Novel chemiluminescent substrates for Factor Xa

Abstract

The present invention relates to chemiluminescent substrates for blood clotting enzyme Factor Xa. The substrates are particularly useful for assaying coagulation factors and for quantifying an anticoagulant in a sample.

Claims

1. A compound of general formula (I-3) or (I-4), ##STR00017## wherein r is 0, 1, 2, or 3; r′ is 0, 1, 2, or 3; d is 0, 1, or 2; g′ is 0 or1; g′ is 0 or 1; and X is a terminal moiety selected from NH.sub.2, OH, O(C.sub.1-6a lkyl), (OCH.sub.2CH.sub.2).sub.1-6OH, (OCH.sub.2CH.sub.2).sub.1-6(C.sub.1-6alkyl), NHC(═O)(O).sub.0-1(C.sub.1-6alkyl), NHC(═O)(O).sub.0-1(C.sub.1-6alkylene)(OCH.sub.2CH.sub.2).sub.1-6OH, NHC(═O)(O).sub.0-1(C.sub.1-6alkylene)(OCH.sub.2CH.sub.2).sub.1-6O(C.sub.1-6alkyl), NHC(═O)(O).sub.0-1(C.sub.1-6alkylene)O(C.sub.1-6alkyl), NHC(═O)(O).sub.0-1(C.sub.1-6alkylene)OH, and NP′ wherein P′ is an amine protecting group; optionally wherein the aminoluciferin moiety is replaced by a different chemiluminescent amine; or a physiologically acceptable salt thereof.

2. The compound according to claim 1, wherein d is 0 or 1-,

3. The compound according to claim 1, wherein r is 1 or 2, or wherein g is 1.

4. The compound according to claim 1, wherein the compound is of general formula (I-3s) or (I-4s), ##STR00018## wherein r is 0, 1, 2, or 3; r′ is 0, 1, 2, or 3; d is 0, 1, or 2; g is 0 or 1; g′ is 0 or 1.

5. The compound according to claim 1, wherein the compound is of general formula (II-3) or (II-4), ##STR00019## wherein r is 0, 1, 2, or 3; r′ is 0, 1, 2, or 3; d is 0, 1, or 2; g is 0 or 1; g′ is 0 or 1; and X is a terminal moiety selected from NH.sub.2, OH, O(C1-6a1kyl), (OCH.sub.2CH.sub.2)1-6OH, (OCH2CH2)1 6O(C1 6alkyl), NHC(═O)(O)0-1(C1-6alkyl), NHC(═O)(O)0 1(C1-6alkylene)(OCH2CH2)1-6OH, NHC(═O)(O)0 1(C1 6alkylene)(OCH2CH2)1 6O(C1 6alkyl), NHC(═O)(O)0-1(C1 6alkylene)O(C1-6alkyl), NHC(═O)(O)0 1(C1-6alkylene)OH, and NP′ wherein P′ is an amine protecting group.

6. The compound according to claim 1, wherein the compound is of general formula (III-3) or (III-4), ##STR00020## wherein d is 0, 1, or 2; r is 0, 1, 2, or 3; r′ is 0, 1, 2, or 3; g is 0 or1; g′ is 0 or 1; m is 0, 1, 2, 3, 4, 5, or 6; p is 0, 1, 2, 3, 4, 5, or 6; s is 0, 1, 2, 3, 4, 5, or 6; and a is 0 or1.

7. The compound according to claim 6, wherein d is 0 or 1, preferably 1; and/or r is 1 or 2, preferably 1; and/or r′ is 1 or 2, preferably 1; and/or g is 0 or 1, preferably 1; and/or g′ is 0 or 1, preferably 1; and/or m is 0 or 1; and/or p is 1, 2, or 3, preferably 2; and/or s is 1 or 2, preferably 1; and/or a is 1.

8. The compound according to claim 1, wherein the compound is selected from MePEG2-IEGR and MePEG2-IDGR ##STR00021##

9. The compound according to claim 1, wherein P′ is selected from the group consisting of trityl, allyl, benzyl (Bn), benzoyl (Bz), 9-fluorenylmethyl oxycarbonyl (Fmoc), t-butyloxycarbonyl (Boc), benzyloxycarbonyl (Z), 2-trimethylsilylethyloxycarbonyl, tosyl (Ts), acetyl (Ac), trifluoroacetyl, phthalimide, benzylideneamine, and allyloxycarbonyl (Alloc), preferably from the group consisting of benzyl (Bn), 9-fluorenylmethyl oxycarbonyl (Fmoc), t-butyloxycarbonyl (Boc), benzyloxycarbonyl (Z), tosyl (Ts), and allyloxycarbonyl (Alloc), more preferably P′ is benzyloxycarbonyl.

10. The compound according to claim 1, wherein the compound is an acid addition salt optionally selected from a HCl salt, an acetic acid salt, a formic acid salt, a TFA salt, and a mesylic acid salt, preferably a HCl salt or a TFA salt, most preferably a TFA salt.

11.-12. (canceled)

13. Method for quantifying a coagulation factor in a sample, the method comprising the steps of: a) contacting the sample with a composition comprising a compound as defined in claim 1 to release aminoluciferin; b) contacting the aminoluciferin with luciferase; and c) determining the relative light intensity generated by the luciferase.

14. The method according to claim 13, wherein the coagulation factor is selected from factor IX, factor IXa, factor VIII, factor VIIIa, factor VII, factor VIIa, factor XI, factor XIa, Factor XII, factor XIIa, factor X, factor Xa, prekallikrein, and kallikrein, optionally wherein during step a) the composition comprises at least one further coagulation factor selected from factor IX, factor IXa, factor VIII, factor VIIIa, factor VII, factor VIIa, factor XI, factor XIa, factor XII, factor XIIa, factor X, prekallikrein, and kallikrein.

15. Method for quantifying an anticoagulant in a sample, the method comprising the steps of: a) contacting the sample with a composition comprising factor Xa and a compound as defined in claim 1 to release aminoluciferin; b) contacting the aminoluciferin with luciferase; and c) determining the relative light intensity generated by the luciferase.

Description

SHORT DESCRIPTION OF DRAWINGS

[0147] FIG. 1-13 Synthesis of compounds according to the invention. A) synthesis towards a shared Gly-Arg/Gly-Orn core; B) conversion of the shared core into a precursor having peptide core Ile-Glu-Gly-Arg; C) conversion of the Ile-Glu-Gly-Arg intermediate into MePEG2-IEGR.

[0148] FIG. 2—Michaelis-Menten kinetics for the enzyme FXa and its substrate MePEG2-IEGR at an FXa concentration of 1.6 nM. A V.sub.max=468000 RLU and a K.sub.M=619 μM were determined, leading to a K.sub.cat=V.sub.max/[FXa]=1.05×10.sup.14 s.sup.−1.

[0149] FIG. 3—Substrate MePEG2-IEGR has minimal cross-reactivity with serine proteases other than FXa. A) 80 nm FXa yielded a signal of 1.000.000 RLU; 52 nM thrombin yielded ˜30.000 RLU; 8 nM plasmin yielded ˜8.000 RLU; 193 IU/mL tPA (the conventional concentration to facilitate fibrinolysis) yielded ˜6.000 RLU; 145 nM FXlla yielded ˜500 RLU; concentrations other than tPA are representative for concentrations in human plasma after activation. B) closer view of a section of panel A wherein the y-axis has been transformed to a logarithmic scale.

[0150] FIG. 4—Apixaban inhibits the conversion of substrate MePEG2-IEGR. FXa (4 nM) in the presence of 100 pg/mL to 100 mg/mL Apixaban reveals inhibition in the 10-1000 ng/ml range.

[0151] FIG. 5—FVIII activity in a sample was determined based on FXa activity by providing a reaction mixture comprising excess components of cascade participants but lacking FVIII. Luminescence as caused by FXa activity thus becomes a measure of FVIII activity; A) RLU measured at different FVIII concentrations expressed as percentage of normal pooled plasma; B) calibration line for the results in panel A. The calibration has a linear regression of y=30331+4376x and r2=0.9935.

[0152] FIG. 6—FIX activity in a sample was determined based on FXa activity by providing a reaction mixture comprising excess components of cascade participants but lacking FIX. Luminescence as caused by FXa activity thus becomes a measure of FIX activity; A) RLU measured at different FIX concentrations expressed as percentage of normal pooled plasma; B) calibration line for the results in panel A. The calibration has a linear regression of y=97471+8547x and r.sup.2=0.9872.

[0153] FIG. 7—FXa activation in a global hemostasis assay using high and low tissue factor concentration (7 μM or 0.28 μM).

[0154] FIG. 8—A) Raw data measured kinetically with a chemiluminescent reader. B) This example uses a fixed time point, in this case 3 minutes, to construct a calibration curve. C) This example calculates the slope between 0.5-3 minutes of each calibrator to construct the calibration curve.

[0155] FIG. 9-13 A) An overlay of 4 calibration curves utilized to measure 31 FVIII clinical samples. The average R.sup.2 value of these four calibration curves combined was 0.9810. B) Recovery of the clinical samples in both the chemiluminescent-based assay as well as the chromogenic FVIII assay using samples containing a recombinant PEGylated FVIII product. C) As for B), but using samples containing a recombinant full-length FVIII product.

[0156] FIG. 10-13 A) Raw data measured kinetically with a chemiluminescent reader with the calibrators containing various FIX levels. B) Constructed calibration curve.

EXAMPLES

Example 1

Provision of Substrates According to the Invention

[0157] As shown in FIG. 1A, substrates were synthesized via key intermediate A1. The synthesis of this intermediate started with the conversion of benzothiazole 1 towards nitrile 2 in 91% yield. Reduction afforded pure aniline 3 in 54% yield, after straight phase and reversed phase flash column chromatography. The coupling with Fmoc-Orn(Boc)-OH (4) was performed with PCI.sub.3 in pyridine. After Fmoc-deprotection compound 6 was obtained successfully. Peptide coupling of 6 with Fmoc-Gly-OH 7 towards 8 and Boc-deprotection gave 9 in 84% yield over these two steps. The reaction with 1,3-bis(tert-butoxycarbonyI)-2-(trifluoromethanesulfonyl)guanidine gave guanidine 10 in good yield and purity after purification by flash column chromatography. For provision of compounds of general formula I-3 or I-4 wherein other amino acids are present, the above protocol is repeated with said other amino acids, or compound 8 is not converted into compound 10.

[0158] As shown in FIG. 1B, Fmoc-deprotection and immediate further coupling with Fmoc-Glu(OtBu)-OH 12 gave 13 in 72% yield over 2 steps. Repeating this sequence, but this time using Fmoc-Ile-OH 15, gave tetrapeptide 16 in 78% yield over 2 steps. Fmoc-deprotection to key building block Al went in 95% yield. For provision of compounds of general formula I-3 or I-4 wherein other amino acids are present, the above protocol is repeated with said other amino acids.

[0159] As shown in FIG. 1C, the synthesis was continued with the peptide coupling of A1 with [2-(2-methoxyethoxy)ethoxy]acetic acid 17 towards 18 in 93% yield. For other moieties X similar couplings can be performed using the appropriate reagents. The coupling with D-cysteine 19 gave aminoluciferin 20 in 76% yield after purification by reversed phase preparative chromatography. The removal of the protecting groups was performed by using trifluoroacetic acid, after which the product, designated MePEG2-IEGR, was isolated by trituration in diethyl ether.

Example 2

Kinetic Behaviour of Substrates According to the Invention

[0160] Michaelis-Menten kinetics were determined for the enzyme FXa and its substrate MePEG2-IEGR (the TFA salt was used in these examples). The following composition was prepared in a microvial on ice:

Composition 1:

[0161] 160 μL Tris buffered saline (TBS) 50 mM Tris-HCI, 150 mM NaCI (pH 7.4)

[0162] 4 μL ATP (final concentration 333 μM)

[0163] 4 μL MgC.sub.2 (final concentration 8.3 mM)

[0164] 12 μL Luciferase (final concentration 0.9 mg/ml)

Substrate compositions 2a-g:

[0165] Microvials were prepared with 30 μL substrate concentrations leading to final concentrations of 2000, 1333, 1000, 666, 333, 167 and 66 μM.

Enzyme composition 3:

[0166] FXa (Coachrom, 16 nM) composition was prepared in a microvial on ice

Subsequently, the following compositions were added together in a white 384-well plate:

[0167] 24 μL Composition 1

[0168] 3 μL Composition 2a-g

[0169] 3 μL Composition 3

Subsequently the well plate was placed in a 37 ° C. thermostated Flexstation 3 (Molecular Devices). Luminescence emission wavelength was with an integration time of 1500 ms, measured for 1s every 30 s during 30 min.
Results are shown in FIG. 2. At an FXa concentration of 1.6 nM a V.sub.max=468000 RLU and a K.sub.M=619 μM were determined, leading to a K.sub.cat=V.sub.max/[FXa]=1.05×10.sup.14 s.sup.−1.

Example 3

Cross Reactivity

[0170] To determine cross-reactivity an assay was performed using a range of coagulation enzymes at representative concentrations in human plasma. The chosen concentrations of the enzymes are comparable to the expected concentrations in vivo after activation. For tissue-type plasminogen activator (tPA) this is the conventional concentration for facilitation of fibrinolysis.
A concentration of 80 nm FXa yielded a signal of 1.000.000 RLU. Other coagulation enzymes resulted in the following signals: 52 nM thrombin yielded ˜30.000 RLU, 8 nM plasmin yielded ˜8.000 RLU, 193 IU/mL tPA yielded ˜6.000 RLU and 145 nM FXlla yielded ˜500 RLU (FIG. 3). These data indicate that the substrate according to the invention is preferentially cleaved by FXa.

Example 4

Quantification of Anticoagulant Activity

[0171] As an example of an anticoagulant, apixaban (CAS Number 503612-47-3) was added at different concentrations into a mixture of FXa and MePEG2-IEGR. Apixaban is an anticoagulant for the treatment of venous thromboembolic events, to be taken orally. It is a direct FXa inhibitor. FIG. 4 shows the inhibition of apixaban on the conversion of the substrate by FXa (4 nM) in the presence of 100 pg/mL up to 100 mg/mL apixaban. Inhibiting effect is shown on FXa activity in the 10-1000 ng/ml range.

Example 5

Quantitative FVIII Assay

[0172] This luminescent FVIII assay is a two-step activity assay that leads to activation of a fixed amount of FVIII from a sample, which subsequently leads to FX activation and thus to stable conversion of the substrate according to the invention. Luminescent factor assays lead to stable substrate conversion due to the fast decay time of the photons, whereas chromogenic or fluorescent substrates accumulate signal (at e.g. 405 nm), leading to increased signal over time (OD/min). For the latter assays the slope must be calculated to determine conversion rate. In luminescent assays the flat output (in RLU) directly indicates the conversion rate, which makes the methods of the present invention very convenient.
In the assay thrombin is added to activate FVIII. Activated Factor VIII forms an enzymatic complex with Factor IXa, phospholipids (PLPs), and calcium. This complex activates Factor X to FXa; FX is supplied in the assay at a constant concentration and in excess. FXa acts on the substrates according to the invention to liberate a substrate for luciferase, leading to luminescent activity. The luminescent activity is thus directly related to the amount of Factor VIII activity, which is the limiting factor in the presence of a constant amount of Factor IXa. Generated Factor Xa is then exactly measured by its activity on the specific Factor Xa luminescent substrate of the invention. Factor Xa cleaves the substrate and leads to release of a photon. The amount of photons generated (expressed as Relative Light Units, RLU) are directly proportional to the Factor Xa activity and consequently the amount of Factor VIII. Chromogenic analogues of such assays are commercially available (for example BIOPHEN Factor VIII assay from Hyphen Biomed, Catalogue Ref. 221402). An important difference with this example is that the substrate according to the invention is used with ATP, Mg.sup.2+, and luciferase, instead of a chromogenic substrate.
Different amounts of FVIII (using normal pooled plasma (NPP) or dilutions thereof) were used in the assay. The samples were mixed with a solution of FX. After about 2 minutes of incubation at 37° C. a solution of FIXa, thrombin, calcium, and phospholipids in distilled water was added, after which the reaction was incubated for about 3 minutes at 37° C. Then MePEG2-IEGR (in this case the TFA salt) was added as substrate for FXa, after which luminescence was determined using a conventional mixture of luciferase, ATP, and Mg.sup.2+. FIG. 5A shows the RLU of FXa activity dependent on the FVIII activity present in the sample, with added FVIII expressed as NPP percentage. FIG. 5B shows the concomitant calibration line. The calibration has a linear regression of y=30331+4376 x and r.sup.2=0.9935.

Example 5.1

Detailed FVIII Assay

Reagents

Reagent 1 (R1):

[0173] Diluted plasma samples used for testing or for the calibration curve. Samples are diluted with buffer containing 50 mM imidazole and 100 mM NaCI (pH 7.4).

Reagent 2 (R2):

[0174] Human Factor IXa, human thrombin, calcium, Gly-Pro-Arg-Pro as fibrin polymerization inhibitor and synthetic phospholipids (28% phosphatidylserine). The phospholipids and calcium are stored at 4-8° C. Other components are stored at −80° C. The reagent mixture is prepared in buffer containing 50 mM imidazole and 100 mM NaCI (pH 7.4).

Reagent 3 (R3):

[0175] Human Factor X, ATP, magnesium, luminescent substrate specific for Factor Xa (in this example, MePEG2-IEGR was used) and the recombinant Luciferase Ultra-Glo from Promega. The luciferase can be exchanged for the QuantiLum luciferase, both types can be applied. Magnesium is stored at 4-8° C. The individual components are stored at −80° C. in aqueous solution. The reagent mixture is prepared in buffer containing 50 mM imidazole and 100 mM NaCI (pH 7.4).

Preparation Calibration Curve Samples

[0176] Table 5 shows the preparation of calibration samples for the FVIII assay. For the preparation of the calibration curve, the following diluted plasma samples are mixed as is shown in table 5. In the 384-well assay plate, 3 μL of the diluted sample or calibrator is used for the 10 μL test volume. Unknown specimens are prepared the same as the 100% samples.

TABLE-US-00006 TABLE 5 Stock solution Working solution Start Volume Final conc. Volume FVIIIdef Total Volume Volume Total conc. Cali- (FVIII start plasma volume stock buffer volume (FVIII brator %) (μL) (μL) (μL) Dilution (μL) (μL).sup.* (μL) %) C1 100 20 0 20 7/30 3.5 11.5 15 100 C2 100 10 10 20 7/30 3.5 11.5 15 50 C3 50 10 10 20 7/30 3.5 11.5 15 25 C4 25 10 10 20 7/30 3.5 11.5 15 12.5 C5 12.5 10 10 20 7/30 3.5 11.5 15 6.25 C6 6.25 10 10 20 7/30 3.5 11.5 15 3.125 C7 3.125 10 90 100 7/30 3.5 11.5 15 0.3 .sup.*buffer is 50 mM imidazole and 100 mM NaCl.

Preparation Protein Reagents

[0177] Tables 6 and 7 describe the preparation of reagent 2 or 3, respectively. These volumes represent one reaction or one sample in a 384 wells plate with a final test volume of 10 μL or 30 μL. The final concentration in each well is described in table 8.

TABLE-US-00007 TABLE 6 Volume in Volume in Component Stock conc. 10 μL (μL) 30 μL (μL) FIXa (human)   274 nM 0.25 0.75 Phospholipids  0.5 mM 0.2 0.6 Gly-Pro-Arg-Pro  11.8 mM 0.675 2.025 Calcium 162.2 nM 0.375 1.125 Thrombin (human)   114 nM 0.06 0.18 Imidazole NaCl buffer   50 mM 1.44 4.32 100 mM Total 3 9

TABLE-US-00008 TABLE 7 Volume in Volume in Component Stock conc. 10 μL (μL) 30 μL (μL) FX (human)  4.8 μM 0.083 0.25 ATP   20 mM 0.85 2.55 Magnesium  500 mM 0.083 0.25 luciferin substrate   10 mM 1 3 rLuciferase (Promega) 12.5 mg/mL 0.483 1.45 Imidazole NaCl buffer   50 mM 1.5 4.5 100 mM Total 4 12

TABLE-US-00009 TABLE 8 Component Final concentration FIXa (human) 6.85 nM Phospholipids   10 μM Gly-Pro-Arg-Pro  0.8 mM Calcium   6 mM Thrombin (human)  0.7 nM FX (human)   40 nM ATP  1.7 mM Magnesium  4.2 mM Luciferin substrate   1 mM rLuciferase (Promega)  0.6 mg/mL

Procedure

[0178] The test is performed using a chemiluminescent reader at 37° C. The three mixtures are separately dispensed in a 384-wells microplate that was incubated at 37° C. Table 9 shows the division of only one well. During the test, the reactants are mixed and Relative Light Units (RLU) intensity is measured kinetically during 20 minutes. How to handle the final results is explained below.

TABLE-US-00010 TABLE 9 Volume in well 10 μL (μL) Volume in well 30 μL (μL) Reactant 1 3  9 Reactant 2 3  9 Reactant 3 4 12

Calibration Curve

[0179] The chemiluminescence-based quantitative FVIII assay can be calibrated to analyze Factor VIII. The assay covers a dynamic range as shown in table 5. There are at least two methods to construct the calibration curve using the kinetic data. The first option is to set a time point after observing the raw data. A very suitable time point is considered when the curves of the individual calibrator samples form a plateau. The second option is determining the slope between approximately 0.5 to 3 minutes. The calibration curve is plotted log-log for both methods.
The calibration curve shown in the figures is obtained on a Flex3 Station (molecular devices, USA) in a final test volume of 10 pL. Based on this figure (raw data) the two options mentioned above were used to construct the final calibration curve.

[0180] An earlier version of this quantitative assay was mostly performed in 30 pL and with the QuantiLum luciferase (Promega). The latter is an import note considering this luciferase does not achieve the same intensity in the same environment as the Ultra-Glo luciferase which was used for the 10 pL structure. Therefore, the FIGS. 1-3 and 4-6 cannot be compared one on one. FIGS. 4 to 6 visualizes the results obtained with the 30 pL assay structure. Validation of this assay was performed with clinical samples of 31 hemophilia patients receiving FVIII replacement treatment. Twenty-two samples contained a recombinant PEGylated FVIII product, three of which were assigned lower than 1% FVIII and where recovered as lower than 1% FVIII. Ten samples contained a recombinant full-length FVIII product, one of which was assigned lower than 1% FVIII and was recovered as lower than 1% FVIII. These samples were measured both with a golden standard test as well as the luminescent-based FVIII assay. The golden standard assay utilized for the validation was a chromogenic FVIII assay containing bovine factors.

Example 6

Quantitative FIX Assay

[0181] Using the same strategy as in Example 5 a luminescent FIX assay was designed. The assay design is comparable to the FVIII assay described above, but more specifically sensitized to FIX by providing an excess amount of FXIa instead of FIXa. FIX is a zymogen that can be cleaved by FXIa to produce FIXa. In the presence of Ca.sup.2+, membrane phospholipids, and FVIIIa, FIXa hydrolyses FX to form FXa. In the reaction mixture the factors of this cascade, except FIX which is to be assayed, were present in excess. The amount of FIX added to the reaction mixture thus leads to a correlated amount of FXa, which in turn leads to the detectable luminescence. Chromogenic analogues of such assays are commercially available (for example BIOPHEN Factor IX assay from Hyphen Biomed, Catalogue Ref. 221802). An important difference with this example is that the substrate according to the invention is used, together with Mg.sup.2+ and ATP and luciferin, instead of a chromogenic substrate.
Different amounts of FIX (using NPP or dilutions thereof) were added to a reaction mixture, after which luminescence was determined. FIG. 6A shows the RLU of FXa activity dependent on the FIX activity present in the sample, with added FIX expressed as NPP percentage. FIG. 6B shows the concomitant calibration line. The calibration has a linear regression of y=97471+8547x and r.sup.2=0.9872.

Example 6.1

Detailed FIX Assay

Reagents

Reagent 4 (R4):

[0182] Diluted plasma samples used for testing or for the calibration curve. Samples are diluted in buffer containing 50 mM imidazole and 100 mM NaCI buffer (pH 7.4).

Reagent 5 (R5):

[0183] Recombinant human Factor VIII, human Factor Xla, human thrombin, synthetic phospholipids (28%), calcium and Gly-Pro-Arg-Pro as fibrin polymerization inhibitor . The individual components are stored at −80° C. in aqueous solution. The reagent mixture is prepared in buffer containing 50 mM imidazole and 100 mM NaCI buffer (pH 7.4).

Reagent 6 (R6):

[0184] Human Factor X, ATP, magnesium, luminescent substrate specific for Factor Xa (in this example, MePEG2-IEGR was used) and the recombinant Luciferase Quantilum from Promega. The luciferase can be exchanged for the Ultra-Glo luciferase, both types can be applied. Magnesium is stored at 4-8° C. The individual components are stored at −80° C. in aqueous solution. The reagent mixture is prepared in buffer containing 50 mM imidazole and 100 mM NaCI (pH 7.4).

Preparation Calibration Curve Samples

[0185] For the preparation of the calibration curve, the following diluted plasma samples are mixed as is shown in table 6 In the 384-well assay plate, 6 μL of the diluted sample or calibrator is used for the 30 μL test volume. Unknown specimens are prepared the same as the 100% samples.

TABLE-US-00011 TABLE 6 Stock solution Start Volume Working solution conc. Volume FVIIIdef Total Volume Volume Total Final Cali- (FVIII start plasma volume stock buffer volume conc. brator %) (μL) (μL) (μL) Dilution (μL) (μL).sup.* (μL) (FVIII %) C1 100 40 0 40 7/60 7 53 60 100 C2 100 20 20 40 7/60 7 53 60 50 C3 50 20 20 40 7/60 7 53 60 25 C4 25 20 20 40 7/60 7 53 60 12.5 C5 12.5 20 20 40 7/60 7 53 60 6.25 C6 6.25 20 20 40 7/60 7 53 60 3.125 C7 3.125 10 90 100 7/60 7 53 60 0.3 .sup.*buffer is 50 mM imidazole and 100 mM NaCl.

Preparation Protein Reagents

[0186] Table 7and 8 show methods for preparing the reagents. These volumes represent 1 sample or 1 well in a 384 wells plate with a final test volume of 30 μL. The final concentration of each component in one sample is shown in table 9.

TABLE-US-00012 TABLE 7 Component Stock conc. Volume in 30 μL (μL) Recombinant FVIII   12 IU/mL 3 FXIa (human)   317 nM 0.15 Thrombin (human)   114 nM 0.26 Phospholipids  0.5 mM 0.6 Calcium 162.2 mM 1.1 GPRP  11.8 mM 2 Imidazole NaCl buffer   50 mM 100 mM 4.89 Total 12

TABLE-US-00013 TABLE 8 Component Stock conc. Volume in 30 μL (μL) FX (human)  4.8 μM 0.25 ATP   20 mM 2.55 Magnesium  500 mM 0.25 luciferin substrate   10 mM 3 rLuciferase (Promega) 13.8 mg/mL 1.5 Imidazole NaCl buffer   50 mM 100 mM 4.45 Total 12

TABLE-US-00014 TABLE 9 Final concentration of each component in one sample Final Component concentration Recombinant FVIII  1.2 IU/mL FX (human)   40 nM Luciferin substrate   1 mM FXIa (human) 1.56 nM Phospholipids   10 μM Calcium   6 mM Thrombin (human)   1 nM ATP  1.7 mM Magnesium  4.2 mM rLuciferase  0.7 mg/mL (Promega)

Procedure

[0187] The test is performed using a chemiluminescent reader at 37° C. The four mixtures are separately dispensed in a 384-wells microplate that was incubated at 37° C. Table 10 shows the division of only one well. During the test, the Relative Light Units (RLU) intensity is measured kinetically.

TABLE-US-00015 TABLE 10 Volume in well 30 μL (μL) Reagent 4  6 Reagent 5 12 Reagent 6 12 Mix and measure the RLU intensity for 20 minutes

Calibration Curve

[0188] The FIX luminescent test can be calibrated for the assay of Factor IX or therapeutic concentrates. The plasma calibrators covering the suggested dynamic range are shown in table 6 and can be used to establish acalibration curve. First the raw data is visually assessed where after a suitable time point is selected to configure the calibration curves with. The calibration curve is plotted log-log.
The calibration curve shown in the figures is obtained on a Flex3 Station (molecular devices, USA). Based on this figure (raw data) the two options mentioned above were used to construct the final calibration curve.

Example 7

FXa Generation Assay

[0189] The substrate was also used in a global hemostasis assay, where FXa is generated in platelet poor plasma by activation of FX in the coagulation process upon initiation by tissue factor, phospholipids, and calcium. The FXa generation assay is initiated by addition of tissue factor and calcium to plasma, leading to FXa generation followed by thrombin generation and subsequent clot formation. FXa generation and its subsequent inhibition by physiological inhibitors leads to a bell-shaped curve (FIG. 7). Different doses of tissue factor were used: 7 μM or 0.28 μM. Measuring the FXa concentration in such a global design gives information about the capacity of the coagulation cascade at the level of factor Xa.