Assembly of alternative prothrombinase by extracellular histones initiates and disseminates intravascular coagulation.

Thrombin generation is pivotal to both physiological blood clot formation and pathological development of disseminated intravascular coagulation (DIC). In critical illness, extensive cell damage can release histones into the circulation, which can increase thrombin generation and cause DIC, but the molecular mechanism is not clear. Typically, thrombin is generated by the prothrombinase complex, comprising activated factor X (FXa), activated cofactor V (FVa), and phospholipids to cleave prothrombin in the presence of calcium. In this study, we found that in the presence of extracellular histones, an alternative prothrombinase could form without FVa and phospholipids. Histones directly bind to prothrombin fragment 1 (F1) and fragment 2 (F2) specifically to facilitate FXa cleavage of prothrombin to release active thrombin, unlike FVa, which requires phospholipid surfaces to anchor the classical prothrombinase complex. In vivo, histone infusion into mice induced DIC, which was significantly abrogated when prothrombin F1 + F2 were infused prior to histones, to act as decoy. In a cohort of intensive care unit patients with sepsis (n = 144), circulating histone levels were significantly elevated in patients with DIC. These data suggest that histone-induced alternative prothrombinase without phospholipid anchorage may disseminate intravascular coagulation and reveal a new molecular mechanism of thrombin generation and DIC development. In addition, histones significantly reduced the requirement for FXa in the coagulation cascade to enable clot formation in factor VIII (FVIII)- and FIX-deficient plasma, as well as in FVIII-deficient mice. In summary, this study highlights a novel mechanism in coagulation with therapeutic potential in both targeting systemic coagulation activation and correcting coagulation factor deficiency.

Heparin Inhibits Cellular Invasion by SARS-CoV-2: Structural Dependence of the Interaction of the Spike S1 Receptor-Binding Domain with Heparin.

The dependence of development and homeostasis in animals on the interaction of hundreds of extracellular regulatory proteins with the peri- and extracellular glycosaminoglycan heparan sulfate (HS) is exploited by many microbial pathogens as a means of adherence and invasion. Heparin, a widely used anticoagulant drug, is structurally similar to HS and is a common experimental proxy. Exogenous heparin prevents infection by a range of viruses, including S-associated coronavirus isolate HSR1. Here, we show that heparin inhibits severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) invasion of Vero cells by up to 80% at doses achievable through prophylaxis and, particularly relevant, within the range deliverable by nebulisation. Surface plasmon resonance and circular dichroism spectroscopy demonstrate that heparin and enoxaparin, a low-molecular-weight heparin which is a clinical anticoagulant, bind and induce a conformational change in the spike (S1) protein receptor-binding domain (S1 RBD) of SARS-CoV-2. A library of heparin derivatives and size-defined fragments were used to probe the structural basis of this interaction. Binding to the RBD is more strongly dependent on the presence of 2-O or 6-O sulfate groups than on N-sulfation and a hexasaccharide is the minimum size required for secondary structural changes to be induced in the RBD. It is likely that inhibition of viral infection arises from an overlap between the binding sites of heparin/HS on S1 RBD and that of the angiotensin-converting enzyme 2. The results suggest a route for the rapid development of a first-line therapeutic by repurposing heparin and its derivatives as antiviral agents against SARS-CoV-2 and other members of the Coronaviridae.

The heparin-binding proteome in normal pancreas and murine experimental acute pancreatitis.

Acute pancreatitis (AP) is acute inflammation of the pancreas, mainly caused by gallstones and alcohol, driven by changes in communication between cells. Heparin-binding proteins (HBPs) play a central role in health and diseases. Therefore, we used heparin affinity proteomics to identify extracellular HBPs in pancreas and plasma of normal mice and in a caerulein mouse model of AP. Many new extracellular HBPs (360) were discovered in the pancreas, taking the total number of HBPs known to 786. Extracellular pancreas HBPs form highly interconnected protein-protein interaction networks in both normal pancreas (NP) and AP. Thus, HBPs represent an important set of extracellular proteins with significant regulatory potential in the pancreas. HBPs in NP are associated with biological functions such as molecular transport and cellular movement that underlie pancreatic homeostasis. However, in AP HBPs are associated with additional inflammatory processes such as acute phase response signalling, complement activation and mitochondrial dysfunction, which has a central role in the development of AP. Plasma HBPs in AP included known AP biomarkers such as serum amyloid A, as well as emerging targets such as histone H2A. Other HBPs such as alpha 2-HS glycoprotein (AHSG) and histidine-rich glycoprotein (HRG) need further investigation for potential applications in the management of AP. Pancreas HBPs are extracellular and so easily accessible and are potential drug targets in AP, whereas plasma HBPs represent potential biomarkers for AP. Thus, their identification paves the way to determine which HBPs may have potential applications in the management of AP.

Circulating Histone Concentrations Differentially Affect the Predominance of Left or Right Ventricular Dysfunction in Critical Illness.

OBJECTIVES: Cardiac complications are common in critical illness and associated with grave consequences. In this setting, elevated circulating histone levels have been linked to cardiac injury and dysfunction in experimental models and patients with sepsis. The mechanisms underlying histone-induced cardiotoxicity and the functional consequences on left ventricle and right ventricle remain unclear. This study aims to examine dose-dependent effects of circulating histones on left ventricle and right ventricle function at clinically relevant concentrations. DESIGN: Prospective laboratory study with in vitro and in vivo investigations. SETTING: University research laboratory. SUBJECTS: Twelve-week old male C57BL/6N mice. INTERVENTIONS: Cultured cardiomyocytes were incubated with clinically relevant histone concentrations, and a histone infusion mouse model was also used with hemodynamic changes characterized by echocardiography and left ventricle/right ventricle catheter-derived variables. Circulating histones and cardiac troponin levels were obtained from serial blood samples. MEASUREMENTS AND MAIN RESULTS: IV histone infusion caused time-dependent cardiac troponin elevation to indicate cardiac injury. At moderate sublethal histone doses (30 mg/kg), left ventricular contractile dysfunction was the prominent abnormality with reduced ejection fraction and prolonged relaxation time. At high doses (≥ 60 mg/kg), pulmonary vascular obstruction induced right ventricular pressure increase and dilatation, but left ventricular end-diastolic volume improved because of reduced blood return from the lungs. Mechanistically, histones induced profound calcium influx and overload in cultured cardiomyocytes with dose-dependent detrimental effects on intracellular calcium transient amplitude, contractility, and rhythm, suggesting that histones directly affect cardiomyocyte function adversely. However, increasing histone-induced neutrophil congestion, neutrophil extracellular trap formation, and thrombosis in the pulmonary microvasculature culminated in right ventricular dysfunction. Antihistone antibody treatment abrogated histone cardiotoxicity. CONCLUSIONS: Circulating histones significantly compromise left ventricular and right ventricular function through different mechanisms that are dependent on histone concentrations. This provides a translational basis to explain and target the spectral manifestations of cardiac dysfunction in critical illness.

Circulating Histones Are Major Mediators of Cardiac Injury in Patients With Sepsis.

OBJECTIVE: To investigate the impact of circulating histones on cardiac injury and dysfunction in a murine model and patients with sepsis. DESIGN: Prospective, observational clinical study with in vivo and ex vivo translational laboratory investigations. SETTING: General ICU and university research laboratory. SUBJECTS: Sixty-five septic patients and 27 healthy volunteers. Twelve-week-old male C57BL/6N mice. INTERVENTIONS: Serial blood samples from 65 patients with sepsis were analyzed, and left ventricular function was assessed by echocardiography. Patients' sera were incubated with cultured cardiomyocytes in the presence or absence of antihistone antibody, and cellular viability was assessed. Murine sepsis was initiated by intraperitoneal Escherichia coli injection (10(8) colony-forming unit/mouse) in 12-week-old male C57BL/6N mice, and the effect of antihistone antibody (10 mg/kg) was studied. Murine blood samples were collected serially, and left ventricular function was assessed by intraventricular catheters and electrocardiography. MEASUREMENTS AND MAIN RESULTS: Circulating histones and cardiac troponins in human and murine plasma were quantified. In 65 patients with sepsis, circulating histones were significantly elevated compared with healthy controls (n = 27) and linearly correlated with cardiac troponin T levels (rs = 0.650; p < 0.001), noradrenaline doses required to achieve hemodynamic stability (rs = 0.608; p < 0.001), Sequential Organ Failure Assessment scores (p = 0.028), and mortality (p = 0.008). In a subset of 36 septic patients without prior cardiac disease, high histone levels were significantly associated with new-onset left ventricular dysfunction (p = 0.001) and arrhythmias (p = 0.01). Left ventricular dysfunction only predicted adverse outcomes when combined with elevated histones or cardiac troponin levels. Furthermore, patients' sera directly induced histone-specific cardiomyocyte death ex vivo, which was abrogated by antihistone antibodies. In vivo studies on septic mice confirmed the cause-effect relationship between circulating histones and the development of cardiac injury, arrhythmias, and left ventricular dysfunction. CONCLUSION: Circulating histones are novel and important mediators of septic cardiomyopathy, which can potentially be utilized for prognostic and therapeutic purposes.