WO1995017420A1 - Analogues de peptides du site de fixation plaquettaire active sur le facteur xi - Google Patents

Analogues de peptides du site de fixation plaquettaire active sur le facteur xi Download PDF

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WO1995017420A1
WO1995017420A1 PCT/US1994/013885 US9413885W WO9517420A1 WO 1995017420 A1 WO1995017420 A1 WO 1995017420A1 US 9413885 W US9413885 W US 9413885W WO 9517420 A1 WO9517420 A1 WO 9517420A1
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peptide
factor
seq
amino acid
conformation
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PCT/US1994/013885
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English (en)
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Peter N. Walsh
Frank A. Baglia
Bradford A. Jameson
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Temple University - Of The Commonwealth System Of Higher Education
Thomas Jefferson University
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Priority to AU13978/95A priority Critical patent/AU1397895A/en
Publication of WO1995017420A1 publication Critical patent/WO1995017420A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6443—Coagulation factor XIa (3.4.21.27)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21027—Coagulation factor XIa (3.4.21.27)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to synthetic peptide analogs of factor XI heavy chain.
  • Human factor XI is a plasma glycoprotein that participates in the contact phase of blood coagulation. Fujikawa et. al., Biochemistry 25. 2417-2424 (1986) (incorporated herein by reference) disclose the amino acid sequence of factor XI, deduced from the sequence of a cDNA insert coding for factor XI.
  • Factor XI circulates in plasma as a complex with its nonenzymatic cofactor, high molecular weight kininogen.
  • the complex of factor XI and kininogen can become bound to an anionic surface, where factor XI can be activated by factor Xlla.
  • An example of an anionic surface to which the complex can become bound is an activated platelet surface.
  • the complex binds specifically to high affinity, saturable receptors on activated platelets.
  • factor XI of the complex becomes bound to an activated platelet, rates of factor XI activation by Xlla can be increased.
  • Activated platelets can promote factor XI activation by factor Xlla in the presence of high molecular weight kininogen.
  • activated platelets can promote factor XI activation in the absence of factor XII.
  • factor XI Activation of factor XI results in a serine protease, factor Xla, consisting of two identical light chains and two identical heavy chains bridged by disulfide bonds.
  • the heavy chain contains four tandem repeat sequences (designated A1, A2, A3 and A4 ), comprising four separate domains.
  • Factor Xla of the complex remains bound to the activated platelet site and recognizes factor IX as its normal macromolecular substrate.
  • Factor Xla catalyzes the activation of factor IX, which can lead to intrinsic coagulation.
  • Two inhibitors of factor Xla enzymatic activity in human plasma are the serpins, 0-1-proteinase inhibitor and antithrombin III.
  • Two other inhibitors are protease nexin II, which is a truncated form of the transmembrane Alzheimer's amyloid ß-protein precursor, and platelet inhibitor of factor XI (PIXI), which is a low molecular weight 8,500 Da protein from platelets. None of these four inhibitors significantly inhibit platelet-bound factor XI.
  • Activated factor IX can be produced by factor Xla enzymatic activity and can bind to a factor IX/IXa binding site on the platelet surface.
  • factor IXa can be produced by factor Xla enzymatic activity and can bind to a factor IX/IXa binding site on the platelet surface.
  • the binding of factors IX/IXa and Villa to their respective sites on the platelet membrane results in a twenty million-fold acceleration in the catalytic efficiency of factor X activation.
  • platelet surface-localized factor IX activation results in enhanced intrinsic coagulation results.
  • factor XI The activation of factor XI and sustained expression of its enzymatic activity at the platelet surface are key biological events in hemostasis. Moreover, the binding of factor XI to the platelet surface protects it from inactivation by both plasma and platelet derived inhibitors. Bound, activated factor XI will continue its protected enzymatic activity at the platelet surface irrespective of the presence of factor Xla inhibitors. Since high molecular weight kininogen is necessary for factor XI to be efficiently bound to platelets (Sinha, et al. J. Clin. Invest. 73 1550-1556, at page 1551, col. 2, 13, and page 1552, col.
  • factor XI binds indi- rectly to platelets through kininogen. See, Greengard et al. Biochem., 25, 3884-3890 (1986).
  • the high molecular weight kininogen binding site on domain A1 of the factor XI heavy chain has been characterized by Baglia et al., J. Biolog. Chem. 267, 4247-4252 (1992); and Baglia et al., J. Biolog. Chem. 265, 4149-4154 (1990) (each incorporated herein by reference).
  • a computer structural model useful for producing constrained peptides capable of inhibiting the binding of factor XI to high molecular weight kininogen was also characterized. Artificially constrained peptides according to the computer model were synthesized, which correspond to amino acids 44 (Thr) to 86 (Ser) in the A1 domain of the intact factor XI heavy chain. See, Baglia et al. J. Biolo ⁇ . Chem. 267, 4247-4252 (1992). The peptides are capable of inhibiting the binding of factor XI to high molecular weight kininogen. Examples of such peptides are SEQ ID NOS: 13, and 17-22.
  • Existing methods of preventing or treating arterial and venous thrombosis involve inhibiting the blood coagulation cascade with oral anticoagulants, heparin or other anticoagulants, or alternatively by pharmacologically inhibiting platelets.
  • oral anticoagulants such as coumarin-like drugs are used to inhibit the synthesis of vitamin K-dependent proteins. They block many coagulation reactions, involving proteins such as prothrombin, factor VII, factor IX and factor X.
  • Heparin by potentiating the action of antithrombin III, accelerates inactivation of thrombin, factor Xa and a variety of other plasma serine proteases.
  • a synthetic peptide comprising an amino acid sequence corresponding to a portion of the sequence of the binding site for activated platelets on the heavy chain of XI.
  • the peptide has an artificially restricted conformation and the ability to inhibit the binding of factor XI to activated platelet surfaces.
  • the invention is directed to a method of designing a peptide analog to the binding site for activated platelets on the factor XI heavy chain.
  • the distance between two parts of a molecular model of the substrate binding site is determined at conformational equilibrium.
  • the primary structure of the binding site is then modified to restrict that distance to the determined distance.
  • a peptide comprising the modified primary structure is then synthesized.
  • a method of producing a peptide having a restricted conformation is provided. Accordingly, a peptide having an amino acid sequence corresponding to a portion of the sequence of the binding site for activated platelets on the factor XI heavy chain is provided.
  • the conformational equilibrium of that portion of the factor XI heavy chain is determined.
  • a covalent modification is introduced into the peptide to restrict a distance between two parts of it to a distance between corresponding parts of the peptide in the equilibrium confirmation determined.
  • the invention further provides pharmaceutical compositions comprising one or more of the peptides according to the invention corresponding to a portion of the sequence of the binding site for activated platelets on the factor XI heavy chain, or a pharmaceutically acceptable salt thereof, in combination with a pharmaceutically acceptable carrier.
  • Preferred pharmaceutical compositions further comprise a second synthetic peptide having an amino acid sequence corresponding to a portion of the sequence of the binding site for high molecular weight kininogen on the heavy chain of XI, or a pharmaceutically acceptable salt of the second peptide; wherein the second peptide has an artificially restricted conformation and the ability to inhibit the binding of factor XI to high molecular weight kininogen.
  • the invention also provides a method of inhibiting the binding of factor XI to activated platelets and factor Xla-induced activation of factor IX on a platelet surface.
  • the activated platelets are contacted with one or more peptides of the invention, corresponding to a portion of the sequence of the binding site for activated platelets on the factor XI heavy chain, which peptide competes with factor XI in the binding of the activated platelets.
  • Activation of factor IX on the platelet surface is thus indirectly inhibited by the peptides of the invention.
  • Inhibition of factor IX activation on the platelet surface in turn inhibits factor IX' s coagulant activity.
  • the peptides of the invention are potent anticoagulants, having antithrombotic utility.
  • a preferred method for inhibiting the binding of factor XI to activated platelets and preventing the factor Xla-induced activation of factor IX on a platelet surface also comprises contacting activated platelets with a second synthetic peptide corresponding to a portion of the sequence of the binding site for high molecular weight kininogen on the heavy chain of XI, or a pharmaceutically acceptable salt of said peptide; wherein the second peptide has an artificially restricted conformation and the ability to inhibit the binding of factor XI to high molecular weight kininogen.
  • platelet binding site or “activated platelet binding site” on factor XI heavy chain is meant the region of the intact factor XI polypeptide chain comprising from about amino acid 193 (Ala) to about amino acid 266 (Arg) of the mature polypeptide, corresponding to amino acid 13 (Ala) to amino acid 86 (Arg) of SEQ ID NO:1.
  • high molecular weight kininogen binding site on factor XI heavy chain is meant the region of the intact factor XI polypeptide chain comprising from about amino acid 44 (Thr) to about amino acid 86 (Ser) of the mature polypeptide, corresponding to SEQ ID NO: 22.
  • sequence corresponds to a portion of an identified binding site
  • sequence which comprises a sequence segment identical to a portion of the identifed binding site sequence or a sequence segment derived from a three-dimensional model of a portion of the identified binding site sequence.
  • Figure 1 is a graph showing the effect of synthetic factor XI domain A3-derived peptides according to the invention on the binding of radio labelled factor XI to activated platelets in the presence of ZnCl 2 (25 ⁇ M), CaCl 2 (2 mM), and high molecular weight kininogen (42 nM).
  • the binding of 125 I-factor XI was compared to control binding in the absence of competing peptides.
  • the percentage of factor XI binding was then plotted against the concentration of the synthetic peptide.
  • the experimental protocol is set forth in detail in Example 13 (d) below.
  • Figure 2 is a graph showing the effect of factor XI and synthetic factor XI heavy chain domain A1-, A2-, A3-, and A4 -derived peptides on the binding of radiolabelled factor XI to activated platelets in the presence of ZnCl 2 (25 ⁇ M), CaCl 2 (2 mM), and high molecular weight kininogen (42 nM).
  • ZnCl 2 25 ⁇ M
  • CaCl 2 (2 mM)
  • high molecular weight kininogen 42 nM
  • the binding of 125 I- factor XI was compared to control binding in the absence of competing XI or competing peptides.
  • the percentage of factor XI binding was then plotted against the concentration of XI or the synthetic peptide.
  • the experimental protocol is set forth in detail in Example 13 (d) below.
  • A1, A2, A3 and A4 Four tandem repeat sequences (designated A1, A2, A3 and A4) comprising four separate domains, are present in the factor XI heavy chain.
  • the binding site consists of the sequence of amino acids Ala 193 to Arg 266 in the A3 domain.
  • the sequence consists of anti-parallel ⁇ -strands connected by ⁇ -turns, forming three stem-loop structures. We have found that these three stem-loop structures together form a continuous surface which is utilized for the binding of platelets.
  • the deduction of the platelet binding site structure was accomplished by computer modeling to calculate a testable three-dimensional structure utilizing the primary amino acid sequence and disulfide linkages within the A3 domain.
  • the calculated structure shows that the three stem- loop structure are defined by amino acid residues Pro 229 - Gln 233, Thr 241 - Leu 246 and Ser 248 - Ser 261, which correspond to SEQ ID NO:1, amino acids 49-53, 61-66, and 68-81, respectively.
  • the modeled A3 domain structure is used as a design template for synthesizing peptides according to the present invention that are expected to adopt a conformational repertoire overlapping that of the native protein.
  • the sequences identified herein from the factor XI heavy chain sequences identified herein from the factor XI heavy chain have not been previously identified as inhibitory of XI binding to platelets, and thus inhibitory of factor IX activation on the platelet surface.
  • the peptides of the invention which mimic the platelet binding site on factor XI and factor Xla, are potent inhibitors on the platelet surface of the enzymatic activity of factor Xla against its macromolecular substrate, factor IX.
  • the peptides are potent anticoagulants, which are believed useful as antithrombotic agents.
  • an antithrombotic agent should interfere with intrinsic coagulation reactions leading to the activation of factors XI and IX, while leaving extrinsic coagulation reactions intact, so that normal hemostatic plug formation can occur at sites of vascular injury.
  • the peptides of the invention by virtue of their specificity for the platelet binding site on factor Xl/XIa, are believed to inhibit factor XIa-catalyzed factor IX activation on the surface of platelets, without affecting the extrinsic pathway of blood coagulation involving factors VII, X and V, and prothrombin.
  • constraints are artificially introduced, e.g., disulfide bonds, to limit the conformational freedom of a synthetic peptide that incorporates the relevant amino acids.
  • Certain conformationally-restricted synthetic peptide analogs having the ability to inhibit the binding of factor XI or factor Xla to platelets correspond to factor XI heavy chain residues 225-236, 229-233, 241-246 and 248-261, according to the numbering of the amino acids of the mature polypeptide.
  • the model disclosed herein may be utilized to prepare additional conformationally-restricted synthetic peptides having similar activity.
  • Appendix 1 included herein contains the set of Brookhaven coordinates and connect statements specifying our equilibrium conformation model of the major portion of factor XI heavy chain domain A3 comprising the 85 amino acids spanning positions Ala 181 to Cys 265, inclusive (SEQ ID NO : 1, amino acids 1-85). The remaining amino acids of the A3 segment, Arg 266 and His 267 - Phe 272 (SEQ ID NO:16), of the factor XI heavy chain were truncated.
  • the corresponding graphic molecular model satisfying these coordinates may be generated by inputting the coordinates and connect statement into any of the many commercially available molecular modeling programs which are capable of reading files in the Brookhaven format.
  • Such programs include, for example, those of BioDesign, Inc., Pasadena, CA; Biosym Technologies, San Diego, CA; Tripos, St. Louis, MO; Polygen, Waltham, MA; and Chemical Design Ltd., Oxford, UK.
  • the data may be entered as an ASCII file.
  • each of the atoms of factor XI heavy chain residues 181-265 is assigned a number and respective X, Y and Z coordinates.
  • the coordinate portion of the listing begins with the alanine residue (ALA 1) at position 181 of the mature factor XI heavy chain.
  • the atom types are identified as “N” for nitrogen, “HN” for hydrogen which is connected to a nitrogen atom, "C” for carbon, “CA” for a carbon, “CB” for ⁇ carbon, "CG” for ⁇ carbon, and so forth.
  • Identical atoms of branched side chains are indicated by numbers.
  • the two ⁇ carbons of VAL 11 are designated “CG 1" and "CG 2" respectively.
  • the data file further comprises a connect statement which begins immediately after the coordinates for atom 771.
  • the connect statement identifies the covalent bonding pattern of each of the 771 atoms.
  • the 68th entry of the connect statement indicates that atom 68, which is the a carbon atom PRO 8 (corresponding to amino acid 188 of the mature factor XI heavy chain sequence), is bonded to atom 67 (the nitrogen of the same residue), atom 69 (the carbonyl carbon of the same amino acid residue), and atom 71 (the ⁇ carbon of the same amino acid residue).
  • the complete data file of 771 coordinates, together with the connect statement for these entries specifies the equilibrium conformation of factor XI heavy chain domain A3.
  • the peptides of the invention generally have an amino acid sequence similar to the native domain A3 sequence in the vicinity of the platelet binding site.
  • a covalent modification is artificially introduced to restrict the analog to the conformation (or one close to it) displayed by the above model.
  • the synthetic peptides consist essentially of peptide having from at least five to about 80 amino acid residues, which peptide has a restricted conformation.
  • the covalent modification is accomplished by determining a distance between two non-contiguous parts of the amino acid chain according to the model. Then a chemical moiety is introduced to fix that determined distance in the analog. For example, a 5-6A distance can be fixed using a disulfide bond.
  • Cysteine residues can be introduced at the appropriate positions in the model and then the new cysteine-containing model is tested for its ability to mimic the structure observed in the model.
  • the disulfide bond can be artificially introduced by generating a disulfide bond between native cysteine residues when this will produce a polypeptide with a restricted conformation corresponding to the above model.
  • the peptide can be internally cross-linked via the side chains of a lysine e -amino group and the carboxylic acid function of a glutamic or aspartic acid side chain, thus creating an amide bond.
  • the peptide is synthesized according to standard procedures on a low substitution (0.2 mmol/gm or less) paramethylbenzhydrylamine resin.
  • the first residue added to the resin is an N- ⁇ -tBOC, ⁇ -fMOC lysine.
  • the rest of the peptide synthesis is continued normally using tBOC chemistry until the final residue is added.
  • the last residue to be added is a Z-protected glutamic acid, where the carboxylic acid moiety is protected with a tert-butyl group.
  • fMOC/tBOC strategies include covalent closure of the peptide between two free amino groups utilizing toluene-2,4-diisocyanate (TDI), a heterobifunctional cross-linker.
  • TDI toluene-2,4-diisocyanate
  • the methyl group of the aromatic ring of TDI prevents the isocyanate group in the 2 position from reacting at a pH 7.5 or below, whereas the isocyanate group in the para position is highly reactive. A shift in pH to greater than 9.0 will initiate a reaction with the isocyanate group in the 2 position, thus enabling highly specific and controlled conditions for covalent closure of the peptide.
  • synthetic peptide analogs can be made and tested for their ability to inhibit factor XI binding to platelets and factor Xla-induced activation of factor IX on the platelet surface.
  • Particularly useful peptide analogs which were derived using the techniques described herein comprise amino acids 181-265, 191-266, 193-199, 226-235, 229-233, 235-266, 241-246, 248-253 and 248-261 of the factor XI heavy chain.
  • the 181-265, 191-266 and 235-266 peptides have an amino acid sequence identical to segments of the native factor XI sequence, i.e., SEQ ID NO:1 amino acids 1-85 and 8-86, and SEQ ID NO: 2, respectively.
  • Each of the three peptides has at least one artif icially introduced disulfide bond, i.e., between their cysteine residues corresponding to positions 242 and 265 in the factor XI mature polypeptide chain.
  • the disulfide bond is artificially introduced in the peptide chain by a chemical reaction step after the synthetic peptide is made and purified.
  • the 193-199, 226-235, 229-233, 241-246 and 248-261 are identical in sequence to the corresponding sequence of native factor XI, except for two modifications in each molecule.
  • A1a 193 and Ser 199 were replaced by cysteine residues to generate SEQ ID NO: 12.
  • This modified 193-199 peptide is designated "Ala 193(C) - Ser 199(C)" to distinguish it from the native 193-199 peptide.
  • the present peptides are relatively short in length and therefore they are easily synthesized by chemical means. Such synthetic peptides have many advantages over the use of the entire A3 domain, or the entire factor XI heavy chain. Large portions of the heavy chain cannot conveniently be made by synthetic techniques and must be made by recombinant DNA techniques, which are expensive and time consuming. Additionally, larger proteins may be insoluble, or may be immunogenic when introduced into a patient. Shorter synthet- ic peptides may be more soluble and less immunogenic than larger proteins.
  • peptide refers to a linear series of no more than about eighty (80) amino acid residues connected to one another by peptide bonds between the alpha-amino groups and carboxy groups of adjacent amino acid residues. Additional covalent bonds between portions of the peptide are also present to restrain the conformation of the molecule, such as amide and disulfide bonds.
  • synthetic peptide means a chemically derived chain of amino acid residues linked together by peptide bonds that is free of naturally occurring proteins and fragments thereof.
  • the term "homology" as describing the relationship between two amino acid sequences means the extent to which the sequences, viewed from the N-terminal to the C-terminal direction, have segments of their sequences which are identical and which occur in the same N-terminal to C-terminal order in the overall sequence.
  • the synthetic peptides according to the invention have an amino acid sequence which is the same as that of the native amino acid sequence, but for inserted, deleted, or interchanged (one or more amino acids is substituted for the same number of other amino acids) portions.
  • the degree of amino acid sequence homology between the amino acid sequence of a synthetic peptide according to the invention and that of the native peptide is expressed as a percentage. This percentage is obtained by determining the number of amino acids in the sequence of the synthetic peptide which occur in segments that are identical to segments of the native amino acid sequence and which occur in the same N-terminal to C-terminal order as the native segments, divided by the total number of amino acids in the native sequence.
  • a "substantial amino acid sequence homology” is any amino acid sequence homology greater that 30 percent. Preferably the homology is greater than 80 percent, most preferably greater than 90 percent.
  • Peptides of the present invention include any analog, fragment or chemical derivative of the peptides capable of inhibiting the binding of factor XI and/or Xla binding to platelets.
  • the term "analog” includes any peptide having substantial amino acid sequence homology to the peptides of the invention in which one or more amino acids have been substituted with other amino acids, and the substituted amino acids allow or require the peptide to assume the equilibrium conformation of the domain of the parent protein.
  • cysteine, lysine and glutamic acid will be used for their side chains which can form covalent linkages to restrict the conformation of a peptide.
  • conservative amino acid changes may be made which do not alter the biological function of the peptide.
  • one polar amino acid such as glycine
  • one acidic amino acid such as aspartic acid
  • another acidic amino acid such as glutamic acid
  • a basic amino acid such as lysine, arginine or histidine
  • a non-polar amino acid such as alanine, leucine or isoleucine may be substituted for another non-polar amino acid.
  • analog shall also include any peptide which has one or more amino acids deleted from or added to an amino acid sequence identical to that of native fragment of the amino acid sequence of factor XI heavy chain domain A3, but which still retains a substantial amino acid sequence homology to the platelet binding site on factor XI or factor Xla, as well as the ability to inhibit the binding of platelets to factor XI or factor Xla.
  • fragment shall refer to any shorter version of the peptides identified herein having at least five amino acid residues, wherein the fragment is a synthetic peptide which is capable of inhibiting the binding of platelets to factor XI or factor Xla.
  • amino acid residues in the peptides of the present invention are those symbols commonly used in the art.
  • the amino acid residues are preferred to be in the "L” isomeric form.
  • residues in the "D” isomeric form may be substituted for any L-amino acid, as long as the desired functional property of inhibition of factor Xla-induced factor IX activation is retained by the peptide.
  • the three-letter symbols used herein refer to the following amino acids: Ser is serine; lle is isoleucine; Gln is glutamine; Phe is phenylalanine; His is histidine; Trp is tryptophan; Lys is lysine; Asn is asparagine; Leu is leucine; Gly is glycine; Thr is threonine; Asp is aspartic acid; Arg is arginine; and Ala is alanine.
  • the peptides of the present invention may be prepared by any of the following known techniques. Conveniently, the peptides may be prepared using the solid-phase synthetic technique initially described by Merrifield, in J. Am. Chem. Soc. 15, 2149-2154 (1963). Other peptide synthesis techniques may be found, for example, in M. Bodanszky et al., Peptide Synthesis, John Wiley & Sons, 2d Ed. (1976); Kent and Clark-Lewis in Synthetic Peptides in Biology and Medicine, eds . Alitalo, K., Partanen, P. and Vakeri, A., (Elsevier Science Publishers, Amsterdam, 1985) p.
  • the present peptides may also be prepared by recombinant DNA techniques. But, such methods are not preferred because of the need for purification and subsequent chemical modifications to conformationally restrain the peptides.
  • these synthetic methods involve the sequential addition of one or more amino acid residues or suitably protected amino acid residues to a growing peptide chain.
  • amino acid residues or suitably protected amino acid residues Normally, either the amino or carboxyl group of the first amino acid residue is protected by a suitable, selectively-removable protecting group.
  • a different, selectively-removable protecting group is utilized for amino acids containing a reactive side group, such as lysine.
  • the protected or derivatized amino acid is attached to an inert solid support through its unprotected carboxyl or amino group.
  • the protecting group of the amino or carboxyl group is then selectively removed and the next amino acid in the sequence having the complementary (amino or carboxyl) group suitably protected is admixed and reacted under conditions suitable for forming the amide linkage with the residue already attached to the solid support.
  • the protecting group of the amino or carboxyl group is then removed from this newly added amino acid residue, and the next amino acid (suitably protected) is then added, and so forth.
  • any remaining terminal and side group protecting groups (and solid support) are removed sequentially or concurrently, to provide the final peptide.
  • the peptides of the invention are devoid of benzylate or methylbenzylated amino acids.
  • Such protecting group moieties may be used in the course of synthesis, but they are removed before the peptides are used. Additional reactions may be necessary, as described elsewhere, to form intramolecular linkages to restrain conformation.
  • the A3 domain-derived peptides of the present invention generally contain at least five (5) amino acid residues and up to eighty (80) amino acid residues, preferably from about five (5) to about forty-five (45) amino acid residues, and as small as about five (5) to about twenty (20) amino acids.
  • These peptides may be linked to an additional sequence of amino acids either or both at the N-terminus and at the C-terminus, wherein the additional sequences are from 1-100 amino acids in length.
  • additional amino acid sequences, or linker sequences can be conveniently affixed to a detectable label or solid matrix, or carrier.
  • Typical amino acid residues used for linking are tyrosine, cysteine, lysine, glutamic acid and aspartic acid, or the like.
  • the A3 domain-derived peptides according to the invention directly inhibit the binding of platelets to factor XI or factor Xla by competing with factor XI for binding sites on the platelet surface. Furthermore, high molecular weight binding to factor XI has been observed to insure the efficiency of factor XI binding to platelets, Sinha, et al. J. Clin. Invest. 73 1550-1556, at 1552, col. 2, ⁇ 2 (1984).
  • Factor XI heavy chain A1 domain-derived peptides are known to inhibit the binding of factor XI or factor Xla to high molecular weight kininogen, thereby indirectly inhibiting the binding of factor Xl/XIa to the platelet surface.
  • A3 domain-derived peptides of the invention may be combined with A1 domain-derived peptides to provide a dual effect.
  • the dual effect is attained when platelets are treated with A3 domain-derived peptides and high molecular weight kininogen is treated with A1 domain-derived peptides prior to adding factor XI/Xla to the platelets and kininogen.
  • the A3 domain peptides directly inhibit factor Xl/XIa binding to platelets by competing with intact factor Xl/XIa.
  • the A1 domain-derived peptides indirectly inhibit factor Xl/XIa binding to platelets by inhibiting high molecular weight kininogen binding to factor Xl/XIa.
  • the modeled A1-domain structure is used as a design template for synthesizing peptides that are expected to adopt a conformational repertoire overlapping that of the native protein in the same manner as described for the modeled A3-domain structure.
  • the model for the A1-domain structure disclosed herein may be utilized to prepare additional conformationally-restricted synthetic peptides having similar activity to the A1-domain derived synthetic peptides described above.
  • Such synthetic A1-domain derived conformationally restricted peptides may be prepared, modified and constrained in essentially the same manner as described above for the A3 domain-derived peptides according to the invention.
  • Appendix 2 included herein contains the set of Brookhaven coordinates and connect statements specifying the equilibrium conformation model of Baglia et al., J. Biolog. Chem. 267, 4247-4252 (1992) (incorporated herein by reference) which characterizes the structure of the high molecular weight kininogen binding site corresponding to amino acids 44 (Thr) to 85 (Ser) of the intact factor XI heavy chain.
  • the major portion of factor XI heavy chain domain A1 comprising the 85 amino acids spanning positions
  • Glu 1 to Cys 85, inclusive (SEQ ID NO:23) is utilized.
  • the corresponding graphic molecular model satisfying these coordinates may be generated by inputting the coordinates and connect statement into any of the many commercially available molecular modeling programs which are capable of reading files in the Brookhaven format.
  • the A1 domain-derived peptide is preferably a synthetic peptide comprising an amino acid sequence from at least five to about fifty amino acids in length, which corresponds to a portion of the sequence of the binding site for high molecular weight kininogen on the heavy chain of XI.
  • the A1 domain-derived peptide has an artificially restricted conformation and the ability to inhibit the binding of factor XI to high molecular weight kininogen.
  • Particularly preferred A1 domain-derived peptides comprise at least one amino acid sequence selected from the group consisting of SEQ ID NO: 13 and SEQ ID NOS: 17-22.
  • the restricted conformation of the A1 domain-derived peptide is determined from the equilibrium conformation model comprising the set of coordinates and connect statements of Appendix 2.
  • the restricted conformation may be provided in the same manner as for the A3 domain-derived peptides.
  • the A3 domain-derived peptide of the present invention and the A1 domain-derived peptide may be used in the form of a pharmaceutically acceptable salt.
  • Suitable acids which are capable of forming salts with the peptides include inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric acid and the like; and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, anthranilic acid, cinnamic acid, naphthalene sulfonic acid, sulfanilic acid or the like.
  • Suitable bases capable of forming salts with the peptides include inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide and the like; and organic bases such a mono-, di- and tri-alkyl and aryl amines (e.g., triethylamine, diisopropyl amine, methyl amine, dimethyl amine and the like) and optionally substituted ethanol-amines (e.g., ethanolamine, diethanolamine and the like).
  • inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide and the like
  • organic bases such a mono-, di- and tri-alkyl and aryl amines (e.g., triethylamine, diisopropyl amine, methyl amine, dimethyl amine and the like) and optionally substituted ethanol-amines (e.g., ethanolamine, diethanolamine and the like).
  • one or more of the synthetic A3 domain derived peptides of the present invention may be present in a pharmaceutical composition in admixture with a pharmaceutically acceptable carrier.
  • Preferred pharmaceutical compositions for inhibiting the binding of platelets to factor XI or factor Xla in a mammal also include a second peptide which inhibits the binding of factor XI or factor Xla to high-molecular weight kininogen to inhibit the binding of factor XI or factor Xla to the platelet surface.
  • the second peptide is an artificially constrained A1 domain-derived synthetic peptide as described above.
  • compositions for oral dosage form may include any of the usual pharmaceutical media, such as, for example, water, oils, alcohols, flavoring agents, preservatives, coloring agents and the like in the case of oral liquid preparations (e.g., suspensions, elixirs and solutions) or carriers such as starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations (e.g., powders, capsules and tablets). Controlled release forms may also be used. Because of their ease in administration, tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be sugar coated or enteric coated by standard techniques.
  • the carrier will usually comprise sterile water, although other ingredients to aid solubility or for preservation purposes may be included.
  • injectable suspensions may also be prepared, in which case appropriate liquid carriers, suspending agents and the like may be employed.
  • the parenteral routes of administration may be intravenous injection, intramuscular injection or subcutaneous injection.
  • the peptides may be dissolved in an appropriate intravenous delivery vehicle containing physiologically compatible substances such as sodium chloride, glycine and the like, having a buffered pH compatible with physiologic conditions.
  • physiologically compatible substances such as sodium chloride, glycine and the like, having a buffered pH compatible with physiologic conditions.
  • the A3 domain-derived peptides of the present invention both alone or in combination with the A1 domain-derived peptides, have utility as anticoagulant and/or antithrombotic agents. It is contemplated that the A3 domain-derived peptides, both alone or in combination with the A1 domain-derived peptides, may be administered to patients either at risk for developing arterial or venous thrombosis, or to patients with established thromboembolism to prevent extension of the thrombi.
  • the A3 domain-derived peptides and optionally the A1 domain-derived peptides may find utility in the prevention and treatment of deep venous thrombosis and pulmonary embolism, treatment and prevention of cerebral vascular thromboembolism, the treatment and prevention of systemic arterial thrombosis and embolism, and the treatment and possibly the prophylaxis of established disseminated intravascular coagulation.
  • Patients suffering from transient ischemic attacks are, in particular, at increased risk of brain damage through thrombus formation.
  • the synthetic peptides will find utility in the prevention of rethrombosis following lytic therapy.
  • lytic agents such as tissue plasminogen activator, urokinase and streptokinase have been utilized to dissolve vascular thrombi, their use is associated with a significant rate of rethrombosis, about 20-30%. This is because lytic therapy results in the exposure of a thrombogenic site, at the location of the prior thrombus.
  • lytic agents are effective in dissolving vascular thrombi, they offer no protection from clot reformation.
  • the A3 domain-derived peptides of the present invention are expected to possess substantial rethrombosis inhibiting activity, by virtue of their inhibition of the binding of platelets to factor XI or factor Xla and thus inhibition of factor Xla-induced activation of factor IX on the platelet surface, are expected to possess substantial rethrombosis inhibiting activity.
  • the peptides may thus be administered as an adjuvant to lytic therapy to prevent reformation of dissolved vascular thrombi.
  • the A3 domain-derived type and A1 domain-derived peptides which respectively directly and indirectly inhibit the binding of factor Xl/XIa to a platelet surface, may be administered by any convenient means which will result in the delivery of each peptide type to the bloodstream in an amount effective to inhibit the binding of factor XI and/or factor Xla to platelets.
  • Intravenous administration is presently contemplated as the preferred administration route.
  • the amount administered will depend on the activity of the particular compound administered, which may be readily determined by those of ordinary skill in the art.
  • the amount may also vary depending on the nature and extent of the lesion which is to be protected from rethrombosis; the size and weight of the patient; the route of administration, the age, sex and health of the patient; and other factors.
  • the A3 domain-derived and A1 domain-derived peptides may each be administered in an amount sufficient to individually or collectively provide a plasma concentration in the range of from about 10 -9 to about 10 -5 M, more preferably in the range of from about 1 ⁇ 10 -8 to about 5 ⁇ 10 -6 M. Plasma concentrations higher or lower than these may be utilized, depending upon the activity of the particular compound being administered, and the nature of the treatment.
  • bolus administration will comprise a dosage of from about 0.1 mg to about 1 gram of each peptide type, per kilogram subject body weight.
  • the bolus administration is most advantageously followed by a continuous infusion of each type of peptide, or a mixture of the two types of peptides, as needed.
  • the amount of each peptide type continuously infused depends on the approximate half-life of that peptide in the circulation.
  • a preferred method for inhibiting thrombosis comprises administering to a mammal in need of such treatment an effective amount of
  • an A3 domain-derived synthetic peptide according to the invention corresponding to a portion of the sequence of the binding site for activated platelets on the factor XI heavy chain, which has an artificially restricted conformation and the ability to compete with factor XI in the binding of the activated platelets, or a pharmaceutically acceptable salt of said A3 domain-derived peptide; and ii) an A1 domain-derived synthetic peptide corresponding to a portion of the sequence of the binding site for high molecular weight kininogen on the heavy chain of XI, which A1 domain-derived peptide has an artificially restricted conformation and the ability to inhibit the binding of factor XI to high molecular weight kininogen, or a pharmaceutically acceptable salt of said A1 domain-derived peptide.
  • the A3 domain-derived peptides of the invention inhibit the activated partial thromboplastin time without affecting the prothrombin time.
  • an amount of each of the A3 domain-derived peptide and A1 domain-derived peptide, shown effective by the in vitro assay described elsewhere herein, is administered to a patient by bolus administration and/or continuous infusion.
  • the potency of each peptide, or the combination, and its clearance from the circulation is then monitored by drawing blood samples at timed intervals and assaying the patient's partial thromboplastin time.
  • the dosage of each peptide is adjusted to provide the desired in vivo effect.
  • a structural model of the A3 domain was constructed using the computational chemis- try package supplied by Molecular Simulations, Inc., Pasadena CA and a Silicon Graphics 4D 280 Parallel Processing Supercomputer. A description of the modeling package and methods has been previously published (Jameson, Nature 349, 465-466 (1989)).
  • the A3 domain was prematurely truncated at Cys 265 because residues Arg 266 and His 267 - Phe 272 (SEQ ID NO: 16) comprise a short connecting peptide not expected to contribute to either the conformation or the function of the A3 domain.
  • Information concerning cysteine disulfide constraints was used to initiate model building, after which extended energy minimization calculations were carried out.
  • peptides were synthesized according to conventional solid phase procedures on an Applied Biosystems 43 OA peptide synthesizer by a modification of the procedure described by Kent and Clark-Lewis in Synthetic Peptides in Biology and Medicine, eds. Alitalo, K., Partanen P., and Vakeri, A. (Elsevier Science Publishers, Amsterdam (pp. 29-58 (1985)), in which dimethyl formamide replaced methylene chloride in the routine wash cycles.
  • the synthesis was carried out using a paramethylbenzhydrylamine resin (United States Biochemical Corp., Cleveland, OH).
  • the solvents and protected amino acids were synthesis grade biotechnology products purchased from Fischer Scientific Co., Pittsburgh, PA.
  • the resulting peptide was refolded by dissolving it in deionized water as a 0.1 mg/ml solution in a flask containing a stir bar.
  • the pH was adjusted to 8.5 with NH 4 OH and the solution was allowed to stir at 5°C for at least three days.
  • the resulting solution was lyophilized.
  • the folded peptides were examined by both reverse phase and gel filtration high performance liquid chromatography (HPLC).
  • HPLC system was the Waters 600 Gradient Module, Model 740 Data Module, Model 46K Universal Injector and Lambda-Max Model 481 Detector.
  • Reverse phase chromatography was performed using a Waters C8 ⁇ Bondapak Column equilibrated with 0.1% (V/V) trifluoroacetic acid. The column was eluted with a linear gradient of aqueous acetonitrile containing 0.1% trifluoroacetic acid with a detector set at a wavelength of 220 nm.
  • Ser 248(C) -Ser 261(C) Peptide Following the procedures of Example 1-3, a synthetic peptide corresponding to factor XI heavy chain residues 248-261 was modeled and prepared, except that the amino acid residues Ser 248 and Ser 261 of the native peptide were replaced with cysteine residues.
  • the resulting modified peptide, Ser 248 (C)-Ser 261(C) had the amino acid sequence of D-Cys-(SEQ ID NO:7)-Cys.
  • the peptide was "refolded" to assume its correct conformation, as described in Examples 2-3.
  • the peptide was reduced with dithiothreitol and alkylated with iodoacetamide as previously described by Sinha et al., J. Biol. Chem. 260, 10714-10719 (1985) .
  • the chromatography results were the same after reduction and alkylation of the peptide, that is, a single peak with retention times identical to the original peptide was observed upon both reverse phase and gel filtration HPLC.
  • the reduced/alkylated and corresponding refolded peptides were examined for free SH groups using the Ellman reagent, 5, 5'-dithiobis[2-nitro-benzoic acid] .
  • a synthetic peptide corresponding to factor XI heavy chain residues 241-246 was modeled and prepared, except that Thr 241 and Leu 246 were both replaced by Cys residues.
  • the modified peptide, Thr 241(C)-Leu 246(C) thus had the amino acid sequence of SEQ ID NO: 8.
  • the refolded peptide and the corresponding reduced/alkylated preparation were again found to have identical retention times by gel filtration and reverse phase HPLC. Moreover, the refolded peptide, as well as the reduced/alkylated peptide, were devoid of free thiols, thus confirming that all free SH groups were either oxidized to disulfides during the refolding procedure, or were reduced and alkylated during alkylation treatment.
  • the refolded peptide and the corresponding reduced/alkylated preparation were again found to have identical retention times by gel filtration and reverse phase HPLC. Moreover, the refolded peptide, as well as the reduced/alkylated peptide, were devoid of free thiols, thus confirming that all free SH groups were either oxidized to disulfides during the refolding procedure, or were reduced and alkylated during alkylation treatment.
  • the refolded peptide and the corresponding reduced/alkylated preparation were again found to have identical retention times by gel filtration and reverse phase HPLC. Moreover, the refolded peptide, as well as the reduced/alkylated peptide, were devoid of free thiols, thus confirming that all free SH groups were either oxidized to disulfides during the refolding procedure, or were reduced and alkylated during alkylation treatment.
  • the refolded peptide and the corresponding reduced/alkylated preparation were again found to have identical retention times by gel filtration and reverse phase HPLC. Moreover, the refolded peptide, as well as the reduced/alkylated peptide, were devoid of free thiols, thus confirming that all free SH groups were either oxidized to disulfides during the refolding procedure, or were reduced and alkylated during alkylation treatment.
  • Peptides corresponding to the A1-domain high molecular weight kininogen binding site in the factor XI heavy chain were synthesized and conformationally constrained in the same general manner as set forth in Examples 1-3. However, the model structure as provided by Baglia et al., J. Biolog. Chem. 267, 4247-4252 (1992), corresponding to the A1 domain was used as a design template instead of the A3 domain.
  • the A1-domain derived peptides were conformationally restricted peptides corresponding to factor XI heavy chain high molecular weight kininogen binding site.
  • the peptides produced have an amino acid sequence according to SEQ ID NOS: 13 and 17-22.
  • Factor XI (specific activity 250 U/mg of protein) was purified from human plasma by immunoaffinity chromatography using a monoclonal antibody to factor XI (Sinha et al., J. Biol. Chem. 260, 10714-10719 (1985)).
  • High molecular weight kininogen (specific activity 15 U/mg) was purified by the method of Kerbiriou et al. (J. Biol. Chem. 254. 12020-12027 (1979)). Factor XI and high molecular weight kininogen were assayed by minor modifications (Scott et al., Blood 63, 42-50 (1984)) of the kaolin-activated partial thromboplastin time (Proctor et al., Am. J. Clin. Pathol. 36, 212-219 (1961)). All purified proteins appeared homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
  • Platelets were incubated with ZnCl 2 (25 ⁇ M), CaCl 2 (2 mM), thrombin (0.1 U/ml) and high molecular weight kininogen (42 nM), and 125 I-factor XI (0.025 ⁇ g/mL) and then mixed with various concentrations of A1-, A2-, A3- or A4- derived synthetic peptides, factor XI or buffer. After 20 minutes, samples were centrifuged. Binding of 125 I- factor XI was compared to control binding in the absence of competing proteins.
  • IC 50 total inhibitor concentration at which the enzyme reaction velocity is 50% of the uninhibited reaction
  • the K i of XI is included in Table 1 for comparison.
  • the factor XI A3 peptide Asn 235 - Arg 266 of SEQ ID NO: 2 is a potent inhibitor of factor XI binding to platelets in the presence of high molecular weight kininogen, CaCl 2 , and ZnCl 2 .
  • the Ki is about 10 nM which is almost identical to the Ki for factor XI binding to platelets (See Table 1).
  • A3 domain all have inhibitory activity in the binding assay.
  • a peptide from the A2 domain e.g., Ala 134-Ala 176 (SEQ ID NO: 14) and from the A4 domain, e.g., Ala 317-Gly 350 (SEQ ID NO.15)
  • a peptide from the A1 domain i.e., Phe 56-Ser 86 (SEQ ID NO:13)
  • the A1 domain peptide inhibits the binding of factor XI to high molecular weight kininogen, which is essential to promote factor XI binding to a platelet receptor.
  • the inhibition of factor XI binding to platelets by the A1 peptides is an indirect inhibition since the A1 peptides do not directly compete with factor XI for a binding site on the platelet surface. Conversely, the A3 peptides directly compete with factor XI for the binding site on the platelet surface.
  • the major binding site for platelets is located on factor XI A3 domain within residues Asn 235-Arg 266.
  • Factor XI heavy chain peptides were assayed for inhibitory effects on blood coagulation.
  • the activated partial thromboplastin time was measured in the presence of activated platelets or phospholipids. Since phospholipids can substitute for platelets in most coagulation reactions, parallel assays were run with the peptides to determine whether their inhibitory effects were specific for their interaction of platelets.
  • Factor XI activity was assayed the method of Scott et al., Blood 63, 42-50 (1984), with minor modifications.
  • the assay determines the kaolin-activated partial thromboplastin time (Proctor et al. Am. J. Clin. Pathol. 36, 212-219 (1961)) using factor XI congenitally deficient substrate plasma. Coagulation mixtures containing kaolin, phospholipids or thrombin-activated platelets and factor XI deficient plasma were incubated at 37 °C for five minutes in the presence of factor and various concentrations of the synthet- ic peptides. The assay results were quantitated on double logarithmic plots of clotting times vs. concentrations of pooled normal plasma.
  • the A1 domain contains a binding site for high molecular weight kininogen
  • the A2 domain contains a substrate binding site for factor IX
  • the A4 domain contains a binding site for factor Xlla.
  • the peptides representing these respective binding sites showed inhibitory effects on intrinsic coagulation in the presence of both phospholipids and platelets, as manifested by the activated partial thromboplastin times.
  • an A3 domain derived peptide according to the invention e.g., Asn 235-Arg 266 (SEQ ID NO:2)
  • Asn 235-Arg 266 SEQ ID NO:2
  • a 100-fold higher concentration of Asn 235-Arg 266 was required to demonstrate a similar inhibitory effect in the presence of phospholipids.
  • the parallel results indicate the specificity of the A3-derived peptides according to the invention for binding to platelets, and not to phospholipids.
  • the factor XI heavy chain A1- and A3- derived artificially constrained peptides Phe 56 - Ser 86 (SEQ ID NO: 13) and Asn 235 - Arg 266 (SEQ ID NO: 2) are assayed for cumulative and synergistic effects by repeating the factor
  • ADDRESSEE Seidel, Gonda, Lavorgna & Monaco, P.C.

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Abstract

Analogues synthétiques de peptides du facteur humain XI limités dans leur conformation par liaison intramoléculaire. Ces peptides concurrencent les facteurs XI ou XIa natifs pour se fixer aux plaquettes et inhibent par là l'activation du facteur XI induite par le facteur XIa à la surface des plaquettes. Les peptides sont réalisés au moyen d'un modèle de conformation équilibré de la chaîne lourde du facteur XI.
PCT/US1994/013885 1993-12-22 1994-12-02 Analogues de peptides du site de fixation plaquettaire active sur le facteur xi WO1995017420A1 (fr)

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US7067117B1 (en) 1997-09-11 2006-06-27 Cambridge University Technical Services, Ltd. Compounds and methods to inhibit or augment an inflammatory response
US7238711B1 (en) 1999-03-17 2007-07-03 Cambridge University Technical Services Ltd. Compounds and methods to inhibit or augment an inflammatory response
US9771417B2 (en) 2014-08-07 2017-09-26 Novartis Ag Angiopoietin-like 4 antibodies and methods of use
US9988443B2 (en) 2014-08-07 2018-06-05 Novartis Ag Angiopoetin-like 4 (ANGPTL4) antibodies and methods of use
US10465011B2 (en) 2015-06-26 2019-11-05 Novartis Ag Factor XI antibodies and methods of use
US10647780B2 (en) 2016-05-25 2020-05-12 Novartis Ag Reversal binding agents for anti-factor XI/XIa antibodies and uses thereof
US10772906B2 (en) 2008-10-15 2020-09-15 Ionis Pharmaceuticals, Inc. Modulation of Factor 11 expression
US11021710B2 (en) 2018-05-09 2021-06-01 Ionis Pharmaceuticals, Inc. Compounds and methods for reducing FXI expression
US11168147B2 (en) 2016-12-23 2021-11-09 Novartis Ag Factor XI antibodies and methods of use

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BIOCHEMISTRY, Volume 25, issued 1986, A.D. TURNER, "rho-Amidino Esters as Irreversible Inhibitors of Factors IXa and Xa and Thrombin", pages 4929-4935. *
BLOOD, Volume 79, No. 2, issued 15 January 1992, R. RAWALA-SHEIKH, "Role of gamma-Carboxyglutamic Acid Residues in the Binding of Factor IXa to Platelets and in Factor-X Activation", pages 398-405. *
THE JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 266, No. 35, issued 15 December 1991, F.A. BAGLIA, "Identification and Chemical Synthesis of a Substrate-binding Site for Factor IX on Coagulation Factor XIa", pages 24190-24197. *
THE JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 267, No. 12, issued 25 April 1992, S.S. AHMAD, "The Role of the First Growth Factor Domain of Human Factor IXa in Binding to Platelets and in Factor X Activation", pages 8571-8576. *
THE JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 267, No. 5, issued 15 February 1992, J. ASTERMARK, "Effects of gamma-Carboxyglutamic Acid and Epidermal Growth Factor-like Modules of Factor IX on Factor X Activation", pages 3249-3256. *

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6989435B2 (en) 1997-09-11 2006-01-24 Cambridge University Technical Services Ltd. Compounds and methods to inhibit or augment an inflammatory response
US7067117B1 (en) 1997-09-11 2006-06-27 Cambridge University Technical Services, Ltd. Compounds and methods to inhibit or augment an inflammatory response
US7700087B2 (en) 1997-09-11 2010-04-20 Cambridge Enterprise Limited Compounds and methods to inhibit or augment an inflammatory response
US7238711B1 (en) 1999-03-17 2007-07-03 Cambridge University Technical Services Ltd. Compounds and methods to inhibit or augment an inflammatory response
US10772906B2 (en) 2008-10-15 2020-09-15 Ionis Pharmaceuticals, Inc. Modulation of Factor 11 expression
RU2739594C2 (ru) * 2008-10-15 2020-12-28 Ионис Фармасьютикалз, Инк. Модуляция экспрессии фактора 11
US11376273B2 (en) 2008-10-15 2022-07-05 Ionis Pharmaceuticals, Inc. Modulation of factor 11 expression
US9988443B2 (en) 2014-08-07 2018-06-05 Novartis Ag Angiopoetin-like 4 (ANGPTL4) antibodies and methods of use
US10577411B2 (en) 2014-08-07 2020-03-03 Novartis Ag Angiopoietin-like 4 antibodies and methods of use
US9771417B2 (en) 2014-08-07 2017-09-26 Novartis Ag Angiopoietin-like 4 antibodies and methods of use
US10465011B2 (en) 2015-06-26 2019-11-05 Novartis Ag Factor XI antibodies and methods of use
US10647780B2 (en) 2016-05-25 2020-05-12 Novartis Ag Reversal binding agents for anti-factor XI/XIa antibodies and uses thereof
US11168147B2 (en) 2016-12-23 2021-11-09 Novartis Ag Factor XI antibodies and methods of use
US11021710B2 (en) 2018-05-09 2021-06-01 Ionis Pharmaceuticals, Inc. Compounds and methods for reducing FXI expression

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