Method for in vitro assay of the circulating tissue factor, and use in the detection of coagulation diseases

1. A method for in vitro assay of the activity of circulating tissue factor (cTF) in a biological sample originating from the prior sampling of a patient, wherein the generation of activated factor X (Xa) is measured in a reaction medium comprising the tested biological sample, an excess of factor VII and of factor X, calcium ions, a fibrin polymerization inhibitor and a TFPI (Tissue Factor Pathway Inhibitor) activity inhibitor.

2. A method for the in vitro measurement of circulating tissue factor activity as claimed in claim 1 , characterized in that the biological sample is a sample of blood or a derivative, especially a plasma sample.

3. A method as claimed in claim 1 , characterized in that the generation of factor Xa in the reaction medium is determined by measuring the hydrolysis activity of said factor on a specific substrate.

4. A method as claimed in claim 3 , characterized in that the specific substrate is an enzymatic substrate.

5. A method as claimed in claim 3 , characterized in that the amidolytic activity of activated factor X (FXa) is assayed using a chromogenic or fluorometric substrate.

6. A method as claimed in claim 5 , characterized in that the liberation of para-nitroaniline by the synthetic chromogenic substrate CBS 5244 is measured.

7. A method as claimed in claim 1 , characterized in that the reaction medium comprises plasma diluted in an appropriate buffer, for example an Owren Koller buffer.

8. A method as claimed in claim 7 , characterized in that the plasma is diluted in a dilution range of 1/2 to 1/20, for example 1/2 to 1/4; in particular, the plasma is diluted by 1/3.

9. A method as claimed in claim 1 , characterized in that the fibrin polymerization inhibitor is a polypeptide inhibitor, for example H-GlyProArgPro-OH.AcOH (GPRP.AcOH) or is an anti-fibrinogen antibody.

10. A method as claimed in claim 1 , characterized in that the calcium ions necessary for the initiation of coagulation are supplied in the form of CaCl 2 .

11. A method as claimed in claim 1 , characterized in that the inhibitor of the activity of TFPI on the circulating tissue factor is constituted by anti-TFPI antibody or functional antibody fragments binding the TFPI that are capable of inhibiting the activity of TFPI on circulating tissue factor.

12. A method as claimed in claim 11 , wherein the TFPI inhibitor is a monoclonal T4E2 antibody or a functional fragment of said antibody.

13. A method as claimed in claim 2 , comprising the steps of:

a) bringing a plasma sample into contact with a reagent 1 comprising a fibrin polymerization inhibitor, an inhibitor of the activity of TFPI on cTF and, if appropriate, a dilution buffer;

b) incubating the reaction medium constituted in step a) with a reagent 2 comprising factor X in excess, factor VII in excess and calcium ions in solution, in order to produce activated factor X (FXa);

c) after incubation, bringing the reaction medium constituted in step b) into contact with an enzymatic substrate for activated factor X (FXa);

d) detecting the transformation of the factor Xa substrate.

14. A method as claimed in claim 12 , characterized in that the reagent 1 also comprises a heparin inhibitor, for example polybrene.

15. A method as claimed in claim 13 , characterized in that the plasma sample is diluted by 1/3, in an Owren Koller buffer supplemented with 1.2 mg/L polybrene and 50 μg/L of anti-TFPI antibody.

16. A method as claimed in claim 13 , characterized in that the quantitative measurement of the transformation of the enzymatic substrate for factor Xa is carried out by reading the optical density in a pre-determined window.

17. A method as claimed in claim 13 , characterized in that the quantity of transformed factor Xa substrate is assayed by referring to a calibration curve.

18. A method as claimed in claim 13 , characterized in that the reaction is carried out under the following conditions:

for step a), the plasma sample diluted by 1/3 in an Owren Koller buffer is brought into contact with 50 μg/L of anti-TFPI antibody and 1.2 mg/L of polybrene and a fibrin polymerization inhibitor designated GPRP.AcOH in a concentration of 10 g/L;

for step b), 25 μl of factor X in a proportion of 80 PEU/mg/mL, 25 μl of factor VII in a proportion of 137 PEU/mg/mL and 50 μl of a 25 mM CaCl 2 solution are added to 50 μl of the mixture of step a), and the reaction mixture obtained is incubated at 37° C. for 500 seconds;

for step c), 100 μl of a solution of the enzymatic substrate for activated factor X is added and the quantity of transformed enzymatic substrate is determined by optical density reading in a reading window of 6 to 600 s, at a wavelength of 405 nm.

19. A method as claimed in claim 1 , characterized in that it further comprises assaying an internal control.

20. A method as claimed in claim 1 , characterized in that the optical density values obtained are compared with those of a calibration curve prepared using a pool of normal plasma enriched in tissue factor or using a NaCl—CaCl 2 solution enriched in tissue factor.

21. A method for the in vitro detection of a coagulation anomaly, comprising measuring the activity of circulating tissue factor as claimed in claim 1 .

22. A method as claimed in claim 21 , comprising comparing the measured circulating tissue factor activity with a normal value for said activity.

23. A method as claimed in claim 22 , wherein the normal value is obtained after assaying the circulating tissue factor activity in a pool of normal plasma samples.

24. A method as claimed in claim 22 , wherein the normal value is established from values for the activity of the circulating tissue factor measured on individually assayed normal plasma samples.

25. A method as claimed in claim 21 , for the detection of an increase in the rate of circulating tissue factor associated with a risk of thrombosis or with a thrombosis, in particular with the symptoms of acute myocardial infarction (AMI), acute coronary syndrome, disseminated intravascular coagulation (DIVC), diabetes, preeclampsia, miscarriage, thrombosis, sepsis, inflammation or cancer.