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Nefrologia 2022
Topics: Humans; Nephrotic Syndrome; Thrombosis; Antithrombin III; Antithrombins
PubMed: 36870820
DOI: 10.1016/j.nefroe.2023.02.003 -
Critical Care (London, England) Feb 2006In disseminated intravascular coagulation (DIC) there is extensive crosstalk between activation of inflammation and coagulation. Endogenous anticoagulatory pathways are... (Review)
Review
In disseminated intravascular coagulation (DIC) there is extensive crosstalk between activation of inflammation and coagulation. Endogenous anticoagulatory pathways are downregulated by inflammation, thus decreasing the natural anti-inflammatory mechanisms that these pathways possess. Supportive strategies aimed at inhibiting activation of coagulation and inflammation may theoretically be justified and have been found to be beneficial in experimental and initial clinical studies. This review assembles the available experimental and clinical data on biological mechanisms of antithrombin in inflammatory coagulation activation. Preclinical research has demonstrated partial interference of heparin--administered even at low doses--with the therapeutic effects of antithrombin, and has confirmed--at the level of cellular mechanisms--a regulatory role for antithrombin in DIC. Against this biological background, re-analyses of data from randomized controlled trials of antithrombin in sepsis suggest that antithrombin has the potential to be developed further as a therapeutic agent in the treatment of DIC. Even though there is a lack of studies employing satisfactory methodology, the results of investigations conducted thus far into the mechanisms of action of antithrombin allow one to infer that there is biological plausibility in the value of this agent. Final assessment of the drug's effectiveness, however, must await the availability of positive, prospective, randomized and placebo-controlled studies.
Topics: Animals; Antithrombin III; Antithrombins; Blood Coagulation; Humans; Sepsis
PubMed: 16542481
DOI: 10.1186/cc4822 -
Journal of Thrombosis and Haemostasis :... Jul 2014Antithrombin, a hemostatic protein and naturally occurring anticoagulant, is a major thrombin inhibitor. The capacity of antithrombin to inhibit thrombin is known to...
BACKGROUND
Antithrombin, a hemostatic protein and naturally occurring anticoagulant, is a major thrombin inhibitor. The capacity of antithrombin to inhibit thrombin is known to increase a 1000-fold whilst in the presence of unfractionated heparin. β-antithrombin is an isoform of antithrombin with a high affinity for unfractionated heparin. This study aimed to determine the differences in the anticoagulant activity of the β-antithrombin isoform in children compared with adults.
METHODS
Plasma samples were obtained from 105 healthy individuals from the following age groups: neonates (day 1 and day 3), 28 days to 1 year, 1-5 years, 6-10 years, 11-16 years and adults. The method utilized to measure the activity of β-antithrombin in plasma is a modified version of the total antithrombin assay routinely used in diagnostic laboratories. The modified version of this assay allows for the specific quantification of the β-antithrombin glycoform anticoagulant activity alone, as the β-antithrombin molecule is activated under a high salt concentration, which in turn does not allow activation of other antithrombin isoforms.
CONCLUSIONS
This study demonstrated that there are no age-specific differences in the activity of β-antithrombin. However, considering that the total AT activity is significantly reduced in neonates, our results suggest that in this population β-antithrombin activity is a major contributor to the overall activity of AT.
Topics: Adolescent; Adult; Anticoagulants; Antithrombins; Blood Coagulation Tests; Child; Child, Preschool; Heparin; Humans; Infant; Infant, Newborn; Pediatrics; Plasma; Protein Isoforms; Thrombin
PubMed: 24801362
DOI: 10.1111/jth.12597 -
The Journal of Biological Chemistry Nov 1996The binding of heparin to antithrombin greatly accelerates the rate of inhibition of the target proteinases thrombin and factor Xa. Acceleration of the rate of...
The binding of heparin to antithrombin greatly accelerates the rate of inhibition of the target proteinases thrombin and factor Xa. Acceleration of the rate of inhibition of factor Xa involves a conformational change in antithrombin that is translated from the heparin binding site to the reactive center loop. A mechanism has been proposed for generation and propagation of the conformational change in which the binding of the negatively charged heparin reduces ionic repulsions between positively charged residues on and adjacent to the D-helix in the heparin binding site of antithrombin (van Boeckel, C. A. A., Grootenhuis, P. D. J., and Visser, A. (1994) Nature Struct. Biol. 1, 423-425). This charge neutralization is proposed to elongate the D-helix and initiate the conformational change which is then translated to the reactive center loop. Several basic residues, including arginine 132 and lysine 133, were predicted to be important both in heparin binding and in this mechanism of heparin activation. To test both the helix extension mechanism and the role of these two residues in heparin binding and factor Xa inhibition, we individually changed arginine 132 and lysine 133 to uncharged methionine by site-directed mutagenesis. The Kd values for binding of R132M and K133M variants to the high affinity pentasaccharide were weakened only 2.3- and 4.5-fold respectively, suggesting a location for R132 and K133 peripheral to the main pentasaccharide binding site. However, the Kd values for long chain high affinity heparin were weakened at least 17-fold for both R132M and K133M, indicating involvement of each residue in binding extended chain heparin species. These reductions in affinity were ionic strength-dependent. The rates of inhibition of factor Xa and thrombin by each variant, however, were indistinguishable from those of control antithrombin, and the accelerations of the rate of inhibition produced by heparin were normal. We conclude that neither arginine 132 nor lysine 133 plays an important role in the binding of heparin pentasaccharide or in the mechanism of heparin activation, suggesting that D-helix extension through charge neutralization is not the mechanism for transmission of conformational change from the heparin binding site to the reactive center region. Arginine 132 and lysine 133 do, however, play a role in tight binding of longer chain heparin species through ionic interactions.
Topics: Antithrombin III; Arginine; Heparin; Humans; Lysine; Mutagenesis, Site-Directed; Protease Inhibitors; Protein Binding
PubMed: 8910598
DOI: 10.1074/jbc.271.46.29353 -
International Journal of Molecular... Dec 2020(1) Background: The endothelial glycocalyx is a primary target during the early phase of sepsis. We previously reported a newly developed recombinant non-fucosylated...
(1) Background: The endothelial glycocalyx is a primary target during the early phase of sepsis. We previously reported a newly developed recombinant non-fucosylated antithrombin has protective effects in vitro. We further evaluated the effects of this recombinant antithrombin on the glycocalyx damage in an animal model of sepsis. (2) Methods: Following endotoxin injection, in Wistar rats, circulating levels of hyaluronan, syndecan-1 and other biomarkers were evaluated in low-dose or high-dose recombinant antithrombin-treated animals and a control group ( = 7 per group). Leukocyte adhesion and blood flow were evaluated with intravital microscopy. The glycocalyx was also examined using side-stream dark-field imaging. (3) Results: The activation of coagulation was inhibited by recombinant antithrombin, leukocyte adhesion was significantly decreased, and flow was better maintained in the high-dose group (both < 0.05). Circulating levels of syndecan-1 ( < 0.01, high-dose group) and hyaluronan ( < 0.05, low-dose group; < 0.01, high-dose group) were significantly reduced by recombinant antithrombin treatment. Increases in lactate and decreases in albumin levels were significantly attenuated in the high-dose group ( < 0.05, respectively). The glycocalyx thickness was reduced over time in control animals, but the derangement was attenuated and microvascular perfusion was better maintained in the high-dose group recombinant antithrombin group ( < 0.05). (4) Conclusions: Recombinant antithrombin maintained vascular integrity and the microcirculation by preserving the glycocalyx in this sepsis model, effects that were more prominent with high-dose therapy.
Topics: Animals; Antithrombin III; Antithrombins; Endothelium, Vascular; Endotoxins; Glycocalyx; Protective Agents; Rats; Rats, Wistar; Recombinant Proteins; Sepsis
PubMed: 33375342
DOI: 10.3390/ijms22010176 -
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 -
Blood Coagulation & Fibrinolysis : An... Jan 2024Preeclampsia is a worldwide contributor to maternal and fetal morbidity and mortality. Women with preeclampsia are in a hyper-coagulable state with increased risk of...
Preeclampsia is a worldwide contributor to maternal and fetal morbidity and mortality. Women with preeclampsia are in a hyper-coagulable state with increased risk of thromboembolic disease later in life compared with normal pregnant women. The contact system (CAS) in plasma can mediate thrombin generation and is an important contributor to thrombus growth, but the activation of CAS during pregnancy complicated by preeclampsia is not yet elucidated, and CAS may play a role in the pathophysiology of preeclampsia. Therefore, the aim of the study is to address thrombin generation, and in particular, the capacity of the CAS-mediated pathway in patients with preeclampsia compared with pregnant controls. One hundred and seventeen women with preeclampsia and matched controls were included. The project was registered at www.clinicaltrials.gov as NCT04825145. CAS and tissue factor induced thrombin generation, proteins C and S, antithrombin, and histidine-rich glycoprotein (HRG) were assessed. Women with preeclampsia had significantly increased CAS and tissue factor-induced endogenous thrombin potential (ETP), and HRG compared with controls, P = 0.022, P = 0.024, and P = 0.02, respectively. The concentrations of protein C and antithrombin were significantly reduced in the preeclampsia group, P = 0.024 and P < 0.0001, respectively. No significant difference in the concentration of protein S was detected, P = 0.06. This study demonstrates a significant increased CAS-induced ETP and an overall decrease of important regulators of coagulation in women with preeclampsia compared with controls. These aspects can contribute to the hyper-coagulable state characterizing preeclampsia.
Topics: Female; Humans; Pregnancy; Pregnant Women; Thrombin; Thromboplastin; Pre-Eclampsia; Anticoagulants; Protein C; Antithrombin III; Antithrombins
PubMed: 38051647
DOI: 10.1097/MBC.0000000000001269 -
Blood Oct 1990Human antithrombin III (AT-III) cDNA was cloned into the cell-free expression phagemid vector pGEM-3Zf(+) and site-directed mutagenesis was used to remove nucleotides...
Human antithrombin III (AT-III) cDNA was cloned into the cell-free expression phagemid vector pGEM-3Zf(+) and site-directed mutagenesis was used to remove nucleotides encoding the signal peptide. AT-III messenger RNA (mRNA) transcripts derived from this construct were translated in an mRNA-dependent rabbit reticulocyte lysate (RRL) system containing (35S)methionine. Immunoprecipitation of the cell-free translation mixture with rabbit polyclonal antibodies to AT-III showed, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), a 47-Kd polypeptide which is the non-glycosylated mature form of plasma AT-III. Densitometric scanning showed that this polypeptide constitutes greater than 90% of the radiolabeled polypeptides produced in this system. Heparin-Sepharose chromatography resulted in the elution of cell-free derived AT-III as a broad peak between 0.2 and 0.7 mol/L NaCl. The cell-free derived AT-III also reacted with human alpha-thrombin. In 2 minutes approximately 20% of the AT-III was found associated with a higher molecular weight species, consistent with the formation of a 1:1 stoichiometric covalent complex between alpha-thrombin and AT-III. Unfractionated heparin accelerated the rate of formation of such complexes. When Ser394 was mutated to Leu to form the AT-III Denver mutant, the cell-free translation product of this mutation did not show any significant complex formation when reacted with alpha-thrombin. A truncated form of AT-III (Met251-Lys432), containing only the putative thrombin-binding domain, was synthesized independently. This 21-Kd polypeptide did not bind heparin; however, it was cleaved by alpha-thrombin presumably at the reactive center Arg393-Ser394. When Ser394 was mutated to Leu the cell-free translation product of this truncated AT-III mutation did not react with alpha-thrombin at the reactive center. This simple cell-free approach, along with site-directed mutagenesis, should allow for the rapid and accurate mapping of the functional domains of human AT-III.
Topics: Animals; Antithrombin III; Cell-Free System; Chromatography; Cloning, Molecular; DNA; Electrophoresis, Polyacrylamide Gel; Gene Expression; Heparin; Humans; Immunosorbent Techniques; Molecular Weight; Mutagenesis, Site-Directed; Protein Biosynthesis; Protein Sorting Signals; RNA, Messenger; Rabbits; Thrombin
PubMed: 2207328
DOI: No ID Found -
Clinical and Applied... May 2014Population pharmacokinetic (PK) analyses were conducted to refine dosing recommendations for recombinant human anti-thrombin therapy in surgery and delivery patients...
Population pharmacokinetic (PK) analyses were conducted to refine dosing recommendations for recombinant human anti-thrombin therapy in surgery and delivery patients with hereditary antithrombin deficiency (HD). Single-dose PK data from patients with HD and nonlinear mixed-effects modeling were used to devise a dosing regimen to target antithrombin (AT) activity levels between 80% and 120% of normal. External validation with data from a phase 3 trial confirmed the correctness of a covariate-free model for surgery patients, but dosing adjustment was necessary for delivery patients. After different covariates were tested, the model was updated to incorporate the influential covariate, delivery. Simulations were used to develop a therapeutic drug-monitoring scenario that results in steady state AT activity levels within the target range as quickly as practically feasible. Data from a second clinical trial provided additional external validation and confirmed the accuracy of the dosing model for both groups of patients.
Topics: Antithrombin III Deficiency; Antithrombins; Drug Monitoring; Female; Humans; Male; Models, Biological; Recombinant Proteins
PubMed: 24335249
DOI: 10.1177/1076029613516188 -
Internal Medicine (Tokyo, Japan) Mar 2023Antithrombin resistance (ATR) is a newly identified strong genetic predisposition to venous thromboembolism (VTE) caused by genetic variations in prothrombin with... (Review)
Review
Antithrombin resistance (ATR) is a newly identified strong genetic predisposition to venous thromboembolism (VTE) caused by genetic variations in prothrombin with substitutions of Arg at position 596 with either Leu, Gln, or Trp. In the present report, we identified a missense variant p.Arg596Gln in 3 patients from 2 families with unprovoked VTE who each experienced their first VTE event at 19, 67, and 19 years old. The three patients did not show any positive markers for thrombophilia on routine testing, suggesting that patients with unprovoked VTE who have negative findings on thrombophilia tests may carry a prothrombin variant with ATR.
Topics: Humans; Venous Thromboembolism; Antithrombins; Prothrombin; Antithrombin III; Anticoagulants; Thrombophilia; Risk Factors
PubMed: 35945029
DOI: 10.2169/internalmedicine.9718-22