The Determination of Factor X Using an Automated Amidolytic Technique

1979 ◽  
Author(s):  
E.M. van Wijk ◽  
L.H. Kahlé ◽  
J.W. ten Cate
Keyword(s):  
Antithrombin Iii ◽  
Oral Anticoagulant Therapy ◽  
Close Correlation ◽  
Bleeding Disorders ◽  
Factor X ◽  
Amidolytic Activity ◽  
One Stage ◽  
Automated Method ◽  
The One

An automated method for the determination of factor X has been developed using a procoagulant fraction from Russell’s Viper Venom (RW), which activates FX within 4 minutes at pH 8.6, resulting in a Xa activity that is assayed using the chromogenic substrate S 2222. The amidolytic activity generated by sufficient RW is proportional to the FX level of the plasma and is not influenced by antithrombin III within the time studied. By using polybrene, the influence of heparin (up to 10 U/ml) can be eliminated. The method has been adapted for performance automatically on an Automated Kinetic Enzyme and Substrate analyser (Vitatron), Comparison of FX activities of 30 normals and 41 patients with bleeding disorders revealed a close correlation of this method with the one stage clotting assay using thromboplastin (r=0.93). Values obtained in a group of 83 patients on oral anticoagulant therapy showed good correlation (r=0.87) but tended to be about 8% higher compared to the one stage method. No significant differences were found when RW was used in the clotting assay (r-0.91). The values obtained by the amidolytic assay correlated well with thrombotest (r=0.87).

The Determination of Factor X Using an Automated Amidolytic Technique

1979 ◽  
Author(s):  
E van Wijk ◽  
L Kahlé ◽  
J ten Cate
Keyword(s):  
Antithrombin Iii ◽  
Oral Anticoagulant Therapy ◽  
Close Correlation ◽  
Bleeding Disorders ◽  
Factor X ◽  
Amidolytic Activity ◽  
One Stage ◽  
Automated Method ◽  
The One

An automated method for the determination of factor X has been developed using a procoagulant fraction from Russell’s Viper Venom (RVV), which activates FX within 4 minutes at pH 8.6, resulting in a Xa activity that is assayed using the chromogenic substrate S 2222. The amidolytic activity generated by sufficient RVV is proportional to the FX level of the plasma and is not influenced by antithrombin III within the time studied. By using polybrene, the influence of heparin (up to 10 U/ml) can be eliminated. The method has been adapted for performance automatically on an Automated Kinetic Enzyme and Substrate analyzer (Vitatron). Comparison of FX activities of 30 normals and 41 patients with bleeding disorders revealed a close correlation of this method with the one stage clotting assay using thromboplastin (r=0.93). Values obtained in a group of 83 patients on oral anticoagulant therapy showed good correlation (r=0.87) but tended to be about 8% higher compared to the one stage method. No significant differences were found when RW was used in the clotting assay (r=0.91). The values obtained by the amidolytic assay correlated well with thrombotest (r=0.87).

The Formation of Intermediate Product I in a Purified System The Role of Factor IX or of its Precursor and of a Hageman Factor-PTA Fraction

1961 ◽  
Vol 6 (02) ◽  
pp. 224-234 ◽  
Author(s):  
E. T Yin ◽  
F Duckert
Keyword(s):  
Intermediate Product ◽  
Factor Ix ◽  
Assay Method ◽  
Lag Phase ◽  
Factor X ◽  
Haemophilia B ◽  
Hageman Factor ◽  
One Stage ◽  
The One

Summary1. The role of two clot promoting fractions isolated from either plasma or serum is studied in a purified system for the generation of intermediate product I in which the serum is replaced by factor X and the investigated fractions.2. Optimal generation of intermediate product I is possible in the purified system utilizing fractions devoid of factor IX one-stage activity. Prothrombin and thrombin are not necessary in this system.3. The fraction containing factor IX or its precursor, no measurable activity by the one-stage assay method, controls the yield of intermediate product I. No similar fraction can be isolated from haemophilia B plasma or serum.4. The Hageman factor — PTA fraction shortens the lag phase of intermediate product I formation and has no influence on the yield. This fraction can also be prepared from haemophilia B plasma or serum.

Two-Stage Procedure for the Quantitative Determination of Autoprothrombin III Concentration and Some Applications

1967 ◽  
Vol 18 (01/02) ◽  
pp. 198-210 ◽  
Author(s):  
Ronald S Reno ◽  
Walter H Seegers
Keyword(s):  
Prothrombin Time ◽  
Factor Vii ◽  
Two Stage ◽  
Pronounced Increase ◽  
One Stage ◽  
Assay Procedure ◽  
Bovine Plasma ◽  
The One ◽  
Autoprothrombin C

SummaryA two-stage assay procedure was developed for the determination of the autoprothrombin C titre which can be developed from prothrombin or autoprothrombin III containing solutions. The proenzyme is activated by Russell’s viper venom and the autoprothrombin C activity that appears is measured by its ability to shorten the partial thromboplastin time of bovine plasma.Using the assay, the autoprothrombin C titre was determined in the plasma of several species, as well as the percentage of it remaining in the serum from blood clotted in glass test tubes. Much autoprothrombin III remains in human serum. With sufficient thromboplastin it was completely utilized. Plasma from selected patients with coagulation disorders was assayed and only Stuart plasma was abnormal. In so-called factor VII, IX, and P.T.A. deficiency the autoprothrombin C titre and thrombin titre that could be developed was normal. In one case (prethrombin irregularity) practically no thrombin titre developed but the amount of autoprothrombin C which generated was in the normal range.Dogs were treated with Dicumarol and the autoprothrombin C titre that could be developed from their plasmas decreased until only traces could be detected. This coincided with a lowering of the thrombin titre that could be developed and a prolongation of the one-stage prothrombin time. While the Dicumarol was acting, the dogs were given an infusion of purified bovine prothrombin and the levels of autoprothrombin C, thrombin and one-stage prothrombin time were followed for several hours. The tests became normal immediately after the infusion and then went back to preinfusion levels over a period of 24 hrs.In other dogs the effect of Dicumarol was reversed by giving vitamin K1 intravenously. The effect of the vitamin was noticed as early as 20 min after administration.In response to vitamin K the most pronounced increase was with that portion of the prothrombin molecule which yields thrombin. The proportion of that protein with respect to the precursor of autoprothrombin C increased during the first hour and then started to go down and after 3 hrs was equal to the proportion normally found in plasma.

The Function of the Platelet Surface in Coagulation

1974 ◽  
Vol 32 (02/03) ◽  
pp. 492-501 ◽  
Author(s):  
J Burnham King
Keyword(s):  
Catalytic Activity ◽  
Density Gradient ◽  
Sole Source ◽  
Assay System ◽  
Factor X ◽  
Platelet Surface ◽  
Size And Shape ◽  
One Stage ◽  
Gradient Technique ◽  
The One

SummaryPlatelets washed by the albumen density gradient technique were used as the sole source of thromboplastin in a new test, and also as the sole source of factor X in a modification of the one-stage factor X assay test of Denson. These platelets were shown to be capable of generating considerable amounts of thrombin during periods of preincubation of up to 8 minutes. For at least 3 minutes of preincubation their size and shape remained invariant, as measured by counting and by sizing on a Coulter channelyser. It is suggested that this test measures a function of the platelet surface, rather than of the platelet after viscous metamorphosis. This function is tentatively named platelet catalytic activity (PCA). Support for this concept was given by the fact that aspirin and phenylbutazone did not affect the test. The factor X assay system confirmed the presence of a small amount of mostly inactive factor X on the surface of the platelet. This factor X could be activated by Russell viper venom.

Biological Properties of the Thromboplastins and Plasmas Included in the ICTH/ICSH Collaborative Study on Prothrombin Time Standardization

1979 ◽  
Vol 42 (04) ◽  
pp. 1115-1127 ◽  
Author(s):  
E A Loeliger ◽  
L P van Halem-Visser
Keyword(s):  
Prothrombin Time ◽  
Collaborative Study ◽  
Biological Properties ◽  
Bovine Brain ◽  
Factor Vii ◽  
Factor X ◽  
One Stage ◽  
Activation Products ◽  
Position Sensitivity ◽  
The One

SummaryThe fourteen types of thromboplastin included in the ICTH/ICSH Collaborative Study on Prothrombin Time Standardization were investigated with respect to their sensitivity for factor VII, activation products, and PIVKAs. All of these thromboplastins displayed sufficient factor VII sensitivity, and all were sensitive to activation products. After correction for the overall sensitivity slope, rabbit plain thromboplastins were the most sensitive preparations and human as well as rabbit diluted the least sensitive; bovine brain thromboplastin lost its exceptional position. Sensitivity to activation products explains why factors II, VII, and X present in artificially prepared abnormal plasmas may be difficult to assess accurately. Also, all thromboplastins appear to be sensitive for both PIVKA VII and PIVKA X. PIVKA VII acts as a procoagulant which increases the amount of factor VII measured by the one-stage assay technique. PIVKA X inhibits and thus reduces the activity of factor X. The procoagulant activity of PIVKA VII is particulary pronounced for the two lung-brain rabbit thromboplastins (Lyoplastin and Simplastin), whereas the anticoagulant action of PIVKA X is strongest with bovine brain thromboplastin (Thrombotest). Considered as a group, rabbit thromboplastins appear to be less sensitive for PIVKA X and more sensitive for PIVKA VII.As judged from thromboplastin calibration results, all three types of lyophilized reference plasmas are to some degree distinguishable from fresh patient plasmas. Primary calibration of a thromboplastin must therefore still be performed with freshly prepared normal as well as patient plasmas.

Mechanized amidolytic technique for determination of factor X and factor-X antigen, and its application to patients being treated with oral anticoagulants.

1980 ◽  
Vol 26 (7) ◽  
pp. 885-890 ◽  
Author(s):  
E M van Wijk ◽  
L H Kahlé ◽  
J W ten Cate
Keyword(s):  
Oral Anticoagulant ◽  
Oral Anticoagulants ◽  
Neutralization Assay ◽  
Factor X ◽  
Extrinsic Pathway ◽  
Oral Anticoagulant Treatment ◽  
Chromogenic Assay ◽  
Russell’S Viper ◽  
The One

Abstract We describe a mechanized chromogenic assay for factor X, the results of which correlate well with those for the one-stage clotting assays for factor X in which it is activated either via the extrinsic pathway by thromboplastin or directly by Russell's viper venom. We purified human factor X and raised monospecific antibodies to it in rabbits. We used our chromogenic assay for factor X to develop a factor-X-inhibitor neutralization assay for determination of factor-X antigen. Patients receiving oral anticoagulant treatment had significantly different factor-X activities after activation via thromboplastin or with Russell's viper venom. The concentration of factor-X antigen, although decreased, significantly exceeded factor-X clotting activity or chromogenic activity in this group of patients. Results of the chromogenic assay for factor X correlated well with results of "Thrombotest," a clotting test introduced by Owren (Lancet ii: 754, 1959) to control anticoagulant therapy. For patients taking oral anticoagulant drugs, the therapeutic range by our assay is 180 to 300 units/L.

Improved Instrumentation for the Determination of Prothrombin Activity

1965 ◽  
Vol 11 (3) ◽  
pp. 409-412 ◽  
Author(s):  
Rex E Sterling ◽  
Alan A Wilcox ◽  
Arnold G Ware ◽  
Mary K Umehara
Keyword(s):  
Prothrombin Activity ◽  
End Point ◽  
One Stage ◽  
The One ◽  
Point Detector

Abstract The one-stage prothrombin method of Ware and Stragnell (2) has been semiautomated. This method employs an automatic pipetter-diluter and an end-point detector. These instruments materially increase the output per technician.

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