Modified polypeptide of activated blood coagulation factor v, and method for measuring activity of blood coagulation control factor using same

Abstract

The present invention provides: a human FVa modified polypeptide useful for a method for measuring the activity of protein S or protein C; and a new method for measuring the activity of protein S or protein C.　The present invention provides: a human FVa modified polypeptide which is a single strand, which does not have a thrombin cleavage site, and which is, in the presence of protein S, cleaved by activated protein C on the C-terminal side of an arginine residue corresponding to position 306 of human FVa; a composition or kit that includes the polypeptide and that is for testing the activity of protein S or protein C; and a method for measuring the activity of protein S or protein C by using the polypeptide.

Description

Modified polypeptide of activated blood coagulation factor V and method for measuring activity of blood coagulation control factor using the same

[0001] The present invention relates to a modified polypeptide of activated blood coagulation factor V (FVa), a composition or kit for measuring the activity of a blood coagulation control factor containing said polypeptide, and a method for measuring the activity of a blood coagulation control factor using said polypeptide.

[0002] Protein S (PS), a blood coagulation regulator, is a single-chain glycoprotein with a molecular weight of approximately 80,000, primarily synthesized in the liver and present in plasma and platelets. It is a vitamin K-dependent plasma protein containing a Gla residue at its NH2-terminus. PS, along with another blood coagulation regulator, protein C (PC), is involved in blood coagulation regulation and plays an important role in anticoagulation, inflammation, cell death, and vascular permeability in humans and other animals. Deficiency of PC or PS, or production of PC or PS without anticoagulant activity, impairs the ability to regulate the coagulation reaction, leading to a hypercoagulable state and thrombosis. Therefore, PC and PS measurements are used to assess suspected thrombosis (primarily deep vein thrombosis, superficial vein thrombosis, and pulmonary thromboembolism), neonatal acute purpura fulminans, severe liver injury, disseminated intravascular coagulation (DIC), and follow-up after initiation of warfarin therapy.

[0003] Current methods for measuring PS or PS activity include the clotting time method and the chromogenic method. The clotting time method observes the effect of prolonging the prothrombin time (PT) or activated partial thromboplastin time (APTT), while the chromogenic method observes the ability to degrade a synthetic substrate. In other words, neither the clotting time method nor the chromogenic method directly quantifies the cleavage of activated factor V (FVa), the substrate for PC / PS. In both PC and PS activity measurements, the clotting time method is affected by the presence of coagulation factors (e.g., factor Va and factor VIIIa), coagulation factor inhibitors (e.g., immunoglobulins against coagulation factors), and direct oral anticoagulants (DOACs), and results may not reflect the PS or PC activity levels in the sample. The chromogenic method may produce falsely elevated values ​​in some patients (e.g., those with PC Gla domain mutations or K193del mutations) or in those with vitamin K deficiency.

[0004] Therefore, a new method for directly measuring the activity of blood coagulation regulators PC and PS, that is, the cleavage of the substrate by PC in the presence of PS, is desired.

[0005] Activated protein C (APC) is known to act on activated blood coagulation factor V (FVa) in the presence of PS, cleaving the A2 domain of FVa and inactivating FVa (Figure 1). Because PS acts as a coenzyme in this reaction, APC alone can degrade FVa. The present inventors have found that a modified FVa polypeptide in which arginine at position 506 of FVa (see SEQ ID NO: 3) is mutated is less susceptible to degradation by APC alone, but is more easily (or more rapidly) degraded in the presence of APC and PS (see SEQ ID NO: 3) than in the absence of PS, via arginine at position 306.

[0006] Accordingly, the present disclosure provides a single-chain polypeptide comprising, in the N-terminal to C-terminal direction, the following: (a) an amino acid sequence having 90% or greater sequence identity with the amino acid sequence represented by positions 1 to 306 of SEQ ID NO: 3, or an amino acid sequence having an N-terminal truncation, wherein the residue corresponding to position 306 of SEQ ID NO: 3 is an arginine residue; (b) an amino acid sequence having 90% or greater sequence identity with the amino acid sequence represented by positions 307 to 709 of SEQ ID NO: 3, wherein the residue corresponding to position 709 of SEQ ID NO: 3 is deleted or is a residue other than arginine, and the residues corresponding to positions 506 and / or 679 of SEQ ID NO: 3 are residues other than arginine; and (c) an amino acid sequence having 90% or greater sequence identity with the amino acid sequence represented by positions 1546 to 1877 of SEQ ID NO: 3, wherein the single-chain polypeptide does not contain a thrombin cleavage site and is cleaved by activated protein C at the C-terminal side of the arginine residue corresponding to position 306 in the presence of protein S.

[0007] The present disclosure also provides a polynucleotide comprising a nucleotide sequence encoding the above polypeptide. The present disclosure also provides a composition for measuring Protein S or Protein C activity, comprising the above polypeptide. Furthermore, the present disclosure provides a kit for measuring Protein S or Protein C activity, comprising the above composition. Furthermore, the present disclosure provides a method for measuring Protein S or Protein C activity, using the above polypeptide as a substrate.

[0008] According to the present invention, a polypeptide useful for measuring the activity of Protein S or Protein C and a novel method for measuring the activity of Protein S or Protein C are provided.

[0009] Figure 1 is a schematic diagram illustrating the structure of human blood coagulation factor V (FV), the generation of an activated form (FVa) by cleavage of FV with thrombin, and the generation of an inactive form (FVi) by cleavage of FVa with activated protein C (APC) and protein S (PS) (inactivation of FVa). Numbers represent amino acid positions according to the numbering of SEQ ID NO: 3 (i.e., when the N-terminal Ala of the mature protein is number 1), and numbers in parentheses represent amino acid positions when the N-terminus of the signal peptide is number 1. Figure 2 is a schematic diagram illustrating the structure of one embodiment of a polypeptide of the present disclosure (FVa-m1; artificial substrate 1) and its cleavage by APC and PS. "FLAG" represents a FLAG tag with a peptide linker sequence, "His" represents a His tag with a peptide linker sequence, and "X" represents an amino acid residue other than arginine (e.g., glutamine Q). Artificial substrate 1 can be cleaved only at R306 by APC and PS. Therefore, the amount of uncleaved artificial substrate 1 can reflect the activity levels of PC and PS. Figure 1 shows a schematic diagram illustrating the structure of another embodiment of a polypeptide of the present disclosure (FVa-m2; artificial substrate 2) and its cleavage by APC and PS. "FLAG" represents a FLAG tag with a peptide linker sequence, "His" represents a His tag with a peptide linker sequence, and "X" represents an amino acid residue other than arginine (e.g., glutamine Q). Artificial substrate 2 can be cleaved only at R306 by APC and PS. Therefore, the amount of uncleaved artificial substrate 2 can reflect the activity levels of PC and PS. Figure 2 shows a schematic diagram illustrating the structure of yet another embodiment of a polypeptide of the present disclosure (FVa-m3; artificial substrate 3) and its cleavage by APC and PS. "FLAG" represents a FLAG tag with a peptide linker sequence, "His" represents a His tag with a peptide linker sequence, and "X" represents an amino acid residue other than arginine (e.g., glutamine Q). Artificial substrate 3 can be cleaved at R306 and R506 by APC and PS, but only cleavage at R306 is dependent on the presence of PS. Therefore, the amount of fragments of artificial substrate 3 that are not cleaved at R306 may reflect the PS activity level. This graph shows the relationship between PS activity and the amount of uncleaved artificial substrate 1 in simulated plasma samples.Uncleaved artificial substrate 1 was detected by a sandwich ELISA using anti-FLAG and anti-His antibodies. The vertical axis represents absorbance, which reflects the amount of captured uncleaved substrate. The horizontal axis represents PS activity in the simulated plasma samples used. This graph shows the relationship between PS activity and the amount of uncleaved artificial substrate 1 in the simulated plasma samples. Uncleaved artificial substrate 1 was detected by an AlphaLISA immunoassay using anti-FLAG and anti-His antibodies. The vertical axis represents signal intensity, which reflects the amount of captured uncleaved substrate. The horizontal axis represents PS activity in the simulated plasma samples used. This graph shows the relationship between PC activity and the amount of uncleaved artificial substrate 1 in the simulated plasma samples. Uncleaved artificial substrate 1 was detected by a sandwich ELISA using anti-FLAG and anti-His antibodies. The vertical axis represents absorbance, which reflects the amount of captured uncleaved substrate. The horizontal axis represents APC concentration in the simulated plasma samples used. This graph shows the relationship between PS activity and the amount of uncleaved artificial substrate 2 in simulated plasma samples. Uncleaved artificial substrate 2 was detected by sandwich ELISA using anti-FLAG and anti-His antibodies. The vertical axis represents absorbance, reflecting the amount of captured uncleaved substrate. The horizontal axis represents PS activity in the simulated plasma samples used. This graph shows the results of Western blotting using FV[307-506] mAb for the simulated plasma samples used in Figure 8, the reaction solutions obtained by reacting artificial substrate 2 with recombinant PS (rPS) solutions and samples without APC, and the reaction solutions obtained by reacting an FVa variant ("QQQ") lacking the APC cleavage site as an APC and PC substrate. This graph shows the correlation between PS activity measurement results using the measurement method of the present disclosure using artificial substrate 2 and PS activity measurement results using the clotting time method. This graph shows the relationship between PC activity in simulated plasma samples and the amount of uncleaved artificial substrate 2. Uncleaved artificial substrate 2 was detected by sandwich ELISA using anti-FLAG and anti-His antibodies. The vertical axis represents absorbance, which reflects the amount of captured uncleaved substrate. The horizontal axis represents the APC concentration in the simulated plasma sample used. The results of Western blotting using FV[307-506] mAb for the reaction solution obtained by reacting the simulated plasma sample used in Figure 11 and a 4 nM simulated plasma sample ("4(P-)") without added phospholipid with artificial substrate 2 are shown.This graph shows the relationship between PC activity and the amount of uncleaved artificial substrate 2 in a simulated plasma sample. Uncleaved artificial substrate 2 was detected by a sandwich ELISA using anti-FLAG and anti-His antibodies. The vertical axis represents absorbance, which reflects the amount of captured uncleaved substrate. The horizontal axis represents PC activity in the simulated plasma sample used. This graph shows the relationship between PS activity in a simulated plasma sample and the amount of the fragment of artificial substrate 3 uncleaved by R306. The substrate fragment uncleaved by R306 was detected by a sandwich ELISA using anti-FLAG and anti-FV[307-506] antibodies. The vertical axis represents absorbance, which reflects the amount of captured substrate fragment uncleaved by R306. The horizontal axis represents PS activity in the simulated plasma sample used. The results of Western blotting using FV[307-506] mAb are shown for the reaction solutions obtained by reacting the simulated plasma sample used in Figure 14, the recombinant PS (rPS) solution, and the sample without APC with artificial substrate 3, and the reaction solutions obtained by reacting the FVa variant ("QQQ") that does not have the APC cleavage site as a substrate for APC and PC.

[0010] <Human Activated Factor V Variant Polypeptide (Artificial Substrate FVa-m)> From a first perspective, the present disclosure provides a human activated Factor V variant polypeptide, a single-chain polypeptide that does not contain a thrombin cleavage site and is cleaved by activated protein C (APC) in the presence of protein S (PS) at the C-terminus of the arginine residue corresponding to position 306 of SEQ ID NO: 3 (hereinafter also referred to as "R306") (hereinafter also referred to as "FVa-m" or simply "polypeptide of the present disclosure"). Thus, the polypeptide of the present disclosure can also be referred to as an artificial substrate that can act as a substrate for PC in the presence of PS. The FVa variant polypeptide (FVa-m) of the present disclosure comprises at least a portion corresponding to at least a part (e.g., the C-terminal portion) of the A1 domain (positions 1 to 306) of human activated factor V (amino acid sequence: SEQ ID NO: 3), a portion corresponding to the A2 domain (positions 307 to 709), and a portion corresponding to the A3 domain (positions 1546 to 1877), and may optionally comprise a portion corresponding to at least a part of the B domain (positions 710 to 1545) and / or at least a portion (e.g., the N-terminal portion) of the C1-C2 domain (positions 1878 to 2196). As used herein, the A1, A2, B, A3, C1, and C2 domains of the polypeptide (FVa-m) of the present disclosure refer to portions of the amino acid sequence corresponding to positions 1 to 306, 307 to 709, 710 to 1545, 1546 to 1877, 1878 to 2036, and 2037 to 2196 of SEQ ID NO: 3 of the polypeptide of the present disclosure.

[0011] In one embodiment, the polypeptide of the present disclosure is a single-chain polypeptide that does not contain a thrombin cleavage site and is cleaved by activated protein C in the presence of protein S at the C-terminus of the arginine residue corresponding to position 306 of SEQ ID NO: 3, and comprises, in the N-terminal to C-terminal direction, the following: (a) an amino acid sequence having 90% or greater sequence identity with the amino acid sequence represented by positions 1 to 306 of SEQ ID NO: 3, or an amino acid sequence having an N-terminal truncation, wherein the residue corresponding to position 306 of SEQ ID NO: 3 is an arginine residue (R306); (b) an amino acid sequence having 90% or greater sequence identity with the amino acid sequence represented by positions 307 to 709 of SEQ ID NO: 3, wherein the residue corresponding to position 709 of SEQ ID NO: 3 is deleted or a residue other than arginine, and the residues corresponding to positions 506 and / or 679 of SEQ ID NO: 3 are residues other than arginine; and (c) an amino acid sequence having 90% or greater sequence identity with the amino acid sequence represented by positions 1546 to 1877 of SEQ ID NO: 3, in that order. Here, the amino acid sequence shown in SEQ ID NO: 3 is the amino acid sequence of human blood coagulation factor V (FV).

[0012] As used herein, the expression "comprise" means that something other than the specified thing (for example, one or more amino acid sequences other than the specified one or more amino acid sequences) may or may not be included. That is, for example, the expression "comprises one or more specific amino acid sequences" also contemplates the case where the something consists of the specified one or more amino acid sequences.

[0013] The polypeptide of the present disclosure may or may not have the coagulation activity and / or anticoagulant activity of activated blood coagulation factor V (FVa), so long as it can be cleaved by activated protein C in the presence of protein S at the C-terminus of the arginine residue (R306) corresponding to position 306 of SEQ ID NO: 3 (i.e., so long as it can act as a substrate for APC in the presence of PS). The polypeptide of the present disclosure is cleaved by activated protein C in the presence of protein S at the C-terminus of the arginine residue (R306) corresponding to position 306, as described above, in the presence of protein S, since the residue corresponding to position 306 of SEQ ID NO: 3 remains an arginine residue and the residues corresponding to positions 506 and / or 679 of SEQ ID NO: 3 are residues other than arginine. Due to this cleavage property, the polypeptide of the present disclosure is useful for measuring the activity of protein S or protein C. In some embodiments, the polypeptide of the present disclosure has an arginine residue corresponding to position 306 of SEQ ID NO: 3 (as is) and a residue other than arginine corresponding to positions 506 and / or 679 of SEQ ID NO: 3, and is therefore cleaved by activated protein C at the C-terminal side of the arginine residue corresponding to position 306 (R306) in the presence of protein S, but is resistant to cleavage in the absence of protein S. Due to this cleavage property, the polypeptide of the present disclosure may be further useful in measuring the activity of protein S or protein C.

[0014] As used herein, with respect to the polypeptides of the present disclosure, and particularly with respect to R306, "resistance to cleavage in the absence of protein S" means that the rate of cleavage by activated protein C in the absence of protein S (or the amount cleaved in a given time period) is significantly slower (or lower) than the rate of cleavage by activated protein C in the presence of protein S (or the amount cleaved in a given time period); more specifically, the rate of cleavage by activated protein C in the absence of protein S may be, for example, 50% or less, more specifically 40% or less, more specifically 30% or less, more specifically 25% or less, more specifically 20% or less, more specifically 15% or less, more specifically 10% or less, more specifically 5% or less, more specifically 2% or less, more specifically 1% or less, more specifically 0.5% or less, and more specifically 0.1% or less of the rate of cleavage by activated protein C in the presence of protein S under the same measurement conditions. The predetermined time period may be, for example, 60 minutes, 30 minutes, 20 minutes, 15 minutes, 10 minutes, 5 minutes, 3 minutes, 2 minutes, 1 minute, or 30 seconds.

[0015] In this specification, the positions of amino acid residues in the amino acid sequence of the polypeptide of the present disclosure, which is derived from the amino acid sequence of blood coagulation factor V, are identified as the positions of the corresponding residues with reference to the amino acid sequence of SEQ ID NO: 3, unless otherwise specified.

[0016] The residues corresponding to positions 506 and / or 679 of SEQ ID NO: 3 may be any natural or unnatural amino acid residue other than an arginine residue (i.e., may be substituted with any residue other than an arginine residue). As used herein, the substitution, insertion, deletion, and / or addition of amino acid residues in the amino acid sequence set forth in SEQ ID NO: 3 does not create new thrombin cleavage sites or additional APC cleavage sites (other than the APC cleavage site present in the amino acid sequence of SEQ ID NO: 3) in the amino acid sequence. Preferably, the substitution of amino acids in the amino acid sequence set forth in SEQ ID NO: 3 does not create unnatural disulfide bonds in the polypeptides of the present disclosure. The residues corresponding to positions 506 and / or 679 of SEQ ID NO: 3 may be substituted with any natural amino acid residue other than arginine or with an unnatural amino acid residue (e.g., an arginine analog, more specifically homoarginine, ornithine, or citrulline), more preferably with any natural amino acid residue other than arginine (more preferably with an amino acid residue selected from the group of 20 amino acids listed below), more preferably with an amino acid residue selected from the group consisting of glutamine, asparagine, glutamic acid, alanine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, histidine, tyrosine, aspartic acid, serine, and glycine, more preferably with a glutamine or asparagine residue.

[0017] As used herein, natural amino acids (or, interchangeably, naturally occurring amino acids) preferably include amino acids encoded by genetic codons and post-translationally modified amino acids thereof (e.g., hydroxyproline, γ-carboxyglutamic acid, and selenomethionine), and more preferably include alanine (Ala, A), leucine (Leu, L), isoleucine (Ile, I), methionine (Met, M), valine (Val, V), phenylalanine (Phe, V), and selenomethionine (Selenomethionine). , F), tryptophan (Trp, W), tyrosine (Tyr, Y), asparagine (Asn, N), cysteine ​​(Cys, C), glutamine (Gln, Q), serine (Ser, S), threonine (Thr, T), aspartic acid (Asp, D), glutamic acid (Glu, E), arginine (Arg, R), histidine (His, H), lysine (Lys, K), glycine (Gly, G), and proline (Pro, P).

[0018] As used herein, unnatural amino acids include derivatives (e.g., analogs and mimetics) of the naturally occurring amino acids, and non-limiting examples include ornithine, citrulline, homoserine, homoglutamine, homoarginine, ω-hydroxy-norarginine, ω-amino-arginine, ω-methyl-arginine, α-methyl-histidine, 2,5-diiodo-histidine, 1-methyl-histidine, 3-methyl-histidine, 4,5-dehydro-lysine, δ-hydroxy-lysine, nitroglutamine, norleucine, norvaline, methionine sulfoxide, methionine methylsulfonium, etc. As used herein, amino acids may be in the L- or D-configuration, but are preferably L-amino acids. The polypeptide FVa-m of the present disclosure is expected to be able to adopt a three-dimensional structure similar to that of native human FVa, since its amino acid sequence (particularly the sequences corresponding to the major domains A2 and A3, and optionally the A1 domain, and further the C1 and C2 domains) has a high sequence identity (90% or more) with that of native human FVa.

[0019] The polypeptides of the present disclosure do not contain a thrombin cleavage site. That is, the polypeptides of the present disclosure do not contain arginine at positions corresponding to positions 709, 1018, and 1545 of SEQ ID NO: 3. The residues corresponding to positions 709, 1018, and 1545 of SEQ ID NO: 3 are either deleted or are residues of any natural or unnatural amino acid other than arginine (e.g., an arginine analog, more specifically homoarginine, ornithine, or citrulline), preferably any natural amino acid residue other than arginine (more preferably an amino acid residue selected from the group of 20 amino acids listed above), more preferably an amino acid residue selected from the group consisting of glutamine, asparagine, glutamic acid, alanine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, histidine, tyrosine, aspartic acid, serine, and glycine, more preferably a glutamine or asparagine residue. The polypeptide of the present disclosure may contain R506 or R679 as an APC cleavage site in addition to R306, but preferably R306 is the only APC cleavage site. In the polypeptide of the present disclosure, R306 is more preferably present in the sequence KKTRNLKK (SEQ ID NO: 17).

[0020] As used herein, sequence identity is calculated as the percentage of amino acids that match at corresponding positions between two amino acid sequences when the two sequences are aligned for maximum identity. That is, the sequence identity between two amino acid sequences can be expressed as follows: sequence identity (%) = (number of matching amino acid residues at corresponding positions between the two amino acid sequences) / (total number of amino acid residues in the reference sequence) × 100. In the polypeptides of the present disclosure, the sequence identity relative to the reference sequence is 90% or more, preferably 91% or more, more preferably 92% or more, more preferably 93% or more, more preferably 94% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more preferably 98% or more, more preferably 99% or more, and most preferably 100%.

[0021] Regarding N-terminal truncation (or N-terminal deletion) and C-terminal truncation (or C-terminal deletion) of an amino acid sequence, the number of amino acids deleted at the N-terminus or C-terminus of each amino acid sequence is not limited, as long as the single-chain polypeptide containing the N-terminal truncation and / or C-terminal truncation is cleaved by APC at the C-terminus of the arginine residue at position 306 in the presence of PS (more specifically, as long as it is cleaved by APC at the C-terminus of the arginine residue at position 306 in the presence of PS but is difficult to cleave in the absence of PS). Such single-chain polypeptides can be easily obtained without excessive trial and error. Such single-chain polypeptides can be obtained, for example, by confirming the cleavage activity of APC on truncated polypeptides obtained by truncating the N- and / or C-terminal amino acids one by one.

[0022] The number of amino acids that may be truncated (or deleted) at the N-terminus of the amino acid sequence (particularly in amino acid sequence (a)) may be, for example, up to 300 amino acids, more particularly up to 250 amino acids, more particularly up to 200 amino acids, more particularly up to 150 amino acids, more particularly up to 100 amino acids, more particularly up to 90 amino acids, more particularly up to 80 amino acids, more particularly up to 70 amino acids, more particularly up to 60 amino acids, more particularly up to 50 amino acids, more particularly up to 40 amino acids, more particularly up to 30 amino acids, more particularly up to 20 amino acids, more particularly up to 10 amino acids, more particularly up to 5 amino acids. The number of amino acids that may be truncated (or deleted) at the C-terminus of the amino acid sequence (particularly in amino acid sequence (d)) may be, for example, up to 200 amino acids, more particularly up to 150 amino acids, more particularly up to 100 amino acids, more particularly up to 90 amino acids, more particularly up to 80 amino acids, more particularly up to 70 amino acids, more particularly up to 60 amino acids, more particularly up to 50 amino acids, more particularly up to 40 amino acids, more particularly up to 30 amino acids, more particularly up to 20 amino acids, more particularly up to 10 amino acids, more particularly up to 5 amino acids.

[0023] The polypeptide of the present disclosure may or may not have an N-terminal signal peptide represented by positions −28 to −1 of SEQ ID NO: 3 on the N-terminal side of amino acid sequence (a).

[0024] In one embodiment, the polypeptide of the present disclosure further comprises, at the C-terminus of the amino acid sequence (c), (d) an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by positions 1878 to 2196 of SEQ ID NO: 3, or said amino acid sequence having a C-terminal truncation. The polypeptide of this embodiment can bind to a phospholipid membrane.

[0025] In one embodiment, the polypeptide of the present disclosure comprises an amino acid sequence having 90% or greater sequence identity with the amino acid sequence represented by positions 1 to 709 of SEQ ID NO: 3, or an amino acid sequence having an N-terminal truncation, wherein the residue corresponding to position 306 of SEQ ID NO: 3 is an arginine residue, the residue corresponding to position 709 of SEQ ID NO: 3 is deleted or is a residue other than arginine, and the residues corresponding to positions 506 and / or 679 of SEQ ID NO: 3 are residues other than arginine. Here, amino acid sequences (a) and (b) are included in the amino acid sequence having 90% or greater sequence identity with the amino acid sequence represented by positions 1 to 709 of SEQ ID NO: 3, or the amino acid sequence having an N-terminal truncation.

[0026] In one embodiment, the polypeptide of the present disclosure comprises an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by positions 1546 to 2196 of SEQ ID NO: 3, or a C-terminal truncation of the amino acid sequence, wherein amino acid sequences (c) and (d) are included in the amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by positions 1546 to 2196 of SEQ ID NO: 3, or the C-terminal truncation of the amino acid sequence.

[0027] In one embodiment, the polypeptide of the present disclosure comprises: (i) an amino acid sequence having 90% or greater sequence identity with the amino acid sequence shown at positions 1 to 709 of SEQ ID NO: 3, or an amino acid sequence having an N-terminal truncation thereof, wherein the residue corresponding to position 306 of SEQ ID NO: 3 is an arginine residue, the residue corresponding to position 709 of SEQ ID NO: 3 is deleted or is a residue other than arginine, and the residues corresponding to positions 506 and / or 679 of SEQ ID NO: 3 are residues other than arginine; and (ii) an amino acid sequence having 90% or greater sequence identity with the amino acid sequence shown at positions 1546 to 2196 of SEQ ID NO: 3, or an amino acid sequence having a C-terminal truncation thereof, wherein amino acid sequences (a) and (b) are included in amino acid sequence (i), and amino acid sequences (c) and (d) are included in amino acid sequence (ii).

[0028] When a polypeptide of the present disclosure comprises a residue corresponding to position 2183 of SEQ ID NO: 3, that residue may be substituted with an amino acid residue other than serine (eg, alanine).

[0029] The polypeptide of the present disclosure further comprises a peptide linker sequence between the amino acid sequence (b) and the amino acid sequence (c). The peptide linker sequence is a flexible amino acid sequence and may be composed of, for example, any natural or unnatural amino acid, preferably any natural amino acid. Natural amino acids and unnatural amino acids are as described above.

[0030] In one embodiment, the peptide linker sequence primarily comprises amino acids selected from glycine, serine, and alanine. Here, "primarily comprises" in relation to the peptide linker sequence means that 50% or more of the total amino acids constituting the peptide linker sequence are selected from glycine, serine, and alanine. The length of this type of peptide linker sequence can be easily determined by one skilled in the art and may be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in length. In one specific embodiment, the polypeptide of the present disclosure further comprises a peptide linker sequence consisting of at least three amino acid residues between amino acid sequence (b) and amino acid sequence (c). This embodiment is preferred when a peptide tag sequence (described below) is included between amino acid sequence (b) and amino acid sequence (c). The peptide linker sequence preferably consists of at least 5 amino acid residues, more preferably at least 8 amino acid residues, more preferably at least 10 amino acid residues, and more preferably at least 15 amino acid residues. When one or more peptide tag sequences are contained between the amino acid sequence (b) and the amino acid sequence (c), the peptide linker sequence may be separated by two or more peptide tag sequences. A peptide tag sequence (e.g., a His tag sequence) may be used as the peptide linker sequence alone or together with a different peptide linker sequence.

[0031] Non-limiting examples of peptide linker sequences include: n , (S) n , (A) n , (GS) n , (SG) n , (GA) n , (AG) n , (AS) n , (SA) n , (G) n S, S(G) n , (GS) n G, (GSG) n , (GGS) n , (SGG)n , (GSS) n , (SSG) n , (SGS) n , (GSA) n , (GSA) n G, (GGS) n G, (GGGS; SEQ ID NO: 18) n , (GGSG; SEQ ID NO: 19) n , (GSGG; SEQ ID NO: 20) n , (SGGG; SEQ ID NO: 21) n , (GSSG; SEQ ID NO: 22) n , (GSAG; SEQ ID NO: 23) n , (GSGGS; SEQ ID NO: 24) n , (GSGSG; SEQ ID NO: 25) n , (GGGSG; SEQ ID NO: 26) n , (GGSGG; SEQ ID NO: 27) n , (GSGGG; SEQ ID NO: 28) n , (GSSGG; SEQ ID NO: 29) n , (SGGSG; SEQ ID NO: 30) n , (GSSSG; SEQ ID NO: 31) n , (GSAGS; SEQ ID NO: 32) n , (GGGGS; SEQ ID NO: 33) n (n is an integer of 1 or more, for example, 1 to 30; the total of multiple n's is, for example, 2 to 30), and any combination thereof.

[0032] In another embodiment, the peptide linker sequence comprises an N-terminal portion and / or a C-terminal portion of an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in positions 710 to 1545 of SEQ ID NO: 3, wherein the residues corresponding to positions 1018 and 1545 of SEQ ID NO: 3 are either independently deleted or are residues other than arginine. In this embodiment, the peptide linker sequence preferably has a deletion of two or more consecutive amino acid residues, including the residue corresponding to position 1018 of SEQ ID NO: 3. When the peptide linker sequence includes only one of the N-terminal portion or the C-terminal portion, the N-terminal portion or the C-terminal portion may be, for example, up to 800 amino acids, more specifically up to 700 amino acids, more specifically up to 600 amino acids, more specifically up to 500 amino acids, more specifically up to 400 amino acids, more specifically up to 300 amino acids, more specifically up to 200 amino acids, more specifically up to 100 amino acids, more specifically up to 90 amino acids, more specifically up to 80 amino acids, more specifically up to 70 amino acids, more specifically up to 60 amino acids, more specifically up to 50 amino acids, more specifically up to 40 amino acids, more specifically up to 30 amino acids, more specifically up to 200 amino acids, more specifically up to 100 amino acids, more specifically up to 90 amino acids, more specifically up to 80 amino acids, more specifically up to 70 amino acids, more specifically up to 60 amino acids, more specifically up to 50 amino acids, more specifically up to 40 amino acids, more specifically up to 30 amino acids, more specifically up to 20 amino acids.

[0033] When the peptide linker sequence includes both the N-terminal portion and the C-terminal portion, the N-terminal portion may be, for example, up to 300 amino acid residues from the N-terminus (from the residue corresponding to position 710 of SEQ ID NO: 3 toward the C-terminus), more specifically up to 250 amino acid residues, more specifically up to 200 amino acid residues, more specifically up to 150 amino acid residues, more specifically up to 100 amino acid residues, more specifically up to 90 amino acids, more specifically up to 80 amino acids, more specifically up to 70 amino acids, more specifically up to 60 amino acids, more specifically up to 50 amino acid residues, more specifically up to 40 amino acids, more specifically up to 30 amino acids, more specifically up to 20 amino acids, or more specifically up to 100 amino acid residues. More specifically, it may be up to 10 amino acids, and the C-terminal portion may be up to 500 amino acid residues from the C-terminus (from the residue corresponding to position 1545 of SEQ ID NO: 3 toward the N-terminus), more specifically up to 400 amino acid residues, more specifically up to 300 amino acid residues, more specifically up to 200 amino acid residues, more specifically up to 100 amino acid residues, more specifically up to 90 amino acids, more specifically up to 80 amino acids, more specifically up to 70 amino acids, more specifically up to 60 amino acids, more specifically up to 50 amino acid residues, more specifically up to 40 amino acids, more specifically up to 30 amino acids, more specifically up to 20 amino acids, and more specifically up to 10 amino acids.

[0034] In one specific embodiment, the polypeptide of the present disclosure comprises, between amino acid sequence (b) and amino acid sequence (c), a peptide linker sequence having 90% or more sequence identity with the amino acid sequences set forth at positions 710 to 810 and / or positions 1492 to 1545 of SEQ ID NO: 3, respectively, wherein the residue corresponding to position 1545 of SEQ ID NO: 3, if present, is a residue other than arginine. In this embodiment, the peptide linker sequence preferably has a deletion of two or more consecutive amino acid residues, including the residue corresponding to position 1018 of SEQ ID NO: 3.

[0035] In one embodiment, the polypeptide of the present disclosure comprises an amino acid sequence having 90% or greater sequence identity with the amino acid sequence set forth at positions 1 to 810 of SEQ ID NO: 3, or an amino acid sequence having an N-terminal truncation thereof, wherein the residue corresponding to position 306 of SEQ ID NO: 3 is an arginine residue, the residue corresponding to position 709 of SEQ ID NO: 3 is deleted or a residue other than arginine, and the residues corresponding to positions 506 and / or 679 of SEQ ID NO: 3 are residues other than arginine. Here, the amino acid sequence (i) is included in the amino acid sequence having 90% or greater sequence identity with the amino acid sequence set forth at positions 1 to 810 of SEQ ID NO: 3, or the amino acid sequence having an N-terminal truncation thereof. In a specific embodiment, the polypeptide of the present disclosure further comprises an amino acid sequence having 90% or greater sequence identity with the amino acid sequence set forth at positions 1492 to 1545 of SEQ ID NO: 3, or an amino acid sequence having an N-terminal truncation thereof, wherein the residue corresponding to position 1545 of SEQ ID NO: 3 is a residue other than arginine.

[0036] In one embodiment, the polypeptide of the present disclosure has a peptide tag sequence on the N-terminal side of the amino acid sequence (a) and / or between the amino acid sequence (b) and the amino acid sequence (c) and / or on the C-terminal side of the amino acid sequence (c). In a specific embodiment, the polypeptide of the present disclosure has a peptide tag sequence on the N-terminal side of the amino acid sequence (a). In another specific embodiment, the polypeptide of the present disclosure has a peptide tag sequence on the N-terminal side of the amino acid sequence (a) and between the amino acid sequence (b) and the amino acid sequence (c), or on the C-terminal side of the amino acid sequence (c). The peptide tag sequence may be linked to the N-terminus of the amino acid sequence (a) and / or the C-terminus of the amino acid sequence (b) and / or the N-terminus of the amino acid sequence (c) and / or the C-terminus of the amino acid sequence (c) and / or the C-terminus of the amino acid sequence (d) with or without a peptide linker sequence (e.g., one described above). The presence of the peptide tag sequence allows the polypeptides of the present disclosure to be conveniently detected by methods known in the art, such as ELISA.

[0037] The peptide tag sequence may be any (poly)peptide tag known in the art, including, but not limited to, a FLAG® tag, an HA tag, a polyhistidine tag (His), etc. n Examples of peptide tags include a polyhistidine glycine tag (n = 6 to 10), a polycysteine ​​tag, a Myc tag, a V5 tag, an S tag, an E tag, a T7 tag, a VSV-G tag, a Glu-Glu tag, a Strep-tag II tag, an HSV tag, an AGIA tag, a PA tag, a CBD (chitin-binding domain) tag, a CBP (calmodulin-binding peptide) tag, a GST (glutathione-S-transferase) tag, a MBP (maltose-binding protein) tag, a Trx (thioredoxin) tag, a GFP (green fluorescent protein) tag, a SOD (superoxide dismutase) tag, an avidin tag, and a streptavidin tag. These peptide tags can be detected or separated by any detection or capture means known in the art, for example, using an antibody (preferably a monoclonal antibody) that specifically recognizes the peptide tag.

[0038] In one embodiment, the polypeptide of the present disclosure comprises an amino acid sequence having 90% or more sequence identity with an amino acid sequence consisting of the sequence shown at positions 1 to 810 of SEQ ID NO: 3 and the sequence shown at positions 1492 to 2196 of SEQ ID NO: 3 linked to the C-terminus of said sequence, wherein the residue corresponding to position 306 of SEQ ID NO: 3 is an arginine residue, and two residues corresponding to positions 709 and 1545 and residues corresponding to positions 506 and / or 679 of SEQ ID NO: 3 are residues other than arginine residues (e.g., the amino acid sequence shown in SEQ ID NO: 4) (see Figure 2). In this embodiment, a peptide tag sequence may be linked to the N-terminus and / or C-terminus (preferably both ends) of the amino acid sequence, directly or via a peptide linker sequence (e.g., one described above).

[0039] In one embodiment, the polypeptide of the present disclosure comprises an amino acid sequence having 90% or more sequence identity with the sequence shown at positions 1 to 709 of SEQ ID NO: 3, wherein the residue corresponding to position 306 of SEQ ID NO: 3 is an arginine residue, the residue corresponding to position 709 of SEQ ID NO: 3 is deleted or is a residue other than arginine, and the residues corresponding to positions 506 and / or 679 of SEQ ID NO: 3 are amino acid residues other than arginine (e.g., the amino acid sequence shown in SEQ ID NO: 5 or 6), and an amino acid sequence having 90% or more sequence identity with the sequence shown at positions 1546 to 2196 of SEQ ID NO: 3 (e.g., the amino acid sequence shown in SEQ ID NO: 7) (see Figures 3 and 4 ). In this embodiment, a peptide tag sequence may be linked directly or via a peptide linker sequence to the N-terminus of the former amino acid sequence and / or the C-terminus of the latter amino acid sequence and / or between both amino acid sequences (preferably the N-terminus of the former amino acid sequence and between both amino acid sequences).

[0040] In a more specific embodiment, the polypeptide of the present disclosure comprises an amino acid sequence having 90% or greater sequence identity with the sequence shown at positions 1 to 709 of SEQ ID NO: 3, wherein the residue corresponding to position 306 of SEQ ID NO: 3 is an arginine residue, the residue corresponding to position 709 of SEQ ID NO: 3 is deleted or is a residue other than arginine, and the residues corresponding to positions 506 and 679 of SEQ ID NO: 3 are amino acid residues other than arginine (e.g., the amino acid sequence shown in SEQ ID NO: 5), and an amino acid sequence having 90% or greater sequence identity with the sequence shown at positions 1546 to 2196 of SEQ ID NO: 3 (e.g., the amino acid sequence shown in SEQ ID NO: 7) (see Figure 3). In this embodiment, a peptide tag sequence can be linked directly or via a peptide linker sequence to the N-terminus of the former amino acid sequence and between the two amino acid sequences.

[0041] In a more specific embodiment, the polypeptide of the present disclosure comprises an amino acid sequence having 90% or greater sequence identity with the sequence shown at positions 1 to 709 of SEQ ID NO: 3, wherein the residue corresponding to position 306 of SEQ ID NO: 3 is an arginine residue, the residue corresponding to position 709 of SEQ ID NO: 3 is deleted or is a residue other than arginine, and the residue corresponding to position 679 of SEQ ID NO: 3 is an amino acid residue other than arginine (e.g., the amino acid sequence shown in SEQ ID NO: 6), and an amino acid sequence having 90% or greater sequence identity with the sequence shown at positions 1546 to 2196 of SEQ ID NO: 3 (e.g., the amino acid sequence shown in SEQ ID NO: 7) (see Figure 4). In this embodiment, a peptide tag sequence may be linked to the N-terminus of the former amino acid sequence directly or via a peptide linker sequence, and a peptide linker sequence may be present between the two amino acid sequences, or the peptide tag sequence may be linked directly or via a peptide linker sequence.

[0042] The polypeptides of the present disclosure may be recombinant polypeptides produced by recombinant expression methods or synthetic polypeptides produced by chemical synthesis methods, but are preferably recombinant polypeptides. The polypeptides of the present disclosure are suitable for use in the methods for measuring PS or PC activity described below.

[0043] (Method for producing artificial substrate FVa-m) The polypeptide of the present disclosure can be produced by recombinant expression methods or chemical synthesis methods. Recombinant expression of the polypeptide of the present invention can be easily carried out by recombinant expression methods known in the art using the amino acid sequence information or nucleotide sequence information described herein. Briefly, the polypeptide can be produced by incorporating a polynucleotide having a nucleotide sequence encoding the amino acid sequence of the polypeptide of the present disclosure into an expression vector, transforming host cells with this expression vector, culturing the transformed cells under conditions suitable for expression of the polypeptide to express the polypeptide, and recovering the expressed polypeptide from the cells or the culture medium.

[0044] Any expression vector known in the art can be used, and can be easily selected taking into consideration an appropriate combination with the host cell to be used. Examples of expression vectors include virus vectors, plasmid vectors, bacteriophage vectors, cosmids, phagemids, and artificial chromosomes. Host cells suitable for expressing polypeptides can be used, including prokaryotic cells such as Escherichia coli cells, and eukaryotic cells such as yeast (e.g., Saccharomyces cerevisiae), insect cells, plant cells, and animal cells.

[0045] Conditions suitable for expressing a polypeptide of the present disclosure (e.g., medium, culture temperature, etc.) can be appropriately determined taking into consideration the host cell, the vector (e.g., plasmid) used for expression, etc. In order to promote extracellular secretion of the expressed polypeptide of the present disclosure from the host cell, the nucleotide sequence to be incorporated into the expression vector may be designed so that the polypeptide of the present disclosure has a signal peptide (consisting of the amino acid sequence shown at positions 1 to 28 of SEQ ID NO: 2). Details of the genetic recombination / expression techniques required in connection with the present disclosure are described in standard textbooks (e.g., Ausubel, F.A. et al. (eds.) (1988), Current Protocols in Molecular Biology, John Wiley and Sons, New York, NY; Sambrook, J. et al. (1989), Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

[0046] The polypeptide of the present disclosure may be expressed in a cell-free expression system. Details of polypeptide expression in a cell-free expression system are described, for example, in Lesly et al. (Methods in Molecular Biology, 37, 265-278, 1995) and Japanese Patent Application Laid-Open No. 2000-175695. Cell-free expression can be carried out using commercially available kits and devices.

[0047] <Polynucleotide Encoding Artificial Substrate FVa-m> From a second perspective, the present disclosure provides a polynucleotide comprising a nucleotide sequence encoding the above-described polypeptide. The polynucleotide of the present disclosure may be DNA or RNA. The polynucleotide of the present disclosure may be incorporated into a vector. The vector can be prepared by methods known in the art. In the vector, the polynucleotide of the present disclosure may be operably linked to a promoter and / or various other expression control elements (e.g., terminator, enhancer, polyadenylation sequence, 5' untranslated region, 3' untranslated region, etc.). The vector may be a recombinant expression vector. Furthermore, this vector may be used to transform or transfect cells. Transformed or transfected host cells are useful, for example, for maintaining and / or propagating the polynucleotide of the present disclosure or expressing the polypeptide of the present disclosure.

[0048] <Composition for Measuring Protein S or Protein C Activity> From a third aspect, the present disclosure provides a composition for measuring the activity of Protein S (PS) or Protein C (PC), comprising the polypeptide described above. If the polypeptide of the present disclosure does not have arginine at both positions 506 and 679 (with reference to SEQ ID NO: 3), it is unlikely to be cleaved by activated protein C (APC) in the absence of protein S, and can therefore be included in the same composition as APC. Therefore, use of the composition of the present disclosure may enable easier measurement of the activity of protein S or protein C. The polypeptide included in the composition of the present disclosure preferably has a peptide tag sequence.

[0049] The composition of the present disclosure may contain APC (preferably in excess relative to the APC activity in the sample) for measuring PS activity, or PS (preferably in excess relative to the PS activity in the sample) for measuring PC activity, optionally together with a phospholipid. The phospholipid may be selected from, for example, glycerophospholipids and sphingophospholipids, preferably phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol, diphosphatidylglycerol, sphingomyelin, and mixtures thereof, and more preferably phosphatidylcholine or a mixture of phosphatidylcholine and phosphatidylserine. The phospholipid may be provided, for example, in the form of a liposome.

[0050] Other components that may be included in the composition of the present disclosure include calcium ions or calcium salts, sodium chloride, buffers, serum albumin, protease inhibitors (those that do not inhibit APC activity), and protein protectants (e.g., glycerol, sucrose, trehalose, gelatin). The calcium salt may be selected from, for example, calcium fluoride, calcium chloride, calcium bromide, calcium iodide, calcium sulfate, calcium nitrate, calcium acetate, calcium lactate, and calcium cyanide. The composition of the present disclosure may also contain a PC activator for measuring PC activity. The composition of the present disclosure may be in liquid form or in lyophilized form. The composition of the present disclosure can be used to carry out the method for measuring PS or PC activity described below.

[0051] <Kit for Measuring Protein S or Protein C Activity> In a fourth aspect, the present disclosure provides a kit for measuring Protein S or Protein C activity, comprising the polypeptide described above or the composition described above. The polypeptide included in the kit of the present disclosure preferably has a peptide tag sequence. The kit of the present disclosure may include one or two antibodies that respectively recognize (preferably specifically) one or two peptide tag sequences carried by the polypeptide of the present disclosure included in the kit, and / or an antibody that recognizes amino acid sequence (a) of the polypeptide or a subsequence thereof, and / or an antibody that recognizes the amino acid sequence corresponding to positions 307 to 506 or positions 307 to 679 of SEQ ID NO: 3 in the polypeptide, or a subsequence thereof.

[0052] In one specific embodiment, the kit of the present disclosure comprises: the polypeptide described above, which has at least a peptide tag sequence on the N-terminal side of amino acid sequence (a) (particularly, a polypeptide in which the residue corresponding to either position 506 or 679 of SEQ ID NO: 3 is other than an arginine residue); an antibody that recognizes the peptide tag sequence on the N-terminal side of amino acid sequence (a) in the polypeptide; and an antibody that recognizes the amino acid sequence corresponding to positions 307 to 506 of SEQ ID NO: 3 or a subsequence thereof in the polypeptide, or an antibody that recognizes the amino acid sequence corresponding to positions 307 to 679 of SEQ ID NO: 3 or a subsequence thereof in the polypeptide.

[0053] In another specific embodiment, the kit of the present disclosure comprises: the polypeptide described above, which has peptide tag sequences at the N-terminus of amino acid sequence (a) and at the C-terminus of amino acid sequence (b) (more specifically, between amino acid sequence (b) and amino acid sequence (c) or at the C-terminus of amino acid sequence (c)) (particularly, a polypeptide in which the residues corresponding to positions 506 and 679 of SEQ ID NO: 3 are residues other than arginine); an antibody that recognizes the N-terminal peptide tag sequence of amino acid sequence (a) in the polypeptide; and an antibody that recognizes the C-terminal peptide tag sequence of amino acid sequence (b) in the polypeptide.

[0054] The antibody may be a known antibody or a newly generated antibody. The antibody may be monoclonal or polyclonal, but is preferably monoclonal. Methods for generating antibodies that bind to a specific peptide are known in the art. Briefly, such antibodies can be obtained by purifying (e.g., as an immunoglobulin fraction or purified antibody) the serum of an animal (e.g., mouse, rat, hamster, guinea pig, rabbit, goat, sheep, pig, horse, camel, chicken, ostrich, etc.) that has been administered once or more times (subcutaneously, intravenously, intraperitoneally, etc.) with the peptide, alone or conjugated to a suitable carrier (e.g., keyhole limpet hemocyanin, diphtheria toxin, tetanus toxin), optionally together with an adjuvant (e.g., complete or incomplete Freund's adjuvant, aluminum hydroxide, or aluminum phosphate (alum)) using a purification method such as affinity chromatography. The whole serum (antiserum) may also be used directly without purification.

[0055] Methods for producing monoclonal antibodies are also known in the art (e.g., Kohler and Milstein (Nature, 256:495-497, 1975; Antibodies: A Laboratory Manual, edited by Harlow and Lane, Cold Spring Harbor Laboratory, 1988). Briefly, spleen cells (B cells) from an animal immunized with the protein or peptide of interest are fused with appropriate myeloma cells by cell fusion to produce immortalized cells (hybridomas), and hybridomas producing antibodies having the desired binding ability are selected. These hybridomas may be grown in vitro or in vivo (e.g., in the peritoneal cavity of a mouse) to obtain the produced antibodies. The antibody is preferably labeled with a detectable label. Antibody labels are known in the art and can be appropriately selected depending on the detection means used. For example, enzymes, radioisotopes, fluorescent substances, chromogenic substances, luminescent substances, etc. can be used as labels. Examples of enzymes include peroxidase (particularly HRP), β-galactosidase, phosphatase (particularly alkaline phosphatase), luciferase, glucose oxidase, β-glucosidase, β-glucuronidase, etc. Examples of radioisotopes include 3 H. 14 C. 32 P. 35 S. 125 I, 131 Examples of fluorescent dyes include fluorescein isothiocyanate (FITC), rhodamine, Cy2, and Cy3. Examples of color-producing substances include 3,3'-diaminobenzidine (DAB) and 3,3',5,5'-tetramethylbenzidine (TMB). Examples of luminescent substances include luminol and luciferin.

[0056] One of the antibodies may be immobilized or coated on a support or substrate, and the immobilization may be by physical adsorption, covalent bonding, or non-covalent bonding (such as ionic bonding, electrostatic bonding, or hydrophobic interaction).

[0057] The kit of the present disclosure may also include a phospholipid, tissue factor, and calcium ions or a calcium salt. In one embodiment, the kit of the present disclosure is a kit for measuring protein S activity and includes activated protein C (APC). In this embodiment, when the kit is a one-reagent system, the reagent may include a polypeptide of the present disclosure, a phospholipid, and APC, and optionally, calcium ions or a calcium salt. When the kit is a two-reagent system, one reagent may include a polypeptide of the present disclosure and the other reagent may include APC, and the phospholipid and optional calcium ions or calcium salt may be included, separately or together, in either one or both reagents.

[0058] In another embodiment, the kit of the present disclosure is a kit for measuring protein C activity, and contains protein S (PS). The kit of this embodiment may also contain a PC activator. This PC activator is used to activate PC in a sample (to convert it into APC). The PC activator may be thrombin, a thrombin and thrombomodulin complex, or snake venom. The snake venom is preferably that of a snake of the Viperidae family, more preferably that of the Agkistrodon genus (an example of a product is Protein S). TM In this embodiment, when the kit is a one-reagent system, the reagent may comprise a polypeptide of the present disclosure, a phospholipid, and PS, and optionally, calcium ions or a calcium salt. When the kit is a two-reagent system, one reagent may comprise a polypeptide of the present disclosure, and the other reagent may comprise PC, and the phospholipid and optional calcium ions or calcium salt may be contained, separately or together, in either one or both reagents.

[0059] The kit of the present disclosure may contain a calcium chelating agent as a substrate degradation reaction stop solution. Examples of calcium chelating agents include ethylenediaminetetraacetic acid (EDTA), dihydroxyethylglycine (DHEG), diaminopropanoltetraacetic acid (DPTA-OH), diethylenetriaminepentaacetic acid (DTPA), ethylenediaminediacetic acid (EDDA), glycol ether diaminetetraacetic acid (GEDTA), ethylenediaminedipropionic acid (EDDP), ethylenediaminetetrakismethylenephosphonic acid (EDTPO), nitrilotriacetic acid (NTA), nitrilotrismethylenephosphonic acid (NTPO), citric acid, gluconic acid, and salts thereof. Examples of EDTA salts include 2Na EDTA, 4Na EDTA, 2K EDTA, and 3K EDTA. Examples of citrate salts include sodium citrate and triNa citrate. The reagents included in the kit of the present disclosure may be in liquid or lyophilized form.

[0060] The kit of the present disclosure may include a solid support. One of the antibodies may be immobilized on the solid support. The form of the solid support may be appropriately selected from those known in the art, such as a multiwell plate, ELISA plate, tube, capillary tube, chip, bead, particle, filter, membrane, disk, stick, strip, slide glass, or chip. The solid support is preferably blocked to prevent nonspecific adsorption. Any blocking agent known in the art can be used, including serum albumin from a mammal other than the target mammal, nonfat powdered milk, and casein. The surface of the blocked solid support to which the antibody is immobilized may be coated with a water-soluble polymer (e.g., polyvinylpyrrolidone, polyvinyl alcohol, or cellulose esters such as hydroxypropylmethylcellulose and hydroxyethylcellulose) or a non-reducing polysaccharide (e.g., sucrose, trehalose, raffinose). Such a coating makes the solid phase containing the antibody on the solid support highly stable and suitable for long-term storage.

[0061] The kit of the present disclosure may contain a labeled signal generator (e.g., a coloring agent) and / or a signal generation reaction stop solution. The labeled signal generator can be appropriately selected from known agents depending on the label. For example, when the label is peroxidase (e.g., HRP), the labeled signal generator may be TMB, DAB, o-phenylenediamine (OPD), 2,2-azino-di-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 10-acetyl-3,7-dihydroxyphenoxazine (ADHP), etc.; when the label is alkaline phosphatase, the labeled signal generator may be p-nitrophenyl phosphate (pNPP), 4-methylumbelliferyl phosphate (4-MUP), etc. The signal generation reaction stop solution may be, for example, sulfuric acid or sodium hydroxide.

[0062] The kit of the present disclosure may also contain a buffer solution as a detergent and / or diluent. The buffer solution can be appropriately selected from those known in the art, such as phosphate buffer, Tris-HCl buffer, citrate buffer, carbonate buffer, borate buffer, succinate buffer, acetate buffer, etc. The buffer solution may contain NaCl, a surfactant (e.g., Tween 20, Triton X-100), and / or a preservative (e.g., sodium azide), as needed. Specific examples of buffer solutions include PBS, PBS-T, TBS, and TBS-T. The kit of the present disclosure may also contain a standard curve (calibration curve), a standard solution for preparing the standard curve (calibration curve), and, optionally, a standard solution diluent. The kit of the present disclosure may also contain a sealant (e.g., a plate seal if the solid support is a plate) for sealing the support during incubation. The kit of the present disclosure can be used to perform the PS or PC activity measurement method described below.

[0063] <Method for Measuring Protein S or Protein C Activity> From a fifth aspect, the present disclosure provides a method for measuring the activity of Protein S (PS) or Protein C (PC), using the above-described polypeptide as a substrate. The sample for measuring PS or PC activity is, for example, a blood sample, specifically, whole blood, serum, or plasma (e.g., citrated plasma). In one embodiment, the method of the present disclosure is a method for measuring PS activity in a sample, characterized by using the above-described polypeptide (artificial substrate FVa-m) as a substrate for activated PC (APC) in the presence of PS. The measurement method of this embodiment of the present disclosure specifically comprises the following steps: (a1) a step of contacting a sample with at least the artificial substrate FVa-m, APC, and calcium ions; (b1) an optional step of contacting a mixed solution of the sample obtained in (a1) and the artificial substrate FVa-m, APC, and calcium ions with a calcium chelator; (c1) a step of detecting the artificial substrate FVa-m uncleaved at R306 or its R306-containing fragment in the sample obtained in (a1) or in the mixed solution obtained in (b1); and (d1) a step of estimating the activity of PS in the sample based on the detected amount of the uncleaved artificial substrate FVa-m or its R306-containing fragment.

[0064] In another embodiment, the method of the present disclosure is a method for measuring PC activity in a sample, using the above-described polypeptide (artificial substrate FVa-m) as an APC substrate. The method of this embodiment specifically includes the following steps: (x2) treating the sample with a PC activator; (a2) contacting the sample treated in (x2) with at least the artificial substrate FVa-m, PS, and calcium ions; (b2) optionally contacting the mixture of the sample obtained in (a2) and the artificial substrate FVa-m, PS, and calcium ions with a calcium chelator; (c2) detecting the artificial substrate FVa-m uncleaved at R306 or its R306-containing fragment in the sample obtained in (a2) or the mixture obtained in (b2); and (d2) estimating the PC activity in the sample based on the amount of the uncleaved artificial substrate FVa-m or its R306-containing fragment detected.

[0065] Step (a) In step (a), a sample and each reagent component (at least artificial substrates FVa-m, Ca 2+ , and APC or PC), a cleavage reaction of the artificial substrate FVa-m by APC in the presence of PS occurs. Here, the amount of APC or PS added is in excess relative to the APC activity or PS activity in the sample. In this manner, the artificial substrate FVa-m is cleaved at R306 by an excess amount of APC in step (a1) depending on the PS activity in the sample, and by an APC in the sample in the presence of an excess amount of PS in step (a2) (i.e., depending on the PC activity in the sample). In other words, the amount of the artificial substrate FVa-m or its R306-containing fragment that is not cleaved at R306 reflects the PS activity level (step (a1)) or PC activity level (step (a2)) in the sample.

[0066] In step (a), the reagent components to be contacted with the sample may include a phospholipid. The phospholipid promotes the formation of a complex between APC and PS. The phospholipid may be provided in the form of a liposome. The sample may be contacted with the reagent components in one batch or in multiple batches. In the latter case, any reagent component or combination of reagent components may be contacted in each batch, and the order of contact may also be arbitrary. For measuring PS activity, it is preferable that the phospholipid is first contacted with the sample together with APC (resulting in the formation of a complex between APC and PS in the sample on the phospholipid), and after appropriate incubation, the artificial substrate FVa-m and calcium ions are contacted with the mixture of phospholipid, APC, and sample (including the APC / PS complex on the phospholipid).

[0067] The contact conditions (or incubation conditions) can be, for example, from about 30 seconds to about 60 minutes at room temperature or preferably at about 37° C. Those skilled in the art can easily determine appropriate conditions.

[0068] When measuring PC activity in a sample, it is preferable to include a step (step (x)) of treating the sample with a PC activator before step (a). This pretreatment activates all functional PC present in the sample (i.e., converts it into APC). This makes it possible to measure PC activity in the sample in subsequent steps, regardless of the degree of PC activation in the sample at the time of collection.

[0069] Step (b) Step (b) is an optional step. When the measurement method of the present disclosure includes step (b), the calcium chelator chelates calcium in the reaction system, thereby stopping the cleavage reaction of the artificial substrate FVa-m by APC.

[0070] Step (c) In step (c), the uncleaved artificial substrate FVa-m or its R306-containing fragment can be detected, for example, by ELISA (particularly, a sandwich assay) using two antibodies that respectively (preferably specifically) recognize two specific amino acid sequences (e.g., the amino acid sequence of a peptide tag or a subsequence thereof) at the N-terminus and C-terminus of R306 in the artificial substrate FVa-m or its R306-containing fragment. Here, "detection" of FVa-m or a fragment thereof includes not only the determination of its presence or absence but also quantification and semi-quantification.

[0071] The antibody may be, for example, an antibody that specifically recognizes a subsequence containing R306 in the amino acid sequence of the artificial substrate FVa-m or its R306-containing fragment (thus, an antibody that binds to the artificial substrate FVa-m or its R306-containing fragment but does not bind to any of the fragments (R306-free fragments) that may be generated by cleavage at R306). Alternatively, the antibody may be a combination of an antibody that (preferably specifically) recognizes a subsequence N-terminal to R306 in the amino acid sequence of the artificial substrate FVa-m or its R306-containing fragment (including the amino acid sequence, if FVa-m contains a peptide tag and / or peptide linker sequence), and an antibody that (preferably specifically) recognizes a subsequence C-terminal to R306 in the amino acid sequence.

[0072] When the subsequence is identical to that in human factor V (hFV), an antibody (including commercially available products) that recognizes the subsequence in the amino acid sequence of the artificial substrate FVa-m or its R306-containing fragment may be used (e.g., FV[307-506] antibody) that recognizes the subsequence in hFV. The antibody used (one of the antibodies when two antibodies are used) is preferably immobilized on a solid phase (e.g., a support such as a well plate). The other antibody is preferably detectably labeled. The antibody label is as described above.

[0073] When the artificial substrate FVa-m is cleaved only at R306 by APC, the uncleaved FVa-m can be captured by a sandwich ELISA using, for example, a combination of: (i) an antibody that recognizes (preferably specifically) the N-terminal subsequence of R306 in amino acid sequence (a), or, if FVa-m has a peptide tag (e.g., a FLAG tag) at the N-terminus, an antibody that recognizes (preferably specifically) the peptide tag (e.g., an anti-FLAG tag antibody); and (ii) an antibody that recognizes (preferably specifically) the C-terminal subsequence of R306 in the amino acid sequence spanning amino acid sequence (a) to amino acid sequence (c), or, if FVa-m has a peptide tag (e.g., a His tag) at the C-terminus or between amino acid sequence (b) and amino acid sequence (c), an antibody that recognizes (preferably specifically) the peptide tag (e.g., an anti-His tag antibody).

[0074] When the artificial substrate FVa-m is cleaved by APC at both R306 and R506 or R679 (arginine at positions 506 or 679, respectively, in the numbering of SEQ ID NO: 3), the FVa-m fragment uncleaved at R306 can be captured by a sandwich ELISA method using, for example, a combination of (i) an antibody that recognizes (preferably specifically) a subsequence N-terminal to R306 in amino acid sequence (a), or, if FVa-m has a peptide tag (e.g., a FLAG tag) at the N-terminus, an antibody that recognizes (preferably specifically) the peptide tag (e.g., an anti-FLAG tag antibody), and (ii) an antibody that recognizes (preferably specifically) a subsequence C-terminal to R306 and N-terminal to R506 or R679 in the amino acid sequence spanning from amino acid sequence (a) to amino acid sequence (b) (e.g., the amino acid sequence at positions 307 to 506 or 307 to 679 in the numbering of SEQ ID NO: 3).

[0075] The signal from the label can be measured using, for example, a spectrophotometer, a fluorometer, a CCD camera, a CMOS camera, a photodiode, a photomultiplier tube, etc., appropriately selected together with a light source and / or a filter as necessary. The amount of the detected signal reflects the PS activity or PC activity in the sample.

[0076] Step (d): Based on the amount of the artificial substrate FVa-m or its R306-containing fragment uncleaved at R306 detected in step (c) (which is reflected in the signal reading from the captured artificial substrate or its fragment), the PS or PC activity in the sample is estimated. The signal reading can generally be converted to the enzyme activity level using a calibration curve. The calibration curve may be provided by the supplier of the reagent used, or may be prepared using a blank and one or more standard solutions with known enzyme activities before, during, or after performing this measurement method.

[0077] The measurement method of the present disclosure can be easily performed using the composition or kit described above. The measurement method of the present disclosure is based on the fact that the polypeptide (artificial substrate) of the present disclosure is easily (or more rapidly) cleaved by APC in the presence of PS, and this cleavage is less affected by the presence of FXa or thrombin. Therefore, unlike coagulation-based assays (e.g., clotting time assays), the measurement method of the present disclosure is thought to be unaffected (specifically, not to produce falsely elevated values) by the coexistence of direct oral anticoagulants (DOACs) (FXa inhibitors and thrombin inhibitors) in the sample. In fact, in an experiment using samples containing different concentrations of the FXa inhibitor rivaroxaban, no correlation was observed between PS activity measured by the measurement method of the present disclosure and rivaroxaban concentration in the sample, whereas a clear correlation was observed between PS activity measured by the clotting time assay and rivaroxaban concentration in the sample.

[0078] <Preparation of human blood coagulation factor Va variants, FVa-m> Three human FVa variants (FVa-m1 to m3) were designed, having the amino acid sequences shown in SEQ ID NOs: 10, 13, and 16, and the structures shown schematically in Figures 2 to 4. These variants are expected to adopt a three-dimensional structure similar to that of native human FVa because their amino acid sequences (particularly the sequences corresponding to the A1, A2, A3, C1, and C2 domains) share a high degree of sequence identity (99% or more) with that of native human FVa.

[0079] (Construction of a vector encoding FVa-m and expression of FVa-m) Human FV cDNA was cloned into human liver polyA + Human FV cDNA was amplified by RT-PCR from RNA (Clontech). Three types of FVa-m cDNA (SEQ ID NOs: 8, 11, and 14, respectively) were constructed by using known site-directed mutagenesis methods (specifically, overlap extension PCR or QuikChange) to introduce the mutations shown in the table below into the encoded amino acid sequence, as well as the peptide tag sequence and peptide linker sequence.

[0080] With reference to the amino acid sequence of SEQ ID NO: 3, substitution of R709 and R1545 with amino acids other than arginine (e.g., glutamine) confers resistance to thrombin cleavage to FVa-m, while substitution of R506 and R679 with amino acids other than arginine (e.g., glutamine) with reference to the amino acid sequence of SEQ ID NO: 3 prevents FVa-m from being easily (or rapidly) cleaved by APC in the absence of PS.

[0081] The cDNA of each FVa-m was cloned into the pcDNA3.1(+) mammalian expression vector (Invitrogen). This expression vector was transfected into COS-1 cells using Lipofectamine 3000 and selected with G418 to establish stable cell lines. Each cell line was cultured in DMEM / F-12 medium supplemented with insulin, transferrin, sodium selenite, 2.5 mM CaCl2, and 1.0 mg / ml Albumax, and the culture supernatant was collected. Each FVa-m was purified from the culture supernatant using Talon Metal Affinity Resin (Clontech) and then dialyzed into a buffer (20 mM HEPES, 150 mM NaCl, 5 mM CaCl2, pH 7.4).

[0082] <Measurement of PS or PC activity using FVa-m> The method for measuring PS or PC activity of the present disclosure uses the artificially created FVa-m with a limited cleavage site as described above as a substrate for APC. Therefore, FVa-m1, FVa-m2, and FVa-m3 will be referred to as artificial substrate 1 (Figure 2 [X=Q]; SEQ ID NO: 10), artificial substrate 2 (Figure 3 [X=Q]; SEQ ID NO: 13), and artificial substrate 3 (Figure 4 [X=Q]; SEQ ID NO: 16), respectively.

[0083] (1-1) Example 1 of PS activity measurement using artificial substrate 1: Mock sample In this activity measurement, a mock plasma sample panel with known PS activity (0-100%) was prepared from Factor Assay Control Plasma (Georage King Bio-Medical) and PS-deficient plasma (Affinity Biologicals) in the following mixing ratio.

[0084] To 15 μL of simulated plasma sample, 10 μL of artificial substrate 1, 10 μL of 200 nM activated PC (APC), 10 μL of liposomes (25 μM phospholipid), and 5 μL of buffer were added and reacted at 37°C for 30-60 seconds. The reaction was then stopped by adding 10 μL of 10 mM EDTA. Uncleaved artificial substrate 1 in the reaction mixture was then captured and semi-quantified by sandwich ELISA using a His-tag antibody and an HRP-labeled FLAG-tag antibody immobilized on a well plate.

[0085] The results are shown in Figure 5. From Figure 5, it can be seen that the higher the PS activity of the simulated plasma sample, the lower the amount of uncleaved substrate, i.e., more artificial substrate 1 is cleaved. Furthermore, the only APC cleavage site in artificial substrate 1 is R306, which is cleaved in the presence of PS, and because APC was added in excess relative to the APC activity in the sample, the decrease in uncleaved artificial substrate 1 is thought to reflect the PS activity in the simulated plasma sample. From the above, it can be seen that PS activity in a sample can be measured by using artificial substrate 1 as an APC substrate.

[0086] (1-2) PS Activity Measurement Example 2 Using Artificial Substrate 1: Mock Sample Uncleaved artificial substrate 1 was semi-quantified as described in Measurement Example 1 in (1-1) above, except that an AlphaLISA immunoassay kit (PerkinElmer) was used for capture and semi-quantification of uncleaved artificial substrate 1. Briefly, biotinylated anti-FLAG tag, streptavidin-coated Alpha donor beads, and anti-His tag-bound AlphaLisa acceptor beads were used. Only when both antibodies bound to uncleaved artificial substrate 1 did the two beads come into close proximity, emitting a specific light (e.g., 615 nm) in response to irradiation with a specific excitation light (e.g., 680 nm). Therefore, the amount of uncleaved substrate could be semi-quantified by detecting the luminescence signal.

[0087] The results are shown in Figure 6. From Figure 6, it can be seen that the higher the PS activity of the simulated plasma sample, the lower the luminescence signal intensity, which reflects the amount of uncleaved substrate present; in other words, a larger amount of artificial substrate 1 is cleaved. Therefore, from these results, it can be seen that PS activity in a sample can be measured by using artificial substrate 1 as a substrate for APC.

[0088] (1-3) PC Activity Measurement Example 1 Using Artificial Substrate 1: Mock Sample. For this activity measurement, a panel of simulated plasma samples with known APC concentrations (0, 1, 2.5, 5, 10, and 20 nM) was prepared from 5 μL of 0-20 nM APC solution and 15 μL of PC-deficient plasma. To 20 μL of the simulated plasma sample, 10 μL of artificial substrate 1, 10 μL of 100 nM PS, and liposomes (25 μM phospholipid) were added and incubated at 37°C for 20 minutes. The reaction was then terminated by the addition of 10 μL of 10 mM EDTA. Subsequently, uncleaved artificial substrate 1 in the reaction mixture was captured and semi-quantified by sandwich ELISA using a His-tag antibody and an HRP-labeled FLAG-tag antibody immobilized on a well plate.

[0089] The results are shown in Figure 7. From Figure 7, it can be seen that the higher the PC concentration in the simulated plasma sample, the lower the amount of polypeptide that can be captured (i.e., uncleaved substrate), i.e., more artificial substrate 1 is cleaved. Furthermore, since the only APC cleavage site in artificial substrate 1 is R306, which is cleaved in the presence of PS, and PS was added in excess relative to the PS activity in the sample, the decrease in uncleaved artificial substrate 1 is thought to reflect the PC activity in the sample. From the above, it can be seen that PC activity in a sample can be measured by using artificial substrate 1 as an APC substrate.

[0090] (2-1) Example 1 of PS Activity Measurement Using Artificial Substrate 2: Mock Samples In this activity measurement, a panel of mock plasma samples with known PS activity (0-200%) was prepared from Factor Assay Control Plasma (Georage King Bio-Medical, #GK-NP2) and PS-deficient plasma (Affinity Biologicals, #PS-DP) as follows.

[0091] To 1 μL of simulated plasma sample, 20 μL of artificial substrate 2, 10 μL of 20 nM APC, 10 μL of liposomes (25 μM phospholipid), and 9 μL of buffer were added, and the mixture was incubated at 37°C for 60 seconds. The reaction was then stopped by adding 10 μL of 10 mM EDTA. Substrate fragments bearing both FLAG and His tags in the reaction mixture were then captured and semi-quantified by sandwich ELISA using a His-tag antibody and an HRP-labeled FLAG-tag antibody immobilized on a well plate.

[0092] The results are shown in Figure 8. Figure 8 indicates that the higher the PS activity of the simulated plasma sample, the lower the amount of polypeptides bearing both peptide tags; that is, more artificial substrate 2 is cleaved between the two peptide tags. Because the only APC cleavage site in artificial substrate 2 is R306, which is cleaved in the presence of PS, the decrease in the amount of polypeptides bearing both peptide tags is thought to reflect the amount of artificial substrate 2 cleaved at R306. Furthermore, because APC was added in excess relative to the APC activity in the sample, the decrease in the amount of polypeptides bearing both peptide tags is thought to reflect the PS activity in the sample. From these results, it can be seen that PS activity in a sample can be measured by using artificial substrate 2 as an APC substrate.

[0093] Figure 9 shows the results of Western blotting using FV[307-506] mAb for reaction mixtures prepared in the same manner as above, using 1.5 nM and 5 nM recombinant PS (rPS) solutions instead of the simulated plasma sample, in addition to the simulated plasma sample, as samples; a reaction mixture prepared in the same manner as above except that APC was not added (PS activity of the simulated plasma sample was 100%); and a reaction mixture prepared in the same manner as above except that the FVa variant ("QQQ") containing three mutations, R306Q, R506Q, and R679Q (i.e., no APC cleavage site) was used as the substrate (PS activity of the simulated plasma sample was 100% or 0%). 9 reveals that (i) as PS activity increases, the concentration of the band corresponding to the uncleaved substrate (with a larger molecular weight) decreases, while the concentration of the band corresponding to the cleaved fragments (with a smaller molecular weight) increases, i.e., the amount of cleaved substrate increases; (ii) artificial substrate 2 is cleaved by the APC / recombinant PS complex but not in the absence of APC; and (iii) the FVa variant ("QQQ") that does not have an APC cleavage site is not cleaved in the FVa cleavage reaction system used. Therefore, it can be seen that artificial substrate 2 functions as a substrate for PS-dependent FVa cleavage by APC and can be used to measure PS activity in samples.

[0094] (2-2) Example 3: PS Activity Measurement Using Artificial Substrate 2: Clinical Samples. For this activity measurement, blood samples (citrated plasma) collected from patients with informed consent were used under the approval of the Research Ethics Committee. Artificial substrate 2 (final concentration: 8 μg / mL), APC (final concentration: 20 nM), liposomes (phospholipids: final concentration: 25 μM), and buffer (to a total volume of 50 μL) were added to 1 μL of clinical plasma sample. The mixture was incubated at 37°C for 1 minute, and then EDTA (final concentration: 10 mM) was added to terminate the reaction. Polypeptides bearing both FLAG and His tags in the reaction mixture were captured by sandwich ELISA using His-tag antibodies and HRP-labeled FLAG-tag antibodies coated on a 96-well plate. Color development was achieved with TMB, and the reaction was terminated with 1 N HCl. The amount of color development was measured using a plate reader to semi-quantitate the captured polypeptides. PS activity was measured using the same sample using the conventional clotting time method.

[0095] The relationship between the measurement results using artificial substrate 2 and the measurement results using the clotting time method is shown in Figure 10. As is clear from Figure 10, there is a strong correlation between the measurements obtained by the measurement method of the present disclosure using artificial substrate 2 and the measurements obtained by the clotting time method. Therefore, it can be seen that the measurement method of the present disclosure using artificial substrate 2 as a substrate for APC is comparable to the conventional clotting time method, and therefore can be used as a substitute.

[0096] (2-3) PC Activity Measurement Example 1 Using Artificial Substrate 2: Mock Sample. In this activity measurement, 0, 0.125, 0.25, 0.5, 1, 2, and 4 nM APC solutions were used as a mock plasma sample panel. 20 μL of artificial substrate 2, 10 μL of 100 nM PS, and liposomes (25 μM phospholipids) were added to 10 μL of mock plasma sample. The reaction was incubated at 37°C for 1 minute, and then 10 μL of 10 mM EDTA was added to terminate the reaction. Subsequently, uncleaved artificial substrate 2 bearing both the FLAG tag and the His tag in the reaction mixture was captured and semi-quantified by sandwich ELISA using a His-tag antibody and an HRP-labeled FLAG-tag antibody immobilized on a well plate.

[0097] The results are shown in Figure 11. Figure 11 shows that the higher the APC concentration in the simulated plasma sample, the lower the amount of polypeptide captured by both antibodies; that is, more artificial substrate 2 is cleaved between the two peptide tags. Because the only APC cleavage site in artificial substrate 2 is R306, which is cleaved in the presence of PS, the decrease in the amount of polypeptide containing both peptide tags is thought to reflect the amount of artificial substrate 2 cleaved at R306. Furthermore, because PS was added in excess relative to the PS activity in the sample, the decrease in the amount of polypeptide containing both peptide tags is thought to reflect the PC activity in the sample. From these findings, it can be seen that PC activity in samples can be measured by using artificial substrate 2 as an APC substrate.

[0098] Figure 12 shows the results of Western blotting using FV[307-506] mAb for reaction mixtures prepared in the same manner as above, except that the simulated plasma sample and a 4 nM simulated plasma sample without added phospholipid were used as the substrate. Figure 12 demonstrates that (i) as the APC concentration increases, the intensity of the band corresponding to the uncleaved substrate (with a larger molecular weight) decreases, while the intensity of the band corresponding to the cleaved fragments (with a smaller molecular weight) increases, i.e., the amount of cleaved substrate increases; and (ii) artificial substrate 2 is not cleaved in the absence of phospholipid. Therefore, artificial substrate 2 functions as a substrate for APC-mediated FVa cleavage and can be used to measure PC activity in samples.

[0099] (2-4) Example 2 of PC Activity Measurement Using Artificial Substrate 2: Mock Samples In this activity measurement, a panel of mock plasma samples with known PC activity (0 to 200%) was prepared from Factor Assay Control Plasma (Georage King Bio-Medical, #GK-NP2) and PC-deficient plasma (Affinity Biologicals, #PC-DP) as follows.

[0100] 1 unit / mL Protac in 25 μL of simulated plasma sample TM25 μL of Pentapharm (a PC activator derived from snake venom) was added and allowed to react at 37°C for 5 minutes. TM To 4 μL of the treated simulated plasma sample, artificial substrate 2 (final concentration 8 μg / mL), PS (final concentration 100 nM), liposomes (phospholipids: final concentration 25 μM), and buffer (to a total volume of 50 μL) were added and incubated at 37°C for 1 minute. The reaction was then stopped by adding EDTA (final concentration 10 mM). Polypeptides bearing both FLAG and His tags in the reaction mixture were captured by sandwich ELISA using a His-tag antibody and an HRP-labeled FLAG-tag antibody coated on a 96-well plate. Color development was achieved with TMB, and the reaction was stopped with 1 N HCl. The amount of color development was measured using a plate reader to semi-quantify the amount of captured polypeptide.

[0101] The results are shown in Figure 13. Figure 13 indicates that the higher the PC activity of the simulated plasma sample, the lower the amount of polypeptide captured by both antibodies, i.e., more artificial substrate 2 is cleaved between the two peptide tags. Because the only APC cleavage site in artificial substrate 2 is R306, which is cleaved in the presence of PS, the decrease in the amount of polypeptide containing both peptide tags is thought to reflect the amount of artificial substrate 2 cleaved at R306. Furthermore, because PS was added in excess relative to the PS activity in the sample, the decrease in the amount of polypeptide containing both peptide tags is thought to reflect the PC activity in the sample. From these findings, it can be seen that PC activity in samples can be measured by using artificial substrate 2 as an APC substrate.

[0102] (3-1) PS Activity Measurement Example 1 Using Artificial Substrate 3: Simulated Sample. This activity measurement used the simulated plasma sample used in "PS Activity Measurement Example 1 Using Artificial Substrate 2" above. To 1 μL of the simulated plasma sample, 20 μL of artificial substrate 3, 10 μL of 10 nM APC, 10 μL of liposomes (25 μM phospholipid), and 9 μL of buffer were added. The mixture was incubated at 37°C for 3 minutes, and then 10 μL of 10 mM EDTA was added to terminate the reaction. Subsequently, a sandwich ELISA was performed using FV[307-506] antibody and HRP-labeled FLAG tag antibody immobilized on a well plate to capture and semi-quantitate polypeptides bearing both the FLAG tag antibody and the FV[307-506] antibody epitopes in the reaction mixture.

[0103] The results are shown in Figure 14. Figure 14 indicates that the higher the PS activity of the simulated plasma sample, the lower the amount of polypeptides containing both epitopes, i.e., more artificial substrate 3 is cleaved between the two epitopes. Artificial substrate 3 has two APC cleavage sites, R306 and R506. However, the presence of PS does not significantly affect cleavage at R506 (Blood, 2008; 111: 3034-3041). Furthermore, because APC was added in excess relative to the PC activity in the sample, almost all of artificial substrate 3 is cleaved at R506, whereas cleavage at R306 between the two epitopes is dependent on PS activity. Therefore, the decrease in polypeptides containing both epitopes is considered to reflect the PS activity in the sample. These findings suggest that the use of artificial substrate 3 as an APC substrate makes it possible to measure PS activity in samples.

[0104] Figure 15 shows the results of Western blotting using FV[307-506] mAb for reaction mixtures prepared in the same manner as above using 1.5 nM and 5 nM rPS solutions in addition to the above simulated plasma sample as samples, as well as reaction mixtures prepared in the same manner as above except that APC was not added (PS activity of the simulated plasma sample was 100%), and reaction mixtures prepared in the same manner as above except that an FVa variant ("QQQ") that is not cleaved by APC was used as the substrate (PS activity of the simulated plasma sample was 100% or 0%). Artificial substrate 3 can be cleaved by the APC / PS complex into four fragments: fragment 1 consisting of a FLAG tag and the amino acid sequence from positions 1 to 306 (with reference to the numbering of SEQ ID NO: 3); fragment 2 consisting of the amino acid sequence from positions 307 to 506 (with reference to the numbering of SEQ ID NO: 3); fragment 3 consisting of a FLAG tag and the amino acid sequence from positions 1 to 506 (with reference to the numbering of SEQ ID NO: 3); and fragment 4 consisting of the amino acid sequence from positions 507 to 2196 (with a His tag in the middle) (Figure 4). Thus, FV[307-506] mAb can bind to fragments 2 and 3 in addition to the uncleaved substrate.

[0105] 15 reveals that (i) as PS activity increases, the concentration of the band corresponding to fragment 3 (with a larger molecular weight) decreases, while the concentration of the band corresponding to fragment 2 (with a smaller molecular weight) increases, i.e., the amount of the fragment cleaved at R306 increases; (ii) artificial substrate 3 is cleaved by APC / rPS but not in the absence of APC; and (iii) the FVa variant ("QQQ") lacking the APC cleavage site is not cleaved in the FVa cleavage reaction system used. Therefore, it can be seen that artificial substrate 3 functions as a substrate for PS-dependent FVa cleavage by APC and can be used to measure PS activity in samples.

[0106] It should be noted that the above-described embodiments and examples are described as examples to facilitate understanding of the present invention, and the present invention is not limited to the specific configurations and arrangements described in this specification or the accompanying drawings. The specific configurations, means, and methods described in this specification can be replaced with many others known in the art.

Claims

1. A single-chain polypeptide comprising, in the N-terminal to C-terminal direction, the following: (a) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown at positions 1 to 306 of SEQ ID NO:3, or an amino acid sequence having an N-terminal truncation, wherein the residue corresponding to position 306 of SEQ ID NO:3 is an arginine residue; (b) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown at positions 307 to 709 of SEQ ID NO:3, wherein the residue corresponding to position 709 of SEQ ID NO:3 is deleted or is a residue other than arginine, and the residues corresponding to positions 506 and / or 679 of SEQ ID NO:3 are residues other than arginine; and (c) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown at positions 1546 to 1877 of SEQ ID NO:3, wherein the single-chain polypeptide does not contain a thrombin cleavage site and is cleaved by activated protein C at the C-terminal side of the arginine residue corresponding to position 306 in the presence of protein S.

2. The polypeptide according to claim 1, further comprising, at the C-terminus of the amino acid sequence (c), (d) an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by positions 1878 to 2196 of SEQ ID NO:3, or said amino acid sequence having a C-terminal truncation.

3. The polypeptide according to claim 1 or 2, comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown at positions 1 to 709 of SEQ ID NO:3 or said amino acid sequence having an N-terminal truncation, wherein the residue corresponding to position 306 of SEQ ID NO:3 is an arginine residue, the residue corresponding to position 709 of SEQ ID NO:3 is deleted or is a residue other than arginine, and the residues corresponding to positions 506 and / or 679 of SEQ ID NO:3 are residues other than arginine, and wherein the amino acid sequences (a) and (b) are contained in the amino acid sequence having 90% or more sequence identity with the amino acid sequence shown at positions 1 to 709 of SEQ ID NO:3 or said amino acid sequence having an N-terminal truncation.

4. The polypeptide according to claim 2, comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown at positions 1546 to 2196 of SEQ ID NO:3 or said amino acid sequence having a C-terminal truncation, wherein the amino acid sequences (c) and (d) are contained in the amino acid sequence having 90% or more sequence identity with the amino acid sequence shown at positions 1546 to 2196 of SEQ ID NO:3 or said amino acid sequence having a C-terminal truncation.

5. The polypeptide according to claim 1 or 2, further comprising a peptide linker sequence consisting of at least 3 amino acid residues between the amino acid sequence (b) and the amino acid sequence (c).

6. The polypeptide of claim 5, wherein the peptide linker sequence comprises an N-terminal and / or C-terminal portion of an amino acid sequence having 90% or greater sequence identity with the amino acid sequence shown in positions 710 to 1545 of SEQ ID NO:3, in which the residues corresponding to positions 1018 and 1545 of SEQ ID NO:3 are either independently deleted or are residues other than arginine residues.

7. The polypeptide of claim 6, wherein the peptide linker sequence has a deletion of two or more consecutive amino acid residues including the residue corresponding to position 1018 of SEQ ID NO:

3.

8. The polypeptide of claim 7, wherein the peptide linker sequence comprises an amino acid sequence having 90% or greater sequence identity with the amino acid sequence shown at positions 710 to 810 and / or positions 1492 to 1545 of SEQ ID NO:3, and the residue corresponding to position 1545 of SEQ ID NO:3, if present, is a residue other than an arginine residue.

9. The polypeptide according to claim 2, comprising: (i) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown at positions 1 to 709 of SEQ ID NO:3, or said amino acid sequence having an N-terminal truncation, in which the residue corresponding to position 306 of SEQ ID NO:3 is an arginine residue, the residue corresponding to position 709 of SEQ ID NO:3 is deleted or is a residue other than arginine, and the residues corresponding to positions 506 and / or 679 of SEQ ID NO:3 are residues other than arginine; and (ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown at positions 1546 to 2196 of SEQ ID NO:3, or said amino acid sequence having a C-terminal truncation, in which the amino acid sequences (a) and (b) are contained in the amino acid sequence (i), and the amino acid sequences (c) and (d) are contained in the amino acid sequence (ii).

10. The polypeptide according to claim 9, comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by positions 1 to 810 of SEQ ID NO:3, or said amino acid sequence having an N-terminal truncation, in which the residue corresponding to position 306 of SEQ ID NO:3 is an arginine residue, the residue corresponding to position 709 of SEQ ID NO:3 is deleted or is a residue other than arginine, and the residues corresponding to positions 506 and / or 679 of SEQ ID NO:3 are residues other than arginine, and the amino acid sequence (i) is included in the amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by positions 1 to 810 of SEQ ID NO:3, or said amino acid sequence having an N-terminal truncation.

11. The polypeptide according to claim 10, further comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown at positions 1492 to 1545 of SEQ ID NO:3, or said amino acid sequence having an N-terminal truncation, wherein the residue corresponding to position 1545 of SEQ ID NO:3 is a residue other than arginine.

12. A polypeptide described in claim 1 or 2, having a peptide tag sequence on the N-terminal side of the amino acid sequence (a) and / or between the amino acid sequence (b) and the amino acid sequence (c) and / or on the C-terminal side of the amino acid sequence (c).

13. The polypeptide of claim 1 or 2, wherein the sequence identities are both 95% or more.

14. The polypeptide of claim 1 or 2, wherein the sequence identities are both 98% or more.

15. A polynucleotide comprising a nucleotide sequence encoding the polypeptide of claim 1 or 2.

16. A vector comprising the polynucleotide of claim 15 or a cell comprising said vector.

17. A composition for measuring protein S or protein C activity, comprising the polypeptide according to claim 1 or 2.

18. A kit for measuring protein S or protein C activity, comprising the composition according to claim 17.

19. The kit according to claim 18, comprising: the polypeptide according to claim 14; an antibody recognizing a peptide tag sequence on the N-terminal side of amino acid sequence (a) in said polypeptide; and an antibody recognizing an amino acid sequence corresponding to positions 307 to 506 of SEQ ID NO: 3 or a subsequence thereof in said polypeptide, or an antibody recognizing an amino acid sequence corresponding to positions 307 to 679 of SEQ ID NO: 3 or a subsequence thereof, or an antibody recognizing a peptide tag sequence on the C-terminal side of amino acid sequence (b).

20. A method for measuring the activity of Protein S or Protein C, which comprises using the polypeptide according to claim 1 or 2 as a substrate.

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