METHOD FOR THE DETERMINATION OF PROTEIN S LEVELS

Abstract

The present invention provides an in vitro method for the assessment of functional protein S levels in a sample. The present invention also provides kits for use in the determination of functional protein S levels in a sample. Also provided is a method of treatment based on the determination of functional protein S levels, followed by administration of a therapeutic agent.

Claims

1. An in vitro method for the determination of functional protein S levels in a sample, the method comprising or consisting of the steps of: (a) contacting a sample obtained from a subject with TFPIα and one or more of: FV-short; or an FV-short variant; or a functionally-equivalent FV-variant; (b) contacting the sample with FXa; and (c) measuring the level of FXa activity in the sample wherein the level of FXa activity is indicative of the level of functional protein S in the sample.

2. The method according to claim 1, wherein the functional protein S level is the level of activity of protein S in the sample, optionally wherein the activity is the ability of the protein S to function as a cofactor for TFPIα.

3. The method according to claim 1, wherein the functional protein S level is the amount of functional protein S in the sample.

4. The method according to claim 1, wherein the functional protein S in the sample is the free protein S in the sample.

5. The method according to claim 1, wherein the functional protein S in the sample is the non-C4BP complexed protein S in the sample.

6. The method according to claim 1, wherein step (a) further comprises contacting the sample with a substrate capable of allowing protein assembly.

7. The method according to claim 6, wherein the substrate capable of allowing protein assembly is phospholipid vesicles.

8. The method according to claim 1, wherein step (a) further comprises calcium.

9. The method according to claim 1, further comprising or consisting of the steps of; (d) providing a standard curve based on functional protein S; and (e) comparing the measurement of step (c) to the standard curve of step (d).

10. The method according to claim 9, wherein the standard curve is generated using plasma samples obtained from healthy individuals, or using known amounts purified protein S, or using a media solution containing a defined amount of protein S.

11. The method according to claim 1, wherein the level of FXa activity measured is indicative of the inhibition of FXa, and wherein the level of inhibition of FXa is indicative of the level of functional protein S in the sample, optionally the level of functional protein S activity in the sample.

12. The method according to claim 1, wherein the sample is plasma, optionally wherein the sample is citrated plasma.

13. The method according to claim 1, wherein the sample has a high dilution factor, for example wherein the dilution factor is between 1/10 and 1/2000, optionally wherein the dilution factor is between 1/50 and 1/400.

14. The method according to claim 1, wherein step (a) further comprises contacting the sample with C4BP.

15. The method according to claim 1, wherein step (b) further comprises contacting the sample with a component capable of emitting a measurable signal in the presence of FXa.

16. The method according to claim 11, wherein the measurable signal emitted in the presence of FXa is fluorescence or colour, optionally wherein the component selected from: S2765, S-2222.

17. The method according to claim 16, wherein the component capable of emitting a measurable signal is S2765, and the concentration of S2765 is between 0.1 to 2 mM, preferably between 0.3 to 1 mM, preferably wherein the concentration of S2765 is 0.8 mM.

18. The method according to claim 1, wherein step (a) and/or step (b) further comprises contacting the sample with a thrombin inhibitor.

19. The method according to claim 18, wherein the thrombin inhibitor is hirudin or Pefa-block.

20. The method according to claim 1, wherein the FV-Short variant or FV-variant is a variant which is resistant to activation by thrombin.

21. The method according to claim 20, wherein the FV short variant or FV-variant contains thrombin cleavage sites which are mutated from Arginine to Glutamine, optionally wherein the FV-short variant is selected from FV-short QQ, FV-short RQ and FV-short QR.

22. The method according to claim 1, wherein the FV-short variant or FV variant is a variant with enhanced or synergistic TFPIα cofactor activity.

23. The method according to claim 22, wherein the FV-short variant is FV-709-1476.

24. The method according to claim 1, wherein the FV-short variant or FV variant is a variant which retains the acidic C-terminal region of the B-domain.

25. The method according to claim 1, wherein the FV-variant or FV-short variant is capable of being cleaved by thrombin at positions 709 and 1018 and/or is not capable of being cleaved by thrombin at position 1545.

26. The method according to claim 25, wherein the FV-variant is FV-1545Q.

27. The method according to claim 1, wherein the method is specific for the free form of protein S.

28. The method according to claim 1, wherein the method is capable of detecting protein S deficiency.

29. The method according to claim 1, for identifying whether the subject is a patient with a protein S deficiency

30. A method of diagnosing a subject as having a protein S deficiency wherein the method comprises determining the level of protein S, optionally level of protein S activity, according to claim 1.

31. The method according to claim 30, wherein the protein S deficiency is a type I protein S deficiency.

32. The method according to claim 30, wherein the protein S deficiency is a type II protein S deficiency.

33. The method according to claim 32, wherein the protein S deficiency is a type II protein S deficiency with defective TFPIα cofactor activity.

34. The method according to claim 30, wherein the protein S deficiency is a type III protein S deficiency.

35. The method according to claim 30, wherein the protein S deficiency is a heterozygous or homozygous protein S deficiency.

36. The method according to claim 30, wherein the protein S deficiency is acquired.

37. The method according to claim 30, wherein the subject is a patient being treated with warfarin.

38. The method according to claim 30, wherein the subject is a patient diagnosed with, or suspected of having, venous thrombo-embolic disease (VTE).

39. The method according to claim 1, wherein the method is capable of detecting low levels of protein S for example wherein the levels of protein S are between 0.1 and 5 nM in a diluted sample, optionally wherein the protein S levels are <3 nM in a diluted sample.

40. The method according to claim 1, wherein the concentration of the FV-Short or FV-Short variant or FV variant is between 0.5 to 20 nM, optionally wherein the concentration is 2 nM.

41. The method according to claim 1, wherein step (a) takes place at 37° C. for between 1 and 15 minutes, optionally wherein the time is 10 minutes.

42. The method according to claim 1, wherein step (c) takes place for between 10 and 30 minutes, optionally wherein the time is 15 minutes.

43. The method according to claim 1, wherein the sample is diluted in a buffer, optionally wherein the buffer has a pKa between 7 and 8 and is compatible with Ca2+.

44. The method according to claim 43, wherein the buffer is HNBSACa2+ buffer or BSA buffer.

45. The method according to claim 1, wherein the ratio of TFPI alpha to FXa is approximately 1:1.

46. The method according to claim 1, wherein the concentration of FXa is between 0.1 to 1 nM, optionally wherein the concentration is between 0.2 to 0.6 nM, optionally wherein the concentration is 0.3 nM.

47. The method according to claim 1, wherein the method is performed in conjunction with a method to determine total protein S levels.

48. The method according to claim 1, wherein the method does not comprise a thrombin generation assay.

49. A method of treatment comprising identifying a subject with protein S deficiency using a method according to claim 1, and administering to said subject a therapeutic agent.

50. A method according to claim 49, wherein the therapeutic agent is an anticoagulant therapy.

51. A therapeutic agent for use in treating a subject with a protein S deficiency, wherein the subject has been diagnosed as having a protein S deficiency according to claim 30, optionally wherein the therapeutic agent is an anticoagulant.

52. A kit for determining functional protein S levels in a sample, comprising any two or more of: FV-short (or an FV-short variant; or a functionally-equivalent FV-variant); TFPIα; FXa; phospholipid vesicles; and an FXa substrate, optionally wherein the FXa substrate is S2765.

53. A kit for determining functional protein S levels in sample using a method according to claim 1.

Description

[0176] Preferred, non-limiting examples which embody certain aspects of the invention will now be described, with reference to the following figures:

[0177] FIG. 1 Effect of protein S as cofactor to TFPIα in inhibition of FXa. FV-Short (2 nM) was incubated with FXa (0.3 nM), 25 uM PL (20/20/60:PS/PE/PC), TFPIα (0.25 nM) in the presence of different concentrations of protein S (above) or dilutions of plasma (below). The substrate (S-2765) conversion was monitored for 900 seconds and a gradually decreased slope of the curve indicates inhibition of FXa amidolytic activity.

[0178] FIG. 2 Specificity for protein S as cofactor to TFPIα in inhibition of FXa. The TFPIα-mediated inhibition of FXa activity was followed as described in methods and in FIG. 1 legend. In this experiment, mixtures of normal and protein S deficient plasma was tested at a 1/100 dilution. At this dilution, normal plasma yields maximum FXa inhibition whereas protein S-deficient plasma has no activity. A dose-dependent effect is observed with increasing ratio of normal/protein S-deficient plasma.

[0179] FIG. 3 Binding of C4BP to protein S results in loss of TFPIα-cofactor activity. The TFPIα-mediated inhibition of FXa (0.3 nM) by TFPIα (0.25 nM) in the presence of FV-Short (2 nM) and protein S (5 nM) was maximum resulting in a low absorbance curve. The addition of increasing concentrations of C4BP, which binds to protein S with high affinity, resulted in a dose-dependent loss of cofactor activity of protein S, at 3.13 nM C4BP much of the cofactor effect of protein S was gone and at 6.25 nM the cofactor activity was completely blocked.

[0180] FIG. 4 Protein S deficient plasma contains little TFPIα-cofactor activity. Inhibition of FXa by TFPIα (0.25 nM) in the presence of FV-Short (2 nM) and dilutions of plasma from either controls (E, F, G and H) or protein S-deficient individuals (A, B, C and D) was monitored with synthetic substrate S-2765

[0181] FIG. 5 Example of standard curve for TFPIα cofactor activity of protein S in plasma. The FXa inhibition by TFPIα plus FV-Short in the presence of different dilutions of normal plasma as source of protein S was monitored for 900 seconds. The final absorbance was used to construct a standard curve where 100% represents the 1/200 dilution, 50% the 1/400 dilution etc.

[0182] FIG. 6 Functional protein S levels in plasma of protein S deficient patients and controls. The results of the assay for TFPIα cofactor activity of plasma protein S from controls and individuals with protein S deficiency demonstrated good separation in protein S values between those with protein S deficiency (left) and those without protein S deficiency (right).

[0183] FIG. 7 Correlation between old test for free protein S and the new functional assay. The results of the new test were compared with those previously determined in an immunological free protein S assay.

[0184] FIG. 8 Correlation between old test for total protein S and the new functional assay. The results of the new test were compared with those previously determined in an immunological total protein S assay.

[0185] FIG. 9 shows a comparison of FV short and FV-short variants. The concentration of the FV-variants are varied whereas the protein S (3 nM), TFPIα (0.25 nM) and FXa (0.3 nM) are constant. The values plotted here are the absorbances reached after 900 seconds.

[0186] FIG. 10 shows the time curves of substrate development with the three mutants. the concentration of the FV-variants is 1 nM, the FXa (0.3 nM) and TFPIα (0.25 nM) are constant as is the FV-variant concentration. The protein S concentration is varied.

[0187] FIG. 11 shows a comparison of FV-short and FV 709-1476, using plasma dilutions as the source of protein S.

[0188] FIG. 12 shows protein S titrations of FV-Short, FV-Short 1545Q, thrombin cleaved FV-Short 1545Q and thrombin cleaved FV-1545Q, all with TFPIα.

EXAMPLES

Example 1—Assay for Functional Protein S

Summary

[0189] The TFPIα-mediated inhibition of FXa in the presence of FV-Short, protein S and negatively charged phospholipid vesicles was monitored in time by synthetic substrate S2765. Diluted plasma was used as source for protein S and a standard curve was constructed using plasma dilutions

[0190] Materials and Methods

[0191] Patients—Individual citrated plasma samples (n=36) from different protein S-deficient families previously characterized were available in the laboratory[26]. Four individuals treated with warfarin with other inherited anticoagulant protein deficiencies were also available; three protein C deficiencies and one antithrombin deficiency. The samples had been stored at −80° C. since the time of collection during the 90-ties. Samples from selected from healthy family (n=37) members with no protein S deficiency or history of thrombosis were used as controls. The values for the plasma concentrations of total and free protein S were available from the published study[26]. The method to determine the free and total protein S in the samples was described previously[27]. A citrated plasma pool collected from healthy individuals was used to create the standard curve, setting the functional protein S concentration in the pool to be 100%.

[0192] Materials—Human FXa was from Hematologic Technologies, Inc (HTI); Protein S-deficient plasma was from Emzyme Research Laboratories; TFPIα expressed in eukaryotic cells was a gift from Dr T Hamuro at the Chemo-Sero-Therapeutic Research Institute, Japan. FV-Short was expressed and purified as previously described[25]-FV709-1476[28] and FV-810[29] was expressed and purified with similar technique. Phosphatidylserine (PS), phosphatidyl ethanolamine (PE) and phosphatidyl choline (PC) were from Avanti Polar Lipids. Phospholipid vesicles were prepared using the LiposoFast basic extruder (Armatis, Germany) as previously described[30]. The phospholipid vesicles were used within 2 days. Synthetic substrate S2765 was provided by Chromogenix Ltd, Milan, Italy. C4BP without bound protein S was purified as described[31].

[0193] Assay for protein S-mediated TFPIα-cofactor activity—The assay is based on the inhibition of FXa by TFPIα in a purified system using a technique we previously described[25]. In this assay, FV-Short (2 nM final concentration) was incubated for 10 minutes at 37° C. with phospholipid (20:20:60 of PS:PE:PC, 25 uM), TFPIα (0.25 nM final concentration), plasma diluted in HNBSACa.sup.2+ buffer (25 Hepes, 0.15 M NaCl, 5 mM CaCl.sub.2) pH 7.7, containing 0.5 mg/ml bovine serum albumin and 5 Units/ml of Hirudin) as source of protein S. The reaction was initiated by addition of S2765 (0.8 mM) and FXa (0.3 nM) and followed for 15 minutes by monitoring absorbance at 405 nM in a Tecan Infinite 200 system. The concentrations given in each experiment are the final concentrations.

[0194] Inhibition of protein S function as TFPIα cofactor by C4BP. To investigate whether both free and C4BP-bound protein S functions as TFPIα cofactor, increasing concentrations of purified C4BP (0-50 nM) were added in a FXa inhibition assay containing 5 nM protein S.

[0195] Results

[0196] The inhibition of FXa by TFPIα in the presence of negatively charged phospholipid vesicles, FV-Short and either purified protein S or diluted pooled plasma was followed in time (FIG. 1). In the absence of protein S or plasma the absorbance curve was almost linear suggesting that there was very little inhibition of FXa by TFPIα in the presence of FV-Short alone. Addition of increasing concentrations of protein S gave a dose-dependent inhibition of FXa with 50% inhibition at 1.25 nM and maximal inhibition at 5 nM protein S. Similarly, the inclusion of diluted plasma in the assay instead of protein S yielded a dose-dependent inhibition with maximum inhibition observed at 1/50 dilution and around 50% inhibition at 1/200. The TFPIα-cofactor effect of protein S in the assay depended on the presence of FV-Short and in the absence of added FV-Short, neither protein S (up to 10 nM) nor plasma (up to 1/100 dilution) yielded any stimulation of FXa inhibition.

[0197] The assay was specific for protein S because addition of protein S-deficient plasma gave no stimulation of FXa inhibition (FIG. 2). Mixtures of normal and protein S deficient plasma were included in the assay at a 1/100 dilution. Increasing the ratio of normal/protein S-deficient plasma resulted in a dose-dependent increased FXa inhibition and close to 50% inhibition was observed at the 3:7 ratio, which provides around the same amount of protein S to the assay as the 1/200 dilution of normal plasma seen in FIG. 1.

[0198] To investigate whether both free and C4BP-complexed forms of protein were active as TFPIα cofactors, increasing concentrations of C4BP (0-50 nM) were included in reactions containing 5 nM protein S (FIG. 3). In the absence of added C4BP, the protein S-mediated stimulation of FXa inhibition was maximal. Addition of increasing concentrations of C4BP yielded a dose-dependent increased absorbance suggesting blockage of the protein S TFPIα-cofactor activity and at 6.25 nM C4BP, no protein S effect was observed. This suggests that the formation of the 1:1 stoichiometric complex of protein S and C4BP results in loss of the TFPIα-cofactor activity.

[0199] A cohort of 36 patients with known inherited protein S deficiency and 37 age and sex matched healthy controls identified from previous family studies was tested in the assay. FIG. 4 illustrates the absorbance readings from four protein S-deficient individuals and four healthy controls. The plasma to be tested was diluted 1/50, 1/100, 1/200 and 1/400 to cover the range between normal concentrations of protein S to the low protein S levels in protein S deficiency.

[0200] The final readings at 900 seconds from an assay using diluted plasma as source of protein S was utilized to construct a standard curve for quantifying the protein S activity as a TFPIα cofactor (FIG. 5). As the 1/200 dilution yielded around 50% inhibition it was set to be 100%. Consequently, the 1/400 reading corresponded to 50% and the 1/100 dilution to 200%. The best absorbance reading range was between 0.1 and 0.25 and as both patients and controls were analyzed at several dilutions, it was possible to get readings within that range.

[0201] The results from the testing of the protein S-deficient individuals and the controls are illustrated in FIG. 6. There was a separation in functional protein S values between the patients and the controls. The mean±SD values of patients and controls were 35±20 and 120±25, respectively. The values for the patients ranged between 8 and 83 and those of controls between 85 and 186. The correlation between the two assays was high with an r-value of 0.93, the slope being 0.82 and the Y-intercept −4.6. This suggests that the assay is measuring the activity of the free form of protein S.

[0202] The functional protein S values were also correlated to the total protein S values (FIG. 8). The correlation (r-value of 0.88) was lower than that to free protein S. The slope was 0.44 and the Y-intercept 46. These results are in agreement with the conclusion that the synergistic TFPIα-cofactor activity is solely associated with the free form of protein S.

[0203] Four of the protein S deficient cases were treated with warfarin. The mean±SD of their functional protein S values was 10.9±4%; range 8.0-16.6%, which agrees well with the results of the free protein S assay (8.9±4%; range 3.2-12.9). Four patients with other inherited anticoagulant deficiencies (three protein C deficiencies and one antihrombin deficiency) were tested to elucidate the effect of warfarin treatment on cases with no protein S abnormality. The mean±SD functional protein S value of these cases was 63.0±23%; range 34.3-98.9, whereas the mean±SD free protein S value was 39.5±19.6%; range 22.6-67.7%. This suggests that the TFPIα-functional test is equally efficient to detect protein S deficiency also in warfarin-treated cases, as is the free protein S assay[26].

[0204] To estimate the intra assay variation of the new test, one normal and one protein S deficient case was analyzed nine times. The mean±SD of the normal case was 85.4±4.3%; range 77.2-92.4%. Corresponding values for the protein S deficient case was 47.8±5.4%; range 40.0-53.7%. Thus, the intra assay coefficient of variation for samples with normal protein S levels was 5.1%, whereas for samples with protein S deficiency it was 10.6%.

Discussion

[0205] Vitamin K-dependent protein S is a multi-functional plasma protein[2]. It is important as anticoagulant regulator of several reactions of blood coagulation. As cofactor to APC, it controls the activity of the cofactors in the tenase (FVIIIa) and the prothrombinse (FVa) complexes. In addition, it serves as a cofactor to TFPIα in the regulation of free FXa[2, 15, 32]. The recent observation that the TFPIα cofactor activity of protein S is stimulated by FV-Short and that FV-Short and protein S function in synergy has added to the complexity but does also provide an opportunity to devise an assay for the function of plasma protein S as TFPIα cofactor[3, 25]. We now report on the creation and characterization of such a functional protein S assay that is based on the rate of inhibition of FXa by TFPIα in the presence of FV-Short, protein S from plasma samples and negatively charged phospholipid vesicles.

[0206] The synergistic TFPIα-cofactor activity of protein S was strictly confined to the free form of protein S. This is consistent with the binding site for C4BP on protein S being located in the SHBG-like region of protein S, a region also known to interact with TFPIα[1, 2, 16, 33]. The APC-cofactor activity of protein S is also preferentially expressed by the free form of protein S and several regions in protein S, including the Gla-domain, the TSR, EGF-domains and the SHBG-like region, have been shown to be important for the APC-cofactor activity. As the TFPIα-mediated FXa-inhibitory reaction takes place on negatively charged phospholipids, the Gla domain of protein S is expected to be important for the TFPIα-cofactor activity of protein S. This is consistent with the low functional protein S levels of several non-protein S deficient warfarin-treated patients.

[0207] The C4BP binds protein S with high affinity, which explains why a decrease in plasma levels of protein S, e.g. in inherited protein S deficiency, preferentially is reflected in decreased levels of free protein S. Therefore, assays for free protein S are superior to those for total protein S for the diagnosis of protein S deficiency[26]. However, the assays for free protein S are not able to detect functional protein S deficiency. Assays for the APC-cofactor activity of protein S have been shown to detect cases with functional defects in the ACP-cofactor activity of protein S, so called type II protein S deficiencies. The now described assay for the function of protein S as TFPIα cofactor will be suitable for detecting not only the level of free protein S but also its functional activity as TFPIα cofactor and type II cases with defects in the TFPIα-cofactor function. Recently, another functional assay for protein S as TFPIα cofactor was described[34]. The assay is based on a TF-initiated thrombin-generation assay in which a fixed amount of TFPIα is added to mixtures of protein S-deficient and patient plasma. This assay is conceptually different from the one we now describe because it does not take advantage of the synergistic TFPIα-cofactor activity between protein S and FV-Short as the amount of any intrinsic FV-Short is too low in the assay. The authors found that the assay detected most cases of protein S deficiency but that the correlation to total and free protein S was relatively low.

[0208] The assay now described is specific for protein S as demonstrated by the lack of TFPIα-cofactor activity of protein S deficient plasma. The assay requires low concentrations (<3 nM) of protein S and a 1/100 dilution of normal plasma yields maximum TFPIα-cofactor activity in the presence of 2 nM FV-Short. Without the addition of the FV-Short, there is little or no TFPIα-cofactor activity at such dilutions of plasma. To detect very low levels of protein S in plasma from protein S deficient patients, lower dilution factor (1/25 or 1/50) can be used. Plasmas from previously characterized protein S deficient families were tested in the new assay and the results compared with the concentrations of both free and total protein S determined with immunological tests. The results of the new functional assay correlated well with the free protein S concentrations an r-value of 0.92. The correlation line had a Y-axis intercept of close to 0 and the slope of the line was slightly below 1. The correlation to total protein S was slightly lower with an r-value of 0.88. Interestingly, the Y-axis intercept was around 46% and the slope of the line was 0.44. The high intercept is consistent with the results showing that the TFPIα-cofactor assay is specific for free protein S.

[0209] The new functional test accurately detected the protein S-deficient cases with a good separation between those with and those without protein S deficiency. In four cases, the values were very close to the lower normal values. These four cases were from families where the protein S-deficient cases had relatively high free protein S levels. One such family is called family 18 in a previous publication and two of the borderline cases were from this family[26]. The other two borderline cases were from two other families with similar phenotype.

[0210] In conclusion, we now describe a new test for the TFPIα-cofactor activity of plasma protein S, which utilizes the recently described synergy between protein S and FV-Short. The test is specific for the TFPIα-cofactor activity of free protein S and discriminates between cases with inherited protein S deficiency and those with normal protein S level and activity. In addition, the test should be able to detect type II protein S deficiency having defective TFPIα-cofactor activity. Such patients are yet to be identified.

Example 2—Properties of FV-Variants and FV-Short Variants

[0211] There are several FV-Short variants that we have been characterized in terms of functional activity. Two such mutants are called FV-709-1476 and FV-810-1491 (as described herein). The FV-709-1476 has increased synergistic TFPIα-cofactor activity as compared to FV-Short (also referred to as FV-756-1458), whereas the FV-810-1491 interestingly has no synergistic cofactor activity with protein S.

[0212] This is illustrated in FIG. 9, where the concentration of the FV-variants are varied whereas the protein S (3 nM), TFPIα (0.25 nM) and FXa (0.3 nM) concentrations are constant. The values plotted here are the absorbances reached after 900 seconds. FV-709-1476 is slightly more efficient than FV-Short whereas the FV-810-1491 is less efficient.

[0213] FIG. 10 shows the time curves of substrate development with the three mutants. in this experiment, the concentration of the FV-variants is 1 nM. The FXa (0.3 nM) and TFPIα (0.25 nM) concentrations are constant as is the FV-variant concentration. The protein S concentration is varied. FIG. 10 shows that the FV-709-1476 is somewhat more efficient as the protein S added has stronger stimulating effect. This has been a consistent finding throughout the experiments. Of particular note is the lack of effect of protein S with the FV-810-1491 showing that this mutant has no synergistic effect with protein S.

[0214] FIG. 11 shows a similar comparison, made using plasma dilutions as source of protein S. FIG. 11 shows the absorbances from the 900 sec point. FIG. 11 shows that the plasma can be diluted approximately twice as much with the FV-709-1476 variant as compared to the FV-Short variant, thus illustrating that the FV-709-1476 variant has higher activity.

[0215] FIG. 12 shows the activity of variant FV-Short 1545Q (resistant to thrombin cleavage at position 1545 because the Arg (R) is replaced with a Gln (Q). In the illustrated time course of FIG. 12, it is evident that this variant is essentially similar to FV-Short and moreover that the activity remains after incubation of the mutant with thrombin. The FV-Short 1545Q mutant will be cleaved at Arg709 but the acidic C-terminal part of the B domain is still attached and thus the mutant retains the synergistic cofactor activity.

[0216] The other variant shown in FIG. 12 is a full length FV variant (bottom part of the figure, on the second page of FIG. 12) that is also mutated at Arg1545 to Gln, referred to as FV-1545Q [35]. The thrombin cleavage sites at positions 709 and 1018 are intact and sensitive to thrombin. This mutant (FV-1545Q) in the uncleaved form is similar to full length FV and in itself has no or little synergistic cofactor activity [25]. However, after cleavage with thrombin (at 709 and 1018) the FV exposes the acidic region that is still attached because the Arg1545 is mutated to Gln (Q). From the time curves it is evident that that the thrombin-cleaved FV-1545Q is equally as efficient as the other variants.

REFERENCES

[0217] 1. Dahlback, B. (2007) The tale of protein S and C4b-binding protein, a story of affection. Thromb Haemost 98 (1), 90-6. [0218] 2. Dahlback, B. (2018) Vitamin K-Dependent Protein S: Beyond the Protein C Pathway. Semin Thromb Hemost 44 (2), 176-184. [0219] 3. Dahlback, B. (2017) Novel insights into the regulation of coagulation by factor V isoforms, tissue factor pathway inhibitoralpha, and protein S. J Thromb Haemost 15 (7), 1241-1250. [0220] 4. Hackeng, T. M. et al. (2009) Regulation of TFPI function by protein S. J Thromb Haemost 7 Suppl 1, 165-8. [0221] 5. Hackeng, T. M. et al. (2006) Protein S stimulates inhibition of the tissue factor pathway by tissue factor pathway inhibitor. Proc Natl Acad Sci USA 103 (9), 3106-11. [0222] 6. Dahlback, B. (2008) Advances in understanding pathogenic mechanisms of thrombophilic disorders. Blood 112 (1), 19-27. [0223] 7. Sailer, F. et al. (2009) Generation and phenotypic analysis of protein S-deficient mice. Blood 114 (11), 2307-14. [0224] 8. Burstyn-Cohen, T. et al. (2009) Lack of protein S in mice causes embryonic lethal coagulopathy and vascular dysgenesis. J Clin Invest 119 (10), 2942-53. [0225] 9. Dahlback, B. (2018) Treating hemophilia by targeting protein S? Blood 131 (12), 1271-1272. [0226] 10. Prince, R. et al. (2018) Targeting anticoagulant protein S to improve hemostasis in hemophilia. Blood 131 (12), 1360-1371. [0227] 11. Dahlback, B. (2000) Blood coagulation. Lancet 355 (9215), 1627-32. [0228] 12. Dahlback, B. and Villoutreix, B. O. (2005) Regulation of blood coagulation by the protein C anticoagulant pathway: novel insights into structure-function relationships and molecular recognition. Arterioscler Thromb Vasc Biol 25 (7), 1311-20. [0229] 13. Ndonwi, M. and Broze, G., Jr. (2008) Protein S enhances the tissue factor pathway inhibitor inhibition of factor Xa but not its inhibition of factor VIIa-tissue factor. J Thromb Haemost 6 (6), 1044-6. [0230] 14. Ndonwi, M. et al. (2010) The Kunitz-3 domain of TFPI-alpha is required for protein S-dependent enhancement of factor Xa inhibition. Blood 116 (8), 1344-51. [0231] 15. Peraramelli, S. et al. (2012) TFPI-dependent activities of protein S. Thromb Res 129 Suppl 2, S23-6. [0232] 16. Reglinska-Matveyev, N. et al. (2014) TFPI cofactor function of protein S: essential role of the protein S SHBG-like domain. Blood 123 (25), 3979-87. [0233] 17. Broze, G. J., Jr. and Girard, T. J. (2012) Tissue factor pathway inhibitor: structure-function. Front Biosci (Landmark Ed) 17, 262-80. [0234] 18. Maroney, S. A. and Mast, A. E. (2015) New insights into the biology of tissue factor pathway inhibitor. J Thromb Haemost 13 Suppl 1, S200-7. [0235] 19. Mast, A. E. (2016) Tissue Factor Pathway Inhibitor: Multiple Anticoagulant Activities fora Single Protein. Arterioscler Thromb Vasc Biol 36 (1), 9-14. [0236] 20. Vincent, L. M. et al. (2013) Coagulation factor V(A2440G) causes east Texas bleeding disorder via TFPIalpha. J Clin Invest 123 (9), 3777-87. [0237] 21. Camire, R. M. (2016) Rethinking events in the haemostatic process: role of factor V and TFPI. Haemophilia 22 Suppl 5, 3-8. [0238] 22. Ndonwi, M. et al. (2012) The C-terminus of tissue factor pathway inhibitor alpha is required for its interaction with factors V and Va. J Thromb Haemost 10 (9), 1944-6. [0239] 23. Wood, J. P. et al. (2013) Tissue factor pathway inhibitor-alpha inhibits prothrombinase during the initiation of blood coagulation. Proc Natl Acad Sci USA 110 (44), 17838-43. [0240] 24. Wood, J. P. et al. (2014) Biology of tissue factor pathway inhibitor. Blood 123 (19), 2934-43. [0241] 25. Dahlback, B. et al. (2017) Factor V-short and protein S as synergistic tissue factor pathway inhibitor (TFPIa) cofactors. Res Pract Throm Haemost 2, 114-124. [0242] 26. Zoller, B. et al. (1995) Evaluation of the relationship between protein S and C4b-binding protein isoforms in hereditary protein S deficiency demonstrating type I and type III deficiencies to be phenotypic variants of the same genetic disease. Blood 85 (12), 3524-31. [0243] 27. Malm, J. et al. (1988) Changes in the plasma levels of vitamin K-dependent proteins C and S and of C4b-binding protein during pregnancy and oral contraception. Br J Haematol 68 (4), 437-43. [0244] 28. Marquette, K. A. et al. (1995) The factor V B-domain provides two functions to facilitate thrombin cleavage and release of the light chain. Blood 86 (8), 3026-34. [0245] 29. Toso, R. and Camire, R. M. (2004) Removal of B-domain sequences from factor V rather than specific proteolysis underlies the mechanism by which cofactor function is realized. J Biol Chem 279 (20), 21643-50. [0246] 30. Happonen, K. E. et al. (2016) The Gas6-Axl Protein Interaction Mediates Endothelial Uptake of Platelet Microparticles. J Biol Chem 291 (20), 10586-601. [0247] 31. Dahlback, B. et al. (1983) Visualization of human C4b-binding protein and its complexes with vitamin K-dependent protein S and complement protein C4b. Proc Natl Acad Sci USA 80 (11), 3461-5. [0248] 32. Hackeng, T. M. and Rosing, J. (2009) Protein S as cofactor for TFPI. Arterioscler Thromb Vasc Biol 29 (12), 2015-20. [0249] 33. Somajo, S. et al. (2015) Amino acid residues in the laminin G domains of protein S involved in tissue factor pathway inhibitor interaction. Thromb Haemost 113 (5), 976-87. [0250] 34. Alshaikh, N. A. et al. (2017) New functional assays to selectively quantify the activated protein C- and tissue factor pathway inhibitor-cofactor activities of protein S in plasma. J Thromb Haemost 15 (5), 950-960. [0251] 35. Thorelli E, Kaufman R J and Dahlbäck B (1997) Cleavage requirement for activation of FV by factor Xa, Eur J Biochem. 247:12-20. [0252] 36. Thompson et al. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nuc. Acid Res. 22:4673-4680 [0253] 37. Caceci. M. S. & Cacheris. W. P. (1984) Fitting curves to data. Byte 9:340-362