WO2000058341A1 - Hookworm platelet inhibitor - Google Patents
Hookworm platelet inhibitor Download PDFInfo
- Publication number
- WO2000058341A1 WO2000058341A1 PCT/US2000/008519 US0008519W WO0058341A1 WO 2000058341 A1 WO2000058341 A1 WO 2000058341A1 US 0008519 W US0008519 W US 0008519W WO 0058341 A1 WO0058341 A1 WO 0058341A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- platelet
- polypeptide
- hpi
- binding
- polypeptide according
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43536—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms
- C07K14/4354—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms from nematodes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P33/00—Antiparasitic agents
- A61P33/10—Anthelmintics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- This invention relates to an isolated, purified, and cloned hookworm polypeptide which is a novel potent inhibitor of platelet aggregation and adhesion.
- hookworm Necator americanus characterized broad inhibition of platelet aggregation in response to multiple agonists, including ADP, collagen, thrombin, and platelet activating factor (39) . They theorized that this hookworm species might secrete an inhibitor of GPIIb/IIIa, the platelet surface ⁇ , Ib ⁇ 3 integrin that mediates adhesion to fibrinogen, as well as activation and aggregation in response to numerous agonists.
- the molecular mechanism(s) of hookworm platelet inhibition are not understood, and the isolation of the responsible parasite-derived compounds have not been heretofor reported.
- the present invention provides a soluble polypeptide originally isolated and purified from Ancyclostoma caninum hookworms, and then cloned.
- the polypeptide inhibits platelet aggregation and adhesion in response to a variety of agonists, by interfering with the binding of at least one cell surface integrin such as glycoprotein (herein denominated "GP") GPIIb/IIIa ( ⁇ IIb ⁇ 3 ) with its respective ligand (fibrinogen for GPIIb/IIIa) and/or the binding of GPIa/IIa ( ⁇ 2 ⁇ 2 ) with collagen.
- GP glycoprotein
- the hookworm platelet inhibitor of the invention not only blocks platelet aggregation, it also prevents adhesion of platelets to fibrinogen and/or collagen.
- a recombinant polypeptide having a predicted molecular weight of about 20.3 to 20.4 kDa is shown in SEQ ID NO: 2.
- the invention further encompasses fragments or variants thereof exhibiting at least about 50%, and in many embodiments, at least about 65 % to about 75 % , sequence homology to the naturally occurring polypep- tide, which exhibit the same biological properties as the native molecule, compositions containing the inhibitor, and methods of using it.
- Figure 1 presents graphs showing the inhibitory effect of hookworm extracts on platelet aggregation and adhesion.
- B Inhibitory effect of hookworm extracts on platelet adhesion to immobilized fibrinogen. Platelets were pre-incubated with increasing amounts of hookworm extracts, then added to fibrinogen (40 ⁇ g/mL) coated microtiter plate wells. Adhesion was measured as described in the examples. Results are expressed as percent of control adhesion values, i.e. , in the absence of hookworm extracts.
- Figure 2 graphs the purification of HPI using anion exchange chromatography. Soluble hookworm extracts from approximately 1000 adult worms was applied to a 25 mL anion exchange column at a flow rate of 2 mL/min. The bound protein (solid line) was eluted with a 0-500 mM NaCl gradient (dashed line). Individual column fractions (5 mL) were collected and tested for inhibition of platelet adhesion to fibrinogen (closed circles) .
- Figure 3 graphs the purification of HPI using size exclusion chromatography. Approximately 0.5 mL of the concentrated pooled anti-platelet activity from the anion exchange column was applied to a Bio-Sil TSK-125 size exclusion column. Individual column fractions (0.5 mL) were collected and tested for inhibition of platelet adhesion and aggregation. Horizontal bar indicates contiguous fractions containing the HPI activity.
- Figure 4 provides bar graphs presenting data on the effect of HPI on platelet aggregation and adhesion.
- HPI (20 ⁇ g/well) blocks aggregation induced by epinephrine (10 ⁇ M), thrombin (10 nM), and ADP (10 ⁇ M). Open bars: platelets pre-incubated with HPI; closed bars: platelet controls.
- Figure 5 shows purification of hookworm platelet inhibitor by reversed- phase HPLC as described in the examples. Following anion exchange and size exclusion chromatographies, the partially purified inhibitor was subjected to reversed-phase HPLC using a C lg column. The bound protein was eluted under a linear gradient of acetonitrile in 0.1 % TFA. Individual peaks of protein were lyophilized, resuspended in aqueous buffer, and tested for inhibition of biotinylated fibrinogen binding to immobilized GPIIb/IIIa and T4 cell binding to collagen.
- This invention is based upon the finding that a hookworm polypeptide is useful for a number of medical and veterinary therapeutic and prophylactic purposes.
- Results reported herein describe the isolation, purification, characterization, and cloning of a protein inhibitor of platelet aggregation and adhesion, hereafter sometimes denominated herein as HPI for "hookworm platelet inhibitor", identified in soluble protein extracts of adult Ancylostoma caninum hookworms and observed in Necator americanus.
- HPI protein inhibitor of platelet aggregation and adhesion
- Platelets respond to a variety of agonists, such as, but not limited to, epinepherine, thrombin, and ADP tested in assays described in the examples section.
- the polypeptide interferes with the interaction of at least one cell surface integrin with its respective ligand, such as GPIIb/IIIa ( ⁇ IIb ⁇ 3 )/fibrinogen or GPIa/IIa ( ⁇ 2 ⁇ *) /collagen, by binding to the integrin.
- a cell surface integrin such as GPIIb/IIIa ( ⁇ IIb ⁇ 3 )/fibrinogen or GPIa/IIa ( ⁇ 2 ⁇ *) /collagen
- GPIIb/IIIa ⁇ IIb ⁇ 3
- GPIa/IIa ⁇ 2 ⁇ *
- hookworm any nematode that sucks blood from the small intestine including, but not limited to, the major hookworms that infect humans, Ancylostoma duodenale, Necator americanus, and, less commonly, A. ceylanicum, as well as hookworms that infect other animals such as Ancylostoma caninum, Bunostomum phlebotomum, Agriostomum vryburgi, B. trigonocephalum, and Gaigeria pachyscelis.
- Other blood-sucking nematodes such as Haemonchus species, e.g. , H. contortus, are also encompassed by this invention.
- Ancylostoma caninum is a preferred source.
- HPI is isolated and purified from hookworms and then cloned and expressed.
- purified is meant essentially homogenous, yielding one polypeptide band on electrophoresis in a system that separates proteins; purified inhibitor is thus substantially free of other hookworm constitu- ents, including associated proteins.
- the isolation preparation from worms is carried out by homogenizing or lysing the nematodes to obtain soluble extracts, and purifying the protein from the extracts, or obtaining extretory or secretory products from live worms. Cloned proteins are obtained from cultures of respective expression organisms.
- Any type of protein purification scheme familiar to the skilled artesan can be employed, such as, for example, affinity, ion-exchange, exclusion, partition, liquid and/or gas-liquid chromatography; zone, paper, thin layer, cellulose acetate membrane, agar gel, starch gel, and/or acryl- amide gel electrophoresis; immunochemical methods; combinations of these with each other and with other separation techniques such as dialysis; and the like.
- affinity, ion-exchange, exclusion, partition, liquid and/or gas-liquid chromatography zone, paper, thin layer, cellulose acetate membrane, agar gel, starch gel, and/or acryl- amide gel electrophoresis; immunochemical methods; combinations of these with each other and with other separation techniques such as dialysis; and the like.
- affinity ion-exchange, exclusion, partition, liquid and/or gas-liquid chromatography
- zone paper, thin layer, cellulose acetate membrane, agar gel, starch gel, and/or
- HPI polypeptides exhibiting activity and structure similar to the isolated and purified protein. Typical procedures employ the amino acid sequence of the native polypeptide to prepare cloned complementary DNA sequences defining HPI, e.g. , SEQ ID NO: 1, which is then be used to transform or transfect a host cell for protein expression using standard means. Also encompassed by this invention are DNA sequences homologous or closely related to complementary DNA described herein, namely DNA sequences which hybridize, particularly under stringent conditions, to HPI cDNA such as that set out in SEQ ID NO: 1, and RNA corresponding thereto. In addition to HPI-encoding sequences, DNA encompassed by this invention may contain additional sequences, depending upon vector construction sequences, that facilitate expression of the gene. Expressed proteins exhibit properties mimicking native proteins.
- DNA starting material which is employed to form DNA coding for HPI of the invention may be natural, recombinant or synthetic.
- DNA starting material isolated from tissue or tissue culture, constructed from oligonucleotides using conventional methods, obtained commercially, or prepared by isolating RNA coding for anticoagulant protein, and using this RNA to synthesize single-stranded cDNA which is used as a template to synthesize the corresponding double stranded DNA can be employed to prepare suitable DNA encoding HPI.
- This DNA, or RNA corresponding thereto, are then inserted into a vector, e.g.
- Host organisms useful in the invention are bacterial (e.g. , E. coli or B. subtilis), yeast (e.g. , Pichia pastoris or S. cervisiae), insect (e.g., S. frugiperda) or mammalian (e.g. , mouse fibroblast).
- This invention thus also provides novel, biologically functional viral and plasmid RNA and DNA vectors incorporating RNA and DNA sequences describing HPI generated by standard means.
- compositions containing HPI useful in a number of applications including, but not limited to, vaccines and pharmaceutical compositions more fully discussed below.
- the invention encompasses the purified polypeptide shown in SEQ ID NO: 2, or fragments or variants thereof, including variants exhibiting at least about 50%, in many cases from about 65% to about 75%, preferably at least about 80% to about 95%, sequence homology to SEQ ID NO: 2, which inhibit platelet function mimicking the native protein.
- HPI of this invention exhibits a number of desirable characteristics.
- the polypeptide inhibits platelet aggregation and adhesion in response to a variety of agonists, including, but not limited to, ADP, thrombin, and epinepherine.
- HPI apparently binds to at least one cell surface integrin such as GPIIb/IIIa ( ⁇ IIb ⁇ 3 ), and possibly also GPIa/IIa (o. 2 ⁇ ,), and mixtures thereof.
- GPIIb/IIIa ⁇ IIb ⁇ 3
- GPIa/IIa o. 2 ⁇ ,
- HPI not only blocks platelet aggregation, it also prevents adhesion of platelets to fibrinogen and possibly also collagen.
- the inhibitor inhibits b-fibrinogen binding to GPIIb/IIIa by 80%, which is comparable to a monoclonal antibody used for comparison in one study.
- the invention provides platelet inhibiting products for veterinary and medical purposes, particularly in vascular biology and cancer therapy.
- Platelet adhesion is critical for formation of primary hemostatic thrombi, and is mediated by adhesion receptors.
- platelet GPIIb/IIIa receptors on the luminal surface of adherent platelets are activated and undergo a conforma- tional change that results in their binding plasma fibrinogen with high affinity (30).
- the bivalent structure of fibrinogen allow proteins that bind to GPIIb/IIIa recep- tors to bind to two or more different platelets simultateously.
- the GPIIb/IIIa receptor is expressed only in megakaryocytes and platelets, and so is uniquely adapted to its role in platelet physiology.
- GPIIb/IIIa receptors on the surface of platelets is extraordinary ( — 80,000 copies spaced ⁇ 20 ⁇ A apart), and there is an additional internal pool of GPIIb/IIIa receptors in ⁇ -granules that can be rapidly mobilized to the surface.
- GPIIb/IIIa ( ⁇ dress b ⁇ 3 ) is the most abundant platelet adhesion receptor (46).
- Agents that are released e.g., ADP
- synthesized and released e.g. , thromoxane A 2
- generated as part of the hemostatic cascade e.g.
- thrombin when vessels are damaged are all able to initiate signals that result in the transformation of the GPIIb/IIIa receptor to a high affinity state.
- HPI of the invention markedly interferes with binding of fibrinogen to receptor GPIIb/IIIa, and very probably inhibits collagen binding to GPIa/IIa, is a unique target for inhibiting platelet activation in anti-integrin receptor therapeutics.
- GPIIb/IIIa antagonists have been suggested and tested for treat- ment of myocardial infarction and unstable angina, alone, and in combination with aspirin, thrombolytic agents, PCI, stents, and anticoagulants (30).
- GPIIb/IIIa antagonist therapy including stroke, cerebral and peripheral arterial angioplasty, thrombotic thrombocytopenic purpura/hemolytic uremic syndrome, heparin- induced thrombosis, micro vascular surgery, and cerebral malaria.
- Administration of HPI of the invention can be local or systemic.
- Systemic administration is preferred in some embodiments.
- Administration can be via any method known in the art such as, for example, oral administration of losenges, tablets, capsules, granules, or other edible compositions; subcutaneous, intrave- nous, intramuscular, or intradermal administration, e.g. , by sterile injections; parenteral administration of fluids and the like.
- Typical administrations involve the use of the inhibitor dispersed or solubilized in a pharmaceutically acceptable carrier.
- Local administration is preferred in other embodiments.
- the inhibitor again preferably in association with a pharmaceutically ac- ceptable carrier in which the inhibitor is dispersed or solubilized, is applied in effective amounts directly to an organ. Combinations of therapies may also be employed.
- compositions or formulations of the invention may also include other carriers, adjuvants, stabilizers, preservatives, dispersing agents, and other agents conventional in the art having regard to the type of formulation in question.
- enhancement of an immune response aimed at HPI of the invention provides a vaccine for reducing the burden of hookworm infection in populations at risk. This typically involves immunizing a mammal (animal or human being) by inoculating the mammal with an effective amount of a product prepared by mixing HPI with a suitable carrier such as isotonic saline. Polyclonal, monoclonal, or fusion phage antibodies are then generated using standard means.
- Human fibrinogen (plasminogen and von Wille- brand factor free) was purchased from Enzyme Research Labs (South Bend, IN). Human ⁇ -thrombin was obtained from Haematologic Technologies (Burlington, VT). Type I collagen, bovine serum albumin (BSA), epinephrine, ADP, and sodium citrate were all purchased from Sigma (St. Louis, MO). RPMI media, penicillin, streptomycin, and gentamicin were purchased from GIBCO BRL (Rockville, MD). All other chemicals were of reagent grade.
- BSA bovine serum albumin
- epinephrine epinephrine
- ADP sodium citrate
- RPMI media penicillin, streptomycin, and gentamicin were purchased from GIBCO BRL (Rockville, MD). All other chemicals were of reagent grade.
- 7E3 which blocks platelet aggregation mediated via GPIIb/IIIa ( ⁇ Iib ⁇ 3 ), as well as the binding of platelets to immobi- lized fibrinogen
- 6F1 which blocks the binding of collagen to platelet GPIa/IIa ( ⁇ 2 ⁇ ,).
- Platelet preparation Using a 20 gauge needle, whole blood was drawn by venipuncture from healthy volunteers into one-tenth volume sodium citrate. Following centrifugation at 700 x g for 4.5 minutes, the platelet-rich plasma (PRP) was removed, and the platelet count was adjusted to approximately 100,000/ ⁇ L using a Ca 2+ -free Tyrode's Buffer (137 mM NaCl, 2.7 mM KC1, 0.5 mM NaH 2 PO 4 , 12 mM NaHCO3, 1 mM MgCl 2 , 5.6 mM glucose, 3.5 g/L BSA). The diluted PRP was kept at room temperature and utilized within 4 hours.
- a Ca 2+ -free Tyrode's Buffer 137 mM NaCl, 2.7 mM KC1, 0.5 mM NaH 2 PO 4 , 12 mM NaHCO3, 1 mM MgCl 2 , 5.6 mM glucose, 3.5 g/L BSA.
- Platelet adhesion to fibrinogen, collagen, or BSA was measured in 96-well microtiter plates (24) . Individual wells were coated with 150 ⁇ L of human fibrinogen (40 ⁇ g/mL), type I collagen (20 ⁇ g/mL) or BSA (2 mg/mL) for 1 hr at 37 ° C. The plates were washed 3 times with PBS, followed by the addition of diluted PRP (100,000 platelets/ ⁇ L) in Ca 2+ -free Tyrode's buffer. For assays of inhibitory activity, diluted PRP was pre-incubated for 10 min with hookworm protein or monoclonal antibody 7E3 or 6F1 (50 ⁇ g/mL).
- the MW of the partially purified inhibitor was estimated by extrapolating from a standard curve (log MW vs R f ) constructed from the elution profile of a mixture of protein standards (Bio-Rad) with known molecular weights (16).
- the pooled fractions containing the an- ti-platelet activity were subjected to SDS polyacrylamide gel electrophoresis (SDS PAGE) using a tricine gel buffer system, and individual protein bands were visualized by silver staining (16).
- SDS PAGE SDS polyacrylamide gel electrophoresis
- ES excretory/secretory products were prepared from live adult A. caninum using a method previously described (25) for infective L 3 hookworm larvae.
- adult hookworms were manually harvested from the intestines of laboratory-infected beagles (26) and washed in PBS.
- Male and female worms were added together in groups of 10 to individual wells of a 24 well tissue culture plate in the presence of 0.5 mL RPMI supplemented with antibiotics (penicillin, streptomycin, and gentamicin) and a low molecular weight (10 kDa) filtrate of canine serum (15% v/v). After incubation overnight at 37° C, 5% CO 2 , the worms were removed and the ES products were sterilized using a 0.2 ⁇ m centrifugal filter (Millipore).
- the ES products Prior to testing for inhibition of platelet aggregation and adhesion, the ES products were concentrated approximately 5 fold using a spin concentrator with a MW cutoff of 5 kDa. As a control for the platelet inhibition assays, the same culture media (RPMI/antibio- tics/ serum filtrate) concentrated in a similar manner was used. There was no difference in the total protein concentrations of the two RPMI solutions, i.e. , with or without ES, based on analysis using a BCA kit (Pierce, North Rockford, IL).
- Fibrinogen binding assay The binding of b-fibrinogen to immobilized GP Ilb/IIIa was measured in an assay that modified the methods from Charo, et al. (50) and Steiner, et al. (51).
- Purified GPIIb/IIIa (Enzyme Research Labs.) was diluted to 5 ⁇ g/mL in 20 mM Tris, 150 mM NaCl, 1 mM CaCl 2 , 0.02% NaN 3 , pH 7.4 (buffer A) and added to 96-well Immulon 1 microtiter plates (Dynatech Laboratories) at 0.1 mL (0.5 ⁇ g) per well.
- the plates were incubated overnight at 4°C and then washed by filling and emptying by suction with 50 mM Tris, 100 mM NaCl, 2 mM CaCl 2 , 0.02% NaN 3 , pH 7.4 (buffer B).
- Bovine serum albumin (BSA) at 20 mg/mL in buffer B was incubated (0.2 mL/well) for 2 hours at room temperature, for blocking of nonspecific binding.
- B-fibrinogen was diluted to varying concentrations (1 - 50 nM) in buffer B containing 1 mg/mL of BSA and was incubated in triplicate wells (0.1 mL/well) for 4 hrs. at 37 °C.
- the plates were then washed (x 3) with buffer B before quantifying b-fibrinogen binding.
- Streptavidin conjugated to horseradish peroxidase (SAV-HRP, Calbiochem) was diluted to 1 ⁇ g/mL in buffer B and 0.1 mL was added per well. The plates were incubated for 1 hour at room temperature and then washed (x 3) with buffer B for removal of unbound SAV-HRP.
- Peroxidase substrate (1-Step Turbo TMB-ELISA, Pierce) was then added (0.15 mL/well) and changes in light absorbance (OD at 630 nm/min) were measured with a kinetic microplate reader (MRX HD, Dyna- tech Labs.).
- Readings were obtained every 30 seconds for 5 minutes, with continuous shaking in between readings.
- Nonspecific binding was determined by measuring the binding of b-fibrinogen to BSA coated wells and it was generally less than 10% of the maximum binding.
- the kinetics of b-fibrinogen binding to GP Ilb/IIIa were characterized by Scatchard analysis (52).
- fibrinogen was incubated at a 10 nM concentration in the presence of various hookworm protein extracts, excretory-secretory (E/S) products and the inhibitory monoclonal antibodies 7E3 and 6F1.
- Collagen binding assay A distinct assay was developed to measure collagen binding to the platelet integrin GP Ia/IIa using T47D mammary carcino- ma cells and based on the work by Keely, et al. (53). An Immulon 2 microtiter plate was coated with type I collagen in PBS and blocked with BSA as described above. Cells at approximately 90% confluence were detached with versene 1 :5000 (0.5 mM EDTA in PBS, Gibco BRL), centrifuged for 5 minutes (4400 RPM), and resuspended in PBS supplemented with 5 mg/mL BSA, 5 mM glucose and 0.3 mM MgCl 2 .
- the cells were resuspended and diluted to 500,000 cells/mL and 0.1 mL was added to each well.
- the plate was incubated for 15 minutes at 37 ° C and non-adherent cells were removed with low suction and rinsing with PBS three times.
- Adherent cells were quantified with the Landegren hexosaminidase assay (54) as described by Haugen et al. (55).
- hexosaminidase substrate solution (3.75 mM /?-itrophenol-N-acetyl-
- the plate was incubated overnight at 37 ° C.
- the color was developed by adding 90 ⁇ L/well of 50 mM glycine, 5 mM EDTA, pH 10.4 and absorbance was measured at 405 nm.
- T47D cells For testing of inhibitory activity, aliquots of T47D cells at the same concentration were preincubated with hookworm proteins or the monoclonal antibodies in an orbital shaker for 20 minutes at room temperature The cells were then transferred to the designated wells and the assay was performed as above. Each sample was done in triplicate and nonspecific binding was again determined by measuring binding of cells to BSA coated wells.
- RNA pellet was washed with 75% and 100% ethanol, air-dried for 10 min, and resuspended in 40 ⁇ L of diethyl pyrocarbonate (DEPC)-treated water.
- DEPC diethyl pyrocarbonate
- Degenerate oligonucleotide primers (5'HPI-l and 3'HPI-l, residues 1 to 23 of SEQ ID NO: 1, and residues 88 to 110 of SEQ ID NO: 1, respectively) were designed based on the amino acid sequence obtained from the native HPI (discussed below). The primers were synthesized by the Keck Foundation Laboratory at Yale. First strand cDNA was synthesized by incubating approximately 1 ⁇ g of A. caninum total RNA, 10 mM DTT and 100 ng of the degenerate antisense strand primer 3'HPI-l in first strand reaction buffer (50 mM Tris-HCl, 75 mM KC1, 3mM MgCl 2 , pH 8.3).
- first strand reaction buffer 50 mM Tris-HCl, 75 mM KC1, 3mM MgCl 2 , pH 8.3
- the solution was heated for 90 seconds at 90 ° C and cooled on ice.
- Each of four deoxynucleotides (1 mM; dATP, dCTP, dGTP, dTTP) was added along with 40 U RNasin (Promega) and 200 U reverse transcrip- tase enzyme (Superscript II; Life Technologies). This mixture was incubated for 1 hr at 42 ° C and cooled on ice.
- the first strand cDNA mixture was combined in thin-walled 0.2 mL tubes with PCR primers 5'HPI-l and 3'HPI-l (100 ng of each), PCR buffer (10 mM Tris-HCl, 50 mM KC1, 1.5 mM MgCl 2 , pH 8.3), and 1 mM of each deoxynucleotide.
- Taq polymerase (5 U; Perkin- Elmer) was then added and samples were placed in a thermal cycler (PCR Sprint, Hybaid) for 40 cycles (94 °C for 15 sec denaturation, 50 ° C for 5 sec annealing, 72 ° C for 30 sec extension), with a final extension at 72 ° C for 2 min.
- the reaction mixture was subjected to 1 % agarose gel electrophoresis and PCR products were visualized by ethidium bromide staining.
- PCR products were ligated into the pCR2.1 TA cloning vector (Invitrogen) by incubating 10 ng of fresh PCR product, 50 ng of vector, T4 DNA ligase, and ligation buffer overnight at 14 °C.
- One Shot E. coli INV ⁇ F' cells were transformed with the ligation product following the manufacturer's protocol. Samples were plated onto LB agar plates containing 30 ⁇ g/mL kanamycin and spread with a 40 mg/mL solution of X-Gal.
- the sequence obtained was used to design an internal primer (5'HPI-l A, residues 66 to 97 of SEQ ID NO: 1) for a 3' Rapid Amplification of cDNA Ends (3 'RACE) protocol used to clone the 3 'end cDNA.
- First strand cDNA was amplified by RT-PCR, as above, using 1 ⁇ g of total RNA and an antisense primer, 3'TTTT.
- the amplified cDNA mixture was then used as a template for PCR using 3'TTTT and 5'HPI-l A. Reaction components were as previously described.
- Amplification conditions were 40 cycles (94 °C for 15 sec denaturation, 55 °C for 5 sec annealing, 72 ° C for 30 sec extension), with a final extension at 72 ° C for 2 min.
- the PCR product was ligated into the pCR2.1 TA cloning vector, One Shot E. coli INV ⁇ F' cells were transformed, and colonies were screened as previously described.
- Miniprep plasmid DNA of positive clones was submitted to the Keck Foundation Laboratory for nucleotide sequencing. The full length cDNA was cloned by performing the 3 'RACE protocol as described above, with the original 5' end primer (5'HPI-l).
- the PCR product containing the full sequence was ligated into the pCR2.1 TA cloning vector, One Shot E. coli INV ⁇ F' cells were transformed, and colonies were screened as before. The sequence was corroborated by analysis of the Miniprep plasmid DNA at the Keck Laboratory.
- Hookworm extracts block platelet aggregation and adhesion. Soluble hookworm extracts blocked platelet aggregation in response to epinephrine, thrombin, and ADP (Figure 1A). Although there was virtually 100% inhibition of aggregation in the presence of 30 ⁇ g of hookworm extracts, the effect was found to be dose-dependent for each of the three agonists studied. Because aggregation in response to a variety of agonists is largely dependent on fibrinogen mediated crosslinking of platelets via GPIIb/IIIa, a cell surface ⁇ IIb ⁇ 3 integrin (27, 28), these data suggested that soluble hookworm extracts might contain a potent inhibitor of this receptor.
- hookworm extracts In order to more specifically characterize the anti-platelet mechanism of hookworm extracts, their effect on platelet adhesion to immobilized fibrinogen, which is also mediated via interaction with GPIIb/IIIa (29, 30), were tested. As shown in Figure IB, hookworm extracts inhibited adhesion of platelets to fibrinogen coated plates in a concentration dependent manner, with 70% inhibition achieved in the presence of 7 ⁇ g of soluble hookworm protein.
- Hookworm Platelet Inhibitor partial purification. Soluble hookworm extracts from approximately 1,000 adult A caninum hookworms were first applied to an anion exchange column. The bound protein was eluted with a gradient of 0-500 mM NaCl ( Figure 2), and column fractions were collected and tested for anti-platelet activity. No inhibition of platelet aggregation or adhesion was ob- served in the flow-through fractions. A single peak of anti-platelet activity was identified in the protein eluted from the column, with complete overlap of the fractions inhibiting aggregation and adhesion to fibrinogen. These five fractions, which contained all of the anti-platelet activity present in the pre-column material, were pooled and concentrated.
- the partially purified inhibitor was then subjected to size exclusion chromatography. Individual fractions were collected and tested for activity using both the adhesion and aggregation assays. Again, all of the anti-platelet activity eluted in a single series of contiguous fractions (Figure 3), which were shown to inhibit aggregation (using ADP as agonist), as well as adhesion to fibrinogen. Based on extrapolation from a standard curve constructed using proteins of known molecular weights (Figure 3, inset), the size of the partially purified active HPI was estimated to be approximately 15 kDa.
- SDS PAGE of the partially purified HPI revealed multiple protein bands detected by silver staining, ranging in estimated MW from 10-25 kDa. There was no obvious enrichment of any single band visible by silver staining when HPI was compared to SDS PAGE of the soluble hookworm extract starting material. However, based on measurements of protein concentration and percent inhibition of platelet aggregation, the HPI activity was estimated to have been enriched by approximately 5-fold after anion exchange and size exclusion chromatographies.
- HPI dual inhibition of GPIIb/IIIa and GPIa/IIa.
- the active fractions from size exclusion chromatography were pooled and again characterized with regard to their inhibitory properties.
- partially purified HPI exhibited a comparable inhibitory profile to soluble hookworm extracts, effectively blocking platelet aggregation in response to epinephrine, thrombin, and ADP.
- HPI The function of HPI was further characterized using the specially developed fibrinogen and collagen binding assays described above. HPI inhibited fibrinogen binding to GP Ilb/IIIa by approximately 80%, which is comparable to the monoclonal 7E3. Inhibitory activity was also detected in E/S products, as well as in extracts from A. ceylanicum and N. americanus (see below). In the collagen binding assay developed, HPI inhibited T47D cells binding to collagen by approximately 80-85%, comparable to the monoclonal 6F1.
- HPI is secreted by adult A. caninum.
- ES products from live adult hookworms removed from an infected dog were tested.
- concentrated hookworm ES products contain an inhibitor of platelet aggregation and adhesion that is comparable to that which was purified from soluble extracts of whole worms.
- concentrated culture media which was comparable to hookworm ES in terms of protein concentration, did not inhibit platelet aggregation or adhesion in vitro.
- Results are expressed as percentage of control values for both assays. Aggregation was measured in response to agonist ADP. ES products and RPMI culture media were both concentrated ⁇ 5-fold prior to use.)
- Hookworm Platelet Inhibitor native protein analysis, cloning and sequencing.
- the partially purified HPI described above was subjected to reversed-phase HPLC using a C lg column. Bound protein was eluted under a linear gradient of acetonitrile in TFA as described above and shown in Figure 5.
- Individual protein peaks were lyophilized, resuspended in aqueous buffer, and tested for inhibition of biotinylated fibrinogen to immobilized GPIIb/IIIa. The two activities were distinctly separated.
- Protein peaks 26 and 27 (designated HPI-2) exclusively inhibited collagen binding to GP Ia/IIa while peaks 28, 29 and 30 (designated HPI-1) inhibited fibrinogen binding to GP Ilb/IIIa.
- HPI-1 Protein analysis of the purified HPI-1 and HPI-2 was performed at the William Keck Foundation Biotechnology Resource Laboratory at Yale School of Medicine. The molecular mass was determined by electrospray ionization mass spectrometry (ESMS) using a Micromass Q-Tof spectrometer and estimated to be approximately 20,314 - 20,365 Da for HPI-1, and approximately 20,546 Da for HPI-2. HPI-1 was then submitted to NH 2 -terminal amino acid sequencing using an Applied Biosystems sequencer equipped with an on-line HPLC system.
- ESMS electrospray ionization mass spectrometry
- the first 40 amino acids were obtained (residues 1 to 40 of SEQ ID NO: 2), and the sequence used to design degenerate oligonucleotide primers for cloning of the cDNA corresponding to the HPI-1 gene. Further analysis of HPI-2 suggests that in fact it could be the same protein as HPI-1, or alternatively, that the two are very closely related.
- NH 2 -terminal amino acid sequencing of HPI-2 revealed that the first six amino acids were identical to those of HPI-1. An internal segment of each protein was sequenced after trypsin digestion, and the amino acid sequence was also identical.
- HPI- 1 Glu-Gly-Asp-Tyr-Ser-Leu
- HPI-2 Glu-Gly-Asp-Tyr-Ser-Leu
- HPI-1 Thr-Ser-Asn-Ile-Ala-Asn-Met-Val
- the NH 2 -terminal amino acid sequence of recombinant HPI isolated and analyzed as described above is set out in residues 1 to 40 of SEQ ID NO: 2.
- Degenerate oligonucleotide primers designed from this sequence were used to obtain the cDNA sequence set out in SEQ ID NO: 1 using cloning procedures described above.
- the full amino acid sequence comprising residues 1 to 40 of the purified native protein and deduced residues 41 to 181 of the cDNA is set out in SEQ ID NO: 2.
- the predicted molecular weight of this recombinant polypeptide is 20,333 Da, and its theoretical isoelectric point, 4.76.
- the estimated molecular weight of native HPI is about 20,314 to 20,365 Da.
- the HPI sequence was compared to other GENBANK sequences (published at www.ncbi.nlm.nih.gov), and found to exhibit little or no homology to previously published sequences.
- Ancylostoma hookworms produce a potent and broad spectrum inhibitor of platelet aggregation.
- the activity present in A. caninum is similar to that identified in the phylogenetically distinct hookworm species N. americanus.
- an HPI-like activity in ES products of live adult hookworms removed from the gut of a permissive host (dog) was identified, strongly suggesting that secretion of the hookworm inhibitor is associated with the bloodfeeding process.
- Preliminary data suggest that the activity is also present in secretory products of infective third stage larvae, suggesting a potentially broad functional role for HPI in the biology of hookworm.
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- General Health & Medical Sciences (AREA)
- General Chemical & Material Sciences (AREA)
- Public Health (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Immunology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- Genetics & Genomics (AREA)
- Toxicology (AREA)
- Zoology (AREA)
- Gastroenterology & Hepatology (AREA)
- Diabetes (AREA)
- Biophysics (AREA)
- Hematology (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
Description
Claims
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2000608041A JP2002539817A (en) | 1999-03-31 | 2000-03-30 | Hookworm platelet inhibitor |
AU39308/00A AU3930800A (en) | 1999-03-31 | 2000-03-30 | Hookworm platelet inhibitor |
CA002367424A CA2367424A1 (en) | 1999-03-31 | 2000-03-30 | Hookworm platelet inhibitor |
EP00918509A EP1165598A4 (en) | 1999-03-31 | 2000-03-30 | Hookworm platelet inhibitor |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US12723999P | 1999-03-31 | 1999-03-31 | |
US60/127,239 | 1999-03-31 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2000058341A1 true WO2000058341A1 (en) | 2000-10-05 |
Family
ID=22429047
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2000/008519 WO2000058341A1 (en) | 1999-03-31 | 2000-03-30 | Hookworm platelet inhibitor |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP1165598A4 (en) |
JP (1) | JP2002539817A (en) |
AU (1) | AU3930800A (en) |
CA (1) | CA2367424A1 (en) |
WO (1) | WO2000058341A1 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7691839B2 (en) | 2005-09-28 | 2010-04-06 | Biovascular, Inc. | Methods and compositions for blocking platelet and cell adhesion, cell migration and inflammation |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1995012615A1 (en) * | 1993-11-02 | 1995-05-11 | The University Of Nottingham | Antihaemostatic agents from necator americanus |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6962795B1 (en) * | 1992-05-11 | 2005-11-08 | Dendreon Corporation | Neutrophil inhibitors |
-
2000
- 2000-03-30 JP JP2000608041A patent/JP2002539817A/en active Pending
- 2000-03-30 WO PCT/US2000/008519 patent/WO2000058341A1/en not_active Application Discontinuation
- 2000-03-30 EP EP00918509A patent/EP1165598A4/en not_active Withdrawn
- 2000-03-30 CA CA002367424A patent/CA2367424A1/en not_active Abandoned
- 2000-03-30 AU AU39308/00A patent/AU3930800A/en not_active Abandoned
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1995012615A1 (en) * | 1993-11-02 | 1995-05-11 | The University Of Nottingham | Antihaemostatic agents from necator americanus |
Non-Patent Citations (2)
Title |
---|
FURMIDGE ET. AL.: "The anti-haemostatic strategies of the human hookworm Necator americanus", PARASITOLOGY, vol. 112, 1995, pages 81 - 87, XP002928966 * |
See also references of EP1165598A4 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7691839B2 (en) | 2005-09-28 | 2010-04-06 | Biovascular, Inc. | Methods and compositions for blocking platelet and cell adhesion, cell migration and inflammation |
US8188034B2 (en) | 2005-09-28 | 2012-05-29 | Biovascular, Inc. | Methods and compositions for blocking platelet and cell adhesion, cell migration and inflammation |
Also Published As
Publication number | Publication date |
---|---|
EP1165598A4 (en) | 2002-10-30 |
JP2002539817A (en) | 2002-11-26 |
CA2367424A1 (en) | 2000-10-05 |
EP1165598A1 (en) | 2002-01-02 |
AU3930800A (en) | 2000-10-16 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US5458874A (en) | Method of increasing monocyte chemotaxis with CAP37 and monocyte chemotactic portions thereof | |
US6040441A (en) | Nematode-extracted serine protease inhibitors and anticoagulant proteins | |
US20240043500A1 (en) | Polypeptides and medical uses thereof | |
US20130052258A1 (en) | Polypeptides and uses thereof | |
WO2000032631A2 (en) | Proteins that bind angiogenesis-inhibiting proteins, compositions and methods of use thereof | |
JPH11504938A (en) | Kunitz-type protease inhibitor | |
Chadderdon et al. | The hookworm platelet inhibitor: functional blockade of integrins GPIIb/IIIa (α IIb β 3) and GPIa/IIa (α 2 β 1) inhibits platelet aggregation and adhesion in vitro | |
AU735427B2 (en) | Bi- or multifunctional molecules based on a dendroaspin scaffold | |
Beresford et al. | Characterization of hemolytic and cytotoxic Gallysins: a relationship with arylphorins | |
US5618675A (en) | Methods and compositions for detecting lipopolysaccharides using CAP18 fragments | |
RU2138275C1 (en) | Thrombin inhibitors, method of their producing and pharmaceutical composition on said | |
US5427937A (en) | Hookworm anticoagulant | |
He et al. | HSC70 from Haemaphysalis flava (Acari: Ixodidae) exerts anticoagulation activity in vitro | |
JP2001517422A (en) | Isolated and recombinant antimicrobial peptides thrombosidine-1 (TC-1) and thrombosidine-2 (TC-2) or variants thereof | |
Kuan-Hong et al. | Identification and characterization of a novel elastase inhibitor from Hirudinaria manillensis | |
JP2703993B2 (en) | Ambriomin with thrombin inhibitory action | |
EP1165598A1 (en) | Hookworm platelet inhibitor | |
US5955294A (en) | Nematode-extracted serine protease inhibitors and anticoagulant proteins | |
Chadderdon et al. | The Hookworm Platelet Inhibitor: Functional Blockade of Integrins GPIIb/IIIa and GPIa/IIa Inhibits Platelet Aggregation and Adhesion in vitro | |
US6036958A (en) | Tsetse thrombin inhibitor | |
US20040014178A1 (en) | Von willebrand factor-binding proteins from staphylococci | |
Levashina et al. | Thioester-containing proteins of protostomes | |
Kazimírová | Pharmacologically active compounds from ticks and other arthropods and their potential use in anticancer therapy | |
Slabá | Characterisation of novel serpin TILIr and its relatives from the superfamily of serine protease inhibitors from Ixodes ricinus tick | |
CN118005776A (en) | Egypt schistosome Kunitz polypeptide variant and application thereof |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DE DK DM EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): GH GM KE LS MW SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG |
|
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
ENP | Entry into the national phase |
Ref document number: 2367424 Country of ref document: CA Ref country code: CA Ref document number: 2367424 Kind code of ref document: A Format of ref document f/p: F |
|
WWE | Wipo information: entry into national phase |
Ref document number: 39308/00 Country of ref document: AU |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2000918509 Country of ref document: EP |
|
ENP | Entry into the national phase |
Ref country code: JP Ref document number: 2000 608041 Kind code of ref document: A Format of ref document f/p: F |
|
WWP | Wipo information: published in national office |
Ref document number: 2000918509 Country of ref document: EP |
|
REG | Reference to national code |
Ref country code: DE Ref legal event code: 8642 |
|
WWW | Wipo information: withdrawn in national office |
Ref document number: 2000918509 Country of ref document: EP |