Plasma Serine Protease Inhibitor Research Articles

Quantification of circulating alpha-1-antitrypsin polymers associated with different SERPINA1 genotypes.

Alpha-1-antitrypsin deficiency is a genetic disorder caused by mutations in the SERPINA1 gene encoding alpha-1-antitrypsin (AAT), the major serine protease inhibitor in plasma. Reduced AAT levels are associated with elevated risk of developing emphysema mainly due to uncontrolled activity of neutrophil elastase in the lungs. The prevalent Z-AAT mutant and many rare pathogenic AAT variants also predispose to liver disease due to their accumulation as polymeric chains in hepatocytes. Part of these polymers are secreted into the bloodstream and could represent biomarkers of intra-hepatic accumulation. Moreover, being inactive, they further lower lung protection against proteases. Aim of our study is to accurately quantify the percentage of circulating polymers (CP) in a cohort of subjects with different SERPINA1 genotypes. CP concentration was measured in plasma or Dried Blood Spot (DBS) by a sensitive sandwich ELISA based on capture by the polymer-specific 2C1 monoclonal antibody. CP were significantly elevated in patients with the prevalent PI*SZ and PI*ZZ genotypes, with considerable intra-genotype variability. Notably, higher percentage of polymers was observed in association with elevated C-reactive protein. CP levels were also increased in carriers of the Mmalton variant, and of Mprocida, I, Plowell and Mherleen in heterozygosity with Z-AAT. These findings highlight the importance of implementing CP quantification in a clinical laboratory. Indeed, the variable amount of CP in patients with the same genotype may correlate with the variable severity of the associated lung and liver diseases. Moreover, CP can reveal the polymerogenic potential of newly discovered ultrarare AAT variants.

Relationship of field and in vitro fertility of dairy bulls with sperm parameters, including DAG1 and SERPINA5 proteins

IntroductionSperm interacts with the female reproductive tract and oocyte through proteins, and these cell-to-cell interactions may play a role in sperm fertility. For consideration of a protein as a potential marker of fertility, there must be variability expressed among animals. The proteins dystroglycan (DAG1) and plasma serine protease inhibitor (SERPINA5) have been reported to play a role in cell-to-cell interactions. Thus, the objectives of this study were to characterize the localization and abundance variability of DAG1 and SERPINA5 in bovine sperm, and to investigate the relationship of DAG1 and SERPINA5 with field fertility (i.e., sire conception rate; SCR), in vitro embryo production (IVP), and sperm parameters.Material and methodsDairy bulls (n = 22) were classified as high-SCR (SCR > 1.0) or low-SCR (SCR < –4.0), and good [blastocyst (BL)-by-cleavage (CL) ratio (BL/CL) > 39%] or poor (BL/CL < 38%) BL/CL. Sperm was evaluated for DAG1 and SERPINA5 immunolocalization, and concentration in two separate ejaculates. Variance between bulls compared with within bulls was evaluated using a generalized linear model (GLM) procedure. The relationship of SCR and IVP classification on DAG1 and SERPINA5 concentrations, percentage of tail labeled for SERPINA5, SCR, sperm total and progressive motility, sperm plasma membrane integrity (PMI), CL, BL, and BL/CL were evaluated with the GLIMMIX procedure, and the correlations between these variables were evaluated.ResultsBoth proteins were localized on the sperm head; however, SERPINA5 was also localized on the sperm tail. There was greater variance in concentration among bulls than within bulls for DAG1 (P < 0.0001; 69.4 vs. 49.1, respectively) and SERPINA5 (P < 0.0001; 325.8 vs. 285.4, respectively). There was a positive correlation between the concentrations of DAG1 and of SERPINA5 (P = 0.01; r = 0.54). In addition, the percentage of tail labeled for SERPINA5 was correlated with PMI (P = 0.05; r = 0.44). There was no relationship between SCR and IVP classifications and DAG1 (P ≥ 0.55), SERPINA5 (P ≥ 0.54), or the percentage of sperm tail labeled for SERPINA5 (P ≥ 0.22).DiscussionIn conclusion, DAG1 and SERPINA5 were localized to the sperm head, and SERPINA 5 was also localized to the tail. Concentrations of DAG1 and SERPINA5 on the sperm head were correlated with each other. The percentage of tail labeled for SERPINA5 was correlated with sperm PMI; however, neither protein was associated with SCR or IVP. Thus, when evaluated by immunofluorescent microscopy, DAG1 and SERPINA5 concentrations are variable and are not good fertility markers for bull sperm.

Connecting single-nucleotide polymorphisms, glycosylation status, and interactions of plasma serine protease inhibitors

Understanding the combined impacts of genetic variances and post-translational modifications requires new approaches. Here, we delineate proteoforms of plasma serine protease inhibitors and relate specific proteoforms to their interactions in complexes through the use of native mass spectrometry (MS). First, we dissect the proteoform repertoire of an acute-phase plasma protein, serine protease inhibitor A1 (SERPINA1), resolving four SERPINA1 variants (M1V, M1A, M2, and M3) with common single-nucleotide polymorphisms (SNPs). Investigating the glycosylation status of these variants and their ability to form complexes with a serine protease, elastase, we find that fucosylation stabilizes the interaction of theSERPINA1 M1V variant through its core fucosylation on Asn271. In contrast, antennary fucosylation on Asn271 destabilizes SERPINA1-elastase interactions. We unveil the same opposing effects of core and antennary fucosylation on SERPINA3 interactions with chymotrypsin. Together, our native MS results highlight the modulating effects of fucosylation with different linkages on glycoprotein interactions.

Acute and chronic blood serum proteome changes in patients with methanol poisoning.

Twenty-four blood serum samples from patients with acute methanol poisoning (M) from the mass methanol poisoning outbreak in the Czech Republic in 2012 were compared with 46 patient samples taken four years after poisoning (S) (overlap of 10 people with group M) and with a control group (C) of 24 samples of patients with a similar proportion of chronic alcohol abuse. When comparing any two groups, tens to hundreds of proteins with a significant change in concentration were identified. Fifteen proteins showed significant changes when compared between any two groups. The group with acute methanol poisoning showed significant changes in protein concentrations for at least 64 proteins compared to the other groups. Among the most important identified proteins closely related to intoxication are mainly those involved in blood coagulation, metabolism of vitamin A (increased retinol-binding protein), immune response (e.g., increased complement factor I, complement factors C3 and C5), and lipid transport (increased apolipoprotein A I, apolipoprotein A II, adiponectin). For blood coagulation, the most affected proteins with significant changes in the methanol poisoning group were von Willebrand factor, carboxypeptidase N, alpha-2-antiplasmin (all increased), inter-alpha-trypsin inhibitor heavy chain H4, kininogen-1, plasma serine protease inhibitor, plasminogen (all decreased). However, heparin administration used for the methanol poisoning group could have interfered with some of the changes in their concentrations. Data are available via ProteomeXchange with the identifier PXD035726.

394 Young Scholar Award Talk: Evaluation of new Markers to Predict Sperm Longevity and Bull Fertility

Abstract Bull fertility is currently evaluated through a breeding soundness exam (BSE) that can detect infertile/sterile bulls with high accuracy; however, subfertile bulls are often classified as satisfactory breeders. In the first study, Angus bulls (n=5) were used to evaluate the effect of different insemination doses (10, 20 and 40 million sperm/dose) on pregnancy/fixed-time AI (P/FTAI; cows n=4,866). The interaction between insemination dose and bull was not significant (P=0.53), and there was no effect of insemination dose on P/FTAI (P=0.31); however, there was an effect of bull on P/FTAI (P <0.01). Two bulls had high, one bull had average, and two bulls had low fertility. Post-thaw sperm analysis was performed by computer assisted sperm analysis (CASA) and flow cytometry; however, analysis performed could not explain field fertility differences. In a second study, Angus and Angus crosses bulls were used to investigate differences in sperm longevity between epididymal and ejaculated sperm. Epididymal and ejaculated semen was incubated at pH 5.8, 6.8, or 7.3. Proteins in epididymal fluid, seminal plasma, and proteins loosely attached to epididymal and ejaculated sperm were also investigated. It was identified that epididymal sperm maintained viability longer compared to ejaculated sperm regardless of media pH (P <0.05). Ejaculated sperm had increased longevity (total motility >20%) at pH 6.8 compared to pH 5.8 or 7.3. There were 458 unique proteins between all samples; 311 and 178 in epididymal fluid and seminal plasma, respectively, and 334 and 298 on epididymal and ejaculated sperm, respectively. Based on comparative proteomics, epididymal sperm energy metabolism is more glycolytic compared to ejaculated sperm. In addition, there were greater numbers of antioxidants available for epididymal sperm likely to maintain reactive oxygen species (ROS) at low concentrations to inhibit premature sperm activation. From this dataset, three proteins that are associated with cell-to-cell interaction were studied [plasma serine protease inhibitor (SERPINA5), dystroglycan (DAG1), and CD9]. Among dairy bulls, SERPINA5 and DAG1 were not associated with field fertility; thus, they are not potential fertility markers. The protein CD9 was evaluated to determine whether induction of sperm capacitation (in vitro) could be used to estimate/predict bulls fertility differences. Semen samples from first study were evaluated for total motility (CASA), and functional sperm analysis (flow cytometry) at pre-wash, post-wash, h 0, 3, 6, and 24 of incubation. Sperm analysis were not able to estimate fertility differences between high and average fertility bulls; however, low fertility bulls had decreased (P <0.05) viability, zinc signature 2, and zinc signature1 + 2, and had increased (P <0.05) dead CD9+ sperm. In summary, the use of an insemination dose of 10-40 million sperm can generate acceptable P/FTAI, and evaluation of CD9, viability and zinc signatures may increase the industry’s ability to estimate fertility of bulls that pass a BSE.

PSVI-2 Relationship of Sperm Plasma Serine Protease Inhibitor (Serpina5) and Dystroglycan (DAG1) Proteins with Dairy Bull Fertility

Abstract It has been reported that plasma serine protease inhibitor (SERPINA5) and dystroglycan (DAG1) are loosely attached to the sperm and SERPINA5 can also be present on sperm tails. The objective of this study was to evaluate whether SERPINA5 and/or DAG1 could be used as a fertility marker in dairy bulls. Frozen semen from dairy bulls (n=19) were evaluated for concentration of SERPINA5 and DAG1 and percentage of sperm tail labeled for SERPINA5 (SERPINA5-tail) by immunofluorescence in a minimum of 200 sperm cells per bull in two different ejaculates. Semen was evaluated for total motility, progressive motility, and viability. Semen was used for in vitro embryo production (~150 oocytes/bull). Bull fertility was classified by two methods, sire conception rates [SCR - High-SCR ( >1.0) or Low-SCR (< -4.0)] and the ratio of embryos that developed into a blastocyst (BL) from those that had cleaved (CL) as Good (BL/CL≥38.5%) or Poor (BL/CL< 38.5%). The GLIMMIX procedure of SAS was used to evaluate fertility classification with SCR, BL/CL, and the interaction in the model with bull as a random effect. Progressive motility, CL, SERPINA5 and DAG1 concentration, and SERPINA5-tail did not differ (P≥0.14) among SCR and BL/CL classifications or their interaction. There was no difference (P>0.33) in total motility between High-SCR and Low-SCR or BL/CL classification; however, the interaction was significant (P=0.02; High-SCR/Good 54.9±2.8%, High-SCR/Poor 39.4±5.4%, Low-SCR/Good 44.8±4.5%, Low-SCR/Poor 52.03±3.2%). High-SCR bulls had decreased BL (P=0.03; 30.6±1.6%, 35.8±1.4%, respectively) and tended to have decreased BL/CL ratio (P=0.10; 38.1±2.0%, 42.9±1.8%, respectively) compared with Low-SCR bulls. In summary, DAG1 and SERPINA5 do not seem to be a putative fertility marker. In vitro embryo production is not a good predictor of SCR, since High-SCR and Low-SCR bulls may have Good or Poor in vitro embryo developments.

ANXA2, SP17, SERPINA5, PRDX2 genes, and sperm DNA fragmentation differentially represented in male partners of infertile couples with normal and abnormal sperm parameters.

This study aims to evaluate the expression of genes associated with the fertilisation potential and embryo development, sperm DNA fragmentation (SDF), and acrosome reaction in male partners of infertile couples with different sperm parameters compared to fertile men. First, male partners of infertile couples with abnormal (N= 25) and normal sperm parameters (N= 25), and fertile men (N= 10) were included in experimental groups I, II, and controls respectively. The mRNA levels of the Annexin A2 (ANXA2), Sperm protein 17 (SP17), Plasma serine protease inhibitor (SERPINA5), and Peroxiredoxin-2 (PRDX2) genes and SDF were evaluated. To evaluate the maturity of the sperm and oxidative stress, the acrosome reaction, the lipid peroxidation, and total antioxidant were measured. As result, SP17 showed a significantly lower expression in both experimental groups. SERPINA5 was significantly down-regulated in experimental group I that was aligned with the low rate of acrosome reaction. Significant overexpression of PRDX2 was found between experimental group II and controls. Significant higher rates of SDF were seen in both experimental groups compared to the controls. Finally, our data suggest that differentially gene expression of SP17 is a potential diagnostic biomarker in infertile men either with normal or abnormal sperm parameters. SDF is one of the causes of male infertility, independent of the sperm parameters.

Plasma proteome changes linked to late phase response after inhaled allergen challenge in asthmatics

BackgroundA subset of individuals with allergic asthma develops a late phase response (LPR) to inhaled allergens, which is characterized by a prolonged airway obstruction, airway inflammation and airway hyperresponsiveness. The aim of this study was to identify changes in the plasma proteome and circulating hematopoietic progenitor cells associated with the LPR following inhaled allergen challenge.MethodsSerial plasma samples from asthmatics undergoing inhaled allergen challenge were analyzed by mass spectrometry and immunosorbent assays. Peripheral blood mononuclear cells were analyzed by flow cytometry. Mass spectrometry data were analyzed using a linear regression to model the relationship between airway obstruction during the LPR and plasma proteome changes. Data from immunosorbent assays were analyzed using linear mixed models.ResultsOut of 396 proteins quantified in plasma, 150 showed a statistically significant change 23 h post allergen challenge. Among the most upregulated proteins were three protease inhibitors: alpha-1-antitrypsin, alpha-1-antichymotrypsin and plasma serine protease inhibitor. Altered levels of 13 proteins were associated with the LPR, including increased factor XIII A and decreased von Willebrand factor. No relationship was found between the LPR and changes in the proportions of classical, intermediate, and non-classical monocytes.ConclusionsAllergic reactions to inhaled allergens in asthmatic subjects were associated with changes in a large proportion of the measured plasma proteome, whereof protease inhibitors showed the largest changes, likely to influence the inflammatory response. Many of the proteins altered in relation to the LPR are associated with coagulation, highlighting potential mechanistic targets for future treatments of type-2 asthma.

Blood Plasma Proteins Associated With Heart Rate Variability in Cosmonauts Who Have Completed Long-Duration Space Missions.

The study presents the results of evaluating the changes in the concentrations of blood plasma proteins associated with heart rate variability (HRV) in cosmonauts who have completed space missions lasting about 6months. The concentrations of 125 proteins were quantified in biological samples of the cosmonauts’ blood plasma. The subgroups of proteins associated with the physiological processes of the HRV autonomic regulation were identified using bioinformatic resources (Immunoglobulin heavy constant mu, Complement C1q subcomponent subunit C, Plasma serine protease inhibitor, Protein-72kDa type IV collagenase, Fibulin-1, Immunoglobulin lambda constant 3). The concentration of these proteins in the blood plasma before the flight, and the dynamics of concentration changes on the 1st and 7th days of the post-flight rehabilitation period differed in the groups of cosmonauts with a predominance of sympathetic or parasympathetic modulating autonomous influences. The dynamics of changes in the concentrations of the identified set of proteins reveal that in cosmonauts with a predominance of sympathetic modulating influences, the mechanisms of autonomic regulation are exposed to significant stress in the recovery period immediately after the completion of the space mission, compared with the cosmonauts with a predominance of parasympathetic modulating influences.

D-Galactose treatment increases ACE2, TMPRSS2, and FURIN and reduces SERPINA1 mRNA expression in A549 human lung epithelial cells.

Several comorbidities including diabetes, immune deficiency, and chronic respiratory disorders increase the risk of severe Covid-19 and fatalities among SARS-CoV-2 infected individuals. Severe Covid-19 risk among diabetes patients may reflect reduced immune response to viral infections. SARS-CoV-2 initially infects respiratory tract epithelial cells by binding to the host cell membrane ACE2, followed by proteolytic priming for cell entry by the host cell membrane serine protease TMPRSS2. Additionally, the protease FURIN facilitates cell exit of mature SARS-CoV-2 virions. Alpha-1 antitrypsin (AAT), the major plasma serine protease inhibitor, encoded by SERPINA1, is known to promote immune response to viral infections. AAT inhibits neutrophil elastase, a key inflammatory serine protease implicated in alveolar cell damage during respiratory infections, and AAT deficiency is associated with susceptibility to lung infections. AAT is implicated in Covid-19 as it inhibits TMPRSS2, a protease essential for SARS-CoV-2 cell entry. Here we show that treatment of A549 human lung epithelial cells for 7 days with 25 mM d-galactose, an inducer of diabetic-like and oxidative stress cellular phenotypes, leads to increased mRNA levels of ACE2, TMPRSS2, and FURIN, along with reduced SERPINA1 mRNA. Together, the dysregulated transcription of these genes following d-galactose treatment suggests that chronic diabetic-like conditions may facilitate SARS-CoV-2 infection of lung epithelial cells. Our findings may in part explain the higher severe Covid-19 risk in diabetes, and highlight the need to develop special treatment protocols for diabetic patients.

CSF Chitinase 3-Like 2 Is Associated With Long-term Disability Progression in Patients With Progressive Multiple Sclerosis.

ObjectiveThis study aimed to identify long-term prognostic protein biomarkers associated with disease progression in patients with progressive multiple sclerosis (MS).MethodsCSF samples were collected from a discovery cohort of 28 patients with progressive MS who participated in a clinical trial with interferon beta. Patients were classified into high and low disability progression phenotypes according to numeric progression rates (NPR) and step-based progression rates (SPR) after a mean follow-up time of 12 years. Protein abundance was measured by shotgun proteomics. Selected proteins from the discovery cohort were quantified by parallel reaction monitoring in CSF samples from an independent validation cohort of 41 patients with progressive MS classified also into high and low disability progression phenotypes after a mean follow-up time of 7 years.ResultsOf 2,548 CSF proteins identified in the discovery cohort, 10 were selected for validation based on their association with long-term disability progression: SPATS2-like protein, chitinase 3–like 2 (CHI3L2), plasma serine protease inhibitor, metallothionein-3, phospholipase D4, beta-hexosaminidase, neurexophilin-1, adipocyte enhancer-binding protein 1, cathepsin L1, and lipopolysaccharide-binding protein. Only CHI3L2 was validated, and patients with high disability progression exhibited significantly higher CSF protein levels compared with patients with low disability progression (p = 0.03 for NPR and p = 0.02 for SPR). CHI3L2 levels showed good performance to discriminate between high and low disability progression in patients with progressive MS (area under the curve 0.73; sensitivity 90% and specificity 63%).ConclusionsAlthough further confirmatory studies are needed, we propose CSF CHI3L2 as a prognostic protein biomarker associated with long-term disability progression in patients with progressive MS.Classification of EvidenceThis study provides Class II evidence that high CSF CHI3L2 levels identified higher disability progression in patients with progressive MS.

Alpha 2-antiplasmin deficiency in a Sudanese child: a case report

BackgroundThe plasma serine protease inhibitor alpha 2-antiplasmin (α2-AP, otherwise known as α2-plasmin inhibitor) is a rapid-acting plasmin inhibitor recently found in human plasma, which seems to have a significant role in the regulation of in vivo fibrinolysis. Congenital deficiency of α2-AP is extremely uncommon.Case presentationWe report here a case of absolute deficiency of α2-AP in an 11-year-old Sudanese boy, who had a lifelong intermittent hemorrhagic tendency (gum bleeding, epistaxis, and exaggerated bleeding after trauma). Coagulation tests including prothrombin time, partial thromboplastin time, thrombin time, bleeding time, platelet count, clot retraction test, antithrombin, and factor VIII levels were within normal limits. Hepatic function tests and complete blood count were also normal. The main interesting finding in this patient was that the whole blood clot lysis was extremely fast, completed within 5–8 hours. The second abnormal finding is that the euglobulin clot lysis time was short. Nevertheless, the concentration of α2-AP in the patient's plasma was 0.2 IU/ml (reference range is 0.80–1.20 IU/ml). The addition of pooled plasma (with normal α2-AP) to the patient's whole blood corrected the accelerated fibrinolysis.ConclusionThe study showed that α2-AP deficiency resulted in uninhibited fibrinolysis that caused the hemorrhagic tendency in this patient. Thus, this report demonstrates the significant role of α2-AP in coagulation.

Therapeutic SERPINs: Improving on Nature.

Serine proteases drive important physiological processes such as coagulation, fibrinolysis, inflammation and angiogenesis. These proteases are controlled by serine protease inhibitors (SERPINs) that neutralize their activity. Currently, over 1,500 SERPINs are known in nature, but only 37 SERPINs are found in humans. Thirty of these are functional protease inhibitors. The inhibitory potential of SERPINs is in perfect balance with the proteolytic activities of its targets to enable physiological protease activity. Hence, SERPIN deficiency (either qualitative or quantitative) can lead to disease. Several SERPIN resupplementation strategies have been developed to treat SERPIN deficiencies, including concentrates derived from plasma and recombinant SERPINs. SERPINs usually inhibit multiple proteases, but only in their active state. Over the past decades, considerable insights have been acquired in the identification of SERPIN biological functions, their inhibitory mechanisms and specificity determinants. This paves the way for the development of therapeutic SERPINs. Through rational design, the inhibitory properties (selectivity and inhibitory potential) of SERPINs can be reformed and optimized. This review explores the current state of SERPIN engineering with a focus on reactive center loop modifications and backbone stabilization. We will discuss the lessons learned from these recombinant SERPINs and explore novel techniques and strategies that will be essential for the creation and application of the future generation of therapeutic SERPINs.

Ethnic differences in alpha-1 antitrypsin deficiency allele frequencies may partially explain national differences in COVID-19 fatality rates.

Infection rates, severity, and fatalities due to COVID‐19, the pandemic mediated by SARS‐CoV‐2, vary greatly between countries. With few exceptions, these are lower in East and Southeast Asian and Sub‐Saharan African countries compared with other regions. Epidemiological differences may reflect differences in border closures, lockdowns, and social distancing measures taken by each county, and by cultural differences, such as common use of face masks in East and Southeast Asian countries. The plasma serine protease inhibitor alpha‐1 antitrypsin was suggested to protect from COVID‐19 by inhibiting TMPRSS2, a cell surface serine protease essential for the SARS‐CoV‐2 cell entry. Here, we present evidence that population differences in alpha‐1 antitrypsin deficiency allele frequencies may partially explain national differences in the COVID‐19 epidemiology. Our study compared reported national estimates for the major alpha‐1 antitrypsin deficiency alleles PiZ and PiS (SERPINA1 rs28929474 and rs17580, respectively) with the Johns Hopkins University Coronavirus Resource Center dataset. We found a significant positive correlation (R = .54, P = 1.98e−6) between the combined frequencies of the alpha‐1 antitrypsin PiZ and PiS deficiency alleles in 67 countries and their reported COVID‐19 mortality rates. Our observations suggest that alpha‐1 antitrypsin deficiency alleles may contribute to national differences in COVID‐19 infection, severity, and mortality rates. Population‐wide screening for carriers of alpha‐1 antitrypsin deficiency alleles should be considered for prioritizing individuals for stricter social distancing measures and for receiving a SARS‐CoV‐2 vaccine once it becomes available.

Plasma proteome atlas for differentiating tumor stage and post-surgical prognosis of hepatocellular carcinoma and cholangiocarcinoma.

Although mass spectrometry-based plasma proteomics enables sensitive and large-scale discovery and validation of biomarkers for various diseases, its integrative application to hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA) is not well investigated. Therefore, we analyzed albumin- and immunoglobulin G-depleted plasma samples from 148 and 60 patients with HCC and CCA, respectively, using liquid chromatography-tandem mass spectrometry. The algorithm used to measure the content of each protein was the percentage of exponentially modified protein abundance index. From 5320 proteins assayed in plasma, 53 and 25 biomarker candidates were identified for HCC and CCA, respectively. The abundance of six and two HCC markers particularly protruded in stage II and III, respectively, whereas plasma serine protease inhibitor was the sole marker the level of which steadily decreased with CCA progression. From a prognostic facet, we showed candidate markers and their cutoff levels for evaluating probability of tumor recurrence and patient survival period. Combination Kaplan-Meier models showed that HCC stage III or IV and both the content of alpha-2-HS-glycoprotein and apolipoprotein CIII <0.2% exhibited the poorest post-surgical recurrence-free and overall survivals. Furthermore, the content of afamin ≥0.2% played a significant role on the poor prognosis in patients with CCA. Our findings, taken together, characterized novel plasma biomarker signatures in dissecting tumor stages and post-surgical outcomes of HCC and CCA.

Novel Associations between Related Proteins and Cellular Effects of High-Density Lipoprotein.

Background and ObjectivesRecent studies have examined the structure-function relationship of high-density lipoprotein (HDL). This study aimed to identify and rank HDL-associated proteins involved in several biological function of HDL.MethodsHDLs isolated from 48 participants were analyzed. Cholesterol efflux capacity, effect of HDL on nitric oxide production, and vascular cell adhesion molecule-1 expression were assessed. The relative abundance of identified proteins in the highest vs. lowest quartile was expressed using the normalized spectral abundance factor ratio.ResultsAfter adjustment by multiple testing, six proteins, thyroxine-binding globulin, alpha-1B-glycoprotein, plasma serine protease inhibitor, vitronectin, angiotensinogen, and serum amyloid A-4, were more abundant (relative abundance ratio ≥2) in HDLs with the highest cholesterol efflux capacity. In contrast, three proteins, complement C4-A, alpha-2-macroglobulin, and immunoglobulin mu chain C region, were less abundant (relative abundance ratio <0.5). In terms of nitric oxide production and vascular cell adhesion molecule-1 expression, no proteins showed abundance ratios ≥2 or <0.5 after adjustment. Proteins correlated with the functional parameters of HDL belonged to diverse biological categories.ConclusionsIn summary, this study ranked proteins showing higher or lower abundance in HDLs with high functional capacities and newly identified multiple proteins linked to cholesterol efflux capacity.

Global serum proteomic changes in water buffaloes infected with Fasciola gigantica

BackgroundThe liver fluke Fasciola gigantica modulates several signaling pathways in infected buffaloes to facilitate its survival and establishment of persistent infection. In response to the parasite invasion, buffaloes activate innate and adaptive immune responses to counter the parasite infection. To detect new proteins that might be involved in the interaction between F. gigantica and the buffaloes, and that also might serve as biomarkers for fasciolosis, we used proteomic techniques to study the serum proteome of buffaloes during F. gigantica infection. Here, we used an isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomic approach to identify serum proteins that are differentially expressed in infected buffaloes compared to uninfected control buffaloes. Additionally, we applied a parallel reaction monitoring (PRM) assay to validate specific proteins identified by the iTRAQ method.ResultsA total of 313, 459 and 399 proteins were identified at 3, 42 and 70 days post-infection, respectively; of these 92, 93 and 138 were differentially abundant proteins. Some of the identified differentially abundant proteins, including complement factor H related 5, complement component C6, complement component C7, amine oxidase, plasma serine protease inhibitor and lysozyme, are known to be involved in complement system activation, blood coagulation, platelet activation, lymphocyte’s adhesion and lysozyme hydrolysis. Analysis of data for all three time points after infection identified six significantly upregulated proteins in infected serum that separated infected and uninfected buffaloes into distinct clusters. Further PRM analysis confirmed the expression of five proteins, namely MHC class I antigen, Beta-2-microglobulin, NID2 protein, Fetuin-B and Fibrinogen gamma-B chain.ConclusionsThese findings provide novel insights into the serum proteomics signature of buffaloes during F. gigantica infection.

Assessment of in vitro effects of direct thrombin inhibitors and activated factor X inhibitors through clot waveform analysis

AimsClot waveform analysis (CWA) has been reported to extend the interpretation of clotting time measurement. The parameters obtained from successive derivatives of the clotting reaction curves reflect the rates of...

Efficacy of Anti-TFPI Antibody PF-06741086 Alone and in Combination with Activated Prothrombin Complex Concentrates in Mouse Hemophilia a Bleeding Model

BACKGROUND: Tissue Factor Pathway inhibitor (TFPI) is a plasma serine protease inhibitor that modulates the initiation of coagulation by directly binding and inhibiting the Tissue Factor (TF)/Factor VIIa/Factor Xa complex. TFPI is a multi-Kunitz domain protein that directly binds to and inhibits both activated Factor Xa (FXa) and FVIIa. Blocking TFPI can act as a bypass therapy by facilitating hemostasis initiated by tissue factor/FVIIa, thereby, compensating for loss of Factor VIII or Factor IX (in hemophilia A or B). PF-06741086, a fully human antibody engineered to inhibit TFPI, exhibits broad cross reactivity to TFPI from numerous species, including mouse. PF-06741086 is being developed as a potential treatment for bleeding disorders including hemophilia A and hemophilia B with and without inhibitors. aPCC (activated Prothrombin complex concentrates or FEIBA, Factor Eight Inhibitor Bypass Agent) is a bypass agent for to control bleed in Hemophilia patients with inhibitors. Since it is a plasma-derived concentrate containing various prothrombin complex coagulation factors in their enzymatic or zymogen form, it is possible that FEIBA could potentially impact the activity of PF-06741086.AIMS: Here, we directly compare the hemostatic effect of PF-06741086 alone, and in combination with aPCC in Hemophilia A mouse model using severe tail vein transection.METHODS: Male hemophilia A mice were dosed with a single intravenous dose of PF-06741086 (0.5, 1, 2 or 6 mg/kg) 30 minutes prior to a 3mm tail clip, or aPCC (50, 100 or 200 U/kg) was administered 5 minutes before the tail clip. Mice were also treated with a combined dose of 0.5 mg/kg anti-TFPI PF-06741086 and aPCC at 50, 100 or 200 U/kg. Blood was collected for 10 minutes and quantified against a standard curve of hemoglobin as volume blood loss.RESULTS: PF-06741086 demonstrated a dose dependent response in improving hemostasis in Hemophilia A mice after tail clip. PF-06741086 was able to restore hemostasis at 1 mg/kg (49%), and higher doses further improved hemostasis at 2 mg/kg (63%), and 6 mg/kg (78%). aPCC also demonstrated a dose dependent reduction in blood loss and improved hemostasis with all tested doses of 50 U/kg (25%), 100 U/kg (23%) and 200 U/kg (66%). At a dose of 0.5 mg/kg, PF-06741086 did not show any improvement in hemostasis over vehicle control. We used this dose for all combination studies with aPCC. Combined use of low dose PF-06741086 (0.5 mg/kg) and 100 U/kg aPCC shows a trend towards improvement in hemostasis compared to either drug alone. A higher dose of aPCC (200 U/kg) combined with low dose PF-06741086 (0.5 mg/kg) significantly reduces blood loss (86%) in Hemophilia A mice in tail clip model compared to saline, TFPI or aPCC alone used at the same dose of 0.5 mg/kg or 200 U/kg respectively.CONCLUSIONS: Prophylactic administration of PF-06741086 exhibits a dose response and improves hemostasis in an injury model in Hemophilia A mice. The addition of aPCC alone restores hemostasis at 200 U/kg and this effect was enhanced in combination with PF-06741086 in this mouse model. DisclosuresBarakat:Pfizer: Employment. Jasuja:Pfizer: Employment. Murphy:Pfizer: Employment. Pittman:Pfizer: Employment.

Serum proteomic pattern in female stress urinary incontinence.

The pathophysiology of Stress Urinary Incontinence (SUI) is poorly understood. The aim of this study was to identify the serum proteomic profile in patients with SUI and to replicate findings from a preceding study in which a significant difference in the urinary proteome was identified. Serum samples were collected from 38patients (19 SUI; 19 matched, continent controls). Sample preparation included serum albumin depletion, in-solution enzymatic digestion of proteins applying a combination of Gluc-C and trypsin and peptide separation using nano High Performance Liquid Chromatography. Label-free quantitation of peptides and proteins was performed after triplicate measurements using quadrupole time-of-flight mass spectrometry. Peptide identification was achieved by searching the Human SwissProt Database using Mascot and X!Tandem. Main outcome measure was the relative abundance of each detected protein in serum. Of 7012 identified proteins, 33 proteins were induced (detected in SUI, not in controls) and five proteins were depleted (detected in controls, not in SUI). All depleted proteins play a role in immune/DNA damage response. Induced proteins are involved in inflammatory response, response to cellular stress, coagulation and cytoskeleton stability/ motility. Plasma serine protease inhibitor (SERPINA5) was found induced and previously also showed a higher abundance in urine samples of SUI patients. Data are available via ProteomeXchange with identifier PXD008553.