Characterization Of Rabbit Antithrombin III

1981 ◽  
Author(s):  
D Estry ◽  
J C Mattson ◽  
T G Bell ◽  
G H Tishkoff
Keyword(s):  
Molecular Weight ◽  
Antithrombin Iii ◽  
Cross Reactivity ◽  
Antigenic Determinants ◽  
Heparin Binding ◽  
Sds Page ◽  
At Iii ◽  
Human Thrombin ◽  
Immunologic Assays

The rabbit is a well established model for studying the disseminated intravascular coagulation (DIC) associated with endotoxic syndromes. In order to establish the role of antithrombin III (AT III) in the modulation of DIC in the rabbit, characterization of rabbit AT III was undertaken. Rabbit antithrombin III, isolated according to modifications of the method of Thaler and Schmer, has a molecular weight comparable to that of human AT III (62,000 daltons) as measured by mobility on SDS-PAGE gels. Mixtures of rabbit and human AT III co-migrate as a single band on 7.5% SDS-PAGE gels. Rabbit AT III possesses both progressive and heparin activated (immediate) antithrombin activity in assays using human thrombin. Antisera raised against rabbit AT III demonstrates no cross reactivity with human AT III suggesting that despite physiologic and molecular weight similarities, antigenic differences are present. Incubation of rabbit antithrombin III with specific antisera, either prior to or after addition of heparin, did not alter the ability of antithrombin III to inhibit thrombin in either the immediate or progressive assays indicating that the antigenic determinants are not found in either the heparin binding or active thrombin binding sites. Crossed immunoelectrophoresis (IEP) demonstrates that antisera to rabbit AT III reacts with both free rabbit antithrombin III and AT III-thrombin complexes and can therefore be used in immunologic assays to quantitate total rabbit AT III (bound and free) and in crossed IEP to demonstrate the mobility of both free and complexed AT III.

Isolation and Partial Characterization of Equine Antithrombin III.

1979 ◽  
Author(s):  
D.W. Estry ◽  
T.G. Bell ◽  
G.H. Tishkoff ◽  
J.C. Mattson ◽  
S.C. Estry
Keyword(s):  
Molecular Weight ◽  
Antithrombin Iii ◽  
Stable Complex ◽  
Sds Page ◽  
At Iii ◽  
Human Thrombin ◽  
Horse Plasma ◽  
Stoichiometric Complex

A protein analogous to human antithrombin III was isolated from fresh horse plasma. The procedure for purification was a modification of Thaler and Schmer’s two-step isolation procedure. The horse protein was homogeneous on 7.5% SDS-PAGE gels and had a molecular weight of 62,000 to 64,000 daltons in both reducing and non-reducing systems (human; 62,300). Rabbit anti-human antithrombin III was used to demonstrate a line of partial identity by Immunoelectrophoresis between the horse and human protein. The horse protein rapidly neutralizes human thrombin (34,000 daltons) and the reaction appears to be greatly potentiated by heparin. In order to establish the formation of 1:1 covalent stoichiometric complex between horse AT III and thrombin (IIa), time studies were run in the presence and absence of heparin. AT III (62,000) at 15 seconds, 2, 5, 10 and 60 minutes formed a stable complex with thrombin (32,000) having a molecular weight of 86,000 daltons. Additional bands developing with time are due to the autolytic capabilities of the uncomplexed IIa. The major autolytic band had a molecular weight of 70,000 daltons. Addition of heparin potentiated the interaction although it did not change the stoichio-metry of the complexes formed. The data accumulated to date demonstrates the similarities between the human and horse protein and the possibilities of using the horse as a model system for the evaluation of AT III replacement therapy in vivo.

Isolation and Partial Characterization of Equine Antithrombin III

1979 ◽  
Author(s):  
D. W. Estry ◽  
T. G. Bell ◽  
G. H. Tishkoff ◽  
J. C. Mattson ◽  
S. C. Estry
Keyword(s):  
Molecular Weight ◽  
Antithrombin Iii ◽  
Stable Complex ◽  
Sds Page ◽  
At Iii ◽  
Human Thrombin ◽  
Horse Plasma ◽  
Stoichiometric Complex

A protein analogous to human antithrombin III was isolated from fresh horse plasma. The procedure for purification was a modification of Thaler and Schmer’s two-step isolation procedure. The horse protein was homogeneous on 7.5% SDS-PAGE gels and had a molecular weight of 62,000 to 64,000 daltons in both reducing and non-reducing systems (human; 62,300). Rabbit anti-human antithrombin III was used to demonstrate a line of partial identity by immunoelectrophoresis between the horse and human protein. The horse protein rapidly neutralizes human thrombin (34,000 daltons) and the reaction appears to be greatly potentiated by heparin. in order to establish the formation of 1:1 covalent stoichiometric complex between horse AT III and thrombin (IIa), time studies were run in the presence and absence of heparin. AT III (62,000) at 15 seconds, 2, 5, 10 and 60 minutes formed a stable complex with thrombin (32,000) having a molecular weight of 86,000 daltons. Additional bands developing with time are due to the autolytic capabilities of the uncomplexed IIa. The major autolytic band had a molecular weight of 70,000 daltons. Addition of heparin potentiated the interaction although it did not change the stoichiometry of the complexes formed. The data accumulated to date demonstrates the similarities between the human and horse protein and the possibilities of using the horse as a model system for the evaluation of AT III replacement therapy in vivo.

The Different Forms of Antithrombin III in Serum

1977 ◽  
Vol 38 (02) ◽  
pp. 0494-0503 ◽  
Author(s):  
D. S Pepper ◽  
D Banhegyi ◽  
J. D Cash
Keyword(s):  
Molecular Weight ◽  
Affinity Chromatography ◽  
Human Serum ◽  
Degradation Product ◽  
Antithrombin Iii ◽  
Gel Filtration ◽  
Free Form ◽  
Polymeric Form ◽  
At Iii ◽  
Human Thrombin

SummaryAntithrombin III (AT III) complexes were isolated from human serum by affinity chromatography and gel filtration. In the first step of the preparation, using heparin-agarose chromatography, we observed that the complexed form of AT III bound less strongly to the gel than the free form and that about half of the AT III was free. With further purification a 2.5 × 105 molecular weight complex was isolated. Using 125I labelled human thrombin, this complex was radioactive indicating the presence of thrombin. Only in a synthetic thrombin-AT III system was a 9 × 104 molecular weight complex detected, but not in serum. These facts suggest that in serum AT III complexes may exist in a polymeric form. Also, an AT III antigen derived from the original AT III molecule, but not complexed, was isolated which may be a degradation product.Abbreviations used: AT-III, antithrombin III. Hepes, N-2-Hydroxyethylpiperazine-N-2-Ethanesulphonic acid.

scholarly journals Antithrombin Milano: a new variant with monomeric and dimeric inactive antithrombin III

Blood ◽  
1985 ◽  
Vol 65 (2) ◽  
pp. 496-500 ◽  
Author(s):  
M Wolf ◽  
C Boyer ◽  
A Tripodi ◽  
D Meyer ◽  
MJ Larrieu ◽  
...  
Keyword(s):  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Reactivity ◽  
Two Dimensional ◽  
Western Blots ◽  
Sds Page ◽  
New Variant ◽  
At Iii ◽  
Monospecific Antisera

Abstract A qualitative defect of antithrombin III (AT III) has been demonstrated over three generations in eight members of an Italian family by the discrepancy between a normal amount of antigen and decreased antithrombin and anti-Xa activity in the presence or in the absence of heparin. By two-dimensional immunoelectrophoresis in the absence of heparin, two peaks of AT III were present in all patients' plasma. AT III was purified from normal and propositus plasma by sulfate dextran precipitation followed by heparin affinity chromatography. The elution profile of the patient's AT III was abnormal and allowed the separation of two populations of AT III, normal and abnormal. The first fraction (normal AT III) contained AT III activity, migrated as a single peak by two-dimensional immunoelectrophoresis and by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE), demonstrated a single band with a molecular weight (mol wt) identical to that of normal AT III (60,000). Conversely, the last fraction, devoid of AT III activity, migrated as a single abnormal peak by two-dimensional immunoelectrophoresis in the absence of heparin. By SDS-PAGE, two bands were observed: one with a mol wt of 60,000 and a second one with a mol wt of 120,000. Western blots clearly demonstrated cross-reactivity of the 120,000 and 60,000 mol wt bands with monospecific antisera to human AT III. Reduction of the 120,000 mol wt band converted it to a single 60,000 mol wt band, suggesting the presence of an abnormal dimeric form of AT III. The name AT III Milano is proposed for this new variant.

scholarly journals The Mode of Action of CY216 and CY222 in Plasma

1992 ◽  
Vol 67 (01) ◽  
pp. 033-041 ◽  
Author(s):  
Suzette Béguin ◽  
Simone Wielders ◽  
J C Lormeau ◽  
H Coenraad Hemker
Keyword(s):  
Molecular Weight ◽  
High Performance ◽  
Time Course ◽  
Antithrombin Iii ◽  
Platelet Rich Plasma ◽  
Gel Permeation Chromatography ◽  
Heparin Binding ◽  
High Affinity ◽  
Binding Material ◽  
At Iii

SummaryThree fractions of the low molecular weight heparin CY216 (fraxiparin, mean molecular weight [MMW] 5,090), with MMWs of respectively, 3,090, 4,400 and 7,910 were prepared by gel permeation chromatography. From CY222 (MMW 3,770) as well as from CY216 and its three fractions the material with high affinity to antithrombin III (AT III) was obtained by chromatography on immobilised AT III. The molecular weight distribution of each of the ten preparations thus obtained was determined by high performance liquid chromatography, while the content of AT III binding material was determined by stoichiometric titration of AT III, monitored by intrinsic fluorescence enhancement.We measured the effect of all heparins on the decay of endogenous thrombin in plasma and on the overall generation of thrombin in plasma, triggered via the extrinsic or via the intrinsic pathway. From these data we calculated the time course of prothrombin conversion, i. e. the course of factor Xa activity as expressed by prothrombinase activity.It was found that in platelet-poor plasma the anticoagulant properties of the heparins are largely dependent on their antithrombin action, which is determined by their content of high affinity material with a MW of 5,400 or higher. The specific antithrombin activity of all heparins, when expressed in terms of material with high affinity to antithrombin III (HAM) with a MW >5,400 is 13.0 min-1/(μg/ml) (range 10.5-15.9). The anticoagulant potency is not influenced by the presence of low-affinity material and hardly by material with MW <5,400.In platelet-rich plasma, however, the presence of non-AT III binding material enhances the inhibition, presumably by neutralising heparin binding material originating from activated platelets. The ultra low MW fractions (<3,400) show a similar activity in PPP and in PRP.

Studies of Heparin Binding to Antithrombin III by Crossed Immunoelectrophoresis

1979 ◽  
Vol 42 (05) ◽  
pp. 1434-1445 ◽  
Author(s):  
Trevor W Barrowcliffe
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Antithrombin Iii ◽  
Heparan Sulphate ◽  
Heparin Binding ◽  
Binding Ability ◽  
Crossed Immunoelectrophoresis ◽  
Binding Material ◽  
At Iii ◽  
Heparin Fractions

SummaryThe technique of crossed immunoelectrophoresis has been used to study the binding to purified antithrombin III (At III) of heparin and other mucopolysaccharides. The technique was unable to detect differences among samples of whole heparin from various manufacturers, but proved useful in studying the binding of heparin fractions; at the same molarities, low and high molecular weight heparin fractions displayed equal binding ability to At III. A semi-synthetic heparin analogue showed no evidence of binding to At III, but a sample of heparan sulphate did interact with At III at a concentration 3 times that of heparin. Samples of purified At III from four different manufacturers all displayed heterogeneity with respect to heparin binding. A proportion of the total At III did not bind to heparin and, in one sample, this non-binding material constituted about 40% of the total. An antiserum made against purified At III contained antibodies with different cross-reactivities against heparin bound and non-heparin bound At III.

CHARACTERIZATION OF HYDROPSYCHE SLOSSONAE (TRICHOPTERA: HYDROPSYCHIDAE) CAPTURE NET POLYPEPTIDES

2000 ◽  
Vol 132 (1) ◽  
pp. 59-68 ◽  
Author(s):  
L. Tessier ◽  
J.L. Boisvert ◽  
L.B-M. Vought ◽  
J.O. Lacoursière
Keyword(s):  
Molecular Weight ◽  
Sequence Data ◽  
Polyacrylamide Gel Electrophoresis ◽  
Current Trend ◽  
Cross Reactivity ◽  
Published Data ◽  
Molecular Weights ◽  
Sds Page ◽  
Terrestrial Insects

AbstractThe aim of this study was to characterize polypeptide components of the capture net spun by trichopteran larvae Hydropsyche slossonae (Banks) (Trichoptera: Hydropsychidae). Thirty-one polypeptide bands were identified by SDS – polyacrylamide gel electrophoresis (SDS–PAGE) from extracted net material, with molecular weights ranging from 8500 to 179 000. Comparison with published data on Bombyx mori (L.) (Lepidoptera: Bombycidae) silk, treated under similar denaturing conditions, shows that six low molecular weight polypeptides ranging between 8500 and 18 800 in the silk of H. slossonae are absent from that of B. mori; furthermore, two high molecular weight polypeptides (210 000 and 220 000) detected in the silk of B. mori are not present in that of H. slossonae. Differences between both groups are probably related to their mode of living and to the specific use of silk (in air versus under water). Our findings are consistent with the current trend in the literature that silk spun by aquatic and terrestrial insects, as well as those spun by different species, is apparently made of different biopolymers according to the protein constituents. Hence, the polypeptide characterization of silk, combined with sequence data and (or) antibodies cross-reactivity data, could represent a potential tool for taxonomic classification improvement of aquatic insects. These results could eventually be used to characterize hydropsychid capture net anomalies induced by environmental pollution.

ANTITHROMBIN III NORTHWICK PARK: CHARACTERIZATION OF AN INACTIVE HIGH MW COMPLEX WITH INCREASED AFFINITY FOR HEPARIN

1987 ◽  
Author(s):  
H Erdjument ◽  
D A Lane ◽  
A M Flynn ◽  
H Ireland ◽  
M Panico ◽  
...  
Keyword(s):  
Antithrombin Iii ◽  
Exchange Chromatography ◽  
Terminal Sequence ◽  
Structural Investigations ◽  
Crossed Immunoelectrophoresis ◽  
Sds Page ◽  
Affected Family Member ◽  
At Iii ◽  
Salt Gradient

It has been shown previously that antithrombin III Northwick Park (AT III NWP) has reduced ability to inactivate thrombin and is characterised by an additional anodal component on crossed immunoelectrophoresis. We have applied plasma from an affected family member to heparin-Sepharose and eluted the AT III with a salt gradient. Evidence will be presented that the anodal component has* higher affinity for heparin than normal AT III. Furthermore, this variant component is present in plasma as a MW >120,000 inactive complex whose tryptic peptide FAB map contains numerous signals not characteristic of normal AT . 111 . This complex can be reduced with dithiothreitol to two non identical bands on SDS PAGE with MW ~60,000, only one of which reacts with anti-AT III. Using ion-exchange chromatography and HPLC these two components have been isolated and separated. The N-terminal sequence of the protein that does not react with anti-AT III is believed to be Asp-Ala-His-Ile-Ser-Glu. Structural investigations on the variant AT III are underway.

scholarly journals Antithrombin Milano: a new variant with monomeric and dimeric inactive antithrombin III

Blood ◽  
1985 ◽  
Vol 65 (2) ◽  
pp. 496-500
Author(s):  
M Wolf ◽  
C Boyer ◽  
A Tripodi ◽  
D Meyer ◽  
MJ Larrieu ◽  
...  
Keyword(s):  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Reactivity ◽  
Two Dimensional ◽  
Western Blots ◽  
Sds Page ◽  
New Variant ◽  
At Iii ◽  
Monospecific Antisera

A qualitative defect of antithrombin III (AT III) has been demonstrated over three generations in eight members of an Italian family by the discrepancy between a normal amount of antigen and decreased antithrombin and anti-Xa activity in the presence or in the absence of heparin. By two-dimensional immunoelectrophoresis in the absence of heparin, two peaks of AT III were present in all patients' plasma. AT III was purified from normal and propositus plasma by sulfate dextran precipitation followed by heparin affinity chromatography. The elution profile of the patient's AT III was abnormal and allowed the separation of two populations of AT III, normal and abnormal. The first fraction (normal AT III) contained AT III activity, migrated as a single peak by two-dimensional immunoelectrophoresis and by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE), demonstrated a single band with a molecular weight (mol wt) identical to that of normal AT III (60,000). Conversely, the last fraction, devoid of AT III activity, migrated as a single abnormal peak by two-dimensional immunoelectrophoresis in the absence of heparin. By SDS-PAGE, two bands were observed: one with a mol wt of 60,000 and a second one with a mol wt of 120,000. Western blots clearly demonstrated cross-reactivity of the 120,000 and 60,000 mol wt bands with monospecific antisera to human AT III. Reduction of the 120,000 mol wt band converted it to a single 60,000 mol wt band, suggesting the presence of an abnormal dimeric form of AT III. The name AT III Milano is proposed for this new variant.

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.

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