Development and characterization of an automated assay of effective heparin activity in plasma.

1987 ◽  
Vol 33 (9) ◽  
pp. 1630-1634 ◽  
Author(s):  
J F Pierson-Perry ◽  
D M Obzansky ◽  
J P Mizzer
Keyword(s):  
Antithrombin Iii ◽  
Factor Xa ◽  
Sole Source ◽  
Chromogenic Substrate ◽  
Blood Components ◽  
Automated Assay ◽  
At Iii ◽  
Assay Result ◽  
Heparin Activity

Abstract We describe a fully automated assay for determining effective heparin activity in plasma, based on heparin-catalyzed inhibition of Factor Xa (EC 3.4.21.6) by antithrombin III (AT III). Residual Factor Xa is determined kinetically by the Du Pont aca discrete clinical analyzer with a chromogenic substrate and is inversely related to heparin activity. Because the test plasma is the sole source of AT III, the assay result is dependent on AT III activity and reflects effective rather than total heparin activity. The assay range is 20-1200 USP units/L, and the assay shows equivalent sensitivity to standard and low-molecular-mass heparins. Within-run reproducibility (CV) is 1.6% at 390 units/L. There was no interference from common blood components or drugs. Results agreed well with those by the Coatest heparin kit (Kabi) adapted to the Cobas-Bio analyzer (r = 0.85, n = 122).

Coagulation Inhibitors Studied with Chromogenic Substrate

1975 ◽  
Author(s):  
U. Abildgaard ◽  
M. Lie ◽  
A. Teien ◽  
O. R. Ødegård
Keyword(s):  
Sensitivity And Specificity ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Combined Effect ◽  
Assay System ◽  
Chromogenic Substrate ◽  
Reversible Inhibition ◽  
Heparin Treatment ◽  
At Iii ◽  
Coagulation Inhibitors

The rapid amidolysis of the chromogenic substrate Bz-Phe-Val-Arg-pNA by thrombin (Svendsen & al. Thromb. Res. 1, 267, 1972) provides a useful tool for the study of coagulation inhibitors. With purified reagents, reversible inhibition of the reaction can be demonstrated with 0.01 U/ml of heparin. The inactivation of thrombin by antithrombin III (At-III) is accelerated by 0.001 U/ml of heparin, making it possible to assay small amounts of heparin. The amidolytic assay system has also been used to study the competition between thrombin and factor Xa for At-III.The sensitivity and specificity of rapid amidolytic methods for assay of the following plasma activités will be reported:1) At-III activity (contaminating heparin neutralized).2) Exogenous heparin (during heparin treatment).3) Combined effect of At-III and exogenous heparin.4) At-III in the presence of optimal concentrations of heparin.

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.

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.

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.

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.

scholarly journals Thrombin Metz: characterization of the dysfunctional thrombin derived from a variant of human prothrombin

Blood ◽  
1984 ◽  
Vol 63 (4) ◽  
pp. 927-934
Author(s):  
MJ Rabiet ◽  
M Jandrot-Perrus ◽  
JP Boissel ◽  
J Elion ◽  
F Josso
Keyword(s):  
High Performance ◽  
Fluorescence Enhancement ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Competitive Inhibitor ◽  
Amidolytic Activity ◽  
Direct Inhibition ◽  
High Affinity ◽  
Factor Va

Thrombin Metz and normal thrombin, resulting from activation of the respective prothrombins by factor Xa in the presence of calcium, phospholipid, and factor Va, were purified by chromatography on sulfopropyl Sephadex. By physicochemical criteria, thrombin Metz is identical to normal thrombin. Its functional properties were investigated in some reactions in which thrombin is classically involved. Thrombin Metz exhibits less than 4% of fibrinogen clotting activity. Both Km and Kcat, determined on S2238, are abnormal. Titration with the high-affinity competitive inhibitor of thrombin, DAPA, shows that fluorescence enhancement of the probe is only 34% in binding to thrombin Metz when compared to that observed in binding to normal thrombin. High-performance liquid chromatography has been used to measure the simultaneous rate of release of fibrinopeptides A and B. A decreased release rate for both fibrinopeptides, more marked for fibrinopeptide B, results in a slow fibrin polymerization, as followed by absorbance at 450 nm. Thrombin Metz is less than 5% as effective as normal thrombin in inducing platelet aggregation. Interaction with antithrombin III is slower than normal when followed by SDS gel electrophoresis and inhibition of the amidolytic activity of thrombin on S2238. This abnormality is not observed in the presence of heparin. However, thrombin Metz binds less tightly to a heparin-Sepharose column, and the direct inhibition of heparin on its activity on S2238 is weaker. From these results, we can predict that the defect in thrombin Metz affects the catalytic site or its vicinity and, jointly or consequently, the region of interaction of thrombin with antithrombin III and heparin.

Prostacyclin Prevents Endothelial & Platelet Damage During Haemodialysis

1981 ◽  
Author(s):  
J H Turney ◽  
N Dodd ◽  
M J Weston
Keyword(s):  
Cardiovascular Disease ◽  
Factor Viii ◽  
Antithrombin Iii ◽  
Endothelial Damage ◽  
Atherosclerotic Cardiovascular Disease ◽  
Vascular Endothelial ◽  
Blood Components ◽  
Prothrombotic State ◽  
At Iii ◽  
Extracorporeal Circuits

We have previously demonstrated that prostacyclin (PGI) enhances the biocompatibility of extracorporeal circuits. Dialysis with & without PGI were studied in 17 patients. We measured platelet count, (β-thromboglobulin, factor VIII related antigen, & Antithrombin III (Bick method). Results are presented as percentage change + SEM of initial values.The change in all values during dialysis with heparin alone was significant (p<0.005). Additional PGI prevented any change (p<0.0001 compared with heparin at 300 minutes). We conclude that platelet activation & consumption persists throughout dialysis with heparin alone. The rise in factor VIII-RA & AT III reflects vascular endothelial damage induced by the reinfusion of activated blood components. Thus PGI not only protects platelets but also prevents dialysis-induced vascular endothelial damage. Longterm use of PGI should tend to reverse the prothrombotic state in dialysed uraemic patients and may therefore reduce their risk of atherosclerotic cardiovascular disease.

Isolation and Partial Characterization of a Novel Circulating Antithrombin

1979 ◽  
Author(s):  
Harry Messmore ◽  
Zaheer Pervez ◽  
Jawed Fareed
Keyword(s):  
Protease Inhibitor ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Serine Protease Inhibitor ◽  
Chromogenic Substrates ◽  
Inhibitor Activity ◽  
Antithrombin Activity ◽  
Life Threatening ◽  
Hydrolysis Of

We have previously reported on a novel circulating anticoagulant (Clin. Res. 22:396 A, 1974. Thromb. and Haem. 38:77, 1977 (abstr) in a 42 year old female who has had repeated episodes of life threatening hemorrhage since the age of 3. Our preliminary studies showed it to be a protein with some of the properties of a heparin activated antithrombin III. This report is on additional studies to further characterize the Inhibitor«The patient’s plasma was fractionated on Sephadex G-200, and the fraction showing immediate acting antithrombin activity was further purified on heparin-sepharose and conconavalin A-sepharose affinity columns. The patient’s inhibitor did not bind to heparin sepharose, and was thus separable from her antithrombin III. It did bind to conconavalin A, and was eluted from the conconavalin A with α - D (+) methylglucoside. It has very broad serine protease inhibitor activity, blocking the hydrolysis of synthetic chromogenic substrates by thrombin, factor Xa, plasmin and trypsin. It did not inhibit Reptilase.Immunochemical assays show it to be α1 antitrypsin. Isoelectric focusing shows it to he a variant of normal, with its isoelectric point being different from a normal control, and pure α1 antitrypsin (commercial, human).The total α1 antitrypsin level in this patient is about 50% of normal, and It has potent immediate acting antithrombin activity. It appears to be similar to a phenotype previously reported as Antithrombin Pittsburgh (Blood 51: 129, 1978).

Effect of Intraperitoneal Administration of Heparin to Patients on Continuous Ambulatory Peritoneal Dialysis (CAPD)

1991 ◽  
Vol 11 (1) ◽  
pp. 81-83 ◽  
Author(s):  
Susumu Takahashi ◽  
Akihito Shimada ◽  
Kazuyoshi Okada ◽  
Tsutomu Kuno ◽  
Yuji Nagura ◽  
...  
Keyword(s):  
Peritoneal Dialysis ◽  
Continuous Ambulatory Peritoneal Dialysis ◽  
Whole Blood ◽  
Blood Clotting ◽  
Antithrombin Iii ◽  
Intraperitoneal Administration ◽  
Clotting Time ◽  
At Iii ◽  
Heparin Activity ◽  
Blood Clotting Time

The effect of intraperitoneal administration of heparin to patients on continuous ambulatory peritoneal dialysis (CAPD) remains obscure. We examined 8 patients on CAPD to investigate its effect. When 2.5 U/ml or 5 U/ml of heparin was given intraperitoneally, t1/2 of heparin activity in the dialysate was 0.5 to 2 hrs, and 6 hrs after administration its activity was 0.5 U/ml and 1.4 U/ml respectively. Whole blood clotting time was hardly affected because the transfer of heparin to the plasma was minimal. The plasma antithrombin III (AT -III) levels were comparable to patients on hemodialysis, but the AT-111level in the dialysate was only 1.5% of those in the plasma. We conclude that the intraperitoneal administration of heparin at these doses is effective in preventing fibrin precipitation when intraperitoneal AT -III levels are expected to be relatively increased such as at the start of CAPD or in the presence of peritonitis.

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.

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