High Molecular Weight Forms of Antithrombin III Complexes in Blood

1983 ◽  
Vol 49 (01) ◽  
pp. 032-036 ◽  
Author(s):  
E Marciniak ◽  
G Gora-Maslak
Keyword(s):  
Antithrombin Iii ◽  
Serum Proteins ◽  
Gel Filtration ◽  
Factor Xa ◽  
Molecular Size ◽  
Molecular Forms ◽  
Factor X ◽  
Monomeric Complex ◽  
At Iii ◽  
Antibody Competition

SummaryA double antibody competition radioimmunoassay was developed that allowed to detect specifically as little as 15 ng antithrombin III (AT III) per ml of the assayed material. In normal plasma examined by this assay, AT III concentration averaged 199 ± 21 μg/ml. Complexes of AT III with thrombin or factor X a crossreacted with free AT III in 87% and 95%, respectively. Molecular forms of AT III produced in plasma treated with coagulation enzymes, or in serum, were assessed by measuring immunoreactive AT III in fractions obtained by gel filtration chromatography on Sephadex G-200. AT III bound by thrombin in fibrinogen free-plasma ranged in molecular size from 160,000 to above 250,000. Similar aggregation occurred when monomeric complex of purified AT III and thrombin, of 90,000 Mr, was added to plasma. Presence of heparin intensified the degree of aggregation. In factor Xa-treated plasma AT III was converted into components with 160,000 Mr, or less. No complexes below 200,000 Mr were present in serum. They decreased in size to 160,000 Mr after affinity chromatography on heparin-Sepharose. These results indicated that blood represents a unique milieu conducive to aggregation of bound AT III. It appears, however, that AT III complexes present in blood may not only aggregate, but also associate with other serum proteins through unstable binding most likely caused by the enzyme component of the complex.

EFFECT OF ANTITHROMBIN III AND HEPARIN ON FACTOR X ACTIVATION BY FACTOR IXa

1987 ◽  
Author(s):  
J Pieters ◽  
G willems ◽  
H C Hemker ◽  
T Lindout
Keyword(s):  
Rate Constant ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Order Rate Constant ◽  
Free Solution ◽  
Factor X ◽  
Order Rate ◽  
Factor Ixa ◽  
At Iii ◽  
Factor X Activation

The heparin-catalyzed inactivation of activated coagulation factors by antithrombin III (AT III) has mostly been studied for isolated serine proteases. However, we decided to study the action of heparin and AT III under more physiological conditions, i.e. during the activation of factor X by factor IXa in the presence of phospholipid and calcium. Thereby we made use of a mathematical model which describes the generation of factor Xa by factor IXa, phospholipid and calcium in the presence of AT III and heparin. Fitting the experimental factor Xa generation curve to a set of equations gave the pseudo-first-order rate constants of factor Xa and factor IXa. In a first approach we examined the effect of AT III alone on factor X activation. We found that the second order rate constant of inhibition of formed factor Xa was 2 x 10 5M-1min-1 , whereas that of factor Xa in free solution was 5 x 10 5M-1min-1 , indicating that phospholipid-bound factor X competes with AT III for factor Xa. The second order rate constant of inhibiton of factor IXa, either in the presence or absence of accessory components, was 8 x 103 M-1min-1. Unfractionated heparin (UFH; 168 USP units/mg) was found to stimulate the inhibition of generated factor Xa by AT III (200 nM) with 0.1 min-1 per nM of UFH, and a synthetic pentasaccharide (PS; 4000 anti-Xa units/mg) stimulated this inhibition with only 0.03 min-1per nM. Due to the presence of phospholipid-bound factor X this stimulation was 4-fold less when compared with factor Xa in free solution. At UFH concentrations higher than 3 nM, and PS concentrations exceeding 10 nM hardly any active factor Xa generation could be measured because of the rapid inactivation of factor Xa whereas factor IXa was not inhibited. Using a factor IXa assay we found that PS, even at relatively high concentrations, had no effect on factor IXa inactivation by AT III (200 nM), both in the presence and absence of accessory components. The inactivation of factor IXa by AT III (200 nM) during factor X activation was stimulated by UFH with 1.6 x 10 -2min-1 per nM of UFH. Surprisingly, this was 4-fold more when compared with factor IXa in the absence of accessory components. We established that calcium stimulates the heparin-dependent inhibition of factor IXa.

Studies Of The Heterogeneity Of Purified Antithrombin III

1981 ◽  
Author(s):  
T W Barrowcliffe ◽  
C A Eggleton ◽  
M Mahmoud
Keyword(s):  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Collaborative Study ◽  
Gel Filtration ◽  
Factor Xa ◽  
Heparin Binding ◽  
Predisposing Factor ◽  
Binding Material ◽  
At Iii ◽  
Immunological Assays

Deficiency of antithrombin III (At III), whether hereditary or acquired, is now recognised as a major predisposing factor for the development of venous thromboembolism. Purified At III concentrates are undergoing clinical trials in various conditions associated with At III deficiency; such concentrates may be given in addition to heparin and their potency is usually assessed by heparin co-factor assays. In an international collaborative study, a reference preparation of purified At III had a lower concentration by heparin co-factor than by immunological assays and this was shown to be due to the presence of non-heparin-binding antigens. In the present study we have examined purified At III from several manufacturers by heparin co-factor (amidolytic), progressive antithrombin (clotting) and immunological assays, and their heparin-binding abilities have been studied by crossed immunoelectrophoresis and heparin-agarose affinity chromatography.There was good agreement between progressive antithrombin and immunological assays, but in some concentrates the heparin co-factor assays gave lower activity. The proportions of non-heparin-binding material varied considerably, from less than 5% to as much as 50% of the total At III antigen in some concentrates. The non-binding material isolated from a heparin column had little heparin co-factor activity, but was able to neutralise thrombin and Factor Xa. Gel filtration and polyacrylamide gel electrophoresis showed no major distinction between heparin-binding and non-binding antigens, indicating the absence of At III-protease complexes.These studies show that some At III concentrates contain substantial amounts of partially denatured molecules, in which the heparin-binding ability of the At III has been impaired but its thrombin and Xa neutralising activity left relatively intact.

Further Investigations on Antithrombin III in the Plasmas of Patients with the Abnormality of ‘Antithrombin III Budapest’

1975 ◽  
Vol 33 (03) ◽  
pp. 564-572 ◽  
Author(s):  
Géza Sas ◽  
Duncan S Pepper ◽  
John D Cash
Keyword(s):  
Electrophoretic Mobility ◽  
Antithrombin Iii ◽  
Gel Filtration ◽  
Molecular Size ◽  
High Concentration ◽  
Abnormal Protein ◽  
Crossed Immunoelectrophoresis ◽  
Normal Plasma ◽  
At Iii ◽  
Cofactor Activity

SummaryAntithrombin III (AT-III) was studied in a thrombophilic family with an abnormal AT-III molecule (antithrombin III Budapest) using a modified crossed Immunoelectrophoresis technique, gel filtration, ‘rocket’ Immunoelectrophoresis and a heparin cofactor assay.When plain agarose was applied in the first phase of the crossed Immunoelectrophoresis, the normal and the pathological AT-III revealed identical electrophoretic mobility. However, when heparin was mixed with agarose in the first phase of electrophoresis, the propositus’ plasma displayed a different AT-III pattern from normal plasma. His plasma contained the first component of the normal plasma (Immune Antithrombin III1, IAT-III1) in a concentration of only 5% of normal, and a protein in high concentration which although immunoreactive to AT-III antisera, had an electrophoretic mobility similar (but not identical) to that of IAT-III2. This ab-normal protein had no heparin cofactor activity and a molecular size greater than normal plasma AT-III. Unlike normal AT-III, the addition of heparin did not change the molecular size of the pathologic AT-III molecule significantly.The abnormal protein was present in lower concentrations in the patient’s children and at the time of study they had no clinical or laboratory evidence of intravascular coagulation.

The Biophysical and Functional Properties of Antithrombin III (AT III) in Vitro and in Vivo

1975 ◽  
Author(s):  
S. Chandra ◽  
N. U. Bang
Keyword(s):  
Antithrombin Iii ◽  
Biological Activities ◽  
Gel Filtration ◽  
Biological Properties ◽  
Molecular Forms ◽  
Step Procedure ◽  
At Iii ◽  
Exclusion Chromatography

Human and rabbit AT III were purified by a two-step procedure of heparin-agarose affinity chromatography and Sephadex G150 gel filtration. The resulting preparation purified approximately 900 fold over plasma was homogeneous by SDS gel electrophoresis (SDSE), immunoelectrophoresis and gel exclusion chromatography (GEC). Both human and rabbit AT III possessed mol wt’s of approximately 68,000 daltons. As previously observed (Rosenberg and Damus: J. Biol. Chern., 248, 0490, 1973), we noted that human as well as rabbit AT III form firm stoichiometric complexes with thrombin demonstrable by SDSE and GEC. 125I labeled rabbit AT III possessing biophysical and biological properties identical to the unlabeled product was injected into rabbits. Radiolabeled AT III displayed complex multiphasic disappearance curves with a half-life of the major component of 36 hours. The apparent mol wt of AT III in vivo was reduced to approximately 30,000 daltons within minutes after its administration into rabbits as evidenced by GEC and SDSE. The 30,000 dalton AT III was as capable as the 68.000 dalton AT III to form firm stoichiometric complexes with thrombin. Thus, AT III may exist in different molecular forms possessing similar biological activities.

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.

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.

GAGs-Potentiated Inhibition of Thrombin, Factor Xa and Plasmin in Plasma and in a Purified System Containing Antithrombin III – Correlation with Total Charge Density

1981 ◽  
Vol 46 (04) ◽  
pp. 749-751 ◽  
Author(s):  
E Cofrancesco ◽  
A Vigo ◽  
E M Pogliani
Keyword(s):  
Charge Density ◽  
Chemical Structure ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Total Charge ◽  
Pure System ◽  
At Iii ◽  
Total Charge Density ◽  
The Difference ◽  
Inhibition Of Thrombin

SummaryThe ability of heparin and related glycosaminoglycans (GAGs) to accelerate the inhibition of thrombin, factor Xa and plasmin in plasma and in a purified system containing antithrombin III (At III) was studied using chromogenic peptide substrate assaysThere was a good correlation between the charge density of the mucopolysaccharides and the activities investigated. While the difference between potentiation of the antithrombin activity by GAGs in plasma and in the purified system was slight, the inhibition of factor Xa in plasma was more pronounced than in the presence of purified At III, indicating the mechanisms for GAGs-potentiated inhibition of thrombin and factor Xa are not identical.For the antiplasmin activity, there was a good correlation between the chemical structure and biological activity only in the pure system, confirming that the antithrombin-GAG complex plays a very limited role in the inactivation of plasmin in plasma.

Bovine Factors X1 and X2: Activation Peptide Based Chromatographic Differences

1979 ◽  
Author(s):  
Takashi Morita ◽  
Craig M. Jackson
Keyword(s):  
Amino Acid ◽  
Gel Filtration ◽  
Factor Xa ◽  
Heart Association ◽  
Personal Communication ◽  
Exchange Chromatography ◽  
Amino Acid Sequences ◽  
Factor X ◽  
Activation Peptide ◽  
Activation Peptides

Bovine Factor X is eluted in two forms (X1 and X2) from anion exchange chromatographic columns. These two forms have indistinguishable amino acid compositions, molecular weights and specific activities. The amino acid sequences containing the γ-carboxyglu-tamic acid residues have been shown to be identical in X1 and X2, (H. Morris, personal communication). An activation peptide is released from the N-terminal region of the heavy chain of Factor X by an activator from Russell’s viper venom. This peptide can be isolated after activation by gel filtration on Sephadex G-100 under nondenaturing conditions. The activation peptides from a mixture of Factors X1 and X2 were separated into two forms by an ion-exchange chromatography. The activation peptide AP1) which eluted first was shown to be derived from Factor X1 while the activation peptide (AP2) which eluted second was shown to be derived from X2 on basis of chromatographic separations carried out on Factors X1 and X2 separately. Factor Xa was eluted as a symmetrical single peak. On the basis of these and other data characterizing these products, we conclude that the difference between X1 and X2 are properties of the structures of the activation peptides. (Supported by a grant HL 12820 from the National Heart, Lung and Blood Institute. C.H.J. is an Established Investigator of the American Heart Association).

RAT HEPAT0CYTES IN CULTURE INACTIVATE FACTOR Xa.

1987 ◽  
Author(s):  
G D Qureshi ◽  
M Sun ◽  
C Gervin ◽  
H Evans
Keyword(s):  
Serine Proteases ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Rat Hepatocytes ◽  
Factor X ◽  
Clotting Factors ◽  
Hepatocyte Cultures ◽  
The Stability ◽  
Stabilization Period

Plasma contains zymogens of clotting factors, which under various stimuli are activated to serine proteases. Whereas much knowledge has been gained about the activation of clotting factors, relatively little is known about inactivation of these proteases. Antithrombin III has been shown to inactivate some activated clotting factors in plasma. Studies in intact animals have suggested that activated clotting factors are mainly inactivated in the liver. To investigate more fully the role of liver in inactivating the activated factors, we studied the stability of activated factor X(Xa) in hepatocyte cultures. Monolayer cultures on non-proliferating rat hepatocytes were prepared according to the method of Bissell et al. The culture medium was chemically defined and was free from serum or serum products. After the 24 h stabilization period, 0.5 units/ml of 100% activated bovine factor Xa was co-cultured with hepatocytes for 8 h. Samples were collected at 0, ½, 1 2, 4 and 8 h and tested for Xa activity using chromogenic substrate S-2222. At the end of 8 h only 41.07% of the initial Xa activity remained. Xa inactivation was not affected by a commercially prepared unfractionated heparin (1 unit/ml) and estradiol at 12.5, 25, 125 nM, a potentiator and inhibitor of antithrombin III, respectively. Inactivation of Xa in hepatocyte cultures was inhibited by the addition of cycloheximide (10-4M). Our data suggests that factor Xa is inactivated in hepatocyte cultures by one or more hepatic derived factors which do not meet the functional characteristics of antithrombin III.

Antithrombin III and Venous Thrombosis

1979 ◽  
Author(s):  
Duncan P. Thomas
Keyword(s):  
Venous Thrombosis ◽  
Antithrombin Iii ◽  
Physiological Role ◽  
Factor Xa ◽  
Original Description ◽  
Clear Cut ◽  
Naturally Occurring ◽  
At Iii ◽  
General Studies

Increasing interest in the physiological role of inhibitors of coagulation has highlighting the role of antithrombin III (AT III) as the most important naturally occurring inhibitor of venous thrombosis. Since Egeberg’s original description in 1965, it has been recognized that inherited deficiency of AT III is associated with an increased incidence of venous thromboembolism. The role of acquired deficiency of AT III in the pathogenesis of thromboembolism remainsless clear-cut, partly due to methodological differences. While low values have been reported in groups of patients with thromboembolism, estimations of AT III in individual patients are not allways abnormal. In general, studies which have measured protein concentration rather than functional activity, or cl otting assays which measure total antithrombin activity and not specific anti-Factor Xa activity have failed to demonstrate a clear relationship between AT III and thromboembolism. However, in two groups of patients, namely women on oral contraceptives and patients undergoing total hipreplacement, an acquired deficiency of AT III, particularly when measured by anti-Xa clotting assays, correlates highly with postoperative venous thrombosis. Although venous thrombosis may develop in patients despite normal AT III values, an activity below approximately 80% in an anti-Xa clotting assay has been found to be of predictive value in patients subjected to the stress of trauma or surgery.

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