Automated Control of Coumarin Therapy by Chromogenic Factor X Assay

1979 ◽  
Author(s):  
R. S. Mibashan ◽  
M.F. Scully ◽  
A.J. Birch ◽  
J.K. Thumpston ◽  
I.R. MacGregor ◽  
...  
Keyword(s):  
Regression Analysis ◽  
Prothrombin Time ◽  
Chromogenic Substrate ◽  
Plasma Samples ◽  
Factor X ◽  
Information Sheet ◽  
Automated Control ◽  
Normal Plasma ◽  
Plasma Factor

A fully automated chromogenic substrate method for measuring plasma factor X has been used to monitor patients attending an anticoagulant clinic. Thirty six patients already established on coumarin therapy were studied by taking four citrate plasma samples from each at monthly intervals. Factor X was assayed in 10 μl aliquots by an adaptation of the method of Aurell & Friberger (Kabi Diagnostica Information Sheet) in an LKB Mark II Analyser with pre-injection facility, allowing 60 s pre-incubation with RW before addition of the substrate S2222. Activity was measured as Δ OD 405/min. Calibration curves were prepared by dilution of lyophilyzed normal plasma, and six coumarin controls were interspersed, with values of 18% (1.09 SD) & 22.2%(1.50 SD) of normal. The F X levels in the 36 patients at each of the monthly clinics were 23.6%. (5.l SD), 23.1 (4.9), 23.1 (5.0) & 23.7% (4.8) of normal, corresponding to RVV/phospholipid coagulant assays of 22.3% (6.3 SD), 22.3 (6.3), 20.7 (5.8) & 21% (6.1) of normal respectively. The prothrombin activities ac each visit, expressed as prothrombin time ratios (PTR), averaged 2.64, 2.80, 2.64 & 2.56. Regression analysis of chromogenic F X values on individual PTR’s gave r=0.65, which led to no change in coumarin dosage when this was repeated blind. The results suggest that rapid, automated F X assays may be suitable clinically, technically & geographically in long-term coumarin control when apparatus & reagents become competitive with established coagulant procedures.

Heparin and Low Molecular Weight Heparins Inhibit Prothrombinase Formation but not its Activity in Plasma

1994 ◽  
Vol 72 (06) ◽  
pp. 862-868 ◽  
Author(s):  
Frederick A Ofosu ◽  
J C Lormeau ◽  
Sharon Craven ◽  
Lori Dewar ◽  
Noorildan Anvari
Keyword(s):  
Factor Xa ◽  
Catalytic Efficiency ◽  
Thrombin Inhibitor ◽  
Lag Phase ◽  
Factor V ◽  
Factor X ◽  
Normal Plasma ◽  
Plasma Factor ◽  
Prothrombin Activation ◽  
Plasma Heparin

SummaryFactor V activation is a critical step preceding prothrombinase formation. This study determined the contributions of factor Xa and thrombin, which activate purified factor V with similar catalytic efficiency, to plasma factor V activation during coagulation. Prothrombin activation began without a lag phase after a suspension of coagulant phospholipids, CaCl2, and factor Xa was added to factor X-depleted plasma. Hirudin, a potent thrombin inhibitor, abrogated prothrombin activation initiated with 0.5 and 1.0 nM factor Xa, but not with 5 nM factor Xa. In contrast, hirudin did not abrogate prothrombin activation in plasmas pre-incubated with 0.5,1.0 or 5 nM α-thrombin for 10 s followed by the coagulant suspension containing 0.5 nM factor Xa. Thus, thrombin activates plasma factor V more efficiently than factor Xa. At concentrations which doubled the clotting time of contact-activated normal plasma, heparin and three low Mr heparins also abrogated prothrombin activation initiated with 0.5 nM factor Xa, but not with 5 nM factor Xa. If factor V in the factor X-depleted plasma was activated (by pre-incubation with 10 nM a-thrombin for 60 s) before adding 0.5,1.0, or 5 nM factor Xa, neither hirudin nor the heparins altered the rates of prothrombin activation. Thus, none of the five anticoagulants inactivates prothrombinase. When 5 or 10 pM relipidated r-human tissue factor and CaCl2 were added to normal plasma, heparin and the three low Mr heparins delayed the onset of prothrombin activation until the concentration of factor Xa generated exceeded 1 nM, and they subsequently inhibited prothrombin activation to the same extent. Thus, hirudin, heparin and low Mr heparins suppress prothrombin activation solely by inhibiting prothrombinase formation.

Characterization of Recombinant Human Coagulation Factor XFriuli

1996 ◽  
Vol 75 (02) ◽  
pp. 313-317 ◽  
Author(s):  
D J Kim ◽  
A Girolami ◽  
H L James
Keyword(s):  
Catalytic Domain ◽  
Coagulation Factor ◽  
Molecular Size ◽  
Binding Pocket ◽  
Site Directed Mutagenesis ◽  
Bleeding Tendency ◽  
Factor X ◽  
Amino Terminal ◽  
Normal Plasma ◽  
Plasma Factor

SummaryNaturally occurring plasma factor XFriuli (pFXFr) is marginally activated by both the extrinsic and intrinsic coagulation pathways and has impaired catalytic potential. These studies were initiated to obtain confirmation that this molecule is multi-functionally defective due to the substitution of Ser for Pro at position 343 in the catalytic domain. By the Nelson-Long site-directed mutagenesis procedure a construct of cDNA in pRc/CMV was derived for recombinant factor XFriuli (rFXFr) produced in human embryonic (293) kidney cells. The rFXFr was purified and shown to have a molecular size identical to that of normal plasma factor X (pFX) by gel electrophoretic, and amino-terminal sequencing revealed normal processing cleavages. Using recombinant normal plasma factor X (rFXN) as a reference, the post-translational y-carboxy-glutamic acid (Gla) and (β-hydroxy aspartic acid (β-OH-Asp) content of rFXFr was over 85% and close to 100%, respectively, of expected levels. The specific activities of rFXFr in activation and catalytic assays were the same as those of pFXFr. Molecular modeling suggested the involvement of a new H-bond between the side-chains of Ser-343 and Thr-318 as they occur in anti-parallel (3-pleated sheets near the substrate-binding pocket of pFXFr. These results support the conclusion that the observed mutation in pFXFr is responsible for its dysfunctional activation and catalytic potentials, and that it accounts for the moderate bleeding tendency in the homozygous individuals who possess this variant procoagulant.

Lack of Agreement of Chromogenic and Glotting Assays for Factor X and Protein C in Warfarinised Plasma

1991 ◽  
Vol 65 (04) ◽  
pp. 360-363 ◽  
Author(s):  
P Han ◽  
K P Fung ◽  
U Rahdakrishnan
Keyword(s):  
Serine Proteases ◽  
Protein C ◽  
Warfarin Therapy ◽  
Intraclass Correlation ◽  
Chromogenic Substrate ◽  
Factor X ◽  
Laboratory Automation ◽  
Quantitative Results ◽  
Assay Methods

SummaryCoagulation serine proteases can be measured with either a chromogenic substrate assay or a clotting assay using deficient plasmas. It is a concern whether both assays give similar quantitative results, in particular in plasma obtained fiom patients on long term warfarin therapy. If these two assay methods were interchangeable, then the chromogenic substrate assay has the advantages of precision as well as laboratory automation. We used the intraclass correlation coefficient (r1) to assess the agreement between the two methods in measuring factor X and protein C levels in warfarinised plasma. The results indicate that the extent and pattern of agreement of the two methods for the measurement of the two variables in warfarinised plasma are poor, despite high Pearson product moment coefficients of correlation.

A Chromogenic Factor X Assay With New Standardized Reagents

1979 ◽  
Author(s):  
P Friberger ◽  
C Lenne
Keyword(s):  
Human Plasma ◽  
Suitable Method ◽  
Chromogenic Substrate ◽  
Factor X ◽  
Normal Human Plasma ◽  
Normal Plasma ◽  
Normal Human

A recently published method for Factor X (FX) assay (1) utilizing Russel's Viper Venom (RVV) and a chromogenic substrate has been further investigated by testing a large number of parameters. This method has been considered as a suitable method for monitoring coumarol treatment (Bergström et al).The conditions for the activation of FX by purified preparations of the RVV have been studied as well as the conditions for FXa determination with a new chromogenic substrate Bz-Ile-Glu(γ-piperidyl)-Gly-Arg-pNA (S-2337). Both purified factors and normal plasma have been used. The effect of plasma inhibitors as well as the selectivity of the method has been studied.The reproducibility and stability of the different reagents and standards have been studied and found to be good.The amount of FXa activity obtained from normal human plasma has been titrated with FXa inhibitors of known purity.1) Aurell L. et al, Thromb. Res., 11, 595 (1977)2) Bergström et al, Thromb. Res., 12, 531 (1978)

Effects of Reagent and Instrument on Prothrombin Times, Activated Partial Thromboplastin Times and Patient/Control Ratios

1988 ◽  
Vol 59 (03) ◽  
pp. 455-463 ◽  
Author(s):  
Freshteh Naghibi ◽  
Yangsook Han ◽  
W Jean Dodds ◽  
Charles E Lawrence
Keyword(s):  
Prothrombin Time ◽  
Proficiency Testing ◽  
Analysis Of Variance ◽  
Plasma Samples ◽  
Error Structure ◽  
Patient Control ◽  
Normal Plasma ◽  
Inherent Error

SummaryActivated partial thromboplastin times accumulated from two proficiency testing surveys were analyzed to determine simultaneously the effects of the method and reagent used. Prothrombin time results were reevaluated concomitantly for comparison.A robust two-way analysis of variance was applied to determine the effect of method and reagent on APTT results. The effect of the reagent and method on the ratio of abnormal to normal plasma clotting times was determined. We found a substantial difference in ratios for the PT using different reagents on the same instrument. There was an even larger effect of reagents on APTT ratios.Our finding of substantial reagent effects for the PT and APTT clearly support the need for standardization. We found standardization to be feasible only for the PT, and only if applied in a form consistent with the inherent error structure of the data. For the APTT the present methodology and plasma samples did not achieve consistent standardization.

An Assessment Of An Amidolytic Assay For Factor VII In The Laboratory Control Of Oral Anticoagulants

1981 ◽  
Author(s):  
A Bodzenta ◽  
Jean M Thomson ◽  
Z S Latallo
Keyword(s):  
Prothrombin Time ◽  
Oral Anticoagulant ◽  
Oral Anticoagulants ◽  
Factor Vii ◽  
Limiting Factor ◽  
Factor X ◽  
Present Evidence ◽  
Chromogenic Assay ◽  
Better Than

An amidolytic assay for factor VII, modified from the method of Seligsohn et al (1978), has been compared with the results of the prothrombin time using British Comparative Thromboplastin, Thronbotest and a clotting assay for factor VII. In ‘long-term’ oral anticoagulant administration agreement with the conventional methods was good and better than in our previous study when amidolytic assays for factors II and X respectively were studied (Latallo et al 1981). The method appeared to be reasonably specific for factor VII.On the present evidence the chromogenic assay for factor VII offers a limited but apparently dependable guide to dosage but it is elaborate to perform and difficult to standardise. The main limiting factor for its routine application is the need to prepare a purified factor X extract.

scholarly journals Production of a High-Titred Sheep Antiserum Against Human Factor IX and Immunological Demonstration of Factor IX Aggregates

1977 ◽  
Author(s):  
K.H. Ørstavik ◽  
A.M. Vennerød
Keyword(s):  
Affinity Chromatography ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Rabbit Antiserum ◽  
Factor Ix ◽  
Factor X ◽  
Weak Band ◽  
Normal Plasma ◽  
Human Factor Ix ◽  
Plasma Factor

Plasma factor IX was purified from a factor IX concentrate by a five step procedure including absorption onto aluminium hydroxide, affinity chromatography on heparin-coupled Sepharose 4B, preparative disc gel electrophoresis, affinity chromatography on an immunosorbent column with rabbit antiserum against factor X and chromatography on DE-52 cellulose. The pooled fractions had a specific activity of approximately 250 U/mg protein. A sheep was immunized with pooled and concentrated fractions. An antiserum was produced which gave one main precipitin band and occasionally an additional weak band against normal plasma in double immunodiffusion. At a dilution of 1/100-1/200 the antiserum neutralized 90% of the factor IX activity in an equal volume of normal plasma.Polyacrylamide disc gel electrophoresis of the fractions from DE-52 cellulose revealed one major and three minor bands with lower electrophoretic mobility and intensity. The three minor bands disappeared on disc gel electrophoresis in the presence of 10 M urea. When the disc electrophoresis gel was submitted to electrophoresis into anagarose gel containing the sheep antiserum or a previously characterized rabbit antiserum against factor IX, four precipitin arcs corresponding to the four bands were seen. A reaction of identity was seen between the four arcs. This study demonstrates that a highly potent antiserum may be produced against factor IX in sheep.

Long-Term Stability Studies on the WHO IRP for Thromboplastin (Human Plain BCT/253)

1994 ◽  
Vol 72 (05) ◽  
pp. 682-684 ◽  
Author(s):  
L Poller ◽  
Jean Pulford ◽  
K J Stevenson ◽  
P Cooper ◽  
G Gamble ◽  
...  
Keyword(s):  
Prothrombin Time ◽  
Second Primary ◽  
Plasma Samples ◽  
Stability Studies ◽  
Term Stability ◽  
Long Term Stability ◽  
Time Performance ◽  
Reference Preparation ◽  
Measurable Change

SummaryLong-term stability studies on the WHO second primary International Reference Preparation (IRP) for thromboplastin (human plain BCT/253) stored at ™20° C have been conducted for ten years.Three centres took part in the exercise using frozen normal and coumarinised plasma samples which were tested throughout. There has been no measurable change in the prothrombin time performance of the human plain IRP over the ten-year period. It can therefore be concluded that new IRP may continue to be calibrated against this preparation in accord with WHO recommendations.

Behaviour of Factors II, VII, IX, and X during Long-Term Treatment with Coumarin

1963 ◽  
Vol 09 (01) ◽  
pp. 074-089 ◽  
Author(s):  
E. A Loeliger ◽  
B van der Esch ◽  
M. J Mattern ◽  
A. S. A den Brabander
Keyword(s):  
Prothrombin Time ◽  
Coagulation Factors ◽  
Term Treatment ◽  
Factor Ix ◽  
Separate Determination ◽  
Factor X ◽  
Warfarin Sodium ◽  
Long Term Treatment ◽  
Significant Difference

SummaryDuring long-term treatment with the long-acting coumarin derivative phenprocoumarol (marcoumar) no statistically significant difference in the depression of the activity of coagulation factors II, VII, IX, and X was found. The shorter-acting anticoagulants acenocoumarol (sintrom), warfarin sodium (coumadin), and dicoumarol, possibly lower factor IX somewhat less and factor X somewhat more than they do factors II and VII.A 2.5-fold prolongation of the “prothrombin” time (using Owren 5 s human brain thromboplastin) and the same prolongation of the thrombotest time appear to correspond with a depression of all four factors from 100% down to approximately 20’%, the normal standard being a mixed population with a mean age of 29 years.Separate determination of one of the four coagulation factors concerned is pointless; “prothrombin” time estimation, and more specifically thrombotest, still remain the most reliable methods for controlling the anti-vitamin K action of anticoagulants during long-term treatment.

A Multicenter Study on Amidolytic Factor X Evaluation in Oral Anticoagulant Therapy

1985 ◽  
Vol 53 (03) ◽  
pp. 433-436 ◽  
Author(s):  
A M Berthier ◽  
M Pommereuil ◽  
P Y Scarabin ◽  
J Conard
Keyword(s):  
Prothrombin Time ◽  
Multicenter Study ◽  
Oral Anticoagulant ◽  
Large Scale ◽  
Oral Anticoagulant Therapy ◽  
Strong Positive Correlation ◽  
Chromogenic Substrate ◽  
Factor X ◽  
Oral Anticoagulant Treatment ◽  
Automated Instruments

SummaryFor laboratory control of oral anticoagulation, amidolytic factor X (F X) determination may offer an alternative to standardization difficulties of prothrombin time (PT). In order to validate this amidolytic assay on a large scale, a multicenter study was undertaken in 6 French laboratories using the same chromogenic substrate (Stachrom X Stago) and different automated instruments. Intra and between laboratory reproducibility of factor X was estimated on fresh and lyophilized patients plasmas and was found to be highly satisfactory. Standardization of the method did not seem to depend on the chromogenic substrate used, as investigated in two different centers. Results of PT and factor X were compared in over 500 patients on a longterm stabilized oral anticoagulant treatment: there was a strong positive correlation between the 2 tests in each center. The therapeutic range for factor X was evaluated from therapeutic PT values reported by Duckert and Marbet for the different thromboplastin reagents: the estimated mean range was 21 to 32%.Pooling the results of the six different centers a concordant information for prothrombin time and factor X amidolytic assay was found in 76% of patients and a fully discordant response was present in 0.6%. The results suggest that amidolytic factor X may be suitable for monitoring long-term anticoagulation. However, prospective trials are needed to evaluate its usefulness as compared to conventional methods.

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