WO1995012615A1 - Antihaemostatic agents from necator americanus - Google Patents

Antihaemostatic agents from necator americanus Download PDF

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Publication number
WO1995012615A1
WO1995012615A1 PCT/GB1994/002406 GB9402406W WO9512615A1 WO 1995012615 A1 WO1995012615 A1 WO 1995012615A1 GB 9402406 W GB9402406 W GB 9402406W WO 9512615 A1 WO9512615 A1 WO 9512615A1
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Prior art keywords
excretory
necator americanus
products
secretory
human
Prior art date
Application number
PCT/GB1994/002406
Other languages
French (fr)
Inventor
David Idris Pritchard
Original Assignee
The University Of Nottingham
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
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Publication of WO1995012615A1 publication Critical patent/WO1995012615A1/en

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    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/56—Materials from animals other than mammals
    • A61K35/62—Leeches; Worms, e.g. cestodes, tapeworms, nematodes, roundworms, earth worms, ascarids, filarias, hookworms, trichinella or taenia
    • 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 present invention relates to antihaemostatic agents for the treatment of human thrombotic disorders.
  • heparin is a common anticoagulant
  • fibrin-rich blood clots may be dissolved using tissue plasminogen activator, streptokinase and urokinase.
  • tissue plasminogen activator streptokinase
  • urokinase urokinase
  • the present invention seeks to provide novel antihaemostatic agents.
  • an excretory-secretory product of the human hookworm Necator americanus for use as an active pharmaceutical substance .
  • the invention further provides for the use of excretory-secretory products of the human hookworm Necator americanus for the manufacture of an antihaemostatic composition.
  • the invention further provides an antihaemostatic composition
  • an antihaemostatic composition comprising a pharmaceutically acceptable diluent or carrier and an excretory-secretory product of the human hookworm Necator americanus.
  • the invention further provides an antihaemostatic composition
  • an antihaemostatic composition comprising a pharmaceutically acceptable diluent or carrier and an active ingredient obtained from an excretory-secretory product of the human hookworm Necator americanus.
  • the active ingredient may be obtained directly, or by chemical synthesis, or by a genetic engineering technique .
  • the invention further provides excretory-secretory products or derivatives thereof of the human hookworm Necator americanus for use as an inhibitor of platelet aggregation .
  • the invention further provides excretory-secretory products or derivatives thereof of the human hookworm Necator americanus for use as an inhibitor of Factor Xa activity.
  • the invention further provides derivatives of excretory-secretory products of the human hookworm Necator americanus for use as an inhibitor of platelet dense granule release.
  • Fig. 1 shows the effect of Necator americanus excretory-secretrory products on human plasma clotting times
  • Fig. 2 shows the effects of Necator americanus excretory-secretory products on human plasma stypven clotting time
  • Fig. 3 shows the effects of Necator americanus on human Factor Xa activity
  • Fig. 4 shows the effects of Necator americanus excretory-secretory products on platelet aggregation in human plasma mediated respectively by collagen, adenosine diphosphate (ADP), thrombin and platelet activating factor (PAF);
  • ADP adenosine diphosphate
  • PAF platelet activating factor
  • Fig. 5 shows the effects of Necator americanus excretory-secretory products on platelet granule release .
  • Fig. 6 shows the degradation of human fibrinogen by Necator americanus excretory-secretory products. PREPARATION OF NECATOR AMERICANUS EXCRETORY-SECRETORY (ES) PRODUCTS
  • Necator americanus is passaged in DSN hamsters. Faecal cultured from infected animals provide infective (L3) larvae, which are then used to infect neonates percutaneously .
  • Adult worms are routinely harvested from the small intestines of infected hamsters 5 weeks post-infection. The ileum of the infected hamster is removed, opened longitudinally, and placed in Hanks' saline at 37°C. As worms release their hold on the mucosa, they are carefully removed, thoroughly washed, and cleansed in Hanks' saline containing 100 iu/ml penicillin and 100 ug/ml streptomycin. Cleansed worms are examined under a dissecting microscope, and undamaged worms retained.
  • worms are added to RPMI 1640, containing penicillin and streptomycin. The worms are then cultured for 16 hrs, and supernates removed for analysis of thro bolytic and anticoagulant activities.
  • Culture supernatants are filtered through 0.2 urn Minisort NML filters (Sartorius) to remove eggs that may have been deposited during the culture period.
  • Concentration of supernatants is carried out using centrifugal separation methods.
  • the bulk of the culture media is removed using Macrosep Centrifugal Concentrators (Flowgen) with a cut off point of 10K.
  • Final concentration and separation into 2 fractions according to MW is carried out using Centricon micro concentrators (Amincon) with cut off points of 30K and 10K.
  • Imidazole saline buffer (Sigma) is used to dilute the supernatant at this stage to aid in separation.
  • platelet rich plasma contains all the factors necessary to activate prothrombin by the intrinsic pathway.
  • the rate of clot ⁇ ting is a measure of the overall coagulant activity dev ⁇ eloped and this will be decreased if there is inhibition of any intrinsic pathway factor or factor-complex.
  • PRP platelet poor plasma
  • platelet substitute phospholipid emulsion
  • the test is carried out in a water bath at 37°C.
  • the 50 ul PPP in a previously unused glass test tube, 25 ul Celite (485 w/v in 0.85? ⁇ NaCl) and 25 ul phospholipid substitute (Rabbit brain cephalin (RVC): 1 vial in 5 ml 0.85? ⁇ NaCl) is added.
  • RVC phospholipid substitute
  • the tube is agitated to disperse the Celite.
  • 50 ul warmed CaClpit (0.025M) is then added and the timing started. At 2-3 second intervals, the tube is tilted and observed for the formation of a clot.
  • the PPP is preincubated for 10 minutes with varying concentrations of ES products (volumes being made up with saline). As delayed clotting time is often followed by the formation of a poor clot, the aggregation of Celite particles (caused by fibrin formation) is taken to indicate clot formation. If no clot is formed within 3 minutes, the timing is terminated.
  • the ES products of N. americanus have a dose-dependent effect on APTT (see figure 1).
  • Tissue thromboplastin tissue factor
  • factor VII a phospholipoprotein
  • the test is carried out in a water bath at 37°C.
  • SUBSTITUTE SHEET (RULE 26 ⁇ Equal volumes of thromboplastin solution and 0.025M CaCl_ are mixed and stored in the water bath.
  • the thromboplastin obtained from Sigma Diagnostics - 1 vial reconstituted with 2 ml dionized water). 50 ul citrated PPP is warmed in a clean unused test tube, 100 ul thromboplastin/CaCl- mixture added, and the timing started. (The tube is tilted every 2-3 seconds and the clotting time noted).
  • the PPP is first incubated for 10 minutes with varying concentrations of ES products (volumes being made up with saline). A prolonged clotting time or no clot formation is taken as an indication of inhibition in the extrinsic pathway by hookworm ES products.
  • the ES products of N. americanus have a dose-dependent effect on PT (see figure 1).
  • Stypven clotting time is the accelerated clotting time of recalcified plasma when mixed with Russell's Viper Venom (RVV). In the presence of brain phospholipid, RVV activates factor X directly.
  • the test is carried out in a water bath at 37°C. 50 ul human PPP is warmed in a clean unused test tube, and 25 ul RVV (in cephalin solution) added.
  • RVV in cephalin solution
  • the RVV in cephalin was obtained from Sigma Diagnostics - 1 vial was dissolved in 3 ml 0.85? ⁇ NaCl). After 3 minutes incubation, 50 ul 0.025M CaClweb is added, and the time s tar ted .
  • hookworm ES products The effect of hookworm ES products on Stypven Time is assessed by adding varying concentrations of ES products to the mixture after incubation with RVV and allowing a further incubation period of 10 minutes. 50 ul of CaCl_ is then added and the timing started. Prolongation of clotting time is taken as an indication of inhibition in the common pathway of coagulation by hookworm ES products.
  • Necator ES products prolong human plasma Stypven clotting time (see figure 2).
  • a synthetic oligopeptide substrate possessing a fluorescent group commercially used to measure activated factor X, was used to confirm factor Xa activity.
  • Factor Xa splits the AMC.HC1 from the carboxyl terminal of this substrate molecule causing it to fluoresce. The enzyme activity can thus be assessed using a fluorimeter .
  • Necator ES products The effect of Necator ES products on fibrinogenolysis was investigated by incubating human fibrinogen and Necator ES products over timed intervals then examining the degradation products using SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE), 40ug aliquots of fibrinogen were incubated with 10ug Necator ES products at 37°C for 1,3,5 and 24 hour periods. At the end of each incubation period, the samples were run under reducing conditions on a 7-12. ⁇ gradient gel. This was run at 30volts overnight, at 200volts until completion, and then stained with Coomassie brilliant blue R-250.
  • SDS-PAGE SDS-Polyacrylamide Gel Electrophoresis
  • Fibrinogenolysis occured during incubation of human fibrinogen with Necator americanus ES products (Fig. 6). After one hour incubation, degradation of fibrinogen was clearly seen. Prolonged incubation times caused increading amounts of degradation, and after 24 hours most of the 40ug protein sample was degraded.
  • excretory-secretory products of the human hookworm prevent haemostasis by various strategies, including inhibition of coagulation factor Xa, a pivotal component of the clotting cascade. Platelet activation is also affected.

Abstract

The invention relates to the use of excretory-secretory (ES) products of the human hookworm Necator americanus as antihaemostatic agents. In particular, the products inhibit the activity of coagulation factor Xa, and inhibit platelet aggregation.

Description

ANTIHAEMOSTATIC AGENTS FROM NECATOR AMERICANUS.
The present invention relates to antihaemostatic agents for the treatment of human thrombotic disorders.
Human thrombotic disorders are widely treated by the use of anticoagulant and thrombolytic drugs. For example, heparin is a common anticoagulant, whilst fibrin-rich blood clots may be dissolved using tissue plasminogen activator, streptokinase and urokinase. However, there are some side effects and other drawbacks associated with the use of the known drugs.
The present invention seeks to provide novel antihaemostatic agents.
According to the invention there is provided an excretory-secretory product of the human hookworm Necator americanus for use as an active pharmaceutical substance .
According to the invention there is provided derivatives of excretory-secretory (ES) products of the human hookworm Necator americanus for use as an active pharmaceutical substance.
The invention further provides for the use of excretory-secretory products of the human hookworm Necator americanus for the manufacture of an antihaemostatic composition.
The invention further provides an antihaemostatic composition comprising a pharmaceutically acceptable diluent or carrier and an excretory-secretory product of the human hookworm Necator americanus.
The invention further provides an antihaemostatic composition comprising a pharmaceutically acceptable diluent or carrier and an active ingredient obtained from an excretory-secretory product of the human hookworm Necator americanus.
The active ingredient may be obtained directly, or by chemical synthesis, or by a genetic engineering technique .
The invention further provides excretory-secretory products or derivatives thereof of the human hookworm Necator americanus for use as an inhibitor of platelet aggregation .
The invention further provides excretory-secretory products or derivatives thereof of the human hookworm Necator americanus for use as an inhibitor of Factor Xa activity. The invention further provides derivatives of excretory-secretory products of the human hookworm Necator americanus for use as an inhibitor of platelet dense granule release.
The invention will be further described for the purposes of illustration only with reference to the following examples and accompanying drawings, in which:
Fig. 1 shows the effect of Necator americanus excretory-secretrory products on human plasma clotting times ;
Fig. 2 shows the effects of Necator americanus excretory-secretory products on human plasma stypven clotting time;
Fig. 3 shows the effects of Necator americanus on human Factor Xa activity;
Fig. 4 shows the effects of Necator americanus excretory-secretory products on platelet aggregation in human plasma mediated respectively by collagen, adenosine diphosphate (ADP), thrombin and platelet activating factor (PAF);
Fig. 5 shows the effects of Necator americanus excretory-secretory products on platelet granule release ; and
Fig. 6 shows the degradation of human fibrinogen by Necator americanus excretory-secretory products. PREPARATION OF NECATOR AMERICANUS EXCRETORY-SECRETORY (ES) PRODUCTS
Necator americanus is passaged in DSN hamsters. Faecal cultured from infected animals provide infective (L3) larvae, which are then used to infect neonates percutaneously . Adult worms are routinely harvested from the small intestines of infected hamsters 5 weeks post-infection. The ileum of the infected hamster is removed, opened longitudinally, and placed in Hanks' saline at 37°C. As worms release their hold on the mucosa, they are carefully removed, thoroughly washed, and cleansed in Hanks' saline containing 100 iu/ml penicillin and 100 ug/ml streptomycin. Cleansed worms are examined under a dissecting microscope, and undamaged worms retained.
Under sterile conditions, worms are added to RPMI 1640, containing penicillin and streptomycin. The worms are then cultured for 16 hrs, and supernates removed for analysis of thro bolytic and anticoagulant activities.
Culture supernatants are filtered through 0.2 urn Minisort NML filters (Sartorius) to remove eggs that may have been deposited during the culture period.
Concentration of supernatants is carried out using centrifugal separation methods. The bulk of the culture media is removed using Macrosep Centrifugal Concentrators (Flowgen) with a cut off point of 10K. Final concentration and separation into 2 fractions according to MW is carried out using Centricon micro concentrators (Amincon) with cut off points of 30K and 10K. Imidazole saline buffer (Sigma) is used to dilute the supernatant at this stage to aid in separation. TESTS TO ASSESS ACTIVITY AGAINST THE INTRINSIC AND EXTRINSIC PATHWAYS OF BLOOD COAGULATION
The Intrinsic Pathway
This involves testing the time taken to form a fibrin polymer clot, with ample prothrombin and fibrinogen present and no activation of factor VII. The test which gives the most consistent results is the activated partial thromboplastin test.
Activated Partial Thromboplastin Test (APTT)
With the exception of Ca , platelet rich plasma (PRP) contains all the factors necessary to activate prothrombin by the intrinsic pathway. The rate of clot¬ ting is a measure of the overall coagulant activity dev¬ eloped and this will be decreased if there is inhibition of any intrinsic pathway factor or factor-complex.
Both the number of platelets present in PRP and the extent of exposure to glass may vary during the test, markedly affecting the test results. To avoid these inconsistencies, platelet poor plasma (PPP) is used for the test, and to this an optimal amount of platelet substitute (phospholipid emulsion) is added. In addition, optimal glass activation is obtained by incubating with Celite.
The test is carried out in a water bath at 37°C. The 50 ul PPP in a previously unused glass test tube, 25 ul Celite (485 w/v in 0.85?ό NaCl) and 25 ul phospholipid substitute (Rabbit brain cephalin (RVC): 1 vial in 5 ml 0.85?ό NaCl) is added. (Both the Celite and RBC were obtained from Sigma Diagnostics). At intervals, during a 6 minute incubation time, the tube is agitated to disperse the Celite. 50 ul warmed CaCl„ (0.025M) is then added and the timing started. At 2-3 second intervals, the tube is tilted and observed for the formation of a clot. To assess the effects of hookworm ES products on APTT, the PPP is preincubated for 10 minutes with varying concentrations of ES products (volumes being made up with saline). As delayed clotting time is often followed by the formation of a poor clot, the aggregation of Celite particles (caused by fibrin formation) is taken to indicate clot formation. If no clot is formed within 3 minutes, the timing is terminated.
Results
The ES products of N. americanus have a dose-dependent effect on APTT (see figure 1).
The Extrinsic Pathway
This involves the time taken to form a fibrin polymer clot without the presence of platelet phospholipid but with tissue factor and ample prothrombin and fibrinogen present. The test used was the Prothrombin Time Test (PT).
Prothrombin Time Test (PT)
Tissue thromboplastin (tissue factor), a phospholipoprotein, was added to human PPP to activate factor VII and to provide the tissue phospholipid needed both for factor X activation and as part of the prothrombinase complex.
The test is carried out in a water bath at 37°C.
SUBSTITUTE SHEET (RULE 26} Equal volumes of thromboplastin solution and 0.025M CaCl_ are mixed and stored in the water bath. The thromboplastin (obtained from Sigma Diagnostics - 1 vial reconstituted with 2 ml dionized water). 50 ul citrated PPP is warmed in a clean unused test tube, 100 ul thromboplastin/CaCl- mixture added, and the timing started. (The tube is tilted every 2-3 seconds and the clotting time noted).
To assess the effects of hookworm ES products on PT, the PPP is first incubated for 10 minutes with varying concentrations of ES products (volumes being made up with saline). A prolonged clotting time or no clot formation is taken as an indication of inhibition in the extrinsic pathway by hookworm ES products.
Results
The ES products of N. americanus have a dose-dependent effect on PT (see figure 1).
MEASUREMENT OF ACTIVITY AGAINST FACTOR Xa
Stypyen Clotting Time Test
Stypven clotting time is the accelerated clotting time of recalcified plasma when mixed with Russell's Viper Venom (RVV). In the presence of brain phospholipid, RVV activates factor X directly.
The test is carried out in a water bath at 37°C. 50 ul human PPP is warmed in a clean unused test tube, and 25 ul RVV (in cephalin solution) added. (The RVV in cephalin was obtained from Sigma Diagnostics - 1 vial was dissolved in 3 ml 0.85?ό NaCl). After 3 minutes incubation, 50 ul 0.025M CaCl„ is added, and the time s tar ted .
The effect of hookworm ES products on Stypven Time is assessed by adding varying concentrations of ES products to the mixture after incubation with RVV and allowing a further incubation period of 10 minutes. 50 ul of CaCl_ is then added and the timing started. Prolongation of clotting time is taken as an indication of inhibition in the common pathway of coagulation by hookworm ES products.
Results
"Necator" ES products prolong human plasma Stypven clotting time (see figure 2).
Confirmatory Fluorogenic Assay for Factor Xa activity
A synthetic oligopeptide substrate, possessing a fluorescent group commercially used to measure activated factor X, was used to confirm factor Xa activity. Factor Xa splits the AMC.HC1 from the carboxyl terminal of this substrate molecule causing it to fluoresce. The enzyme activity can thus be assessed using a fluorimeter .
Boc . Ile.Glu.Gly .Arg. AMC.HC1 , the substrate used for the measurement of factor Xa activity, was purchased from Novabiochem (UK) Ltd., Nottingham. This is made up to a 5 M solution with distilled water and stored in small aliquots at -20°C. Human Factor Xa was obtained from Diagnostics Astho, Deeside, Clywd.
Assays are conducated at 37°C using 50 mM Tris-HCl buffer pH 8.0 containing 100 mM NaCl and 10 mM CaCl2. Res u l t s
The release of platelet dense granules were inhibited by the preincubation of platelet products with ES products (Fig. 5)
Fibrinoqenolysis
The effect of Necator ES products on fibrinogenolysis was investigated by incubating human fibrinogen and Necator ES products over timed intervals then examining the degradation products using SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE), 40ug aliquots of fibrinogen were incubated with 10ug Necator ES products at 37°C for 1,3,5 and 24 hour periods. At the end of each incubation period, the samples were run under reducing conditions on a 7-12.ό gradient gel. This was run at 30volts overnight, at 200volts until completion, and then stained with Coomassie brilliant blue R-250.
Results
Fibrinogenolysis occured during incubation of human fibrinogen with Necator americanus ES products (Fig. 6). After one hour incubation, degradation of fibrinogen was clearly seen. Prolonged incubation times caused increading amounts of degradation, and after 24 hours most of the 40ug protein sample was degraded.
It is thus shown that excretory-secretory products of the human hookworm prevent haemostasis by various strategies, including inhibition of coagulation factor Xa, a pivotal component of the clotting cascade. Platelet activation is also affected.
Products derived from a human parasite would not be expected to give rise to compatability problems when administered to humans.
Whilst endeavouring in the foregoing specification to draw attention to those features of the invention believed to be of particular importance it should be understood that the Applicant claims protection in respect of any patentable feature or combination of features hereinbefore referred to and/or shown in the drawings whether or not particular emphasis has been placed thereon.

Claims

Claims : -
1. An excretory-secretory product of the human hookworm Necator americanus for use as an active pharmaceutical substance.
2. Derivatives of excretory-secretory (ES) products of the human hookworm Necator americanus for use as an active pharmaceutical substance.
3. The use of excretory-secretory products of the human hookworm Necator americanus for the manufacture of an antihaemostatic composition.
4. An antihaemostatic composition comprising a pharmaceutically acceptable diluent or carrier and an excretory-secretory product of the human hookworm Necator americanus.
5. An antihaemostatic composition comprising a pharmaceutically acceptable diluent or carrier and an active ingredient obtained from an excretory-secretory product of the human hookworm Necator americanus.
6. A composition according to Claim 5, wherein the active ingredient is obtained either directly or by chemical synthesis or by a genetic engineering technique .
7. Excretory-secretory products or derivatives thereof of the human hookworm Necator americanus for use as an inhibitor of platelet aggregation.
8. Excretory-secretory products or derivatives thereof of the human hookworm Necator americanus for use as an inhibitor of Factor Xa activity.
9. Excretory-secretory products of the human hookworm Necator americanus for use as an inhibitor of platelet dense granule release.
10. Any novel subject matter or combination including novel subject matter disclosed, whether or not within the scope of or relating to the same invention as any of the preceding Claims.
PCT/GB1994/002406 1993-11-02 1994-11-02 Antihaemostatic agents from necator americanus WO1995012615A1 (en)

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996032479A1 (en) * 1995-04-10 1996-10-17 Yale University Hookworm vaccine
US5864009A (en) * 1994-10-18 1999-01-26 Corvas International, Inc. Nematode-extracted anticoagulant protein
US5863894A (en) * 1995-06-05 1999-01-26 Corvas International, Inc. Nematode-extracted anticoagulant protein
US5866542A (en) * 1994-10-18 1999-02-02 Corvas International, Inc. Nematode-extracted anticoagulant protein
US5866543A (en) * 1995-06-05 1999-02-02 Corvas International, Inc. Nematode-extracted anticoagulant protein
US5872098A (en) * 1995-06-05 1999-02-16 Corvas International, Inc. Nematode-extracted anticoagulant protein
US5955294A (en) * 1994-10-18 1999-09-21 Corvas International, Inc. Nematode-extracted serine protease inhibitors and anticoagulant proteins
US6040441A (en) * 1994-10-18 2000-03-21 Corvas International, Inc. Nematode-extracted serine protease inhibitors and anticoagulant proteins
WO2000041706A1 (en) * 1999-01-15 2000-07-20 University Of Nottingham Pro-apoptotic agents
WO2000058341A1 (en) * 1999-03-31 2000-10-05 Yale University Hookworm platelet inhibitor

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
BEHNKE ET AL: "HUMAN PARASITIC DISEASES.VOLUME 4.HOOKWORM INFECTIONS (GILLES AND BALL,EDS.).9.AN OVERVIEW.PAGES 217-237", 1991, ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM *
CARR ET AL: "ANTIGEN EXPRESSION DURING DEVELOPMENT OF THE HUMAN HOOKWORM,NECATOR AMERICANUS (NEMATODA)", PARASITE IMMUNOLOGY, vol. 9, no. 2, March 1987 (1987-03-01), OXFORD,GB, pages 219 - 234 *
CARR ET AL: "IDENTIFICATION OF HOOKWORM (NECATOR AMERICANUS) ANTIGENS AND THEIR TRANSLATION IN VITRO", MOLECULAR AND BIOCHEMICAL PARASITOLOGY, vol. 19, no. 3, 1986, AMSTERDAM, pages 251 - 258 *
KUMAR ET AL: "THE PARTIAL CHARACTERIZATION OF PROTEASES PRESENT IN THE EXCRETORY/SECRETORY PRODUCTS AND EXSHEATHING FLUID OF THE INFECTIVE (L3) LARVA OF NECATOR AMERICANUS", INTERNATIONAL JOURNAL FOR PARASITOLOGY, vol. 22, no. 5, 1992, OXFORD,GB, pages 563 - 572 *
PRITCHARD ET AL: "EPIDEMIOLOGY AND IMMUNOLOGY OF NECATOR AMERICANUS INFECTION IN A COMMUNITY IN PAPUA NEW GUINEA:HUMORAL RESPONSES TO EXCRETORY-SECRETORY AND CUTICULAR COLLAGEN ANTIGENS", PARASITOLOGY, vol. 100, no. PT.2, April 1990 (1990-04-01), CAMBRIDGE,GB, pages 317 - 326 *
PRITCHARD ET AL: "NECATOR AMERICANUS SECRETORY ACETYLCHOLINESTERASE AND ITS PURIFICATION FROM EXCRETORY-SECRETORY PRODUCTS BY AFFINITY CHROMATOGRAPHY", PARASITE IMMUNOLOGY, vol. 13, no. 2, March 1991 (1991-03-01), OXFORD,GB, pages 187 - 199 *

Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5945275A (en) * 1994-10-18 1999-08-31 Corvas International, Inc. Nematode-extracted anticoagulant protein
US5864009A (en) * 1994-10-18 1999-01-26 Corvas International, Inc. Nematode-extracted anticoagulant protein
US6872808B1 (en) 1994-10-18 2005-03-29 Corvas International, Inc. Nematode-extracted serine protease inhibitors and anticoagulant proteins
US5866542A (en) * 1994-10-18 1999-02-02 Corvas International, Inc. Nematode-extracted anticoagulant protein
US6534629B1 (en) 1994-10-18 2003-03-18 Corvas International, Inc. Nematode-extracted serine protease inhibitors and anticoagulant proteins
US6096877A (en) * 1994-10-18 2000-08-01 Corvas International, Inc. Nematode-extracted serine protease inhibitors and anticoagulant proteins
US5955294A (en) * 1994-10-18 1999-09-21 Corvas International, Inc. Nematode-extracted serine protease inhibitors and anticoagulant proteins
US6040441A (en) * 1994-10-18 2000-03-21 Corvas International, Inc. Nematode-extracted serine protease inhibitors and anticoagulant proteins
US6046318A (en) * 1994-10-18 2000-04-04 Corvas International, Inc. Nematode-extracted serine protease inhibitors and anticoagulant proteins
US6087487A (en) * 1994-10-18 2000-07-11 Corvas International, Inc. Nematode-extracted serine protease inhibitors and anticoagulant proteins
US6090916A (en) * 1994-10-18 2000-07-18 Corvas International, Inc. Nematode-extracted serine protease inhibitors and anticoagulant proteins
US6121435A (en) * 1994-10-18 2000-09-19 Corvas International, Inc. Nematode-extracted serine protease inhibitors and anticoagulant proteins
WO1996032479A1 (en) * 1995-04-10 1996-10-17 Yale University Hookworm vaccine
US5866543A (en) * 1995-06-05 1999-02-02 Corvas International, Inc. Nematode-extracted anticoagulant protein
US5872098A (en) * 1995-06-05 1999-02-16 Corvas International, Inc. Nematode-extracted anticoagulant protein
US5863894A (en) * 1995-06-05 1999-01-26 Corvas International, Inc. Nematode-extracted anticoagulant protein
WO2000041706A1 (en) * 1999-01-15 2000-07-20 University Of Nottingham Pro-apoptotic agents
WO2000058341A1 (en) * 1999-03-31 2000-10-05 Yale University Hookworm platelet inhibitor

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