Chromogenic Substrates

 

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CHROMOGENIC SUBSTRATES | METHODS | FACTOR X


Determination of factor X in plasma with Chromogenic Substrate S-2765

Measurement Principle
The method is based on a two-stage principle. In stage one, Factor X is activated in the presence of calcium to Factor Xa (FXa) using the activator Russell’s Viper venom (RVV). In stage two, the generated FXa hydrolyses the chromogenic substrate Z-D-Arg-Gly-Arg-pNA (S-2765), thus liberating the chromophoric group pNA (p-nitroaniline). The colour is then read photometrically at 405 nm. The generated FXa (and thus the intensity of colour) is proportional to the FX activity of the sample.

Factor X   RVV
Factor Xa
Z-D-Arg-Gly-Arg-pNA + H2O FXa
Z-D-Arg-Gly-Arg-OH + pNA

Reagents

  1. S-2765, 25 mg   Art. No. 82 14 13
    Reconstitute the substrate S-2765 (MW: 714.6) with 20 ml sterile water.
  2. Russell’s Viper Venom (RVV)
    Prepare a solution of Russell’s Viper Venom at a concentration of 0.087 mg/ml.
  3. CaCl2
    0.1 mol/l calcium chloride solution.
  4. Tris EDTA Buffer Art. No. 82 36 66
    Dilute the buffer 1:10 with distilled water according to the insert sheet instructions.
  5. Normal Plasma
    Calibrated, lyophilised or fresh frozen human plasma is used for the standardisation of the assay. A pooled normal plasma can be prepared by taking samples from 20 healthy donors. 10-30 ml citrate blood (9 vol blood and 1 vol 0.1 mol/l sodium citrate) from each donor is centrifuged at 2000 x g for 20 minutes at 15-25°C. The plasma is pooled and subsequently dispensed in small volumes, which are frozen rapidly at -20°C or below. To avoid low temperature activation of prekallikrein the plasma is kept at 15-25°C before use or freezing. Thawing of plasma should be performed at 37°C and then kept at 15-25°C until used.
  6. RVV + CaCl2
    Before use, mix 1 volume of RVV with 1 volume of CaCl 2 . The mixture is stable for 48 hours at 2-8°C.
  7. Acetic acid 20%
    Acetic acid is used as a stopper solution in the end-point method.

Specimen collection
Blood (9 vol) is mixed with 0.1 mol/l sodium citrate (1 vol) and centrifuged at 2000 x g for 20 minutes at 20-25°C. Storage: 1 week at 2-8°C or 3-4 months at -20°C.

Standard curve

  Predilution Final dilution
FX
%
Normal plasma ml Buffer
ml
Predil.
plasma
ml
Buffer
ml
0 - - - 1000
25 25 75 50 1000
50 50 50 50 1000
75 75 25 50 1000
100 - - 50 1000
124 - - 50 800

Method

Sample dilution

Buffer 1000 ml
Test plasma or standard 50 ml
Mix  

 

Acid stopped method

A

B

Diluted sample

200 ml

50 ml

Incubate at 37°C

3-4 min

3-4 min

Substrate (37°C)

200 ml

50 ml

Mix and add within 30 sec

RVV + CaCl2

200 ml

50 ml

Mix and incubate at 37°C

3 min

3 min

Acetic acid 20%

200 ml

50 ml

A= test tube method
B= microplate method

Sample blank activities should be determined and subtracted when analysing strongly coloured plasma, e.g. lipemic and hemolytic. The sample blanks are prepared by mixing the diluted sample, acetic acid 20% and water instead of the reagents (400 µl for test tubes and 100 µl for microplates). Read the absorbance of the samples and blanks at 405 nm. The colour is stable for at least four hours. When possible, use a dual wavelength mode with 490 nm as the reference wavelength.

Initial rate method:
When performing the initial rate method, transfer the microplate to a microplate reader immediately after the addition of RVV+CaCl 2 and read the change in A/min. The microplate reader must be pre-incubated at 37°C.

Calculation
Plot A or DA/min for the standards against their concentration of Factor X. Read the Factor X value for the corresponding A or DA/min of the unknown test sample from the standard curve.

Bibliography

  1. Kiesel W et al. Factor X activating enzyme from Russell’s Viper Venom; isolation and characterisation. Biochemistry 15, 4901-4905 (1976).
  2. Lindhout MJ et al. Activation of decarboxyfactor X by a protein from Russell’s Viper Venom. Purification and partial characterisation of activated decarboxyfactor X. Biochem Biophys Acta 533, 327-341, (1978).
  3. Bergström K and Egberg N. Determination of vitamin K sensitive coagulation factors in plasma. Studies on three methods using synthetic chromogenic substrates. Thromb Res 12, 531-547 (1978).
  4. Van Wijk EM et al. A rapid manual chromogenic factor X assay. Thromb Res 22, 681-686 (1981). Egberg N and Heedman PA. Simplified performance of amidolitic factor X assay. Thromb Res 25, 437-440 (1982).
  5. Chabbat J et al. Aprotinin is a competitive inhibitor of the factor VIIa-tissue factor complex. Thromb Res 71, 205-215 (1993).
  6. Mielicki WP and Gordon SG. Three stage chromogenic assay for the analysis of activation properties of factor X by cancer procoagulant. Blood Coagul Fibrinol 4, 441-446 (1993).
  7. Koppaka V et al. Soluble phospholipids enhance factor Xa-catalyzed prothrombin activation in solution. Biochemistry 35, 7482-7491 (1996).
  8. Riesbeck K et al. Human tissue factor pathway inhibitor fused to CD4 binds both FXa and TF/FVIIa at the cell surface. Thromb Haemost 78, 1488-1494 (1997).
  9. Romisch J et al. Comparative in vitro investigation of prothrombin complex concentrates. Semin Thromb Hemost 24, 175-181 (1998)
  10. Faria F, et al. A new factor Xa inhibitor (lefaxin) from the Haementeria depressa leec.Thromb Haemost 82, 1469-73 (1999).
 

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