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Biochemistry. Biokhimiia Jul 2013Heparan sulfate (HS) represents a large class of linear polysaccharides that are required for the function of all mammalian physiological systems. HS is characterized by... (Review)
Review
Heparan sulfate (HS) represents a large class of linear polysaccharides that are required for the function of all mammalian physiological systems. HS is characterized by a repeating disaccharide backbone that is subject to a wide range of modifications, making this class of macromolecules arguably the most information dense in all of biology. The majority of HS functions are associated with the ability to bind and regulate a wide range of proteins. Indeed, recent years have seen an explosion in the discovery of new activities for HS where it is now recognized that this class of glycans functions as co-receptors for growth factors and cytokines, modulates cellular uptake of lipoproteins, regulates protease activity, is critical to amyloid plaque formation, is used by opportunistic pathogens to enter cells, and may even participate in epigenetic regulation. This review will discuss the current state of understanding regarding the specificity of HS-protein binding and will describe the concept that protein binding to HS depends on the overall organization of domains within HS rather than fine structure.
Topics: Animals; Antithrombin III; Fibroblast Growth Factors; Heparitin Sulfate; Humans; Leukocyte Elastase; Protein Binding; Proteins
PubMed: 24010836
DOI: 10.1134/S0006297913070055 -
The Journal of Biological Chemistry Jan 1983Antithrombin III mRNA was enriched from a baboon liver by specific polysome immunoprecipitation. The partially purified antithrombin III mRNA preparation was used for...
Antithrombin III mRNA was enriched from a baboon liver by specific polysome immunoprecipitation. The partially purified antithrombin III mRNA preparation was used for cDNA synthesis and cloning. Candidate antithrombin III cDNA clones were identified by differential hybridization using as probes [32P]cDNAs synthesized from the polysome-enriched and -depleted RNA fractions, respectively. The candidate clones were further analyzed by hybrid-selected translation. The authenticity of a cDNA clone positive to both tests was unambiguously confirmed by matching its nucleotide sequence with the known amino acid sequence of human antithrombin III. The baboon antithrombin III cDNA clone hybridized well with human antithrombin III mRNA and can be used as a probe to isolate the corresponding human gene.
Topics: Animals; Antithrombin III; Base Sequence; Cell-Free System; Cloning, Molecular; DNA; Humans; Nucleic Acid Hybridization; Papio; Protein Biosynthesis; RNA, Messenger
PubMed: 6687384
DOI: No ID Found -
Journal of Thrombosis and Haemostasis :... Feb 2016ESSENTIALS: Antithrombin III (AT)β binds heparin with higher affinity than ATα. A conformation-specific antibody against ATβ, TPP2009, was made to investigate ATβ in...
UNLABELLED
ESSENTIALS: Antithrombin III (AT)β binds heparin with higher affinity than ATα. A conformation-specific antibody against ATβ, TPP2009, was made to investigate ATβ in hemostasis. TPP2009 bound specifically to heparin-ATβ and greatly reduced the anticoagulant effect of AT. This antibody was effective in elucidating the importance of ATβ in hemostasis.
BACKGROUND
Antithrombin III (AT)β is an isoform of AT that lacks the post-translational carbohydrate modification at Asn135. This isoform binds heparin with greater affinity than ATα, and has been shown to target antithrombotic function to the extracellular vascular endothelial injury site.
OBJECTIVES
To characterize a conformation-specific antibody against ATβ and begin to investigate the role of ATβ in maintaining hemostasis.
METHODS
Surface plasmon resonance (SPR), antigen binding and functional assays were conducted to characterize the mode of action of antibodies generated against heparin-bound ATβ (ATβ*H) by the use of phage display.
RESULTS
SPR and binding studies showed that one of the antibodies, TPP2009, bound specifically to ATβ*H and glycosaminoglycan-associated ATβ on endothelial cells. In diluted prothrombin and activated factor X (FXa)-induced clotting assays, TPP2009 dose-dependently reduced the anticoagulant effect of heparin in non-hemophilic and FVIII-deficient human plasma, with half-maximal effective concentrations (EC50 ) of 10.5 nm and 4.7 nm, respectively. In AT-deficient human plasma, TPP2009 dose-dependently inhibited the effects of exogenously added ATβ and heparin. In purified systems with ATβ and pentasaccharide, TPP2009 restored > 91% of FXa activity. TPP2009 dose-dependently reversed the effects of heparin in rabbit (EC50 , 25.7 nm) and cynomolgus monkey (EC50 , 21.5 nm) plasma, but not in mouse plasma. TPP2009 was also effective in partially restoring FXa activity in rabbit and cynomolgus monkey plasma treated with FVIII function-neutralizing antibodies.
CONCLUSIONS
TPP2009 specifically targets a unique conformational epitope on ATβ*H and blocks ATβ-mediated anticoagulation. It effectively promotes coagulation in plasma, indicating the importance of ATβ in hemostasis.
Topics: Animals; Antibodies; Antibody Specificity; Antithrombin III; Binding Sites, Antibody; Blood Coagulation; Blood Coagulation Tests; Cell Line; Cell Surface Display Techniques; Coagulants; Dose-Response Relationship, Drug; Endothelial Cells; Epitope Mapping; Humans; Protein Binding; Protein Structure, Secondary; Rabbits; Structure-Activity Relationship; Surface Plasmon Resonance; Time Factors
PubMed: 26581031
DOI: 10.1111/jth.13198 -
Journal of Biochemistry Dec 19791. Human, porcine, rabbit, and rat antithrombin III have been purified by affinity chromatography using heparin-agarose. The amino acid and carbohydrate compositions,... (Comparative Study)
Comparative Study
1. Human, porcine, rabbit, and rat antithrombin III have been purified by affinity chromatography using heparin-agarose. The amino acid and carbohydrate compositions, amino-terminal sequences, immunological cross-reactivities, and inhibitions of human thrombin were studied. 2. Human, porcine, rabbit, and rat antithrombin III are single-chain glycoproteins containing hexose, glucosamine, and neuraminic acid. 3. The total carbohydrate contents were 17, 16, 14, and 15% for human, porcine, rabbit, and rat antithrombin III, respectively. 4. Molecular weights estimated from the migration in sodium dodecyl sulfate (SDS)-poly-acrylamide gel electrophoresis were 59,000, 58,000, 63,000, and 63,000 for human, porcine rabbit, and rat antithrombin III, respectively. 5. These four proteins have similar amino acid compositions, although some minor differences were noted. 6. Human, porcine, and rabbit antithrombin III have a histidine residue at the amino-terminus, while rat antithrombin III contains an amino-terminal asparagine residue. 7. The amino-terminal sequences up to the first 17 residues showed high homology among the four proteins. 8. Some immunological cross-reactivity was observed only between human and porcine antithrombin III. 9. The apparent dissociation constants (KI) for the complexes between human thrombin and human, porcine, rabbit, and rat antithrombin III were about 1.2 x 10(-10) M, 9.5 X 10 (-9) M, 1.4 X 10(-7) M, and 2.8 X 10(-9) M, respectively.
Topics: Amino Acid Sequence; Amino Acids; Animals; Antithrombin III; Carbohydrates; Cattle; Horses; Immunoassay; Immunodiffusion; Molecular Weight; Rabbits; Rats; Species Specificity; Swine
PubMed: 118968
DOI: 10.1093/oxfordjournals.jbchem.a132706 -
The Journal of Biological Chemistry Jan 1985Two distinct forms of antithrombin III were isolated by chromatography of normal human plasma on heparin-Sepharose. The predominant antithrombin species present (AT-III...
Two distinct forms of antithrombin III were isolated by chromatography of normal human plasma on heparin-Sepharose. The predominant antithrombin species present (AT-III alpha), which eluted from the affinity column in 1 M NaCl, was identified as the antithrombin III form which has been previously characterized. Ionic strength of the buffer was increased to elute a variant form of antithrombin III, designated as AT-III beta. The molecular weight of AT-III beta is less than that of AT-III alpha, but physicochemical studies do not indicate measureable differences in the polypeptide portion of the proteins. Carbohydrate determination revealed the sole detectable structural difference in the two antithrombins: levels of hexosamine, neutral sugars, and sialic acid in AT-III beta were all 25-30% less than in AT-III alpha. Kinetic studies of thrombin inactivation by both antithrombins, in the presence of nonsaturating amounts of heparin, indicated that AT-III beta inhibited thrombin more rapidly. AT-III beta is also distinguishable from AT-III alpha on the basis of heparin-binding affinity estimated from titration of protein fluorescence with heparin. Thus, antithrombin III exists as two molecular entities in human plasma which differ both structurally, in carbohydrate content, and functionally, in their heparin-binding behavior.
Topics: Amino Acids; Antithrombin III; Carbohydrates; Enzyme-Linked Immunosorbent Assay; Genetic Variation; Heparin; Humans; Kinetics; Protein Binding
PubMed: 3965464
DOI: No ID Found -
Kidney International Oct 2015Antithrombin III, encoded by SerpinC1, is a major anti-coagulation molecule in vivo and has anti-inflammatory effects. We found that patients with low antithrombin III...
Antithrombin III, encoded by SerpinC1, is a major anti-coagulation molecule in vivo and has anti-inflammatory effects. We found that patients with low antithrombin III activities presented a higher risk of developing acute kidney injury after cardiac surgery. To study this further, we generated SerpinC1 heterozygous knockout rats and followed the development of acute kidney injury in a model of modest renal ischemia/reperfusion injury. Renal injury, assessed by serum creatinine and renal tubular injury scores after 24 h of reperfusion, was significantly exacerbated in SerpinC1(+/-) rats compared to wild-type littermates. Concomitantly, renal oxidative stress, tubular apoptosis, and macrophage infiltration following this injury were significantly aggravated in SerpinC1(+/-) rats. However, significant thrombosis was not found in the kidneys of any group of rats. Antithrombin III is reported to stimulate the production of prostaglandin I2, a known regulator of renal cortical blood flow, in addition to having anti-inflammatory effects and to protect against renal failure. Prostaglandin F1α, an assayable metabolite of prostaglandin I2, was increased in the kidneys of the wild-type rats at 3 h after reperfusion. The increase of prostaglandin F1α was significantly blunted in SerpinC1(+/-) rats, which preceded increased tubular injury and oxidative stress. Thus, our study found a novel role of SerpinC1 insufficiency in increasing the severity of renal ischemia/reperfusion injury.
Topics: Acute Kidney Injury; Aged; Animals; Antithrombin III; Antithrombin III Deficiency; Apoptosis; Biomarkers; Cardiac Surgical Procedures; Creatinine; Disease Models, Animal; Female; Gene Knockdown Techniques; Genetic Predisposition to Disease; Heterozygote; Humans; Kidney; Macrophages; Male; Middle Aged; Oxidative Stress; Phenotype; Prostaglandins F; Rats, Transgenic; Reperfusion Injury; Risk Factors; Severity of Illness Index; Signal Transduction; Time Factors
PubMed: 26108065
DOI: 10.1038/ki.2015.176 -
The Journal of Clinical Investigation Mar 1986A hereditary (three family members) deficiency of antithrombin III (AT-III) in which AT-III antigen (AT-III ag) is normal in spite of low heparin cofactor and...
A hereditary (three family members) deficiency of antithrombin III (AT-III) in which AT-III antigen (AT-III ag) is normal in spite of low heparin cofactor and antithrombin activity is described. Plasma levels were: AT-III ag, 0.92-0.96 U/ml; AT-III heparin cofactor activity, 0.54-0.62 U/ml; progressive antithrombin activity index, 0.13-0.18; anti-Xa activity, 0.50-0.56 U/ml. Plasma crossed immunoelectrophoresis (CIE) patterns performed with and without added heparin were normal, but serum CIE revealed a decreased complex peak. Purification of the patient's plasma AT-III by heparin-sepharose affinity chromatography showed a normal protein recovery and elution profile, but the purified AT-III fraction showed only 50% of the normal progressive thrombin neutralization and anti-Xa activity. When thrombin-antithrombin (TAT) complexes were formed by incubating with excess thrombin, SDS-polyacrylamide gel electrophoresis (PAGE) analysis revealed that half the patient AT-III formed TAT complexes while the remainder migrated as free AT-III. All the control AT-III formed TAT complexes. The patient's nonreacting AT-III (AT-III "Denver"), isolated by affinity chromatography, showed CIE and SDS-PAGE migration patterns characteristic of normal AT-III but failed to bind thrombin or Xa. Calculations from turnover studies in one patient and normal subjects with autologous 131I-AT-III suggested that AT-III "Denver" is removed from the plasma slightly more rapidly than normal. These studies indicate that the patients' variant AT-III molecule was characterized by normal heparin interaction but defective binding and inhibition of thrombin and Xa. These characteristics allow isolation of the nonreactive variant molecule by heparin-sepharose affinity chromatography.
Topics: Antithrombin III; Antithrombin III Deficiency; Endopeptidases; Humans; Immunoelectrophoresis, Two-Dimensional; Mutation; Pedigree; Protein Binding; Serine Endopeptidases
PubMed: 3512602
DOI: 10.1172/JCI112386 -
BMC Veterinary Research Mar 2022Diabetes mellitus (DM) often leads to dangerous thromboembolic complications in humans. DM is also a relatively common endocrinopathy of dogs. There is scarce...
BACKGROUND
Diabetes mellitus (DM) often leads to dangerous thromboembolic complications in humans. DM is also a relatively common endocrinopathy of dogs. There is scarce information regarding procoagulant and anticoagulant plasma indicators in this disease. The aim of the study was to evaluate the levels of the selected plasma haemostatic parameters in dogs suffering from diabetes. The study group consisted of 20 dogs meeting all the inclusion criteria, with fasting glycaemia exceeding 11.1 mmol/l. The control group consisted of 15 healthy dogs presented for routine examination. An evaluation of the prothrombin time (PT); and fibrinogen, D-dimer and antithrombin III (ATIII) levels was performed.
RESULTS
Except for ATIII activity, the haemostatic parameter differences were not statistically significant. High values of ATIII activity were observed in 90% of diabetic dogs. On average, the values amounted to 166.6% and were 31.4% higher than those in the control group. The ATIII activity in the diabetic group was significantly higher than that in the control group (p = 0.0004).
CONCLUSIONS
Here, we report elevated levels of ATIII in diabetic dogs. This finding may suggest the protective role of ATIII against potential thrombotic events. However, the exact role of ATIII in dog diabetes remains unclear.
Topics: Animals; Anticoagulants; Antithrombin III; Diabetes Mellitus; Dog Diseases; Dogs; Fibrinogen; Hemostasis
PubMed: 35305618
DOI: 10.1186/s12917-022-03179-7 -
The Journal of Biological Chemistry Mar 1986The molecular interactions between components of the heparin-catalyzed antithrombin III/thrombin reaction were investigated by light scattering. When heparin was added...
The molecular interactions between components of the heparin-catalyzed antithrombin III/thrombin reaction were investigated by light scattering. When heparin was added to antithrombin III, the molecular weight increased to a maximum and then decreased to that of a 1:1 (antithrombin III X heparin) complex. The initial molecular weights at low heparin to antithrombin III ratios were consistent with the formation of a 2:1 (antithrombin III X heparin) complex in which only one antithrombin III molecule had undergone the conformational change measured by protein fluorescence enhancement. The peak molecular weight never reached that of a complete 2:1 complex. This behavior was observed for bovine and human antithrombin III in the presence of both unfractionated heparin and high molecular weight-high affinity heparin. Pentosane polysulfate also caused some multiple associations. Bovine antithrombin III and thrombin formed a 1:1 complex that underwent further aggregation within minutes, while the human proteins did not aggregate on this time scale after forming the 1:1 complex. In the presence of stoichiometric amounts of heparin, the bovine proteins formed an initial complex of Mr = 230,000 (corresponding to a dimer of heparin-antithrombin III-thrombin) which underwent further aggregation. The human proteins, however, formed a 1:1 (antithrombin III X thrombin) initial complex in the presence of heparin, followed by aggregation. These interactions of thrombin and antithrombin with heparin suggest complex interactions that could relate to heparin function.
Topics: Animals; Antithrombin III; Cattle; Heparin; Humans; Macromolecular Substances; Molecular Weight; Thrombin; Ultracentrifugation
PubMed: 3949807
DOI: No ID Found -
Polish Archives of Internal Medicine Jan 2022
Topics: Antithrombin III; Antithrombin III Deficiency; Fibrin; Humans; Mutation
PubMed: 34851072
DOI: 10.20452/pamw.16158