Crossed Immunoelectrofocusing Of Antithrombin III In Healthy And In Congenital And Acquired Deficiency

1981 ◽  
Author(s):  
G Leone ◽  
V M Valori ◽  
G Traisci ◽  
B Bizzi
Keyword(s):  
Isoelectric Focusing ◽  
Hepatic Cirrhosis ◽  
Antithrombin Iii ◽  
Congenital Defect ◽  
Plasma Pool ◽  
Normal Plasma ◽  
Isoelectric Points ◽  
At Iii ◽  
Two Peaks ◽  
Normal Pool Plasma

Isoelectrofocusing was carried out in LKB Multiphor apparatus with pH 4-6.5 carrier ampholites using poly- acrilamide gels. Specimens of human purified antithrombin, normal plasma pool, plasma with congenital and acquired AT- III deficiency were isoelectric focused. Purified Antithrombin showed two peaks. The isoelectric points of these antithrombin peaks were 4.6 and 4.9. Next the poly- acrilamide gel slabs were placed on glass plates coated with agarose containing 4% anti human antithrombin antibody; crossed electrophoresis was then carried out. Normal pool plasma exhibits two peaks, which correspond to the isoelectric focusing position of the purified AT-III. Plasmas of two families with congenital AT-III deficiency exhibit only one peak at pH lower. On the contrary plasmas of patients with acquired AT-III deficiency (hepatic cirrhosis, asparaginase therapy) exhibit two peaks. Considering that microheterogeneity is fundamentally due to a difference in glycosylation (Borsodi and Nerasimhan, Brit. J. Haemat. 1978, 39,121) it is possible to hypothesize a defect in glycosylation in congenital defect.

Isolation of Antithrombin III from Normal and α1-Antitrypsin-Deficient Human Plasma

1977 ◽  
Vol 38 (02) ◽  
pp. 0475-0485 ◽  
Author(s):  
Anna D. Borsodi ◽  
Ralph A. Bradshaw
Keyword(s):  
Enzymatic Activity ◽  
Isoelectric Focusing ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Antithrombin Activity ◽  
Bovine Trypsin ◽  
Normal Plasma ◽  
Antitrypsin Deficiency ◽  
Simplified Procedure ◽  
Stimulation Of

SummaryThe plasma of individuals, hetero- or homozygous for α1-antitrypsin deficiency, contains greatly decreased amounts of antithrombin activity as assayed against factor Xa. However, heparin stimulation of the residual antithrombin activity is observed, which is comparable to that of normal plasma. Antithrombins isolated from both normal and α1-antitrypsin deficient plasma by a simplified procedure are indistinguishable in both properties and yields. The microheterogeneity observed on isoelectric focusing of both preparations can be eliminated by treatment with neuraminidase. Neither purified human antithrombin nor α1-antitrypsin, when assayed against bovine trypsin, is stimulated by heparin. These results clearly establish the unique natures of antithrombin and α1-antitrypsin and show that about 75% of the antithrombin activity measured in normal plasma is due to α1-antitrypsin. Estimates of anti thrombin III activity in normal plasma by assays dependent on enzymatic activity can probably be obtained only in the presence of heparin.

Antithrombin III in Full-Term and Pre-Term Newborn Infants : Three Cases of Neonatal Diagnosis of AT III Congenital Defect

1987 ◽  
Vol 57 (03) ◽  
pp. 329-331 ◽  
Author(s):  
V De Stefano ◽  
G Leone ◽  
M P De Carolis ◽  
R Ferrelli ◽  
S De Carolis ◽  
...  
Keyword(s):  
Plasma Levels ◽  
Antithrombin Iii ◽  
Full Term ◽  
Congenital Defect ◽  
Preterm Neonates ◽  
Control Group ◽  
Term Infants ◽  
Term Neonates ◽  
Congenital Deficiency ◽  
At Iii

SummaryAntithrombin III (AT III) plasma levels were investigated in 18 full term neonates and 14 healthy preterm neonates. A control group of 20 healthy adults was also studied. AT III was measured as antigen concentration (Ag) and antithrombin or anti-factor Xa heparin cofactor (H. C.) activities. Crossed immunoelectrophoresis on heparin-agarose (H-CIE) was carried out on plasma samples; moreover the distribution of isoantithrombins was investigated on whole plasma by a technique of crossed immunoelectrofocusing (CIEF). AT III plasma levels in full term infants were significantly lower as compared to the adult values. The preterm newborns group showed a further significant decrease in AT III levels as compared to the full term neonates. In all infants AT III H-CIE runs displayed a single fast moving anodal peak, so that a normal binding to heparin was demonstrated. The CIEF AT III plasma pattern of the adults as well as of all neonates displayed three major peaks at pH range 5.2-4.9, a small amount of AT III at pH 4.9-4.8 and a minor peak at pH 4.8-4.6, so that it was concluded that the isoantithrombins plasma distribution in neonatal age is identical to that of the adult subjects. Four neonates whose mothers were affected by AT III congenital defect were also investigated: diagnosis of congenital deficiency was established in three cases.

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.

Immunchemical Investigation on Antithrombin III/AT-III/in Presence of Heparin

1979 ◽  
Author(s):  
D. Bánuergyi ◽  
G. Sas
Keyword(s):  
Affinity Chromatography ◽  
Antithrombin Iii ◽  
Original Method ◽  
Normal Plasma ◽  
At Iii ◽  
Different Sources

Various components of AT-III car, be differentiated by crossed ioununo-eleetrophoresis in the presence of heparin as we described previously / Sas et al. 1975 /. The migration of heparin is discontinuous in this original method, i.e. at the end of the electrophoresis the plate does not contain heparin at all. This fact rises the possibility pf the artefactual nature of these AT-III components. To exclude this effect we introduced a modification using double plates incorporated with heparin. The second plate was used as a heparin “reservoir”. By this modification we obtained constant heparin flow in the course of electrophoresis. We found four different AT-III components in normal plasma comparing with three AT-III eomportints of the original method.Investigating heparins originated from different sources we found a higher mobility of AT-III in the pnsenee of mucous heparin than that of lung heparin. This observation corresponds well with the results obtamed by heparin-agarose affinity chromatography.

scholarly journals Transferrin plays a central role to maintain coagulation balance by interacting with clotting factors

2019 ◽  
Author(s):  
Xiaopeng Tang ◽  
Zhiye Zhang ◽  
Mingqian Fang ◽  
Yajun Han ◽  
Sheng Wang ◽  
...  
Keyword(s):  
Plasma Concentration ◽  
Mouse Models ◽  
Antithrombin Iii ◽  
Fine Tuning ◽  
Coagulation Cascade ◽  
Molar Ratio ◽  
Clotting Factor ◽  
Clotting Factors ◽  
Normal Plasma ◽  
At Iii

SummaryCoagulation balance is maintained through fine-tuning interactions among clotting factors. Physiological concentrations of clotting factors are huge difference. Especially, coagulation proteases’ concentration (pM to nM) is much lower than their natural inactivator antithrombin III (AT-III, ∼3 μM). Here we show that transferrin (normal plasma concentration ∼40 μM) interacts with fibrinogen, thrombin, FXIIa and AT-III with different affinity to maintain coagulation balance. Normally, transferrin is sequestered by binding with fibrinogen (normal plasma concentration ∼10 μM) with a molar ratio of 4:1. In atherosclerosis, abnormally up-regulated transferrin interacts with and potentiates thrombin/FXIIa and blocks AT-III’s inactivation on coagulation proteases by binding to AT-III, and thus induces hypercoagulability. In mouse models, transferrin-overexpression aggravated atherosclerosis while transferrin-knockdown, anti-transferrin antibody or designed peptides interfering transferrin-thrombin/FXIIa interactions alleviated it. Collectively, these findings identify transferrin as a clotting factor and an adjuster for maintaining coagulation balance and modify the coagulation cascade.

A New Variant of Antithrombin III. Study of three Related Cases.

1979 ◽  
Author(s):  
M. Wolf ◽  
C. Boyer ◽  
J.M. Lavergne ◽  
M.J. Larrieu
Keyword(s):  
Electrophoretic Mobility ◽  
Antithrombin Iii ◽  
Chromogenic Substrates ◽  
Double Peak ◽  
Enzymatic Assays ◽  
Crossed Immunoelectrophoresis ◽  
New Variant ◽  
At Iii ◽  
Cofactor Activity ◽  
Two Peaks

A qualitative abnormality of antithrombin III (AT III) was demonstrated in three member from the same family. The mother (58 y.) had recurrent episodes of venous thrombosis an pulmonary embolism whereas the affected daughters (20 and 29 y.) are asymptomatic. AT III was investigated in presence and absence Of heparin hy enzymatic assays using chromogenic substrates (S-2238 and S-2160) and by rocket - and crossed - immunoelectro phoresis using two different monospecific anti-AT III antisera. In the three patients the qualitative abnormality of AT III was suggested by the discrepancy between a norml level of AT III antigen and decreased heparin-cofactor activity (49, 51, 54%). By crossed immunoe1ectrophoresis, patients’ AT III migrated as a single peak in absence of heparin, with the same electrophoretic mobility as that of the control. In presence of heparin (25 u/ml), crossed immunoelectrophoresis demonstrated the presence of two peaks, one with a normal, and the other with a decreased electrophoretic mobility. A double peak was also observed by rocket immunoelectrophoresis in presence of heparin. In this family with a variant of AT III, the three heterozygous affected cases demonstrate two popu1ations of AT III, with a different affinity for heparin.

Further Investigations on Antithrombin III in the Plasmas of Patients with the Abnormality of ‘Antithrombin III Budapest’

1975 ◽  
Vol 33 (03) ◽  
pp. 564-572 ◽  
Author(s):  
Géza Sas ◽  
Duncan S Pepper ◽  
John D Cash
Keyword(s):  
Electrophoretic Mobility ◽  
Antithrombin Iii ◽  
Gel Filtration ◽  
Molecular Size ◽  
High Concentration ◽  
Abnormal Protein ◽  
Crossed Immunoelectrophoresis ◽  
Normal Plasma ◽  
At Iii ◽  
Cofactor Activity

SummaryAntithrombin III (AT-III) was studied in a thrombophilic family with an abnormal AT-III molecule (antithrombin III Budapest) using a modified crossed Immunoelectrophoresis technique, gel filtration, ‘rocket’ Immunoelectrophoresis and a heparin cofactor assay.When plain agarose was applied in the first phase of the crossed Immunoelectrophoresis, the normal and the pathological AT-III revealed identical electrophoretic mobility. However, when heparin was mixed with agarose in the first phase of electrophoresis, the propositus’ plasma displayed a different AT-III pattern from normal plasma. His plasma contained the first component of the normal plasma (Immune Antithrombin III1, IAT-III1) in a concentration of only 5% of normal, and a protein in high concentration which although immunoreactive to AT-III antisera, had an electrophoretic mobility similar (but not identical) to that of IAT-III2. This ab-normal protein had no heparin cofactor activity and a molecular size greater than normal plasma AT-III. Unlike normal AT-III, the addition of heparin did not change the molecular size of the pathologic AT-III molecule significantly.The abnormal protein was present in lower concentrations in the patient’s children and at the time of study they had no clinical or laboratory evidence of intravascular coagulation.

An International Collaborative Study Establishing a Reference Preparation for Antithrombin III

1980 ◽  
Vol 43 (01) ◽  
pp. 010-015 ◽  
Author(s):  
T B L Kirkwood ◽  
T W Barroweliffe ◽  
D P Thomas
Keyword(s):  
Antithrombin Iii ◽  
Collaborative Study ◽  
World Health ◽  
Normal Plasma ◽  
Freeze Dried ◽  
At Iii ◽  
Reference Preparation ◽  
International Collaborative ◽  
Suitable Reference ◽  
International Collaborative Study

SummaryAn international collaborative study involving 12 laboratories in 7 countries was carried out to establish a suitable reference preparation of antithrombin III (At III). The amount of At III present in two purified preparations, a freeze-dried normal plasma and local normal plasma pools was measured by clotting, immunological, and amidolytic assays. 120 assays were submitted of which 105 were accepted as valid for inclusion in subsequent analyses. Less laboratory to laboratory variation was found when At III was assayed in freeze-dried normal plasma, as compared to purified preparations of At III, and there was also less method to method variation when At III was measured in freeze-dried plasma. When measured as heparin co-factor activity, the two purified preparations contained only about half the level of At III found by immunoassay or progressive At III clotting assays. In contrast, the use of freeze-dried plasma provided results which showed excellent agreement between the various laboratories by all assays; accordingly, this material has been established by the World Health Organization as the International Reference Preparation for At III, with an assigned potency of 0.9 i. u. per ml.

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