ABSTRACT
Thromboembolic diseases are a major cause of mortality in human and the currently available anticoagulants are associated with various drawbacks, therefore the search for anticoagulants that have better safety profile is highly desirable. Compounds that are part of the dietary routine can be modified to possibly increase their anticoagulant potential. We show mannose 2,3,4,5,6-O-pentasulfate (MPS) as a synthetically modified form of mannose that has appreciable anticoagulation properties. An in silico study identified that mannose in sulfated form can bind effectively to the heparin-binding site of antithrombin (ATIII) and heparin cofactor II (HCII). Mannose was sulfated using a simple sulfation strategy-involving triethylamine-sulfur trioxide adduct. HCII and ATIII were purified from human plasma and the binding analysis using fluorometer and isothermal calorimetry showed that MPS binds at a unique site. A thrombin inhibition analysis using the chromogenic substrate showed that MPS partially enhances the activity of HCII. Further an assessment of in vitro blood coagulation assays using human plasma showed that the activated partial thromboplastin time (APTT) and prothrombin time (PT) were prolonged in the presence of MPS. A molecular dynamics simulation analysis of the HCII-MPS complex showed fluctuations in a N-terminal loop and the cofactor binding site of HCII. The results indicate that MPS is a promising lead due to its effect on the in vitro coagulation rate.Communicated by Ramaswamy H. Sarma.
Subject(s)
Heparin Cofactor II , Mannose , Humans , Heparin Cofactor II/chemistry , Heparin Cofactor II/metabolism , Mannose/pharmacology , Blood Coagulation , Anticoagulants/pharmacology , Anticoagulants/chemistry , Heparin/pharmacology , Antithrombin III/pharmacology , Antithrombin III/physiology , Antithrombins/pharmacology , Thrombin/chemistryABSTRACT
Objective: Detailed 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. Methods: A 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. Results: Thirty-four proteins were measured. At baseline, 4 of 18 were found to differ in T2D versus controls 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 changes versus baseline. 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)]. Conclusion: Seven of 34 proteins suggested to be biomarkers of COVID-19 severity within the platelet degranulation and complement and coagulation cascades differed in T2D versus controls, 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 Registration: https://clinicaltrials.gov/, identifier NCT03102801.
Subject(s)
Blood Coagulation Factors/metabolism , COVID-19/metabolism , Diabetes Mellitus, Type 2/metabolism , Hypoglycemia/metabolism , Aged , Biomarkers/metabolism , Blood Coagulation , Case-Control Studies , Complement Activation , Factor IX/metabolism , Female , Glucose Clamp Technique , Heparin Cofactor II/metabolism , Humans , Kininogens/metabolism , Male , Middle Aged , Peptides/metabolism , Platelet Activation , Platelet Factor 4/metabolism , Prospective Studies , Protein C/metabolism , Proteomics , SARS-CoV-2 , Severity of Illness Index , Thrombospondin 1/metabolism , beta-Thromboglobulin/metabolismABSTRACT
Polycyclic aromatic hydrocarbons (PAHs) are considered as an external factor that induces atherosclerotic cardiovascular disease. Although miR-155 is known to be involved in cardiovascular disease, whether it is involved in PAH-induced arteriosclerosis remains unclear. We evaluated the effects of PAHs on vascularization, permeability, and miR-155 expression in HUVECs. We found that PAHs-induced sclerosis of HUVECs was characterized by increasing permeability, decreasing proliferation, and vascular lumen number. The expression of miR-155 was upregulated by PAHs treatment, and transfection with miR-155 inhibitor could reverse above effect of PAHs-induced sclerosis. Meanwhile, transcriptome sequencing revealed that 63 genes were downregulated in the group of PAHs treatment alone, and were then upregulated in the miR-155 inhibitor group. These genes were mainly involved in complement and coagulation cascades, cytokine-cytokine receptor interaction, TNF signaling pathway, and NF-kappa B signaling pathway. Among these 63 genes, SERPIND1 was directly targeted and regulated by miR-155. Further in vivo experiments in ApoE-/- mice confirmed that PAH accelerates the development of arteriosclerosis by promoting the expression of miR-155 to downregulate the SERPIND1. Therefore, PAH exaggerates atherosclerosis by activating miR-155-dependent endothelial injury. This study provides a fundamental insight on the miR-155 mechanism for PAHs enhancing atherosclerosis and miR-155 potentially serving as a novel drug target.
Subject(s)
Atherosclerosis/pathology , Gene Expression Regulation/drug effects , Heparin Cofactor II/metabolism , Human Umbilical Vein Endothelial Cells/pathology , MicroRNAs/genetics , Polycyclic Aromatic Hydrocarbons/toxicity , Animals , Atherosclerosis/chemically induced , Atherosclerosis/genetics , Atherosclerosis/metabolism , Heparin Cofactor II/genetics , Human Umbilical Vein Endothelial Cells/drug effects , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Male , Mice , Mice, Knockout, ApoEABSTRACT
Heparin cofactor II (HCII) is predominantly expressed in the liver and inhibits thrombin in blood plasma to influence the blood coagulation cascade. Its deficiency is associated with arterial thrombosis. Its cleavage by neutrophil elastase produces fragment that helps in neutrophil chemotaxis in the acute inflammatory response in human. In the present study, we have identified a novel alternatively spliced transcript of the HCII gene in human liver. This novel transcript includes an additional novel region in continuation with exon 3 called exon 3b. Exon 3b acts like an alternate last exon, and hence its inclusion in the transcript due to alternative splicing removes exon 4 and encodes for a different C-terminal region to give a novel protein, HCII-N. MD simulations of HCII-N and three-dimensional structure showed a unique 51 amino acid sequence at the C-terminal having unique RCL-like structure. The HCII-N protein purified from bacterial culture showed a protein migrating at lower molecular weight (MW 55 kDa) as compared to native HCII (MW 66 kDa). A fluorescence-based analysis revealed a more compact structure of HCII-N that was in a more hydrophilic environment. The HCII-N protein, however, showed no inhibitory activity against thrombin. Due to large conformational variation observed in comparison with native HCII, HCII-N may have alternate protease specificity or a non-inhibitory role. Western blot of HCII purified from large plasma volume showed the presence of a low MW 59 kDa band with no thrombin activity. This study provides the first evidence of alternatively spliced novel isoform of the HCII gene.
Subject(s)
Heparin Cofactor II/chemistry , Heparin Cofactor II/genetics , Heparin Cofactor II/metabolism , Liver/metabolism , Alternative Splicing , Factor Xa/metabolism , Humans , Models, Molecular , Molecular Dynamics Simulation , Protein Isoforms , Spectrometry, Fluorescence , Thrombin/metabolism , Tissue Plasminogen Activator/antagonists & inhibitors , Tissue Plasminogen Activator/metabolismABSTRACT
Tyrosine sulfation is an important post-translational modification found in higher eukaryotes. Here we report an engineered tyrosyl-tRNA synthetase/tRNA pair that co-translationally incorporates O-sulfotyrosine in response to UAG codons in Escherichia coli and mammalian cells. This platform enables recombinant expression of eukaryotic proteins homogeneously sulfated at chosen sites, which was demonstrated by expressing human heparin cofactor II in mammalian cells in different states of sulfation.
Subject(s)
Protein Engineering/methods , Somatomedins/chemistry , Tyrosine/analogs & derivatives , Animals , Codon, Terminator/metabolism , Escherichia coli/metabolism , Genetic Code , Heparin Cofactor II/metabolism , Humans , Mammals , Protein Processing, Post-Translational , Proteins/chemistry , Tyrosine/chemistry , Tyrosine-tRNA Ligase/metabolismABSTRACT
Premature ovarian insufficiency (POI) is a primary ovarian defect characterized by premature depletion of ovarian follicles before 40â¯years of age. The disorder has been attributed to various causes, but the study of altered proteins in serum levels as the cause is rare. Additionally, identifying novel biomarkers can contribute to more accurate diagnosis or prognosis of POI. In the present study, a solid-phase antibody array simultaneously detecting multiple proteins was used to analyze POI serum with menopausal and healthy fertile subjects as control groups. As a result, compared to the menopause and healthy fertile groups, eleven proteins, including Neurturin, Frizzled-5, Serpin D1, MMP-7, ICAM-3, IL-17F, IFN-gamma R1, IL-29, IL-17R, IL-17C and Soggy-1, were uniquely down-regulated, and Afamin was particularly up-regulated in POI serum. More importantly, all of these factors were firstly found to be associated with POI in this study, suggesting that these proteins may participate in the pathogenesis of POI and may be novel serum biomarkers for POI.
Subject(s)
Biomarkers/blood , Menopause, Premature/blood , Primary Ovarian Insufficiency/blood , Adult , Antibodies , Carrier Proteins/blood , Down-Regulation , Estradiol/blood , Female , Frizzled Receptors/blood , Glycoproteins/blood , Heparin Cofactor II/metabolism , Humans , Intercellular Adhesion Molecule-3/blood , Intercellular Signaling Peptides and Proteins/blood , Interferon-gamma/blood , Interferons/blood , Interleukin-17/blood , Interleukins/blood , Matrix Metalloproteinase 7/blood , Middle Aged , Neurturin/blood , Primary Ovarian Insufficiency/immunology , Primary Ovarian Insufficiency/pathology , Protein Array Analysis , Receptors, Interleukin-17/blood , Serum Albumin, Human , Up-RegulationABSTRACT
The glycosaminoglycan dermatan sulfate (DS) is a well-known activator of heparin cofactor II-dependent inactivation of thrombin. In contrast to heparin, dermatan sulfate has never been prepared recombinantly from material of non-animal origin. Here we report on the enzymatic synthesis of structurally well-defined DS with high anticoagulant activity. Using a microbial K4 polysaccharide and the recombinant enzymes DS-epimerase 1, dermatan 4-O-sulfotransferase 1, uronyl 2-O-sulfotransferase and N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase, several new glycostructures have been prepared, such as a homogenously sulfated IdoA-GalNAc-4S polymer and its 2-O-, 6-O- and 2,6-O-sulfated derivatives. Importantly, the recombinant highly 2,4-O-sulfated DS inhibits thrombin via heparin cofactor II, approximately 20 times better than heparin, enabling manipulation of vascular and extravascular coagulation. The potential of this method can be extended to preparation of specific structures that are of importance for binding and activation of cytokines, and control of inflammation and metastasis, involving extravasation and migration.
Subject(s)
Dermatan Sulfate/pharmacology , Heparin Cofactor II/metabolism , Serine Proteinase Inhibitors/pharmacology , Thrombin/antagonists & inhibitors , Carbohydrate Conformation , Dermatan Sulfate/chemical synthesis , Dermatan Sulfate/chemistry , Humans , Models, Molecular , Serine Proteinase Inhibitors/chemical synthesis , Serine Proteinase Inhibitors/chemistry , Thrombin/metabolismABSTRACT
Globally, depression is one of the most serious debilitating psychiatric mental disorders. In this study, we validated the expression levels of fibrinogen alpha (FGA), fibrinogen beta (FGB), fibrinogen gamma (FGG), Complement factor B (CFB) and serpin family D member 1(SERPIND1) in the acute phase response signaling pathway in plasma samples using enzyme-linked immunosorbent assay (ELISA).Then illuminate the roles of FGA, FGB, FGG, CFB, SERPIND1 in depression using microarray data. Gene expression dataset GSE98793 was downloaded from the Gene Expression Omnibus database. There were 128 whole blood samples included 64 patients with major depressed patients and 64 healthy controls. Differentially expressed genes (DEGs) were identified, and then protein-protein interaction (PPI) network was constructed to screen crucial genes associated with FGA, FGB, FGG, CFB and SERPIND1. Moreover, gene ontology (GO) biological processes analyses was performed. The ELISA data showed that the expression levels of FGA, FGB, FGG, CFB and SERPIND1 were up-regulated in depressed patients. The enriched GO terms were predominantly associated with the biological processes including more genes were inflammation related. The PPI network was found these five genes interacted with 11 genes. FGA, FGB, FGG, CFB and SERPIND1 may be important in the pathogenesis of depression.
Subject(s)
Acute-Phase Reaction/blood , Acute-Phase Reaction/diagnosis , Complement Factor B/metabolism , Depression/blood , Depression/diagnosis , Heparin Cofactor II/metabolism , Acute-Phase Reaction/psychology , Adult , Biomarkers/blood , Depression/psychology , Female , Fibrinogen/metabolism , Humans , Male , Middle AgedABSTRACT
Fucosylated chondroitin sulfate (FCS), an unusual glycosaminoglycan with fucose side chains, is a promising anticoagulant agent. To assess the effect of its structure on anticoagulant activity, its derivatives with various degrees of fucosylation (DF), molecular weights (Mw) and sulfation patterns were prepared and characterized. Biological tests showed that their APTT (activated partial thromboplastin time) prolonging activity and intrinsic factor Xase complex (factor IXa-VIIIa-Ca2+-PL complex) inhibitory activity were both reduced in FCS derivatives with lower Mw and DF. However, FCSs with DF at least 16% resulted in greater heparin cofactor II (HCII)-dependent thrombin inhibitory activity in response to decreasing DF, and these activities did not depend on Mw (Mwâ¯>â¯5.2â¯kDa). Solution competition binding assay further suggested that modulating the DF of FCS derivatives might enhance inhibition of thrombin by activating HCII. These findings imply that FCS derivatives with suitable chain length and DF value may be novel anticoagulants by activating HCII.
Subject(s)
Anticoagulants/pharmacology , Chondroitin Sulfates/pharmacology , Heparin Cofactor II/metabolism , Thrombin/antagonists & inhibitors , Anticoagulants/chemistry , Anticoagulants/isolation & purification , Chondroitin Sulfates/chemistry , Chondroitin Sulfates/isolation & purification , Dose-Response Relationship, Drug , Heparin Cofactor II/chemistry , Humans , Molecular Structure , Structure-Activity Relationship , Thrombin/metabolismABSTRACT
Oversulfated chondroitin sulfate (OSCS) was identified as a contaminant in certain heparin preparations as the cause of adverse reactions in patients. OSCS was found to possess both plasma anticoagulant activity and the ability to activate prekallikrein to kallikrein. Differentially sulfated chondroitin sulfates were prepared by synthetic modification of chondroitin sulfate and were compared to the activity of OSCS purified from contaminated heparin. Whilst chondroitin sulfate was found to have minimal anticoagulant activity, increasing sulfation levels produced an anticoagulant response which we directly show for the first time is mediated through heparin cofactor II. However, the tetra-sulfated preparations did not possess any higher anticoagulant activity than several tri-sulfated variants, and also had lower heparin cofactor II mediated activity. Activation of prekallikrein was concentration dependent for all samples, and broadly increased with the degree of sulfation, though the di-sulfated preparation was able to form more kallikrein than some of the tri-sulfated preparations. The ability of the samples to activate the kinin system, as measured by bradykinin, was observed to be through kallikrein generation. These results show that whilst an increase in sulfation of chondroitin sulfate did cause an increase in anticoagulant activity and activation of the kinin system, there may be subtler structural interactions other than sulfation at play given the different responses observed.
Subject(s)
Anticoagulants/chemical synthesis , Bradykinin/metabolism , Chondroitin Sulfates/chemical synthesis , Heparin/chemistry , Kallikreins/metabolism , Animals , Anticoagulants/chemistry , Anticoagulants/pharmacology , Chondroitin Sulfates/chemistry , Chondroitin Sulfates/pharmacology , Dose-Response Relationship, Drug , Drug Contamination , Enzyme Activation/drug effects , Heparin Cofactor II/metabolism , Humans , Structure-Activity RelationshipABSTRACT
Glycosaminoglycan (GAG) sequences that selectively target heparin cofactor II (HCII), a key serpin present in human plasma, remain unknown. Using a computational strategy on a library of 46 656 heparan sulfate hexasaccharides we identified a rare sequence consisting of consecutive glucuronic acid 2-O-sulfate residues as selectively targeting HCII. This and four other unique hexasaccharides were chemically synthesized. The designed sequence was found to activate HCII ca. 250-fold, while leaving aside antithrombin, a closely related serpin, essentially unactivated. This group of rare designed hexasaccharides will help understand HCII function. More importantly, our results show for the first time that rigorous use of computational techniques can lead to discovery of unique GAG sequences that can selectively target GAG-binding protein(s), which may lead to chemical biology or drug discovery tools.
Subject(s)
Glucuronates/pharmacology , Heparin Cofactor II/agonists , Heparitin Sulfate/pharmacology , Drug Discovery , Glucuronates/chemistry , Heparin Cofactor II/metabolism , Heparitin Sulfate/chemistry , Humans , Protein Binding , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacologyABSTRACT
Rupture of carotid atherosclerotic plaque may cause stroke, while few biomarker in clinic can evaluate carotid plaque vulnerability. In this study, we divided the recruited participants into no plaque, stable plaque, and vulnerable plaque group according to carotid ultrasonography, and screened the differentially expressed proteins in plasma of these participants using isobaric tags for relative and absolute quantitation labeling coupled with liquid chromatography-tandem mass spectrometry. 28 proteins were identified differentially expressed, among which alpha-2-macroglobulin (α2M) and heparin cofactor II (HCII) were found to be at hub position in the interactions of these proteins by STRING analysis and were selected for enzyme-linked immunosorbent assay measurement to assess their relevance with carotid plaques vulnerability and diagnostic efficiency. The plasma level of α2M was found positively correlated, while HCII level was negatively correlated with higher vulnerability of carotid plaques. Both proteins were efficient in differentiating stable and vulnerable carotid plaques. These findings provide potential new targets for the research of carotid plaque vulnerability. Plasma α2M and HCII may be potential biomarkers for evaluation of the vulnerability of carotid plaques if further studied.
Subject(s)
Carotid Stenosis/blood , Heparin Cofactor II/metabolism , Plaque, Atherosclerotic/blood , alpha-Macroglobulins/metabolism , Adult , Aged , Aged, 80 and over , Biomarkers/blood , Blood Chemical Analysis/methods , Female , Humans , Male , Middle AgedABSTRACT
Heparin is a sulfated polysaccharide of animal origin showing excellent anticoagulant properties. Although it strongly inhibits the coagulation cascade, its interaction with multiple sites results in several side effects. An ideal alternative compound should not only possess anticoagulant and antithrombotic activities, but also provide specific binding to components of the coagulation cascade to decrease side effects and facilitate the control of pharmacologic actions in patient's body. In this work, we performed a scan of potential targets for chemically sulfated pectin from Citrus sinensis (SCP) that shows an efficient anticoagulant activity by combining proteomics and molecular docking techniques. Defining the interaction partners of SCP is fundamental to evaluate if its pharmacological side effects can be as harmful as those from heparin. SCP interacts directly with heparin cofactor II, probably favoring its interaction with thrombin. SCP interaction with antithrombin depends likely on its association with thrombin or factor Xa. In addition to the interaction with factors related to homeostasis, SCP may also act on the renin-angiotensin and on the complement systems. BIOLOGICAL SIGNIFICANCE: The knowledge of potential molecular targets of SCP provides clues to understand its mechanism of action in order to guide molecular changes in this compound to increase its specificity.
Subject(s)
Anticoagulants/chemistry , Citrus sinensis/chemistry , Pectins/chemistry , Antithrombins/metabolism , Heparin Cofactor II/metabolism , Humans , Molecular Docking Simulation , Pectins/metabolism , Pectins/therapeutic use , Protein Binding , Proteomics , Sulfates/chemistry , Thrombin/chemistry , Thrombin/metabolismABSTRACT
Currently, there are emerging multiple studies on human epididymis protein 4 (HE4) in ovarian cancer. HE4 possesses higher sensitivity and specificity than CA125 in the confirmative early diagnosis for ovarian cancer. Although much attention has been given to explore its clinical application, research of the basic mechanisms of HE4 in ovarian cancer are still unclear. In the present study, we provide fundamental data to identify full-scale differentially expressed genes (DEGs) in response to HE4 by use of human whole-genome microarrays in human epithelial ovarian cancer cell line ES-2 following overexpression and silencing of HE4. We found that a total of 717 genes were upregulated and 898 genes were downregulated in the HE4-overexpressing cells vs. the HE4-Mock cells, and 166 genes were upregulated and 285 were downregulated in the HE4-silenced cells vs. the HE4-Mock cells. An overlap of 16 genes consistently upregulated and 8 genes downregulated in response to HE4 were noted. These DEGs were involved in MAPK, steroid biosynthesis, cell cycle, the p53 hypoxia pathway, and focal adhesion pathways. Interaction network analysis predicted that the genes participated in the regulatory connection. Highly differential expression of the FOXA2, SERPIND1, BDKRD1 and IL1A genes was verified by quantitative real-time PCR in 4 cell line samples. Finally, SERPIND1 (HCII) was validated at the protein level by immunohistochemistry in 107 paraffin-embedded ovarian tissues. We found that SERPIND1 may act as a potential oncogene in the development of ovarian cancer. The present study displayed the most fundamental and full-scale data to show DEGs in response to HE4. These identified genes may provide a theoretical basis for investigations of the underlying molecular mechanism of HE4 in ovarian cancer.
Subject(s)
Gene Expression Regulation, Neoplastic/genetics , Heparin Cofactor II/genetics , Neoplasms, Glandular and Epithelial/genetics , Ovarian Neoplasms/genetics , Proteins/metabolism , Blotting, Western , Carcinoma, Ovarian Epithelial , Cell Line, Tumor , Female , Gene Knockdown Techniques , Heparin Cofactor II/metabolism , Humans , Immunohistochemistry , Kaplan-Meier Estimate , Neoplasms, Glandular and Epithelial/mortality , Oligonucleotide Array Sequence Analysis , Oncogenes/genetics , Ovarian Neoplasms/mortality , Proteins/genetics , Real-Time Polymerase Chain Reaction , Transcriptome , WAP Four-Disulfide Core Domain Protein 2ABSTRACT
Follicular fluid (FF) accumulates in the antrum of the ovarian follicle and provides the microenvironment for oocyte development. FF plays an important role in follicle growth and oocyte maturation. The FF provides a unique window to investigate the processes occurring during buffalo follicular development. The observed low quality of buffalo oocytes may arise from the poor follicular microenvironment. Investigating proteins found in buffalo FF (BFF) should provide insight into follicular development processes and provide further understanding of intra-follicular maturation and oocytes quality. Here, a proteomic-based approach was used to analyze the proteome of BFF. SDS-PAGE separation combined with mass spectrometry was used to generate the proteomic dataset. In total, 363 proteins were identified and classified by Gene Ontology terms. The proteins were assigned to 153 pathways, including signaling pathways. To evaluate difference in proteins expressed between BFF with different follicle size (small, <4 mm; and large, >8 mm), a quantitative proteomic analysis based on multi-dimensional liquid chromatography pre-fractionation tandem Orbitrap mass spectrometry identification was performed. Eleven differentially expressed proteins (six downregulated and five upregulated in large BFF) were identified and assigned to a variety of functional processes, including serine protease inhibition, oxidation protection and the complement cascade system. Three differentially expressed proteins, Vimentin, Peroxiredoxin-1 and SERPIND1, were verified by Western blotting, consistent with the quantitative proteomics results. Our datasets offers new information about proteins present in BFF and should facilitate the development of new biomarkers. These differentially expressed proteins illuminate the size-dependent protein changes in follicle microenvironment.
Subject(s)
Buffaloes/metabolism , Ovarian Follicle/metabolism , Proteome/analysis , Proteomics , Animals , Blotting, Western , Chromatography, High Pressure Liquid , Estradiol/analysis , Female , Follicular Fluid/metabolism , Heparin Cofactor II/metabolism , Ovarian Follicle/growth & development , Peptides/analysis , Peroxiredoxins/metabolism , Progesterone/analysis , Tandem Mass Spectrometry , Vimentin/metabolismABSTRACT
OBJECTIVE: Thrombin signaling promotes atherosclerosis by initiating inflammatory events indirectly through platelet activation and directly via protease-activated receptors. Therefore, endogenous thrombin inhibitors may be relevant modulators of atheroprogression and cardiovascular risk. In addition, endogenous thrombin inhibitors may affect the response to non-vitamin K-dependent oral anticoagulants. Here, the question was addressed whether the small leucine-rich proteoglycan biglycan acts as an endogenous thrombin inhibitor in atherosclerosis through activation of heparin cofactor II. APPROACH AND RESULTS: Biglycan concentrations were elevated in the plasma of patients with acute coronary syndrome and in male Apolipoprotein E-deficient (ApoE(-/-)) mice. Biglycan was detected in the glycocalyx of capillaries and the subendothelial matrix of arterioles of ApoE(-/-) mice and in atherosclerotic plaques. Thereby a vascular compartment is provided that may mediate the endothelial and subendothelial activation of heparin cofactor II through biglycan. ApoE and Bgn double-deficient (ApoE(-/-)/Bgn(-/0)) mice showed higher activity of circulating thrombin, increased platelet activation and platelet adhesion in vivo, supporting a role of biglycan in balancing thrombin activity. Furthermore, concentrations of circulating cytokines and aortic macrophage content were elevated in ApoE(-/-)/Bgn(-/0) mice, suggesting a proinflammatory phenotype. Elevated platelet activation and macrophage accumulation were reversed by treating ApoE(-/-)/Bgn(-/0) mice with the thrombin inhibitor argatroban. Ultimately, ApoE(-/-)/Bgn(-/0) mice developed aggravated atherosclerosis. CONCLUSIONS: The present results indicate that biglycan plays a previously unappreciated protective role during the progression of atherosclerosis by inhibiting thrombin activity, platelet activation, and finally macrophage-mediated plaque inflammation.
Subject(s)
Aorta/metabolism , Aortic Diseases/metabolism , Apolipoproteins E/deficiency , Atherosclerosis/metabolism , Biglycan/deficiency , Inflammation/metabolism , Thrombin/metabolism , Acute Coronary Syndrome/blood , Animals , Antithrombins/pharmacology , Aorta/drug effects , Aorta/pathology , Aortic Diseases/blood , Aortic Diseases/genetics , Aortic Diseases/prevention & control , Apolipoproteins E/genetics , Atherosclerosis/blood , Atherosclerosis/genetics , Atherosclerosis/prevention & control , Biglycan/blood , Biglycan/genetics , Cytokines/blood , Disease Models, Animal , Genotype , Heparin Cofactor II/metabolism , Humans , Inflammation/blood , Inflammation/genetics , Inflammation/prevention & control , Macrophages/metabolism , Male , Mice, Inbred C57BL , Mice, Knockout , Phenotype , Plaque, Atherosclerotic , Platelet Activation , Time FactorsABSTRACT
Sulfated fucans, the complex polysaccharides, exhibit various biological activities. Herein, we purified two fucans from the sea cucumbers Holothuria edulis and Ludwigothurea grisea. Their structures were verified by means of HPGPC, FT-IR, GC-MS and NMR. As a result, a novel structural motif for this type of polymers is reported. The fucans have a unique structure composed of a central core of regular (1â2) and (1â3)-linked tetrasaccharide repeating units. Approximately 50% of the units from L. grisea (100% for H. edulis fucan) contain sides of oligosaccharides formed by nonsulfated fucose units linked to the O-4 position of the central core. Anticoagulant activity assays indicate that the sea cucumber fucans strongly inhibit human blood clotting through the intrinsic pathways of the coagulation cascade. Moreover, the mechanism of anticoagulant action of the fucans is selective inhibition of thrombin activity by heparin cofactor II. The distinctive tetrasaccharide repeating units contribute to the anticoagulant action. Additionally, unlike the fucans from marine alga, although the sea cucumber fucans have great molecular weights and affluent sulfates, they do not induce platelet aggregation. Overall, our results may be helpful in understanding the structure-function relationships of the well-defined polysaccharides from invertebrate as new types of safer anticoagulants.
Subject(s)
Anticoagulants/isolation & purification , Drug Discovery , Polysaccharides/isolation & purification , Sea Cucumbers/chemistry , Animals , Anticoagulants/chemistry , Anticoagulants/pharmacology , Blood Coagulation/drug effects , Brazil , Carbohydrate Sequence , Chemical Phenomena , China , Heparin Cofactor II/antagonists & inhibitors , Heparin Cofactor II/metabolism , Holothuria/chemistry , Humans , Kinetics , Molecular Weight , Polysaccharides/chemistry , Polysaccharides/pharmacology , Sea Cucumbers/growth & development , Species Specificity , Structure-Activity RelationshipABSTRACT
OBJECTIVE: To explore the relationship between activity of plasma heparin cofactor II (HC II) and the incidence of in-stent restenosis aft er the intervention of arteriosclerosis obliterans in lower extremity. METHODS: A total of 62 patients with arteriosclerosis obliterans in lower extremity underwent femoropopliteal stent implantation. They were divided into 2 groups: A high HC II activity group (≥100%, n=40) and a low HC II activity group (<100%, n=22). All patients filled in follow up tables and conducted body examination. Possible risk factors resulting in restenosis were collected. Patients were followed up for 6 months after femoropopliteal stent implantation. RESULTS: Baseline clinical characteristics were not significantly different between the 2 groups. The degree and incidence of angiographic restenosis at the end of the 6th month after the implantation in the high HC II activity group were all significantly lower than those in the low HC II activity group (P<0.05). Multivariate analysis demonstrated that high plasma HC II activity was an independent factor in reducing the incidence of angiographic restenosis (OR=0.982, P=0.048, 95%CI, 0.966, 0.998). CONCLUSION: High plasma HC II activity is an independent factor in reducing the degree of in-stent restenosis. The lower the plasma HC II activity, the severer the degree of in-stent restenosis.
Subject(s)
Arteriosclerosis Obliterans/surgery , Heparin Cofactor II/metabolism , Stents , Constriction, Pathologic , Humans , Incidence , Lower Extremity , Risk FactorsABSTRACT
Heparan sulfate normally binds to heparin cofactor II and modulates the coagulation pathway by inhibiting thrombin. However, when human heparin cofactor II was incubated with heparan sulfate, heparin cofactor II became degraded. Other glycosaminoglycans were tested, including hyaluronic acid, chondroitin sulfates, dermatan sulfate, and heparin, but only dextran sulfate also degraded heparin cofactor II. Pretreatment of heparan sulfate with heparinase reduced its heparin cofactor II-degrading activity. Heparan sulfate and dextran sulfate diminished the thrombin inhibitory activity of heparin cofactor II. Other serpins, including antithrombin III and pigment epithelium-derived factor, were also degraded by heparan sulfate. This is the first evidence of acidic polysaccharides exhibiting protein-degrading activity without the aid of other proteins.
Subject(s)
Antithrombins/metabolism , Dextran Sulfate/metabolism , Heparin Cofactor II/metabolism , Heparitin Sulfate/metabolism , Proteolysis , Animals , Antithrombin III/metabolism , Antithrombins/pharmacology , Cattle , Flavobacterium/enzymology , Heparin Cofactor II/pharmacology , Heparin Lyase/metabolism , Humans , Indicators and Reagents/metabolismABSTRACT
The purpose of this study was to investigate a possible correlation between single-nucleotide polymorphisms (SNPs) of the antithrombin (gene, SERPINC1, and perioperative sensitivity to heparin in patients receiving heart surgery. The SERPINC1 genotype and allele frequency, coagulation parameters 24 hours before and after surgery, and clinical findings were compared among 3 ethnic groups, Han, Uighur, and Kazakh, patientswho received heart surgery. In Han patients, longer coagulation time as well as higher heparin and protamine dosage was observed. SERPINC1 gene sequencing identified 2 mutations in exon 5, g.981A>G (rs5877) and g.1011A>G (rs5878). The minor allele frequency of allele (A>G) for rs5877 and rs5878 was higher in the Han patients and was significantly different among the ethnic groups (P = .004 and P = .006, respectively). The increased SERPINC1 SNP frequency among Han patients receiving heart surgery might contribute to the differences in their perioperative sensitivity to heparin.