WO2006031226A1 - Region du domaine des proteases du facteur ixa en interaction avec le facteur viiia, et procedes correspondants - Google Patents

Region du domaine des proteases du facteur ixa en interaction avec le facteur viiia, et procedes correspondants Download PDF

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Publication number
WO2006031226A1
WO2006031226A1 PCT/US2004/029755 US2004029755W WO2006031226A1 WO 2006031226 A1 WO2006031226 A1 WO 2006031226A1 US 2004029755 W US2004029755 W US 2004029755W WO 2006031226 A1 WO2006031226 A1 WO 2006031226A1
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factor
ixa
polypeptide
villa
seq
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PCT/US2004/029755
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English (en)
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Gary Bledsoe
Berton Moed
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Saint Louis University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0819—Tripeptides with the first amino acid being acidic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/101—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1021—Tetrapeptides with the first amino acid being acidic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates generally to the prevention of coagulation. More particularly, this invention relates to compositions and methods for preventing coagulation by inhibiting binding of factor IXa to factor Villa, and applications utilizing these compositions and methods, including treating patients in need of anti-coagulants, preventing coagulation in blood samples, and detecting factor Villa.
  • Factor IX a vitamin K-dependent protein
  • hepatocytes As a precursor molecule of 461 residues containing a 28 residue signal propeptide and an IS residue leader propeptide (Yoshitake et al., 1985, Biochemistry 24, 3736-3750).
  • the nascent protein undergoes several posttranslational modifications, resulting in a single-chain protein consisting of 415 amino acids and containing 17% carbohydrate by weight (DiScipio et al., 1978, 7. CHn. Invest. 61, 1528-1538).
  • the mature protein circulates in blood as a zymogen of Mr 57,000.
  • Factor IX is activated during physiologic clotting to the two-chain, disulfide-linked serine protease, factor IXa, by VIIa/Ca 2+ /tissue factor (TF) or by factor XIa/Ca 2+ (Davie et al., 1991, Biochemistry 29, 10363-10370).
  • the domain organization of factor IXa is similar to those of the other two enzymes (factors Vila and Xa) involved in the TF-induced coagulation and to that of an anticoagulant enzyme termed activated protein C.
  • the light chain of IXa consists of an amino-terminal ⁇ -carboxyglutamic acid domain ("GIa domain", residues 1-40 out of which 12 are ⁇ -carboxyglutamic acid residues), a short hydrophobic segment (residues 41-46), and two epidermal growth factor (EGF)-like domains (EGFl residues 47-85, and EGF2 residues 86-127) whereas the heavy chain contains the carboxy-terminal serine protease domain with trypsin-like specificity (Id.; Brandstetter et al., 1995, Proc. Natl. Acad. ScL USA 92, 9796-9800).
  • Activation peptide (AP) of residues 145-180 which is released upon conversion of factor IX to IXa is rich in carbohydrate and is the least conserved region in IX from different species (Sarkar et al., 1990, Genomics 6, 133-134).
  • Factor IXa hence formed converts factor X to Xa in the coagulation cascade; for a biologically significant rate, this reaction requires Ca 2+ , phospholipid and factor Villa.
  • the Ca 2+ -loaded conformer of the GIa domain binds to phospholipid vesicles (Freedman et al., 1996, J. Biol. Chem. 271, 16227- 16236) and the EGF 1 domain of IX is required for its activation by VIIa/Ca 2+ /TF
  • the identification of the factor VlIIa binding site on factor IXa would be useful for identifying agents with anti-coagulation activity, as well as for designing treatments which prevent excessive thrombosis in a patient, and preventing coagulation of blood samples.
  • compositions which comprise critical amino acids of this region, but which do not activate factor X bind to factor Villa, preventing the binding of factor IXa, thus preventing coagulation.
  • These compositions can thus be used to prevent thrombosis in patients in need thereof.
  • the compositions can also be used to prevent coagulation in a blood sample, and to detect and quantify factor VIIIa in a sample.
  • one embodiment of the present invention is directed to a polypeptide, or derivative thereof, including peptidomimetics and nonpeptidomimetics, comprising DRX aa T or derivatives, where X aa is any amino acid, wherein the polypeptide or derivative has anti ⁇ coagulation activity.
  • This polypeptide or derivative comprises the three amino acids essential to the factor Villa binding site in factor IXa.
  • this polypeptide, or derivative binds to factor Villa but does not activate factor X.
  • the polypeptide or derivative also preferably comprises from 4 to 20 contiguous amino acids.
  • Preferred polypeptides or derivatives comprise SEQ ID NO:2 (DRAT) or SEQ ID NO:1 (LVDRATCLR), which are sequences in the factor Villa binding site in factor IXa.
  • the polypeptide consists essentially of SEQ ID NO:2 (DRAT). More preferably, the polypeptide consists of SEQ ID NO:2 (DRAT).
  • Another embodiment of the present invention is directed to an isolated and substantially purified polypeptide or derivative thereof, comprising DRX aa T where X aa is any amino acid other than alanine.
  • Preferred polypeptides or derivatives here have from 4 to 20 amino acids.
  • the polypeptide or derivative also preferably binds to factor Villa but does not activate factor X.
  • Another embodiment of the invention is directed to an isolated and substantially purified polypeptide or derivative thereof consisting of from 4 to 9 amino acids, wherein the polypeptide is capable of inhibiting the activation of coagulation factor X in the presence of coagulation factor Villa, and the polypeptide is derived from helix 330 of factor IXa.
  • Preferred polypeptides consist of SEQ ID NO:2 (DRAT), SEQ ID NO:5 (L VX aa ,X 332 AT), wherein X aal is either aspartic acid or tyrosine and X aa2 is either arginine or glutamine, or SEQ ID NO:9 (LVDRATX aa LR), wherein X aa is any amino acid except cysteine.
  • More preferred polypeptides consist of SEQ ID NO:2 (DRAT), SEQ ID NO:6 (LVDRAT), SEQ ID NO:7 (LVYRAT), SEQ ID NO:8 (LVDQAT) or SEQ ID NO: 10 (LVDRATALR).
  • DRAT SEQ ID NO:2
  • LDDRAT SEQ ID NO:6
  • LVYRAT SEQ ID NO:7
  • LPDQAT SEQ ID NO:8
  • SEQ ID NO: 10 LVDRATALR
  • the most preferred polypeptide consists of SEQ ID NO:2 (DRAT).
  • the present invention is directed to an isolated and purified nucleic acid molecule, comprising a nucleotide sequence encoding a polypeptide comprising DRX aa T, where X aa is any amino acid, wherein the polypeptide or derivative has anti-coagulation activity, or the complement thereof.
  • the polypeptide comprises SEQ ID NO: 1 (LVDRATCLR) or SEQ ID NO:2 (DRAT).
  • the present invention is directed to an isolated and purified nucleic acid molecule, comprising a nucleotide sequence encoding a polypeptide or derivative thereof that has anti-coagulation activity, or the complement thereof.
  • the polypeptide comprises SEQ ID NO:2 (DRAT), SEQ ID NO:5 (LVX aa iX aa2 AT), wherein X aal is either aspartic acid or tyrosine and X aa2 is either arginine or glutamine, or SEQ ID NO:9 (LVDRATX aa LR), wherein X aa is any amino acid except cysteine.
  • the nucleic acid molecules encodes a polypeptide comprising SEQ ID NO:2 (DRAT), SEQ ID NO:6 (LVDRAT), SEQ ID NO:7 (LVYRAT), SEQ ID NO:8 (LVDQAT) or SEQ ID NO: 10 (LVDRATALR).
  • DRAT SEQ ID NO:2
  • LDDRAT SEQ ID NO:6
  • LVYRAT SEQ ID NO:7
  • LPDQAT SEQ ID NO:8
  • SEQ ID NO: 10 LVDRATALR
  • the most preferred nucleic acid encodes a polypeptide comprising SEQ ID NO:2 (DRAT).
  • the present invention is directed to a method for identifying an agent having anti-coagulation activity.
  • the method comprises determining whether a candidate agent displaces the binding of a polypeptide comprising SEQ ID NO:2 (DRAT), SEQ ID NO:5 (LVX aa iX aa2 AT), wherein X aa i is either aspartic acid or tyrosine and X aa2 is either arginine or glutamine, or SEQ ID NO:9 (LVDRATX aa LR), wherein X aa is any amino acid except cysteine from factor Villa, wherein the polypeptide preferably has from 4 to 20 amino acids, but may be longer, up to and including the entire factor IXa molecule.
  • DRAT SEQ ID NO:5
  • LVX aa iX aa2 AT SEQ ID NO:5
  • X aa i is either aspartic acid or tyrosine
  • X aa2 is either arginine or
  • the polypeptide is a labeled polypeptide and the determining step comprises detecting the labeled polypeptide displaced from factor Villa.
  • Preferred labels comprise a radioactive or a fluorescent moiety.
  • the present invention is directed to a method of treatment to prevent thrombosis in a patient in need thereof. The method comprises administering to the patient a polypeptide or derivative thereof comprising DRX aa T, where X aa , is any amino acid, wherein the polypeptide or derivative has anti-coagulation activity.
  • the preferred polypeptide or derivative binds to factor Villa but does not activate factor X. More preferably, the polypeptide has from 4 to 20 amino acids.
  • polypeptide comprise SEQ ID NO:2 (DRAT) or SEQ ID NO:1 (LVDRATCLR).
  • the polypeptide comprises SEQ ID NO:5 (LVX aa iX aa2 AT), wherein X aa i is either aspartic acid or tyrosine and X aa2 is either arginine or glutamine, or SEQ ID NO:9 (LVDRATX aa LR), wherein X aa is any amino acid except cysteine.
  • the nucleic acid molecules encodes a polypeptide comprising SEQ ID NO:2 (DRAT), SEQ ID NO:6 (LVDRAT), SEQ ID NO:7 (LVYRAT), SEQ ID NO:8 (LVDQAT) or SEQ ID NO: 10 (LVDRATALR).
  • DRAT SEQ ID NO:2
  • LEO SEQ ID NO:6
  • LVYRAT SEQ ID NO:7
  • LPDQAT SEQ ID NO:8
  • SEQ ID NO: 10 LVDRATALR
  • the most preferred nucleic acid encodes a polypeptide comprising SEQ ID NO:2 (DRAT).
  • An antibody that specifically binds to these sequences would also be expected to prevent thrombosis. Such an antibody could also be utilized in place of the above polypeptides or derivatives in the embodiments described below.
  • the present invention is directed to a method of treatment to prevent thrombosis in a patient in need thereof.
  • the method comprises selecting an agent having anti-coagulation activity, wherein the selecting comprises testing the agent for activity in displacing the binding of Factor IXa from Factor Villa without activating Factor X, and administering the agent to the patient.
  • An additional embodiment of the present invention is a method of preventing coagulation in a blood sample.
  • the method comprises adding to the sample, in sufficient quantity to prevent coagulation, a polypeptide or derivative thereof comprising DRX aa T, where X aa is any amino acid, wherein the polypeptide or derivative has anti-coagulation activity.
  • a preferred polypeptide or derivative is capable of binding to factor Villa but does not activate factor X. More preferably, the polypeptide or derivative has from 4 to 20 amino acids or derivatives. Most preferably, the polypeptide or derivative comprises SEQ ID NO:2 (DRAT) or SEQ ID NO:1 (LVDRATCLR).
  • the polypeptide comprises SEQ ID NO:5 (LVX aa iX aa2 AT) ; wherein X aa i is either aspartic acid or tyrosine and X aa2 is either arginine or glutamine, or SEQ ID NO:9 (LVDRATX aa LR), wherein X aa is any amino acid except cysteine.
  • the nucleic acid molecules encodes a polypeptide comprising SEQ ID NO:2 (DRAT), SEQ ID NO:6 (LVDRAT), SEQ ID NO:7 (LVYRAT), SEQ ID NO:8 (LVDQAT) or SEQ ID NO: 10 (LVDRATALR).
  • the most preferred nucleic acid encodes a polypeptide comprising SEQ ID NO:2 (DRAT).
  • the present invention is directed to a method of detecting factor Villa in a sample.
  • the method comprises (a) contacting the sample with a polypeptide or derivative thereof comprising a covalently attached detectable moiety and DRX aa T, where X aa is any amino acid, and wherein the polypeptide or derivative has anti-coagulation activity, and (b) determining whether the polypeptide or derivative is binding factor Villa from the sample.
  • the detectable moiety is radioactive or fluorescent.
  • Figure IA depicts an SDS gel electrophoretic analysis of various protease domain mutants of Factor IX, and wild-type Factor IX (WT) before activation with factor XIa and Ca 2+ , where approximately 2.3 ⁇ g of protein was applied to each lane.
  • Figure IB depicts the same analysis as in Figure IA, after the factor IX protein was activated for 90 min at 37 0 C, where "IX" indicates the migration position of a single chain IX of residues 1-415, H ⁇ is the migration of the heavy chain of factor IX activation intermediate comprised of residues 146-415, Hp is the migration of the heavy chain of factor IXa comprised of residues 181-415, L is the migration of the light chain of factor IXa comprised of residues 1-145, and the two digit numbers indicate the migration of molecular weight markers.
  • Activation peptide (AP) which is comprised of residues 146-180, stains poorly and was not observed on these gels.
  • Figure 2A depicts the time course of factor X activation by each protease domain IXa mutant in a system containing Ca 2+ and phospholipid (PL), in an activation mixture without Villa, where Xa generated was measured by S-2222 hydrolysis, and where the Factor IXa proteins are: WT (o), L330I ( * ), V331A ( ⁇ ), D332Y ( ⁇ ), R333L (A), R333Q (D), T335A (0), L337I ( ⁇ ), R338Q (•), and IXa hel , x vi, (X).
  • Figure 2B depicts the time course of the factor Villa mediated potentiation of factor
  • Figure 3 A depicts the time course of factor X activation under conditions as in Figure 2A, but without phospholipid, wherein the symbols for the IXa proteins are the same as in Figure 2A.
  • Figure 3B depicts the time course of factor X activation under conditions as in Figure 3 A, but including Factor Villa, wherein the symbols for the IXa proteins are the same as in Figure 2A.
  • Figure 4 A depicts the inhibition of factor Xa generation by the active site-blocked protease domain IXa mutants in the presence of factor Villa and phospholipid, wherein the symbols for the IXa proteins are the same as in Figure 2A.
  • Figure 4B depicts the inhibition of factor Xa generation by the active site-blocked protease domain IXa mutants in the presence of factor Villa but without phospholipid, wherein the symbols for the IXa proteins are the same as in Figure 2A.
  • Figure 5A depicts factor Villa mediated potentiation of factor X activation by the EGFl domain IXa mutants in the presence of phospholipid, where the factor IXa proteins are WT (o), Q50P (•), and IX PCE G FI ( ⁇ ).
  • Figure 5B depicts factor Villa mediated potentiation of factor X activation by the
  • Figure 6A depicts the inhibition of factor Xa generation by the active-site blocked (DEGR) EGFl domain IXa mutants in the presence of phospholipid, where the DEGR-IXa proteins are WT (o); Q50P (•); and IX PCEGFI (A) and control proteins are protein C zymogen (D) and DEGR-activated protein C (APC) (A).
  • DEGR-IXa proteins WT (o); Q50P (•); and IX PCEGFI (A) and control proteins are protein C zymogen (D) and DEGR-activated protein C (APC) (A).
  • Figure 6B depicts the inhibition of factor Xa generation by the active-site blocked (DEGR) EGFl domain IXa mutants in the absence of phospholipid, where the symbols for the IXa proteins are the same as in Figure 6A.
  • Figure 7A depicts the position of various regions of factor IXa, including the helix-
  • Figure 7B depicts the orientation of the amino acid side chains of the helix-330 [162] of protease domain of factor IXa, where the point mutants investigated in the present study are given in parenthesis, and where the direction of the helix is from bottom to top.
  • Figure 8 A depicts a schematic representation of the interactions and location of factor IXa residue L330[162] in a hydrophobic pocket surrounded by F349[181] and L300[131], where the van der Waals contact distances (A) are shown by dashed lines.
  • Figure SB depicts a schematic representation of the interactions of residues V331[163], D332[164], and T335[167] of factor IXa protease domain, where both the hydrogen bonds and the van der Waals contacts are shown by dashed lines.
  • Figure 8C depicts a schematic representation of the location and interactions of factor IXa residue L337[169] in a hydrophobic pocket, where the van der Waals distances of residues located in this hydrophobic pocket are shown by dashed lines.
  • Figure SD depicts a schematic representation of the effect of the factor IXa mutation of L330 to 1330 in a patient with hemophilia B, where the hydrophobic interaction between 1330 with F349 which is present in wild-type factor IXa ( Figure 8A), is eliminated.
  • Figure 9 depicts a schematic representation of the relative positions of the helix-330 in IXapcEGF and IXayuEGFi with respect to its position in normal IXa.
  • Figure 10 depicts the inhibition of factor X activation by SEQ ID NO: 6 (LVDRAT), SEQ ID NO:7 (LVYRAT), and SEQ ID NO:8 (LVDQAT).
  • Figure 11 demonstrates that the peptide of SEQ ID NO:6 has no inhibitory effect on factor IXa's ability to activate factor X in the absence of factor Villa.
  • factor X can be inhibited from factor IXa activation by the introduction of compositions capable of binding to factor Villa at the factor IXa binding site.
  • the compositions comprise polypeptides or polypeptide derivatives which comprise certain residues (or derivatives) of the factor Villa binding site on factor IX.
  • the utilization of these compositions in vitro or in vivo effectively inhibits the coagulation cascade.
  • the factor Villa binding site on factor IXa is in the 330 helix of factor IXa and has the sequence
  • residues 162-170 using the chymotrypsin numbering system corresponds to residues 162-170 using the chymotrypsin numbering system.
  • residues 332 (D), 333 (R), and 335(T) are most crucial, since those resides are present in the hydrophilic portion of the helix in this binding site and are thus expected to interact most predominantly with factor Villa.
  • X aa is any amino acid or derivative
  • the polypeptide or derivative will bind factor Villa at the factor IXa binding site and prevent wild-type factor IXa from binding, thus preventing coagulation and factor X activation.
  • the present invention provides a composition that comprises a polypeptide or derivative that includes the sequence DRX aa T, wherein X 30 is independently any amino acid, or a derivative thereof. Since this composition has the crucial amino acid residues present in factor IXa as D332, R333, and T335, or derivatives of those resides, it will bind to factor Villa at the factor IXa binding site and prevent coagulation and factor X activation.
  • Preferred compositions comprise the entire 9-mer binding site
  • LVDRATCLR SEQ ID NO: 1 or derivatives, or the 4-mer within that sequence which contains the three crucial amino acid residues, DRAT (SEQ ID NO:2).
  • DRAT SEQ ID NO:2
  • the most preferred polypeptides have 4-20 amino acid residues or derivatives, but can comprise much larger sequences, including, for example, factor IXa which has been altered to eliminate its serine protease activity.
  • the polypeptide comprises SEQ ID NO:5 (LVX 1 X 2 AT), wherein X 1 is either D or Y, and X 2 is either R or Q, SEQ ID NO:6 (LVDRAT), SEQ ID NO:7 (LVYRAT), SEQ ID NO:8 (LVDQAT), SEQ ID NO:9 (LVDRATXLR), wherein X is any amino acid other than C, or SEQ ID NO: 10 (LVDRATALR).
  • the term "derivative" includes any non-peptide compound, including peptidomimetics or nonpeptidomimetics, that can substitute for a particular amino acid or polypeptide.
  • a non-peptide derivative includes any non-peptide chemical compound that can bind to factor Villa and prevent coagulation.
  • the compound can also be tested functionally by methods known in the art, e.g., by its ability to reduce or abolish activity of factor Villa in a coagulation based assay or in factor X activation assay. See, e.g., the Example 1 for such methods.
  • peptidomimetics and nonpeptidomimetics are often superior to analogous peptides in therapeutic applications because the mimetics are generally more resistant to digestion than peptides.
  • included within the derivatives contemplated as part of the invention are the polypeptides disclosed above, wherein individual amino acids in the claimed sequence are substituted with linkers which are not amino acids but which allow other amino acids in the sequence to be spaced properly to allow binding to factor Villa.
  • the X aa of DRX aa T can be a linker to allow the D, R, and T to align properly to bind to factor Villa.
  • linkers are well known in the art and their design in this context would not require undue experimentation.
  • the factor Villa binding site on factor IXa forms a helix in the native IXa protein (see, e.g., Figure 7B).
  • Short polypeptides (ca. 4-15 amino acids) comprising the essential amino acid residues of this sequence (DRX aa T), or derivatives, are therefore most effective in binding to factor Villa when the polypeptide or derivative retains the native helical shape. It is known in the art that certain substitutions may be made in the sequence of short polypeptide helical sequences such as SEQ ID NO:1 to retain the helical shape and prevent undesirable secondary interactions.
  • valine (V) the native amino acid that immediately precedes the aspartic acid (D) in the native sequence can advantageously be substituted with an alanine (A).
  • the cysteine (C) residue immediately following the threonine residue in this helix can also be substituted with an alanine (A) to prevent disulfide bridges from forming between two C residues in two polypeptides.
  • other polypeptides or derivatives thereof within the scope of the present invention include ADRAT (SEQ ID NO:3) and DRATA (SEQ ID NO:4).
  • polypeptides or derivatives that comprise DRX aa T the X aa can also be glycine (G).
  • G can substitute for amino acids or spacers in many compositions since such a substitution allows increased flexibility in the compound. This is because G has hydrogen as its side chain, allowing flexability of rotation in its Ramachandran ⁇ and ⁇ angles.
  • amino acids in a polypeptide which retains maximal factor Villa binding will be the same as, or conservative substitutions for, the amino acids at analogous positions of SEQ ID NO: 1.
  • Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains.
  • Conservatively substituted amino acids can be grouped according to the chemical properties of their side chains.
  • one grouping of amino acids includes those amino acids that have neutral and hydrophobic side chains (A, V, L, I, P, W, F, and M); another grouping is those amino acids having neutral and polar side chains (G, S, T, Y, C, N, and Q); another grouping is those amino acids having basic side chains (K, R, and H); another grouping is those amino acids having acidic side chains (D and E); another grouping is those amino acids having aliphatic side chains (G, A, V, L, and I); another grouping is those amino acids having aliphatic- hydroxyl side chains (S and T); another grouping is those amino acids having amine- containing side chains (N, Q, K, R, and H); another grouping is those amino acids having aromatic side chains (F, Y, and W); and another grouping is those amino acids having sulfur- containing side chains (C and M).
  • A, V, L, I, P, W, F, and M amino acids having neutral and polar side chains
  • amino acids in SEQ ID NO: 1 preferred conservative amino acid substitutions groups are: R-K; E-D, L-M, and V-I. Based on the results disclosed in the Example 1, it is believed that retention of the leucine at position 337 is particularly important in retaining optimal retention of the binding site helix (see, e.g., Table
  • Polypeptides or derivatives comprising an amino acid sequence or derivative that prevents binding of factor IXa to factor Villa can be produced by a number of methods known in the art.
  • the peptide can be produced by standard synthetic procedures such as the "classical” Merrifield method of solid phase peptide synthesis or by using the FMOC strategy on a RAMPS multiple peptide synthesis system (DuPont Co., Wilmington Del.) as described in Caprino and Han, J, Org. Chem. 37:3404, 1972.
  • the polypeptide can be produced using standard molecular biological methods. See, e.g., Fredrick M. Ausubel et al. (1995), "Short Protocols in Molecular Biology", John Wiley and Sons.
  • factor IX mutants are made which alter amino acid residues being tested.
  • the amino acid mutations can consist of amino acid replacements or deletions, or replacements or deletions of an entire domain being tested.
  • the ability of those mutants to interfere with normal factor Villa binding and/or factor X activation is then determined, e.g., by determining the ability of the mutant to mediate clot formation, or, preferably, by measuring the apparent Kd (Kd, app) of binding of each mutant to Villa.
  • Comparisons of the mutants with wild type factor IXa are also preferably made in both the presence and the absence of phospholipid, since regions which affect phospholipid position in relation to factor IXa would affect Villa binding in the presence, but not in the absence, of phospholipid, whereas mutants in a region where Villa binding takes place would affect the binding whether phospholipid was present or not (see Example 1 ).
  • the present invention also provides nucleic acid molecules that comprise a nucleotide sequence, or the complement thereof, which encodes a polypeptide having anti-coagulation activity.
  • the polypeptide comprises the sequence DRX aa T, wherein X aa is independently any amino acid or derivative thereof.
  • the polypeptide comprises SEQ ID NO: 5 (LVXiX 2 AT, wherein X, is either D or Y, and X 2 is either R or Q), SEQ ID NO:6 (LVDRAT), SEQ ID NO:7 (LVYRAT), SEQ ID NO:8 (LVDQAT), SEQ ID NO:9 (LVDRATXLR, wherein X is any amino acid other than C), or SEQ ID NO: 10 (LVDRATALR).
  • LVXiX 2 AT wherein X, is either D or Y, and X 2 is either R or Q
  • SEQ ID NO:6 LVDRAT
  • SEQ ID NO:7 LVYRAT
  • SEQ ID NO:8 LVDQAT
  • SEQ ID NO:9 LPDRATXLR, wherein X is any amino acid other than C
  • SEQ ID NO: 10 LVDRATALR
  • the nucleic acid molecules can also encode a fusion protein comprising the polypeptide and a component, such as a histidine tag, to facilitate purification.
  • a fusion protein comprising the polypeptide and a component, such as a histidine tag, to facilitate purification.
  • the nucleotide sequence preferably encodes a polypeptide comprising SEQ ID NO:2 (DRAT) or SEQ ID NO: 10 (LVDRATALR).
  • the present invention provides methods for identifying agents having anti-coagulation activity.
  • the method comprises determining whether a candidate agent displaces the binding of a polypeptide or derivative that binds to factor Villa but does not activate factor X.
  • Suitable polypeptides or derivatives for these methods include any polypeptide or derivative which includes the sequence DRX aa T, wherein X aa is independently any amino acid or derivative thereof, and which will bind to factor Villa at the factor IXa binding site and prevent coagulation and factor X activation.
  • the preferred polypeptides or derivatives comprise the entire 9-mer binding site LVDRATCLR (SEQ ID NO:1), or the 4-mer within that sequence which contains the three crucial amino acid residues, DRAT (SEQ ID NO:2), or derivatives of those sequences.
  • the polypeptide comprises SEQ ID NO: 5 (LVXiX 2 AT, wherein Xi is either D or Y, and X 2 is either R or Q), SEQ ID NO:6 (LVDRAT), SEQ ID NO:7 (LVYRAT), SEQ ID NO:8 (LVDQAT), SEQ ID NO:9 (LVDRATXLR, wherein X is any amino acid other than C), or SEQ ID NO: 10 (LVDRATALR).
  • polypeptides or derivatives have 4-20 amino acid residues or the equivalent, but can comprise much larger sequences, including, for example, factor IXa which has been altered to eliminate its serine protease activity, e.g., by changing the active site serine to alanine.
  • the methods for identifying agents having anti-coagulation activity include mixing the candidate agent with factor Villa and the polypeptide or derivative, then determining whether less of the polypeptide or derivative binds to the factor Villa than the amount that binds in the absence of the polypeptide or derivative.
  • the candidate agent is mixed with factor Villa before adding the polypeptide or derivative in order to allow the candidate agent to optimally compete for factor Villa binding sites with the polypeptide or derivative.
  • the amount of binding of the polypeptide or derivative to the factor Villa can be determined by any of a number of methods that are well known in the art.
  • the polypeptide or derivative can be labeled with a radioactive agent or a dye such as a fluorescent dye, and unbound vs.
  • bound polypeptide or derivative can be determined by methods such as chromatography or electrophoresis, where the chromatographic or electrophoretic conditions are selected where unbound polypeptide migrates differently than polypeptide bound to factor Villa.
  • bound vs. unbound polypeptide or derivative can be determined by dialysis, using a membrane which allows the passage of unbound labeled polypeptide or derivative but not polypeptide or derivative bound to factor Villa.
  • Another alternative method for determining polypeptide or derivative bound to factor Villa is by the determination of displacement of labeled polypeptide from factor Villa that is adsorbed to a solid phase.
  • the present invention provides methods of treatment to prevent thrombosis in patients in need thereof.
  • the methods comprise administering to the patient a polypeptide or derivative which includes the sequence DRX aa T, wherein X aa is any amino acid or derivative thereof, and which will bind to factor Villa at the factor IXa binding site and prevent coagulation and factor X activation.
  • the preferred polypeptides or derivatives comprise the entire 9-mer binding site LVDRATCLR (SEQ ID NO:1), or the 4-mer within that sequence which contains the three crucial amino acid residues, DRAT (SEQ ID NO:2), or the equivalent derivatives.
  • the polypeptide comprises SEQ ID NO: 5 (LVXiX 2 AT, wherein Xi is either D or Y, and X 2 is either R or Q), SEQ ID NO:6 (LVDRAT), SEQ ID NO:7 (LVYRAT), SEQ ID NO:8 (LVDQAT), SEQ ID NO:9 (LVDRATXLR, wherein X is any amino acid other than C), or SEQ ID NO: 10 (LVDRATALR).
  • the most preferred compositions have 4-20 amino acid residues or derivatives, but can comprise much larger sequences, including, for example, factor IXa which has been altered to eliminate its serine protease activity.
  • Antibodies that specifically bind to the above polypeptides would also be expected to prevent thrombosis through their ability to bind the same region in factor IXa. Such antibodies can be produced without undue experimentation by the skilled artisan.
  • the patients in these methods can be any vertebrate animal. While the factor Villa binding region of factor IXa is invariant among all mammals tested (Table 2, in Example 1), it is believed that factor IXa from all vertebrates would have the same factor Villa binding region. However, preferred patients in these methods are mammals; most preferred patients are humans at risk for undesired thrombosis. Nonetheless, the utility of the methods with any vertebrate can be determined without undue experimentation by mixing the polypeptide or derivative with blood of the vertebrate and determining whether a clot forms as quickly as in the absence of the polypeptide. Alternatively, the utility of the method can also be assessed by evaluating binding of the polypeptide or derivative or by measuring formation of factor Xa, as described herein and/or as is well known in the art.
  • polypeptides or derivatives that bind to factor Villa at the factor IXa binding site to prevent coagulation and factor X activation can be administered to a vertebrate by any suitable route known in the art including, for example, intravenous, subcutaneous, intramuscular, transdermal, intrathecal, or intracerebral. Administration can be either rapid as by injection or over a period of time as by slow infusion or administration of a slow release formulation.
  • polypeptides or derivatives of the present invention are usually employed in the form of pharmaceutical preparations. Such preparations are made in a manner well known in the pharmaceutical art.
  • One preferred preparation utilizes a vehicle of physiological saline solution, but it is contemplated that other pharmaceutically acceptable carriers such as physiological concentrations of other non-toxic salts, five percent aqueous glucose solution, sterile water or the like may also be used. It may also be desirable that a suitable buffer be present in the composition.
  • Such solutions can, if desired, be lyophilized and stored in a sterile ampoule ready for reconstitution by the addition of sterile water for ready injection.
  • the primary solvent can be aqueous or alternatively non-aqueous.
  • the carrier can also contain other pharmaceutically-acceptable excipients for modifying or maintaining the pH, osmolarity, viscosity, clarity, color, sterility, stability, rate of dissolution, or odor of the formulation.
  • the carrier may contain still other pharmaceutically- acceptable excipients for modifying or maintaining release or absorption or penetration across the blood-brain barrier.
  • excipients are those substances usually and customarily employed to formulate dosages for parenteral administration in either unit dosage or multi-dose form or for direct infusion by continuous or periodic infusion. It is also contemplated that certain formulations comprising the polypeptides or derivatives are to be administered orally. Such formulations are preferably encapsulated and formulated with suitable earners in solid dosage forms.
  • suitable carriers include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, gelatin, syrup, methyl cellulose, methyl- and propylhydroxybenzoates, talc, magnesium, stearate, water, mineral oil, and the like.
  • the formulations can additionally include lubricating agents, wetting agents, emulsifying and suspending agents, preserving agents, sweetening agents or flavoring agents.
  • compositions may be formulated so as to provide rapid, sustained, or delayed release of the active ingredients after administration to the patient by employing procedures well known in the art.
  • the formulations can also contain substances that diminish proteolytic and nucleic acid degradation and/or substances that promote absorption such as, for example, surface active agents.
  • the polypeptide or derivative is administered to vertebrates in an amount effective to decrease thrombosis within the vertebrate.
  • the specific dose is calculated according to the approximate body weight or body surface area of the patient or the volume of body space to be occupied.
  • the dose will also be calculated dependent upon the particular route of administration selected. Further refinement of the calculations necessary to determine the appropriate dosage for treatment is routinely made by those of ordinary skill in the art. Such calculations can be made without undue experimentation by one skilled in the art in light of the activity disclosed herein in thrombosis assays. Exact dosages are determined in conjunction with standard dose-response studies.
  • the amount of the composition actually administered will be determined by a practitioner, in the light of the relevant circumstances including the condition or conditions to be treated, the choice of composition to be administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the chosen route of administration. Dose administration can be repeated depending upon the pharmacokinetic parameters of the dosage formulation and the route of administration used.
  • methods of treatment for preventing thrombosis in a patient. These methods comprise selecting an agent having anti- coagulation activity, wherein the selecting comprises testing the agent for activity in displacing the binding of factor IXa from factor Villa without activating factor X, and administering the agent to the patient.
  • the selecting step in this embodiment is identical to the method of identifying an agent having anti-coagulation activity previously disclosed, and can be performed in the same manner. Additionally, the treatment step in this embodiment can be performed as with the polypeptide or derivative treatment step disclosed above.
  • methods for preventing coagulation in a blood sample.
  • the methods comprise mixing a freshly drawn blood sample with a polypeptide or derivative which includes the sequence DRX aa T or derivatives, wherein X aa is any amino acid or derivative thereof, and which will bind to factor Villa at the factor IXa binding site and prevent coagulation and factor X activation.
  • the preferred polypeptides or derivatives comprise the entire 9-mer binding site LVDRATCLR (SEQ ID NO:1), or the 4-mer within that sequence which contains the three crucial amino acid residues, DRAT (SEQ ID NO:2), or derivatives of these sequences.
  • the polypeptide comprises SEQ ID NO: 5 (LVXiX 2 AT, wherein Xi is either D or Y, and X 2 is either R or Q), SEQ ID NO: ⁇ (LVDRAT), SEQ ID NO:7 (LVYRAT), SEQ ID NO:8 (LVDQAT), SEQ ID NO:9 (LVDRATXLR, wherein X is any amino acid other than C), or SEQ ID NO: 10 (LVDRATALR).
  • the most preferred compositions have 4-20 amino acid residues or derivatives, but can comprise much larger sequences, including, for example, factor IXa which has been altered to eliminate its serine protease activity. An antibody that specifically binds to these sequences would also be expected to prevent coagulation in a blood sample. Such antibodies can be produced without undue experimentation by a skilled artisan.
  • the polypeptide or derivative can be added to the blood sample as a liquid or dried preparation.
  • the polypeptide can be present in the container that receives the blood sample (for example a vacutainer), in order for the blood sample to be exposed to the polypeptide when the sample enters the container.
  • the quantity of the polypeptide or derivative added to the container can be determined without undue experimentation, merely by determining the quantity of the polypeptide or derivative necessary to prevent coagulation of the quantity of blood which is to be drawn in the sample.
  • Other embodiments of the invention include the provision of methods for detecting factor Villa in a sample.
  • these methods comprise contacting the sample with a polypeptide or derivative which will bind to factor Villa at the factor IXa binding site and prevent coagulation and factor X activation, then determining whether the polypeptide or derivative has bound factor Villa.
  • the polypeptide or derivative comprises a covalently attached detectable moiety, as previously disclosed.
  • the polypeptide comprises the sequence DRX aa T or derivative, wherein X aa is any amino acid or derivative thereof.
  • the preferred polypeptides comprise the entire 9-mer binding site LVDRATCLR (SEQ ID NO: 1), or the 4-mer within that sequence which contains the three crucial amino acid residues, DRAT (SEQ ID NO:2), or derivatives of these sequences.
  • the polypeptide comprises SEQ ID NO: 5 (LVXiX 2 AT, wherein Xi is either D or Y, and X 2 is either R or Q), SEQ ID NO:6 (LVDRAT), SEQ ID NO:7 (LVYRAT), SEQ ID NO:8 (LVDQAT), SEQ ID NO:9 (LVDRATXLR, wherein X is any amino acid other than C), or SEQ ID NO: 10 (LVDRATALR).
  • the most preferred polypeptides have 4-20 amino acid residues, but can comprise much larger sequences, including, for example, factor IXa which has been altered to eliminate its serine protease activity.
  • the determination of whether the polypeptide or derivative has bound factor Villa can be accomplished by any method known in the art, for example by methods such as chromatography or electrophoresis, where the chromatography or electrophoretic conditions are selected such that labeled polypeptide or derivative that is bound to factor Villa will have a different migration rate than unbound polypeptide, or dialysis, using a membrane that allows passage of unbound, labeled polypeptide but not labeled polypeptide or derivative bound to factor Villa.
  • activation of factor X can be evaluated in the presence or absence of the polypeptide or derivative, as previously discussed. See also Example 1. Industrial Application
  • compositions and methods of the present invention provide novel treatments to prevent thrombosis, methods for preventing coagulation in blood samples, and methods for identifying agents that have anti-coagulation activity.
  • This example describes the determination and characterization of the factor Villa binding region of factor IXa.
  • the following reagents were used. Benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide (S-2222) was purchased from Helena Laboratories. Dansyl-Glu-Gly-Arg-chloromethyl ketone (DEGR-ck) was obtained from Calbiochem. Phosphatidylcholine, phosphatidylserine, recombinant hirudin, and fatty acid free bovine serum albumin (BSA) were obtained from Sigma Chemical Co.
  • BSA fatty acid free bovine serum albumin
  • Factor IX and factor VlII deficient plasmas were purchased from Sigma Chemical Co.
  • IX NP normal human plasma factor IX
  • factor Xa Factor Xa
  • Purified human factor XIa, protein C, activated protein C, and ⁇ -thrombin (Ha) were purchased from Enzyme Research Laboratories (South Bend, IN).
  • Recombinant human tissue factor of aa 1-243 containing the transmembrane domain was generously provided by Genentech Inc.
  • the preparation was free of all other coagulation factors and contained human albumin as a stabilizing agent.
  • Purification of a mouse monoclonal antibody (mAb) that inhibits the interaction of factor IXa with factor Villa was as described in Bajaj et al., 1985, J. Biol. Chem. 260:11574-1 1580.
  • factor IXa sites were generated as follows.
  • the pRc/CMV vector (Invitrogen) was used for expression of wild-type and each mutant factor IX.
  • Hind III and Xba I sites in the multiple cloning sites of the vector were used for ligation of the DNA.
  • the mutant primer was based upon the factor IX gene sequence (Yoshitake et al., supra) and corresponded to six codons (18 bases) with a mutant base at the desired position involving the third codon.
  • the base substitution for each point mutant was: L330I (CTT ⁇ ATT), V331 A (GTT ⁇ GCT), D332Y (GAC ⁇ TAC), R333L (CGA ⁇ CTA), R333Q (CGA ⁇ CAA), T335A (ACA ⁇ GCA), L337I (CTT ⁇ ATT) and R338Q (CGA ⁇ CAA).
  • Factor IX he i, x vii in which 330-338 residues of factor IX [chymotrypsin 162-170] were replaced by the corresponding residues of factor VII, was constructed using a 63-base primer. The first 18 bases of this primer corresponded to factor IX gene sequence coding for residues 324 to 329 [chymotrypsin 156 to 161] followed by 27 bases from the factor VII gene sequence (O'Hara et al., 1987, Proc. Natl. Acad. Sci.
  • Coomassie Brilliant Blue staining is shown in Fig IA.
  • ⁇ -carboxyglutamic acid concentration of these proteins was also determined by Commonwealth Biotechnologies, Inc., Richmond, VA. Automated Edman degradation of each factor IX protein (-0.5 nmol) was performed using an Applied Biosystems gas phase sequencer, ⁇ -carboxy glutamic acid analysis of each sample was performed by alkaline hydrolysis followed by HPLC analysis. The amount of ⁇ -carboxyglutamic acid was quantitated based upon the 46 residues of Asp and Asn present per mol of factor IX. Plasma factor IX and each recombinant protein had 11.5 to 12.5 ⁇ -carboxyglutamic acid residues per mol.
  • the N-terminal sequence of each protein was also determined. All recombinant proteins revealed a major and a minor N-terminal sequence. The major sequence in each case was Tyr-Asn-Ser-Gly-Lys and the minor sequence in each case was Thr-Val-Phe. The major sequence corresponds to the sequence of mature protein in plasma, and the minor sequence corresponds to the protein in which the prosequence has not been cleaved (Yoshitake et al., supra). The minor sequence was not detected in plasma factor IX and it amounted to less than 5% in each recombinant protein.
  • the relative coagulant activity of each protein was: IXNP, 100% (I SO ⁇ IO units/mg); IX W ⁇ , ⁇ 90%; IXL 330 I, ⁇ %; IX V S S I A , ⁇ 6%; IX 0332 Y, ⁇ 2%; IXR333L, ⁇ 0.3%; IXR 333 Q, -0.5%; IX 1335 A, -40%; IX L337[) -1%; IX R338Q , -65%; and IX he ,,w ⁇ , not measurable.
  • Factor XWCa 2+ was also determined, under conditions described in Mathur et al., 1997, supra, either by VIIa/TF/Ca 2+ or by factor XIaZCa 2+ .
  • the rates of the mutants were similar to that of IXNP as analyzed by SDS gel electrophoresis.
  • the 90 min activation sample of IX W T and each mutant is shown in Fig IB.
  • coagulant activity of XIa- activated IXa WT was -95%
  • of IXa L 33oi was -7%
  • of IXa V 33iA was -6%
  • Of IXa 0332 Y was -2%
  • of IXa R333L was -0.4%
  • of IXaR 333 Q was -0.6%
  • of IXa T3 35A was -35%
  • of IXaL 3371 was 0.8%
  • IXaR 338 Q was -80%
  • IXaheiixvn was not measurable.
  • each activated mutant to activate factor X in the presence and absence of phospholipid and factor Villa was determined as follows. The activation was earned out at 37 0 C in a 50 ⁇ l reaction volume, in TBS/BSA pH 7.4, for various time periods. At the end of the incubation time period, each reaction mixture received 1 ⁇ l of 0.5 M EDTA to stop further generation of factor Xa. A 40 ⁇ L aliquot was then added to 0.1 ml quartz cuvette containing S-2222 in 75 ⁇ L of TBS/BSA, pH 7.4. The final concentration of S-2222 was 100 ⁇ M.
  • Factor Xa generated was calculated from a standard curve constructed using factor Xa prepared from insolubilized Russell's viper venom.
  • Factor X activation measurements were made under four experimental regimes. In one, Ca 2+ and phospholipid were present. In this system activation was carried out for 5-15 minutes and the concentration of each IXa protein was 20 nM, phospholipid was 25 ⁇ M, and factor X was 100 nM. In the second regime, Ca 2+ , phospholipid and factor Villa were present. Activation was carried out here for 15-120 sec and the concentration of each IXa protein was 0.5 nM, phospholipid was 10 ⁇ M, Villa was 0.07 nM and factor X was 15 nM. In the third regime, only Ca 2+ was present.
  • activation was carried out for 2-20 min and concentration of each IXa protein was 400 nM and factor X was 1 ⁇ M.
  • concentration of each IXa protein was 400 nM and factor X was 1 ⁇ M.
  • Ca 2+ and Villa were present.
  • activation was carried out for 15-120 sec and the concentration of each IXa protein was 2 ⁇ M, Villa was 14 nM, and factor X was 400 nM.
  • Rates of factor X activation versus phospholipid showed a bell shaped curve with a broad optima between 20-40 ⁇ M phospholipid. The rate increased linearly from 0-20 ⁇ M and after 40 ⁇ M it showed a gradual decrease. Therefore, phospholipid concentration in the absence of factor Villa was fixed at 25 ⁇ M. In the presence of factor Villa, 10 ⁇ M phospholipid was used based upon previous observations (Mathur et al., 1997, supra; van Dieijen et al. ; 1981, J. Biol. Chem. 256, 3433- 3442).
  • the concentrations of factor X selected for each set of reaction conditions are those which are below or at the Km values (van Dieijen et al., supra; Fay and Koshibu, 1998, J. Biol. Chem. 273, 19049-19054).
  • the rate of formation of factor Xa is proportional to the substrate factor X and therefore to the affinity of the factor IXa enzyme for factor X.
  • IXa R338Q it was the same as IXa W ⁇ and for other mutants (see Table 1) it could not be measured.
  • factor VIHa concentration was increased from 70 pM to 14 nM, the following rates of factor X activation were obtained — IXa D332Y -4.1 nM/min, IXa R333 L ⁇ 2.7 nM/min, IXa R333 Q -2.4 riM/min, and IXa L337 i ⁇ 3.2 nM/min; for IXa he i.wir it was still not measurable.
  • the IXa mutants also activated factor X in the Ca 2+ /VIIIa system at rates that were slower than those obtained with IXa W ⁇ (or IXa NP ).
  • Table 1 Considering a kcat value of 1.1/niin, a km of 380 nM and an ECs 0 (functional Kd of IXa: Villa interaction) value of 2.2 ⁇ M (16) at 400 nM factor X concentration used in our system, the expected rate of Xa formation using 14 nM Villa and 2 ⁇ M IXa would be 3.3 nM/min.
  • the experimental rate obtained with IXa W ⁇ (or IXa NP ) was -2.75 nM/min, a value close to the expected value.
  • the rate of activation by IXa L33O i was -0.94 nM/min
  • by IXa V3 3i A was -0.54 nM/min
  • by IXa T33 5A was -1.83 nM/min
  • by IXa R338Q was ⁇ 2.22 nM/min. Under these conditions, i.e., in the presence of limiting concentrations of Villa, the rates of factor X activation by other mutants could not be measured.
  • reaction mixtures (50 ⁇ l) in the presence of phospholipid contained 0.2 nM factor IXawT, 0.48 ⁇ M factor X, 0.07 nM factor Villa, 5 mM Ca 2+ , 10 ⁇ M phospholipid and varying concentrations of DEGR-IXa proteins.
  • reaction mixtures in the absence of phospholipid contained 0.1 nM factor IXa W ⁇ , 2 ⁇ M factor X, 14 nM factor Villa, 5 mM Ca 2+ , and varying concentrations of DEGR-IXa proteins. Reactions were carried out for 2 min and the rates of factor X activation were measured as outlined above.
  • DEGR-IXa NP , DEGR-IXa WT1 and various DEGR-IXa mutant proteins were prepared as described in Mathur et al., 1997, supra, and free DEGR-ck was removed as described in Sabharwal et al., 1995, supra and Krishnaswamy, 1992, J. Biol. Chem. 267, 23696-23706.
  • DEGR-activated protein C was prepared similarly.
  • factor IXa W ⁇ concentrations are below the EC 50 values at the factor X concentrations used. Further, in each case ⁇ 10% of factor IXa is bound to factor Villa in the absence of the competitor and no measurable rates of activation of factor X were observed under these conditions in the absence of added factor Villa.
  • IC 50 values concentration of DEGR-IXa yielding 50% inhibition
  • y is the rate of Xa formation in the presence of a given concentration of DEGR-IXa protein represented by x
  • a is the maximum rate of Xa formation in the absence of DEGR- IXa
  • s is the slope factor.
  • y is the rate of Xa formation in the presence of a given concentration of DEGR-IXa protein represented by x
  • a is the maximum rate of Xa formation in the absence of DEGR- IXa
  • s is the slope factor.
  • IXa V ii h eii x mutant failed to bind to factor Villa at concentrations -140,000- fold greater than the Kd,app for IXa W ⁇ in the presence of phospholipid and at concentrations ⁇ 150- fold greater than the Kd,app in the absence of phospholipid.
  • both the kinetic and the binding data provide strong evidence that the helix-330 in the protease domain of factor IXa provides a critical binding site for factor Villa.
  • Both mutants had the same N-terminal sequence and the ⁇ -carboxyglutamic acid content as normal IX and could be readily activated to IXa-like molecules by factor XIa. As discussed above, these mutants also bind to the mAb (that interferes with the IXa: Villa interaction) with the same affinity (kd ⁇ 15 nM) as normal IX. In the Ca 2+ / phospholipid system (same conditions as in figure 2A, i.e., 20 nM IXa and 100 nM X), IXaQ 50P activated factor X at 0.07 nM/min and IXa PC EGFi at 0.06 nM/min.
  • helix-330 In order to identify the region in the protease domain for IXa: Villa interaction, the role of surface-exposed helix-330 was studied, as reported above. The position of this helix is shown in Figure 7A. The sequence in this helix is identical in factor IX from all species (Bajaj et al., 1993, supra) and is different from all other homologous blood coagulation serine proteases (Table 2). Further, helix-330 is located 12 residues away from the autolysis loop cleavage site to which it is connected via a single ⁇ -strand, marked 2 in Figure 7A. Moreover, point mutations in eight of the nine residues in this helix are reported to cause hemophilia B (Giannelli et al., 1998, Nucleic Acids Res. 26, 265-268).
  • a modeled structure of human factor IXa was obtained using a homology model building approach described earlier (Bajaj, 1993, supra).
  • the starting template used was the structure of porcine factor IXa (Ref. 2, code IPFX). Since the model structure of human factor IXa for all purposes as it relates to this paper was the same as porcine factor IXa and the residues involved at the mutational sites are identical between the two proteins, the X-ray structure of porcine factor IXa was also utilized for analysis of the above data.
  • the model of IXa PCEGF i protein was constructed by replacing the EGFI domain of factor IXa with that of activated protein C (Mather et al., 1996, EMBO J. 15, 6822-6831 ) and is more fully described in Bajaj, 1999, Thrombos. Haenwstas.82: ⁇ 663- ⁇ 612, incorporated herein by reference.
  • change of L337[169] to 1337 may result in the disruption of another hydrophobic pocket and a slight shift of the helix.
  • change of R338[170] to Q338[170] resulted in a minimal loss of biologic activity and affinity for factor Villa (Table 1). This is consistent with a slightly increased activity of R338A mutant without an impairment in factor Villa binding described by Chang and coworkers (Chang et al., 1998, J. Biol. Chem. 273, 12089- 12094). Note that all of the helix-330 point mutants have impaired interaction with factor Villa both in the presence and absence of phospholipid.
  • IXa h eii x vn mutant failed to interact with factor Villa even at very high concentrations both in the presence or absence of phospholipid.
  • Hemophilia B mutants not expressed in the present study in the helix-330 may have impaired interactions with factor Villa due to the following reasons.
  • a change of L330[162] to proline may result in a turn accompanying a subtle directional change in the propagation of the helix, and a change of V33 If 163] to aspartic acid is expected to disrupt the hydrophobic interactions depicted in Figure SB.
  • Replacement of R333[165] by glycine, and of A334[166] by aspartic acid or threonine could disrupt the direct binding of factor IXa to factor Villa.
  • a change of C336 to any other residue will disrupt the disulfide bond and a possible change in the local tertiary structure.
  • helix-330 in IXa provides a crucial binding site for Villa and that the EGFl domain in this context primarily serves to correctly position the protease domain above the phospholipid surface for optimal interaction with Villa.
  • IXaV 33 IA 0.55 ⁇ 0 0.54 ⁇ 0.02 1.35 ⁇ 0.31
  • IXapCEGFl NM 5.71 ⁇ 0.82 2.71 ⁇ 0.27 0.21 ⁇ 0.01 Table 2. Sequence of helix-330 in vitamin K dependent four coagulant and one anticoagulant (Protein C) serine proteases. The sequence of helix-330 is identical in factor IX from human, bovine, porcine, canine, rabbit, sheep, guinea pig, mouse and rat. For comparison, the residue number for each protein corresponding to residue 162 in chymotrypsin is given in parenthesis. A hyphen indicates the same residue as in factor IX. All sequences are taken from Bajaj and Birktoft, 1993, Methods Enzymol. 222, 96-128.
  • Factor VlII was activated at a concentration of 40 units/mL with 0.2 nM thrombin in TBS (Tris-HCl pH 7.4) containing 1 mg/mL BSA and 5 mM CaCl 2 . The reaction was incubated at 37° C for 2 minutes at which time hirudin was added.
  • Factor LX Peptide Competition Assay in the presence of factor Villa was activated at a concentration of 40 units/mL with 0.2 nM thrombin in TBS (Tris-HCl pH 7.4) containing 1 mg/mL BSA and 5 mM CaCl 2 . The reaction was incubated at 37° C for 2 minutes at which time hirudin was added.
  • Factor LX Peptide Competition Assay in the presence of factor Villa
  • Each reaction contained 0.2 nM factor IXa, 480 nM factor X, 0.07 nM factor Villa, various concentrations of peptide, 10 ⁇ M phospholipid, and 5 mM CaCl 2 in TBS (Tris-HCl, pH 7.4) containing 1 mg/mL BSA. Each reaction was incubated for either 30, 60, 90, or 120 seconds and the reaction was stopped by adding 1.5 ⁇ L of 0.5 M EDTA and placed on ice.
  • reaction mixtures were incubated for 2.5, 5, 10, 15, and 20 minutes at 37° C and were stopped with the addition of 1.5 ⁇ L of 0.5 M EDTA and placed on ice. Forty ⁇ L of each reaction was added to 100 ⁇ L of TBS/BSA in a 96 well microtiter plate and S-2222 was added to a final concentration of 100 ⁇ M. The change in absorbance at 405 nM was then observed using a Bio Rad microplate reader. Table 3

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Abstract

La présente invention concerne de nouveaux polypeptides ou dérivés comprenant sur le facteur IXa le site de liaison au facteur VIIIa. Ces nouveaux polypeptides ou dérivés ont une activité anti-coagulation. L'invention concerne également des acides nucléiques codant ces polypeptides. L'invention concerne aussi des procédés permettant d'identifier un agent à activité anti-coagulation en déterminant si l'agent déplace le polypeptide ou dérivé de son site de liaison au facteur VIIIa. L'agent ainsi identifié convient également pour la prévention de la thrombose chez un patient par administration de cet agent au patient. L'invention concerne aussi d'autres procédés de préventifon de la thrombose chez un patient, par administration à ce patient d'un polypeptide ou dérivé comprenant, sur le facteur IXa, le site de liaison au facteur IXa. L'invention concerne en outre des procédés de prévention de la coagulation dans un prélèvement sanguin, par adjonction des polypeptides ou dérivés de l'invention. L'invention concerne enfin des procédés permettant de détecter le facteur VIIIa dans un prélèvement. A cet effet, on met l'échantillon en contact avec le polypeptide ou dérivé de l'invention qui comprend également un groupe fonctionnel détectable attaché par covalence, puis on vérifie si le polypeptide ou dérivé se lie au facteur VIIIa du prélèvement.
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Publication number Priority date Publication date Assignee Title
US9982248B2 (en) 2008-09-15 2018-05-29 Uniqure Biopharma B.V. Factor IX polypeptide mutant, its uses and method for its production

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Publication number Priority date Publication date Assignee Title
WO1995026979A1 (fr) * 1994-04-05 1995-10-12 The Board Of Trustees Of The Leland Stanford Junior University Regulation de l'activite des lymphocytes t cytotoxiques par des peptides de cmh de classe i
US6624289B1 (en) * 1999-06-16 2003-09-23 Saint Louis University Region of factor IXa protease domain that interacts with factor VIIIa and methods therefor

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995026979A1 (fr) * 1994-04-05 1995-10-12 The Board Of Trustees Of The Leland Stanford Junior University Regulation de l'activite des lymphocytes t cytotoxiques par des peptides de cmh de classe i
US6624289B1 (en) * 1999-06-16 2003-09-23 Saint Louis University Region of factor IXa protease domain that interacts with factor VIIIa and methods therefor

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9982248B2 (en) 2008-09-15 2018-05-29 Uniqure Biopharma B.V. Factor IX polypeptide mutant, its uses and method for its production
US10465180B2 (en) 2008-09-15 2019-11-05 Uniqure Biopharma B.V. Factor IX polypeptide mutant, its uses and method for its production

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