Isolation of Antithrombin III Without Interfering with Ethanol Fractionation System

1979 ◽  
Author(s):  
M. Wickerhauser ◽  
C. Williams
Keyword(s):  
Polyethylene Glycol ◽  
Large Scale ◽  
Antithrombin Iii ◽  
Plasma Fraction ◽  
Peg 4000 ◽  
At Iii ◽  
Elution Step ◽  
Final Purification

We described previously the isolation of antithrombin III (AT III) from the 20% polyethylene glycol (PEG 4000) supernatant of plasma or of Cohn Fraction IV-1 (Vox Sang., in press). The first of these two methods gives good recoveries of AT III but cannot be integrated with the conventional ethanol fractionation system due to the presence of PEG in the remaining plasma fraction, while Cohn Fr action IV-1, a byproduct of routine fractionation, is a poor source of AT III in terms of yield. Our modified’method involves batchwise adsorption of AT III from plasma (cryosup-ernatant) with heparin-Sepharose, using one volume of gel for each 50 volumes of plasma. The unadsorbed plasma can be used for ethanol fractionation. The AT III eluate is further purified by precipitation of some impurities including HB Ag, if present, with 20% PEG. Final purification of AT III and removal of PEG is achieved by a second adsorption-elution step on heparin-Sepharose. This method is economical and suitable for large scale application. Recovery of a highly purified AT III was 25%.

scholarly journals Large Scale Method for the Isolation of Antithrombin III.

1977 ◽  
Author(s):  
M. Wickerhauser ◽  
C. Williams
Keyword(s):  
Polyethylene Glycol ◽  
Hepatitis B ◽  
Gel Electrophoresis ◽  
Large Scale ◽  
Antithrombin Iii ◽  
Scale Method ◽  
Sterile Filtration ◽  
Peg 4000 ◽  
At Iii ◽  
Hepatitis B Antigen

Antithrombin III (AT III) concentrates may be of value in the treatment of various hypercoagulable states associated with congenital or acquired AT III deficiency. We have developed a large scale method for isolation of AT III from plasma or from Cohn fraction IV-1, an unused byproduct of routineplasma fractionation. The method consists of the following steps:(a) precipitation of the starting plasma or Cohn fraction IV-1 extract with 2 0% polyethylene glycol (PEG)4000 to remove a number of impurities including hepatitis B antigen if present;(b) batchwise adsorption to and elution from heparin-Sepharose using one volume of gel for each 50 liters of the 20% PEG supernatant; and (c) concentration of the eluted AT III by bulk lyophilization, followed by desalting on Sephadex G-50, sterile filtration and final lyophilization. In two large scale experiments, 7.1 and 8.7 grams of AT III, determined as antigen by radial immunodiffusion, corresponding to 25,000 and 31,000 plasma equivalents, were recovered from 100 liter plasma and 15 kg Cohn fraction IV-1 batches respectively. Both preparations were over 95% pure by disc gel electrophoresis and had an activity to antigen ratio close to that of AT III in plasma. Both preparations were nonpyrogenic and met all other FDA requirements for biologic products.

Antithrombin III Progressive Function: A Biochemical Analysis

1981 ◽  
Author(s):  
E D Gomperts ◽  
P Izadi
Keyword(s):  
Biochemical Analysis ◽  
Antithrombin Iii ◽  
Partial Purification ◽  
Thrombin Inhibition ◽  
Electrophoretic Patterns ◽  
Peg 4000 ◽  
At Iii ◽  
Specific Antisera ◽  
Slow Moving ◽  
Heparin Affinity

Antithrombin III (AT III) was measured in the various fractions obtained during the partial purification of AT III-Heparin Cofactor (AT III-HCF) via a heparin-bound affinity chromatography system. AT III antigen and HCF activity was present to some extent in all fractions, but significant progressive function was observed only in that obtained via PEG-4000 precipitation. This precipitate was applied to a Sephacryl-200 column. Fractions were collected and those demonstrating maximum AT III antigen and progressive thrombin inhibition were pooled and reapplied to the washed Sephacryl S-200 column. Fractions were collected and assayed via specific antisera for AT III, α1AT, α2M and α1 acid glycoprotein (α1AG) . AT III antigen and progressive function were confined primarily to one peak containing virtually no α2M, a low level of α1AT, and moderate quantities of α1AG. PAGE of the component showed AT III antigen separating into two bands. Assessment of the two other prominent bands showed no reactivity with antisera to α2M, α1AT or α1AG. PAGE-SDS showed a different pattern with the AT III doublet moving as a single band. These electrophoretic patterns were identical with that of the AT III-HCF purified by the heparin-affinity system. AT III antigen-two dimensional immunoelectrophoresis in the presence of heparin of both the Sephacryl and heparin- affinity purified components was very different with the Sephacryl purified AT III protein showing both a fast peak and a very prominent slow moving hump. The AT III heparin purified component showed primarily a fast component. These two fractions differed in one other respect in that AT III inhibition of thrombin was activated, heparin-like, by EDTA. This effect was totally absent in the heparin affinity purified AT III-HCF. On the basis of these observations it is postulated that AT III purified by the affinity system is biochemically altered resulting in an inability to respond functionally to non-heparin activating stimuli.

PURIFICATION AND CHARACTERIZATION OF GENTECHNOLOGICALLY PREPARED ANTITHROMBIN III

1987 ◽  
Author(s):  
H E Karges ◽  
G Zettlemeiβl ◽  
H Naumann ◽  
U Eberhard ◽  
M Bröker
Keyword(s):  
Large Scale ◽  
Antithrombin Iii ◽  
Calf Serum ◽  
Yeast Cells ◽  
Growth Media ◽  
Isolation And Purification ◽  
Authentic Material ◽  
At Iii ◽  
Human Proteins

Isolation and purification of antithrombin III (AT III) by affinity chromatography on immobilized heparin is a standard method for the large scale preparation of this protein from human or animal plasma. Hence, after AT III became available by gentechnological methods, we tried to adapt this procedure for the isolation of AT III from supernatants of mammalian- and yeast-cells. Indeed, it was possible to use this method also for the isolation of the recombinant gene products. Since, however, the cell growth media contain heterologous protein or peptide mixtures like fetal calf serum, the method had to be improved to avoid the adsorption of non human proteins or peptides. We are now able to purify AT III from CHO-cell-superna-tants to more than 95 % purity. The characterization of this AT III-product by double immuno diffusion revealed that it is immunologically totally identical with the authentic material from plasma. AT III antigen content, progressive inhibitor activity and heparin cofactor activity compare very well in the final product; hence, it is totally active compared to AT III from plasma.In polyacrylamidegel electrophoresis most of the material migrated differently to the authentic material showing 9 bands in equal distance to each other, instead four in the At III from plasma. After degradation with sialinidase from both AT III preparations identical cleavage products were obtained migrating predominantly as a single band. Hence, the electrophoretic heterogeneity seems to be due to a different degree of sialinyla-tion of the products.

Molekularbiologische Grundlagen und Diagnostik der hereditären Defekte von Antithrombin III, Protein C und Protein S

2002 ◽  
Vol 22 (02) ◽  
pp. 57-66
Author(s):  
I. Witt
Keyword(s):  
Protein C ◽  
Antithrombin Iii ◽  
Protein S ◽  
Klinische Relevanz ◽  
At Iii

ZusammenfassungDie enormen Fortschritte in der Molekularbiologie in den letzten Jahren ermöglichten sowohl die Aufklärung der Nukleotidsequenzen der Gene für Antithrombin III (AT III), Protein C (PROC) und Protein S (PROS) als auch die Identifizierung zahlreicher Mutationen bei hereditären Defekten dieser wichtigen Inhibitoren des plasmatischen Gerinnungssystems. Da die Gene für AT III (13,8 kb) und PROC (11,2 kb) nicht groß und relativ leicht zu analysieren sind, gibt es bereits umfangreiche »databases« der Mutationen (50, 73). Für AT III sind 79 und für PROC 160 unterschiedliche Mutationen beschrieben.Sowohl beim AT-III-Mangel als auch beim Protein-C-Mangel hat die Mutationsaufklärung neue Erkenntnisse über die Struktur-Funktions-Beziehung der Proteine gebracht. Beim Protein-C-Mangel steht die klinische Relevanz der DNA-Analyse im Vordergrund, da die Diagnostik des Protein-C-Mangels auf der Proteinebene nicht immer zuverlässig möglich ist.Das Protein-S-Gen ist für die Analytik schwer zugänglich, da es groß ist (80 kb) und außerdem ein Pseudogen existiert. Es sind schon zahlreiche Mutationen bei Patienten mit Protein-S-Mangel identifiziert worden. Eine Database ist bisher nicht publiziert. Die klinische Notwendigkeit zur Mutationsaufklärung besteht ebenso wie beim Protein-C-Mangel. Es ist zu erwarten, dass zukünftig die Identifizierung von Mutationen auch beim Protein-S-Mangel beschleunigt vorangeht.

Immunologic Studies of Antithrombin III Heparin Cofactor in the Newborn

1978 ◽  
Vol 39 (03) ◽  
pp. 624-630 ◽  
Author(s):  
W E Hathaway ◽  
L L Neumann ◽  
C A Borden ◽  
L J Jacobson
Keyword(s):  
Gestational Age ◽  
Antithrombin Iii ◽  
Newborn Infants ◽  
Term Infant ◽  
Sick Newborn ◽  
At Iii ◽  
Thrombotic Complications ◽  
Low Levels ◽  
Collection Methods ◽  
Made In

SummarySerial quantitative immunoelectrophoretic (IE) measurements of antithrombin III heparin cofactor (AT III) were made in groups of well and sick newborn infants classified by gestational age. Collection methods (venous vs. capillary) did not influence the results; serum IE measurements were comparable to AT III activity by a clotting method. AT III is gestational age-dependent, increasing from 28.7% of normal adult values at 28-32 weeks to 50.9% at 37-40 weeks, and shows a gradual increase to term infant levels (57.4%) by 3-4 weeks of age. Infants with the respiratory distress syndrome (RDS) show lower levels of AT III in the 33-36 week group, 22% vs. 44% and in the 37-40 week group, 33.6% vs. 50.9%, than prematures without RDS. Infants of 28-32 week gestational age had only slight differences, RDS = 24%, non-RDS = 28.7%. The lowest levels of AT III were seen in patients with RDS complicated by disseminated intravascular coagulation and those with necrotizing enterocolitis. Crossed IE on representative infants displayed a consistent pattern which was identical to adult controls except for appropriate decreases in the amplitude of the peaks. The thrombotic complications seen in the sick preterm infant may be related to the low levels of AT III.

Antithrombin III and Heparin Cofactor II in Patients with Chronic Renal Failure Undergoing Regular Hemodialysis

1987 ◽  
Vol 57 (03) ◽  
pp. 263-268 ◽  
Author(s):  
P Toulon ◽  
C Jacquot ◽  
L Capron ◽  
M -O Frydman ◽  
D Vignon ◽  
...  
Keyword(s):  
Renal Failure ◽  
Chronic Renal Failure ◽  
Plasma Proteins ◽  
Antithrombin Iii ◽  
Vascular Wall ◽  
Relative Increase ◽  
Heparin Cofactor Ii ◽  
Normal Ranges ◽  
At Iii ◽  
Regular Hemodialysis

SummaryHeparin enhances the inhibition rate of thrombin by both antithrombin III (AT III) and heparin cofactor II (HC II). We studied the activity of these two plasma proteins in patients with chronic renal failure (CRF) undergoing regular hemodialysis as their heparin requirements varied widely. In 77 normal blood donors, normal ranges (mean ± 2 SD) were 82-122% for AT III and 65-145% for HC II. When compared with these controls 82 dialyzed CRF patients had a subnormal AT III activity and a significantly (p <0.001) lower HC II activity. To evaluate the effect of hemodialysis we compared AT III, HC II and total proteins in plasma before and after dialysis in. 24 patients (12 with normal and 12 with low basal HC II activity). AT III and HC II activities significantly (p <0.001) increased in absolute value. When related to total plasma proteins, in order to suppress the influence of hemoconcentration induced by dialysis, AT III decreased significantly (p <0.01) whereas HC II increased slightly but significantly (p <0.01) in the 12 patients with low initial HC II activity. The decrease of AT III induced by heparin administrated during dialysis is likely to account for this relative decrease of AT III activity. A modification of the distribution of both HC II and heparin between the vascular wall and the circulating blood is evoked to explain the relative increase in HC II activity and the need for higher heparin dosage in patients with low HC II levels.

Severe Antithrombin III Deficiency in an Infant Associated with Multiple Arterial and Venous Thromboses

1976 ◽  
Vol 36 (03) ◽  
pp. 495-502 ◽  
Author(s):  
Geoffrey Mendelsohn ◽  
Edward D. Gomperts ◽  
Dennis Gurwitz
Keyword(s):  
Antithrombin Iii ◽  
Adult Life ◽  
Rare Condition ◽  
Male Infant ◽  
Liver Cells ◽  
Liver Pathology ◽  
Fixed Tissue ◽  
Formalin Fixed Tissue ◽  
First Case ◽  
At Iii

SummaryInherited antithrombin III (AT-II, heparin cofactor) deficiency is a rare condition, presenting with thrombotic disease in adult life. This paper reports an 8 months old South African Black male infant with multiple large vessel venous and arterial thromboses, and E. coli septicaemia. This was associated with an extremely low plasma AT-II level. Micronodular cirrhosis and intracytoplasmic hyaline globules in the liver cells were present. These globules were eosinophilic, and PAS-positive after diastase. They measured approximately 5 μ to 30 μ in diameter, occurred singly in the liver cells and were located mainly in the periportal areas. The histological findings in the liver are similar to those observed in α1-antitrypsin (AAT) deficiency in which the intracytoplasmic globules represent accumulation of altered AAT. Immunochemical studies carried out on formalin fixed tissue failed to detect cross reaction material with anti-α1 antitrypsin or anti-AT III antiserum. This is the first case report of AT-III deficiency presenting in infancy. It is also the first case associated with distinctive liver pathology.The available data presented are insufficient to distinguish between an inborn defect and acquired causes of the severely depressed AT-III plasma level and the distinctive liver pathology.

Predictive Value of Decreasing Antithrombin III in Deep-Vein Thrombosis

1980 ◽  
Vol 44 (03) ◽  
pp. 135-137 ◽  
Author(s):  
Thorkild Lund Andreasen
Keyword(s):  
Deep Vein Thrombosis ◽  
Predictive Value ◽  
Coronary Care Unit ◽  
Antithrombin Iii ◽  
Predictive Values ◽  
Vein Thrombosis ◽  
At Iii ◽  
Coronary Care ◽  
Time Of Admission ◽  
Deep Vein

SummaryAntithrombin III (At-III) was measured at the time of admission and two days later in 131 patients laid up in a coronary care unit. The patients were examined for deep-vein thrombosis (DVT) clinically and by means of 125I-fibrinogen scanning. 19 patients developed DVT. In 11 subjects with and 25 without DVT At-III decreased more than 10%. And in 7 with and 17 without DVT At-III decreased more than 15%. One person with DVT had subnormal At-III. By using decrease of At-III or subnormal initial At-III to predict DVT the following predictive value (PV) were found. Decrease ≤ 10%, PV pos.= 0.32 and PV neg. = 0.93. Decrease ≤ 15%, PV pos. = 0.32 and PV neg. = 0.90. The positive predictive values obtained were too low to let decreasing At-III give occasion for prophylactic anticoagulant treatment.

Reactivity of a Hereditary Abnormal Antithrombin III Fraction in the Inhibition of Thrombin and Factor Xa

1980 ◽  
Vol 44 (02) ◽  
pp. 092-095 ◽  
Author(s):  
T H Tran ◽  
C Bondeli ◽  
G A Marbet ◽  
F Duckert
Keyword(s):  
Antithrombin Iii ◽  
Factor Xa ◽  
Heparin Binding ◽  
Thrombin Inhibition ◽  
Superficial Thrombophlebitis ◽  
Heparin Concentration ◽  
Heparin Binding Site ◽  
At Iii ◽  
The Difference ◽  
Inhibition Of Thrombin

SummaryTwo different AT-III fractions were purified from the plasma of a patient with recurrent superficial thrombophlebitis. The abnormal AT-III fraction (A-AT) was compared to the normal AT-III fraction (N-AT) in the inhibition of thrombin and factor Xa. Without heparin, both inactivate proteases in a similar manner and at the same rate. However, at low heparin concentration the thrombin inhibition proceeds more slowly with A-AT than with N-AT. At high heparin concentration the difference between A-AT and N-AT becomes very small. The inhibition of factor Xa follows a similar pattern. It is suggested that the heparin binding site of A-AT differs from that of N-AT resulting in a decreased heparin cofactor activity.

Respective Roles of Antithrombin III and Alpha 2 Macroglobulin in Thrombin Inactivation

1981 ◽  
Vol 45 (01) ◽  
pp. 051-054 ◽  
Author(s):  
A M Fischer ◽  
J Tapon-Bretaudiere ◽  
A Bros ◽  
F Josso
Keyword(s):  
Antithrombin Iii ◽  
Practical Interest ◽  
High Plasma ◽  
At Iii ◽  
Human Material ◽  
Cofactor Activity

SummaryIn order to investigate the mechanism of thrombin inactivation in the presence of both antithrombin III (AT III) and α 2-macroglobulin (α 2 M), thrombin and the inhibitors have been purified from human material and thrombin inactivation studied using purified reagents either alone or added to defibrinated plasma. Comparison of clotting and amidolytic activities of residual thrombin allowed to measure the amount of thrombin bound to α 2 M. In a purified reagent system as well as in plasma, part of exogenous thrombin is bound to α 2 M. The amount of bound thrombin is related to α 2 M concentration. Conversely, previous plasma α 2 M depletion by immunoabsorption increases the consumption of heparin-cofactor activity by exogenous thrombin. Thus AT III and α 2 M compete for thrombin inactivation. This finding could be of practical interest in clinical situations associating high plasma α 2 M levels and a decrease of AT III concentration.

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