constitute an underlying mechanism of aspiration-induced lung injury, a major factor of the low utilization of donor lungs in lung transplantation (LTx). METHODS: To determine whether NET removal during ex vivo lung perfusion (EVLP) can restore lung function and morphology in aspiration-damaged lungs, gastric aspiration lung injury was induced in 12 pigs. After confirmation of acute respiratory distress syndrome, the lungs were explanted and assigned to NET removal connected to EVLP (treated) (n = 6) or EVLP only (nontreated) (n = 6). Hemodynamic measurements were taken, and blood and tissue samples were collected to assess lung function, morphology, levels of cell-free DNA, extracellular histones, and nucleosomes as markers of NETs, as well as cytokine levels. RESULTS: After EVLP and NET removal in porcine lungs, PaO2/FiO2 ratios increased significantly compared to those undergoing EVLP alone (p = 0.0411). Treated lungs had lower cell-free DNA (p = 0.0260) and lower levels of extracellular histones in EVLP perfusate (p= 0.0260) than nontreated lungs. According to histopathology, treated lungs showed less immune cell infiltration and less edema compared with nontreated lungs, which was reflected in decreased levels of proinflammatory cytokines in EVLP perfusate and bronchoalveolar lavage fluid. CONCLUSIONS: To conclude, removing NETs during EVLP improved lung function and morphology in aspiration-damaged donor lungs. The ability to remove NETs during EVLP could represent a new therapeutic approach for LTx and potentially expand the donor pool for transplantation. J Heart Lung Transplant 2024;43:1919-1929 (c) 2024 The Authors. Published by Elsevier Inc. on behalf of International Society for Heart and Lung Transplantation. This is an open access article under the CC BY license (http://creativecommons.org/ licenses/by/4.0/).

Microclots have been associated with various conditions, including postacute sequelae of severe acute respiratory syndrome coronavirus 2 infection. They have been postulated to be amyloid-fibrin(ogen) aggregates, but their role as a prognostic biomarker remains unclear. To examine their possible clinical utility, blood samples were collected for the first 96 hours from critically ill patients (n = 104) admitted to the intensive care unit (ICU). Detection was by staining platelet-poor plasma samples with thioflavin T and visualized by fluorescent microscopy. Image J software was trained to identify and quantify microclots, which were detected in 44 patients (42.3%) on ICU admission but not in the remaining 60 (57.7%) or the 20 healthy controls (0.0%). Microclots on admission to ICU were associated with a primary diagnosis of sepsis (microclots present in sepsis, 23/44 [52.3%] vs microclots absent in sepsis, 19/60 [31.7%]; P = .044). Multicolor immunofluorescence demonstrated that microclots consisted of amyloid-fibrinogen aggregates, which was supported by proteomic analysis. Patients with either a high number or larger-sized microclots had a higher likelihood of developing disseminated intravascular coagulation (odds ratio [OR], 51.4; 95% confidence interval [CI], 6.3-6721.1; P < .001) and had an increased probability of 28-day mortality (OR, 5.3; 95% CI, 2.0-15.6; P < .001). This study concludes that microclots, as defined by amyloid-fibrin(ogen) aggregates, are potentially useful in identifying sepsis and predicting adverse coagulopathic and clinical outcomes.

Vaccination has proven to be a valuable tool to combat SARS-CoV-2. However, reports of rare adverse reactions such as thrombosis/thrombocytopenia syndrome after ChAdOx1 nCoV-19 vaccination have caused scientific, public and media concern. ChAdOx1 was vectorised from the Y25 chimpanzee adenovirus, which was selected due to low human seroprevalence to circumvent pre-existing immunity. In this study, we aimed to explore patterns of T-cell activation after SARS-CoV-2 COVID-19 vaccine exposure in vitro using PBMCs collected from pre-pandemic ChAdOx1 nCoV-19 naïve healthy donors (HDs), and ChAdOx1 nCoV-19 and Pfizer vaccinated controls. PBMCs were assessed for T-cell proliferation using the lymphocyte transformation test (LTT) following exposure to SARS-CoV-2 COVID-19 vaccines. Cytokine analysis was performed via intracellular cytokine staining, ELISpot assay and LEGENDplex immunoassays. T-cell assays performed in pre-pandemic vaccine naïve HDs, revealed widespread lymphocyte stimulation after exposure to ChAdOx1 nCoV-19 (95%), ChAdOx-spike (90%) and the Ad26.COV2. S vaccine, but not on exposure to the BNT162b2 vaccine. ICS analysis demonstrated that CD4+ CD45RO+ memory T-cells are activated by ChAdOx1 nCoV-19 in vaccine naïve HDs. Cytometric immunoassays showed ChAdOx1 nCoV-19 exposure was associated with the release of proinflammatory and cytotoxic molecules, such as IFN-γ, IL-6, perforin, granzyme B and FasL. These studies demonstrate a ubiquitous T-cell response to ChAdOx1 nCoV-19 and Ad26.COV2. S in HDs recruited prior to the SARS-CoV-2 pandemic, with T-cell stimulation also identified in vaccinated controls. This may be due to underlying T-cell cross-reactivity with prevalent human adenoviruses and further study will be needed to identify T-cell epitopes involved.

Journal Article Corrected proof Scientific Business Abstracts Get access Keith Siew, Keith Siew University College London, London, United Kingdom Search for other works by this author on: Oxford Academic PubMed Google Scholar Vaksha Patel, Vaksha Patel University College London, London, United Kingdom Search for other works by this author on: Oxford Academic PubMed Google Scholar Jasminka Zimmermann, Jasminka Zimmermann University College London, London, United Kingdom Search for other works by this author on: Oxford Academic PubMed Google Scholar Michael Vaughan, Michael Vaughan University of Cork, Eire Search for other works by this author on: Oxford Academic PubMed Google Scholar Christopher Cheshire, Christopher Cheshire Crick Institute, London, United Kingdom Search for other works by this author on: Oxford Academic PubMed Google Scholar Angela Kubik, Angela Kubik NASA Ames Research Centre, USA Search for other works by this author on: Oxford Academic PubMed Google Scholar Rebecca Finch, Rebecca Finch University of Staffordshire, United Kingdom Search for other works by this author on: Oxford Academic PubMed Google Scholar Zhongwang Li, Zhongwang Li University College London, London, United Kingdom Search for other works by this author on: Oxford Academic PubMed Google Scholar Selin Altinok, Selin Altinok University North Carolina, Chapel Hill, USA Search for other works by this author on: Oxford Academic PubMed Google Scholar Willian De Silvera, Willian De Silvera University of Staffordshire, United Kingdom Search for other works by this author on: Oxford Academic PubMed Google Scholar ... 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BACKGROUND:Adenoviral vector-based COVID-19 vaccine-induced immune thrombotic thrombocytopenia (VITT) is rare but carries significant risks of mortality and long-term morbidity. The underlying pathophysiology of severe disease is still not fully understood. The objectives were to explore the pathophysiological profile and examine for clinically informative biomarkers in patients with severe VITT. METHODS:Twenty-two hospitalized patients with VITT, 9 pre- and 21 post-ChAdOx1 vaccine controls, were recruited across England, United Kingdom. Admission blood samples were analyzed for cytokine profiles, cell death markers (lactate dehydrogenase and circulating histones), neutrophil extracellular traps, and coagulation parameters. Tissue specimens from deceased patients were analyzed. RESULTS:There were strong immune responses characterized by significant elevations in proinflammatory cytokines and T helper 1 and 2 cell activation in patients with VITT. Markers of systemic endothelial activation and coagulation activation in both circulation and organ sections were also significantly elevated. About 70% (n = 15/22) of patients met the International Society for Thrombosis and Haemostasis criteria for disseminated intravascular coagulation despite negligible changes in the prothrombin time. The increased neutrophil extracellular trap formation, in conjunction with marked lymphopenia, elevated lactate dehydrogenase, and circulating histone levels, indicates systemic immune cell injury or death. Both lymphopenia and circulating histone levels independently predicted 28-day mortality in patients with VITT. CONCLUSION:The coupling of systemic cell damage and death with strong immune-inflammatory and coagulant responses are pathophysiologically dominant and clinically relevant in severe VITT.

The cell-based model of coagulation remains the basis of our current understanding of clinical hemostasis and thrombosis. Its advancement on the coagulation cascade model has enabled new prohemostatic and anticoagulant treatments to be developed. In the past decade, there has been increasing evidence of the procoagulant properties of extracellular, cell-free histones (CFHs). Although high levels of circulating CFHs released following extensive cell death in acute critical illnesses, such as sepsis and trauma, have been associated with adverse coagulation outcomes, including dissemi-nated intravascular coagulation, new information has also emerged on how its local effects contribute to physiological clot formation. CFHs initiate coagulation by tissue factor exposure, either by destruction of the endovascular barrier or induction of endoluminal tissue factor expression on endothelia and monocytes. CFHs can also bind prothrombin directly, generating thrombin via the alternative prothrombinase pathway. In amplifying and augmenting the procoagulant signal, CFHs activate and aggregate platelets, increase procoagulant material bioavailability through platelet degranulation and Weibel-Palade body exocytosis, activate intrinsic coagulation via platelet poly-phosphate release, and induce phosphatidylserine exposure. CFHs also inhibit protein C activation and downregulate thrombomodulin expression to reduce anti-inflammatory and anticoagulant effects. In consolidating clot formation, CFHs augment the fibrin polymer to confer fibrinolytic resistance and integrate neutrophil extracellular traps into the clot structure. Such new information holds the promise of new therapeutic developments, including improved targeting of immunothrombotic pathologies in acute critical illnesses.

Reduction in cardiac contractility is common in severe sepsis. However, the pathological mechanism is still not fully understood. Recently it has been found that circulating histones released after extensive immune cell death play important roles in multiple organ injury and disfunction, particularly in cardiomyocyte injury and contractility reduction. How extracellular histones cause cardiac contractility depression is still not fully clear. In this work, using cultured cardiomyocytes and a histone infusion mouse model, we demonstrate that clinically relevant histone concentrations cause significant increases in intracellular calcium concentrations with subsequent activation and enriched localization of calcium-dependent protein kinase C (PKC) α and βII into the myofilament fraction of cardiomyocytes in vitro and in vivo. Furthermore, histones induced dose-dependent phosphorylation of cardiac troponin I (cTnI) at the PKC-regulated phosphorylation residues (S43 and T144) in cultured cardiomyocytes, which was also confirmed in murine cardiomyocytes following intravenous histone injection. Specific inhibitors against PKCα and PKCβII revealed that histone-induced cTnI phosphorylation was mainly mediated by PKCα activation, but not PKCβII. Blocking PKCα also significantly abrogated histone-induced deterioration in peak shortening, duration and the velocity of shortening, and re-lengthening of cardiomyocyte contractility. These in vitro and in vivo findings collectively indicate a potential mechanism of histone-induced cardiomyocyte dysfunction driven by PKCα activation with subsequent enhanced phosphorylation of cTnI. These findings also indicate a potential mechanism of clinical cardiac dysfunction in sepsis and other critical illnesses with high levels of circulating histones, which holds the potential translational benefit to these patients by targeting circulating histones and downstream pathways.