scholarly journals Characterization of the cellular binding domain and the effects of monoclonal antibodies and thrombin inhibitors on the binding and internalization of the antithrombin-III - thrombin complex by cultured cells

1991 ◽  
Vol 195 (3) ◽  
pp. 801-806 ◽  
Author(s):  
Sarah KNOLLER ◽  
Naphtali SAVION
Keyword(s):  
Monoclonal Antibodies ◽  
Antithrombin Iii ◽  
Cultured Cells ◽  
Binding Domain ◽  
Thrombin Inhibitors

scholarly journals Monoclonal antibodies against plasma protease inhibitors: production and characterization of 15 monoclonal antibodies against human antithrombin III. Relation between antigenic determinants and functional sites of antithrombin III

Blood ◽  
1985 ◽  
Vol 65 (5) ◽  
pp. 1201-1207 ◽  
Author(s):  
P Herion ◽  
M Francotte ◽  
D Siberdt ◽  
GG Soto ◽  
J Urbain ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Antithrombin Iii ◽  
Antigenic Determinants ◽  
Mouse Strains ◽  
High Avidity ◽  
Functional Sites ◽  
Crude Preparation ◽  
Alpha 1 Antitrypsin ◽  
Antithrombin Iii Activity

Abstract Fifteen hybridomas secreting monoclonal antibodies against human antithrombin III, originating from two mouse strains, have been produced by the cell fusion technique. Eight monoclonal antibodies belong to the class IgG1, five to the class IgG2a, and two to the class IgG2b. All light chains belong to the kappa group. No cross-reaction of the monoclonal antibodies have been observed with a crude preparation of albumin nor with alpha 1-antitrypsin and alpha 2-antiplasmin. Five of these monoclonal antibodies exhibit a relatively high avidity for antithrombin III. Inhibition experiments showed that the 15 monoclonal antibodies define seven more or less independent antigenic regions on the antithrombin III molecule. Examination of the effects of these antibodies on the inhibitory capacity of antithrombin III toward thrombin activity, either in the presence or in the absence of heparin, showed that several monoclonal antibodies inhibit the antithrombin III activity and allowed to relate some of the antigenic determinants to functional sites on the antithrombin III molecule.

scholarly journals Monoclonal antibodies against plasma protease inhibitors: production and characterization of 15 monoclonal antibodies against human antithrombin III. Relation between antigenic determinants and functional sites of antithrombin III

Blood ◽  
1985 ◽  
Vol 65 (5) ◽  
pp. 1201-1207
Author(s):  
P Herion ◽  
M Francotte ◽  
D Siberdt ◽  
GG Soto ◽  
J Urbain ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Antithrombin Iii ◽  
Antigenic Determinants ◽  
Mouse Strains ◽  
High Avidity ◽  
Functional Sites ◽  
Crude Preparation ◽  
Alpha 1 Antitrypsin ◽  
Antithrombin Iii Activity

Fifteen hybridomas secreting monoclonal antibodies against human antithrombin III, originating from two mouse strains, have been produced by the cell fusion technique. Eight monoclonal antibodies belong to the class IgG1, five to the class IgG2a, and two to the class IgG2b. All light chains belong to the kappa group. No cross-reaction of the monoclonal antibodies have been observed with a crude preparation of albumin nor with alpha 1-antitrypsin and alpha 2-antiplasmin. Five of these monoclonal antibodies exhibit a relatively high avidity for antithrombin III. Inhibition experiments showed that the 15 monoclonal antibodies define seven more or less independent antigenic regions on the antithrombin III molecule. Examination of the effects of these antibodies on the inhibitory capacity of antithrombin III toward thrombin activity, either in the presence or in the absence of heparin, showed that several monoclonal antibodies inhibit the antithrombin III activity and allowed to relate some of the antigenic determinants to functional sites on the antithrombin III molecule.

MODULATION OF ANTITHROMBIN III ACTIVITY AND ANTITHROMBIN III-THROMBIN COMPLEXES BINDING TO CULTURED CELLS BY MONOCLONAL ANTIBODIES AGAINST ANTITHROMBIN III

1987 ◽  
Author(s):  
S Knoller ◽  
N Savion
Keyword(s):  
Monoclonal Antibodies ◽  
Binding Site ◽  
Conformational Changes ◽  
Antithrombin Iii ◽  
Cultured Cells ◽  
Inhibitory Effect ◽  
National Council ◽  
Corneal Endothelial Cells ◽  
Inhibitory Effects ◽  
Antithrombin Iii Activity

Two monoclonal antibodies (mAb's) against antithrombin III (ATIII) were characterized with respect to their ability to interfere with ATIII activity. AT III activity was measured by its ability to inhibit the amidolitic activity of thrombin on the substrate BCP-100. Incubation of 150 ng of ATIII with 28pg mAb A36R2 prior to addition of 50 ng thrombin totally abolishes the inhibitory effect of ATIII on thrombin. Incubation of 200ng of ATIII with 10 μg of mAb B26R4 prior to addition of 75 ng thrombin raises the inhibitory effects of ATIII from 37% to 100%. We examined the effect of these mAb's on binding of antithrombin III-thrombin (ATIII-Th) complexes to bovine corneal endothelial cells. 120 pg/ml mAb's are reacted with 2 μg/ml ATIII-Th complexes prior to their addition to the cells. mAb A36R2 completely blocks ATIII-Th complexes binding. In contrast, mAb B26R4 enhances binding up to 250% of the control binding.We conclude that mAb A36R2 prevents binding of thrombin to ATIII by recognizing an epitope on ATIII close to thrombin binding site or that its binding to ATIII induces a conformational change in the thrombin binding site thus it no longer recognizes thrombin. mAb B26R4 has a heparin-like effect on ATIII: Its binding to ATIII induces conformational changes which improve thrombin binding to ATIII. There is a correlation between inhibition and enhancement of thrombin binding to ATIII and of ATIII-Th complexes binding to cells by the two mAb's. These mAb's may provide a new tool to control the activity of ATIII and to identify the cellular binding site on the ATIII-Th complex.This research was supported by a grant from the National Council for Research and Development, Israel and G.S.F. München, Germany.

Isolation and characterization of two monoclonal antibodies that recognize remote epitopes on the cell-binding domain of human fibronectin

1989 ◽  
Vol 185 (1) ◽  
pp. 229-236 ◽  
Author(s):  
Masahiko Katayama ◽  
Fumitsugu Hino ◽  
Yoichi Odate ◽  
Shoichi Goto ◽  
Fusao Kimizuka ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Binding Domain ◽  
Cell Binding ◽  
Isolation And Characterization ◽  
Human Fibronectin ◽  
Cell Binding Domain

Characterization of Monoclonal Anti-human Factor VII Antibody (B101/B1) that Recognized Three-dimensional Structures near Arg at Position 79 in the First EGF-like Domain

1998 ◽  
Vol 79 (01) ◽  
pp. 104-109 ◽  
Author(s):  
Osamu Takamiya
Keyword(s):  
Monoclonal Antibodies ◽  
Tissue Factor ◽  
Human Factor ◽  
Solid Phase ◽  
Three Dimensional ◽  
Coagulation Factors ◽  
Factor Vii ◽  
Dimensional Structure ◽  
Epidermal Growth

SummaryMurine monoclonal antibodies (designated hVII-B101/B1, hVIIDC2/D4 and hVII-DC6/3D8) directed against human factor VII (FVII) were prepared and characterized, with more extensive characterization of hVII-B101/B1 that did not bind reduced FVIIa. The immunoglobulin of the three monoclonal antibodies consisted of IgG1. These antibodies did not inhibit procoagulant activities of other vitamin K-dependent coagulation factors except FVII and did not cross-react with proteins in the immunoblotting test. hVII-DC2/D4 recognized the light chain after reduction of FVIIa with 2-mercaptoethanol, and hVIIDC6/3D8 the heavy chain. hVII-B101/B1 bound FVII without Ca2+, and possessed stronger affinity for FVII in the presence of Ca2+. The Kd for hVII-B101/B1 to FVII was 1.75 x 10–10 M in the presence of 5 mM CaCl2. The antibody inhibited the binding of FVII to tissue factor in the presence of Ca2+. hVII-B101/B1 also inhibited the activation of FX by the complex of FVIIa and tissue factor in the presence of Ca2+. Furthermore, immunoblotting revealed that hVII-B101/B1 reacted with non-reduced γ-carboxyglutaminic acid (Gla)-domainless-FVII and/or FVIIa. hVII-B101/B1 showed a similar pattern to that of non-reduced proteolytic fragments of FVII by trypsin with hVII-DC2/D4 on immunoblotting test. hVII-B101/B1 reacted differently with the FVII from the dysfunctional FVII variant, FVII Shinjo, which has a substitution of Gln for Arg at residue 79 in the first epidermal growth factor (1st EGF)-like domain (Takamiya O, et al. Haemosta 25, 89-97,1995) compared with normal FVII, when used as a solid phase-antibody for ELISA by the sandwich method. hVII-B101/B1 did not react with a series of short peptide sequences near position 79 in the first EGF-like domain on the solid-phase support for epitope scanning. These results suggested that the specific epitope of the antibody, hVII-B101/B1, was located in the three-dimensional structure near position 79 in the first EGF-like domain of human FVII.

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.

Characterization of an Abnormal Antithrombin (Milano 2) with Defective Thrombin Binding

1986 ◽  
Vol 56 (03) ◽  
pp. 349-352 ◽  
Author(s):  
A Tripodi ◽  
A Krachmalnicoff ◽  
P M Mannucci
Keyword(s):  
Venous Thromboembolism ◽  
Active Site ◽  
Inhibitory Activity ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Molecular Defect ◽  
Affinity Adsorption ◽  
Italian Family ◽  
Crossed Immunoelectrophoresis

SummaryFour members of an Italian family (two with histories of venous thromboembolism) had a qualitative defect of antithrombin III reflected by normal antigen concentrations and halfnormal antithrombin activity with or without heparin. Anti-factor Xa activities were consistently borderline low (about 70% of normal). For the propositus’ plasma and serum the patterns of antithrombin III in crossed-immunoelectrophoresis with or without heparin were indistinguishable from those of normal plasma or serum. A normal affinity of antithrombin III for heparin was documented by heparin-sepharose chromatography. Affinity adsorption of the propositus’ plasma to human α-thrombin immobilized on sepharose beads revealed defective binding of the anti thrombin III to thrombin-sepharose. Hence the molecular defect of this variant appears to be at the active site responsible for binding and neutralizing thrombin, thus accounting for the low thrombin inhibitory activity.

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