scholarly journals Purified radiolabeled antithrombin III metabolism in three families with hereditary AT III deficiency: application of a three-compartment model

Blood ◽  
1986 ◽  
Vol 67 (1) ◽  
pp. 93-98
Author(s):  
EA Knot ◽  
E de Jong ◽  
JW ten Cate ◽  
AH Iburg ◽  
CP Henny ◽  
...  
Keyword(s):  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Compartment Model ◽  
Control Group ◽  
Surface Binding ◽  
Catabolic Rate ◽  
At Iii ◽  
Three Compartment Model ◽  
The Mean

Purified human radioiodinated antithrombin III (125I-AT III) was used to study its metabolism in six members from three different families with a known hereditary AT III deficiency. Six healthy volunteers served as a control group. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and crossed immunoelectrophoresis (CIE) showed the purified AT III to be homogeneous. Amino acid analysis of the protein revealed a composition identical to a highly purified internal standard. The specific activity was 5.6 U/mg. Analysis of plasma radioactivity data was performed, using a three-compartment model. Neither plasma disappearance half-times nor fractional catabolic rate constants differed significantly between patients and control subjects. The mean absolute catabolic rate in the patient group was significantly lower than that of the control group at 2.57 +/- 0.44 and 4.46 +/- 0.80 mg/kg/day, respectively. In addition, the mean patient alpha 1-phase, flux ratio (k1,2 and k2,1) of the second compartment alpha 2-phase and influx (k3,1) of the third compartment were significantly reduced as compared with control values. It has been tentatively concluded that the observed reduction in the second compartment may be caused by a decrease in endothelial cell surface binding.

scholarly journals Purified radiolabeled antithrombin III metabolism in three families with hereditary AT III deficiency: application of a three-compartment model

Blood ◽  
1986 ◽  
Vol 67 (1) ◽  
pp. 93-98 ◽  
Author(s):  
EA Knot ◽  
E de Jong ◽  
JW ten Cate ◽  
AH Iburg ◽  
CP Henny ◽  
...  
Keyword(s):  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Compartment Model ◽  
Control Group ◽  
Surface Binding ◽  
Catabolic Rate ◽  
At Iii ◽  
Three Compartment Model ◽  
The Mean

Abstract Purified human radioiodinated antithrombin III (125I-AT III) was used to study its metabolism in six members from three different families with a known hereditary AT III deficiency. Six healthy volunteers served as a control group. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and crossed immunoelectrophoresis (CIE) showed the purified AT III to be homogeneous. Amino acid analysis of the protein revealed a composition identical to a highly purified internal standard. The specific activity was 5.6 U/mg. Analysis of plasma radioactivity data was performed, using a three-compartment model. Neither plasma disappearance half-times nor fractional catabolic rate constants differed significantly between patients and control subjects. The mean absolute catabolic rate in the patient group was significantly lower than that of the control group at 2.57 +/- 0.44 and 4.46 +/- 0.80 mg/kg/day, respectively. In addition, the mean patient alpha 1-phase, flux ratio (k1,2 and k2,1) of the second compartment alpha 2-phase and influx (k3,1) of the third compartment were significantly reduced as compared with control values. It has been tentatively concluded that the observed reduction in the second compartment may be caused by a decrease in endothelial cell surface binding.

scholarly journals Antithrombin Milano: a new variant with monomeric and dimeric inactive antithrombin III

Blood ◽  
1985 ◽  
Vol 65 (2) ◽  
pp. 496-500 ◽  
Author(s):  
M Wolf ◽  
C Boyer ◽  
A Tripodi ◽  
D Meyer ◽  
MJ Larrieu ◽  
...  
Keyword(s):  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Reactivity ◽  
Two Dimensional ◽  
Western Blots ◽  
Sds Page ◽  
New Variant ◽  
At Iii ◽  
Monospecific Antisera

Abstract A qualitative defect of antithrombin III (AT III) has been demonstrated over three generations in eight members of an Italian family by the discrepancy between a normal amount of antigen and decreased antithrombin and anti-Xa activity in the presence or in the absence of heparin. By two-dimensional immunoelectrophoresis in the absence of heparin, two peaks of AT III were present in all patients' plasma. AT III was purified from normal and propositus plasma by sulfate dextran precipitation followed by heparin affinity chromatography. The elution profile of the patient's AT III was abnormal and allowed the separation of two populations of AT III, normal and abnormal. The first fraction (normal AT III) contained AT III activity, migrated as a single peak by two-dimensional immunoelectrophoresis and by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE), demonstrated a single band with a molecular weight (mol wt) identical to that of normal AT III (60,000). Conversely, the last fraction, devoid of AT III activity, migrated as a single abnormal peak by two-dimensional immunoelectrophoresis in the absence of heparin. By SDS-PAGE, two bands were observed: one with a mol wt of 60,000 and a second one with a mol wt of 120,000. Western blots clearly demonstrated cross-reactivity of the 120,000 and 60,000 mol wt bands with monospecific antisera to human AT III. Reduction of the 120,000 mol wt band converted it to a single 60,000 mol wt band, suggesting the presence of an abnormal dimeric form of AT III. The name AT III Milano is proposed for this new variant.

Antithrombin III: Biodistribution in Healthy Volunteers

1987 ◽  
Vol 58 (04) ◽  
pp. 1008-1011 ◽  
Author(s):  
E A R Knot ◽  
E de Jong ◽  
J W ten Cate ◽  
Liem Kian Gie ◽  
E A van Royen
Keyword(s):  
Healthy Volunteers ◽  
Antithrombin Iii ◽  
Compartment Model ◽  
Fixed Time ◽  
Whole Body ◽  
Catabolic Rate ◽  
At Iii ◽  
Great Vessels ◽  
Static Images ◽  
Liver Region

SummaryFive healthy volunteers were injected intravenously with 73-90 uCi purified human 131I-Antithrombin III (AT III), specific biological activity 5.6 U/mg. The tracer data were analysed using a three compartment model. The plasma radioactivity half life was 66.2 ± 1.2 (sem) h, the fractional catabolic rate constant of the plasma pool was 0.025 ± 0.002 (sem) h-1. These data were comparable with those described in the literature. Because of the difficulty in translating the mathematical analysis of various compartments into the biological model, biodistribiition was monitored by a gamma camera linked to a DEC PDF 11/34 computer system. Dynamic and static images were obtained at fixed time intervals following the injection of 131I-AT III.Whole body scanning at intervals between the time of injection (t=0)and t=24.5 h showed 131I-AT III distribution over the heart, lungs, liver, spleen and great vessels. Dynamic scanning was performed over the heart, spleen and liver. Overlayed frames in the first ten minutes after the 131I-AT III injection showed the following radioactivity expressed as percentage of the injected dose; 5.9% ± 0.3(sem) over the heart, 10.6% ± 0.9 (sem) over the liver and 1.1% ⊥ 0.1(sem) over the spleen.A slower decline of the radioactivity between t = 0 and t = 24 h; (19%) was measured over the liver compared with the radioactivity disappearance over the heart region. This shows, in combination with the fact that the radioactivity disappearance over the heart was identical with the radioactivity decline measured in the plasma samples that retention of 131I-AT III occurred in the liver. Heparin iv injected 6 h after the 131I-AT III injection in two volunteers induced a sharp increase of radioactivity over the liver region during the scanning demonstrating that heparin enhances the 131I-AT III uptake in the liver.

scholarly journals Antithrombin III Activity, Measured with a Chromogenic Substrate, in Patients with Hepatic Cirrhosis, with Prosthetic Heart Valves and During Parenteral Administration of Medroxyprogesterone Acetate as a Contraceptive Agent

1977 ◽  
Author(s):  
G. Baele ◽  
E. Matthys ◽  
G. De Cock ◽  
M. Thiery ◽  
F. Barbier
Keyword(s):  
Medroxyprogesterone Acetate ◽  
Heart Valves ◽  
Hepatic Cirrhosis ◽  
Antithrombin Iii ◽  
Prosthetic Heart Valve ◽  
Control Group ◽  
Prosthetic Heart Valves ◽  
Chromogenic Substrate ◽  
At Iii ◽  
The Mean

Antithrombin III (AT III) activity was assayed on heat defibrinated plasma using a synthetic chromogenic substrate, benzoyl-Phe-Val-Arg-p. nitroanilide. AT III activity in 30 normal subjects averaged 95% ± 18 (± 1 SD). As AT III is synthesized in the liver, we measured its activity in 72 samples from 44 patients with hepatic cirrhosis. The mean activity (49% ± 23) was significantly lower than in the control group. AT III activity was also measured in 32 patients with prosthetic heart valves receiving sodium warfarin therapy. The mean activity in this group (90% ± 23) fell in the normal range. It also did not differ significantly from the mean activity (95% ± 29) in a similar (matched for sex and age) group of 32 subjects, also treated with sodium warfarin but for other reasons than bearing a prosthetic heart valve. As low AT III levels have been reported in women using a combined oral contraceptive, we also measured AT III in 19 women receiving trimonthly injections of 150 mg of medroxyprogesterone acetate as contraception. AT III levels in this group of women (102% ± 23) were found to be normal. So the oestrogen in the combined contraceptive may be responsible for the reported fall in serum AT III activity.

scholarly journals Iodine-mediated inactivation of lipid- and nonlipid-enveloped viruses in human antithrombin III concentrate

Blood ◽  
1995 ◽  
Vol 86 (2) ◽  
pp. 791-796 ◽  
Author(s):  
F Highsmith ◽  
H Xue ◽  
X Chen ◽  
L Benade ◽  
J Owens ◽  
...  
Keyword(s):  
Sindbis Virus ◽  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Human Albumin ◽  
Enveloped Viruses ◽  
Complete Inactivation ◽  
Ensure Patient Safety ◽  
Sds Page ◽  
At Iii

Human plasma-derived protein concentrates intended for clinical use must be treated for viral inactivation to ensure patient safety. This study explored the use of liquid iodine for inactivation of several lipid- and nonlipid-enveloped viruses in an antithrombin III (AT-III) concentrate. Iodine at levels of 0.01% to 0.02% caused between 43% and 94% loss of AT-III activity, as well as degradation of AT-III as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. However, addition of up to 0.1% human albumin protected the AT-III against both inactivation and fragmentation. At albumin levels sufficient to retain greater than 75% of AT-III activity, greater than 6 logs of sindbis, encephalomyocarditis, and vesicular stomatitis viruses, greater than 4 logs of pseudorabies, and greater than 3 logs of human immunodeficiency virus were inactivated. Except with sindbis virus, this represented complete inactivation of all the viruses spiked into the AT-III concentrate.

scholarly journals Expression in a cell-free system of normal and variant forms of human antithrombin III. Ability to bind heparin and react with alpha-thrombin

Blood ◽  
1990 ◽  
Vol 76 (8) ◽  
pp. 1521-1529
Author(s):  
RC Austin ◽  
RA Rachubinski ◽  
F Fernandez-Rachubinski ◽  
MA Blajchman
Keyword(s):  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Translation Product ◽  
Free System ◽  
Reactive Center ◽  
At Iii ◽  
Free Translation

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.

scholarly journals Expression in a cell-free system of normal and variant forms of human antithrombin III. Ability to bind heparin and react with alpha-thrombin

Blood ◽  
1990 ◽  
Vol 76 (8) ◽  
pp. 1521-1529 ◽  
Author(s):  
RC Austin ◽  
RA Rachubinski ◽  
F Fernandez-Rachubinski ◽  
MA Blajchman
Keyword(s):  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Translation Product ◽  
Free System ◽  
Reactive Center ◽  
At Iii ◽  
Free Translation

Abstract 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.

scholarly journals Antithrombin Milano: a new variant with monomeric and dimeric inactive antithrombin III

Blood ◽  
1985 ◽  
Vol 65 (2) ◽  
pp. 496-500
Author(s):  
M Wolf ◽  
C Boyer ◽  
A Tripodi ◽  
D Meyer ◽  
MJ Larrieu ◽  
...  
Keyword(s):  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Reactivity ◽  
Two Dimensional ◽  
Western Blots ◽  
Sds Page ◽  
New Variant ◽  
At Iii ◽  
Monospecific Antisera

A qualitative defect of antithrombin III (AT III) has been demonstrated over three generations in eight members of an Italian family by the discrepancy between a normal amount of antigen and decreased antithrombin and anti-Xa activity in the presence or in the absence of heparin. By two-dimensional immunoelectrophoresis in the absence of heparin, two peaks of AT III were present in all patients' plasma. AT III was purified from normal and propositus plasma by sulfate dextran precipitation followed by heparin affinity chromatography. The elution profile of the patient's AT III was abnormal and allowed the separation of two populations of AT III, normal and abnormal. The first fraction (normal AT III) contained AT III activity, migrated as a single peak by two-dimensional immunoelectrophoresis and by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE), demonstrated a single band with a molecular weight (mol wt) identical to that of normal AT III (60,000). Conversely, the last fraction, devoid of AT III activity, migrated as a single abnormal peak by two-dimensional immunoelectrophoresis in the absence of heparin. By SDS-PAGE, two bands were observed: one with a mol wt of 60,000 and a second one with a mol wt of 120,000. Western blots clearly demonstrated cross-reactivity of the 120,000 and 60,000 mol wt bands with monospecific antisera to human AT III. Reduction of the 120,000 mol wt band converted it to a single 60,000 mol wt band, suggesting the presence of an abnormal dimeric form of AT III. The name AT III Milano is proposed for this new variant.

Antithrombin III, Heparin Cofactor and Antifactor Xa in Relation to Age, sex And Pathological Conditions

1979 ◽  
Author(s):  
F. Panicucci ◽  
A. Sacripanti ◽  
E. Pinori ◽  
M. Vispi ◽  
B. Conte ◽  
...  
Keyword(s):  
Oral Contraceptive ◽  
Antithrombin Iii ◽  
Normal Subjects ◽  
Cerebral Thrombosis ◽  
Positive Correlation ◽  
Pathological Conditions ◽  
At Iii ◽  
The Mean ◽  
Antifactor Xa ◽  
Cofactor Activity

Determinations of AT-III activity, heparin cofactor activity, antifactor Xa activity and AT-III protein were carried out in 200 healthy adults, evenly distributed within age and sex groups, in 60 patients with cerebral thrombosis and in 20 oral contraceptive users. There was a positive correlation between AT-III protein and its activitiesin normal subjects and in patients with cerebral thrombosis. In oral contraceptive users the positive correlation was between AT-III protein and its activities, antifactor Xa activity excepted. The mean AT-III protein and heparin cofactor activity values decreased in males with age and were significantly lower in the groups between 50 and 70 years. The mean AT-III protein and heparin cofactor activity values decreased slightly in women in fertile age and were lower in the 40 to 50 age-group. The mean AT-III protein and its activities values did not show any variation in the patients with cerebral thrombosis. The mean antifactor Xa activity value in the women, taking the pill for 3 months, decreased, whereas the other AT-III activities and AT-III protein were unchanged.

scholarly journals In Vitro Characterization of Rabbit Thrombin, Antithrombin III, and Thrombin-Antithrombin III Complex, and Determination of Their Survival Times in Vivo

1977 ◽  
Author(s):  
Christine N. Vogel ◽  
Kingdon S. Henry ◽  
Roger L. Lundblad
Keyword(s):  
Gel Electrophoresis ◽  
Half Life ◽  
Antithrombin Iii ◽  
Specific Activity ◽  
Sodium Dodecyl ◽  
In Vivo Studies ◽  
Survival Times ◽  
At Iii

Our intention is to study the interaction of rabbit thrombin with antithrombin III (AT-III) in vitro and in vivo. After activation of crude prothrombin with tissue thromboplastin and CaCl2, thrombin was purified and showed two species of thrombin with molecular weights of 36,000 and 39,000 daltons as determined by sodium dodecyl sulfate discontinuous gel electrophoresis. Rabbit AT-III was purified using a heparin agarose column and had a molecular weight of 55,000 daltons. The inhibition of thrombin by AT-III was followed by fibrinogen clotting assays and an AT-III-thrombin complex was observed on gel electrophoresis. For the in vivo studies both thrombin and AT-III were radiolabelled with Na125i using the solid state lactoperoxidase method and retained 99% of the pre-iodinated specific activity. Radiolabelled thrombin and a radiolabelled AT-III-thrombin complex were injected into different rabbits. The rate of removal of both was very similar with a half-life of approximately 9 hours. When radiolabelled AT-III was injected, the half-life was approximately 60 hours. Since the disappearance rate of thrombin more closely approximates that of the preformed AT-III-thrombin complex and is clearly shorter than the turnover rate of AT-III, the possibility is raised that thrombin combines in vivo with a native inhibitor such as AT-III and may in fact be removed from the circulation as a complex rather than as a native molecule.

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