Measurement of Antithrombin III in Normal and Pathologic States Using Chromogenic Substrate S-2238: Comparison with Immunoelectrophoretic and Factor Xa Inhibition Assays

1980 ◽  
Vol 73 (5) ◽  
pp. 639-647 ◽  
Author(s):  
Scott H. Goodnight ◽  
Janis L. Schaeffer ◽  
Kirtikant Sheth
Keyword(s):  
Antithrombin Iii ◽  
Factor Xa ◽  
Chromogenic Substrate ◽  
Factor Xa Inhibition

An Antithrombin III Assay Based on Factor Xa Inhibition Provides a More Reliable Test to Identify Congenital Antithrombin III Deficiency Than an Assay Based on Thrombin Inhibition

1993 ◽  
Vol 69 (03) ◽  
pp. 231-235 ◽  
Author(s):  
Christine Demers ◽  
Penny Henderson ◽  
Morris A Blajchman ◽  
Michael J Wells ◽  
Lesley Mitchell ◽  
...  
Keyword(s):  
Dna Analysis ◽  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Factor Xa ◽  
Chromogenic Substrate ◽  
Cross Sectional ◽  
Thrombin Inhibition ◽  
Factor Xa Inhibition ◽  
At Iii ◽  
The Difference

SummaryObjectives: To determine whether functional antithrombin III (AT-III) levels measured by a factor Xa inhibition (AT-III-Xa) assay identifies AT-III deficient individuals more reliably than functional AT-III levels measured by a thrombin inhibition (AT-III-IIa) assay.Study design: Cross-sectional study.Patient population: Sixty-seven members of a large family with type 2 AT-III deficiency.Intervention: DNA analysis was used as the reference diagnostic standard for AT-III status and subjects were classified as AT-III deficient or non deficient according to these results. Functional AT-III levels were measured in all subjects using: 1) a chromogenic substrate for thrombin and added human thrombin (AT-III-IIa), and 2) a chromogenic substrate for factor Xa and added bovine factor Xa (AT-III-Xa). Functional heparin cofactor II (HC-II) levels were measured using a commercially available kit. The proportions of 125I-α-thrombin complexed to AT-III and HC-II were measured by polyacrylamide gel electrophoresis and autoradiography.Results: Thirty-one (46%) individuals were classified as AT-III deficient and 36 (54%) as AT-III non deficient. AT-III-Xa assay measured a significantly lower mean AT-III value and a narrower range for individuals classified as AT-III deficient than the AT-III-IIa assay. Using the AT-III-IIa assay, six subjects had borderline AT-III levels compared to none with the AT-III-Xa assay. Thrombin inhibition by HC-II likely accounts for the AT-III-IIa assay giving higher values than the AT-III-Xa assay since 1) there was a significant correlation between the difference in AT-III-IIa and AT-III-Xa levels and HC-II levels, 2) the mean level of HC-II was significantly higher for individuals who had a positive difference between AT-III-IIa and AT-III-Xa levels compared to those who had a negative difference and 3) there was a significant correlation between the difference in AT-III-IIa and AT-III-Xa levels and the percentage of 125I-α-thrombin complexed to HC-II.Conclusion: The AT-III-Xa assay is a better discriminant between AT-III deficient and AT-III non deficient individuals than the AT-III-IIa assay.

Antithrombin III Avranches, a New Variant with Defective Serine-Protease Inhibition - Comparison with Antithrombin III Charleville

1988 ◽  
Vol 60 (01) ◽  
pp. 094-096 ◽  
Author(s):  
M Aiach ◽  
M Roncato ◽  
G Chadeuf ◽  
P Dezellus ◽  
L Capron ◽  
...  
Keyword(s):  
Antithrombin Iii ◽  
Factor Xa ◽  
Kinetic Studies ◽  
Exchange Chromatography ◽  
Protease Inhibition ◽  
Sds Page ◽  
Recurrent Venous Thromboembolism ◽  
Factor Xa Inhibition ◽  
New Variant ◽  
At Iii

SummaryA decreased plasma antithrombin activity in presence or in absence of heparin was discovered in a 47-year-old patient presenting with recurrent venous thromboembolism. The immunoreactive material (AT ΠΙ-IR) was normal. The same biological abnormalities were found in two relatives of the patient, leading to the diagnosis of hereditary qualitative AT III deficiency.The propositus’ AT III was coeluted with normal AT III from an heparin-sepharose column. An additional step of ion-exchange chromatography on a Mono Q column using a FPLC system (Pharmacia, St-Quentin en Yvelines, France) allowed the purification of a protein which was homogenous in SDS-10% polyacrylamide electrophoresis gel (PAGE). AT III purified from propositus’ plasma, normal plasma and the plasma of the patient known to have an AT III variant with defective protease binding (AT III Charleville) were compared. The specific activities measured as heparin cofactor anti thrombin or factor Xa inhibition in absence of heparin were decreased to half the normal value.Kinetic studies confirmed a decreased rate of thrombin inhibi-tion for both abnormal AT III preparations. SDS-PAGE experi-ments performed in purified system and immunoblots obtained from plasma showed that the two variants have different behaviour: in the case of AT III Charleville thrombin induced an apparent 5 Δ increase in molecular mass, probably due to a conformational change. AT III Avranches did not form stoechiometric complexes with thrombin, but was unmodified by the protease.

Development and characterization of an automated assay of effective heparin activity in plasma.

1987 ◽  
Vol 33 (9) ◽  
pp. 1630-1634 ◽  
Author(s):  
J F Pierson-Perry ◽  
D M Obzansky ◽  
J P Mizzer
Keyword(s):  
Antithrombin Iii ◽  
Factor Xa ◽  
Sole Source ◽  
Chromogenic Substrate ◽  
Blood Components ◽  
Automated Assay ◽  
At Iii ◽  
Assay Result ◽  
Heparin Activity

Abstract We describe a fully automated assay for determining effective heparin activity in plasma, based on heparin-catalyzed inhibition of Factor Xa (EC 3.4.21.6) by antithrombin III (AT III). Residual Factor Xa is determined kinetically by the Du Pont aca discrete clinical analyzer with a chromogenic substrate and is inversely related to heparin activity. Because the test plasma is the sole source of AT III, the assay result is dependent on AT III activity and reflects effective rather than total heparin activity. The assay range is 20-1200 USP units/L, and the assay shows equivalent sensitivity to standard and low-molecular-mass heparins. Within-run reproducibility (CV) is 1.6% at 390 units/L. There was no interference from common blood components or drugs. Results agreed well with those by the Coatest heparin kit (Kabi) adapted to the Cobas-Bio analyzer (r = 0.85, n = 122).

scholarly journals Elimination of glycosylation heterogeneity affecting heparin affinity of recombinant human antithrombin III by expression of a β-like variant in baculovirus-infected insect cells

1995 ◽  
Vol 310 (1) ◽  
pp. 323-330 ◽  
Author(s):  
E Ersdal-Badju ◽  
A Lu ◽  
X Peng ◽  
V Picard ◽  
P Zendehrouh ◽  
...  
Keyword(s):  
Insect Cells ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Structural Basis ◽  
Heparin Binding ◽  
Factor Xa Inhibition ◽  
Infected Insect ◽  
Spectroscopy Studies ◽  
High Degree ◽  
Good Agreement

In order to promote homogeneity of recombinant antithrombin III interactions with heparin, an asparagine-135 to alanine substitution mutant was expressed in baculovirus-infected insect cells. The N135A variant does not bear an N-linked oligosaccharide on residue 135 and is therefore similar to the beta isoform of plasma antithrombin. Purified bv.hat3.N135A is homogeneous with respect to molecular mass, charge and elution from immobilized heparin. Second-order rate constants for thrombin and factor Xa inhibition determined in the absence and presence of heparin are in good agreement with values established for plasma antithrombin and these enzymes. Based on far- and near-UV CD, bv.hat3.N135A has a high degree of conformational similarity to plasma antithrombin. Near-UV CD, absorption difference and fluorescence spectroscopy studies indicate that it also undergoes an identical or very similar conformational change upon heparin binding. The Kds of bv.hat3.N135A for high-affinity heparin and pentasaccharide were determined and are in good agreement with those of the plasma beta-antithrombin isoform. The demonstrated similarity of bv.hat3.N135A and plasma antithrombin interactions with target proteinases and heparins suggest that it will be a useful base molecule for investigating the structural basis of antithrombin III heparin cofactor activity.

Measurement of Markers of Activated Coagulation in Antithrombin III Deficient Subjects

1992 ◽  
Vol 67 (05) ◽  
pp. 542-544 ◽  
Author(s):  
Christine Demers ◽  
Jeffrey S Ginsberg ◽  
Penny Henderson ◽  
Fred A Ofosu ◽  
Jeffrey I Weitz ◽  
...  
Keyword(s):  
Antithrombin Iii ◽  
Factor Xa ◽  
Large Family ◽  
Cross Sectional Study ◽  
Cross Sectional ◽  
Mean Values ◽  
Factor Xa Inhibition ◽  
Antithrombin Iii Deficiency ◽  
The Mean

SummaryFunctional antithrombin III levels were measured by factor Xa inhibition in 63 members of a large family with type 2 antithrombin III deficiency and individuals were classified as antithrombin III deficient or non-deficient according to the results. FI+2 and TAT complexes were measured using an ELISA and FPA levels were measured by radioimmunoassay.Thirty subjects (48%) were classified as antithrombin III deficient and 33 (52%) as antithrombin III non-deficient. The mean level of FI+2 was significantly higher in the deficient adults (0.87 ± 0.26) compared to both the non-deficient adults (0.70 ± 0.21) (p = 0.03) and the deficient adults receiving warfarin (0.16 ± 0.01) (p <0.001). The differences in the mean values of TAT complexes and FPA between deficient and non-deficient individuals were not statistically significant.These findings suggest that untreated antithrombin III deficient subjects generate more thrombin than their non-deficient family members and that warfarin inhibits this thrombin formation. In this cross-sectional study, it is not possible to correlate the levels of the surrogate makers with future clinical outcome.

Coagulation Inhibitors Studied with Chromogenic Substrate

1975 ◽  
Author(s):  
U. Abildgaard ◽  
M. Lie ◽  
A. Teien ◽  
O. R. Ødegård
Keyword(s):  
Sensitivity And Specificity ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Combined Effect ◽  
Assay System ◽  
Chromogenic Substrate ◽  
Reversible Inhibition ◽  
Heparin Treatment ◽  
At Iii ◽  
Coagulation Inhibitors

The rapid amidolysis of the chromogenic substrate Bz-Phe-Val-Arg-pNA by thrombin (Svendsen & al. Thromb. Res. 1, 267, 1972) provides a useful tool for the study of coagulation inhibitors. With purified reagents, reversible inhibition of the reaction can be demonstrated with 0.01 U/ml of heparin. The inactivation of thrombin by antithrombin III (At-III) is accelerated by 0.001 U/ml of heparin, making it possible to assay small amounts of heparin. The amidolytic assay system has also been used to study the competition between thrombin and factor Xa for At-III.The sensitivity and specificity of rapid amidolytic methods for assay of the following plasma activités will be reported:1) At-III activity (contaminating heparin neutralized).2) Exogenous heparin (during heparin treatment).3) Combined effect of At-III and exogenous heparin.4) At-III in the presence of optimal concentrations of heparin.

Biological Activrty and Safety of the Subcutaneous Administration of High Doses of Low Molecular Weight Heparin for 8 Days in Human Volunteers

1989 ◽  
Vol 61 (03) ◽  
pp. 357-362 ◽  
Author(s):  
J Harenberg ◽  
Ch Giese ◽  
C E Dempfle ◽  
G Stehle ◽  
D L Heene
Keyword(s):  
Molecular Weight ◽  
Factor Xa ◽  
Low Molecular Weight ◽  
Chromogenic Substrate ◽  
Clotting Time ◽  
Human Volunteers ◽  
Factor Xa Inhibition ◽  
Group 2 ◽  
High Doses ◽  
Group 1

SummaryThis study reports on the biological activity and safety of high dose low molecular weight (LMW) heparin therapy administered by two subcutaneous (s.C.) injections daily for 8 days in healthy human volunteers. Group 1 received 2 × 30 aPTT units LMW heparin/kg bodyweight, and group 2 received 2 × 50 aPTT units/kg per day.In group 1, activated partial thromboplastin time (aPTT) and thrombin clotting time (TCT) were uniformly prolonged by 3-5 sec 4 hrs after s.c. administration of heparin. Heptest coagulation time values were prolonged consistently as well by 57 sec on day 1 to 68 sec on day 8. Factor Xa inhibition measured by the S 2222 chromogenic substrate method continuously increased from 0.16 units/ml on day 1 to 0.28 units/ml on day 8.In group 2 prolongation of a aPTT and TCT values increased from 6 sec on day 1 to 15 sec on day 8 and of Heptest time from 70 sec on day 1 to 110 sec on day 8. S 2222 method showed factor Xa inhibitory activity which increased from 0.5 units/ml on day 1 to 0.75 units/ml on day 8. The clinical tolerance of the treatment was good. No changes in clinical chemistry parameters were detected, except for a reversible increase of serum transaminases.The coagulation studies demonstrate accumulation of LMW heparin when high doses are given twice daily. The half life of LMW heparin of factor Xa inhibition increases with increasing doses.Heptest coagulation values were prolonged to 4-6 times the normal values during administration of heparin. S 2222 chromogenic substrate values were in the same range as upon application of high doses of unfractionated heparin. aPTT and thrombin clotting time values ranged from 1.2 to 1.5 times the starting values.

Increased concentrations of heparin cofactor II in diabetic patients, and possible effects on thrombin inhibition assay of antithrombin III.

1989 ◽  
Vol 35 (1) ◽  
pp. 52-55 ◽  
Author(s):  
J Gram ◽  
J Jespersen
Keyword(s):  
Antithrombin Iii ◽  
Factor Xa ◽  
Diabetic Patients ◽  
Type I ◽  
Inhibition Assay ◽  
Heparin Cofactor Ii ◽  
Thrombin Inhibition ◽  
Factor Xa Inhibition ◽  
At Iii ◽  
High Concentrations

Abstract We compared concentrations of antithrombin III (AT-III) in plasma, as determined by an immunological method and by a functional thrombin inhibition method, in the presence of heparin in 160 blood samples from Type I diabetics. Although the correlation was highly significant (P less than 0.001) between the results obtained by the two methods, our data demonstrated that results by the thrombin inhibition assay, 121 (SD 15)%, expressed as percentages of the results for a normal plasma pool, were significantly (P less than 0.001) higher than by the immunoreactive method, 104 (SD 15)%, indicating an overestimation of functionally active AT-III. Concentrations of functionally active AT-III determined by a factor Xa inhibition assay, 105 (SD 13)%, were in the same range as immunoreactive AT-III. Addition of IgG antiserum to normal pooled plasma quenched only about 90% of the AT-III activity determined by the thrombin inhibition assay, but all of the AT-III activity determined by a factor Xa inhibition assay. These results demonstrate that the factor Xa inhibition assay is more specific for the determination of AT-III than the thrombin inhibition assay. We suggest that the high concentrations of heparin cofactor II, 117 (SD 17)%, might have caused an overestimation of AT III in this group of patients with diabetes Type I, and should not be overlooked in other clinical situations.

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