The Rivanol Method for the Quantitative Determination of Antithrombin III in Plasma

1974 ◽  
Vol 31 (03) ◽  
pp. 439-451
Author(s):  
Robert H. Yue ◽  
Toby Starr ◽  
Menard M. Gertler
Keyword(s):  
Quantitative Method ◽  
Antithrombin Iii ◽  
Order Reaction ◽  
Routine Laboratory ◽  
Second Order Reaction ◽  
Experimental Conditions ◽  
Thrombin Activity ◽  
Assay Procedure ◽  
Inhibition Of Thrombin

SummaryA simple quantitative method for the determination of antithrombin III in plasma is described. It is based on the preferential precipitation of plasma antithrombin activities by a chemical compound 2,5-diamino-7-ethoxyacridin lactate (rivanol). When the incubation time of plasma with rivanol took place within 0.5 and 2 hours under specifically controlled experimental conditions, rivanol precipitated α2-macro-globulin selectively and quantitatively without any effect on antithrombin III. In the presence of rivanol, the inhibition of thrombin by plasma antithrombin III followed a second-order reaction with a rate constant of 80 1/M/sec. In this assay procedure, only the initial thrombin activity and the 30 minutes residual thrombin activity are required to evaluate the amount of antithrombin III present in the plasma quantitatively. “With proper routine laboratory care, this assay system was found to be accurate and reproducible.

Studies in the Vulcanization of Rubber. III—Kinetics of Vulcanization of Rubber with Sulfur and Selenium

1930 ◽  
Vol 3 (4) ◽  
pp. 650-659
Author(s):  
John T. Blake
Keyword(s):  
Molecular Weight ◽  
Order Reaction ◽  
Order Equation ◽  
Second Order Reaction ◽  
Experimental Conditions ◽  
Mathematical Basis ◽  
First Order ◽  
Kinetics Of ◽  
Kinetics Of Vulcanization

Abstract A procedure for the determination of combined selenium in rubber has been evolved. The rate of combination of selenium and rubber has been ascertained under certain conditions and shown to follow a first-order equation. A minimum value for the molecular weight of rubber has been estimated. The formation of hard rubber under chosen experimental conditions has been put on a mathematical basis and has been shown to follow a second-order reaction. The soft- and hard-rubber reactions have been shown qualitatively to be successive reactions and the function of accelerators has been discussed. The theory explains the anomalous results obtained by previous investigators.

A Method to Correct for the Continuing Activation during the Second Stage in a Two-Stage Assay

1968 ◽  
Vol 19 (01/02) ◽  
pp. 161-168
Author(s):  
W Berg
Keyword(s):  
Kinetic Data ◽  
Quantitative Methods ◽  
Order Reaction ◽  
Second Order Reaction ◽  
Plasmin Activity ◽  
Second Stage ◽  
Lysis Time ◽  
The Moment ◽  
Coagulation And Fibrinolysis

SummaryIn coagulation and fibrinolysis, kinetic data are difficult to obtain with ordinary quantitative methods because no simple means are available to stop the reaction before the determination of the activity formed.This work describes how to calculate, from the data recorded, the amount of activity at the moment the determination is started.A formula is given for a zero, a first and a second-order reaction.The method is exemplified by urokinase activation of plasminogen into plasmin. The determination of the plasmin activity is done by means of the lysis time method.

Routine Laboratory Investigation of Urinary Catecholamine Metabolites in Sick Children

1979 ◽  
Vol 16 (1-6) ◽  
pp. 38-43 ◽  
Author(s):  
C. E. Niehaus ◽  
R. S. Ersser ◽  
Sheila M. Atherden
Keyword(s):  
Thin Layer ◽  
Quantitative Method ◽  
Mandelic Acid ◽  
Normal Subjects ◽  
Routine Laboratory ◽  
Urinary Catecholamine ◽  
Routine Laboratory Investigation ◽  
Catecholamine Metabolites ◽  
Sick Children

Simple and rapid thin-layer chromatographic methods have been used to investigate the catecholamine metabolites present in the urine of sick children. A semi-quantitative method for 4-hydroxy-3-methoxy-mandelic acid (HMMA) has been devised and compared with the quantitative spectrophotometric procedure. The methods have been performed on both normal subjects and children with catecholamine-secreting tumours, without dietary restriction, and have led to a 50% reduction in the number of samples requiring laborious quantitative determination of HMMA excretion.

The Use of a Commercial ELISA for Assay of Thrombin-Antithrombin Complexes in Purified Systems

1990 ◽  
Vol 63 (03) ◽  
pp. 435-438 ◽  
Author(s):  
G Elgue ◽  
B Pasche ◽  
M Blombäck ◽  
p Olsson
Keyword(s):  
Human Plasma ◽  
Immunosorbent Assay ◽  
Antithrombin Iii ◽  
Thrombin Inhibitor ◽  
Enzyme Linked Immunosorbent Assay ◽  
Excellent Correlation ◽  
Elisa Method ◽  
Inhibition Of Thrombin

SummaryInhibition of thrombin by antithrombin III (AT) results in the formation of stable thrombin-AT complexes (TAT). An enzyme-linked immunosorbent assay (ELISA) following the sandwich principle is available for the determination of TAT complexes in human plasma, however, this ELISA method could not be used in purified systems containing thrombin and AT. It has therefore been modified for use in purified systems and an excellent correlation was found between the disappearance of thrombin and AT and the recovery of TAT complexes. Addition of thrombin inhibitor hirudin and heparin inhibitor polybrene into the reacting thrombin-AT mixture did not interfere with the assay of TAT. It was found that the use of siliconised tubes was necessary for the conservation of the TAT complexes.

scholarly journals Changes in Thermodynamic Parameters in the Inhibition of Thrombin by Bovine Antithrombin III

1977 ◽  
Author(s):  
Robert H. Yue ◽  
Toby Starr ◽  
Menard M. Gertler
Keyword(s):  
Thermodynamic Parameters ◽  
Rate Constant ◽  
Antithrombin Iii ◽  
Order Rate Constant ◽  
Second Order ◽  
Kcal Mole ◽  
Experimental Conditions ◽  
Reaction Proceeds ◽  
Entropy Of Activation ◽  
Inhibition Of Thrombin

The inhibition of thrombin by isolated bovine antithrombin III (1, 200 units/mg of protein) was studied under a variety of experimental conditions. This inactivation reaction follows a second-order reaction and the rate constant depends on a number of parameters. The second-order rate constant decreases with an increase of thrombin concentration. However, at a constant initial concentration of thrombin, the measured amount of antithrombin III present in a sample is directly proportional to the aliquot of the sample. The rate of inactivation was investigated with changes of temperature. For example, at an initial thrombin concentration of 6. 8 units/ml and antithrombin III concentration of 4. 2 units/ml, the reaction proceeds with Ea of 25 Kcal/mole and Δ S‡ of 42 e. u. /mole. In the presence of 0. 0015 unit of heparin/ml and with similar concentrations of thrombin and antithrombin III, the reaction proceeds with Ea of 16 Kcal/mole and Δ S‡ of 14 e.u. /mole. Therefore, the presence of heparin causes a lowering of the activation energy and a concomitant decrease in the entropy of activation. This change in the thermodynamic parameters allows the inactivation reaction to proceed much faster in the presence of heparin. The effect of heparin may be the result of solvent macromolecular interaction in this inhibition reaction.

Is Quantitative Determination of Fibrin(ogen) Degradation Products and Thrombin-Antithrombin III Complexes Useful to Diagnose Deep Venous Thrombosis in Outpatients?

1989 ◽  
Vol 62 (04) ◽  
pp. 1043-1045 ◽  
Author(s):  
Paul F M M van Bergen ◽  
Eduard A R Knot ◽  
Jan J C Jonker ◽  
Auke C de Boer ◽  
Moniek P M de Maat
Keyword(s):  
Quantitative Determination ◽  
Venous Thrombosis ◽  
Deep Venous Thrombosis ◽  
Antithrombin Iii ◽  
Degradation Products ◽  
Family Doctor ◽  
Diagnostic Value ◽  
Impedance Plethysmography ◽  
Fibrin Degradation Products

SummaryWe studied the diagnostic value of recently introduced ELISA’s for the determination of thrombin-antithrombin III (TAT) complexes, fibrin degradation products (FbDP), fibrinogen degradation products (FgDP) and total degradation products (TDP) for deep venous thrombosis (DVT) in plasma of 239 consecutive outpatients, suspected for DVT by their family doctor. DVT was confirmed by impedance plethysmography in 60 patients. Using the 95th percentile range of 42 healthy volunteers the sensitivity for the detection of DVT was: 37% for TAT, 95% for TDP, 92% for FbDP and 90% for FgDP. Specificity was: 88% for TAT, 16% for TDP, 20% for FbDP and 25% for FgDP.We conclude that these assays are of little value in the diagnosis of DVT in outpatients.

Antithrombin-mediated Inhibition of Factor Vlla-Tissue Factor Complex by the Synthetic Pentasaccharide Representing the Heparin Binding Site to Antithrombin

1996 ◽  
Vol 76 (01) ◽  
pp. 005-008 ◽  
Author(s):  
Jean Claude Lormeau ◽  
Jean Pascal Herault ◽  
Jean Marc Herbert
Keyword(s):  
Binding Site ◽  
Tissue Factor ◽  
Residual Activity ◽  
Coagulation Factors ◽  
Heparin Binding ◽  
Experimental Conditions ◽  
First Order ◽  
Heparin Binding Site ◽  
Coagulant Activity

SummaryWe examined the effect of the synthetic pentasaccharide representing the minimal binding site of heparin to antithrombin on the antithrombin-mediated inactivation of factor Vila bound to tissue factor. This effect was compared to the effect of unfractionated heparin. Using purified recombinant human coagulation factors and either a clotting or an amidolytic assay for the determination of the residual activity of factor Vila, we showed that the pentasaccharide was an efficient antithrombin-dependent inhibitor of the coagulant activity of tissue factor-factor Vila complex. In our experimental conditions, assuming a mean MW of 14,000 for heparin, the molar pseudo-first order rate constants for ATIII-mediated FVIIa inhibition by ATIII-binding heparin and by the synthetic pentasaccharide were found to be similar with respective values of 104,000 ± 10,500 min-1 and 112,000 ± 12,000 min-1 (mean ± s.e.m., n = 3)

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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