-
The Journal of Biological Chemistry Apr 1988Antithrombin Northwick Park and antithrombin Glasgow are functionally variant antithrombins with impaired abilities to interact with thrombin. Thrombosis is associated...
Single amino acid substitutions in the reactive site of antithrombin leading to thrombosis. Congenital substitution of arginine 393 to cysteine in antithrombin Northwick Park and to histidine in antithrombin Glasgow.
Antithrombin Northwick Park and antithrombin Glasgow are functionally variant antithrombins with impaired abilities to interact with thrombin. Thrombosis is associated with their inheritance. Both of the purified, reduced, and S-carboxymethylated variant antithrombins were treated with cyanogen bromide and the major pools of each containing the amino acid sequence Gly339-Met423 were isolated. Following treatment of these pools with trypsin, fast atom bombardment mass spectrometry identified tryptic peptides (found also in normal antithrombin treated in the same way) that corresponded to amino acid sequences Gly339-Lys370 and Val400-Met423. The tryptic peptides, corresponding to amino acid sequences Ala371-Arg393 and Ser394-Arg399 were present in both variant preparations in greatly reduced amounts compared to a normal antithrombin preparation. However, two novel tryptic peptides of molecular mass (M + H)+ 2976 and 2952 were identified in the digests of antithrombin Northwick Park and Glasgow, respectively. Further analyses of these novel tryptic peptides were carried out by V8 protease treatment and sequential Edman degradation coupled with mass spectrometric analysis of the shortened peptides. This established that these peptides comprised the amino acid sequence Ala371-Arg399, but with single amino acid substitutions at the reactive site, Arg393 replaced by Cys (in antithrombin Northwick Park) and by His (in antithrombin Glasgow).
Topics: Amino Acid Sequence; Antithrombin III; Arginine; Cyanogen Bromide; Cysteine; Genetic Variation; Histidine; Humans; Mass Spectrometry; Molecular Sequence Data; Peptide Fragments; Serine Endopeptidases; Thrombosis; Trypsin
PubMed: 3162733
DOI: No ID Found -
Critical Care Medicine Dec 2013To test the hypothesis that restoration of antithrombin plasma concentrations attenuates vascular leakage by inhibiting neutrophil activation through syndecan-4 receptor...
OBJECTIVE
To test the hypothesis that restoration of antithrombin plasma concentrations attenuates vascular leakage by inhibiting neutrophil activation through syndecan-4 receptor inhibition in an established ovine model of acute lung injury.
DESIGN
Randomized controlled laboratory experiment.
SETTING
University animal research facility.
SUBJECTS
Eighteen chronically instrumented sheep.
INTERVENTIONS
Following combined burn and smoke inhalation injury (40% of total body surface area, third-degree flame burn; 4 × 12 breaths of cold cotton smoke), chronically instrumented sheep were randomly assigned to receive an IV infusion of 6 IU/kg/hr recombinant human antithrombin III or normal saline (n = 6 each) during the 48-hour study period. In addition, six sham animals (not injured, continuous infusion of vehicle) were used to obtain reference values for histological and immunohistochemical analyses.
MEASUREMENTS AND MAIN RESULTS
Compared to control animals, recombinant human antithrombin III reduced the number of neutrophils per hour in the pulmonary lymph (p < 0.01 at 24 and 48 hr), alveolar neutrophil infiltration (p = 0.04), and pulmonary myeloperoxidase activity (p = 0.026). Flow cytometric analysis revealed a significant reduction of syndecan-4-positive neutrophils (p = 0.002 vs control at 24 hr). Treatment with recombinant human antithrombin III resulted in a reduction of pulmonary nitrosative stress (p = 0.002), airway obstruction (bronchi: p = 0.001, bronchioli: p = 0.013), parenchymal edema (p = 0.044), and lung bloodless wet-to-dry-weight ratio (p = 0.015). Clinically, recombinant human antithrombin III attenuated the increased pulmonary transvascular fluid flux (12-48 hr: p ≤ 0.001 vs control each) and the deteriorated pulmonary gas exchange (12-48 hr: p < 0.05 vs control each) without increasing the risk of bleeding.
CONCLUSIONS
The present study provides evidence for the interaction between antithrombin and neutrophils in vivo, its pathophysiological role in vascular leakage, and the therapeutic potential of recombinant human antithrombin III in a large animal model of acute lung injury.
Topics: Acute Lung Injury; Airway Obstruction; Animals; Antithrombin III; Antithrombins; Burns; Capillary Permeability; Cell Movement; Disease Models, Animal; Edema; Female; Lung; Neutrophil Activation; Neutrophils; Peroxidase; Pulmonary Gas Exchange; Random Allocation; Receptors, G-Protein-Coupled; Recombinant Proteins; Sheep; Smoke Inhalation Injury; Syndecan-4
PubMed: 24107637
DOI: 10.1097/CCM.0b013e318298ad3a -
Anaesthesia May 1998
Review
Topics: Antithrombin III; Antithrombin III Deficiency; Disseminated Intravascular Coagulation; Humans; Postoperative Complications; Serine Proteinase Inhibitors; Shock, Septic; Thrombophlebitis
PubMed: 9659067
DOI: 10.1111/j.1365-2044.1998.tb15155.x -
Blood Apr 2023Factor IXa (FIXa) plays a pivotal role in coagulation by contributing to FX activation via the intrinsic pathway. Although antithrombin (AT) and other plasma inhibitors...
Factor IXa (FIXa) plays a pivotal role in coagulation by contributing to FX activation via the intrinsic pathway. Although antithrombin (AT) and other plasma inhibitors are thought to regulate FIXa procoagulant function, the impact of FIXa inhibition on thrombin generation and clot formation in vivo remains unclear. Here, we generated FIXa variants with altered reactivity to plasma inhibitors that target the FIXa active site but maintain procoagulant function when bound to its cofactor, FVIIIa. We found that selected FIXa variants (eg, FIXa-V16L) have a prolonged activity half-life in the plasma due, in part, to AT resistance. Studies using hemophilia B mice have shown that delayed FIXa inhibition has a major impact on reducing the bleeding phenotype and promoting thrombus formation following administration of FIX protein. Overall, these results demonstrate that the regulation of FIXa inhibition contributes in a major way to the spatial and temporal control of coagulation at the site of vascular injury. Our findings provide novel insights into the physiological regulation of FIXa, enhance our understanding of thrombus formation in vivo via the intrinsic pathway, and suggest that altering FIXa inhibition could have therapeutic benefits.
Topics: Animals; Mice; Factor IXa; Blood Coagulation; Anticoagulants; Blood Coagulation Tests; Hemophilia B; Antithrombin III
PubMed: 36724452
DOI: 10.1182/blood.2022018083 -
The Journal of Biological Chemistry Oct 1988We investigated the kinetics of the inhibitory action of antithrombin III and antithrombin III plus heparin during the activation of factor X by factor IXa. Generation...
We investigated the kinetics of the inhibitory action of antithrombin III and antithrombin III plus heparin during the activation of factor X by factor IXa. Generation and inactivation curves were fitted to a three-parameter two-exponentional model to determine the pseudo first-order rate constants of inhibition of factor IXa and factor Xa by antithrombin III/heparin. In the absence of heparin, the second-order rate constant of inhibition of factor Xa generated by factor IXa was 2.5-fold lower than the rate constant of inhibition of exogenous factor Xa. It appeared that phospholipid-bound factor X protected factor Xa from inactivation by antithrombin III. It is, as yet, unclear whether an active site or a nonactive site interaction between factor Xa and factor X at the phospholipid surface is involved. The inactivation of factor IXa by antithrombin III was found to be very slow and was not affected by phospholipid, calcium, and/or factor X. With unfractionated heparin above 40 ng/ml and antithrombin III at 200 nM, the apparent second-order rate constant of inhibition of exogenous and generated factor Xa were the same. Thus, in this case phospholipid-bound factor X did not protect factor Xa from inhibition. In the presence of synthetic pentasaccharide heparin, however, phospholipid-bound factor X reduced the rate constant about 5-fold. Pentasaccharide had no effect on the factor IXa/antithrombin III reaction. Unfractionated heparin (1 micrograms/ml) stimulated the antithrombin III-dependent inhibition of factor IXa during factor X activation 400-fold. In the absence of reaction components this stimulated was 65-fold. We established that calcium stimulated the heparin-dependent inhibition of factor IXa.
Topics: Algorithms; Antithrombin III; Calcium; Factor IXa; Factor X; Factor Xa; Heparin; Humans; Phospholipids; Serine Proteinase Inhibitors
PubMed: 3262615
DOI: No ID Found -
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 -
Clinical and Applied... 2022The efficacy of antithrombin (AT) supplementation against septic disseminated intravascular coagulation (DIC) may depend on various pre-existing factors, particularly...
INTRODUCTION
The efficacy of antithrombin (AT) supplementation against septic disseminated intravascular coagulation (DIC) may depend on various pre-existing factors, particularly the AT dose and multiple organ dysfunction severity. This study aimed to identify the impactful factors for early DIC recovery.
METHODS
Patients' clinical records, including AT therapy and septic DIC data, were retrospectively extracted from January 2015 to December 2020. The patients were divided into those with early DIC recovery (n = 34) and those without (n = 37). Multivariate logistic regression analysis determined significant independent factors. Time-to-event analysis confirmed how these factors affected the DIC recovery time.
RESULTS
The AT dose per patient body weight (odds ratio [95% confidence interval]: 2.879 [1.031-8.042], = 0.044) and pre-existing organ dysfunction severity (0.333 [0.120-0.920], = 0.034) were significant independent factors affecting early DIC recovery. A higher AT dose significantly shortened the DIC recovery time among patients with severe organ dysfunction ( < 0.01), but not among non-severe patients ( = 0.855).
CONCLUSION
The therapeutic efficacy of AT treatment for septic DIC might depend on the severity of pre-existing organ failure and the AT dose per patient body weight.
Topics: APACHE; Age Factors; Aged; Aged, 80 and over; Antithrombins; Body Weight; Comorbidity; Disseminated Intravascular Coagulation; Female; Humans; Logistic Models; Male; Middle Aged; Multiple Organ Failure; Organ Dysfunction Scores; Patient Acuity; Retrospective Studies; Sepsis; Sex Factors
PubMed: 35187966
DOI: 10.1177/10760296221080942 -
Clinical and Applied... 2022Recombinant antithrombin gamma (rAT) is reported as an effective drug for patients with disseminated intravascular coagulation (DIC) in Japan. As the appropriate dose...
Recombinant antithrombin gamma (rAT) is reported as an effective drug for patients with disseminated intravascular coagulation (DIC) in Japan. As the appropriate dose and targeted AT activity remain unknown, this study aimed to determine these aspects for sepsis-induced DIC. Thirty-one patients with septic shock and DIC with AT levels <70% were treated with rAT between May 2018 and December 2020. The recovery rates from DIC were 32.2% and 63.3% on day 3 and 5 post administration, respectively. Recovery and survival rates were significantly higher in patients who achieved AT activity ≥70% or 80% on day 3 post administration. Receiver operating characteristic curve analysis revealed that the cutoff values of post-treatment AT activity on day 3 for 28-day survival and 5-day recovery from DIC were 79.5% and 81.5%, respectively. Patients who did not achieve AT activity ≥80% on day 3 presented a lower base level of AT activity and lower dose supplementation. Our results suggest that targeted AT activity should be at least 70%, and ideally 80%, and sufficient doses to maintain this activity are required to achieve better outcomes.
Topics: Humans; Disseminated Intravascular Coagulation; Antithrombins; Treatment Outcome; Antithrombin III; Anticoagulants; Sepsis
PubMed: 36380520
DOI: 10.1177/10760296221135790 -
The Journal of Extra-corporeal... Jun 2022Hematologic complications are a source of morbidity and mortality for patients receiving extracorporeal membrane oxygenation (ECMO) support. There is no consensus... (Observational Study)
Observational Study
Hematologic complications are a source of morbidity and mortality for patients receiving extracorporeal membrane oxygenation (ECMO) support. There is no consensus strategy for monitoring anticoagulation for children supported with ECMO. This study evaluated a novel measurement of anticoagulation for children on ECMO. This was a single-center observational study of children supported with ECMO from 2015 to 2020. Each patient's current unfractionated heparin dose was multiplied by the current antithrombin III (AT) level to obtain a novel anticoagulation value, the heparin-antithrombin product (HAP). This value was compared with the heparin dose, AT, and activated clotting time (ACT) to predict anti-Xa value using linear correlation and decision tree methods. Data were obtained from 128 patients supported with ECMO. The HAP value was more highly correlated with anti-Xa level than heparin dose, AT level, and ACT. This correlation was highest in the neonatal population ( = .7). The variable importance metrics from the regression tree and random forest models both identified the HAP value as the most influential predictor variable for anti-Xa value. The HAP value is more highly correlated with the anti-Xa level than heparin dose, AT level, or ACT. Further research is needed to evaluate the effectiveness of the HAP value as a measurement of anticoagulation for children on ECMO.
Topics: Anticoagulants; Antithrombin III; Antithrombins; Child; Extracorporeal Membrane Oxygenation; Heparin; Humans; Infant, Newborn; Retrospective Studies
PubMed: 35928336
DOI: 10.1182/ject-115-122 -
The Journal of Biological Chemistry Jan 1978Human antithrombin III was found to contain covalently linked N-acetylglucosamine, mannose, galactose, and sialic acid in a molar ratio of approximately 1:1:0.6:1....
Human antithrombin III was found to contain covalently linked N-acetylglucosamine, mannose, galactose, and sialic acid in a molar ratio of approximately 1:1:0.6:1. Sialic acid was released upon treatment with neuraminidase. The modified glycoprotein retained the capability to inhibit thrombin and to bind with heparin. Antithrombin III isolated by different procedures was also found to contain glucose in an approximately equimolar ratio with N-acetylglucosamine. Th" glucose-containing component was extractable with lipid solvents and shown to be beta-glucosylceramide. This glycolipid is tightly complexed with antithrombin III and could not be separated by fractional precipitations or ion exchange gels. Although it remains to be established whether the inhibitory actions of antithrombin III are affected by glucosylceramide, the relative amounts which are bound suggest that antithrombin III may be a significant carrier of the glycolipid.
Topics: Antithrombins; Cerebrosides; Glucose; Glycolipids; Heparin; Hexoses; Humans; Neuraminidase; Sialic Acids; Thrombin
PubMed: 618863
DOI: No ID Found