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There has been an expansion in our understanding of the multifaceted roles of circulating blood cells in regulating haemostasis and contributing to thrombosis. Notably, there is greater recognition of the interplay between coagulation with inflammation and innate immune activation and the contribution of leucocytes. The full blood count (FBC) is a time-honoured test in medicine; however, its components are often viewed in isolation and without consideration of their haemostatic and thrombotic potential. Here, we review how the individual components of the FBC, that is, haemoglobin, platelets and leucocytes, engage with the haemostatic system and focus on both their quantitative and qualitative attributes. We also explore how this information can be harnessed into better management of people with multiple long-term conditions because of their higher risk of adverse clinical events.
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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.
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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.
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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.
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COVID-19 , Fibrinogênio , Histonas , Ligação Proteica , Humanos , Fibrinogênio/metabolismo , Histonas/sangue , Histonas/metabolismo , COVID-19/sangue , Animais , Masculino , Feminino , Pessoa de Meia-Idade , Estudos Retrospectivos , Idoso , Camundongos , Agregação Plaquetária/efeitos dos fármacos , Plaquetas/metabolismo , SARS-CoV-2 , Células Endoteliais/metabolismo , AdultoRESUMO
It is increasingly apparent that the pathologic interplay between coagulation and innate immunity, ie, immunothrombosis, forms the common basis of many challenges across the boundaries of specialized medicine and cannot be fully explained by the conventional concepts of cascade and cell-based coagulation. To improve our understanding of coagulation, we propose a model of coagulation that converges with inflammation and innate immune activation as a unified response toward vascular injury. Evolutionarily integral to the convergent response are damage-associated molecular patterns, which are released as a consequence of injury. Damage-associated molecular patterns facilitate diverse interactions within and between systems, not only to complement and reinforce cell-based clot formation but also to steer the response toward clot resolution and wound healing. By extending coagulation beyond its current boundaries, the convergent model aims to deliver novel diagnostics and therapeutics for contemporary and unexpected challenges across medicine, as exposed by COVID-19 and vaccine-induced immune thrombotic thrombocytopenia.
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Coagulação Sanguínea , COVID-19 , Imunidade Inata , Humanos , COVID-19/imunologia , COVID-19/sangue , Trombose/imunologia , Trombose/sangue , Animais , Inflamação/imunologia , SARS-CoV-2/imunologia , Plaquetas/imunologia , Plaquetas/metabolismo , Alarminas/metabolismo , Alarminas/imunologiaRESUMO
ABSTRACT: 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.
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Amiloide , COVID-19 , Coagulação Intravascular Disseminada , Fibrinogênio , Humanos , Coagulação Intravascular Disseminada/sangue , Coagulação Intravascular Disseminada/mortalidade , Coagulação Intravascular Disseminada/etiologia , Coagulação Intravascular Disseminada/diagnóstico , Feminino , Masculino , Pessoa de Meia-Idade , Idoso , Amiloide/metabolismo , Fibrinogênio/análise , Fibrinogênio/metabolismo , COVID-19/sangue , COVID-19/mortalidade , COVID-19/complicações , Sepse/mortalidade , Sepse/sangue , Prognóstico , SARS-CoV-2/isolamento & purificação , Biomarcadores , Agregados Proteicos , Estado TerminalRESUMO
In their paper, Hannan et al. suggest that new approaches to the management of the acute and remission phases of thrombotic thrombocytopenic purpura should be considered to address white matter changes seen in patients undergoing magnetic resonance imaging. Timely intervention may have significant implications for the long-term physical and mental health of patients. Commentary on: Hannan et al. Cognitive decline in thrombotic thrombocytopenic purpura survivors: The role of white matter health as assessed by MRI. Br J Haematol 2024;204:1005-1016.
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Disfunção Cognitiva , Púrpura Trombocitopênica Trombótica , Humanos , Púrpura Trombocitopênica Trombótica/patologia , Encéfalo/patologiaRESUMO
Dysregulated innate immunity participates in the pathomechanisms of disseminated intravascular coagulation (DIC) in trauma-induced coagulopathy. Accidental and regulated cell deaths and neutrophil extracellular traps release damage-associated molecular patterns (DAMPs), such as histones, nuclear and mitochondrial DNA, and high-mobility group box 1, into circulation immediately after trauma. DAMP-induced inflammation activation releases tissue factor-bearing procoagulant extracellular vesicles through gasdermin D-mediated pore formation and plasma membrane rupture by regulated cell death. DAMPs also evoke systemic inflammation, platelet, coagulation activation, and impaired fibrinolysis associated with endothelial injury, leading to the dysfunction of anticoagulation systems, which are the main pathophysiological mechanisms of DIC. All these processes induce systemic thrombin generation in vivo, not restricted to the injury sites immediately after trauma. Thrombin generation at the site of injury stops bleeding and maintains homeostasis. However, DIC associated with endothelial injury generates massive thrombin, enhancing protease-activated, receptor-mediated bidirectional interplays between inflammation and coagulation, aggravating the diverse actions of thrombin and disturbing homeostasis. Insufficiently regulated thrombin causes disseminated microvascular thrombosis, resulting in tissue hypoxia due to reduced oxygen delivery, and mitochondrial dysfunction due to DAMPs causes tissue dysoxia. In addition, DAMP-induced calcium influx and overload, as well as neutrophil activation, play a role in endothelial cell injury. Tissue hypoxia and cytotoxicity result in multiple organ dysfunction in DIC after trauma. Controls against dysregulated innate immunity evoking systemic inflammation, thrombin generation, and cytotoxicity are key issues in improving the prognosis of DIC in trauma-induced coagulopathy.
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Transtornos da Coagulação Sanguínea , Coagulação Intravascular Disseminada , Humanos , Trombina , Imunidade Inata , Inflamação/complicações , HipóxiaRESUMO
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.
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Linfopenia , Púrpura Trombocitopênica Idiopática , Trombocitopenia , Trombose , Vacinas , Humanos , Histonas , Vacinas contra COVID-19/efeitos adversos , Lactato DesidrogenasesRESUMO
ABSTRACT: Advancements in the conceptual thinking of hemostasis and thrombosis have been catalyzed by major developments within health research over several decades. The cascade model of coagulation was first described in the 1960s, when biochemistry gained prominence through innovative experimentation and technical developments. This was followed by the cell-based model, which integrated cellular coordination to the enzymology of clot formation and was conceptualized during the growth period in cell biology at the turn of the millennium. Each step forward has heralded a revolution in clinical therapeutics, both in procoagulant and anticoagulant treatments to improve patient care. In current times, the COVID-19 pandemic may also prove to be a catalyst: thrombotic challenges including the mixed responses to anticoagulant treatment and the vaccine-induced immune thrombotic thrombocytopenia have exposed limitations in our preexisting concepts while simultaneously demanding novel therapeutic approaches. It is increasingly clear that innate immune activation as part of the host response to injury is not separate but integrated into adaptive clot formation. Our review summarizes current understanding of the major molecules facilitating such a cross talk between immunity, inflammation and coagulation. We demonstrate how such effects can be layered upon the cascade and cell-based models to evolve conceptual understanding of the physiology of immunohemostasis and the pathology of immunothrombosis.
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Trombocitopenia , Trombose , Humanos , Pandemias , Imunidade Inata , Coagulação Sanguínea , Anticoagulantes/uso terapêutico , Trombocitopenia/tratamento farmacológicoRESUMO
Randomised trials are the best method to determine the efficacy and safety of health technologies. A recent report by Lord O'Shaughnessy highlighted many of the current challenges to delivering trials in the UK and proposed potential solutions. Among these, making trials the business of all NHS institutions and a valued part of all doctors' work, while leveraging the potential of the data that the NHS collects routinely, offers an opportunity to improve NHS efficiency, doctors' job satisfaction and population health simultaneously.
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Médicos , Medicina Estatal , Humanos , Atitude do Pessoal de Saúde , Satisfação no EmpregoRESUMO
BACKGROUND: The quality of warfarin anticoagulation among Sub-Saharan African patients is suboptimal. This is due to several factors, including a lack of standardized dosing algorithms, difficulty in providing timely international normalized ratio (INR) results, a lack of patient feedback on their experiences with treatment, a lack of education on adherence, and inadequate knowledge and training of health care workers. Low quality of warfarin anticoagulation, expressed as time in therapeutic range (TTR), is associated with higher adverse event rates, including bleeding and thrombosis, and ultimately, increased morbidity and mortality. Processes and interventions that improve this situation are urgently needed. OBJECTIVE: This study aims to evaluate the implementation of the "warfarin bundle," a package of interventions to improve the quality of anticoagulation and thereby clinical outcomes. The primary outcome for this study is TTR over the initial 3 months of warfarin therapy. METHODS: Patients aged 18 years or older who are newly initiated on warfarin for venous thromboembolism, atrial fibrillation, or valvular heart disease will be enrolled and followed up for 3 months at clinics in Cape Town, South Africa, and Kampala, Uganda, where the warfarin bundle is implemented. A retrospective review of the clinical records of patients on warfarin treatment before implementation (controls) will be used for comparison. This study uses a mixed methods approach of the implementation of patient- and process-centered activities to improve the quality of anticoagulation. Patient-centered activities include the use of clinical dosing algorithms, adherence support, and root cause analysis, whereas process-centered activities include point-of-care INR testing, staff training, and patient education and training. We will assess the impact of these interventions by comparing the TTR and safety outcomes across the 2 groups, as well as the cost-effectiveness and acceptability of the package. RESULTS: We started recruitment in June 2021 and stopped in August 2022, having recruited 167 participants. We obtained ethics approval from the University of Cape Town Faculty of Health Sciences Human Research Ethics Committee, the Provincial Health Research Committees in South Africa, the Joint Clinical Research Centre Institutional Review Board, Kampala, and the University of Liverpool Research Ethics Committee. As of February 2023, data cleaning and formal analysis are underway. We expect to publish the full results by December 2023. CONCLUSIONS: We anticipate that the "bundle of care," which includes a clinical algorithm to guide individualized dosing of warfarin, will improve INR control and TTR of patients in Uganda and South Africa. We will use these findings to design a larger, multisite clinical trial across several Sub-Saharan African countries. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/46710.
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Acute thrombosis and thrombocytopenia pose challenges to the clinician. Thrombocytopenia is naturally viewed as a risk factor for bleeding, and an association with acute thrombosis appears paradoxical. It presents typically as a medical emergency and requires treatment to be started before having confirmatory results. This review supports the attending clinician to recognise and manage conditions that are part of the thrombotic thrombocytopenic syndrome through four illustrative clinical cases. Common themes linking the underlying pathology and treatment are explored to highlight the continued relevance of this rare, but often devastating, presentation.
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Púrpura Trombocitopênica Trombótica , Trombose , Humanos , Púrpura Trombocitopênica Trombótica/complicações , Púrpura Trombocitopênica Trombótica/terapia , Trombose/diagnóstico , Trombose/terapia , Trombose/etiologia , Síndrome , Fatores de RiscoRESUMO
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 disseminated 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 polyphosphate 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.
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Histonas , Trombose , Humanos , Histonas/metabolismo , Tromboplastina/metabolismo , Estado Terminal , Coagulação Sanguínea/fisiologia , Trombose/metabolismo , AnticoagulantesRESUMO
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 ßII 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 PKCßII revealed that histone-induced cTnI phosphorylation was mainly mediated by PKCα activation, but not PKCßII. 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.