scholarly journals Disruption of a Tight Cluster Surrounding Tyrosine 131 in the Native Conformation of Antithrombin III Activates It for Factor Xa Inhibition

2006 ◽  
Vol 281 (42) ◽  
pp. 31668-31676 ◽  
Author(s):  
Richard Glenn C. dela Cruz ◽  
Mohamad Aman Jairajpuri ◽  
Susan C. Bock
Keyword(s):  
Antithrombin Iii ◽  
Factor Xa ◽  
Native Conformation ◽  
Tight Cluster ◽  
Factor Xa Inhibition

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.

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.

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.

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.

Effects Of Heparin On The Rates Of Inhibition Of Thrombin And Factor Xa By Antithrqmbin III

1981 ◽  
Author(s):  
M J Griffith
Keyword(s):  
Mathematical Modeling ◽  
Enzyme Inhibition ◽  
Maximum Rate ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Thrombin Inhibition ◽  
Heparin Concentration ◽  
Factor Xa Inhibition ◽  
Direct Binding ◽  
Inhibition Of Thrombin

The rate of inhibition of thrombin and Factor Xa by antithrombin III has been investigated as a function of heparin concentration. Three general observations were made. First, the amount of heparin required to maximally enhance the inhibition of thrombin is much less than the amount of heparin required for Factor Xa inhibition by anti thrombin III. Second, the maximum rate of thrombin inhibition was approximately 5-fold higher than the maximum rate of Factor Xa inhibition with optimal heparin levels. Third, heparin concentrations greater than 10-6M decreased the rates of inhibition of both Factor Xa and thrombin. Mathematical modeling was attempted to describe the mechanism of action of heparin. At low heparin concentrations (< 10-6M) the inhibition of thrombin and Factor Xa could best be described by a model which presumed that binding of heparin to the enzyme accelerates enzyme inhibition by antithrombin III. The effect of heparin at concentrations < 10-6 on the rates of enzyme inhibition could not be explained by direct binding of heparin to either antithrcmbin III or thrombin (Factor Xa). This observation has led to the hypothesis that the interaction of thrombin and Factor Xa with heparin is altered at relatively high heparin concentration which results in a decrease in the effectiveness of heparin in accelerating enzyme inhibition by anti thrombin III.

The Effect of Antithrombin III on the Activity of the Coagulation Factors VII, IX and X

1976 ◽  
Vol 35 (02) ◽  
pp. 295-304 ◽  
Author(s):  
B Østerud ◽  
M Miller-Andersson ◽  
U Abildgaard ◽  
H Prydz
Keyword(s):  
Antithrombin Iii ◽  
Factor Xa ◽  
Coagulation Factors ◽  
Disc Electrophoresis ◽  
Polyacrylamide Gel ◽  
Factor Vii ◽  
Native Form ◽  
Factor Ixa ◽  
Plasma Factor

SummaryAntithrombin III, purified to homogeneity according to Polyacrylamide gel disc electrophoresis and immunoelectrophoresis, inhibited the activity of purified factor IXa and Xa, whereas factor VII was not inhibited either in the active or in the native form.Antithrombin III is the single most important inhibitor of factor Xa in plasma. Factor Xa does not, however, reduce the activity of antithrombin III against thrombin.

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