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.

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.

Antithrombin ‘DREUX’ (Lys114Glu): A Variant with Complete Loss of Heparin Affinity

2002 ◽  
Vol 88 (09) ◽  
pp. 436-443 ◽  
Author(s):  
James Huntington ◽  
Jacqueline Conard ◽  
Robin Carrell ◽  
Alec Mushunje ◽  
Aiwu Zhou
Keyword(s):  
Factor Xa ◽  
Fold Increase ◽  
Complete Loss ◽  
Factor X ◽  
Heparin Binding ◽  
High Affinity ◽  
The Core ◽  
Factor Xa Inhibition ◽  
Heparin Affinity ◽  
Father And Son

SummaryHere we report the finding of a new natural antithrombin mutation that confirms the critical contribution of lysine 114 to the binding of the core heparin pentasaccharide, with the replacement of lysine 114 by glutamate causing a complete loss in affinity. The variant was identified in a father and son, the father having been investigated for an episode of cerebral ischaemia associated with hypercholesterolaemia. The variant forms SDS-stable complexes with activated factor X (fXa) and its thermal stability and rate of factor Xa inhibition in the absence of heparin are identical to those of normal antithrombin. Normal antithrombin binds to the high affinity heparin pentasaccharide with a Kd of 1nM, as detected by a 45% change in intrinsic fluorescence, resulting in a 230-fold increase in rate of factor Xa inhibition. However, no change in fluorescence was detected for the variant when titrated with heparin or the heparin pentasaccharide, nor was there detectable activation towards factor Xa, indicating a complete loss of heparin binding.

The Heparin Binding Site Of Antithrombin: Identification Of A Critical Tryptophan Residue Within The Amino Acid Sequence

1981 ◽  
Author(s):  
M Blackburn
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Tryptophan Fluorescence ◽  
Tryptophan Residue ◽  
Affinity Column ◽  
Heparin Binding ◽  
High Affinity ◽  
Heparin Binding Site

Chemical modification of antithrombin III with the tryptophan reagent, dimethyl (2-hydroxy-5-nitrobenzyl) sulfonium bromide, results in the incorporation of one hydroxynitrobenzyl (HNB) moiety per molecule of antithrombin III. Heparin protects against tryptophan modification, particularly at low reagent concentrations. Unlike native antithrombin, which has high affinity for heparin, HNB-anti- thrombin does not bind to a heparin-agarose affinity column. Furthermore, the heparin-induced increase in tryptophan fluorescence, obtained with native antithrombin, is not observed with the singly modified inhibitor. HNB-anti- thrombin does not exhibit heparin-promoted rate enhancement in the inactivation of thrombin and Factor Xa. However, in the absence of heparin, HNB-antithrombin and native antithrombin possess progressive antithrombin activity, inactivating these proteases at identical rates. These results indicate that the integrity of a specific tryptophan residue is required for the binding of heparin to antithrombin III. Chemical and enzymatic cleavage techniques have been used to isolate peptides containing this tryptophan from both HNB-labeled and native antithrombin and to identify this critical tryptophan residue within the amino acid sequence of the antithrombin molecule.

scholarly journals NMR structure determination of Ixolaris and factor X(a) interaction reveals a noncanonical mechanism of Kunitz inhibition

Blood ◽  
2019 ◽  
Vol 134 (8) ◽  
pp. 699-708 ◽  
Author(s):  
Viviane S. De Paula ◽  
Nikolaos G. Sgourakis ◽  
Ivo M. B. Francischetti ◽  
Fabio C. L. Almeida ◽  
Robson Q. Monteiro ◽  
...  
Keyword(s):  
Factor Xa ◽  
Coagulation Factor ◽  
Structural Basis ◽  
Factor X ◽  
Heparin Binding ◽  
Nmr Structure Determination ◽  
High Resolution Structure ◽  
Function Relationship ◽  
Affinity Interaction ◽  
Relationship Of

Abstract Ixolaris is a potent tick salivary anticoagulant that binds coagulation factor Xa (FXa) and zymogen FX, with formation of a quaternary tissue factor (TF)/FVIIa/ FX(a)/Ixolaris inhibitory complex. Ixolaris blocks TF-induced coagulation and PAR2 signaling and prevents thrombosis, tumor growth, and immune activation. We present a high-resolution structure and dynamics of Ixolaris and describe the structural basis for recognition of FX. Ixolaris consists of 2 Kunitz domains (K1 and K2) in which K2 is strikingly dynamic and encompasses several residues involved in FX binding. This indicates that the backbone plasticity of K2 is critical for Ixolaris biological activity. Notably, a nuclear magnetic resonance–derived model reveals a mechanism for an electrostatically guided, high-affinity interaction between Ixolaris and FX heparin-binding (pro)exosite, resulting in an allosteric switch in the catalytic site. This is the first report revealing the structure-function relationship of an anticoagulant targeting a zymogen serving as a scaffold for TF inhibition.

scholarly journals Heparin Binding Proteins as Therapeutic Target: An Historical Account and Current Trends

Medicines ◽  
2019 ◽  
Vol 6 (3) ◽  
pp. 80 ◽  
Author(s):  
Giancarlo Ghiselli
Keyword(s):  
Small Molecules ◽  
Biological Function ◽  
Antithrombin Iii ◽  
Biological Effects ◽  
Structural Flexibility ◽  
Heparin Binding ◽  
New Class ◽  
High Affinity ◽  
Current Trends ◽  
High Degree

The polyanionic nature and the ability to interact with proteins with different affinities are properties of sulfated glycosaminoglycans (GAGs) that determine their biological function. In designing drugs affecting the interaction of proteins with GAGs the challenge has been to generate agents with high binding specificity. The example to emulated has been a heparin-derived pentasaccharide that binds to antithrombin-III with high affinity. However, the portability of this model to other biological situations is questioned on several accounts. Because of their structural flexibility, oligosaccharides with different sulfation and uronic acid conformation can display the same binding proficiency to different proteins and produce comparable biological effects. This circumstance represents a formidable obstacle to the design of drugs based on the heparin scaffold. The conceptual framework discussed in this article is that through a direct intervention on the heparin-binding functionality of proteins is possible to achieve a high degree of action specificity. This objective is currently pursued through two strategies. The first makes use of small molecules for which in the text we provide examples from past and present literature concerning angiogenic factors and enzymes. The second approach entails the mutagenesis of the GAG-binding site of proteins as a means to generate a new class of biologics of therapeutic interest.

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.

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.

EVIDENCE FOR A HOMOLOGY BETWEEN THE HEPARIN AND HEPARAN SULFATE BINDING REGIONS TO ANTITHROMBIN III

1987 ◽  
Author(s):  
A M Fischer ◽  
J Tapon-Bretaudière ◽  
M D Dautzenberg ◽  
C Sternberg ◽  
J Choay
Keyword(s):  
Heparan Sulfate ◽  
Antithrombin Iii ◽  
Competitive Inhibition ◽  
Factor Xa ◽  
Inhibitory Effect ◽  
Heparin Binding ◽  
At Iii ◽  
Binding Regions ◽  
Partial Homology ◽  
Type 3

As we have previously demonstrated, heparan sulfate, a glycosaminoglycan physiologically present on the endothelial wall, is, like heparin, unable to potentiate the inhibitory effect against Factors IIa and Xa of two abnormal type 3 antithrombin III (AT III) variants. We report here that a synthetic pentasaccharide which constitutes the sequence of the heparin binding site to AT III is also unable to potentiate these two AT III variants in an anti-Factor Xa assay. According to these data, we speculated the existence of a homology between the heparin and heparan sulfate binding regions to normal AT III. We thus studied the competitive inhibition by the pentasaccharide of heparin and heparan sulfate in their potentiation of AT III activity. Such a competitive inhibition can be observed in an AT III antiFactor IIa assay because the pentasaccharide which exhibits a high anti-Factor Xa activity is devoid of any anti-Factor IIa activity. In the absence of pentasaccharide, with both heparin and heparan sulfate, a plateau is reached in AT III potentiation for concentrations of respectively 2.5 ng and 17.2 μg (corresponding to the same anti-Factor IIa activity of 0.4 u/ml for both glycosaminoglycans). In the presence of 5.5 μg of pentasaccharide, the inhibition of heparin cofactor activity is 70%. With heparan sulfate, the inhibition by the same amount of pentasaccharide is less pronounced, being only 30%. These results strongly suggest the existence of a partial homology between heparin and heparan sulfate binding sites to AT III. For heparan sulfate, the exact sequence of this site remains to be identified .

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.

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