Optimization of plasma-based BioID identifies plasminogen as a ligand of ADAMTS13.

ADAMTS13, a disintegrin and metalloprotease with a thrombospondin type 1 motif, member 13, regulates the length of Von Willebrand factor (VWF) multimers and their platelet-binding activity. ADAMTS13 is constitutively secreted as an active protease and is not inhibited by circulating protease inhibitors. Therefore, the mechanisms that regulate ADAMTS13 protease activity are unknown. We performed an unbiased proteomics screen to identify ligands of ADAMTS13 by optimizing the application of BioID to plasma. Plasma BioID identified 5 plasma proteins significantly labeled by the ADAMTS13-birA* fusion, including VWF and plasminogen. Glu-plasminogen, Lys-plasminogen, mini-plasminogen, and apo(a) bound ADAMTS13 with high affinity, whereas micro-plasminogen did not. None of the plasminogen variants or apo(a) bound to a C-terminal truncation variant of ADAMTS13 (MDTCS). The binding of plasminogen to ADAMTS13 was attenuated by tranexamic acid or ε-aminocaproic acid, and tranexamic acid protected ADAMTS13 from plasmin degradation. These data demonstrate that plasminogen is an important ligand of ADAMTS13 in plasma by binding to the C-terminus of ADAMTS13. Plasmin proteolytically degrades ADAMTS13 in a lysine-dependent manner, which may contribute to its regulation. Adapting BioID to identify protein-interaction networks in plasma provides a powerful new tool to study protease regulation in the cardiovascular system.

IL-6/STAT3 axis dictates the PNPLA3-mediated susceptibility to non-alcoholic fatty liver disease.

BACKGROUND & AIMS: A number of genetic polymorphisms have been associated with susceptibility to or protection against non-alcoholic fatty liver disease (NAFLD), but the underlying mechanisms remain unknown. Here, we focused on the rs738409 C>G single nucleotide polymorphism (SNP), which produces the I148M variant of patatin-like phospholipase domain-containing protein 3 (PNPLA3) and is strongly associated with NAFLD. METHODS: To enable mechanistic dissection, we developed a human pluripotent stem cell (hPSC)-derived multicellular liver culture by incorporating hPSC-derived hepatocytes, hepatic stellate cells, and macrophages. We first applied this liver culture to model NAFLD by utilising a lipotoxic milieu reflecting the circulating levels of disease risk factors in affected individuals. We then created an isogenic pair of liver cultures differing only at rs738049 and compared NAFLD phenotype development. RESULTS: Our hPSC-derived liver culture recapitulated many key characteristics of NAFLD development and progression including lipid accumulation and oxidative stress, inflammatory response, and stellate cell activation. Under the lipotoxic conditions, the I148M variant caused the enhanced development of NAFLD phenotypes. These differences were associated with elevated IL-6/signal transducer and activator of transcription 3 (STAT3) activity in liver cultures, consistent with transcriptomic data of liver biopsies from individuals carrying the rs738409 SNP. Dampening IL-6/STAT3 activity alleviated the I148M-mediated susceptibility to NAFLD, whereas boosting it in wild-type liver cultures enhanced NAFLD development. Finally, we attributed this elevated IL-6/STAT3 activity in liver cultures carrying the rs738409 SNP to increased NF-κB activity. CONCLUSIONS: Our study thus reveals a potential causal link between elevated IL-6/STAT3 activity and 148M-mediated susceptibility to NAFLD. IMPACT AND IMPLICATIONS: An increasing number of genetic variants manifest in non-alcoholic fatty liver disease (NAFLD) development and progression; however, the underlying mechanisms remain elusive. To study these variants in human-relevant systems, we developed an induced pluripotent stem cell-derived multicellular liver culture and focused on a common genetic variant (i.e. rs738409 in PNPLA3). Our findings not only provide mechanistic insight, but also a potential therapeutic strategy for NAFLD driven by this genetic variant in PNPLA3. Our liver culture is therefore a useful platform for exploring genetic variants in NAFLD development.

Variants in STXBP3 are Associated with Very Early Onset Inflammatory Bowel Disease, Bilateral Sensorineural Hearing Loss and Immune Dysregulation.

BACKGROUND AND AIMS: Very early onset inflammatory bowel disease [VEOIBD] is characterized by intestinal inflammation affecting infants and children less than 6 years of age. To date, over 60 monogenic aetiologies of VEOIBD have been identified, many characterized by highly penetrant recessive or dominant variants in underlying immune and/or epithelial pathways. We sought to identify the genetic cause of VEOIBD in a subset of patients with a unique clinical presentation. METHODS: Whole exome sequencing was performed on five families with ten patients who presented with a similar constellation of symptoms including medically refractory infantile-onset IBD, bilateral sensorineural hearing loss and, in the majority, recurrent infections. Genetic aetiologies of VEOIBD were assessed and Sanger sequencing was performed to confirm novel genetic findings. Western analysis on peripheral blood mononuclear cells and functional studies with epithelial cell lines were employed. RESULTS: In each of the ten patients, we identified damaging heterozygous or biallelic variants in the Syntaxin-Binding Protein 3 gene [STXBP3], a protein known to regulate intracellular vesicular trafficking in the syntaxin-binding protein family of molecules, but not associated to date with either VEOIBD or sensorineural hearing loss. These mutations interfere with either intron splicing or protein stability and lead to reduced STXBP3 protein expression. Knock-down of STXBP3 in CaCo2 cells resulted in defects in cell polarity. CONCLUSION: Overall, we describe a novel genetic syndrome and identify a critical role for STXBP3 in VEOIBD, sensorineural hearing loss and immune dysregulation.

Characterization of ADAMTS13 and von Willebrand factor levels in septic and non-septic ICU patients.

Sepsis is a life-threatening disease characterized by excessive host response to infection that can lead to activation of the coagulation system. Von Willebrand Factor (VWF) and ADAMTS13 are important regulators of hemostasis and their dysregulation during sepsis progression is not well understood. Herein we characterize ADAMTS13 and VWF in septic and non-septic patients. ADAMTS13 activity, ADAMTS13 antigen, VWF antigen, myeloperoxidase, and protein C, were measured in plasma collected from 40 septic patients (20 non-survivors and 20 survivors) and 40 non-septic patients on the first and last day of their ICU stay. ADAMTS13 activity and ADAMTS13 antigen were reduced, whereas VWF antigen was elevated among septic patients compared to non-septic patients and healthy controls. Non-septic patients also exhibited elevated VWF antigen and reduced ADAMTS13 activity, but to a lesser extent than septic patients. Non-survivor septic patients exhibited the lowest levels of ADAMTS13 activity. ADAMTS13 activity:antigen ratio was similar across all patient cohorts suggesting that the specific activity of ADAMTS13 remains unchanged. Therefore, reduced ADAMTS13 function in circulation is likely due to a reduction in circulating levels. We suggest that massive release of VWF in response to inflammation consumes limited circulating ADAMTS13, resulting in the imbalance observed between VWF and ADAMTS13 among septic and to a lesser extent in non-septic ICU patients. Changes to ADAMTS13 did not correlate with myeloperoxidase or protein C levels. Reduced ADAMTS13 activity and antigen, and elevated VWF antigen observed among all patient cohorts on admission remained unchanged in survivors at ICU discharge. Prolonged reduction in ADAMTS13 activity and antigen in septic patients coincides with elevated levels of VWF. The persistent abnormalities in ADAMTS13 and VWF in sepsis patients discharged from the ICU may contribute to a sustained prothrombotic state.

Identification of hemostatic markers that define the pre-DIC state: A multi-center observational study.

BACKGROUND: A limitation of diagnostic scoring systems for disseminated intravascular coagulation (DIC) is that once DIC is identified, it may be in a state of irreversible deterioration. OBJECTIVES: To identify hemostatic markers that can identify the pre-DIC state. METHODS: This was a multi-center observational study of 357 septic patients. The incidence of DIC was determined using the International Society on Thrombosis and Haemostasis (ISTH) DIC Score. Markers of interest include components of the DIC score: protein C (PC), antithrombin (AT), and citrullinated histones (H3Cit), which is a marker of NETosis. RESULTS: Out of 357 sepsis patients, 236 patients did not develop DIC (without-DIC), 79 patients had DIC on Day 1 (overt-DIC), and 42 patients developed DIC after Day 1 (pre-DIC). Compared to without-DIC patients, pre-DIC patients had decreased platelet count, increased international normalized ratio (INR), decreased PC and AT, and increased H3Cit. In contrast, D-dimer and fibrinogen levels did not differ between pre-DIC and without-DIC patients. Using receiver operating characteristics (ROC) analysis, we found that platelet count and INR in combination with PC and AT could discriminate pre-DIC from without-DIC. The area under the curve in the ROC analysis was 0.83 (95% confidence interval, 0.76 to 0.89). CONCLUSION: Our study suggests that platelets and INR in combination with PC and AT can identify the pre-DIC state in septic patients. In contrast, D-dimer increased and fibrinogen decreased in the late (ie, overt) stages of DIC. Our data also suggest that NETosis contributes to the onset of DIC in sepsis.

Comparison of the source and prognostic utility of cfDNA in trauma and sepsis.

BACKGROUND: Circulating cell-free DNA (cfDNA) may contribute to the pathophysiology of post-injury inflammation and coagulation in trauma. However, the source and mechanism of release of cfDNA in trauma is not well understood. One potential source of cfDNA is from Neutrophil Extracellular Traps (NETs), released by activated neutrophils during the process of NETosis. The primary objective of our study was to determine if cfDNA has prognostic utility in trauma. The secondary objective of this study was to determine the source of cfDNA in trauma compared to sepsis. METHODS: We studied trauma patients from two prospective observational cohort studies: the DNA as a Prognostic Marker in ICU Patients (DYNAMICS) study and the Endotoxin in Polytrauma (ENPOLY) study. We also studied septic patients from the DYNAMICS study. Citrated plasma samples were collected longitudinally from the patients (days 1 to 7). The following molecules were measured in the plasma samples: cfDNA, protein C (PC), myeloperoxidase (MPO) (a marker of neutrophil activation), citrullinated Histone H3 (H3Cit, a marker of NETosis), cyclophilin A (a marker of necrosis), and caspase-cleaved K18 (a marker of apoptosis). RESULTS: A total of 77 trauma patients were included (n = 38 from DYNAMICS and n = 39 from ENPOLY). The median age was 49 years; 27.3% were female, and mortality was 16.9% at 28 days. Levels of cfDNA were elevated compared to healthy values but not significantly different between survivors and non-survivors. There was a positive correlation between MPO and cfDNA in septic patients (r = 0.424, p < 0.001). In contrast, there was no correlation between MPO and cfDNA in trauma patients (r = - 0.192, p = 0.115). Levels of H3Cit, a marker of NETosis, were significantly elevated in septic patients compared to trauma patients (p < 0.01) while apoptosis and necrosis markers did not differ between the two groups. CONCLUSION: Our studies suggest that the source and mechanism of release of cfDNA differ between trauma and sepsis patients. In sepsis, cfDNA is likely primarily released by activated neutrophils via the process of NETosis. In contrast, cfDNA in trauma appears to originate mainly from injured or necrotic cells. Although cfDNA is elevated in trauma and sepsis patients compared to healthy controls, cfDNA does not appear to have prognostic utility in trauma patients. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01355042 . Registered May 17, 2011.

Antiviral resistance of stem cells.

Stem cells are important for growth and regeneration given their ability to self-renew and differentiate into mature cells. Resistance to certain viral infections has been established as a phenotype of stem cells, a protection in line with their important physiological function. Antiviral resistance is critical to all cells, but it is differentially regulated between stem cells and differentiated cells. Stem cells utilize antiviral RNA interference, interferon-independent repression of endogenous retroviruses and intrinsic expression of antiviral interferon-stimulated genes. Differentiated cells often rely on the interferon-associated protein-based response to induce a local antiviral state. This review outlines the antiviral resistance mechanisms of stem cells and discusses some ideas as to why stem cells and differentiated cells may have evolved to utilize distinct mechanisms.

Body temperature and mouse scoring systems as surrogate markers of death in cecal ligation and puncture sepsis.

BACKGROUND: Despite increasing ethical standards for conducting animal research, death is still often used as an endpoint in mouse sepsis studies. Recently, the Murine Sepsis Score (MSS), Mouse Clinical Assessment Score for Sepsis (M-CASS), and Mouse Grimace Scale (MGS) were developed as surrogate endpoint scoring systems for assessing pain and disease severity in mice. The objective of our study was to compare the effectiveness of these scoring systems and monitoring of body temperature for predicting disease progression and death in the cecal ligation and puncture (CLP) sepsis model, in order to better inform selection of surrogate endpoints for death in experimental sepsis. METHODS: C57Bl/6J mice were subjected to control sham surgery, or moderate or severe CLP sepsis. All mice were monitored every 4 h for surrogate markers of death using modified versions of the MSS, M-CASS, and MGS scoring systems until 24 h post-operatively, or until endpoint (inability to ambulate) and consequent euthanasia. RESULTS: Thirty percent of mice subjected to moderate severity CLP reached endpoint by 24 h post-CLP, whereas 100% undergoing severe CLP reached endpoint within 20 h. Modified MSS, M-CASS, and MGS scores all increased, while body temperature decreased, in a time-dependent and sepsis severity-dependent manner, although modified M-CASS scores showed substantial variability. Receiver operating characteristic curves demonstrate that the last recorded body temperature (AUC = 0.88; 95% CI 0.77-0.99), change in body temperature (AUC = 0.89; 95% CI 0.78-0.99), modified M-CASS (AUC = 0.93; 95% CI 0.85-1.00), and modified MSS (AUC = 0.95; 95% CI 0.88-1.01) scores are all robust for predicting death in CLP sepsis, whereas modified MGS (AUC = 0.78; 95% CI 0.63-0.92) is less robust. CONCLUSIONS: The modified MSS and body temperature are effective markers for assessing disease severity and predicting death in the CLP model, and should thus be considered as valid surrogate markers to replace death as an endpoint in mouse CLP sepsis studies.