Analysis of coagulation factors in angioedema/urticaria: increased values of D-dimer and fibrinogen in isolated angioedema.

INTRODUCTION: Recent research has shown that blood coagulation and the extrinsic coagulation cascade are involved in the pathogenesis of chronic spontaneous urticaria (CSU), but little is known about the coagulation factors in angioedema. METHODS: This study included 58 participants: 29 patients with chronic angioedema (14 with isolated angioedema and 15 with angioedema with wheals) and 29 healthy controls (HCs). We compared the values of coagulation factors in patients with isolated angioedema to those with wheals. Plasma levels of D-dimer, fibrinogen, and factor VII were measured by enzyme-linked immunosorbent assay (ELISA) for all participants. RESULTS: Significantly higher D-dimer (p = 0.016; ε² = 0.381) and fibrinogen (p = 0.044; ε² = 0.331) levels were recorded in patients with angioedema (both groups) than in the HCs, with higher levels for angioedema with wheals. Factor VII and fibrinogen levels did not differ significantly between the groups with angioedema, but coagulation factors were more often elevated in both angioedema groups than in HCs. CONCLUSIONS: One characteristic of angioedema is an elevated blood coagulation potential, which may help produce fibrin and may be important in controlling angioedema attacks.

Predictors of bleeding complications during catHeter-dirEcted thrombolysis for peripheral arterial occlusions (POCHET).

INTRODUCTION: The risk of major bleeding complications in catheter directed thrombolysis (CDT) for acute limb ischemia (ALI) remains high, with reported major bleeding complication rates in up to 1 in every 10 treated patients. Fibrinogen was the only predictive marker used for bleeding complications in CDT, despite the lack of high quality evidence to support this. Therefore, recent international guidelines recommend against the use of fibrinogen during CDT. However, no alternative biomarkers exist to effectively predict CDT-related bleeding complications. The aim of the POCHET biobank is to prospectively assess the rate and etiology of bleeding complications during CDT and to provide a biobank of blood samples to investigate potential novel biomarkers to predict bleeding complications during CDT. METHODS: The POCHET biobank is a multicentre prospective biobank. After informed consent, all consecutive patients with lower extremity ALI eligible for CDT are included. All patients are treated according to a predefined standard operating procedure which is aligned in all participating centres. Baseline and follow-up data are collected. Prior to CDT and subsequently every six hours, venous blood samples are obtained and stored in the biobank for future analyses. The primary outcome is the occurrence of non-access related major bleeding complications, which is assessed by an independent adjudication committee. Secondary outcomes are non-major bleeding complications and other CDT related complications. Proposed biomarkers to be investigated include fibrinogen, to end the debate on its usefulness, anti-plasmin and D-Dimer. DISCUSSION AND CONCLUSION: The POCHET biobank provides contemporary data and outcomes of patients during CDT for ALI, coupled with their blood samples taken prior and during CDT. Thereby, the POCHET biobank is a real world monitor on biomarkers during CDT, supporting a broad spectrum of future research for the identification of patients at high risk for bleeding complications during CDT and to identify new biomarkers to enhance safety in CDT treatment.

Glycine by enteral route does not improve major clinical outcomes in severe COVID-19: a randomized clinical pilot trial.

There is a worrying scarcity of drug options for patients with severe COVID-19. Glycine possesses anti-inflammatory, cytoprotective, endothelium-protective, and platelet-antiaggregant properties, so its use in these patients seems promising. In this open label, controlled clinical trial, inpatients with severe COVID-19 requiring mechanical ventilation randomly received usual care (control group) or usual care plus 0.5 g/kg/day glycine by the enteral route (experimental group). Major outcomes included mortality, time to weaning from mechanical ventilation, total time on mechanical ventilation, and time from study recruitment to death. Secondary outcomes included laboratory tests and serum cytokines. Patients from experimental (n = 33) and control groups (n = 23) did not differ in basal characteristics. There were no differences in mortality (glycine group, 63.6% vs control group, 52.2%, p = 0.60) nor in any other major outcome. Glycine intake was associated with lower fibrinogen levels, either evaluated per week of follow-up (p < 0.05 at weeks 1, 2, and 4) or as weighted mean during the whole hospitalization (608.7 ± 17.7 mg/dl vs control 712.2 ± 25.0 mg/dl, p = 0.001), but did not modify any other laboratory test or cytokine concentration. In summary, in severe COVID-19 glycine was unable to modify major clinical outcomes, serum cytokines or most laboratory tests, but was associated with lower serum fibrinogen concentration.Registration: ClinicalTrials.gov NCT04443673, 23/06/2020.

Comparison between XGboost model and logistic regression model for predicting sepsis after extremely severe burns.

OBJECTIVE: To compare an Extreme Gradient Boosting (XGboost) model with a multivariable logistic regression (LR) model for their ability to predict sepsis after extremely severe burns. METHODS: For this observational study, patient demographic and clinical information were collected from medical records. The two models were evaluated using area under curve (AUC) of the receiver operating characteristic (ROC) curve. RESULTS: Of the 103 eligible patients with extremely severe burns, 20 (19%) were in the sepsis group, and 83 (81%) in the non-sepsis group. The LR model showed that age, admission time, body index (BI), fibrinogen, and neutrophil to lymphocyte ratio (NLR) were risk factors for sepsis. Comparing AUC of the ROC curves, the XGboost model had a higher predictive performance (0.91) than the LR model (0.88). The SHAP visualization tool indicated fibrinogen, NLR, BI, and age were important features of sepsis in patients with extremely severe burns. CONCLUSIONS: The XGboost model was superior to the LR model in predictive efficacy. Results suggest that, fibrinogen, NLR, BI, and age were correlated with sepsis after extremely severe burns.

Diagnostic value and clinical significance of lncRNA LINC01123 combined with fibrinogen in acute cerebral infarction.

OBJECTIVE: To explore the diagnostic value and clinical significance of lncRNA LINC01123 (LINC01123) binding fibrinogen in acute cerebral infarction (ACI) by evaluating the expression and potential molecular mechanism of LINC01123 in patients with acute cerebral infarction. METHODS: The clinical data of all the volunteers were collected. The level of serum LINC01123 in ACI patients was detected by RT-qPCR. The relationship between LINC01123 and fibrinogen was studied via Pearson's correlation analysis. ROC curve was used to evaluate the diagnostic value of LINC01123 and fibrinogen for ACI. The risk factors of ACI were investigated by Binary Logistic regression analysis. And the targeting relationship between LINC01123 and downstream miR-361-3p was verified through luciferase activity assay. RESULTS: Serum LINC01123 and fibrinogen levels were upregulated in ACI patients compared with healthy controls (P < 0.001), and there was a positive correlation between them (r = 0.6537, P < 0.001). In predicting the occurrence of ACI, LINC01123 and fibrinogen have high diagnostic value, and the AUC of combined diagnosis was 0.961, and the sensitivity and specificity (92.54%, 85.82%) were more significant. Meanwhile, LINC01123 and fibrinogen were confirmed to be independent risk factors for ACI (P < 0.0001). Mechanistically, miR-361-3p is the target of LINC01123. The expression of miR-361-3p was low in the serum of ACI patients, which was negatively correlated with the LINC01123 expression (r = -0.6885, P < 0.0001). CONCLUSION: LINC01123 combined with fibrinogen may have important reference value in the diagnosis of ACI as serum markers, which may become clinical indicators to predict the occurrence of ACI.

U-shaped relationship found between fibrinogen-to-albumin ratio and systemic inflammation response index in osteoporotic fracture patients.

The relationship between the Systemic Inflammatory Response Index (SIRI) and the Fibrinogen-to-albumin ratio (FAR) has not been extensively investigated. The objective of this study was to determine the independent relationship between FAR and SIRI in people with osteoporotic fractures (OPF). A cross-sectional study was conducted using retrospective data from 3431 hospitalized OPF patients. The exposure variable in this study was the baseline FAR, while the outcome variable was the SIRI. Covariates, including age, gender, BMI, and other clinical and laboratory factors, were adjusted. Cross-correlation analysis and linear regression models were applied. The generalized additive model (GAM) investigated non-linear relationships. Adjusted analysis revealed an independent negative association between FAR and SIRI in OPF patients (ß = - 0.114, p = 0.00064, 95% CI - 0.180, - 0.049). A substantial U-shaped association between FAR and SIRI was shown using GAM analysis (p < 0.001). FAR and SIRI indicated a negative association for FAR below 6.344% and a positive correlation for FAR over 6.344%. The results of our study revealed a U-shaped relationship between SIRI and FAR. The lowest conceivable FAR for a bone-loose inflammatory disease might be 6.344%, suggesting that this has particular significance for the medical diagnosis and therapy of persons with OPF. Consequently, the term "inflammatory trough" is proposed. These results offer fresh perspectives on controlling inflammation in individuals with OPF and preventing inflammatory osteoporosis.

Rapid Separation and Display of Active Fibrinogenolytic Agents in Sipunculus nudus through Fibrinogen-Polyacrylamide Gel Electrophoresis.

Fibrinogenolytic agents that can dissolve fibrinogen directly have been widely used in anti-coagulation treatment. Generally, identifying new fibrinogenolytic agents requires the separation of each component first and then checking their fibrinogenolytic activities. Currently, polyacrylamide gel electrophoresis (PAGE) and chromatography are mostly used in the separating stage. Meanwhile, the fibrinogen plate assay and reaction products based PAGE are usually adopted to display their fibrinogenolytic activities. However, because of the spatiotemporal separation of those two stages, it is impossible to separate and display the active fibrinogenolytic agents with the same gel. To simplify the separating and displaying processes of fibrinogenolytic agent identification, we constructed a new fibrinogen-PAGE method to rapidly separate and display the fibrinogenolytic agents of peanut worms (Sipunculus nudus) in this study. This method includes fibrinogen-PAGE preparation, electrophoresis, renaturation, incubation, staining, and decolorization. The fibrinogenolytic activity and molecular weight of the protein can be detected simultaneously. According to this method, we successfully detected more than one active fibrinogenolytic agent of peanut wormhomogenate within 6 h. Moreover, this fibrinogen-PAGE method is time and cost-friendly. Furthermore, this method could be used to study the fibrinogenolytic agents of the other organisms.

Potential clinical value of fibrinogen-like protein 1 as a serum biomarker for the identification of diabetic cardiomyopathy.

Diabetic individuals with diabetic cardiomyopathy (DbCM) present with abnormal myocardial structure and function. DbCM cannot be accurately diagnosed due to the lack of suitable diagnostic biomarkers. In this study, 171 eligible participants were divided into a healthy control (HC), type 2 diabetes mellitus (T2DM) patients without DbCM (T2DM), or DbCM group. Serum fibrinogen-like protein 1 (FGL-1) and other biochemical parameters were determined for all participants. Serum FGL-1 levels were significantly higher in patients with DbCM compared with those in the T2DM group and HCs. Serum FGL-1 levels were negatively correlated with left ventricular fractional shortening and left ventricular ejection fraction (LVEF) and positively correlated with left ventricular mass index in patients with DbCM after adjusting for age, sex and body mass index. Interaction of serum FGL-1 and triglyceride levels on LVEF was noted in patients with DbCM. A composite marker including serum FGL-1 and triglycerides could differentiate patients with DbCM from those with T2DM and HCs with an area under the curve of 0.773 and 0.789, respectively. Composite marker levels were negatively correlated with N-terminal B-type natriuretic peptide levels in patients with DbCM. Circulating FGL-1 may therefore be a valuable index reflecting cardiac functions in DbCM and to diagnose DbCM.

The role of preoperative FPR and FAR in prognostic evaluation of stages II and III radical colorectal cancer: A single-center retrospective study.

The inflammatory and nutritional states of body are 2 important causes associated with the initiation and progression of colorectal cancer (CRC). The aim of this study is to investigate the prognostic evaluation value of preoperative fibrinogen-to-prealbumin ratio (FPR) and preoperative fibrinogen-to-albumin ratio (FAR) in CRC. The clinical data of 350 stages II and III patients with CRC who received radical resection were retrospectively analyzed. All patients were followed up for 5 years to observe the overall survival and disease-free survival of 5 years and analyze the relationship between preoperative FPR and FAR and prognosis of all enrolled patients. In addition, we analyzed the diagnostic and application value of combined biomarkers. This study showed high-level preoperative FPR and FAR were significantly associated with poor overall survival and disease-free survival of stages II and III patients with CRC. The elevated preoperative FPR and FAR level was significantly related to age, tumor differentiation level, TNM stage, vascular infiltration, carcinoembryonic antigen, carbohydrate antigen199, etc. The combination of FPR, FAR, neutrophil-to-lymphocyte ratio, and carbohydrate antigen199 had the maximum area under curve (AUC = 0.856, 95% CI: 0.814-0.897, Sen = 78.20%, Spe = 82.49%, P < .05) under the receiver-operating characteristics curve. The preoperative FPR and FAR have important prognostic value and they can be used as independent prognostic marker for patients with stages II and III CRC undergoing radical resection. Moreover, the combination of biomarkers could further enhance the diagnostic and prognostic efficacy of CRC.

Fibrinogen: Structure, abnormalities and laboratory assays.

Fibrinogen is the primary precursor protein for the fibrin clot, which is the final target of blood clotting. It is also an acute phase reactant that can vary under physiologic and inflammatory conditions. Disorders in fibrinogen concentration and/or function have been variably linked to the risk of bleeding and/or thrombosis. Fibrinogen assays are commonly used in the management of bleeding as well as the treatment of thrombosis. This chapter examines the structure of fibrinogen, its role in hemostasis as well as in bleeding abnormalities and measurement thereof with respect to clinical management.

Exosomes Secreted by Wharton's Jelly-Derived Mesenchymal Stem Cells Promote the Ability of Cell Proliferation and Migration for Keratinocyte.

Mesenchymal stem cells (MSCs) isolated from Wharton's jelly (WJ-MSCs) and adipose tissue (AD-MSCs) are alternative sources for bone marrow-derived MSCs. Owing to their multiple functions in angiogenesis, immune modulation, proliferation, migration, and nerve regeneration, MSC-derived exosomes can be applied in "cell-free cell therapy". Here, we investigated the functional protein components between the exosomes from WJ-MSCs and AD-MSCs to explain their distinct functions. Proteins of WJ-MSC and AD-MSC exosomes were collected and compared based on iTRAQ gel-free proteomics data. Results: In total, 1695 proteins were detected in exosomes. Of these, 315 were more abundant (>1.25-fold) in AD-MSC exosomes and 362 kept higher levels in WJ-MSC exosomes, including fibrinogen proteins. Pathway enrichment analysis suggested that WJ-MSC exosomes had higher potential for wound healing than AD-MSC exosomes. Therefore, we treated keratinocyte cells with exosomes and the recombinant protein of fibrinogen beta chain (FGB). It turned out that WJ-MSC exosomes better promoted keratinocyte growth and migration than AD-MSC exosomes. In addition, FGB treatment had similar results to WJ-MSC exosomes. The fact that WJ-MSC exosomes promoted keratinocyte growth and migration better than AD-MSC exosomes can be explained by their higher FGB abundance. Exploring the various components of AD-MSC and WJ-MSC exosomes can aid in their different clinical applications.

Nonlinear association of fibrinogen levels with functional prognosis in patients with acute ischemic stroke: a prospective cohort study.

OBJECTIVE: Fibrinogen, essential in primary hemostasis, platelet aggregation, and leukocyte-endothelial interactions, is also associated with a heightened risk of acute ischemic stroke (AIS). However, its influence on AIS patient outcomes is unclear. This study examines the correlation between fibrinogen levels and the risk of unfavorable outcomes three months post-AIS. METHODS: This is a secondary analysis of a prospective cohort study conducted in Korea. The sample consisted of 1851 AIS patients who received treatment at a Korean hospital between January 2010 and December 2016. Statistical models were established to understand the relationship between fibrinogen levels(mg/dL) and unfavorable outcomes(mRs ≥ 3), including logistic regression models, Generalized Additive Models (GAM), and smooth curve fitting (penalized splines). The log-likelihood ratio test has been utilized to evaluate the best fit. To ensure the robustness of the results, sensitivity analyses were conducted by reanalyzing the relationship after excluding participants with TG > 200 mg/dl and BMI > 25 kg/m2. Subgroup analyses were also performed to assess whether influencing factors modify the association between fibrinogen levels and unfavorable outcomes. RESULTS: After adjusting for multiple covariates including age, BMI, sex, LDL-c, TG, HGB, HDL-c, BUN, FPG, ALB, PLT, AF, hypertension, smoking, DM, mRs score at admission, the binary logistic regression model demonstrated revealed a significant positive association between fibrinogen levels and the risk of unfavorable outcomes in AIS patients (OR = 1.215, 95% CI: 1.032-1.429, p = 0.019). Sensitivity analyses supported these findings, with similar ORs observed in subsets of patients with TG < 200 mg/dL (OR = 1.221, 95% CI: 1.036-1.440) and BMI < 25 kg/m2 (OR = 1.259, 95% CI: 1.051-1.509). Additionally, the relationship between fibrinogen levels and outcomes was nonlinear, with a critical threshold of 2.74 g/L. Below the inflection point, the OR for unfavorable outcomes was 0.666 ((95% CI: 0.360, 1.233, p = 0.196), whereas above it, the OR increased to 1.374 (95% CI: 1.138, 1.659). CONCLUSIONS: This study has provided evidence of a positive and nonlinear correlation between fibrinogen levels and 3-month poor functional outcomes in patients with AIS. When fibrinogen levels exceeded 2.74 g/L, a significant and positive association was observed with the risk of poor outcomes. This study provides a further reference for optimizing rehabilitation exercises and facilitating clinical counseling in patients with acute ischemic stroke.

Significance of serum protein electrophoresis in the diagnosis of Tsukayama type IV periprosthetic joint infection.

Objectives: To investigate the efficacy of serum protein electrophoresis (SPE) in the diagnosis of periprosthetic joint infection (PJI) after hip and knee arthroplasty. Methods: The medical records of patients undergoing hip and knee arthroplasty at a class A tertiary hospital between August 2013 and January 2021 were retrospectively investigated. A total of 179 patients were included and divided into two groups: 66 patients in the PJI group and 113 patients in the aseptic loosening (AL) group. Serum C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), D-dimer, Fibrinogen, Serum albumin and the proportion of serum protein in SPE were compared between the two groups. The diagnostic sensitivity and specificity were determined using the receiver operating characteristic (ROC) curve, and the diagnostic value was compared using the area under the ROC curve (AUC). Results: There was no significant difference in age, sex and body mass index (BMI) between PJI group and AL group (P>0.05), but there was significant difference in the ratio of hip to knee (X2 = 22.043, P<0.001). The CRP, ESR, D-dimer, Fibrinogen and the proportion of α1 globulin band in PJI group was 22.99(10.55,40.58) mg/L, 37.00(23.00,61.70) mm/h, 790.00(500.00,1500.00) ng/ml, 4.84(3.81,5.55) g/L and 5.80(5.00,7.73) % which was higher than that in AL group [1.89(0.50,4.12) mg/L, U=7.984, P<0.001; 10.10(7.00,16.90) mm/h, U=8.095, P<0.001; 570.00(372.50,780.00) ng/ml, U=3.448, P<0.001; 2.84(2.45,3.43) g/L, U=8.053, P<0.001 and 4.20(3.90,4.80) %, U=8.154, P<0.001]. The Serum albumin and the proportion of Albumin band in PJI group was 36.10(33.10,39.00) g/L and 49.00(44.95,52.20) % which was lower than that in AL group [38.10(34.00,41.10) g/L, U=-2.383, P=0.017 and 54.40(51.55,56.70) %, U=-6.162, P<0.001]. The proportion of In PJI group, the AUC of proportion of α1 globulin was 0.8654, which was equivalent to CRP (0.8698), ESR (0.8680) and outperformed that of fibrinogen (0.8025). Conclusions: Elevated proportion of α1 globulin in SPE presented with good diagnostic value for Tsukayama type IV PJI, and its accuracy was comparable to those of ESR and CRP. And α1 globulin can assist with CRP and ESR to determining the timing of second-stage revision.

Selective binding of cationic fibrinogen-mimicking chitosan nanoparticles to activated platelets and efficient drug release for antithrombotic therapy.

Thrombosis is the main cause of catastrophic events including ischemic stroke, myocardial infarction and pulmonary embolism. Acetylsalicylic acid (ASA) therapy offers a desirable approach to antithrombosis through a reduction of platelet reactivity. However, major bleeding complications, severe off-target side effects, and resistance or nonresponse to ASA greatly attenuate its clinical outcomes. Herein, we report a cationic fibrinogen-mimicking nanoparticle, denoted as ASA-RGD-CS@TPP, to achieve activated-platelet-targeted delivery and efficient release of ASA for safer and more effective antithrombotic therapy. This biomimetic antithrombotic system was prepared by one-pot ionic gelation between cationic arginine-glycine-aspartic acid (RGD)-grafted chitosan (RGD-CS) and anionic tripolyphosphate (TPP). The platform exhibited selective binding to activated platelets, leading to efficient release of ASA and subsequent attenuation of platelet functions, including the remarkable inhibition of platelet aggregation through a potent blockage of cyclooxygenase-1 (COX-1). After intravenous administration, ASA-RGD-CS@TPP displayed significantly prolonged circulation time and successful prevention of thrombosis in a mouse model. ASA-RGD-CS@TPP was demonstrated to significantly enhance antithrombotic therapy while showing minimal coagulation and hemorrhagic risks and excellent biocompatibility in vivo as compared to free ASA. This platform provides a simple, safe, effective and targeted strategy for the development of antithrombotic nanomedicines.

Controlling nanoparticle-induced endothelial leakiness with the protein corona.

Understanding nanoparticle-cell interaction is essential for advancing research in nanomedicine and nanotoxicology. Apart from the transcytotic pathway mediated by cellular recognition and energetics, nanoparticles (including nanomedicines) may harness the paracellular route for their transport by inducing endothelial leakiness at cadherin junctions. This phenomenon, termed as NanoEL, is correlated with the physicochemical properties of the nanoparticles in close association with cellular signalling, membrane mechanics, as well as cytoskeletal remodelling. However, nanoparticles in biological systems are transformed by the ubiquitous protein corona and yet the potential effect of the protein corona on NanoEL remains unclear. Using confocal fluorescence microscopy, biolayer interferometry, transwell, toxicity, and molecular inhibition assays, complemented by molecular docking, here we reveal the minimal to significant effects of the anionic human serum albumin and fibrinogen, the charge neutral immunoglobulin G as well as the cationic lysozyme on negating gold nanoparticle-induced endothelial leakiness in vitro and in vivo. This study suggests that nanoparticle-cadherin interaction and hence the extent of NanoEL may be partially controlled by pre-exposing the nanoparticles to plasma proteins of specific charge and topology to facilitate their biomedical applications.

The Effect of Reduced Fibrinogen on Cerebrovascular Permeability during Traumatic Brain Injury in Fibrinogen Gene Heterozygous Knockout Mice.

Vascular contribution to cognitive impairment and dementia (VCID) is a term referring to all types of cerebrovascular and cardiovascular disease-related cognitive decline, spanning many neuroinflammatory diseases including traumatic brain injury (TBI). This becomes particularly important during mild-to-moderate TBI (m-mTBI), which is characterized by short-term memory (STM) decline. Enhanced cerebrovascular permeability for proteins is typically observed during m-mTBI. We have previously shown that an increase in the blood content of fibrinogen (Fg) during m-mTBI results in enhanced cerebrovascular permeability. Primarily extravasated via a transcellular pathway, Fg can deposit into the parenchyma and exacerbate inflammatory reactions that can lead to neurodegeneration, resulting in cognitive impairment. In the current study, we investigated the effect of a chronic reduction in Fg concentration in blood on cerebrovascular permeability and the interactions of extravasated Fg with astrocytes and neurons. Cortical contusion injury (CCI) was used to generate m-mTBI in transgenic mice with a deleted Fg γ chain (Fg γ+/-), resulting in a low blood content of Fg, and in control C57BL/6J wild-type (WT) mice. Cerebrovascular permeability was tested in vivo. Interactions of Fg with astrocytes and neurons and the expression of neuronal nuclear factor-кB (NF-кB) were assessed via immunohistochemistry. The results showed that 14 days after CCI, there was less cerebrovascular permeability, lower extravascular deposition of Fg, less activation of astrocytes, less colocalization of Fg with neurons, and lower expression of neuronal pro-inflammatory NF-кB in Fg γ+/- mice compared to that found in WT mice. Combined, our data provide strong evidence that increased Fg extravasation, and its resultant extravascular deposition, triggers astrocyte activation and leads to potential interactions of Fg with neurons, resulting in the overexpression of neuronal NF-кB. These effects suggest that reduced blood levels of Fg can be beneficial in mitigating the STM reduction seen in m-mTBI.

Immune system-related plasma extracellular vesicles in healthy aging.

Objectives: To identify age-related plasma extracellular vehicle (EVs) phenotypes in healthy adults. Methods: EV proteomics by high-resolution mass spectrometry to evaluate EV protein stability and discover age-associated EV proteins (n=4 with 4 serial freeze-thaws each); validation by high-resolution flow cytometry and EV cytokine quantification by multiplex ELISA (n=28 healthy donors, aged 18-83 years); quantification of WI-38 fibroblast cell proliferation response to co-culture with PKH67-labeled young and old plasma EVs. The EV samples from these plasma specimens were previously characterized for bilayer structure, intra-vesicle mitochondria and cytokines, and hematopoietic cell-related surface markers. Results: Compared with matched exo-EVs (EV-depleted supernatants), endo-EVs (EV-associated) had higher mean TNF-α and IL-27, lower mean IL-6, IL-11, IFN-γ, and IL-17A/F, and similar mean IL-1ß, IL-21, and IL-22 concentrations. Some endo-EV and exo-EV cytokine concentrations were correlated, including TNF-α, IL-27, IL-6, IL-1ß, and IFN-γ, but not IL-11, IL-17A/F, IL-21 or IL-22. Endo-EV IFN-γ and exo-EV IL-17A/F and IL-21 declined with age. By proteomics and confirmed by flow cytometry, we identified age-associated decline of fibrinogen (FGA, FGB and FGG) in EVs. Age-related EV proteins indicated predominant origins in the liver and innate immune system. WI-38 cells (>95%) internalized similar amounts of young and old plasma EVs, but cells that internalized PKH67-EVs, particularly young EVs, underwent significantly greater cell proliferation. Conclusion: Endo-EV and exo-EV cytokines function as different biomarkers. The observed healthy aging EV phenotype reflected a downregulation of EV fibrinogen subpopulations consistent with the absence of a pro-coagulant and pro-inflammatory condition common with age-related disease.

Fibrin promotes oxidative stress and neuronal loss in traumatic brain injury via innate immune activation.

BACKGROUND: Traumatic brain injury (TBI) causes significant blood-brain barrier (BBB) breakdown, resulting in the extravasation of blood proteins into the brain. The impact of blood proteins, especially fibrinogen, on inflammation and neurodegeneration post-TBI is not fully understood, highlighting a critical gap in our comprehension of TBI pathology and its connection to innate immune activation. METHODS: We combined vascular casting with 3D imaging of solvent-cleared organs (uDISCO) to study the spatial distribution of the blood coagulation protein fibrinogen in large, intact brain volumes and assessed the temporal regulation of the fibrin(ogen) deposition by immunohistochemistry in a murine model of TBI. Fibrin(ogen) deposition and innate immune cell markers were co-localized by immunohistochemistry in mouse and human brains after TBI. We assessed the role of fibrinogen in TBI using unbiased transcriptomics, flow cytometry and immunohistochemistry for innate immune and neuronal markers in Fggγ390-396A knock-in mice, which express a mutant fibrinogen that retains normal clotting function, but lacks the γ390-396 binding motif to CD11b/CD18 integrin receptor. RESULTS: We show that cerebral fibrinogen deposits were associated with activated innate immune cells in both human and murine TBI. Genetic elimination of fibrin-CD11b interaction reduced peripheral monocyte recruitment and the activation of inflammatory and reactive oxygen species (ROS) gene pathways in microglia and macrophages after TBI. Blockade of the fibrin-CD11b interaction was also protective from oxidative stress damage and cortical loss after TBI. CONCLUSIONS: These data suggest that fibrinogen is a regulator of innate immune activation and neurodegeneration in TBI. Abrogating post-injury neuroinflammation by selective blockade of fibrin's inflammatory functions may have implications for long-term neurologic recovery following brain trauma.

Global O-glycoproteome enrichment and analysis enabled by a combinatorial enzymatic workflow.

A comprehensive analysis of site-specific protein O-glycosylation is hindered by the absence of a consensus O-glycosylation motif, the diversity of O-glycan structures, and the lack of a universal enzyme that cleaves attached O-glycans. Here, we report the development of a robust O-glycoproteomic workflow for analyzing complex biological samples by combining four different strategies: removal of N-glycans, complementary digestion using O-glycoprotease (IMPa) with/without another protease, glycopeptide enrichment, and mass spectrometry with fragmentation of glycopeptides using stepped collision energy. Using this workflow, we cataloged 474 O-glycopeptides on 189 O-glycosites derived from 79 O-glycoproteins from human plasma. These data revealed O-glycosylation of several abundant proteins that have not been previously reported. Because many of the proteins that contained unannotated O-glycosylation sites have been extensively studied, we wished to confirm glycosylation at these sites in a targeted fashion. Thus, we analyzed selected purified proteins (kininogen-1, fetuin-A, fibrinogen, apolipoprotein E, and plasminogen) in independent experiments and validated the previously unknown O-glycosites.

Central subfield thickness of diabetic macular edema: Correlation with the aqueous humor proteome.

Purpose: Diabetic macular edema (DME) is a sight-threatening complication of diabetes. Consequently, studying the proteome of DME may provide novel insights into underlying molecular mechanisms. Methods: In this study, aqueous humor samples from eyes with treatment-naïve clinically significant DME (n = 13) and age-matched controls (n = 11) were compared with label-free liquid chromatography-tandem mass spectrometry. Additional aqueous humor samples from eyes with treatment-naïve DME (n = 15) and controls (n = 8) were obtained for validation by enzyme-linked immunosorbent assay (ELISA). Best-corrected visual acuity (BCVA) was evaluated, and the severity of DME was measured as central subfield thickness (CST) employing optical coherence tomography. Control samples were obtained before cataract surgery. Significantly changed proteins were identified using a permutation-based calculation, with a false discovery rate of 0.05. A human donor eye with DME and a control eye were used for immunofluorescence. Results: A total of 101 proteins were differentially expressed in the DME. Regulated proteins were involved in complement activation, glycolysis, extracellular matrix interaction, and cholesterol metabolism. The highest-fold change was observed for the fibrinogen alpha chain (fold change = 17.8). Complement components C2, C5, and C8, fibronectin, and hepatocyte growth factor-like protein were increased in DME and correlated with best-corrected visual acuity (BCVA). Ceruloplasmin and complement component C8 correlated with central subfield thickness (CST). Hemopexin, plasma kallikrein, monocyte differentiation antigen CD14 (CD14), and lipopolysaccharide-binding protein (LBP) were upregulated in the DME. LBP was correlated with vascular endothelial growth factor. The increased level of LBP in DME was confirmed using ELISA. The proteins involved in desmosomal integrity, including desmocollin-1 and desmoglein-1, were downregulated in DME and correlated negatively with CST. Immunofluorescence confirmed the extravasation of fibrinogen at the retinal level in the DME. Conclusion: Elevated levels of pro-inflammatory proteins, including the complement components LBP and CD14, were observed in DME. DME was associated with the loss of basal membrane proteins, compromised desmosomal integrity, and perturbation of glycolysis.