Defibrinogenation Procedures for Antithrombin III (AT-III) Biological Assay

1975 ◽  
Author(s):  
M. C. Tonolli ◽  
M. B. Donati
Keyword(s):  
Biological Activity ◽  
Incubation Period ◽  
Research Council ◽  
Antithrombin Iii ◽  
The Other ◽  
Radial Immunodiffusion ◽  
Biological Assay ◽  
Immunological Reactivity ◽  
Antithrombin Activity ◽  
At Iii

Since fibrinogen has itself an antithrombin activity, defibrinogenation of the sample is a prerequisite for AT-III determination. Five different defibrinogenation procedures were tested: heat precipitation for either 3 or 10 minutes, ristocetin precipitation, clotting with either Reptilase-R or Botropase-R. AT-III was measured as biological activity according to Howie et al. (Br. J. Haemat. 25, 101, 1973) and assayed immunologically by radial immunodiffusion. Defibrinogenation was complete with all the procedures used (< 16 μg/ml F. R.-antigen being left). In the samples defibrinogenated by ristocetin, the biological assay could not be performed, since some ristocetin remained in the supernatant and provoked a rapid precipitation of the substrate fibrinogen used for the assay. The possibility that Botropase-R or Reptilase-R had themselves some antithrombin activity was preliminarly ruled out. In samples definbriogenated by heating (3 min), Reptilase-R or Botropase-R the same biological AT-III activity was found; in addition, no differences in immunological reactivity of the protein were observed as compared with the corresponding-plasma before defibrinogenation. In contrast, a statistically significant (P < 0.001) reduction of both biological and immunological AT-III activity was found in the samples defibrinogenated by heating (10 min), as compared with the other 3 procedures. These results indicate that Reptilase-R and Boropase-R are suitable reagents for defibrinogenation of plasma prior to AT-III assay; heat precipitation can also be used, provided the duration of the incubation period is accurately controlled.(Supported by Grant N. 73.00400.04 of the Italian Research Council (C. N. R.).)

scholarly journals Differential Determination of Antithrombin Activities of Plasma Antithrombin III and α2-Macroglobulin

1977 ◽  
Author(s):  
T. Matsuda ◽  
M. Ogawara ◽  
N. Hirabayashi ◽  
T. Seki ◽  
M. Murakami
Keyword(s):  
Healthy Subjects ◽  
Antithrombin Iii ◽  
Radial Immunodiffusion ◽  
Thrombin Inhibitors ◽  
Healthy Individuals ◽  
Single Radial Immunodiffusion ◽  
Antithrombin Activity ◽  
Α2 Macroglobulin ◽  
At Iii

Among several thrombin inhibitors in normal plasma, antithrombin III (AT-III) and α2-macroglobulin (α2-M) are especially important.However, there has been no appropriate method to determine AT-III and α2-M separately. This study was carried out to differentiate the actions of these two inhibitors of thrombin in plasma. By using single radial immunodiffusion method, we observed that AT-III was adsorbed to bentonite, although α2-M was not. It was necessary to incubate 1 ml of oxalated plasma with 300 mg of bentonite at 37°C for 10 minutes to remove AT-III completely. Supernated plasma contains neither AT-III nor fibrinogen which affects antithrombin activity of plasma. From these results, it is evident that antithrombin activity of plasma adsorbed with bentonite was attributed to that of α2-M. Antithrombin activities of plasma adsorbed with bentonite in 20 healthy subjects were significantly correlated with concentrations of α2-M in plasma, measured immunologically (r=+0.87, p<0.001). It is reasonable to presume that difference between antithrombin activity of defibrinated plasma by heating at 56°C for 2 minutes and that of bentonite treated plasma mainly indicates activity of AT-III. These differences measured in 20 healthy individuals were significantly correlated with plasma AT-TH levels, assayed immunochemically (r=+0.53, p<0.01).

Comparison of Progressive Antithrombin Activity and the Concentration of Three Thrombin Inhibitors in Nephrotic Syndrome

1981 ◽  
Vol 46 (03) ◽  
pp. 623-625 ◽  
Author(s):  
B Boneu ◽  
F Bouissou ◽  
M Abbal ◽  
P Sie ◽  
C Caranobe ◽  
...  
Keyword(s):  
Nephrotic Syndrome ◽  
Antithrombin Iii ◽  
Heparin Therapy ◽  
Thrombin Inhibitors ◽  
Compensatory Mechanism ◽  
Dramatic Reduction ◽  
Antithrombin Activity ◽  
Α2 Macroglobulin ◽  
At Iii ◽  
A Prospective Study

SummaryIn order to compare the plasmatic progressive antithrombin activity to the concentration of three thrombin inhibitors, antithrombin III (AT III), α2 macroglobulin (α2, M), α1 anti-trypsin (α1 AT) in nephrotic syndrome, a prospective study was carried out on a group of 28 children affected with the disease. A dramatic reduction of the level of AT III and of α1 AT, two inhibitors of molecular weight close to that of albumin, was observed. The decreased level of AT III was counterbalanced by an increase in α2 M. This phenomenon accounts for the increased progressive antithrombin activity observed in all the affected children. It is suggested that the above compensatory mechanism explains the absence of thrombotic accidents in this series and that the benefit of heparin therapy is doubtful in these conditions.

INTERACTION BETWEEN CULTURED ENDOTHELIAL CELLS AND ABNORMAL ANTITHROMBIN III "TOYAMA"

1987 ◽  
Author(s):  
N Sakuragawa ◽  
S Saitoh ◽  
K Takahashi
Keyword(s):  
Endothelial Cells ◽  
Room Temperature ◽  
Antithrombin Iii ◽  
Cultured Cells ◽  
Binding Activity ◽  
Endothelial Cell Culture ◽  
Antithrombin Activity ◽  
Thrombin Activity ◽  
Cultured Endothelial Cells ◽  
At Iii

Purpose: Abnormal antithrombin III(AT-III)Toyama showed non-affinity to heparin and heparinoid to show loss of immediate antithrombin activity. On the endothelial cells, there are heparinoids including heparan sulfate. We investigated on the interaction between cultured endothelial cells and abnormal AT-III"Toyama" from the viewpoint of antithrombin activity.Materials and methods: (1) Endothelial cell culture:^125I-labelled normal and abnormal AT-III were placed on the washed endothelial cultured cells in 0.2 ml of RPMI-1640 medium for 15 min at 37°C. The medium was suctioned off and the cell layer was washed with Hank's balanced salt solution. The cells were incubated with 1 ml of heparin(3 ug/ml) for 15 min at 4°C. The radioactivity in the supernatant was counted, and represented AT-III which bound to the cells surface. (2) Antithrombin activity: 0.23 ml of thrombin solution^ U/ml) and 0.03 ml of normal or abnormal AT-III plasma were mixed, and incubated on the cultured cell surface for 5 min at room temperature. The residual thrombin activity was assayed by 0.3 ml of the substrate (S-2238) solution(0.8mM)for 5 min. After these procedures,2 ml of 2% citric acid solution was added to stop the reaction, and 0D(405 nm) was recorded.Results: Abnormal AT-III showed reduced binding-activity to cultured cells to one fifth compared with normal AT-III, and the residual thrombin activity in the abnormal was higher compared with that in normal plasma.Conclusion: Abnormal AT-III showed less binding activity to the cultured endothelial cells, and less thrombin neutralizing activity to show thrombogenic tendency.

Biological Characteristics Op Antithrombin III In An Antithrombin III Deficient Family

1981 ◽  
Author(s):  
S Kondo ◽  
T Matsuo ◽  
Y Ohoki ◽  
O Matsuo
Keyword(s):  
Antithrombin Iii ◽  
Recurrent Thrombosis ◽  
Normal Range ◽  
Japanese Family ◽  
Neutralization Activity ◽  
Normal Value ◽  
Antithrombin Activity ◽  
At Iii ◽  
Molecular Abnormality ◽  
Antifactor Xa

In the familial AT III deficiency of a Japanese family, the propositus (a-39-yr old female) and her mother had episodes of recurrent thrombosis and their AT III levels as measured immunologically and biologically were below the normal value. In the plasma of her brother, the AT III concentration as measured immunologically was half of the normal value, but his biological antithrombin activity was within the normal range. The progressive antithrombin activity and antifactor Xa activity of plasma samples in this familial AT III deficiency were within the normal range. Measurements of the rate of thrombin neutralization activity revealed that the brother’s plasma was in the normal range, but the plasma of the propositus and of her mother showed rates of thrombin neutralization activity which were somewhat below the normal value. The rate of thrombin neutralization activity per mg protein of AT III was highest in the plasma of the brother, and became slower in the mother, propositus, and pooled normal plasma in that order. In the plasma of this familial AT III deficiency, the rate of Xa neutralization activity was much slower than the normal value. It is postulated that since the antithrombin of the brother of the propositus was found to react as normal in the neutralization of thrombin, he does not have episodes of thrombosis. Such characteristic hyperfunction of antithrombin in the plasma of the brother may be due to some molecular abnormality of AT III within this hereditary deficient family.

Coagulation Inhibitors in Plasma and Serum

1975 ◽  
Author(s):  
O. R. Ødegård ◽  
U. Abildgaard
Keyword(s):  
Antithrombin Iii ◽  
Close Correlation ◽  
The Other ◽  
At Iii ◽  
Coagulation Inhibitors ◽  
Patient Groups ◽  
Cofactor Activity ◽  
The Difference ◽  
Immuno Assay ◽  
Activity Method

Heparin cofactor activity and antithrombin III (At-III) activity measured with amidolytic methods; antifactor Xa by a clotting method (Biggs et al., Brit. J. Haemat. 19, 287, 1970) and immunoassay of At-III (Fagerhol & Abildgaard, Scand. J. Haemat. 7, 10, 1970) in plasma and serum showed:1) There was a close correlation between the plasma values as measured by all these methods (r = 0.84–0.93).2) The difference between plasma and serum values (“consumption”) was lower in warfarin treated and in haemophiliacs than in the other groups.3) The difference between plasma and serum was greater when measured by the heparin cofactor activity method than by the other methods. The reason for this discrepancy will be discussed. The results in different patient groups will be reported.4) As the heparin cofactor activity assay can be completed within 10 minutes after blood sampling, and has a higher precision than clotting assay and immuno assay, it is preferable for clinical use.

Pasteurization Of Antithrombin III: Stabilization By Heparin And Lyotropic Anions

1981 ◽  
Author(s):  
Thomas F Busby ◽  
Donald H Atha ◽  
Kenneth C Ingham
Keyword(s):  
Sodium Citrate ◽  
Antithrombin Iii ◽  
Antithrombin Activity ◽  
At Iii ◽  
Exclusion Chromatography ◽  
Direct Binding ◽  
Chaotropic Anions ◽  
Increasing Temperature ◽  
Denaturation Curve ◽  
Lyotropic Anions

Pasteurization of Antithrombin III (AT III) for 10 hours at 60°C is necessary to reduce the risk of transfusion hepatitis. Addition of appropriate stabilizers can largely prevent the loss of antithrombin activity which otherwise occurs during pasteurization. Studies of the mechanism of denaturation and stabilization have been facilitated by the use of 1,8-anilinonaphthalene sulfonate (ANS) which binds weakly to the inhibitor and whose fluorescence undergoes a sigmoidal response to increasing temperature as the protein unfolds. The extent of the increase in ANS fluorescence correlated roughly with the loss of antithrombin activity and with the extent of protein aggregation as determined by high pressure exclusion chromatography. The midpoint, Td, of the thermal denaturation curve increased by 13 and 19°C in the presence of 0.5 M and 1.0 M sodium citrate respectively. Phosphate, sulfate, and EDTA were also strong stabilizers while the chaotropic anions, iodidé and thiocyanate were potent destabilizers. Heparin, at 10 mg/ml, increased Td by 7°, presumbly through a direct binding mechanism. Reducing agents increased ANS fluorescence by an amount similar to that seen with thermally denatured samples, an effect which was inhibited by heparin but not by citrate. Furthermore, incorporation of 14C-iodoacetamide into AT III during thermal titration was coincident with the increase in ANS fluorescence suggesting that disulfide cleavage is the event which triggers the unfolding of the protein. Samples pasteurized for 10 hours at 60°C in the presence of 0.5 M and 1.0 M citrate retained full antithrombin activity but exhibited evidence of minor alterations in the ability to bind heparin.

The Presence of Antithrombin Activity in Platelets

1979 ◽  
Author(s):  
E. E. Czapek ◽  
H. C. Kwaan
Keyword(s):  
Antithrombin Iii ◽  
Human Platelet ◽  
Platelet Rich Plasma ◽  
Clot Formation ◽  
Chromogenic Substrate ◽  
Antithrombotic Activity ◽  
Platelet Poor Plasma ◽  
Antithrombin Activity ◽  
At Iii ◽  
Platelet Pellet

The platelet has been implicated as the nidus around which clot formation occurs. We therefore decided to investigate whether any antithrombotic activity was associated with platelets. Thrombin activity was measured using the chromogenic substrate S-2238. Freshly prepared human platelet rich plasma (PRP) and platelet poor plasma (PPP) were found to have similar amounts of antithrombin activity. This antithrombin activity could be easily removed from a platelet pellet by repeated washing. However, sonicating PRP resulted in the appearance of additional antithrombin activity. After 6 minutes of sonication, the antithrombin activity of PRP decreased to 35% of presonication levels, whereas the antithrombin activity of PPP remained 100%. When subjected to Laurell Immunoelectrophoresis using an antibody to antithrombin III (AT-III), a greater amount of antigen was detected in PRP than in PPP. We conclude that platelets contain antithrombin material which is probably AT-III.

Determination of the Biological Activity of Heparin by Use of a Chromogenic Substrate

1979 ◽  
Vol 42 (05) ◽  
pp. 1446-1451 ◽  
Author(s):  
Knut Bartl ◽  
Elfriede Dorsch ◽  
Helmut Lill ◽  
Joachim Ziegenhorn
Keyword(s):  
Biological Activity ◽  
Therapeutic Range ◽  
Antithrombin Iii ◽  
Photometric Determination ◽  
Chromogenic Substrate ◽  
Standard Samples ◽  
Spectrum Line ◽  
At Iii ◽  
Specific Determination

SummaryWe describe a new method for determining the biological activity of heparin in plasma with use of thrombin and the substrate Tos-Gly-Pro-Arg-pNA. The procedure is based on the photometric determination of the inactivation of thrombin after incubation with plasma in the presence of endogenous antithrombin III (At III). The method allows the specific determination of heparin concentrations from 0.02 USP to 0.8 USP/ml of plasma in the presence of normal At III levels. It has been carried out manually by use of an Eppendorf spectrum line photometer or automatically by use of a Vitatron Akes analyzer. For evaluation, the results were compared with two standard samples which contained heparin in the low and high therapeutic range, respectively.

Biological Activity Of Antithrombin III In Blood Plasma And Edematous Fluids Of Patients With A Nephrotic Syndrome

1981 ◽  
Author(s):  
L V Podorolskaya ◽  
G V Andreenko ◽  
L R Polyantseva
Keyword(s):  
Biological Activity ◽  
Nephrotic Syndrome ◽  
Blood Plasma ◽  
Prognostic Value ◽  
Antithrombin Iii ◽  
Venous Occlusion ◽  
Heparin Therapy ◽  
Renal Lesions ◽  
At Iii ◽  
In Blood Plasma

The biological activity of antithrombin (AT) III was tested by the Abilgaard method in blood plasma of 149 patients with renal lesions of various etiology, of 27 donors and in 17 samples of edematous fluid of patients with nephrotic syndrome (NS). The majority of patients had a decreased AT III content in blood plasma, which was especially well pronounced in NS patients irrespective of etiology. In edematous fluids the level of AT III was 10% of that in the plasma. The functional activity of AT III in the plasma was well-correlated with IIS manifestations, i.e, 24 hr-proteinuria, hypoalbuminemia, hyperchole- sterinemia) and with hemocoagulation and fibrinolysis parei:.eters (SFMC content, plasma tolerancy to heparin, contents of heparin, plasminogen activator and kallikrein and total antitryptic activity). A reactive increase of AT III in NS patients with venous occlusion is of essential prognostic value. Heparin therapy results in an increase of the originally low AT III content and in positive dynamics of NS.

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