WO1998055504A1 - PEPTIDES A BASE DE hsPLA2 DU GROUPE II PRODUISANT UN EFFET ANTICOAGULANT - Google Patents

PEPTIDES A BASE DE hsPLA2 DU GROUPE II PRODUISANT UN EFFET ANTICOAGULANT Download PDF

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WO1998055504A1
WO1998055504A1 PCT/IB1998/000869 IB9800869W WO9855504A1 WO 1998055504 A1 WO1998055504 A1 WO 1998055504A1 IB 9800869 W IB9800869 W IB 9800869W WO 9855504 A1 WO9855504 A1 WO 9855504A1
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peptide
hspla
fxa
fva
activity
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PCT/IB1998/000869
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English (en)
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Carine Mounier
Tilman Hackeng
John Griffin
Cassian Bon
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Institut Pasteur
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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • 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
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • HsPLA 2 GR II PEPTIDES EXHIBITING AN ANTICOAGULANT EFFECT
  • the present invention relates to hsPLA 2 gr II and to specific hsPLA 2 gr II peptides exhibiting an anticoagulant effect, to antibodies which are directed against said peptides and to pharmaceutical compositions comprising said peptides or antibodies.
  • the present invention further relates to methods of regulating the coagulant effect and to methods of treating or preventing thrombus formation and limiting platelet activation in vivo in human or in animal comprising the step of administering an effective amount of said peptides.
  • the present invention further relates to methods of screening new pharmaceutical compounds which may be used for the prevention or treatment of hemostatic disorders and to kits for the determination of hemostatic disorders.
  • hsPLA 2 grll has been detected in various cellular types including macrophages, eosinophiles and blood platelets
  • hsPLA2 grll In platelets, hsPLA2 grll is associated with the ⁇ -granules and is released into the extracellular medium upon activation (Horigome et al . , 1987; Kramer et al . , 1989) .
  • the hsP 2 grll shares common characteristics with other group II secretory PLA 2 (sPLA 2 ) . In particular, its polypeptide sequence is homologous to that of these enzymes and its mechanism of action is identical
  • Blood platelet activation plays a central role during hemostasis leading to primary plug formation and increasing the efficiency of coagulation process (Mann et al . , 1990; Davie et al., 1991; Zwall et al . , 1992; Davie, E.W. , 1995). Blood platelets are also a source of factor V (Tracy et al . , 1982) .
  • hsPI-A 2 grll On blood coagulation, once secreted by activated platelets, The prothrombinase complex composed of FVa, FXa, phospholipids, and Ca++ plays a central role in the coagulation cascade (Mann et al . , 1990; Rosing et al . , 1988) .
  • the hsPLA 2 grll is shown to exert a moderate anticoagulant effect on plasma (Cirino et al . , 1993) and to inhibit prothrombinase activity (Inada et al . , 1994).
  • the present invention is based upon the discovery of the precise hsPI-A 2 grll region which is specifically involved in the inhibitory effects on prothrombinase activity and its anticoagulant mechanism. Further, the Applicant has clearly identified the proteinaceous target of hsPI-A 2 grll, the prothrombinase complex component which is affected, and under which conditions these effects can be achieved.
  • the invention relates to a peptide comprising at least eleven amino acids numbered 51 to 62 of hsPL-A 2 gr II sequence shown in table 2.
  • the invention relates to a peptide according to the invention, comprising amino acids numbered 51 to 74 of hsPI-A 2 gr II sequence.
  • the present invention also relates to a peptide exhibiting an anticoagulant effect corresponding to an amino acid chain containing at least a seven consecutive amino acid from the numbered 51 to 62 amino acid sequence of hsPLA 2 gr II.
  • the invention further comprises a peptide exhibiting an anticoagulant effect corresponding to an amino acid chain containing at least 14 amino acids having at least
  • Peptides according to the invention include those peptides mentioned above and peptides with minor amino acid variations from the natural amino acid sequence of the peptide; in particular, conservative amino acid replacements are contemplated.
  • the invention relates to a peptide according to the invention, wherein said peptide is able to inhibit prothrombinase activity.
  • the invention also relates to a peptide according to the invention, wherein the molecular target for the anticoagulant action of said peptide is Factor Xa (Fxa) .
  • the invention further relates to a peptide according to the invention, wherein the presence of Factor Va (Fva) is capable of reversing the activity of said peptide, particularly under suboptimal conditions.
  • Fva Factor Va
  • the invention further relates to peptide according to the invention, wherein said peptide is a Fva competitor.
  • the peptides according to the invention can be prepared by peptide synthesis or by recombinant DNA techniques, which are known to the person skilled in the art.
  • the nucleotide sequences (RNA or DNA) coding for the peptide according to the invention are part of the invention.
  • the invention further relates to anticoagulant compound, preferably prothrombinase activity inhibitor compound, more preferably fXa inhibitor compound, and Fva competitor compound selected in a group comprising the peptides according to the invention.
  • anticoagulant compound preferably prothrombinase activity inhibitor compound, more preferably fXa inhibitor compound, and Fva competitor compound selected in a group comprising the peptides according to the invention.
  • Fva competitor compound refers to compound according to the invention which either may bind to FXa at the same site(s) as FVa, or that the binding of FVa to FXa may modify the structure of FXa leading to the dissociation and the removal of said Fva competitor compound .
  • the invention comprises a monoclonal or polyclonal antibody, or fragments thereof, characterized in that it binds a peptide according to the invention.
  • said monoclonal or polyclonal antibody, or fragments thereof according to claim 9 are characterized in that it inhibits hsPLA 2 gr II anticoagulant effect.
  • the peptides according to the invention may also be used as antigenic models for the preparation of antibodies or antibodies like proteins, which may be used to inhibit excess activity of endogenous substances.
  • the monoclonal or polyclonal antibody according to this invention includes any naturally or non-naturally occurring polypeptide having the binding specificity of peptides according to the invention, that is, a polypeptide which binds to an epitope on said peptides, inhibits said peptides binding with Fxa or antagonizes the said peptides anticogulant effect.
  • examples of such antibody include a half antibody molecule (a single heavy: light chain pair) , or a fragment, such as the univalent fragments Fab or Fab 1 and the divalent fragment F(ab')2 ("FAB" meaning fragment antigen binding) , that possess the same specificity for binding as complete antibody.
  • a fragment, according to the present invention may also be a single chain Fv fragment produced by methods well known in the art. See Skerra et al., Science, 240: 1038-1041 (1988) and King et al . , Biochemical J., 290: 723-729 (1991), each of which is hereby incorporated by reference.
  • the antibody of the present invention also includes anti-idiotypic antibodies produced by methods well-known to the art of the invention. See, e.g. Cozenza, Eur. J. Immunol. 6: 114 (1976) and Harlow et al . , Antibodies: A Laboratory Manual, Cold Spring Harbor publications pp. 726 (1988) , each of which is hereby incorporated by reference.
  • epitopic determinants includes any determinant of peptides according to the invention responsible for the specific interaction with an antibody molecule.
  • Epitopic determinants usually consist of chemically active surface groupings of amino acids and have specific three-dimensional structural characteristics, as well as specific charge characteristics .
  • the antibody according to this invention also includes antibody conjugates, which are for example, enzymes, fluorescent markers, radiolabels.
  • the monoclonal antibody of the present invention is a "humanized" antibody, produced by techniques well-known in the art. Carter et al . , PNAS 89: 4285-4289 (1992); Singer et al . , J. Immun. 150: 2844-2857 (1992) and Mountain et al . Biotechnol . Genet. Eng . Rev. 10: 1-142 (1992) , each of which is hereby incorporated by reference.
  • Monoclonal antibodies can be produced in various ways using techniques well -understood by those having ordinary skill in the art. Details of these techniques are described in Antibodies : A Laboratory Manual, Harlow et al . , Cold Spring Harbor Publications, p. 726 (1988) , which is hereby incorporated by reference.
  • the subject of the present invention is also a pharmaceutical composition comprising a peptide or an antibody according to the invention in combination with a pharmaceutically acceptable vehicle.
  • the invention relates to the use of a peptide or an antibody according to the invention in a manufacture of a medicament for the prevention or the treatment of hemostatic disorders.
  • the human type PLA 2 grll and derivatives of the present invention can be used for therapeutic purposes as anticoagulants.
  • the peptides can be used alone, or they can be used in combination with other drugs.
  • Another subject of the present invention is a method of regulating the coagulant effect in vivo in human or in animal comprising the step of administering an effective amount of an active peptide, of an antibody or of a pharmaceutical composition according to the invention.
  • the invention relates to a method of treating or preventing thrombus formation and limiting platelet activation in vivo in human or in animal comprising the step of administering an effective amount of an active peptide or of a pharmaceutical composition according to the invention.
  • the pharmaceutical composition of the present invention may be administered in the form of oral, intravenous, intraperitoneal , or intra muscular administration, transdermal diffusion, and others.
  • a typical composition for such purpose comprises a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carriers include, water, saline, buffers, solutions, non-toxic excipients, including salts, preservatives, buffers and the like, as described, for instance, in Reminington ' s Pharmaceutical Sciences, 15th Ed. Easton: Mack Publishing Co.
  • non-aqueous solvents examples include propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters such as ethyloleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, and other compounds described, e.g., in Merck Index, Merck & Co., Rahway, New Jersey.
  • Intravenous vehicles include fluid and nutrient replenishers .
  • Preservatives include antimicrobial, anti-oxidants, chelating agents and inert gases.
  • pH and exact concentration of the various components the pharmaceutical composition are adjusted according to routine skills in the art. See Goodman and Gilman's, The Pharmacological Basis for Therapeutics (7th Ed.) .
  • the quantity of the peptide or antibody of the present invention necessary for effective therapy will depend upon many different factors, including the means of administration, target site, physiological state of the patient, other medicants administered, etc. Thus treatment dosages should be titrated to optimize safety and efficacy.
  • dosages used in vitro may provide useful guidance in the amounts useful for in situ administration of the peptide or the antibody, and as noted above, animal models may be used to determine effective dosages for treatment of particular disorders.
  • Various considerations are described, e.g. in Gilman et al . (eds.) (1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics, 8th Ed., Pergamon Press; and Remington's Pharmaceutical Sciences, 17th Ed. (1990), Mack Publishing Co., Easton, Pa., each of which is herein incorporated by reference.
  • the peptides of the present invention may also be used to characterize new drugs, either as molecular models or as tools for screening.
  • the present invention relates to method of screening new compounds for their use as medicament for the prevention or the treatment of coagulation disorders, comprising the use of a peptide according to the invention.
  • a method of screening according to the invention comprises the steps of : a) contacting a sample containing said test compound with a peptide according tothe invention; b) detecting the binding of said test compound with said peptide ; and c) selecting said test compound which is able to bind with said peptide.
  • a method of screening according to the invention comprises the steps of : a) contacting a sample containing said test compound with a peptide according to the invention in conditions permitting the measure of said peptide anticoagulant effect; b) measuring the said peptide anticoagulant effect ; and c) selecting said test compound which is able to modify said peptide anticoagulant effect, particularly test compound which is able to inhibit said peptide anticoagulant effect.
  • the peptides of the present invention may also be use to develop kits for detecting hemostatic disorders.
  • kits for the determination of a hemostatic disorder in a sample from human or animal comprising a peptide according to the invention.
  • Figure 1 Effect of hsPLA 2 grll on FXa and on FVa activities in clotting assays.
  • (A) FXa-1-stage clotting assay 10 nM FXa in Hepes buffer is incubated at 37°C in the absence (-0-, control) or in the presence of 3.5 ⁇ M hsPLA2 grll (-•-). After indicated times, 20 ⁇ l of the incubation mixture is used to perform an FXa-l-stage assay as described in "Methods". The amount of active FXa is determined using a calibration curve of purified FXa. The mean of duplicate experiments are shown.
  • Figure 2 Effect of hsPLA 2 on FXa and on FVa activities in coagulation assays.
  • FXa-one-stage coagulation assay 20 nM FXa in Hepes buffer is incubated for 2 min at 37°C, in the absence (-0-) or in the presence (-•-) of 5 mM CaCl2, with the indicated amounts of hsPLA 2 . Then, 20 ⁇ l of the incubation mixture is used to perform an FXa-one- stage assay as described in " Methods " .
  • the amount of active FXa is determined using a calibration curve of purified FXa.
  • the activity of FVa is measured using prothrombin time coagulation assays, performed as described in "Methods", in the presence of 5 mM CaC12 and the indicated amounts of hsPLA2 (-A-) .
  • the FVa activity is determined using a calibration curve of purified FVa. The means ⁇ SEM of three independent experiments are shown.
  • Figures 3 and 3 bis Effect of hsPLA2 grll on prothrombinase activity.
  • Prothrombinase assays are performed as described in "Methods", after hsPLA 2 grll is preincubated with 120 pMFV and 20 pM FXa (A), with 20 pM FXa(B), or with 120 pM FV
  • Figure 4 Effect of hsPLA 2 grll on prothrombinase activity measured under suboptimal conditions. Prothrombinase assays are performed as described in "Methods" in the absence (-•-) or in the presence of 3.5 ⁇ M hsPLA 2 grll (-0-).
  • A Prothrombinase activity (20 nM FXa, 120 pM FV, 5 mM Ca++) measured in the absence of PL;
  • B prothrombinase activity (2 nM FXa, 5 ⁇ M phospholipid (PL) , 5 mM Ca++) measured in the absence of FV or FVa.
  • the means ⁇ SEM of three independent experiments are shown.
  • Figure 5 Effect of hsPLA 2 on prothrombinase activity in the absence of PL. Prothrombinase assays are performed as described in "Methods" in the presence of the indicated amount of hsPLA 2 .
  • Prothrombinase activity (20 nM FXa, 120 pM FVa, 5 mM Ca++) is measured in the absence of PL (-0-) and 20 nM FXa with 5 mM Ca++ served as control (-•-) .
  • Figures 6 and 6 bis Reversal of hsPLA 2 grll inhibition on prothrombinase activity by the addition of FVa during the prothrombinase assay.
  • Prothrombinase activity assays (20 pM FXa, 120 pM FV, 5 ⁇ M
  • PL, 5 mM Ca++ are performed as described in "Methods", either in the absence of hsPLA2 grll (-•-), or, in the presence of 3.5 ⁇ M hsPLA 2 grll with 200 pM (-0-), 20 pM
  • Figure 7 Inhibition of prothrombinase activity by peptide 51-74.
  • Prothrombinase activity assays are performed as described in "Methods" with 20 pM FXa, 120 pM FV, 5 ⁇ M PL and 5 mM
  • FIG. 8 Calorimetric titration of FXa with hsPLA2 at 37°C.
  • the top panel shows the heat signal (after subtraction of base line) for 21 injections of 8 ⁇ L aliquots of buffer (20 mM Tris-HCl, pH 7.4, 0.125 M NaCl and 5 mM CaCl2) with 45.7 ⁇ M hsPLA 2 into a 1.35 mL cell containing the same buffer with 4.1 ⁇ M FXa.
  • the bottom panel shows the integrated heat of each injection after correction for the heat of dilution of hsPLA 2 and normalization to the amount of hsPLA 2 injected (filled rectangles) .
  • the curve through the points represents the best fit to a model involving a single set of independent binding sites.
  • FII Human prothrombin
  • FV Human factor V
  • FVIII activated factor X
  • CBS 34,47 The chromogenic substrate for thrombin amidolytic activity, CBS 34,47, is from Diagnostica Stago (Asnieres, France) and Chromogenix (Cincinnati, OH) .
  • FV-deficient plasma is from George King Bio-Medical (Overland Park, KS) .
  • Innovin is from DADE (Miami, FL)
  • BSA bovine serum albumin
  • fraction V is from Sigma (St Louis, MO) .
  • the synthetic scrambled peptide containing hsPLA 2 residues 51-74 as well as peptides 51-62, 59-70, 63-74, 62-51 (reverse) and D-51-62 (all residues in the D-configuration) are from Neosystem, Isochem SA (Strasbourg, France) .
  • the synthetic peptide 51- 74 of the hsPLA 2 grll (human group II secretory phospholipase A ) is purchased to the organic chemistry unit of Pasteur Institute (Paris, France) .
  • the peptides are purified by HPLC (purity ⁇ 95%) , their purity and sequence checked by mass spectroscopy . All these peptides have a N-terminal acetyl group and a C-terminal amide group .
  • the expression plasmid used is pT7-7 (Pharmacia) which is then transfected into the BL21 [DE3] Coli strain [Studies & Moffat, 1986] .
  • the BamHl/Hindlll fragment encoding hsPLA 2 grll [Franken et al . , 1992] is cloned in the similar cut expression vector.
  • the hsPLA 2 grll is expressed as a fusion protein with a 6 amino acids N- terminal extension ending in an arginine residue for tryptic liberation of the PLA 2 (phospholipase A 2 ) moiety.
  • Cells are grown in LB medium enriched with M9 salts.
  • the hsPLA 2 grll is purified on two subsequent SP- sephadex columns at pH 6 and pH 7.5.
  • the purified hsPLA 2 grll is then tested for its activity, using a fluorescent substrate, and checked for its purity by FPLC chromatography and SDS-PAGE as already reported [Mounier et al. , 1994] .
  • Phospholipid (PL) vesicles are prepared essentially as described by de Kruijff et al . [1974]. Solutions of phosphatidylserine and phosphatidylcholine (ratio of 1:9, PS: PC) in chloroform are mixed vigorously and dry under nitrogen. The dried PL are resuspended as vesicles in 0.1 M Tris-HCl, 0.05 M NaCl , pH 7.4 by sonication for 10 minutes .
  • Prothrombinase complexes are reconstituted using purified components, at 37°C and in Tris-buffered saline (0.1 M Tris-HCl, 0.05 M NaCl , 0,5% BSA, 5 mM CaCl 2 , pH 7.4) under the following conditions: a) FV, FXa and PL:
  • FV-preincubation conditions 10 pM FV is incubated for 4 min, then the reaction is started with 1 nM FXa, 5 ⁇ M PL and 200 nM FII.
  • FXa-preincubation conditions 10 pM FXa is incubated for 4 min, then the reaction is started with 1 nM FV, 5 ⁇ M PL and 200 nM FII.
  • FV, FVa and FXa 20 nM FXa is incubated for 4 min with 120 pM FV (or 120 pM FVa) , then the reaction is started with 1 ⁇ M FII (prothrombin) .
  • FXa and PL 2 nM FXa is incubated for 4 min, then the reaction is started with 5 ⁇ M PL and 1 ⁇ M FII.
  • FXa/FV-preincubation conditions are used as described above (a) . Briefly, 20 pM FXa is incubated with 120 pM FV for 4 min, then the reaction is started with 5 ⁇ M PL and 200 nM FII. After 6 min, the indicated amounts of FVa are added. The formation of thrombin as a function of time is followed as described above. Intrinsic tenase assay
  • Intrinsic tenase purified protein components are mixed at 37°C using purified components in Tris-buffered saline under the following conditions. 25 nM FIXa is incubated for 5 min with 1 nM FVIII in the absence or in the presence of defined hsPLA 2 amounts, then FX activation is started by addition of 1 ⁇ M FX (final concentrations) . Aliquots are then taken at various time points and the reaction is immediately stopped by the addition of aliquots containing 50 mM EDTA (final concentration) . The level of FXa activity is determined by hydrolysis of the chromogenic substrate S-2222 (200 ⁇ M, final concentration) monitored at 405 nm, in comparison to a standard curve using purified FXa .
  • FXa (20 nM) is incubated at 37°C in Hepes-buffered saline (50 mM Hepes pH 7.4, 0.1% BSA, 0.1 M NaCl) in the absence or in the presence of 5 mM CaCl 2 for different limes and in the absence (control) or in the presence of various concentrations of hsPLA 2 grll.
  • FXa-one-stage coagulation assays are performed as follows: 20 ⁇ l of the incubation mixture is added to a prewarmed mixture of 25 ⁇ l FV-deficient plasma and 30 ⁇ l PS: PC at 166 ⁇ M .
  • coagulation is started by the addition of 50 ⁇ l CaCl2 at 20 mM .
  • Clotting time is recorded using an ST4 coagulometer (Diagnostica stago, Asnieres, France) .
  • FVa activity measured in a prothrombin time clotting assay Fva (1 nM) is incubated for 2 min at 37°C in Hepes- buffered saline containing 5 mM CaCl 2 in the absence
  • a prothrombin time assay is performed as follows: 5 ⁇ l of the incubation mixture is added to a prewarmed mixture of 50 ⁇ l FV-deficient plasma and 45 ⁇ l Hepes-buffered saline. After 1 minute, coagulation is started by the addition of 50 ⁇ l innovin. Clotting times are recorded using an ST4 coagulometer . Measurement of FV activation by thrombin or FXa
  • FV 300 nM
  • Hepes- buffered saline 50 mm Hepes pH 7.4 , 0.1% BSA, 0.1 M NaCl, 5 mM CaCl 2
  • the activation is started by the addition of 1 nM thrombin.
  • the amounts of generated FVa are determined by performing a prothrombin time clotting assay as described above, and FV proteolysis is also analysed by SDS-PAGE.
  • FV (3 nM) is incubated for 30 minutes in Hepes-buffered saline (50 mm Hepes pH 7.4, 0.1% BSA, 0.1 M NaCl, 5 mM CaCl2) at 37°C in the presence of 25 ⁇ M PL, and with or without 3.5 ⁇ M hsPLA 2 grll, then the activation is started by the addition of 6 nM FXa. During 120 minutes, the amounts of generated FVa are determined by performing a prothrombin time clotting assay as described above. Isothermal titration calorimetry (ITC)
  • the FXa sample is thermostated at 37.0 ⁇ 0.1°C in a stirred (410 rpm) reaction cell (1.3514 ml) , and 31 injections, each of 8 ⁇ l volume and 5 s duration, with a 3.5 min interval between injections, are carried out using a 250- ⁇ l syringe filled with a hsPLA solution.
  • An injection series is preceeded by a 2 ⁇ l calibration injection.
  • the reference cell of the calorimeter contained water plus 0.01% sodium azide. Data points are averaged and stored at 2-s intervals.
  • FXa 14 ⁇ g/ml in 10 mM sodium acetate, pH 4.8
  • the immobilization run is performed at a flow of 5 ⁇ l/min at 25°C.
  • the SPR signal for immobilized FXa (three different flow cells with three different quantities of FXa) are found to be: 4,400 resonance units (RU) , 2,000 RU and 1,000 RU, where 1 RU corresponds to an immobilized protein concentration of ⁇ 1 pg/mm2. Unreacted moieties on the surface are blocked by ethanolamine .
  • One independent flow cell of the same sensor chip, used as a control flow cell, is subjected to a "blank immobilization", i.e. with no FXa added. All experiments are carried out in 10 mM HEPES, pH 7.4, 0.005 % surfactant P20 and 150 mM NaCl.
  • hsPLA 2 varying from 0-25 ⁇ g/ml , is injected in the same buffer in the presence or in the absence of CaCl2 at 5 mM, with a flow of 10 ⁇ l/min. Between each injection, surfaces are regenerated with 10 ⁇ l of 1 M NaCl. Analyses are performed at 25°C.
  • FIG. 1A shows a time-dependent inhibition of FXa activity by 3.5 ⁇ M hsPLA 2 grll, the inhibition being even faster when 5 mM Ca++ is present in the preincubation mixture (see Fig.2).
  • hsPLA 2 grll The effect of hsPLA 2 grll is next investigated on FVa activity measured with a prothrombin time clotting assay, using FV-deficient plasma. Fixed amounts of FVa are preincubated for different times with or without hsPLA 2 grll before addition to the assay. In these conditions, clotting efficiency is related to the activity of added FVa.
  • Figure IB shows that 3.5 ⁇ M hsPLA 2 grll is unable to significantly reduce the activity of FVa. Effect of hsPLA ? grll on FXa and FVa activities measured in coagulation assays (FIGURE 2)
  • FXa-one-stage coagulation assays performed with FV deficient plasma and fixed amounts of FXa that had been preincubated with varying amounts of hsPLA 2 , showed a dose-dependent inhibition of FXa activity by hsPLA (Fig. 2) .
  • the inhibition is strongly reduced in the absence of Ca++ in the preincubation mixture, even though Ca++ is later present during FXa-one-stage assays (Fig. 2) .
  • prothrombinase complex composition is varied to create optimal (FXa, FVa, PL and Ca++) and different suboptimal conditions (i . e . using FV instead of FVa, as well as in the absence of FVa or PL) .
  • prothrombinase complex When the prothrombinase complex is reconstituted from 20 pM FXa, 120 pM FV, 5 ⁇ M PL (phospholipid) and 5 mM Ca++, after that hsPLA 2 grll is preincubated with FXa and FV, a strong inhibition of prothrombinase activity is observed by low concentrations of hsPLA 2 grll (Fig. 3A and 3A bis) . When PL concentrations are decreased to 1 ⁇ M, or increased to 50 ⁇ M, we did not observe any modification of the inhibitory activity of the hsPLA 2 grll (data not shown) .
  • Figures 4A and 4B represent the results obtained in the absence of PL (20 nM FXa, 120 pM FV, 5mM Ca++) and in the absence of FV or FVa (2 nM FXa, 5 ⁇ M PL, 5mM Ca++ ) .
  • hsPLA 2 grll induces an 50%-inhibition of the prothrombinase activity at 3.5 ⁇ M .
  • Figures 5A and 5B represent the results obtained in the absence of PL (20 nM FXa, 120 pM FVa, 5mM Ca++ ; or 1 nM FXa, 1 nM FVa, 5mM Ca++) and in the absence of both PL and FVa (20 nM FXa, 5mM Ca++ ; or 1 nM FXa, 5mM Ca++) .
  • the effect of hsPLA 2 on prothrombinase activity is then tested in the absence of PL.
  • a potent inhibitory effect of hsPLA2 in the presence of FVa (Fig. 5A and 5B) can be seen.
  • prothrombinase (20 nM FXa, 120 pM FVa, 5 mM Ca++) by hsPLA 2 is never complete in the absence of PL, even at high concentrations of hsPLA 2 (10 ⁇ M) , and as can be seen, the rate of prothrombin activation decreased until a value corresponding the same as that obtained with FXa alone, i.e. 0.16 ⁇ 0.008 nM thrombin. min- 1 compared to 0.14 + 0.004 nM thrombin. min- 1 (Fig. 5A) .
  • Intrinsic tenase purified components include 25 nM FIXa, 1 nM FVIII and 5 mM Ca++.
  • the substrate, FX is used at 1 ⁇ M. Any inhibitory effect of the hsPLA 2 has been observed on the ability of these components to activate FX under these conditions, even at hsPLA 2 concentrations up to 10 ⁇ M (data not shown) .
  • the PL- independent anticoagulant action of hsPLA2 is specific for the prothrombinase complex. An inhibition of the prothrombinase generation has been observed in the absence of FVa but in the presence of
  • FVa is determined as a function of time in a prothrombin time assay using FV deficient plasma. It has been observed that hsPLA 2 is unable to inhibit FV- activation by either thrombin or FXa/PL (data not shown) . This suggests that hsPLA 2 may specifically inhibit prothrombin activation by inhibiting the formation of the prothrombinase complex.
  • hsPLA 2 grll by affecting FXa, is a potent inhibitor of prothrombinase activity, under conditions where the prothrombinase complex activity is suboptimal.
  • the prothrombinase complex is reconstituted with FV instead of FVa (20 pM FXa, 120 pM FV, 5 ⁇ M PL, Ca++) , and the effect of 3.5 ⁇ M hsPLA2 grll, preincubated with FV and Fxa, is followed during 6 minutes. At this time, various amounts of FVa (4 pM, 20 pM or 200 pM) are added to the prothrombinase complex. Consistent with previous experiments (Fig.
  • hsPLA ⁇ grll is able to inhibit prothrombinase activity in the presence of FXa, FVa, Ca++, and in the absence of PL.
  • a 50%-inhibition is observed at 3.5 ⁇ M hsPLA grll (data not shown) comparable to the effect of hsPLA 2 grll in prothrombinase assays performed in the presence of FXa, FV, Ca++, and in the absence of PL.
  • hsPLA ⁇ grll is thus able to inhibit the prothrombinase activity in all suboptimal conditions tested even if FVa is present.
  • prothrombinase complex reaches optimal conditions (FXa, FVa, PL, Ca++) the presence of FVa reverses the inhibitory effect of hsPLA 2 grll.
  • FIG. 8 shows original data from the calorimetric titration of a 1.4 ml solution containing 4.1 ⁇ M of FXa with a solution containing 45.7 ⁇ M of hsPLA at 37°C.
  • Two kinetic phases are associated with each injection. Immediately following injection, an initial exothermic phase (negative numbers) , which will refer to as site binding, is observed. This is followed by a smaller and slower endothermic phase (positive numbers) suggesting that a slow conformational rearrangement takes place after binding.
  • the inhibitory effect of hsPLA 2 on FXa activity is mainly observed in the presence of Ca++ (Fig.l and 2). Based on this observation, the influence of Ca++ on FXa/hsPLA 2 binding affinity is tested using surface plasmon resonance (SPR) , studies which require less protein material than ITC. SPR allows measurements of the association rate constant (k on ) and of the dissociation rate constant (k 0f f ) of hsPLA 2 to immobilized FXa.
  • SPR surface plasmon resonance
  • K d app value is usually smaller than the equilibrium K d value as SPR rate constants measurements are performed far from equilibrium, and with one immobilized protein which decreases the overall entropy of the association reaction with respect to free protein association in solution.
  • Table 1 Determination of k Q n and k ⁇ ff rate constants for hsPLA2 interaction with immobilized FXa using surface plasmon resonance.
  • Table 1 shows that hsPLA binds to FXa immobilized onto the sensor chip surface in the absence of Ca++.
  • 5 mM Ca++ in the screening buffer decreases koff nearly ten fold and increases kon nearly 3 fold, leading to a 30 fold increase in K d app . Therefore, data in Table 3 (see example 6) demonstrate that Ca++ allows the formation a higher affinity FXa/hsPLA complex.
  • Table 3 The scrambled peptide 51-74 is used as control.
  • Table 2 Amino acids sequences of hsPLA grll and two synthetic peptides.
  • hsPLA grll 1 10 20 30 40 50 NLVNFHRMIKLTTGKEAALSYGFYGCHCGVGGRGSPKDATDRCCVTHDCC
  • peptide 51-74 Ac-YKRLEKRGSGTKFLSYKFSNSGSR-NH 2 (net charge +6)
  • Prothrombinase activity assays are performed as described in "Methods”. Reactants (20 pM FXa, 120 pM FV, 5 ⁇ M PL and 5 mM Ca++) are preincubated in the presence of various concentrations of the indicated peptides. After 8 min preincubations, the prothrombinase activity is determined and expressed in percent of the prothrombinase activity measured in the absence of peptide. Then, the percent of residual prothrombinase activity is analyzed as a function of the peptide concentration, and the IC50 value is calculated. ND : not determined
  • the scrambled peptide shows a nonspecific inhibitory effect when testing with 0.1 M NaCl (IC50 of 70 ⁇ 5 ⁇ M) since it is lost when higher salt concentration (0.22 M NaCl) is used (Table 2) .
  • IC50 IC50 of 70 ⁇ 5 ⁇ M
  • higher salt concentration 0.22 M NaCl
  • Table 2 This supports the idea that the inhibitory effect of scrambled peptide at 0.1 M NaCl concentration is due to its basic nature (net charge +6) but does not imply specific residues.
  • the inhibition of prothrombinase activity by peptides 51-74 and 51-62 is specific since it is maintained with similar efficiency under 0.22 M NaCl concentration (Table 3).
  • a 12-mer reversed peptide 62-51 is devoid of inhibitory effect, as is the control peptide D-51-62 with all amino acid residues in a D-configuration.
  • the stereospecific L- conformation of amino acids in peptide 51-62 is required for the inhibition of FXa/FVa complex formation.
  • residues 51-74 of hsPLA2 specifically inhibits prothrombinase activity, and is most likely responsible for the inhibition of FXa/FVa complex formation.
  • These basic region may therefore represent the part of the molecule being involved in the inhibition of FXa. It is important to note that residues 51-74 of the molecule are not implicated in the catalytic activity and that the same region has been postulated to be involved in the anticoagulant effect of sPLA2 from snake venom.
  • hsPLA 2 grll secreted during platelet activation, exhibits anticoagulant activity. In this way, it might exert a negative feedback regulation on coagulation, which would avoid an excessive procoagulant effect of activated platelets (Mounier et al . , 1996) . It has been demonstrated that this inhibitory effect does not require the enzymatic activity of the enzyme, indicating that hsPLA2 grll may interact and have pharmacological effects on non-phospholipid targets. This is in good agreement with results of Ouyang et al . (1978), Stefansson et al .
  • hsPLA 2 grll does not inhibit FVa activity
  • FXa-one-stage coagulation assays show an inhibition of FXa activity, particularly in the presence of Ca++.
  • hsPLA 2 grll is able to inhibit FXa activity in clotting assays.
  • the inhibitory action of hsPLA 2 grll on prothrombinase activity was also observed by Inada et al .
  • hsPLA 2 grll is able to inhibit prothrombinase activity, especially when the conditions are not optimal for prothrombinase complex activity or assembly. It has been demonstrated that in the absence of FVa or PL, dependent on the preincubation of hsPLA2 grll with FXa, hsPLA 2 grll effectively downregulates prothrombinase activity (Fig. 3 and 4) and that in the absence of PL and in the presence of FVa, preincubation of hsPLA 2 with FXa effectively downregulates prothrombinase generation (Fig. 5).
  • hsPLA 2 grll inhibites prothrombinase activity only until there is enough in situ FVa generated by traces of thrombin/FXa to optimize prothrombinase activity conditions and overcome hsPLA 2 (Fig. 6 and 6 bis) .
  • the activity of the intrinsic tenase complex (FIXa, FVIII, Ca++) is unaffected by hsPLA 2 , although this coagulation complex shares common characteristics with the prothrombinase complex (associated with the structural homology of FVa and FVIIIa, and of FIXa and FXa) .
  • hsPLA 2 activation of FV by either FXa or thrombin is not inhibited by hsPLA 2 .
  • hsPLA 2 grll may bind to FXa at the same site(s) as FVa, but with a lower affinity and thereby may decrease the prothrombinase activity by inhibiting the formation of a FXa/FVa complex.
  • This is supported by the demonstration that hsPLA 2 binds to FXa with a 1:1 stoichiometry and a K d value of 230 nM (Fig.8).
  • the Ca++ increases the k on rate constant and decreases the k 0 f f rate constant, leading to a higher affinity of hsPLA 2 for FXa (Table 1) .
  • the prothrombinase complex has a catalytic efficiency in the activation of prothrombin that is several orders of magnitude higher than FXa acting alone.
  • FVa-FXa interaction is governed by a K d of 0.8 ⁇ M and is dependent on the presence of Ca++ (Pryzdial and Mann, 1991) .
  • Ca++ the K of the FVa-FXa complex decreased to approximatively 1 nM (Krishnaswany et al . , 1990).
  • hsPLA2 is thus able to produce an inhibitory effect on blood coagulation under experimental conditions occurring during clot formation. Moreover, it is well established that the level of hsPLA2 in serum is strongly increased, from 0.35 nM up to 0.6 ⁇ M, under various pathological states associated with inflammation, as in the case of acute pancreatitis, multiple organ failure, septic shock or rheumatoid arthritis (Nevalainen et al . , 1993 ; Nyman et al., 1996 ; Rintala et al .
  • hsPLA 2 grll released by activated platelets during primary hemostasis may act as a negative modulator of thrombin generation by preventing the initial prothrombinase complex assembly. It has been already mentioned that the K d value of hsPLA 2 /FXa interaction (230 nM) is higher than the one of FVa/FXa interaction (1 nM) . For equal concentrations of hsPLA 2 and FVa, these K d values would favor FXa/FVa interactions.
  • the hsPLA grll have seven basic amino acids (K or R) located between residues 51-74, and three-dimensional studies have shown that this part of the hsPLA grll is exposed at the surface of the molecule and is not associated with the catalytic site (White at al . , 1991 ; Wery et al . , 1991). Thus, this region appears to be a good candidate to be involved in the inhibition of FXa by hsPLA 2 grll. To test this hypothesis, the inhibitory effect of a synthetic peptide 51-74 has been examined on prothrombinase generation. The peptide 51-74 shows an inhibitory effect with an IC50 of 8 ⁇ M.
  • Reversed peptide 62-51 and peptide (D) 51-62 are unable to inhibit prothrombinase activity up to 200 ⁇ M, supporting the specificity of the interaction of peptide 51-62 with FXa.
  • the region 51-74, and more precisely the region 51- 62, of the hsPLA2 grll is independently able to inhibit prothrombinase activity duo to a specific mechanism.
  • the importance of the 51-62 region in the binding of hsPLA2 on FXa points out the presence of basic clustered residues which might be critical for the interaction.
  • the small enthalpic contribution may account for the short amino acid sequence size at the binding site, while the favorable entropic contribution to binding may reflect binding induced desolvation and/or anion release upon binding at the level of the basic sequence of residues 51-62 of hsPLA2.
  • Heeb et al . (1996) suggested that one binding site for FXa involves residues 493-506 in FVa (GLLLICKSRSLDRR) , which shows some similarity (bold letters) to the peptide 51-62 (YKRLEKRGSGTK) when basic residues are compared.
  • this part of the hsPLA 2 grll molecule (CYKRLEKRGSGTK) also shares similarities with the equivalent region on FVIII (LLICYKESVDQRG) . It is thus plausible to speculate that hsPLA 2 grll would be able to inhibit the tenase activity by similar mechanism with FVIII and FIXa.
  • hsPLA2 acts by competing with FVa for binding to FXa. It is not at all clear which parts of the FXa molecule is targetted by FVa, and thus it is also very difficult to establish which FXa regions interact with hsPLA2.
  • the most probable hypothesis is that hsPLA2 binds to the same site as FVa on FXa, but a noncompetitive mechanism can not be exclude due to the binding of clustered basic residues of hsPLA2 to specific negatively charged residues present in ⁇ -carboxyglutamate-rich domain of FXa.
  • Platelet secretory phospholipase A 2 fails to induce rabbit platelet activation and to release arachidonic acid in contrast with venom phospholipases A 2 . Biochem. Biophys. Acta 1214, 88- 96.
  • Verheij H.M., Boffa, M.C, Rothen, C, Bryckaert, M.C, Verger, R. & deHaas, G.H. (1980) Correlation of enzymatic activity and anticoagulant properties of phospholipase A 2 . Eur. J. Biochem. 112, 25-32.

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Abstract

Cette invention se rapporte à des peptides à base de hsPLA2 du groupe II et à base de hsPLA2 du groupe II spécifique, qui produisent un effet anticoagulant, à des anticorps qui sont dirigés contre ces peptides et à des compositions pharmaceutiques comprenant ces peptides ou ces anticorps. Cette invention se rapporte également à des procédés de régulation de l'effet coagulant et à des procédés de traitement ou de prévention de la formation de thrombus et de limitation de l'activation des plaquettes in vivo chez l'homme ou chez les animaux, qui consistent à administrer une quantité efficace de ces peptides. Cette invention se rapporte en outre à des procédés de sélection de nouveaux composés pharmaceutiques pouvant être utilisés dans la prévention ou le traitement des troubles hémostatiques et à des kits pour la détermination des troubles hémostatiques.
PCT/IB1998/000869 1997-06-05 1998-06-04 PEPTIDES A BASE DE hsPLA2 DU GROUPE II PRODUISANT UN EFFET ANTICOAGULANT WO1998055504A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1745145A2 (fr) * 2004-05-11 2007-01-24 Heptest Laboratories, Inc. Compositions, kit et procede en une etape permettant de surveiller des composes possedant des activites d'antifacteur xa et/ou d'antifacteur iia
US8158127B2 (en) 2003-09-22 2012-04-17 Pentracor Gmbh Compounds for neutralizing the effects of secreted PLA2 IIA
CN117534731A (zh) * 2021-09-24 2024-02-09 江西康之康中药科技有限公司 一种抗凝血肽及其在抗凝血产品上的应用

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WO1989009818A1 (fr) * 1988-04-15 1989-10-19 Biogen, Inc. Procede de purification de phospholipase a2 et de production de polypeptides semblables a la phospholipase a2
US5019508A (en) * 1987-08-27 1991-05-28 Biotechnology Research Partners, Ltd. Synovial phospholipases
WO1993001215A1 (fr) * 1991-07-04 1993-01-21 Garvan Institute Of Medical Research Composes inhibant l'activite enzymatique des phospholipases a2 (pla2)
EP0687685A1 (fr) * 1993-03-03 1995-12-20 Teijin Limited Peptide physiologiquement actif

Patent Citations (4)

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Publication number Priority date Publication date Assignee Title
US5019508A (en) * 1987-08-27 1991-05-28 Biotechnology Research Partners, Ltd. Synovial phospholipases
WO1989009818A1 (fr) * 1988-04-15 1989-10-19 Biogen, Inc. Procede de purification de phospholipase a2 et de production de polypeptides semblables a la phospholipase a2
WO1993001215A1 (fr) * 1991-07-04 1993-01-21 Garvan Institute Of Medical Research Composes inhibant l'activite enzymatique des phospholipases a2 (pla2)
EP0687685A1 (fr) * 1993-03-03 1995-12-20 Teijin Limited Peptide physiologiquement actif

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Title
C. MOUNIER ET AL: "The anticoagulant effect of the human secretory phospholipase A2 on blood plasma and on a cell-free system is due to a phospholipid-independent mechanism of action anvolving the inhibition of factor Va", EUR. J. BIOCHEM., vol. 237, 1996, pages 778 - 785, XP002082787 *
D.L. SCOTT ET AL.: "Structure of free and inhibited human secretory phospholipase A2 from inflammatory exudate", SCIENCE, vol. 254, November 1991 (1991-11-01), pages 1007 - 1010, XP002082622 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8158127B2 (en) 2003-09-22 2012-04-17 Pentracor Gmbh Compounds for neutralizing the effects of secreted PLA2 IIA
EP1745145A2 (fr) * 2004-05-11 2007-01-24 Heptest Laboratories, Inc. Compositions, kit et procede en une etape permettant de surveiller des composes possedant des activites d'antifacteur xa et/ou d'antifacteur iia
EP1745145A4 (fr) * 2004-05-11 2008-03-26 Heptest Lab Inc Compositions, kit et procede en une etape permettant de surveiller des composes possedant des activites d'antifacteur xa et/ou d'antifacteur iia
CN117534731A (zh) * 2021-09-24 2024-02-09 江西康之康中药科技有限公司 一种抗凝血肽及其在抗凝血产品上的应用

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