Search

Objectives Current treatments are effective only in 30% of lupus nephritis patients emphasizing the need for novel therapeutic strategies. To develop mechanistic hypotheses and explore novel biomarkers, we analyzed the longitudinal urinary proteomic profiles in patients with lupus nephritis undergoing treatment. Methods We quantified 1,000 urinary proteins in 30 patients with lupus nephritis at the time of the diagnostic renal biopsy and after 3, 6, and 12 months. The proteins and molecular pathways detected in the urine proteome were then analyzed with respect to baseline clinical features and longitudinal trajectories. The intrarenal expression of candidate biomarkers was evaluated using single cell transcriptomics of renal biopsies from lupus nephritis patients. Results Our analysis revealed multiple biological pathways including chemotaxis, neutrophil activation, platelet degranulation, and extracellular matrix organization that could be noninvasively quantified and monitored in the urine. We identified 237 urinary biomarkers associated with lupus nephritis as compared to controls without SLE. IL-16, CD163, and TGF-β mirrored intrarenal nephritis activity. Response to treatment was paralleled by a reduction of urinary IL-16, a CD4 ligand with proinflammatory and chemotactic properties. Single cell RNA sequencing independently demonstrated that IL16 is the second most expressed cytokine by most infiltrating immune cells in lupus nephritis kidneys. IL-16 producing cells were found at key sites of kidney injury. Conclusion Urine proteomics may profoundly change the diagnosis and management of lupus nephritis by noninvasively monitor active intrarenal biological pathways. These findings implicate IL-16 in lupus nephritis pathogenesis designating it as a potentially treatable target and biomarker.

Background The role of platelets in the pathogenesis of venous thromboembolism (VTE) is receiving increasing attention; however, limited information is available on platelet function in the acute phase of the disease. Objective To characterize platelet function according to VTE phenotypes. Patients/Methods In total, 154 subjects (isolated pulmonary embolism [iPE], n = 28; isolated deep vein thrombosis [iDVT], n = 35; DVT+PE, n = 91) were included. In this study platelet function analyzer (PFA)-200, light transmission aggregometry (LTA), thrombin generation (TG) in presence (PRP) and absence (PFP) of platelets and platelet flow cytometry were investigated. LASSO regression was used to select clinical and platelet biomarkers that distinguish between VTE phenotypes. Results PFA-200 results did not differ between VTE phenotypes. LTA from DVT+PE subjects showed lowest maximum aggregation after epinephrine and adenosine diphosphate compared to iPE and iDVT. Lower % of PAC-1-positive platelets after in-vitro trigger were present in DVT+PE and iPE compared to iDVT. TG in PRP had lower peak height and velocity in DVT+PE and iPE against iDVT. The results of LASSO regression for the distinction between DVT+PE vs iDVT identified 18 variables (AUC =0.93) of which 72% were platelet biomarkers. For distinction between iPE and iDVT, 10 variables were selected (AUC = 0.96) of which 50% were platelet-related. Obesity was the only variable weakly discriminating between DVT+PE vs iPE (AUC = 0.66). Conclusion This explorative study suggests an important distinction between PE-related phenotypes and iDVT when considering clinical and platelet function data. Lower platelet-dependent TG along with reduced platelet reactivity suggest higher platelet degranulation in PE-dependent phenotypes compared to iDVT.

Background. Hepatic fibrosis is a severe liver disease that has threatened human health for a long time. In order to undergo timely and adequate therapy, it is important for patients to obtain an accurate diagnosis of fibrosis. Laboratory inspection methods have been efficient in distinguishing between advanced hepatic fibrosis stages (F3, F4), but the identification of early stages of fibrosis has not been achieved. The development of proteomics may provide us with a new direction to identify the stages of fibrosis. Methods. We established serum proteomic maps for patients with hepatic fibrosis at different stages and identified differential expression of proteins between fibrosis stages through ultra-high-performance liquid chromatography tandem mass spectrometry proteomic analysis. Results. From the proteomic profiles of the serum of patients with different stages of liver fibrosis, a total of 1,338 proteins were identified. Among three early fibrosis stages (control, F1, and F2), 55 differential proteins were identified, but no proteins simultaneously exhibited differential expression between control, F1, and F2. Differential proteins were detected in the comparison between different fibrosis stages. Significant differences were found between advanced fibrosis stages (F2-vs.-F3 and F3-vs.-F4) through a series of statistical analysis, including hierarchical clustering, Gene Ontology (GO) functional annotation, Kyoto Encyclopedia of Genes and Genomes pathway, and protein-protein interaction network analysis. The differential proteins identified by GO annotation were associated with biological processes (mainly platelet degranulation and cell adhesion), molecular functions, and cellular components. Conclusions. All potential biomarkers identified between the stages of fibrosis could be key points in determining the fibrosis staging. The differences between early stages may provide a useful reference in addressing the challenge of early fibrosis staging.

Objectives Patients with Gaucher disease (GD) are at increased risk of bleeding and have varying degrees of thrombocytopenia, making the analysis of platelet function difficult. This study aimed to provide a clinically relevant quantitative assessment of platelet function and determine its relationship with bleeding and GD-related data. Methods Unstimulated and stimulated platelet function was measured by whole blood flow cytometry of platelet surface-activated αIIbβ3 integrin (detected with monoclonal antibody PAC1), P-selectin (CD62P), and lysosomal-associated membrane protein (LAMP3/CD63) in 149 GD patients. Results GD patients had a higher level of unstimulated CD63 expression than healthy subjects, which was mildly correlated with glucosylsphingosine (lyso-Gb1) levels (r = 0.17, p-value = 0.042). Splenectomized GD patients had a higher level of unstimulated αIIbβ3 integrin and P-selectin expression. Reduced platelet reactivity (−2 standard deviation of reference range) was found in 79 (53%, 95% confidence interval [CI]: 44–61%) patients, of whom 10 (6.7%, 95% CI: 3.3–12%) had more severe platelet dysfunction. In a multivariate model, only lyso-Gb1 levels were associated with the more severe platelet dysfunction. Fifty-four (49%) of 128 adult patients who completed the bleeding tendency questionnaire reported positive bleeding history. In a multivariate logistic model, older age (odds ratio [OR]: 1.05, 95% CI: 1.01–1.1) and low P-selectin reactivity (OR: 2.03, 95% CI: 1.25–3.35) were associated with more than one bleeding manifestation. Conclusion Flow cytometry enables the study of platelet function in thrombocytopenic GD patients. A platelet degranulation defect, but not αIIbβ3 integrin activation defect, is associated with clinical bleeding. In vivo increased CD63 expression may be related to GD-related inflammation.

N-Glycosylation of proteins, an important post-translational modification in eukaryotic cells, plays an essential role in the regulation of cell adhesion, migration, signal transduction, and apoptosis. Abnormal changes in protein glycosylation are closely related to the occurrence of many critical diseases, including diabetes, tumors, and neurological, kidney, and inflammatory diseases. A non-invasive type of liquid biopsy, urine sampling has the advantage of reducing the complexity of proteomic analysis. This facilitates the design of large-scale and continuous or multi-time point sampling strategies. However, the dynamic range of urinary protein abundance is relatively large, owing to individual differences and physiological conditions. Currently, there is a lack of specialized research on individual differences, physiological fluctuations, and physiological abundance ranges of urinary N-glycoproteins in large healthy populations. Therefore, it is difficult to accurately distinguish individual differences and normal physiological fluctuations from changes caused by disease; this poses a great challenge in disease marker research. Liquid chromatography-mass spectrometry (LC-MS) is an analytical technique widely used for the large-scale profiling of proteomes in biological systems, and the enrichment of N-glycopeptides is a prerequisite for their detection by MS.In this study, we established an approach based on hydrophilic interaction chromatography (HILIC) by optimizing the activation, cleaning, and elution processes of the enrichment method, for instance through the optimization of particle size and solvent composition, and investigated the identification number, selectivity, and stability of N-glycoprotein/N-glycopeptide enrichment under different experimental conditions. We found that N-glycoproteins and N-glycopeptides were highly enriched in a trifluoroacetic acid system with 5-μm filling particles in the HILIC column. On this basis, we analyzed the levels of N-glycoproteins/N-glycopeptides in urine samples. The consistency of N-glycoprotein/N-glycopeptide levels in urine samples taken from the same healthy person for five consecutive days was investigated by correlation analysis. This analysis revealed that the urinary N-glycoproteome of the same healthy person was relatively stable over a short period of time. Next, urinary samples from 20 healthy male volunteers and 20 healthy female volunteers were enriched for N-glycoproteins/N-glycopeptides, which were profiled by MS through qualitative and quantitative analyses. Screening and functional analysis of differential proteins were then carried out. A total of 1016 N-glycoproteins and 2192 N-glycopeptides were identified in the mid-morning urine samples of the 40 healthy volunteers. A label-free quantitation strategy was used to investigate the fluctuation range of the physiologically abundant urinary N-glycopeptides. The abundance of urinary N-glycopeptides spanned across approximately five orders of magnitude. Subsequently, gender differences in the N-glycosylation levels of urinary proteins were also explored in healthy people. Functional analysis of the N-glycoproteins that exhibited gender differences in abundance was performed. Based on multivariate statistical analysis, 206 differentially expressed proteins (p 4) were identified. In females, we found 175 significantly down-regulated N-glycoproteins and 31 significantly up-regulated N-glycoproteins with respect to males. The expression levels of N-glycopeptides between the two groups suggested a clear gender difference. To investigate the biological processes and functions of these proteins, gene ontology (GO) analysis was performed on the N-glycoproteins/N-glycopeptides differentially expressed between males and females. Metabolic pathway analysis was also carried out based on the kyoto encyclopedia of genes and genomes (KEGG). Differentially expressed N-glycoproteins were mostly associated with platelet degranulation, extracellular region, and ossification. The top three relevant pathways were glycan biosynthesis and metabolism, metabolism of cofactors and vitamins, and lipid metabolism. Overall, sex may be an important factor for urinary N-glycoproteome differences among normal individuals and should be considered in clinical applications. This study provides relevant information regarding the function and mechanisms of the urinary glycoproteome and the screening of clinical biomarkers.

Bullous pemphigoid is an autoimmune blistering disease caused by autoantibodies against components of the cutaneous basement membrane zone. Autoantibodies lead to complement-dependent and -independent inflammation and blistering. Blister fluid is a valuable biologic resource, as it provides insight into both systemic and local microenvironment responses. Here, we utilized liquid chromatography with tandem mass spectrometry to characterize the bullous pemphigoid blister fluid proteome. We then depleted exosomes to better understand the exosomal versus non-exosomal proteome. We identified 339 proteins in the blister fluid of bullous pemphigoid patients. Gene ontology demonstrated enrichment of several key biologic processes including innate immune response, neutrophil degranulation, platelet degranulation, and complement activation. Exosome depletion resulted in a significant decrease in normalized reporter intensities of 192 proteins, consistent with our observation of a large number of exosomal proteins found in the blister fluid. We then compared the bullous pemphigoid blister fluid proteome to prior proteomic datasets in suction blister fluid, snake bites, and thermal burns, identifying 76 proteins unique to bullous pemphigoid. These include major basic protein, eosinophil peroxidase, galectin-10, and the immunoglobulin epsilon heavy constant region, consistent with tissue eosinophilia. We lastly validated several previously reported blister fluid exosomal components. Blister fluid in bullous pemphigoid contains a mixture of numerous biologic processes. While many of these processes are shared with blistering from alternative causes, we have identified several notable features unique to bullous pemphigoid.

Background The aim of this study was to apply proteomic methodology for the analysis of proteome changes in women with polycystic ovary syndrome (PCOS). Material and methods All the participators including 31 PCOS patients and 31 healthy female as controls were recruited, the clinical characteristics data was recorded at the time of recruitment, the laboratory biochemical data was detected. Then, a data-independent acquisition (DIA)-based proteomics method was performed to compare the serum protein changes between PCOS patients and controls. In addition, Western blotting was used to validate the expression of identified proteomic biomarkers. Results There were 80 proteins differentially expressed between PCOS patients and controls significantly, including 54 downregulated and 26 upregulated proteins. Gene ontology and Kyoto Encyclopedia of Genes and Genomes analysis showed that downregulated proteins were enriched in platelet degranulation, cell adhesion, cell activation, blood coagulation, hemostasis, defense response and inflammatory response terms; upregulated proteins were enriched in cofactor catabolic process, hydrogen peroxide catabolic process, antioxidant activity, cellular oxidant detoxification, cellular detoxification, antibiotic catabolic process and hydrogen peroxide metabolic process. Receiver operating characteristic curves analysis showed that the area under curve of Histone H4 (H4), Histone H2A (H2A), Trem-like transcript 1 protein (TLT-1) were all over than 0.9, indicated promising diagnosis values of these proteins. Western blotting results proved that the detected significant proteins, including H4, H2A, TLT-1, Peroxiredoxin-1, Band 3 anion transport protein were all differently expressed in PCOS and control groups significantly. Conclusion These proteomic biomarkers provided the potentiality to help us understand PCOS better, but future studies comparing systemic expression and exact role of these candidate biomarkers in PCOS are essential for confirmation of this hypothesis.

Cheng Zhou,1 Chunlin Song,1 Xiang Huang,1 Shufen Chen,1 Yan Long,2 Shanshui Zeng,2 Hongling Yang,2 Min Jiang2 1Laboratory of Molecular Diagnostics, Southern Medical University Affiliated Maternal & Child Health Hospital of Foshan, Foshan, 528000, People’s Republic of China; 2Department of Laboratory, Guangzhou Women and Children’s Medical Centre, Guangzhou Medical University, Guangzhou, 510623, People’s Republic of ChinaCorrespondence: Hongling Yang; Min JiangDepartment of Laboratory, Guangzhou Women and Children’s Medical Centre, Guangzhou Medical University, No. 9, Jinsui Road, Guangzhou, 510623, People’s Republic of ChinaTel +86-20-38857723; +86-20-38076256Email hlyang62@163.com; jiangmin2011@126.comBackground: Gestational hypertension (GH), a hypertensive disorder of pregnancy (HDP), is a leading cause of maternal and fetal mortality due to the lack of clarity on its exact etiology and clinically feasible prediction models. This study was performed to discover novel biomarkers before 20 weeks gestation and thereby construct an early GH prediction model.Methods: This study was designed based on differentially expressed protein screening followed by clinical validation. In the screening phase, a nested case-controlled study was conducted by plasma proteomic analyses using label-free LC-MS/MS and plasma samples from seven pre-GH cases before 20-week gestation and seven age- and gestational week-matched controls. In the validation phase, 10 proteins with differential expression in the screening phase were validated by ELISA or electrochemiluminescence in an independent study consisting of 29 pre-GH cases before 20-week gestation and 29 matched controls.Results: In the screening phase, 149 proteins were found to be differentially expressed between the two groups and were predominantly involved in complement and coagulation cascades, platelet degranulation and positive regulation of cell motility. Further validation showed that serpin family C member 1 (SERPINC1), serpin family A member 5 (SERPINA5), complement factor H-related protein 5 (CFHR5), clusterin, cytokeratin 18 (CK18) and histidine-rich glycoprotein (HRG) levels were significantly higher in women who later developed GH compared to women with uncomplicated pregnancies (P< 0.05). Binary logistic regression analysis was used to determine the combination efficacy of models for early prediction of GH. The model with a combination of SERPINC1, CK18 and HRG had a significantly better discriminatory power (AUC = 0.91, 95% CI 0.83– 0.98) compared to the models with those proteins alone as independent predictors of GH.Conclusion: Plasma levels of SERPINC1, SERPINA5, CFHR5, clusterin, CK18 and HRG are potential novel predictive biomarkers of GH, and a prediction model using a combination of SERPINC1, CK18 and HRG has good discriminatory performance for GH before 20 weeks gestation.Keywords: label-free LC-MS/MS, gestational hypertension, biomarkers

Objective: Mechanical circulatory support has emerged as lifesaving therapy for patients with advanced heart failure. However, mechanical circulatory support remains limited by a paradoxical coagulopathy accompanied by both thrombosis and bleeding. While mechanisms of mechanical circulatory support thrombosis are increasingly defined, mechanical circulatory support-related bleeding, as related to shear-mediated alteration of platelet function, remains poorly understood. We tested the hypothesis that platelet exposure to elevated shear stress, while a defined prothrombotic activator of platelets, coordinately induces downregulation of key platelet adhesion receptors GPIb-IX-V, α IIb β 3 , and P-selectin, thus decreasing platelet functional responsiveness to physiological stimuli. Approach and Results: Human gel-filtered platelets were exposed to continuous or pulsatile shear stress in vitro. Surface expression of platelet receptors and platelet-derived microparticle generation were quantified by flow cytometry. Shedding of receptor soluble forms were assessed via ELISA, and platelet aggregation was measured by optical aggregometry. We demonstrate that platelet exposure to elevated shear stress led to a downregulation of GPIb and α IIb β 3 receptors on platelets with a progressive increase in the generation of platelet-derived microparticles expressing elevated levels of α IIb β 3 and GPIb on their surface. No shear-mediated shedding of GPIb and β 3 subunit soluble fragments was detected. Soluble P-selectin was extensively shed from platelets, while surface expression of P-selectin on platelets was not significantly altered by shear. Shear-mediated downregulation of GPIb and α IIb β 3 on platelets was associated with an evident decrease of platelet aggregatory response induced by ADP and TRAP 6 (thrombin receptor activating peptide 6). Conclusions: Our data clearly indicate that accumulation of shear stress, consistent with supraphysiologic conditions characterizing device-supported circulation (1) induces adequate platelet degranulation, yet (2) causes downregulation of primary platelet adhesion receptors via ejection of receptor-enriched platelet-derived microparticles, thus mechanistically limiting platelet activation and the aggregatory response.

One of the hallmarks of live cells is the asymmetric distribution of lipids across their plasma membrane. Changes in this asymmetry due to lipid “scrambling” result in phosphatidylserine exposure at the cell surface that is detected by annexin V staining. This alteration is observed during cell death processes such as apoptosis, and during physiological responses such as platelet degranulation and membrane repair. Previous studies have shown that activation of NK cells is accompanied by exposure of phosphatidylserine at the cell surface. While this response was thought to be indicative of ongoing NK cell death, it may also reflect the regulation of NK cell activation in the absence of cell death. Herein, we found that NK cell activation was accompanied by rapid phosphatidylserine exposure to an extent proportional to the degree of NK cell activation. Through enforced expression of a lipid scramblase, we provided evidence that activation-induced lipid scrambling in NK cells is reversible and does not lead to cell death. In contrast, lipid scrambling attenuates NK cell activation. This response was accompanied by reduced cell surface expression of activating receptors such as 2B4, and by loss of binding of Src family protein tyrosine kinases Fyn and Lck to the inner leaflet of the plasma membrane. Hence, lipid scrambling during NK cell activation is, at least in part, a physiological response that reduces the NK cell activation level. This effect is due to the ability of lipid scrambling to alter the distribution of membrane-associated receptors and kinases required for NK cell activation.

Background Topical platelet-rich plasma (PRP) must demonstrate stability to insure biologic activity in aesthetic medicine. Objective The objective of this research was to evaluate the role of platelet homeostasis in a novel PRP topical cosmetic formulation to provide facial appearance improvement. Methods The stability of the topical PRP formulation was evaluated in vitro followed by clinical in vivo testing. The in vitro evaluation examined platelet stability and morphology over a 90-day period within the preservative cosmetic base utilizing ELISA and light microscopy (LM)/scanning electron microscopy (SEM). The in vivo clinical study enrolled 20 subjects in a 120-day double blind split face study to evaluate the effect of 5n7x concentrated PRP compared to 2n3x concentrated PRP on facial photoaging. Cosmetic effect was evaluated by the subject and the dermatologist investigator on a 5-point ordinal scale at baseline, week 8, and week 16. Results 90-day stability for the topical PRP formulation was verified via ELISA and LM/SEM. ELISA showed the PRP was more inactive than control conditions via analyte concentration curves (PDGF-AB, EGF, and P-Selectin). LM/SEM demonstrated the PRP had less aggregation/activation over time within the cosmetic base and that refrigeration is superior to room-temperature storage thus delaying full platelet degranulation. The in vivo clinical study demonstrated parity between 20ml and 60ml PRP in terms of clinical efficacy. Conclusion Platelets remain viable for up to 90 days in a refrigerated cosmetic vehicle with demonstrated topical clinical PRP facial benefits. PRP kits of 20ml and 60ml volumes for topical PRP are equally efficacious. J Drugs Dermatol. 2020;19(12): doi:10.36849/JDD.2020.5495.

We hypothesize that first trimester circulating micro particle (CMP) proteins will define preeclampsia risk while identifying clusters of disease subtypes among cases. We performed a nested case–control analysis among women with and without preeclampsia. Cases diagnosed p p = 0.049), increased protein excretion (p = 0.01), more extreme laboratory derangement (p p = 0.09). We conclude that CMP-associated proteins at 12 weeks’ gestation predict the overall risk of developing early preeclampsia and indicate distinct subtypes of pathophysiology and clinical morbidity.

Background Antiplatelet therapy is the mainstay of secondary prevention of cardiovascular events. Studies suggest that women do not obtain equal therapeutic benefit from antiplatelet therapy compared with men. The link between sex differences in platelet biology and response to antiplatelet therapies is unclear. We therefore investigated the role of sex differences in platelet reactivity in a cohort of outpatients with chest pain, in response to treatment with antiplatelet agents. Methods Platelet reactivity was measured in 382 randomly selected patients participating in the Myocardial Ischemia Detection by Circulating Biomarkers (MYOMARKER) study, an observational cohort study of outpatients suspected of myocardial ischemia. In all patients, blood was collected during diagnostic workup, and platelet reactivity was assessed with a flow cytometry–based platelet activation test that quantifies both platelet degranulation (P‐selectin expression) and platelet aggregation (fibrinogen binding to integrin αIIbβ3) in whole blood. Results Platelet reactivity was higher in women compared with men when activated with protease activating receptor 1–activating peptide SFLLRN (PAR1‐AP) and adenosine 5′‐phosphate (ADP), independent of age, basal activation status, estimated glomerular filtration rate

Study of changes in the proteome of extracellular body fluids under conditions of simulated weightlessness of medium duration (21 days) remains relevant for clarifying the physiological mechanisms of homeostasis regulation and is important for gravitational physiology and medicine. Plasma samples of 8 healthy volunteers participating in head down bed rest (HDBR –6°) experiment were studied using semi-quantitative proteomics methods. Each volunteer participated both in the control session and in the session with physical training for negative changes prevention. By the end of HDBR, significant changes in the concentration of proteins involved in platelet degranulation, blood coagulation, fibrinolysis, proteolysis regulation, complement activation, and the immune response were detected in both sessions. The following changes in biochemical parameters of carbohydrate metabolism and regulation of circulating blood volume were shown: a significant increase in renin concentration and a tendency to an increase in aldosterone; an increase in fasting insulin concentration and a tendency to an increase in the insulin resistance index. Physical training did not have a significant effect on biochemical parameters, except for the representation of cholesterol fractions, un which a significant decrease in the concentration of high-density lipoprotein cholesterol observed in HDBR became statistically insignificant in session with physical training. However, the protein composition in blood plasma of volunteers in the prophylaxis session changed less compared to HDBR. Physical training resulted in increase in the concentration of proteins involved in the normalization of carbohydrate and lipid metabolism, such as a macrophage stimulating protein and phosphatidylinositol-glycan-specific phospholipase D, which indicates the effectiveness of preventive measures.

Objectives: The analysis of annotated transcripts from genome-wide expression studies may help to understand the pathogenesis of complex diseases, such as systemic sclerosis (SSc). We performed a whole blood (WB) transcriptome analysis on RNA collected in the context of the European PRECISESADS project, aiming at characterising the pathways that differentiate SSc from controls and that are reproducible in geographically diverse populations. Methods: Samples from 162 patients and 252 controls were collected in RNA stabilisers. Cases and controls were divided into a discovery (n=79+163; Southern Europe) and validation cohort (n=83+89; Central-Western Europe). RNA sequencing was performed by an Illumina assay. Functional annotations of Reactome pathways were performed with the Functional Analysis of Individual Microarray Expression (FAIME) algorithm. In parallel, immunophenotyping of 28 circulating cell populations was performed. We tested the presence of differentially expressed genes/pathways and the correlation between absolute cell counts and RNA transcripts/FAIME scores in regression models. Results significant in both populations were considered as replicated. Results: Overall, 15 224 genes and 1277 functional pathways were available; of these, 99 and 225 were significant in both sets. Among replicated pathways, we found a deregulation in type-I interferon, Toll-like receptor cascade, tumour suppressor p53 protein function, platelet degranulation and activation. RNA transcripts or FAIME scores were jointly correlated with cell subtypes with strong geographical differences; neutrophils were the major determinant of gene expression in SSc-WB samples. Conclusions: We discovered a set of differentially expressed genes/pathways validated in two independent sets of patients with SSc, highlighting a number of deregulated processes that have relevance for the pathogenesis of autoimmunity and SSc., This work was supported by eU/eFPia/innovative Medicines initiative Joint Undertaking PrecisesaDs Grant no. 115 565.

Inherited platelet function disorders are rare hemorrhagic diseases. The gold standard for their exploration is optical aggregometry; however, investigations by flow cytometry (FCM) are being increasingly used. In this review, the physiology of platelets is first recalled, setting the stage for the compartments of platelets that can be apprehended by specific and appropriate labeling. As this requires some pre-analytical precautions and specific analytical settings, a second part focuses on these characteristic aspects, based on literature and on the authors' experience in the field, for qualitative or quantitative explorations. Membrane labeling with antibodies to CD42a or CD41, respectively, useful to assess the genetic-related defects of Glanzmann thrombocytopenia and Bernard Soulier syndrome are then described. Platelet degranulation disorders are detailed in the next section, as they can be explored, upon platelet activation, by measuring the expression of surface P-Selectin (CD62P) or CD63. Mepacrin uptake and release after activation is another test allowing to explore the function of dense granules. Finally, the flip-flop anomaly related to Scott syndrome is depicted. Tables summarizing possible FCM assays, and characteristic histograms are provided as reference for flow laboratories interested in developing platelet exploration.

The use of mass spectrometry for protein identification and quantification in cerebrospinal fluid (CSF) is at the forefront of research efforts to identify and explore biomarkers for the early diagnosis and prognosis of neurologic disorders. Here we implemented a 4-plex N,N-dimethyl leucine (DiLeu) isobaric labeling strategy in a longitudinal study aiming to investigate protein dynamics in children with B-cell acute lymphoblastic leukemia (B-cell ALL) undergoing chemotherapy. The temporal profile of CSF proteome during chemotherapy treatment at weeks 5, 10-14, and 24-28 highlighted many differentially expressed proteins, such as neural cell adhesion molecule, neuronal growth regulator 1, and secretogranin-3, all of which play important roles in neurodegenerative diseases. A total of 63 proteins were significantly altered across all of the time points investigated. The most over-represented biological processes from gene ontology analysis included platelet degranulation, complement activation, cell adhesion, fibrinolysis, neuron projection, regeneration, and regulation of neuron death. We expect that results from this and future studies will provide a means to monitor neurotoxicity and develop strategies to prevent central nervous system injury in response to chemotherapy in children.

Supplemental Digital Content is available in the text., Despite growing use of mechanical circulatory support, limitations remain related to hemocompatibility. Here, we performed a head-to-head comparison of the hemocompatibility of a centrifugal cardiac assist system—the Centrimag, with that of the latest generation of an intravascular microaxial system—the Impella 5.5. Specifically, hemolysis, platelet activation, microparticle (MP) generation, and von Willebrand factor (vWF) degradation were evaluated for both devices. Freshly obtained porcine blood was recirculated within device propelled mock loops for 4 hours, and alteration of the hemocompatibility parameters was monitored over time. We found that the Impella 5.5 and Centrimag exhibited low levels of hemolysis, as indicated by minor increase in plasma free hemoglobin. Both devices did not induce platelet degranulation, as no alteration of β-thromboglobulin and P-selectin in plasma occurred, rather minor downregulation of platelet surface P-selectin was detected. Furthermore, blood exposure to shear stress via both Centrimag and Impella 5.5 resulted in a minor decrease of platelet count with associated ejection of procoagulant MPs, and a decrease of vWF functional activity (but not plasma level of vWF-antigen). Greater MP generation was observed with the Centrimag relative to the Impella 5.5. Thus, the Impella 5.5 despite having a lower profile and higher impeller rotational speed demonstrated good and equivalent hemocompatibility, in comparison with the predicate Centrimag, with the advantage of lower generation of MPs.

Intimal injury is an early stage of several cardiovascular diseases. Endothelial progenitor cells (EPCs) play a significant role in endothelial repair following vascular injury. Once the intima is damaged, EPCs are mobilized from the bone marrow to the injury site. Meanwhile, the injury to the intimal surface triggers platelet degranulation, aggregation, and adhesion to the damaged endothelium, and exposed collagen stimulates platelet to secrete platelet-derived microvesicles (PMVs). However, the role of PMVs in EPC function during this process remains unknown. In an in vivo study, EPCs and platelets were found to adhere to the injury site in Sprague-Dawley (SD) rat vascular injury model. In vitro, collagen stimulation induced the release of PMVs, and collagen-activated PMVs (ac.PMVs) significantly promoted EPC proliferation. Transforming growth factor-β1 (TGF-β1) content was increased in ac.PMVs. Activated PMVs significantly upregulated Smad3 phosphorylation in EPCs and increased Smad3 nuclear translocation from the cytoplasm. TGF-β1 knockdown ac.PMVs downregulated EPC proliferation. Recombinant TGF-β1 enhanced EPC proliferation. The TGF-β1 inhibitor SB431542 significantly repressed the intracellular signal triggered by ac.PMVs. Furthermore, the Smad3-specific phosphorylation inhibitor SIS3 effectively reversed the cell proliferation induced by ac.PMVs. Smad3 translocated to the nucleus and enhanced EPC proliferation via its downstream genes tenascin C (TNC), CDKN1A, and CDKN2A. r-TGF-β1 promoted reendothelialization and EPC proliferation in vivo. Our data demonstrate that activated PMVs deliver TGF-β1 from collagen-activated platelets to EPCs, which in turn activates Smad3 phosphorylation and regulates TNC, CDKN1A, and CDKN2A expression to promote EPC proliferation, suggesting that PMVs act as a key transporter and a potential therapeutic target for vascular injury.

The aim of the study was to monitor and analyse injured and diseased loggerhead sea turtles (Caretta caretta) plasma proteome profiles and biochemistry parameters during their recovery period in rescue centre within different age and recovery period groups, and determine the potential biomarkers that can be used in diagnostics. The plasma biochemical parameters of total protein and glucose content, accompanied by aspartate aminotransferase (AST) and N-acetyl-cystein-activated creatinine kinase (CK-NAC) are highlighted as valuable and potential biomarkers of turtle's health status and condition. Using high throughput tandem mass tag (TMT)-based proteomic approach we identified 913 plasma proteins, 12 of which shown to be modulated in loggerheads age groups, and identified as a part of (i) platelet degranulation, (ii) neutrophil degranulation, and (iii) innate immune system pathways. The neurofascin (NFASC) is shown to be differentially abundant among all the age groups, and alpha-1-acid glycoprotein 2-like (ORM2) and alpha-1-antitrypsin-like (SERPINA1) proteins were recognized as members of all three above mentioned REACTOME pathways. Furthermore, 29 of plasma proteins were significantly differentially abundant in loggerheads age and recovery period groups. Out of 15 recognized pathways, those proteins were mostly included in three specific REACTOME pathways: (i) post-translational phosphorylation, (ii) regulation of Insulin-like Growth Factor (IGF) transport and uptake by Insulin-like Growth Factor Binding Proteins (IGFBPs), and (iii) platelet degranulation. The alpha-fetoprotein (AFP) was the only protein which showed statistically significant up-regulation patterns in all loggerhead age groups before release from the rescue centre, and the complement component 3 (C3) protein was the only protein modulated in all recovery period groups. Furthermore, C3 protein takes part in 9; and followed up with apolipoprotein A-I (APOA1) in 7; complement component 4 (C4), complement component 5 (C5) and kininogen-1 (KNG1) in 6 REACTOME pathways. Thereby, those proteins are highlighted and recommended as potential biomarkers of turtle's health status. Data are available via ProteomeXchange with identifier PXD029569. Finally, based on our results, we believe that comprehensive omics approach and routine plasma biochemical analysis, accompanied by proteins of acute phase, acid-base status and immune-response indicator analysis may significantly and reliably improve assessment of captive loggerheads rehabilitation and medication. SIGNIFICANCE: Monitoring and comparison of loggerhead sea turtles (C. caretta) blood plasma biochemistry parameters and plasma proteome profiles in relation to the age, and recovery period pointed out significantly differentially abundant proteins, along with certain biochemical parameter contents as potential biomarkers of turtle's fitness, health status and physiology.

Introduction: Radiation therapy (RT) as a common method for cancer treatment could result in some side effects. The molecular investigation is one of the approaches that could assist in decrypting the molecular mechanisms of this incident. For this aim, protein-protein interaction (PPI) network analysis as a complementary study of the proteome is conducted to explore the RT effect on brain cancer after the early stage of exposure prior to the appearance of the skin lesion. Methods: Cytoscape 3.7.2 and its plug-ins were used to analyze the network of differential expression of proteins (DEPs) in the treatment condition, and the centrality and pathway enrichment was conducted by the use of NetworkAnalyzer and ClueGO+CluePedia. Results: A network of 15 DEPs indicated that 6 nodes were key players in the network stability and SERPINC1 and F5 were from the query proteins. The pathways of post-translational protein phosphorylation, platelet degranulation, and complement and coagulation cascades were the most highlighted ones for the central nodes that could be affected in RT. Conclusion: The central proteins of the network of early-stage treatments could have additional importance in the mechanisms of radiotherapy response prior to skin lesions. Introduced biomarkers can be used for the patients’ follow-up. These candidates are worth precise attention for this type of therapy after approving by validation studies.

Dogs infected with the cardiopulmonary nematode Angiostrongylus vasorum may suffer from respiratory distress and/or bleeding disorders. Descriptions of clinical signs in foxes are rare, despite high prevalence. To evaluate the impact of infection on coagulation and immune response, serum proteins from eight experimentally infected foxes before and after inoculation (day 0, 35, 84, 154) were subjected to differential proteomic analyses based on quantitative data and compared to available data from dogs. The number of proteins with differential abundance compared to the uninfected baseline increased with chronicity of infection. Bone marrow proteoglycan, chitinase 3-like protein 1 and pulmonary surfactant-associated protein B were among the most prominently increased proteins. The abundance of several proteins involved in coagulation was decreased. Enriched pathways obtained from both increased and decreased proteins included, among others, "platelet degranulation " and "haemostasis ", and indicated both activation and suppression of coagulation. Qualitative comparison to dog data suggests some parallel serum proteomic alterations. The comparison, however, also indicates that foxes have a more adequate immunopathological response to A. vasorum infection compared to dogs, facilitating persistent infections in foxes. Our findings imply that foxes may be more tolerant to A. vasorum infection, as compared to dogs, reflecting a longer evolutionary host-parasite adaptation in foxes, which constitute a key wildlife reservoir., Pathogens, 10 (11), ISSN:2076-0817

Synovial hyperplasia is the main cause of chronic rheumatoid arthritis (RA), but the mechanism of synovial hyperplasia is still unclear. Etodolac (ETD) is a selective COX-2 inhibitor for relieving pain and stiffness in RA, but the disease modifying effect is still lack of evidence. Proteomics method was used to study the differential proteome of synovial tissue in collagen induced arthritis (CIA) in rats. With the help of STRING analysis, the upregulated proteins enriched in the cluster of complement and coagulation cascades and platelet degranulation were highlighted, these proteins with fibrogenic factors Lum, CIV, CXI and Tgfbi participated in the synovial inflammation, fibrosis and hyperplasia in CIA. Based on KOG function class analysis, the proteins involved in the events of the central dogma was explored. They might be hyperplasia related proteins for most of them are related to the proliferation of cancer. ETD significantly attenuated synovial inflammation, fibrosis and hyperplasia in CIA rats by downregulating these proteins. Several proteins have not been observed in RA so far, such as Tmsb4x, Pura, Nfic, Ruvbl1, Snrpd3, U2af2, Srrm2, Srsf7, Elavl1, Hnrnph1, Wars, Yars, Bzw2, Mcts1, Eif4b, Ctsh, Lamp1, Dpp7, Ptges3, Cdc37 and Septin9, they might be potentials targets for RA. Blood biochemistry tests showed the safety of 7 months use of ETD on rats. In conclusion, present study displayed a comprehensive mechanism of synovial hyperplasia in CIA rats, on this basis, the clinical value of ETD in the treatment of RA was well confirmed. Supplementary Information The online version contains supplementary material available at 10.1186/s43556-021-00052-1.

Accelerated healing in wild or captive South American sea lions (Otaria flavescens) is a key tool to help minimize infection and complications associated with open wounds, dental disease, and ocular pathology. Platelet-rich plasma (PRP) is an autogenous source for growth factors based on platelet concentration, which can be obtained by centrifuging whole blood collected in sodium citrate anticoagulant. Currently, there are well-defined PRP concentration protocols for humans and most domestic companion animal species. However, there is no clear centrifugation protocol for obtaining PRP in most marine mammal species. This study aimed to optimize the platelet concentration protocol based on whole blood centrifugation using speeds ranging from 500 to 5,000 rpm and times ranging from 3 to 6 min. Blood was drawn from seven adult South American sea lions, placed into 1-ml sodium citrate tubes, and centrifuged following 12 different centrifugation protocols. PRP was designated as the lower third fraction of the centrifuged plasma. Platelet counts were performed using flow cytometry and statistical analysis was carried out to establish a well-defined protocol for efficient PRP production. Transmission electron microscopy (TEM) analysis was performed to evaluate possible platelet degranulation during the different centrifugation protocols and measure platelet areas. Maximum concentration of platelets in PRP was 4.73-fold higher than the number of platelets in equal volume of whole blood, and significant differences in the concentrations obtained were found between the 12 centrifugation protocols evaluated using different speed and time combinations. The best one-step centrifugation protocol resulted from using 900-rpm speed for 3 min. The highest-fold increase was achieved using a two-step centrifugation protocol, which combined the most efficient one-step centrifugation protocol (900 rpm, 3 min) with a second centrifugation using 2,000-rpm speed for 6 min. TEM analysis confirmed that platelets were complete and maintained integrity after the proposed protocol.

Adequate endometrial stromal cell (ESC) decidualization is vital for endometrial health. Given the importance of extracellular vesicles (EVs) in intercellular communication, we investigated how their protein landscape is reprogrammed and dysregulated during decidual response. Small EVs (sEVs) from human ESC-conditioned media at Day-2 and -14 following decidual stimuli were grouped as well- (WD) or poorly decidualized (PD) based on their prolactin secretion and subjected to mass spectrometry-based quantitative proteomics. On Day 2, in PD- versus WD-ESC-sEVs, 17 sEV- proteins were down-regulated (C5, C6; complement/coagulation cascades, and SERPING1, HRG; platelet degranulation and fibrinolysis) and 39 up-regulated (FLNA, COL1A1; focal adhesion, ENO1, PKM; glycolysis/gluconeogenesis, and RAP1B, MSN; leukocyte transendothelial migration). On Day 14, in PD- versus WD-ESC-sEVs, FLNA was down-regulated while 21 proteins were up-regulated involved in complement/coagulation cascades (C3, C6), platelet degranulation (SERPINA4, ITIH4), B-cell receptor signalling and innate immune response (immunoglobulins). Changes from Days 2 to 14 suggested a subsequent response in PD-ESC-sEVs with 89 differentially expressed proteins mostly involved in complement and coagulation cascades (C3, C6, C5), but no change in WD-ESC-sEVs ESC. Poor decidualization was also associated with loss of crucial sEV-proteins for cell adhesion and invasion (ITGA5, PFN1), glycolysis (ALDOA, PGK1) and cytoskeletal reorganization (VCL, RAC1). Overall, this study indicates varied ESC response even prior to decidualization and provides insight into sEVs-proteomes as a benchmark of well-decidualized ESC. It shows distinct variation in sEV-protein composition depending on the ESC decidual response that is critical for embryo implantation, enabling and limiting trophoblast invasion during placentation and sensing a healthy embryo.

Objective. To explore the therapeutic targets, network modules, and coexpressed genes of Radix Rhei Et Rhizome intervention in cerebral infarction (CI), and to predict significant biological processes and pathways through network pharmacology. To explore the differential proteins of Radix Rhei Et Rhizome intervention in CI, conduct bioinformatics verification, and initially explain the possible therapeutic mechanism of Radix Rhei Et Rhizome intervention in CI through proteomics. Methods. The TCM database was used to predict the potential compounds of Radix Rhei Et Rhizome, and the PharmMapper was used to predict its potential targets. GeneCards and OMIM were used to search for CI-related genes. Cytoscape was used to construct a protein-protein interaction (PPI) network and to screen out core genes and detection network modules. Then, DAVID and Metascape were used for enrichment analysis. After that, in-depth analysis of the proteomics data was carried out to further explore the mechanism of Radix Rhei Et Rhizome intervention in CI. Results. (1) A total of 14 Radix Rhei Et Rhizome potential components and 425 potential targets were obtained. The core components include sennoside A, palmidin A, emodin, toralactone, and so on. The potential targets were combined with 297 CI genes to construct a PPI network. The targets shared by Radix Rhei Et Rhizome and CI include ALB, AKT1, MMP9, IGF1, CASP3, etc. The biological processes that Radix Rhei Et Rhizome may treat CI include platelet degranulation, cell migration, fibrinolysis, platelet activation, hypoxia, angiogenesis, endothelial cell apoptosis, coagulation, and neuronal apoptosis. The signaling pathways include Ras, PI3K-Akt, TNF, FoxO, HIF-1, and Rap1 signaling pathways. (2) Proteomics shows that the top 20 proteins in the differential protein PPI network were Syp, Syn1, Mbp, Gap43, Aif1, Camk2a, Syt1, Calm1, Calb1, Nsf, Nefl, Hspa5, Nefh, Ncam1, Dcx, Unc13a, Mapk1, Syt2, Dnm1, and Cltc. Differential protein enrichment results show that these proteins may be related to synaptic vesicle cycle, vesicle-mediated transport in synapse, presynaptic endocytosis, synaptic vesicle endocytosis, axon guidance, calcium signaling pathway, and so on. Conclusion. This study combined network pharmacology and proteomics to explore the main material basis of Radix Rhei Et Rhizome for the treatment of CI such as sennoside A, palmidin A, emodin, and toralactone. The mechanism may be related to the regulation of biological processes (such as synaptic vesicle cycle, vesicle-mediated transport in synapse, presynaptic endocytosis, and synaptic vesicle endocytosis) and signaling pathways (such as Ras, PI3K-Akt, TNF, FoxO, HIF-1, Rap1, and axon guidance).

ObjectiveIdiopathic short stature (ISS), an endocrine-related disease, is difficult to diagnose. Previous studies have shown that many children with some inflammation-related diseases often have short stature, but whether inflammation is the underlying mechanism of ISS has not been studied. Here, we attempt to explore the role of inflammation in the occurrence and development of ISS and to demonstrate an available clinical diagnostic model of ISS.MethodsFrozen serum samples were collected from ISS patients (n = 4) and control individuals (n = 4). Isobaric tags for relative and absolute quantitation (iTRAQ) combined with LC-MS/MS analysis were applied to quantitative proteomics analysis. To assess clusters of potentially interacting proteins, functional enrichment (GO and KEGG) and protein-protein interaction network analyses were performed, and the crucial proteins were detected by Molecular Complex Detection (MCODE). Furthermore, serum levels of two selected proteins were measured by ELISA between ISS patients (n = 80) and controls (n = 80). In addition, experimentsin vitrowere used to further explore the effects of crucial proteins on endochondral ossification.ResultsA total of 437 proteins were quantified, and 84 DEPs (60 upregulated and 24 downregulated) were identified between patients with ISS and controls. Functional enrichment analysis showed that the DEPs were primarily enriched in blood microparticle, acute inflammatory response, protein activation cascade, collagen-containing extracellular matrix, platelet degranulation, etc. According to the results of top 10 fold change DEPs and MCODE analysis, C1QA and C1QB were selected to further experiment. The expression levels of C1QA and C1QB were validated in serum samples. Based on the logistic regression analysis and ROC curve analysis, we constructed a novel diagnostic model by serum levels of C1QA and C1QB with a specificity of 91.2% and a sensitivity of 75% (AUC = 0.900, p ConclusionOur study is the first to demonstrate the significant role of inflammation in the development of ISS. In addition, we identify C1QA and C1QB as novel serum biomarkers for the diagnosis of ISS.

Bariatric surgery restores glucose tolerance in many, but not all, severely obese subjects with type 2 diabetes (T2D). We aimed to evaluate the plasma protein profiles associated with the T2D remission after obesity surgery. We recruited seventeen women with severe obesity submitted to bariatric procedures, including six non-diabetic patients and eleven patients with T2D. After surgery, diabetes remitted in 7 of the 11 patients with T2D. Plasma protein profiles at baseline and 6 months after bariatric surgery were analyzed by two-dimensional differential gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization-time-of-flight/time-of-flight coupled to mass spectrometry (MALDI-TOF/TOF MS). Remission of T2D following bariatric procedures was associated with changes in alpha-1-antichymotrypsin (SERPINA 3, p &lt, 0.05), alpha-2-macroglobulin (A2M, p &lt, 0.005), ceruloplasmin (CP, p &lt, 0.05), fibrinogen beta chain (FBG, p &lt, 0.05), fibrinogen gamma chain (FGG, p &lt, 0.05), gelsolin (GSN, p &lt, 0.05), prothrombin (F2, p &lt, 0.05), and serum amyloid p-component (APCS, p &lt, 0.05). The resolution of diabetes after bariatric surgery is associated with specific changes in the plasma proteomic profiles of proteins involved in acute-phase response, fibrinolysis, platelet degranulation, and blood coagulation, providing a pathophysiological basis for the study of their potential use as biomarkers of the surgical remission of T2D in a larger series of severely obese patients.

ObjectiveHere, we aimed to identify protein biomarkers that could rapidly and accurately diagnose multiple sclerosis (MS) using a highly sensitive proteomic immunoassay.MethodsTandem mass tag (TMT) quantitative proteomic analysis was performed to determine the differentially expressed proteins (DEPs) in cerebrospinal fluid (CSF) samples collected from 10 patients with MS and 10 non-inflammatory neurological controls (NINCs). The DEPs were analyzed using bioinformatics tools, and the candidate proteins were validated using the ELISA method in another cohort comprising 160 samples (paired CSF and plasma of 40 patients with MS, CSF of 40 NINCs, and plasma of 40 healthy individuals). Receiver operating characteristic (ROC) curves were used to determine the diagnostic potential of this method.ResultsCompared to NINCs, we identified 83 CSF-specific DEPs out of a total of 343 proteins in MS patients. Gene ontology (GO) enrichment analysis revealed that these DEPs are mainly involved in platelet degranulation, negative regulation of proteolysis, and post-translational protein modification. Pathway enrichment analysis revealed that the complement and coagulation cascades, Ras signaling pathway, and PI3K-Akt signaling pathway are the main components. Insulin-like growth factor-binding protein 7 (IGFBP7), insulin-like growth factor 2 (IGF2), and somatostatin (SST) were identified as the potential proteins with high scores, degree, and centrality in the protein-protein interaction (PPI) network. We validated the expression of these three proteins using ELISA. Compared to NINCs, the level of CSF IGFBP7 was significantly upregulated, and the level of CSF SST was significantly downregulated in the MS group.ConclusionOur results suggest that SST and IGFBP7 might be associated with the pathogenesis of MS and would be helpful in diagnosing MS. Since IGFBP7 was used to classify relapsing remitting MS (RRMS) and secondary progressive MS (SPMS) patients, therefore, it may act as a potential key marker and therapeutic target in MS.

Human milk (HM) is the golden standard of infant nutrition that can protect immature body function and enhance nutrition metabolism to ensure infant growth. Region specificity and lactation period could change the protein composition in HM. In this research, proteomics analysis was used to compare proteomes across eight cities, namely Harbin, Lanzhou, Guangzhou, Chengdu, Jinhua, Weihai, Zhengzhou, and Beijing, which represented the northeast, northwest, southeast, southwest, east, and north and central regions of China,. Proteins varied significantly among the cities. These different proteins were mainly involved in the process of platelet degranulation, innate immune response, and triglyceride metabolic process, which might be due to different living environments. These differences also lead to variation in protection and fat metabolism from mothers to infants in different cities. Four proteins were expressed differently during 6 months of lactation, namely Dipeptidyl peptidase 1, Lysozyme C, Carbonic anhydrase 6, and Chordin-like protein 2. The changes in these proteins might be because of the change of growth needs of the infants. The findings from our results might help to improve the understanding of HM as well as to design infant formula.

Hemolysis is a pathological component of many diseases and is associated with thrombosis and vascular dysfunction. Hemolytic products, including cell-free hemoglobin and free heme directly activate platelets. However, the effect of hemolysis on platelet degranulation, a central process in not only thrombosis, but also inflammatory and mitogenic signaling, remains less clear. Our group showed that hemoglobin-induced platelet activation involved the production of mitochondrial reactive oxygen species (mtROS). However, the molecular mechanism by which extracellular hemolysis induces platelet mtROS production, and whether the mtROS regulate platelet degranulation remains unknown. Here, we demonstrate using isolated human platelets that cell free heme is a more potent agonist for platelet activation than hemoglobin, and stimulates the release of a specific set of molecules from the α-granule of platelets, including the glycoprotein thrombospondin-1 (TSP-1). We uncover the mechanism of heme-mediated platelet mtROS production which is dependent on the activation of platelet TLR4 signaling and leads to the downstream phosphorylation of complex-V by the serine kinase Akt. Notably, inhibition of platelet TLR4 or Akt, or scavenging mtROS prevents heme-induced granule release in vitro. Further, heme-dependent granule release is significantly attenuated in vivo in mice lacking TLR4 or those treated with the mtROS scavenger MitoTEMPO. These data elucidate a novel mechanism of TLR4-mediated mitochondrial regulation, establish the mechanistic link between hemolysis and platelet degranulation, and begin to define the heme and mtROS-dependent platelet secretome. These data have implications for hemolysis-induced thrombo-inflammatory signaling and for the consideration of platelet mitochondria as a therapeutic target in hemolytic disorders.Key pointsHeme induces platelet mtROS production by inhibiting complex-V activity via TLR4 signaling.Heme stimulated platelet granule secretion is regulated by mtROS.

Lipoprotein(a) (Lp(a)) is one of the most important risk factors for the development of calcific aortic valve stenosis (CAVS). However, the mechanisms through which Lp(a) causes CAVS are currently unknown. Our objectives were to characterize the Lp(a) proteome and to identify proteins that may be differentially associated with Lp(a) in patients with versus without CAVS. Our second objective was to identify genes that may be differentially regulated by exposure to high versus low Lp(a) levels in explanted aortic valves from patients with CAVS. We isolated Lp(a) from the blood of 21 patients with CAVS and 22 volunteers and performed untargeted label-free analysis of the Lp(a) proteome. We also investigated the transcriptomic signature of calcified aortic valves from patients who underwent aortic valve replacement with high versus low Lp(a) levels (n = 118). Proteins involved in the protein activation cascade, platelet degranulation, leukocyte migration, and response to wounding may be associated with Lp(a) depending on CAVS status. The transcriptomic analysis identified genes involved in cardiac aging, chondrocyte development, and inflammation as potentially influenced by Lp(a). Our multi-omic analyses identified biological pathways through which Lp(a) may cause CAVS, as well as key molecular events that could be triggered by Lp(a) in CAVS development.

ObjectiveDetailed proteomic analysis in a cohort of patients with differing severity of COVID-19 disease identified biomarkers within the complement and coagulation cascades as biomarkers for disease severity has been reported; however, it is unclear if these proteins differ sufficiently from other conditions to be considered as biomarkers.MethodsA prospective, parallel study in T2D (n = 23) and controls (n = 23). A hyperinsulinemic clamp was performed and normoglycemia induced in T2D [4.5 ± 0.07 mmol/L (81 ± 1.2 mg/dl)] for 1-h, following which blood glucose was decreased to ≤2.0 mmol/L (36 mg/dl). Proteomic analysis for the complement and coagulation cascades were measured using Slow Off-rate Modified Aptamer (SOMA)-scan.ResultsThirty-four proteins were measured. At baseline, 4 of 18 were found to differ in T2Dversuscontrols for platelet degranulation [Neutrophil-activating peptide-2 (p = 0.014), Thrombospondin-1 (p = 0.012), Platelet factor-4 (p = 0.007), and Kininogen-1 (p = 0.05)], whilst 3 of 16 proteins differed for complement and coagulation cascades [Coagulation factor IX (p < 0.05), Kininogen-1 (p = 0.05), and Heparin cofactor-2 (p = 0.007)]; STRING analysis demonstrated the close relationship of these proteins to one another. Induced euglycemia in T2D showed no protein changesversusbaseline. At hypoglycemia, however, four proteins changed in controls from baseline [Thrombospondin-1 (p < 0.014), platelet factor-4 (p < 0.01), Platelet basic protein (p < 0.008), and Vitamin K-dependent protein-C (p < 0.00003)], and one protein changed in T2D [Vitamin K-dependent protein-C, (p < 0.0002)].ConclusionSeven of 34 proteins suggested to be biomarkers of COVID-19 severity within the platelet degranulation and complement and coagulation cascades differed in T2Dversuscontrols, with further changes occurring at hypoglycemia, suggesting that validation of these biomarkers is critical. It is unclear if these protein changes in T2D may predict worse COVID-19 disease for these patients.Clinical Trial Registrationhttps://clinicaltrials.gov/, identifier NCT03102801.

Platelet accumulation at the site of vascular injury is regulated by soluble platelet agonists, which induce various types of platelet responses, including integrin activation and granule secretion. The interplay between local biochemical cues, mechanical interactions between platelets and macroscopic thrombus dynamics is poorly understood.Here we describe a novel computational model of microvascular thrombus formation for detailed analysis of thrombus mechanics. Adopting a previously developed two-dimensional particle-based model focused on the thrombus shell formation, we revise it to introduce platelet agonists. Blood flow is simulated via computational fluid dynamics approach. In order to model soluble platelet activators, we apply Langevin dynamics to a large number of non-dimensional virtual particles. Taking advantage of the available data on platelet dense granule secretion kinetics, we model platelet degranulation as a stochastic agonist-dependent process.The new model qualitatively reproduces enhanced thrombus formation due to granule secretion in line with in vivo findings and provides a mechanism for thrombin confinement at the early stages of aggregate formation. Our calculations also predict that release of dense granules results in additional mechanical stabilization of the inner layers of the thrombus. Distribution of the inter-platelet forces throughout the aggregate reveals multiple weak spots in the outer regions of thrombus, which are expected to result in mechanical disruptions at the later stages of thrombus formation.

COVID-19 has emerged as global health threats. Chronic kidney disease (CKD) patients are immune-compromised and may have a high risk of infection by the SARS-CoV-2. We aimed to detect common transcriptomic signatures and pathways between COVID-19 and CKD by systems biology analysis. We analyzed transcriptomic data obtained from peripheral blood mononuclear cells (PBMC) infected with SARS-CoV-2 and PBMC of CKD patients. We identified 49 differentially expressed genes (DEGs) which were common between COVID-19 and CKD. The gene ontology and pathways analysis showed the DEGs were associated with "platelet degranulation", "regulation of wound healing", "platelet activation", "focal adhesion", "regulation of actin cytoskeleton" and "PI3K-Akt signalling pathway". The protein-protein interaction (PPI) network encoded by the common DEGs showed ten hub proteins (EPHB2, PRKAR2B, CAV1, ARHGEF12, HSP90B1, ITGA2B, BCL2L1, E2F1, TUBB1, and C3). Besides, we identified significant transcription factors and microRNAs that may regulate the common DEGs. We investigated protein-drug interaction analysis and identified potential drugs namely, aspirin, estradiol, rapamycin, and nebivolol. The identified common gene signature and pathways between COVID-19 and CKD may be therapeutic targets in COVID-19 patients with CKD comorbidity.

We have shown that overexpression of human alpha-1 antitrypsin (hAAT) in mesenchymal stromal cells (hAAT-MSCs) improves mobility and function of MSCs. In this study we further characterized hAAT MSCs and control MSCs and compared their therapeutic effects in the prevention of type 1 diabetes in the spontaneous non-obese diabetic (NOD) mice. Bone marrow specimens from three individual donors were purchased from StemExpress. Cells were grown to passage 3-4 and infected with hAAT-containing virus for hAAT-overexpression. Single Cell RNAseq was performed by the 10X Genomic System. Proteomic Analysis of exosomes was performed at the MUSC Proteomic Core. MSCs and hAAT-MSCs from one donor were injected into the NOD mice at 8 weeks of age. Mice blood glucose levels were measured twice per week. Cell infiltration into islets was analyzed by H&E staining of pancreatic tissues. Single cell RNAseq analysis showed that hAAT-MSCs and control MSCs have different gene expression signatures, in which hAAT-MSCs showed increased expressions of chemokine (C-X-C motif) ligands (CXCL1, 3, 8), HES5 and others. Exosome proteomic analysis indicates that exosomes from these two types of cells have distinct protein patterns. In hAAT-MSCs cells, platelet degranulation proteins were upregulated and proteins involved in cell-cell adhesion, and cell division were been downregulated. In the NOD mice, a single dose of hAAT-MSCs led to delayed onset of T1D, associated with reduced cell infiltration into the islets. hAAT-MSC therapy showed more profound protection compared to control MSCs. It is likely that overexpression of hAAT enhanced the anti-inflammatory function (via regulating chemokines) promoting the Notch signaling pathway (via regulating HES5) and lead to improved protection in the NOD mice. Disclosure H. Wei: None. W. Gou: None. C. Strange: None. H. Wang: None. Funding National Institutes of Health (1R01DK105183, DK120394, DK118529); U.S. Department of Veterans Affairs (I01BX004536)

We studied morphofunctional properties of human blood cells and diploid culture cells exposed to different types of magnesium materials: pure magnesium (Mg), magnesium-yttrium-neodymium-zirconium alloy (Mg-Y-Nd-Zr) and magnesium-zinc-calcium alloy (Mg-Zn-Ca). The materials were incubated with donor blood and mesenchymal multipotent stromal cells over 3 days. The studied materials did not induce massive lysis of human erythrocytes and leukocytes in vitro, but gradually impaired their structural integrity. In all cases, spontaneous platelet aggregation was observed in 6 h. In the presence of pure Mg and Mg-Zn-Ca alloy, this was accompanied by a decrease in the number of platelets with granules. In 24 h, substantial platelet degranulation occurred in all cases and in 72 h, the platelets did not contain granules. In parallel, the formation of large aggregates (60 μ) was observed. In the culture of stromal cells, all Mg-based materials reduced structural integrity of cells in 24 h, but did not significantly inhibit cell proliferation. Structural integrity of stromal cells partially recovered by day 3 in culture. The studied materials (Mg, Mg-Y-Nd-Zr, and Mg-Zn-Ca) seemed to be low-toxic for human cells during short-term contact, but could stimulate platelet aggregation and spontaneous degranulation and reduced the viability of diploid cells in vitro.