Use of antidotes to coagulation inhibitors indicated in the prevention or treatment of thromboembolic pathologies

Abstract

The use of factor Xa in the prevention or treatment in a patient, in particular a human being or an animal, of haemorrhagic events induced by taking anticoagulants.

Claims

1. A method of treating in a patient of haemorrhagic events induced by taking anticoagulants comprising administering to a patient in need thereof an effective amount of factor Xa having a procoagulant activity due to a prothrombin activation.

2. The method according to claim 1, in which the anticoagulants are low-molecular-weight heparins.

3. The method according to claim 2, in which the low-molecular-weight heparins are selected from bemiparin, certoparin, deltaparin, enoxaparin, nadroparin, parnaparin, reviparin or tinzaparin.

4. The method according to claim 1, in which the anticoagulants are specific inhibitors of factor Xa.

5. The method according to claim 4, in which the specific inhibitors of factor Xa are selected from apixaban, betrixaban, edoxaban, otamixaban or rivaroxaban.

6. The method according to claim 1, in which the anticoagulants are the oligosaccharides fondaparinux or idraparinux.

7. The method according to claim 1, in which factor Xa restores procoagulant activity without the latter exceeding the basal level of the procoagulant activity measured in a group of healthy patients.

8. The method according to claim 1, in which factor Xa is a native protein isolated from plasma or a recombinant protein.

9. The method according to claim 1, in which factor Xa has an increased circulating half-life compared with native factor Xa isolated from plasma.

10. The method according to claim 1, in which said patient is a human being or an animal.

11. A method for treating haemorrhagic events induced by taking anticoagulants, in a mammal, comprising a step of administration of factor Xa having a procoagulant activity due to a prothrombin activation to said mammal in a sufficient quantity making it possible to restore basal procoagulant activity.

12. The method according to claim 11, in which the anticoagulants are specific inhibitors of factor Xa.

13. The method according to claim 12, in which the specific inhibitors of factor Xa are selected from apixaban, betrixaban, edoxaban, otamixaban or rivaroxaban.

14. The method according to claim 13, in which factor Xa is administered to the mammal during or after taking specific inhibitors of factor Xa.

15. The method according to claim 14, in which factor Xa is administrated to human.

16. The method according to claim 11, in which said mammal is human.

Description

CAPTIONS TO THE FIGURES

(1) FIG. 1-A: Thrombinogram obtained from the addition of 10 g/mL of FX to a normal plasma pool following activation with tissue factor: on the x-axis: time (in minutes) on the y-axis: the maximum concentration of thrombin observed (in nM)
The normal plasma pool sample is represented by the black curve (PT=11.9 seconds).
Factor X was added at a rate of 10 g/mL (dotted grey curve) to the normal plasma pool sample (PT=11.1 seconds).

(2) FIG. 1-B: Thrombinogram obtained from the addition of 10 g/mL of FX to a plasma pool overloaded with 0.5 g/mL of rivaroxaban following activation with tissue factor: on the x-axis: time (in minutes) on the y-axis: the maximum concentration of thrombin observed (in nM)
The plasma pool sample overloaded with 0.5 g/mL of rivaroxaban is represented by the grey curve (PT=32.5 seconds).
Factor X was added at a rate of 10 g/mL (black curve) to the sample overloaded with 0.5 g/mL of rivaroxaban (PT=29.3 seconds).

(3) FIG. 1-C: Thrombinogram obtained from the addition of 25 g/mL of FX to a normal plasma pool overloaded with 0.5 g/mL of rivaroxaban following activation with tissue factor: on the x-axis: time (in minutes) on the y-axis: the maximum concentration of thrombin observed (in nM)
The normal plasma pool sample is represented by the black curve (1).
The normal plasma pool sample overloaded with 0.5 g/mL of rivaroxaban is represented by the curve (2).
Factor X was added at a rate of 25 g/mL to the normal plasma pool sample (curve (3)) and to the normal plasma pool sample overloaded with 0.5 g/mL of rivaroxaban (curve (4)).

(4) FIG. 1-D: Thrombinogram obtained from the addition of 100 g/mL of FX to a normal plasma pool overloaded with 0.5 g/mL of rivaroxaban following activation with tissue factor: on the x-axis: time (in minutes) on the y-axis: the maximum concentration of thrombin observed (in nM)
The normal plasma pool sample is represented by the thick black curve (1).
The normal plasma pool sample overloaded with 0.5 g/mL of rivaroxaban is represented by the thin black curve (2).
Factor X was added at a rate of 100 g/mL to the normal plasma pool sample (grey curve (3)) and to the normal plasma pool sample overloaded with 0.5 g/mL of rivaroxaban (light grey curve (4)).

(5) FIG. 2: Thrombinogram obtained from the addition of 10 g/mL of FX to a normal plasma pool overloaded with 0.35 g/mL of rivaroxaban following induction with cephalin: on the x-axis: time (in minutes) on the y-axis: the maximum concentration of thrombin observed (in nM)
The normal plasma pool sample is represented by the black curve (1).
The normal plasma pool sample overloaded with 0.35 g/mL of rivaroxaban is represented by the curve (3).
Factor X was added at a rate of 10 g/mL to the normal plasma pool sample (curve (2)) and to the normal plasma pool sample overloaded with 0.35 g/mL of rivaroxaban (curve (4)).

(6) FIG. 3-A: Thrombinogram obtained from the addition of increasing doses of FXa to a normal plasma pool: on the x-axis: time (in minutes) on the y-axis: the maximum concentration of thrombin observed (in nM)
The normal plasma pool sample is represented by the black curve (1).
Factor Xa was added at a rate of 0.5 g/mL (curve (2)), 1.0 g/mL (curve (3)), 1.5 g/mL (curve (4)), 2.0 g/mL (curve (5)), 2.5 g/mL (curve (6)), 5.0 g/mL (curve (7)) to the normal plasma pool sample.

(7) FIG. 3-B: Thrombinogram obtained from the addition of increasing doses of FXa to a normal plasma pool overloaded with 0.35 g/mL of rivaroxaban: on the x-axis: time (in minutes) on the y-axis: the maximum concentration of thrombin observed (in nM)
The normal plasma pool sample is represented by the black curve (1).
0.35 g/mL of rivaroxaban was added to the normal plasma pool sample (curve (2)).
Factor Xa was added at a rate of 0.5 g/mL (curve (3)), 1.0 g/mL (curve (4)), 1.5 g/mL (curve (5)), 2.0 g/mL (curve (6)), 2.5 g/mL (curve (7)), 5.0 g/mL (curve (8)) to the normal plasma pool sample overloaded with 0.35 g/mL of rivaroxaban.

(8) FIG. 4: Thrombinograms obtained from the addition of 0.01 to 1 g/mL of activated FX to a normal plasma pool without activation: on the x-axis: time (in minutes) on the y-axis: the maximum concentration of thrombin observed (in nM)
The normal plasma pool sample is represented by the curve .circle-solid.. The effect of the concentrations of FXa added to normal plasma is represented by the following curves: 0.01 g/ml (); 0.05 g/ml (.square-solid.); 0.1 g/ml (); 0.5 g/ml (.box-tangle-solidup.) and 1 g/ml (). The controls illustrating thrombin generation following the induction of coagulation either by tissue factor/phospholipids (x) or by cephalin () are indicated.

(9) FIG. 5: Thrombinograms obtained from the addition of 0.03 to 0.5 g/mL of activated FX to a normal plasma pool following activation with tissue factor: on the x-axis: time (in minutes) on the y-axis: the maximum concentration of thrombin observed (in nM)
The normal plasma pool sample is represented by the curve .circle-solid.. The effect of the concentrations of FXa added to normal plasma is represented by the following curves: 0.0312 g/ml (); 0.0625 g/ml (.square-solid.); 0.125 g/ml (); 0.25 g/ml (.box-tangle-solidup.) and 0.5 g/ml ().

(10) FIG. 6: Thrombinograms obtained from the addition of 0.03 to 0.5 g/mL of activated FX to a normal plasma pool in the presence of Rivaroxaban (0.35 g/ml) following activation with tissue factor: on the x-axis: time (in minutes) on the y-axis: the maximum concentration of thrombin observed (in nM)
The normal plasma pool sample without Rivaroxaban (0.35 g/ml) is represented by the curve .circle-solid.. The same plasma activated under the same conditions but in the presence of Rivaroxaban (0.35 g/ml) is represented by the curve x. The effect of the concentrations of FXa added to normal plasma in the presence of Rivaroxaban (0.35 g/ml) is represented by the following curves: 0.0312 g/ml (); 0.0625 g/ml (.square-solid.); 0.125 g/ml (); 0.25 g/ml (.box-tangle-solidup.) and 0.5 g/ml ().

(11) FIG. 7: Thrombinograms obtained from the addition of 0.03 to 0.5 g/mL of activated FX to a normal plasma pool following activation with cephalin: on the x-axis: time (in minutes) on the y-axis: the maximum concentration of thrombin observed (in nM)
The normal plasma pool sample is represented by the curve .circle-solid.. The effect of the concentrations of FXa added to normal plasma is represented by the following curves: 0.0312 g/ml (); 0.0625 g/ml (.square-solid.); 0.125 g/ml (); 0.25 g/ml (.box-tangle-solidup.) and 0.5 g/ml ().

(12) FIG. 8: Thrombinograms obtained from the addition of 0.03 to 0.5 g/mL of activated FX to a normal plasma pool following activation with cephalin in the presence of Rivaroxaban (0.35 g/ml): on the x-axis: time (in minutes) on the y-axis: the maximum concentration of thrombin observed (in nM)
The normal plasma pool sample without Rivaroxaban (0.35 g/ml) is represented by the curve .circle-solid.. The same plasma activated under the same conditions but in the presence of Rivaroxaban (0.35 g/ml) is represented by the curve x. The effect of the concentrations of FXa added to normal plasma is represented by the following curves: 0.0312 g/ml (); 0.0625 g/ml (.square-solid.); 0.125 g/ml (); 0.25 g/ml (.box-tangle-solidup.) and 0.5 g/ml ().

EXAMPLES

Example 1: Extrinsic Coagulation Pathway (TF 1 pM/PL 4 M)

(13) 1Protocol

(14) 1.1Reagents Thrombin calibrator (Stago) TC1103 PPP reagent 5 pM (Stago) PPP 1009 PPP reagent low (Stago) PPL1101 CK-Prest (Stago) 106025 Fluo-buffer (Stago) FLB1012 Fluo-substrate (Stago) FLS1012 Rivaroxaban at 436 g/mL NPP (Cryopep) A1112 human FX at 6.8 mg/mL (HTI) AA1108 bovine FXa at 2.15 mg/mL (Hyphen Biomed) 110728C

(15) 1.2Experimental Protocol

(16) The stock solution of rivaroxaban at 436 g/mL is diluted in 5% DMSO in order to obtain intermediate concentrations which, added to the NPP (Normal Pool Plasma), give final concentrations of rivaroxaban in g per mL of plasma.

(17) FX or FXa are introduced into the plasma without an incubation step.

(18) It is considered that 1 unit of FX=10 g/mL in the plasma, corresponding to a 100% level of FX in the plasma.

(19) The thrombin generation test consists of activating coagulation ex vivo either using a mixture of tissue factor and phospholipids, or using cephalin and then measuring the concentration of thrombin generated over time.

(20) The thrombin generation tests are carried out on 80 l of a plasma pool optionally containing FX and/or Rivaroxaban in the presence either of 20 l of PPP reagent (Stago) finally containing 5 pM of Tissue Factor (TF) and 4 M of phospholipids (PL), or in the presence of 20 L of cephalin (CK-Prest reconstituted with 5 mL of distilled H.sub.2O).

(21) The reaction is started by the addition of 20 L of Fluca-kit (substrate+CaCl.sub.2) which constitutes the start of the measurement of the appearance of thrombin. The appearance of fluorescence is measured on a fluorometer of the Fluoroskan Ascent type (ThermoLabsystems) at an excitation wavelength of 390 nm and emission wavelength of 460 nm. The thrombinograms (curves representing the intensity of fluorescence as a function of time) are then analyzed using the Thrombinoscope software which converts the fluorescence value to nM of thrombin by comparative calculation.

(22) 2Results

(23) 2.1Effects of FX (10 g/Ml) on a Plasma Overloaded with Rivaroxaban

(24) All of the results are shown in Table 1 below and are illustrated by the thrombinograms of FIGS. 1-A and 1-B:

(25) TABLE-US-00001 TABLE 1 Effect of factor X on a normal plasma or plasma supplemented with 0.5 g/mL of Rivaroxaban. TGT Fluoroskan (FT = 1 pM and PL = 4 M final) FX (HTI) in LT ETP Peak TTP ST mVRI PT (RecombiPlastin g/mL final (min) (nM min) (nM) (min) (min) (nM/min) ISI: 1.0) in seconds PNP 0 8 1190 127.15 12.33 38 29.36 11.9 10 6.33 1244 136.38 11.17 35.5 28.18 11.1 PNP + 0 25.5 nd 7.42 49.33 nd 0.31 32.5 rivaroxaban at 10 25.83 nd 8 53.67 nd 0.29 29.3 0.5 g/ml LT = lag time, ETP = endogenous thrombin potential, TTP = time to peak, ST = start time, mVRI = mean velocity rate index, PT = prothrombin time.

(26) The addition of 10 g/mL of FX to a normal plasma slightly increases (+13%) the total quantity of thrombin generated (ETP), the height of the peak (+7.3%) and reduces the period of time comprised between triggering the reaction and the point of maximum thrombin concentration (TTP) with respect to values that can be observed for a normal plasma (FIG. 1-A).

(27) The addition of 0.5 g/mL of Rivaroxaban completely destroys the ability to generate thrombin from normal plasma, following activation by tissue factor and phospholipids. Supplementation with 10 g/mL of FX from a normal plasma overloaded with Rivaroxaban at 0.5 g/mL does not make it possible to restore a thrombin generation different from that obtained during overloading with Rivaroxaban (FIG. 1-B).

(28) In conclusion, commercial human FX (HTI) at 10 g/mL final does not correct a plasma overloaded with rivaroxaban at 0.5 g/mL in the extrinsic pathway at 1 pM of TF.

(29) 2.2Effects of FX (25 g/Ml or 100 g/ml) on a Plasma Overloaded with Rivaroxaban (0.5 g/ml)

(30) All of the results are shown in Table 2 below and are illustrated by the thrombinograms of FIGS. 1-C and 1-D:

(31) TABLE-US-00002 TABLE 2 Dose-effect of factor X on a normal plasma containing 0.5 g/mL of Rivaroxaban. TGT Fluoroskan (FT = 1 pMet and PL = 4 M final) final concentration rivaroxaban LT ETP Peak TTP ST mVRI Names of the graphs FX (HTI) (g/mL) (g/mL) (min) (nM min) (nM) (min) (min) (nM/min) PNP alone PNP 7.55 1145 108.42 12.39 41.5 22.40 PNP diluted 1/2 FBS 0 7.05 1948 161.58 13.56 45.5 24.82 Riva 0.5 g/mL 0.5 30.59 nd 6.83 58.81 nd 0.24 PNP + FX (HTI) 25 g/mL 25 0 5.55 1285 118.82 11.06 36.5 21.56 R0.5 + FX (HTI) 25 g/mL 0.5 29.09 nd 11.22 58.64 nd 0.38 PNP + FX (HTI) 100 g/mL 100 0 5.88 1327 149.34 10.22 35.5 34.41 R0.5 + FX (HTI) 100 g/mL 0.5 58.31 nd 1.77 62.15 nd 0.46 LT = lag time, ETP = endogenous thrombin potential, TTP = time to peak, ST = start time, mVRI = mean velocity rate index.

(32) The addition of FX (25 g/ml or 100 g/ml) to a normal plasma increases the total quantity of thrombin generated (+13 and +16% respectively), the height of the peak (+9.5% and 37% respectively) and reduces the period of time comprised between triggering the reaction and the appearance of thrombin but is incapable of reversing the effects of the Rivaroxaban.

(33) In conclusion, commercial human FX (HTI) increases, in a dose-dependent manner, the thrombin generation of a normal plasma pool in the extrinsic pathway at 1 pM of TF final. By contrast it does not correct thrombin generation of a plasma overloaded with Rivaroxaban to 0.5 g/mL up to concentrations of 100 g/ml i.e. 10 U/ml.

Example 2: Intrinsic Coagulation Pathway (Cephalin Alone)

(34) 1Protocol

(35) The reagents, the automated equipment and the experimental protocol are identical to those described in Example 1.

(36) The thrombin generation tests are carried out on 80 l of a normal plasma pool optionally containing FX and/or Rivaroxaban in the presence of 20 l of cephalin (CK-Prest reconstituted with 5 mL of distilled H.sub.2O) and 20 L fluca-kit (substrate+CaCl.sub.2).

(37) 2Results

(38) 2.1Effect of FX on a Normal Plasma Overloaded with Rivaroxaban

(39) All of the results are shown in Table 3 below and are illustrated by the thrombinograms of FIG. 2:

(40) TABLE-US-00003 TABLE 3 Effect of factor X on a normal plasma containing 0.35 g/mL of Rivaroxaban. CAT (cephalin) FX (HTI) in LT ETP Peak TTP ST mVRI g/mL final (min) (nM min) (nM) (min) (min) (nM/min) PNP 0 22 1213 188.63 25.33 42 56.65 10 17 1441 225.75 20.17 35.5 71.21 PNP + 0 48.17 230.5 8.49 54 116 1.46 rivaroxaban at 10 nd nd nd nd nd nd 0.35 g/ml LT = lag time, ETP = endogenous thrombin potential, TTP = time to peak, ST = start time, mVRI = mean velocity rate index.

(41) The addition of 10 g/mL of FX to a normal plasma increases the total quantity of thrombin generated by 19%. Moreover, the addition of 10 g/mL of FX to a normal plasma reduces the period of time comprised between triggering the reaction and the start of clot formation (LT) and the period of time comprised between triggering the reaction and the point of maximum thrombin concentration (TTP).

(42) The addition of 0.35 g/mL of Rivaroxaban completely destroys the ability to generate thrombin from normal plasma following activation with Cephalin. The addition of 10 g/mL of FX to a normal plasma overloaded with Rivaroxaban at 0.35 g/mL does not make it possible to restore thrombin generation.

(43) In conclusion, commercial human FX (HTI) at 10 g/mL final does not correct a plasma overloaded with Rivaroxaban at 0.35 g/mL in the intrinsic pathway.

(44) In the intrinsic pathway, as in the extrinsic pathway, FX (HTI) does not correct a plasma overloaded with Rivaroxaban (at 0.35 g/ml).

Example 3: Generation of Thrombin by Activation of the Extrinsic Coagulation Pathway (TF 5 pM/PL 401)

(45) 1Protocol

(46) The reagents, the automaton and the experimental protocol are identical to those described in Example 1.

(47) The thrombin generation tests are carried out on 80 l of a normal plasma pool containing FXa and/or Rivaroxababn in the presence of 20 l of PPP reagent low finally containing 1 pM of Tissue Factor (TF) and 4 M of phospholipids (PL), and 20 L fluca-kit (substrate+CaCl2).

(48) 2Results

(49) All of the results are shown in Table 4 below and are illustrated by the thrombinograms of FIGS. 3-A and 3-B:

(50) TABLE-US-00004 TABLE 4 Effect of factor Xa on a normal plasma containing 0.35 g/mL of Rivaroxaban. TGT Fluoroskan (FT = 5 pM and PL = 4 M final) PT (sec) with LT ETP Peak TTP ST mVRI RecombiPlastin aPTT (sec) (min) (nM min) (nM) (min) (min) (nM/min) 2G (ISI: 1.0) with PTT-A PNP 0 3.83 1612 293 6.78 21.67 101.38 11.7 31.3 bovine FXa in g/mL final PNP 0 4.33 1782 297 7 23.67 111.24 11.7 31.3 0.5 3.67 1762 299 6.44 23 107.94 11.3 23.1 1 3 1725 315.2 5.67 21.67 118.05 10.8 20.6 1.5 2.67 1708 332.69 5.22 20 130.47 10.6 20.4 2 2.89 1873 322 5.44 22.67 126.27 10.4 19.6 2.5 2.67 1929 330.37 5.22 22.67 129.56 10.2 19.3 5 2.33 2033 353.39 4.67 21.67 151.02 9 17.1 PNP + 0 11.11 772 31.32 25.33 67 2.20 26 50.5 rivaroxaban 0.5 6.78 1513 155.96 11.33 31.33 34.28 25.8 21 at 0.35 g/mL 1 6.33 1577 192.53 10.22 28.33 49.49 24.8 19.1 1.5 5.56 1500.67 211.74 9 25.33 61.55 24.8 19.3 2 5.67 1490.67 199.24 9.33 26.67 54.44 24.4 16.9 2.5 5.67 1548 199.81 9.44 26.67 53.00 25 18.5 5 5 1549 208.59 8.78 25.67 55.18 22.6 14.8 LT = lag time, ETP = endogenous thrombin potential, TTP = time to peak, ST = start time, mVRI = mean velocity rate index, PT = prothrombin time, aPTT = activated partial thromboplastin time, PTT-A = activated prothromboplastin.

(51) The addition of FXa to a normal plasma reduces the period of time comprised between triggering the reaction by successive stages in a dose-dependent manner. By contrast, the addition of 0.35 g/mL of rivaroxaban to a normal plasma increases this period of time by a factor comprised between 2 and 3 and completely eliminates the formation of thrombin (FIGS. 3-A and 3-B).

(52) The addition of FXa to the normal plasma reduces the period of time comprised between triggering the reaction and the point of maximum thrombin concentration (TTP) by successive stages in a dose-dependent manner. By contrast, the addition of 0.35 g/mL of rivaroxaban to a normal plasma increases this period of time by a factor comprised between 3 and 4.

(53) The addition of FXa to the normal plasma increases by approximately 14% the total quantity of thrombin generated (ETP) for the highest dose of FXa (5 g/mL). By contrast, the addition of 0.35 g/mL of rivaroxaban to a normal plasma reduces the total quantity of thrombin generated.

(54) The addition of increasing quantities of FXa to a normal plasma overloaded with Rivaroxaban at 0.35 g/mL restores the two parameters LT and ETP to the level observed for normal plasma alone.

(55) FXa at 0.5 to 5 g/mL final partially corrects a plasma overloaded with Rivaroxaban at 0.35 g/mL in a dose-dependent manner up to 2.5 g/mL in the extrinsic pathway, at 5 pM of TF. Factor Xa also improves thrombin generation from a normal plasma pool in a dose-dependent manner.

(56) In conclusion, it is noted that the addition of FXa to a normal plasma appears to provide a moderate thrombogenic effect by allowing the generation of larger quantities of thrombin earlier than those obtained from a non-supplemented plasma. On the other hand, the inhibitory effect of Rivaroxaban at 0.35 g/ml is largely eliminated by the addition of FXa with a plateau effect at 2.5 g/ml which does not however make it possible to achieve the thrombin values generated in normal plasma.

Example 4: Assessment of the Useful Doses of Factor Xa (FXa) Inducing Coagulation or Required to Counterbalance the Effect of Rivaroxaban

(57) 1Induction of Thrombin Generation by Factor Xa Alone

(58) The effect of an overdose of FXa in a normal plasma was assessed by measuring thrombin generation (TGT) without additional induction of coagulation (FIG. 4). Doses from 0.01 g/ml to 1 g/ml of FXa were compared. The quantity of thrombin generated depends on the dose of FXa added. The effect of FXa was compared to normal plasma without induction of coagulation (.circle-solid.), activated by tissue factor (X) or cephalin (). The doses of FXa from 0.01 g/ml to 0.05 g/ml produce quantities of thrombin similar to that of a normal plasma activated by TF or cephalin and follow the same appearance kinetics. These results suggest that under these experimental conditions, FXa at a dose of 50 g/ml should not be thrombogenic. The dose of 50 ng/ml would therefore correspond to the lowest useable dose of FXa making it possible to restore coagulation.

(59) 2Induction of Thrombin Generation by Tissue Factor

(60) The effect of an overdose of FXa in normal plasma was assessed by measuring thrombin generation (TGT) following induction by tissue factor (0.5 pM) (FIG. 5, Table I). Doses of 0.03 g/ml to 0.5 g/ml of FXa were compared. The quantity of thrombin generated depends on the dose of FXa added. Doses of FXa from 0.03 g/ml to 0.125 g/ml produce quantities of thrombin similar to that of an activated normal plasma (.circle-solid.) and follow the same appearance kinetics. These results suggest that under these experimental conditions, FXa does not appear to induce thrombogenicity additional to that provided by the inducer in the case of doses from 0.03 g/ml to 0.125 g/ml.

(61) A similar experiment was repeated in the presence of a therapeutic dose of 0.35 g/ml of Rivaroxaban (FIG. 6, Table I). The dose of Rivaroxaban used reduces thrombin generation very significantly (approximately 75%; curve x) compared with that obtained in an untreated plasma (.circle-solid.). FXa supplementation of 0.03 g/ml () does not make it possible to recover significant thrombin generation. On the other hand, a correction of thrombin generation is obtained starting from 0.06 g/ml (.square-solid.) up to a dose of 0.25 g/ml (.box-tangle-solidup.). In the case of the higher dose, the quantity of thrombin generated exceeds that of the control suggesting an FXa overdose and therefore a possible thrombogenic effect despite the Rivaroxaban.

(62) TABLE-US-00005 TABLE I Values of lag time, thrombin potential, peak height and time to peak thrombin generation induced by tissue factor in a normal plasma in the presence or absence of factor Xa and/or Rivaroxaban (data extracted from FIGS. 4 and 5). Lag time ETP Peak ttPeak Name of group (min) (nM) (nM) (min) Unicalibrator 3.83 1179 123.96 8.33 Unicalibrator + 0.5 g/ml hFXa 1.67 1462 211.77 5.33 Unicalibrator + 0.25 g/ml hFXa 2.33 1339 172.05 6.33 Unicalibrator + 0.125 g/ml hFXa 2.67 1238.5 146.77 7 Unicalibrator + 0.0625 g/ml 3 1189.5 134.13 7.33 hFXa Unicalibrator + 0.03125 g/ml 3.33 1147.5 119.98 8 hFXa Unicalibrator + 0.35 g/ml 10.17 282 10.69 20.5 Rivaroxaban Unicalibrator + 0.35 g/ml 2.67 1368.5 165.27 6.33 Rivaroxaban + 0.5 g/ml hFXa Unicalibrator + 0.35 g/ml 3 1317.5 127.63 7.67 Rivaroxaban + 0.25 g/ml hFXa Unicalibrator + 0.35 g/ml 3.33 1217.5 88.16 9.67 Rivaroxaban + 0.125 g/ml hFXa Unicalibrator + 0.35 g/ml 3.83 1189.5 73.14 10.83 Rivaroxaban + 0.0625 g/ml hFXa Unicalibrator + 0.35 g/ml 4 712 28.83 14.5 Rivaroxaban + 0.03125 g/ml hFXa

(63) 3Induction of Thrombin Generation by Cephalin

(64) A series of similar experiments is carried out by inducing the initiation of coagulation by cephalin. The series of preliminary experiments mimics the extrinsic activation of coagulation. This second series of experiments mimics the intrinsic activation of coagulation. The presence of FXa in an increasing quantity in normal plasma (.circle-solid.) reduces the speed of appearance of thrombin without changing the total quantity of thrombin generated (FIG. 7, Table II). The speed of appearance of the thrombin is correlated with the dose of FXa added. Shorter times to peak are obtained in the presence of FXa. This difference is significant and suggests a risk of thrombosis starting from 0.25 g/ml of FXa.

(65) The effect of FXa following the induction of thrombin generation by cephalin is then measured in the presence of Rivaroxaban at 0.35 g/ml (FIG. 8, Table II). The Rivaroxaban completely inhibits thrombin generation induced by cephalin (curve x). The presence of FXa in an increasing concentration makes it possible to increase the quantity of thrombin generated in a dose-dependent manner, as well as to reduce the time to peak. A maximum quantity of thrombin is obtained in the case of a concentration comprised between 0.125 and 0.25 g/ml.

(66) TABLE-US-00006 TABLE II Values of lag time, thrombin potential, peak height and time to peak of thrombin generation induced by the cephalin of a normal plasma in the presence or absence of factor Xa and/or rivaroxaban (data extracted from FIGS. 7 and 8). Lag time ETP Peak ttPeak Name of group (min) (nM) (nM) (min) Unicalibrator 12.33 1277.5 283.42 14.67 Unicalibrator + 0.5 g/ml hFXa 3.67 1291 283.25 6 Unicalibrator + 0.25 g/ml hFXa 5.83 1234.5 287.96 7.83 Unicalibrator + 0.125 g/ml hFXa 8.5 1229 297.26 10.33 Unicalibrator + 0.0625 g/ml hFXa 10 1233 285.93 12 Unicalibrator + 0.03125 g/ml 10.67 1225 272.54 13.17 hFXa Unicalibrator + 0.35 g/ml 0 0 0 0 Rivaroxaban Unicalibrator + 0.35 g/ml 3 1232.5 235.61 5.5 Rivaroxaban + 0.5 g/ml hFXa Unicalibrator + 0.35 g/ml 3.67 1221.5 183.27 7.17 Rivaroxaban + 0.25 g/ml hFXa Unicalibrator + 0.35 g/ml 4.83 1079 121.62 9.33 Rivaroxaban + 0.125 g/ml hFXa Unicalibrator + 0.35 g/ml 6.33 1060.5 84.19 12.17 Rivaroxaban + 0.0625 g/ml hFXa Unicalibrator + 0.35 g/ml 8.33 867.5 59.78 15 Rivaroxaban + 0.03125 g/ml hFXa

(67) In conclusion, the data presented show that FXa has the possibility of correcting the presence of Rivaroxaban in normal plasma following the induction of the intrinsic pathway by cephalin or of the extrinsic pathway by tissue factor. For doses less than or equal to 0.25 g/ml of factor Xa and in the case of a dose of Rivaroxaban of 0.35 g/ml, the data obtained in terms of TGT do not identify a major risk of thrombogenicity. In the case of doses greater than 0.25 g/ml, thrombin generation time is very short and could reveal a thrombogenic risk.