WO2002017711A2 - Traitements et diagnostics selon un nouveau systeme de transduction du signal dans les thrombocytes - Google Patents

Traitements et diagnostics selon un nouveau systeme de transduction du signal dans les thrombocytes Download PDF

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WO2002017711A2
WO2002017711A2 PCT/US2001/026936 US0126936W WO0217711A2 WO 2002017711 A2 WO2002017711 A2 WO 2002017711A2 US 0126936 W US0126936 W US 0126936W WO 0217711 A2 WO0217711 A2 WO 0217711A2
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thrombin
platelets
platelet
agent
aggregation
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PCT/US2001/026936
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WO2002017711A9 (fr
WO2002017711A3 (fr
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Vanitha Ramakrishnan
David Phillips
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Cor Therapeutics, Inc.
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Priority to AU2001286900A priority Critical patent/AU2001286900A1/en
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Publication of WO2002017711A3 publication Critical patent/WO2002017711A3/fr
Publication of WO2002017711A9 publication Critical patent/WO2002017711A9/fr

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    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
    • A01K67/027—New breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0276—Knockout animals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/035—Animal model for multifactorial diseases
    • A01K2267/0375—Animal model for cardiovascular diseases
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70546—Integrin superfamily, e.g. VLAs, leuCAM, GPIIb/GPIIIa, LPAM
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/974—Thrombin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • the present invention relates to the field of hematology.
  • the present invention relates to a novel signal transduction system in platelets and to therapeutic and diagnostic methods based thereon.
  • the GP Ib-IX-N complex is a large multimeric protein complex on the platelet surface which consists of 4 different subunits GP Ib ⁇ , GP Ib ⁇ , GP IX and GP N in the ratio of 2:2:2:1. Absence of some or all of the subunits of this complex results in a severe recessive bleeding disorder known as Bernard-Soulier syndrome (Blood (1998) 91(12):4397-4418).
  • the GP Ib ⁇ subunit contains the high affinity binding site for thrombin and the binding site for vWf.
  • GP N is a platelet and endothelial cell specific glycoprotein, and that it is a substrate for thrombin. It also is known that the activation of platelets by thrombin results in the loss of surface GP N. However, the precise role that GP N plays in the function of the GP Ib-LX-V complex has not been described. The Role of GP V in Platelet Biology
  • Platelet thrombosis and hemostasis are complex reactions dependent upon adhesive interactions mediated by specific receptors.
  • a major platelet complex is GP Ib-LX-V.
  • the initial adhesion of platelets is primarily mediated by binding of platelet membrane GP Ib-LX-V to von Willebrand factor (vWf) found on damaged vessel walls (Baumgartner et al, 1978).
  • vWf von Willebrand factor
  • binding of other agonists such as thrombin, adenosine diphosphate (ADP), and collagen, induce signaling events that ultimately activate the receptor function of ⁇ llb ⁇ 3 for soluble fibrinogen, leading to platelet aggregation (Clemetson et al, 1995).
  • platelet aggregates are required for normal hemostasis, they can in addition cause arterial thrombosis in atherosclerotic arteries, e.g. acute myocardial infarction and stroke, inducing ischemic complications of cardiovascular disease (Davies et al, 1994; Jang et al, 1994).
  • GP Ib-LX-V complex in normal platelet function is underscored by the study of Bernard-Soulier syndrome (BSS), an inherited bleeding disorder characterized by large platelets that are defective in adhesion to damaged vessel walls (Baumgartner et al, 1978). This genetic disorder is caused by a lack of functional GP Ib-LX-V, and has been linked to defects in either GP lb or GP LX (Lopez et al, 1998).
  • BSS Bernard-Soulier syndrome
  • the activities mapped to the GPIb subunit of the GP Ib-LX-V complex include vWf (Marchese et al, 1995) and thrombin binding (Handa et al, 1986; De Marco et al, 1994) on the extracellular domain and actin binding protein (Andrews et al, 1991; Andrews et al, 1992; Xu et al, 1998) and 14-3-3 (Du et al, 1994; Du et al, 1996; Calverley et al, 1998; Andrews et al, 1998) binding on the cytoplasmic domain.
  • GP V has been show to be cleaved by thrombin f om the platelet surface during thrombin-mediated platelet stimulation (Berndt et al. , 1981), but the role of GP V cleavage in this thrombin-induced platelet response is unresolved (Bienz et al, 1986).
  • the signaling protein 14-3-3 has been shown to bind to the cytoplasmic domain of GP V (Calverley et al, 1998; Andrews et al, 1998), raising the possibility that GP V may be involved in platelet signaling during adhesion to vWf.
  • GP V has been shown to promote the expression of GP Ib-LX in heterologous cells (Calverley et al, 1995; Li et al, 1995; Meyer et al, 1995), suggesting that the synthesis of all subunits of GP Ib-V-IX are required for optimal expression of the complex.
  • the Mechanism of Action of GP Ib-LX-V in Thrombin Induced Platelet Aggregation The proteolytic activity of thrombin causes a wide spectrum of biological responses. These include clot formation and inhibition (Leung et al, 1997; Wu, Q. Y. et al, 1991), stimulation of mitosis, inflammation and migration in vascular cells, and aggregation of platelets (Shuman, M. A. 1986). The mechanism by which thrombin induces platelet aggregation, via the platelet glycoprotein (GP) Ib-LX-V complex was not known until it was elucidated by the inventors of the present invention.
  • GP platelet glycoprotein
  • the present invention provides nonhuman transgenic animals, preferably mammals, that contain or comprise a modified GP V gene.
  • the genomic GP V gene of such transgenic animals has been modified in the sense that it has been deleted, in whole or in part, or that it has been altered, substituted or mutated in some way.
  • the particular modification is not critical so long as the cells of the transgenic animal do not express a GP V protein, do not express a functional GP V protein or express a GP V protein that demonstrates a modified (i.e., reduced) functionality as compared with the same type of cell that expresses the native or wild- type GP V protein.
  • mammals contemplated by the present invention include sheep, goats, mice, pigs, dogs, cats, monkeys, chimpanzees, hamsters, rats, rabbits, cows and guinea pigs.
  • the present invention also provides cells isolated from such animals, including platelets and other blood cells isolated from the blood of the transgenic non-human animals according to the present invention.
  • the present invention also provides methods of preparing a nonhuman transgenic animal, preferably a mammal, with a modified GP V gene. Such modification may be accomplished by techniques that are known in the art and that are discussed below.
  • the present invention also provides methods of comparing a characteristic between two mammals of the same species, or strain, wherein one mammal has, for example, a wild-type GP V gene and the other mammal has a modified GP V gene.
  • the present invention similarly provides methods for comparing cells isolated from such mammals.
  • the present invention also provides methods of determining the effect of various agents on selected biological characteristics of a genetically engineered animal expressing a modified GP V gene, wherein the methods comprise: a) administering the agent to the genetically engineered animal; b) maintaining the animal for a desired period of time after administration of the agent; and c) determining whether a characteristic of the animal that is attributable to the expression of the modified GP V gene has been affected by the administration of said agent.
  • the present invention similarly provides methods for determining the effects of various agents on the phenotypical, physiological, or biological characteristics of cells isolated from such genetically engineered animals.
  • Some embodiments of the invention provide a method of identifying an agent that modulates thrombin activity, wherein the activity is modulated by GP V, comprising administering a test agent and thrombin to a GP V null non-human transgenic animal and monitoring aggregation of platelets of the animal.
  • the tlrrombin may be inactive.
  • preferred inactivated thrombins include-but are not limited to-PPACK- inactivated thrombin, S205A thrombin, and DlP-thrombin.
  • the thrombin may be active, h some embodiments, the agents tested may bind GP Ib-LX. Examples of agents that bind GP Ib-LX include-but are not limited to-antibodies that bind specifically to GP Ib-LX.
  • Some embodiments of the present invention provide methods of identifying an agent that modulates thrombin-induced activation, wherein the activation is modulated by GP V, comprising administering a test agent and thrombin to a platelet derived from a GP V null non- human transgenic animal and monitoring aggregation of the platelets, h some embodiments, the thrombin may be inactive. Examples of preferred inactivated thrombins include-but are not limited to-PPACK-inactivated thrombin, S205 A thrombin, and DlP-thrombin. In other embodiments, the thrombin may be active. In some embodiments, the agents tested may bind GP Ib-LX.
  • agents that bind GP Ib-LX include-but are not limited to-antibodies that bind specifically to GP Ib-LX.
  • the modulation to be identified may be an inhibition of thrombin-induced activation of platelets. Inhibition can be assayed in both a whole animal whose platelets do not display GP V on their surface and in platelets isolated from the animal, h some embodiments, the present invention provides compositions comprising a platelet that does not display GP V on its surface and an inactive thrombin. Platelets of this type do not include platelets that display GP V on their surface wherein the GP V has been cleaved from the surface by proteolytic action.
  • compositions may further comprise an agent that modulates an activity of the platelet induced by thrombin.
  • Example s of this type of activity include-but are not limited to-plateltet activation, platelet aggregation and thrombosis.
  • Inactive thrombins include-but are not limited to-PPACK-inactivated thrombin, S205A thrombin, and DlP-thrombin.
  • the compositions of the present invention may be used for various purposes including the formulation of kits to practice the methods of the present invention.
  • K ts may comprise one or more containers comprising one or more of plates that do not express GP V on their surface and inactive thrombin.
  • the present invention provides a method of determining predisposition to thrombosis in a subject, comprising determining a level of GP Vfl in a blood sample from the subject wherein GP Vfl presence in the sample indicates a predisposition to thrombosis.
  • the subject may preferably be mammalian, for example, human. Determining the level of GP Vfl may be accomplished by any means known to those skilled in the art. In some embodiments, determining may be accomplished using immunological assays such as ELISA assays and/or Western blot assays, hi some embodiments, determining is performed using an antibody specific for GP Vfl.
  • the methods of invention provide a diagnostic methodology for determining predisposition to thrombosis in a subject, comprising determining a level of GP V on platelets derived from the subject, wherein a decrease in the level of GP V on the platelets is indicative of a predisposition to thrombosis.
  • the determining may be performed by fluorescence activated cell sorting (FACS).
  • FACS fluorescence activated cell sorting
  • determining may be performed by Western blot.
  • the invention provides a method of inhibiting thrombosis in a subject, comprising administering to the subject an inhibitor of GP Ib-LX signal transduction.
  • the agent may act at any stage of the signal transduction process, hi some embodiments, the agent may bind to exosite JJ of thrombin.
  • Some embodiments of the present invention provide a method of screening for antithrombotic agents, comprising contacting a platelet with an agent, contacting the platelet with a GP Ib-IX signaling activator, and dete ⁇ nining GP Ib-LX mediated signal transduction level, wherein a reduced level is indicative of an antithrombotic agent.
  • Some embodiments of the invention may employ a platelet that does not display GP V on its surface.
  • Some embodiments of the present invention may utilize thrombin as the GP Ib-LX activator, hi some embodiments, the thrombin may be inactive.
  • inactivated thrombins examples include-but are not limited to-PPACK-inactivated thrombin, S205A tlrrombin, and DLP- thrombin.
  • the thrombin may be active.
  • the signal transduction level is determined by measuring platelet aggregation.
  • the present invention provides a method of preventing platelet activation in a subject, comprising administering to the subject an agent, wherein the agent prevents interaction of thrombin exosite II with GP Ib-LX.
  • Figure 1 shows the murine DNA sequence of GP V.
  • Figure 2 shows the murine GP V amino acid sequence.
  • Figure 3 shows the human DNA sequence of GP V.
  • Figure 4 shows the human GP V amino acid sequence.
  • Figure 5 shows the genomic organization of the mouse GP V gene and structure of the targeting vector pGP VKO.
  • Mouse GP V was mapped from a BAC ESI 29 library and a 5' homology region (8Kb) consisting of the Xmal(blunt)-Bam HI fragment was cloned into the Ace 651 (blunt)-BamHI sites on the vector pPN2T, and the 3 ' homology region (1.4Kb) consisting of the Xhol-Hmdi ⁇ was cloned into the corresponding sites around the Neo r cassette, as shown.
  • 8Kb 5' homology region
  • 1.4Kb consisting of the Xhol-Hmdi ⁇
  • Figure 6 shows binding of GP V -/- platelets to immobilized human vWf Pooled washed platelets (WP) from wt ( ⁇ ), +/- (V) and -/- (o) mice were incubated as described in the Examples. The data shown is the average of duplicates and is representative of 3 such experiments.
  • Figure 7 shows binding of FITC ⁇ vWf to GP V-/- platelets.
  • Pooled platelet-rich plasma (PRP) from GP V wt ( ⁇ ), +/ - (dashed) and -/- ( ⁇ ) mice were incubated with F ⁇ TC ⁇ vWf and botrocetin and analyzed by flow cytometry. The data is representative of 3 experiments done in duplicate.
  • PRP platelet-rich plasma
  • Figure 8 shows thrombin-induced FITC-fibrinogen binding in washed platelets from GP V wt, +/- and -/- mice.
  • Mouse WP from individual mice [wt ( ⁇ ) +/ - (V) and -/- (o)] were stimulated with the indicated amounts of thrombin.
  • the platelets were incubated with FITC-labeled fibrinogen for 30 min fixed and analyzed by flow cytometry. The data is representative of 3 experiments done in duplicate.
  • Figure 9 shows thrombin-induced aggregation in washed platelets from GP V wt, +/- and -/- mice.
  • WP were prepared from 6-10 mice of each genotype, which were littermate controls. Platelet aggregation was determined in an aggregometer (Chrono-Log Corp). Five such experiments were carried out, and 4 out of five worked in the manner shown.
  • FIGS 10A-C show Dff -thrombin-induced aggregation in washed mouse platelets.
  • A Pooled washed platelets from GP V null mice were aggregated with either (line 1) DIP- thrombin (lOOnM) or (line 2) thrombin (lOnM). Inset. EC 50 for DIP-thrombin in GP V null platelets. Washed platelets from 4 mice were aggregated with DIP -thrombm. The data is the average of duplicate experiments.
  • B Pooled washed wt mouse platelets were (line 1) pre- treated with thrombin as described and aggregation was initiated by the addition of DIP- thrombin (lOOnM).
  • Line 2 shows the response of wt platelets not pre-treated with thrombin to DIP-thrombin with 40pM thrombin added simultaneously.
  • GP V fl is released from wt platelets treated with 50pM thrombin.
  • Lane 1 and 2 are the supematants of control platelets, and lanes 3 and 4 are the supematants of 50pM thrombin treated platelets.
  • Lanes 1 and 3 are the IPs with control IgG, and lanes 2 and 4 are the IPs with Ab 808.
  • the arrow shows the position of cleaved GP V (GP Vfl).
  • GP V null platelets were aggregated with S205A- thrombin (l ⁇ M) in the absence (line 1) or presence (line 2) of a ⁇ llb ⁇ 3 inhibitor.
  • the tracings represent experiments done at least 3 times with pooled platelets from 4 mice each.
  • Figures 11 A-D show Dff -thrombin-induced aggregation is inhibited by anti-GP lb antibody.
  • Washed GP V null platelets were pre-incubated with either anti-GP Ib-LX Ab 3584 (2 ⁇ M, line 2) or control rabbit IgG (2 ⁇ M, line 1) for 10 min at room temp.
  • Glycocalicin-purified Ab 3584 IgG (1.89 ⁇ M) was incubated with GP V null platelets for 10 min prior to the initiation of aggregation with 500nM DIP- thrombm (line 2).
  • Control (line 1) shows the aggregation response with 500nM DIP-thrombin following incubation with control rabbit IgG.
  • D Washed GP V null platelets were pre- incubated with either control IgG (2 ⁇ M, line 1) or Ab 3584 (2 ⁇ M, line 2) for 10 min and aggregation was initiated by the addition of l ⁇ M S205A-thrombin.
  • Figures 12A-H show effect of various inhibitors on aggregation. Pooled, washed platelets were treated with the indicated concentrations of unfractionated heparin (0.3u/ml, Panels A-D), or prostacyclin (PGI ) (4.45 ⁇ M, Panels E-H), and aggregation responses were measured compared to that obtained in the absence of heparin or PGI 2 .
  • heparin 0.3u/ml
  • PGI 2 prostacyclin
  • Line 1 in each tracing represents the control untreated platelets and line 2 the platelets treated with inhibitor. The tracings are typical of results from experiments performed at least 3 times with pooled platelets from 4 mice each.
  • Figures 13A-F show thrombosis in wt and GP V null mice.
  • the mice were injected with either lOOnM DIP-thrombin (Panel A), InM thrombin (Panel B), lOnM thrombin (Panel C), 0.46 ⁇ M CHO-expressed wt DIP-thrombin (Panel D), 0.75 ⁇ M CHO-expressed DIP-
  • Figure 14 shows thrombin-induced platelet activation.
  • the novel pathway involves the initial cleavage of GP V from the GP Ib-LX-V complex at low thrombin concentrations, resulting in a hyper-responsive platelet. Occupancy of the binding site on GP Ib ⁇ by thrombin results in a signalling response which leads to ⁇ llb ⁇ 3 activation. Additionally, thrombin can cleave the PARs on platelets that will then also stimulate platelet aggregation.
  • the present invention relates to the production and use of nonhuman transgenic animals, preferably mammals, that contain or comprise a modified GP V gene.
  • the genomic GP V gene of such transgenic animals has been modified in the sense that it has been deleted, in whole or in part, or that it has been altered, substituted or mutated in some way.
  • Particularly contemplated are those modifications in which the cells of the transgenic animal do not express a functional GP V protein or express a GP V protein that demonstrates a reduced functionality as compared with the same type of cell that expresses the native or wild- type GP V protein.
  • the nature of a particular modification is not critical so long as transgenic animal or transgenic cells contain or express such a modified GP V gene.
  • the present invention also relates to cells isolated from such transgenic animals. Particularly contemplated are platelets and other blood cells isolated from the blood of the transgenic non- human animals according to the present invention.
  • Figure 1 provides the DNA sequence of the mouse GP V gene.
  • transgenic animals containing or expressing modified sequences of this GP V-encoding DNA sequence can be generated using knock-out procedures that are known in the art to disrupt the genomic gene. A variety of known procedures are contemplated, such as targeted recombination.
  • a transgenic or genetically-engineered animal for example, a "knock-out mouse”
  • a transgenic nonhuman animal may be prepared by producing a vector containing an appropriately modified GP V genomic sequence and then transfecting such a vector into embryonic stem cells (ES Cells) of that animal species.
  • ES Cells embryonic stem cells
  • transfected mouse ES Cells that had undergone a homologous recombination event at the GP V locus were then identified by restriction analysis Southern blotting.
  • the desired modified ES cells were then injected into blastocysts in order to generate chimeric mice which were bred to wild-type mice to produce heterozygote animals expressing one normal and one modified GP V allele (as assessed by Southern blotting of tail genomic DNA).
  • heterozygotic (or chimeric) females may then be crossed with chimeric males to generate homozygotes.
  • Platelets from such transgenic mice can be used in a number of assays to identify agents that modulate GP V function, in particular, modulate platelet activation and thrombosis.
  • the bleeding time of modified or transgenic mice can be monitored in a screening assay to identify agents that improve or restore the wild-type clotting phenotype.
  • Such assays also may help to elucidate the extent to which GP V is critical for normal hemostasis.
  • the effects of agents on the activation/aggregation of isolated platelets can be used to identify agents that inhibit thrombin-induced activities such as aggregation, hi a preferred embodiment, platelets that do not display a GP V on their surface can be contacted with a GP Ib-IX activator-such as inactivated thrombin-in order to induce platelet activation.
  • the platelets may be contacted with various agents either before, concurrently or after contact with the activating agent in order to identify those agents that inhibit the activation response.
  • Agents may be of any type known to those of skill in the art, including enzymes, proteins, peptides, small molecules, carbohydrates, lipids and/or nucleic acids.
  • a mouse was generated in which the GP V gene was modified by targeted disruption of the GP V coding region to inactivate the GP V gene.
  • the platelets from these animals may be expected to show certain phenotypic effects resulting from the loss of a fully-functional GP V gene (and expression product) on both the expression and function of the GP Ib-LX-V complex.
  • the intact transgenic animals and cells derived from such animals maybe used to evaluate the activity of various agents that modulate GP V function.
  • Such animals and cells also provide a model system useful in evaluating the consequences of GP V cleavage on the function of the GP Ib-LX-V complex and in identifying agents useful in modulating these consequences.
  • thrombin-induced activation is activon mediated by the GP Ib-LX signal transduction system. Activities induced by thrombin include-but are not limited to- thrombosis and platelet activation.
  • the term "animal” is used herein to include all vertebrate animals including mammals, except humans. It also includes an individual animal in all stages of development, including embryonic and fetal stages.
  • a "transgenic animal” is any animal containing one or more cells bearing genetic information altered or received, directly or indirectly, by deliberate genetic manipulation at a subcellular level, such as by targeted recombination or microinjection or infection with recombinant virus.
  • the term "transgenic animal” is not intended to encompass an animal produced by classical cross-breeding alone or by in vitro fertilization alone, but rather is meant to encompass animals in which one or more cells are altered by or receive a recombinant, exogenous or cloned DNA molecule. This molecule may be specifically targeted to a defined genetic locus, be randomly integrated within a chromosome, or it may be extrachromosomally replicating DNA.
  • a "subject” is any animal to be treated or tested. Preferred subjects include mammals, particularly humans.
  • knock-out generally refers to mutant organisms, usually mice, which contain a null or non-functional allele of a specific gene.
  • knock-in generally refers to mutant organisms, also usually mice, into which a gene has been inserted through homologous recombination.
  • the knock-in gene may be a mutant form of a gene which replaces the endogenous, wild-type gene.
  • Non-functional GP V genes include GP V genes which have been modified or inactivated, in whole or in part, by mutation or via any available method so that GP V expression is prevented, disrupted or altered so as to disrupt the wild type GP V phenotype.
  • transgenic mammals of the present invention may display non-normal platelet aggregation and/or other effects.
  • the transgenic animals of the present invention can also be used to identify agents that modulate (i.e., either promote or further inhibit) platelet aggregation or other effects that are mediated by the GP Ib-LX-V complex.
  • agents that modulate i.e., either promote or further inhibit
  • the evaluation of such agents can be conducted either in vitro, in situ, or in vivo by techniques known to those skilled in the art.
  • Agents that can be tested include various anticoagulant, thrombolytic and antiplatelet therapeutics and drugs.
  • agents include glycosaminoglycans such as heparin; oral anticoagulants such as dicumarol, anisindione, and bromadkiolone; tissue plasminogen activator (t-PA); urokinase; aspirin; dipyridamole; and ticlopidine.
  • transgenic animals of the present invention may also be used to investigate gene regulation, expression and organization in animals.
  • transgenic mammals especially transgenic mice, see U.S. Patent No. 5,569,824.
  • Genes that are modified, truncated or replaced in whole or in part can be introduced into a target cell in a site directed fashion using homologous recombination.
  • homologous recombination techniques may be used to introduce a DNA sequence into the cells of an organism where a particular gene has been deleted from its native position in that sequence. Papers discussing homologous recombination are discussed in R. Kucherlapati et al, (1995) U.S. Patent No. 5,413,923. Through these technique, for example, a DNA sequence in which the GP V gene has been modified or deleted can be introduced.
  • Such methods result in the creation of a transgenic animal, wherein the animal's genome has been modified, and the phenotype of the modified animal or cells from the modified animal can be studied for purposes of drug screening, investigating physiologic processes, developing new products and the like.
  • embryonic stem cells or a stem cell line may be obtained.
  • Cells other than embryonic stem cells can be utilized ⁇ e.g., hematopoietic stem cells, etc.) See, for more examples, J.G. Seidman et al, (1994) U.S. Patent No. 5,589,369.
  • the cells may be grown on an appropriate fibroblast fetal layer or grown in the presence of leukemia inhibiting factor (LIF) and then used. Once transformed, the embryonic stem cells may be injected into a blastocyst that has been previously obtained, to provide a chimeric animal.
  • LIF leukemia inhibiting factor
  • the main advantage of the embryonic stem cell technique is that the cells transfected with the "transgene" can be tested, prior to reimplantation into a female animal for gestation, to assess integration of the transgene and the effect of the transgene.
  • the homologous respective endogenous gene can be removed from a chromosome by homologous recombination with the transgene.
  • animals can be bred which carry the transgene on both chromosomes. If mutations are incorporated into the transgenes which block expression of the normal gene, the endogenous genes can be eliminated by this technique and functional studies can thus be performed for purposes described above.
  • Homologous recombination can also proceed extrachromasomally, which may be of benefit when handling large gene sequences (e.g., larger than 50 kb).
  • Methods of perfonning extrachromosomal homologous recombination are described in R.M Kay et al, (1998) U.S. Patent No. 5,721,367.
  • Transgenic animals are genetically modified animals into which recombinant, exogenous or cloned genetic material has been experimentally transferred. Such genetic material is often referred to as a "transgene".
  • the nucleic acid sequence of the transgene may be integrated either at a locus of a genome where that particular nucleic acid sequence is not otherwise normally found or at the normal locus for the transgene.
  • the transgene may consist of nucleic acid sequences derived from the genome of the same species or of a different species than the species of the target animal.
  • germ cell line transgenic animal refers to a transgenic animal in which the genetic alteration or genetic information was introduced into a germ line cell, thereby conferring the ability of the transgenic animal to transfer the genetic information to offspring.
  • the alteration or genetic information may be foreign to the species of animal to which the recipient belongs, or foreign only to the particular individual recipient, or may be genetic information already possessed by the recipient. In the last case, the altered or introduced gene may be expressed differently than the native gene.
  • transgenic technology allows investigators to create animals of virtually any genotype and to assess the consequences of introducing specific foreign nucleic acid sequences on the physiological and morphological characteristics of the transformed animals, hi general, the availability of transgenic animals permits cellular processes to be influenced and examined in a systematic and specific manner not achievable with most other test systems.
  • the development of transgenic animals provides biological and medical scientists with models that are useful in the study of disease. Such animals are also useful for the testing and development of new pharmaceutically active substances.
  • Transgenic animals can be produced by a variety of different methods including transfection, electroporation, microinjection, gene targeting in embryonic stem cells and recombinant viral and retroviral infection (see, e.g., U.S. Patent No. 4,736,866; U.S. Patent No.
  • mice A number of recombinant or transgenic mice have been produced, including those which express an activated oncogene sequence (U.S. Patent No. 4,736,866); express simian SV
  • mice and rats remain the animals of choice for most transgenic experimentation, in some instances it is preferable or even necessary to use alternative animal species.
  • Transgenic procedures have been successfully utilized in a variety of non-murine animals, including sheep, goats, pigs, dogs, cats, monkeys, chimpanzees, hamsters, rabbits, cows and guinea pigs (see, e.g., Kim et al., 1997 Mol. Reprod. Dev. 46, 515-526; Houdebine, 1995
  • the method of introduction of nucleic acid fragments into recombination competent mammalian cells can be by any method which favors co-transformation of multiple nucleic acid molecules.
  • Detailed procedures for producing transgenic animals are readily available to one skilled in the art, including the disclosures in U.S. Patent No. 5,489,743 and U.S. Patent No. 5,602,307.
  • GP V gene deletion results in hyper-aggregable platelets which have increased thrombin responsiveness and shorter bleeding times.
  • GP V functions as a negative modulator of platelet activation. Since GP V is the thrombin substrate in the GP Ib-LX-V complex and GP lb binds thrombin and catalytically inactive thrombin (termed DIP-thrombin) with equal affinity, and since the GP V -/- platelets lack this thrombin substrate, we tested the ability of D -thrombin to activate GP V-/- platelets.
  • GP V -/- platelets can aggregate in response to catalytically-inactive DIP-thrombin (Di-isopropylfluoro-phosphothrombin, also termed dead thrombin).
  • DIP-thrombin Di-isopropylfluoro-phosphothrombin, also termed dead thrombin.
  • This aggregation is physiologically relevant since aggregation occurs by a GP IJb-IJJa mediated process.
  • wild type platelets can be converted to this DIP-responsive phenotype using sub-threshold (very low) doses of active thrombin which are capable of cleaving GP V from the platelets, but which do not induce platelet aggregation.
  • thrombin-mediated platelet aggregation can occur via the GP Ib-LX-V complex, and that this complex is responsive to catalytically-inactive forms of thrombin.
  • thrombin can induce platelet aggregation in a manner independent of the catalysis of G-protein coupled PARs.
  • This novel role of thrombin in platelet aggregation indicates that inhibition of the binding of thrombin to GP Ib-LX-V complex or prevention of GP V cleavage by thrombin provides new strategies for therapeutic regulation of clinical processes such as but not limited to thrombosis and stroke events dependent on platelet aggregation.
  • diagnostic screens based on the measurement of cleaved GP V can be used as indicators of ongoing thrombotic events and/or a predisposition to thrombotic events.
  • platelets lacking the GP Vfl portion can be used to assay for ongoing thrombotic events and/or a predisposition to such events.
  • Rabbit antibodies against peptides based on mouse GP V generated by standard methods include Ab #810 (against mouse GP V residues C 472 -A 490 ) and Ab#808 (against mouse GP V residues L 432 -R 450 ).
  • Anti-GP Ib-IX (rabbit polyclonal Ab #3584) was kindly provided by Dr. Beat Steiner, Hoffinan-LaRoche (Poujol et al, 1998).
  • Anti- ⁇ llb ⁇ 3 (Ab #41) was described previously (Law et al, 1999).
  • Human fibrinogen Enzyme Research Labs
  • human vWf Human vWf (Haematologic technologies) were FITC-labeled in 0.1M NaHCO 3 (lmg/mL) using FITC-celite (Molecular Probes). Labeling conditions were designed to obtain F/P ratios between 1 and 4.
  • Snake venom peptide botrocetin was purified as described from the venom of Bothrops jaracaca (Andrews et al, 1989) and kindly provided by J. Rose. Human glycocalicin was purified from outdated platelets as described (Vicente et al, 1988) by affinity chromatography using mouse anti-human glycocalicin (MAb 5A12; kindly provided by Dr. Burt Adelman). Generation of the GP V -/- Mouse.
  • Rat platelet RNA was isolated using RNAzol from fresh rat platelets and the GP V coding region (700bp) obtained by RT-PCR using degenerate primers based on the human GP V sequence (Lanza et al, 1993), was cloned into pCR2.1 (hivitrogen) and sequenced.
  • the rat GP V insert was used to screen a mouse 129 BAG library (Genome Systems) and 2 positive clones (11487 and 11488) were identified.
  • Genomic DNA was isolated and a detailed map of ⁇ 22Kb of the mouse GP V generated ( Figure 5).
  • the 5' Xmal fragment (11Kb) and the EcoRl fragment (4Kb, containing the mouse GP V gene) were isolated from BAC plasmid DNA, subcloned into BlueScript (Stratagene), and sequenced.
  • the mouse 129 GP V coding sequence showed 91% homology to the rat coding sequence, compared to 71%) to human GP V.
  • the -8Kb XmaI(blunt)-BamHI fragment from the Xmal plasmid was first subcloned into the vector pPN2T (Tybulewicz et al, 1991; Morrison et al, 1996) at the Acc651(blunt)-BamHI sites to generate the 5' homology region (HR) downstream of the Neo r cassette, followed by the 1.4Kb Xhol-Hindi ⁇ fragment from the EcoRI plasmid generating the 3' HR upstream of the Neo r cassette.
  • Neo r cassette oriented transcriptionally in the opposite direction ( Figure 5).
  • pGP VKO was electroporated into the ES cell line RW4 (Hug et al, 1996. Neo r clones were identified by positive selection in G418 media.
  • Recombinants were rnicroinjected into embryos from C57BL/6J mice using standard techniques (Dr. R. Wesselschmidt, Genome Systems). One of several chimeric males generated was bred with C57BL/6J females.
  • Blood from anaesthetized mice was obtained by cardiac puncture diluted into saline containing 1/10 vol of either (a) TSC buffer (3.8% trisodium citrate, 0.11 IM glucose, pH7, 0.4 ⁇ M prostaglandin El (PGE1)) for platelet-rich plasma (PRP) or (b) Acid-Citrate-Dextrose (ACD, 85mM sodium citrate, 0.11 IM glucose, 714mM citric acid, 0.4 ⁇ M PGE1), for washed platelets (WP). Diluted blood was centrifuged at 82xg for 10 min.
  • the supernatant from (b) was pooled with a second obtained by centrifugation after the repeat addition of 137mM NaCl, and centrifuged at 325xg for 10 min.
  • Platelets were washed twice in CGS buffer (12.9mM sodium citrate, 33.33mM glucose, and 123.2mM NaCl, ⁇ H7) and resuspended in calcium-free Tyrodes-Hepes buffer (CFTH;10mM Hepes, 5.56mM glucose, 137mM NaCl, 12mM NaHCO 3 , 2.7mM KC1, 0.36mM NaH 2 PO 4 , ImM MgCl 2 , pH7.4). Platelets were normalized to 2xl0 8 /ml. PRP or WP were incubated at room temp for 30 min prior to use.
  • Flow cytometry was carried out as follows: PRP (lO ⁇ l) was incubated with primary rabbit antibody in CFTH containing 0.1 % BSA for 1 hour at 4°C, followed by phycoerythrin (PE)-conjugated donkey anti-rabbit IgG (H+L) F(ab') 2 (1 :200, Jackson ImmunoResearch) for
  • FITC ⁇ vWf and botrocetin (4 ⁇ M-40 ⁇ M) for lOmin Samples were diluted in CFTH buffer prior to analysis.
  • WP were isolated from individual mice and incubated in duplicate (lxlO 6 ) ⁇ -thrombin for 10 min. The reaction was terminated with PPACK (phenylalaninylprolylargininylchloromethylketone; 50 ⁇ M final).
  • PPACK phenylalaninylprolylargininylchloromethylketone
  • the platelets were incubated with FITC-labeled fibrinogen (lOO ⁇ gs/ml) for 30 min, fixed with ⁇ -formaldehyde (10%)) for 20 min and diluted into P/o ⁇ -formaldehyde in CFTH and analyzed by flow cytometry.
  • 96-well plates were coated with various concentrations (25-500 ng/well) of human vWf overnight at 4°C.
  • Pooled WP from mice were resuspended in Mg 2+ -free CFTH buffer (1.2xl0 8 /mL) containing botrocetin (4 ⁇ M), and incubated immobilized human vWf for lhour at room temp.
  • bound platelets were lysed and intracellular acid phosphatase activity was quantitated colorimetrically using the substrate pNPP (Sigma).
  • Bleeding time measurements were obtained using the tail cut model (Hodivala-Dilke et al, 1999) on littermate mice generated from heterozygous breeding. Since complete litters were not used the numbers of wt to +/- and -/- do not reflect Mendelian ratios. All experiments were blinded.
  • mice were transected at the 5mm mark from the tip of tail and incubated in warm (37 °C) saline. The time for cessation of bleeding was noted as the primary endpoint. If bleeding did not stop in 15 min, the tail was cauterized and 900 sec noted as the bleeding time. Data are presented as mean ⁇ sem, and statistical significance was assessed using both Student's t-test analysis and Mann- Whitney nonparametric analysis.
  • Coding sequences 5' GGCATGACCGTC(CT)TGCA(GA)CG 3' (SEQ ED NO: 1) which corresponds to human GP V residues GMTVLQR (SEQ DD NO: 2).
  • 5'GGCCCCA(AG)(TG)CC(AG)CA(AG)TC(AG)CAGA(AG)CCA(AG)GA 3' corresponds to the complement of the human GP V sequence encoding SWRCDCGLG (SEQ ED NO: 10).
  • Fresh rat platelets were isolated by standard techniques and RNA was isolated using RNAzol. PolyA + RNA was generated using the Oligo-Tex system. cDNA was prepared from the polyA + RNA using the In-Vitrogen cDNA cycle kit. The cDNA was then used in PCR reactions with each combination of the primers listed above. All PCR reaction products were then cloned into pCR2.1 cloning vectors from the Ln-Vitrogen TA cloning kit. GEBCO SURE competent cells were transformed using the manufacturer's protocol and white (transformant) colonies were selected. Miniprep DNA was generated by the rapid boiling method and restriction analysis was used to identify the clones containing inserts of the right size ( ⁇ 700bp).
  • the vector pPN2T (10.15Kb) is a modified version of the pPNT vector (Tybulewicz et al, 1991; Morrison et al, 1996) which, in addition to the Neo resistance (Neo 1 ) cassette, has 2 contiguous herpes simplex virus thymidine kinase (TK) cassettes (instead of the single one in pPNT) and a pUC vector backbone.
  • BAC plasmid DNA was isolated from clone 11488 using protocols supplied by Genome Systems. We isolated a 11 -16Kb Xmal fragment and a -4Kb EcoRl fragment which were subcloned into BlueScript.
  • the Xmal containing plasmid was used to generate a -8Kb Xmal-BamHl fragment which was blunted using Klenow at the Xmal site. This fragment was then subcloned into the targeting vector pPN2T at the
  • the 1.4Kb Xhol-HindHI 3' homology region was isolated from the BAC plasmid DNA using the same methodology, and subcloned into BlueScript and the cut out using Xhol-Notl. This Xhol-Notl fragment was then inserted into the targeting vector at the 5' end of the Neo r cassette.
  • the final targeting vector pGP VKO had the mouse GP V homology regions in the opposite orientation from that of the Neo gene.
  • Example 2 Generation of ES Cells.
  • the targeting vector was inserted into the ES cell line RW4 by electroporation by standard techniques (Genome Systems® and Hug et al. 1996).
  • Neo r clones were identified by positive selection in G418 media. Identification of the targeted ES cells which had undergone recombination was done using the restriction enzyme Sph 1 to digest the genomic DNA from the clones and Southern blotting with a probe designed to show linkage (outside probe).
  • Clone 367 was shown to be recombinant since Southern blot analysis showed the expected 2 bands, one at 6Kb (wild type allele) and one at 2Kb (recombinant) and was then micro-injected into embryos from C57B16 mice using standard techniques.
  • Several chimeric males were generated which were then bred with C57B16 females to determine germline transmission.
  • Recombinants were selected based on Southern analysis using the probe shown in Figure 5 and two recombinants were used to generate the founder chimeras.
  • One of these founder males (>85% > chimeric) was successfully mated with C57BL/6J females to produce +/- offspring, which were bred to generate homozygotes.
  • Deficiency in the GP V gene has not affected viability at birth as evidenced by findings that the litters have expected Mendelian ratios of-/- offspring (1:4) and that the GP V-/- animals are fertile with no gross observable defects.
  • Platelets were isolated from the -/- animals to confirm that gene deletion resulted in absence of GP V protein expression and analyzed for GP V expression using GP V antibodies. No GP V protein was detectable either on the intact platelet surface using FACS analysis or in total platelet lysates as determined by Western blotting.
  • GP V is usually expressed in platelets as a complex with GP Ib-LX (Meyer et al, 1995; Modderman et al. , 1992).
  • Two assays were used to determine whether the GP lb expressed on GP V -/- platelets was functional.
  • One assay measured the adhesion of platelets to immobilized vWf that was activated by botrocetin to bind GP lb.
  • Figure 6 shows that GP V -/- platelets bound to immobilized, botrocetin-activated human vWf in a manner indistinguishable from wt platelets.
  • Example 5 Effect of GP V gene deletion on thrombin-induced platelet function.
  • Agents can be incubated with the platelets to be assayed before, during or after being contacted with a GP Ib-EX signaling activator, for example, thrombin.
  • a GP Ib-EX signaling activator for example, thrombin.
  • the ability of the platelets to bind FITC-labeled fibrinogen can be determined as above, hi addition to thrombin, other GP Ib-EX signaling activators may be used in place of thrombin. Examples include-but are not limited to-proteolytically inactive thrombin.
  • platelets lacking GP V exhibited an increased aggregation response to thrombin compared to wt platelets.
  • platelets from GP V -/- mice aggregated when treated with sub-threshold concentrations of thrombin (0.5nM) that did not induce a significant response in wt platelets ( Figure 9).
  • This assay can be readily adapted to screen for agents that modulate platelet activation. Agents can be incubated with the platelets to be assayed before, during or after thrombin activation and then the ability of the platelets to aggregate can be determined as above.
  • GP Ib-IX signaling activators may be used in place of thrombin.
  • examples include-but are not limited to-proteolytically inactive thrombin.
  • platelets from GP V+/- heterozygous animals gave an intermediate response in the aggregation assays. We determined if this increased responsiveness was related to increased expression of ⁇ llb ⁇ 3, using an antibody specific for the mouse fibrinogen receptor.
  • the bleeding time in the +/- animals was intermediate (224 ⁇ 25 sec) but not statistically different from either wt or -/- mice.
  • 70% of the -/- mice had bleeding times less than 120 sec, compared to 50%> of the wt and +/- mice.
  • 21.6% of the wt mice had bleeding times greater than 500sec, compared to 9.5% in the +/- mice and 8.5% in the -/- mice.
  • Transgenic animals preferably mice, that do not display a GP V on the surfaces of their platelets can be injected with agents and then the effect of the agent on bleeding time can be determined.
  • This assay can be used to screen for agents that inhibit or promote clot formation.
  • This assay can also be used to screen for agents that inhibit clot formation by injectin the animal with an agent to be screened before, concomitantly with, or after injecting the animal with a second agent that is known to induce clot formation.
  • An increase in clotting time indicates that the first agent have the property of inhibiting clot formation.
  • GP V is a negative regulator of platelet function.
  • Gene targeting of GP V resulted in hyper-responsive platelets as detected by enhanced fibrinogen binding and enhanced aggregation, resulting in enhanced hemostatic activity in the mice harboring this deletion.
  • the data suggest a novel role for GP V in decreasing thrombin responsiveness of platelets, with removal of GP V from the platelet surface contributing to platelet stimulation by thrombin.
  • GP V- /- platelets had normal amounts of GP Ib-IX and the vWf binding function of GP V-/- platelets was normal. These results are consistent with the fact that mutations in GP lb and GP IX only have been observed in BSS.
  • Thrombin is the most potent platelet agonist and is known to function by a proteolytic mechanism (Davey et al, 1967). The elegant work of Coughlin and coworkers established that thrombin initiates platelet stimulation by cleaving one or more of the protease activated receptor (PAR) family of G-protein coupled receptors (Coughlin et al, 1992; Kahn et al, 1998; Kahn et al, 1999).
  • PAR protease activated receptor
  • the GP lb subunit of the GP Ib-EXN complex is a second thrombin binding site on platelets, providing the moderate affinity, high capacity binding site (Hayes et al, 1999) and thus regulating surface bound thrombin in human platelets (Mazzucato et al, 1998).
  • the GP V subunit is a thrombin substrate and is cleaved during thrombin-induced platelet aggregation.
  • thrombosis is dependent upon platelet aggregation and is typically associated with a systemic increase in the generation of thrombin as reflected by increases in thrombin- antithrombin complex (TAT), fibrin degradation product (FDP) and markers indicative of thrombin-induced platelet activation such as P-selectin and PGF2 .
  • TAT thrombin- antithrombin complex
  • FDP fibrin degradation product
  • Example 7 A Novel Thrombin Receptor Function for Platelet GP lb-IX Unmasked by Cleavage of GP V.
  • Plasma derived thrombin and DIP-thrombin was purchased from Haematologic Technologies, VT. Activity of plasma DIP-thrombin was between 0-0.03% by chromogenic assay with Chromozyme TH (Boerhinger-Mannheim, EN) as the substrate. Plasma derived DEP- thrombin binding of hirudin was measured by fluorimetry, and was found to be similar to proteolytically-active thrombin. CHO-expressed prothrombins (wt, S205 A, R89/R93/E94 and R98A) were expressed and purified as described (Hall et al. 1999).
  • Activation to thrombin was carried out using the prothrombinase complex (for wt and S205A) by a modification of the procedure of Malhotra et al (Malhotra et al, 1985), or using Echis carinatus venom as described (Hall et al. 1999) (for R89/R93/E94 and R98A).
  • CHO-expressed wt thrombin was compared to plasma-derived thrombin using fibrinogen clotting assays with lO ⁇ M purified fibrinogen (Enzyme Research Labs, EN), and was found to have 70%> less activity.
  • DFP- treatment of CHO-derived proteins was carried out as described (Ramakrishnan, V. et al, 1990). Loss of proteolytic activity was determined by chromogenic assay with ChromozymeTM and S2238. Affinity Purification of Ab 3584.
  • Ab 3584 was kindly provided by Drs. B. Steiner and S. Meyer. We first purified the human extracellular domain of GP Ib ⁇ (Glycocalicin) as described (Vicente et al, 1988). Ab 3584 was then affinity purified on a glycocalicin column. The affinity purified Ab 3584 IgG recognised human glycocalicin as assessed by Western blotting and by FACS analysis of mouse platelets.
  • Washed platelets were isolated as described previously (Ramakrishnan, V. et al, 1999).
  • Platelets were re-suspended in Tyrode-Hepes buffer and rested for 15 min prior to use in aggregation. Wt platelets were incubated with repeated doses (4) of lOpM thrombin in calcium-free Tyrodes-Hepes buffer with gentle mixing. Platelets were rested for at least 2 min prior to the addition of DIP-thrombin. Aggregation was measured as the change in transmitttance obtained following the addition of agonist using a Chronolog lumi- aggregometer.
  • GP Vfl present in blood will be identified using a similar procedure.
  • Cells will be removed from blood samples by centrifugation and the supernatant assayed for the presence of the GP Vfl .
  • the supernatant may be concentrated using conventional means prior to being assayed.
  • the presence or absence of the fl fragment can be used to assay for a predisposition to thrombosis since platelets that have cleaved GP V are more readily activated than those having a full length GP V.
  • mice were anaesthetised and the jugular vein was exposed surgically.
  • a retro-orbital bleed (125 ⁇ L) was taken using heparinised tubes, which was transferred into tubes containing 0.9%> saline/50mM EDTA (125 ⁇ L), to determine the baseline platelet counts.
  • various agonists were injected into the jugular vein in a volume of lOO ⁇ l, such that the concentration noted is the final circulating concentration. After 55 sec, another retro-orbital bleed was obtained.
  • the platelet counts in PRP obtained in duplicate from the 2 bleeds were used to determine the loss, if any, in platelet number. Loss in platelet counts in this model represents ongoing thrombosis. Blood volumes were assumed to be 10% of the weight of the animal, and this volume was used to calculate molar concentrations for the various thrombins in circulation. Animals were age and weight matched in separate experiments, and ranged in weight between 25-50g.
  • This model system will be used to assay for agents that modulate thrombosis.
  • Animals may be injected with agents to be assayed before, concurrently with, or after injection of a thrombotic agonist. Blood samples will then be taken and assayed for loss of platelet count. A smaller decrease-or no decrease- in platelet count compared to the decrease caused by agonist alone indicates that the agent has anti-thrombotic properties
  • thrombin a family of G-protein coupled receptors termed PARs (protease-activated receptors) (Kahn, M. L. et al. 1998). Nevertheless, proteolytically-inactive thrombin can potentiate the activity of sub-optimal concentrations of thrombin in platelets (Phillips, 1974), suggesting a non- proteolytic function for thrombin. GP Ib ⁇ can bind thrombin (De Marco et al, 1991) and could therefore be important for this effect.
  • PARs proteolytically-inactive thrombin
  • thrombin pre-treatment hydrolysed GP V from the platelet surface, as determined by the release of GP Vfl, the thrombin hydrolytic fragment of GP V (Fig 10B inset).
  • Antibody 3584 recognises GP Ib-EX on human and mouse platelets (Ramakrishnan, V. et al., 1999). Because GP Ib ⁇ is a candidate receptor for thrombin, we tested whether the antibody could inhibit platelet aggregation in response to proteolytically- inactive thrombin.
  • Ab 3584 (Fig 11 A) and affinity purified Ab 3584 which recognises only the GP Ib ⁇ sub-unit (Fig 11 C; see Methods), effectively blocked aggregation of GP V-deficient platelets caused by DEP-thrombin or S205A-thrombin response (Fig 1 ID), but had only a slight effect on aggregation induced by native untreated thrombin (Fig 1 IB).
  • Ab 3584 also inhibited DEP-thrombin-mediated aggregation of wt mouse platelets that had been pre-treated with sub- optimal doses of thrombin (not shown).
  • MAb LJ-1B10 which inhibits thrombin binding to GP Ib ⁇ (De Marco et al, 1994), inhibits the aggregation in human platelets rendered GP V deficient by thrombin pre-treatment (not shown).
  • the exosite II of thrombin binds heparin and may also be involved in the interaction with GP Ib ⁇ (De Cristofaro et al., 2000). We therefore examined whether aggregation induced by DEP-thrombin could be inhibited by heparin.
  • heparin significantly inhibited DEP-thrombin-induced platelet aggregation and completely reversed the response of both GP V-null (Fig 12 A) and thrombin-pretreated wt platelets (Fig 12C) within 5 minutes, hi contrast, this concentration of heparin provided only marginal inhibition of aggregation caused by native thrombin applied to either GP V null (Fig 12B) or wt platelets (Fig 12D).
  • GP V null mice showed a significant platelet loss when injected with lOOnM DEP -thrombin compared to wt mice (Fig 13 A), in which platelet loss was minimal.
  • mutant CHO- expressed thrombins (Hall et al. 1999) in which the exosite Et has been mutated (R89/R93/E94 and R98A). These exosite JJ mutants were inactivated using DFP, and then injected into either GP V null or wt mice.
  • GP Ib-EX can signal in response to the binding of vWf (Oda et al. 1995; Asazuma. et al, 1997) and induce platelet activation, probably through 14-3-3 (Du et al, 1994; Du et al, 1996), a signalling molecule constitutively associated with the cytoplasmic tail of GP Ib ⁇ that is phosphorylated at Ser609 (Bodnar et al, 1999).
  • Other studies have shown that agents like PGI 2 and VGEi negatively regulate GP Ib-EX signalling.
  • adenylyl cyclase activators appear to be through the activation of the cAMP-dependent kinase and phosphorylation of the cytoplasmic domain of GP Ib ⁇ (Fox et al, 1987; Fox et al, 1989).
  • thrombin signalling function of GP Ib-EX-V was similarly regulated.
  • Fig. 12E shows that PGI 2 completely inhibited DEP -thrombin-induced shape change and aggregation of GP V -/- platelets (IC 50 ⁇ 70nM).
  • PGE ls another activator of adenylyl cyclase, also inhibited DEP-thrombin induced aggregation in GP V -/- platelets (IC 50 «300nM).
  • PGI 2 inhibited DEP -thrombin-induced aggregation in both mouse (Fig 12G) and human platelets pre- treated with sub-optimal doses of active thrombin.
  • thrombin-induced aggregation was not affected significantly by PGI 2 even at high concentrations (4.45 ⁇ M, Fig 12F and H).
  • PG- 2 -treated platelets that fail to respond to DEP-thrombin were fully responsive to lOnM ⁇ -thrombin (data not shown).
  • thrombin signalling function of GP Ib-EX-V is ablated in the presence of adenylyl cyclase activators. This finding also suggests that incubation of isolated platelets with PGI 2 or PGE 1 could suppress this signalling pathway. Ln previous studies reported by Kahn and co-workers (Kahn et al, 1999; Kahn et al, 1999), only PAR signalling was observed in the platelet response to thrombin. The inclusion of PGE 1 during platelet isolation and desensitisation studies described therein may have inhibited signalling through GP Ib-EX-V.
  • thrombin-induced platelet activation involves not only the established pathway mediated by the PARs, but also a novel pathway in which the presence of GP V in the GP Ib-EX-V complex inhibits the ability of thrombin to function as a receptor ligand.
  • thrombin binding to GP Ib ⁇ results in activation of ⁇ llb ⁇ 3 and consequently in aggregation.
  • the GP Ib ⁇ -bound thrombin need not be catalytically functional for this response to occur.
  • thrombin concentrations can be as high as 1.4 ⁇ M, which encompasses the range described in these studies (van 't Veer et al, 1997; Mann et al, 1999).
  • the binding of thrombin to GP Ib ⁇ occurs via the heparin binding exosite and prevents the inactivation of tlrrombin by antithrombin HE (De Cristofaro et al, 2000).
  • Our data indicate that this GP Ib ⁇ -bound thrombin can itself initiate additional signalling responses in platelets.
  • the two signalling pathways may have evolved to exploit the high local thrombin concentrations for a more robust aggregation response particularly under conditions of arterial flow.
  • Effective anti-thrombotic therapies targeting thrombin-induced platelet activation would thus require the inhibition of both pathways.
  • the findings therefore reveal a new arena for therapeutic intervention in cardiovascular disease.
  • Example 8 Identification and use of agents that modulate platelet activation and thrombosis.
  • Agents that modulate the activation of platelets and/or thrombosis can be identified using the assays described above. Agents thus identified can be use to formulate pharmaceutical compositions for the treatment of pathological conditions associated with platelet activation and/or thrombosis.
  • Compositions comprising platelets that do not display a GP V molecule on their surface will be particularly useful for performing assays to identify agents that modulate thrombin-induced platelet activities.
  • These compositions may comprise, in addition to platelets, one or more buffers or salts, one or more GP Ib-IX signal activators, one or more agents that modulate thrombin-induced platelet activity.
  • the compositions of the present invention may be prepared into kits for diagnostic or therapeutic purposes.
  • kits may include one or more containers containing platelets that do not display a GP V on their surface. Kits may also comprise one or more containers comprising one or more of buffers and/or salts, GP Ib-IX signaling activators and/or one or more agents that modulated thrombin-induced platelet activities.
  • an agent is said to modulate platelet activation and/or thrombosis when the presence of the agent decreases or prevents the GP Ib-IX mediated activation or aggregation of platelets.
  • Agents are preferably compounds that have previously not been identified as having a function in platelet activation, hi particular, heparin is not included in the set of agents contemplated by this invention.
  • agents will reduce or block the association by binding to the GP Ib-LX complex while another class of agents will reduce or block the association by binding to the exosite JJ of thrombin.
  • Other classes of agents include those that block the cytoplasmic signaling mediated by the GP Ib-EX complex.
  • Agents that are assayed in the above methods can be randomly selected or rationally selected or designed.
  • an agent is said to be randomly selected when the agent is chosen randomly without considering the specific sequences involved in the association of thrombin with GP Ib-EX.
  • An example of randomly selected agents is the use a chemical library, a peptide combinatorial library or a growth broth of an organism.
  • an agent is said to be rationally selected or designed when the agent is chosen on a nonrandom basis which takes into account the sequence of the target site and/or its conformation in connection with the agent's action.
  • Agents can be rationally selected or rationally designed by utilizing the peptide sequences that make up the contact sites of the thrombin-GP Ib-EX complex pair.
  • a rationally selected peptide agent can be a peptide whose amino acid sequence is identical to the heparin binding site exosite II of thrombin.
  • the agents of the present invention can be, as examples, peptides, small molecules, vitamin derivatives, as well as carbohydrates.
  • Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of the template peptide. These types of non-peptide compound are termed "peptide imetics" or “peptidomimetics” (Fauchere, J. (1986) Adv. Drug Res. 15:29; Veber and Freidinger (1985) TENS p.392; and Evans et al. (1987) J. Med. Chem. 30:1229, which are incorporated herein by reference). A skilled artisan can readily recognize that there is no limit as to the structural nature of the agents of the present invention.
  • agents of the present invention are peptide agents whose amino acid sequences are chosen based on exosite EC of thrombin. Ln addition to recombinant expression of the desired peptide by the methods well known in the art, the peptide agents of the invention can be prepared using standard solid phase (or solution phase) peptide synthesis methods, as is known in the art.
  • Another class of agents of the present invention are antibodies immunoreactive with critical positions of the GP Ib-EX complex or thrombin.
  • Antibody agents are obtained by irnmunization of suitable mammalian subjects with peptides, containing as antigenic regions, those portions of the GP Ib-EX complex or thrombin intended to be targeted by the antibodies. Examples of such peptides would include peptides from thrombin encompassing serine 205. Other peptides encompassing critical regions, such as the regions encompassing contact sites involved in the association of the GP Ib-EX complex with thrombin, may be used.
  • Antibody agents are prepared by immunizing suitable mammalian hosts in appropriate immunization protocols using the peptide haptens alone, if they are of sufficient length, or, if desired, or if required to enhance immunogenicity, conjugated to suitable carriers. Methods for preparing immunogenic conjugates with carriers such as BSA, KLH, or other carrier proteins are well known in the art. hi some circumstances, direct conjugation using, for example, carbodiimide reagents may be effective; in other instances linking reagents such as those supplied by Pierce Chemical Co., Rockford, EL, may be desirable to provide accessibility to the hapten.
  • the hapten peptides can be extended at either the amino or carboxy terminus with a Cys residue or interspersed with cysteine residues, for example, to facilitate linking to a carrier.
  • Administration of the immunogens is conducted generally by injection over a suitable time period and with use of suitable adjuvants, as is generally understood in the art.
  • titers of antibodies are taken to determine adequacy of antibody formation.
  • Immortalized cell lines which secrete the desired monoclonal antibodies may be prepared using the standard method of Kohler and Milstein or modifications which effect immortalization of lymphocytes or spleen cells, as is generally known.
  • the immortalized cell lines secreting the desired antibodies are screened by immunoassay in which the antigen is the peptide hapten or is thrombin or the GP Ib-EX signaling complex itself.
  • the cells can be cultured either in vitro or by production in ascites fluid.
  • the desired monoclonal antibodies are then recovered from the culture supernatant or from the ascites supernatant. Fragments of the monoclonals or the polyclonal antisera which contain the immunologically significant portion can be used as antagonists, as well as the intact antibodies. Use of immunologically reactive fragments, such as the Fab, Fab', of F(ab') 2 fragments is often preferable, especially in a therapeutic context, as these fragments are generally less immunogenic than the whole immunoglobulin.
  • the antibodies or fragments may also be produced, using current technology, by recombinant means. Regions that bind specifically to the desired regions of the targets can also be produced in the context of chimeras with multiple species origin.
  • agents of the present invention can be provided alone, or in combination with other agents that modulate a particular pathological process.
  • an agent of the present invention that reduces thrombosis by blocking GP Eb-EX-thrombin association can be administered in combination with other anti-thrombotic agents.
  • two agents are said to be administered in combination when the two agents are admimstered simultaneously or are administered independently in a fashion such that the agents will act at the same time.
  • the agents of the present invention can be administered via parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, or buccal routes. Alternatively, or concurrently, administration may be by the oral route.
  • the dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.
  • the present invention further provides compositions containing one or more agents which modulate thrombin-induced platelet activity. While individual needs vary, determination of optimal ranges of effective amounts of each component is within the skill of the art. Typical dosages comprise 0.1 to 100 ⁇ g/kg body wt. The preferred dosages comprise 0.1 to 10 ⁇ g/kg body wt. The most preferred dosages comprise 0.1 to 1 ⁇ g/kg body wt.
  • the compositions of the present invention may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically for delivery to the site of action.
  • Suitable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form, for example, water-soluble salts.
  • suspensions of the active compounds as appropriate oily injection suspensions may be administered.
  • Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension include, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran.
  • the suspension may also contain stabilizers. Liposomes can also be used to encapsulate the agent for delivery into the cell.
  • the pharmaceutical formulation for systemic administration according to the invention may be formulated for enteral, parenteral or topical administration. Indeed, all three types of formulations may be used simultaneously to achieve systemic administration of the active ingredient.
  • Suitable formulations for oral administration include hard or soft gelatin capsules, pills, tablets, including coated tablets, elixirs, suspensions, syrups or inhalations and controlled release forms thereof.
  • the compounds of this invention may be used alone or in combination, or in combination with other therapeutic or diagnostic agents, hi certain preferred embodiments, the compounds of this invention may be coadministered along with other compounds typically prescribed for these conditions according to generally accepted medical practice, such as anticoagulant agents, thrombolytic agents, or other antithrombotics, including platelet aggregation inhibitors that target a different activation system from the thrombin-GP Ib-IX activation system described herein, for example, the PAR mediated signal transduction system, tissue plasminogen activators, urokinase, prourokinase, streptokinase, heparin, aspirin, or warfarin.
  • the compounds of this invention can be utilized in vivo, ordinarily in mammals, such as humans, sheep, horses,
  • glycoprotein lb beta as one of the major proteins phosphorylated during exposure of intact platelets to agents that activate cyclic AMP-dependent protein kinase. J Biol Chem 262, 12627-31 (1987).

Abstract

La présente invention concerne des traitements et des diagnostics qui utilisent un nouveau mécanisme de transduction du signal dans les thrombocytes. De plus, la présente invention concerne des procédés permettant d'identifier des agents qui modulent des activités induites par le nouveau mécanisme de transduction, telles que l'activation de thrombocytes et la thrombose.
PCT/US2001/026936 2000-08-31 2001-08-31 Traitements et diagnostics selon un nouveau systeme de transduction du signal dans les thrombocytes WO2002017711A2 (fr)

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WO2019035055A1 (fr) 2017-08-16 2019-02-21 Janssen Pharmaceutica Nv Molécules d'anticorps anti-thrombine et procédés d'utilisation avec des agents antiagrégants plaquettaires

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9518128B2 (en) 2011-12-14 2016-12-13 Janssen Pharmaceuticals, Inc. Thrombin-binding antibody molecules
US9518129B2 (en) 2011-12-14 2016-12-13 Janssen Pharmaceuticals, Inc. Thrombin-binding antibody molecules and uses thereof
US9605082B2 (en) 2011-12-14 2017-03-28 Janssen Pharmaceuticals, Inc. Thrombin-binding antibody molecules and uses thereof
US9988463B2 (en) 2011-12-14 2018-06-05 Janssen Pharmaceuticals, Inc. Thrombin-binding antibody molecules and uses thereof
US9988461B2 (en) 2011-12-14 2018-06-05 Janssen Pharmaceuticals, Inc. Thrombin-binding antibody molecules and uses thereof
US10287363B2 (en) 2011-12-14 2019-05-14 Janssen Pharmaceuticals, Inc. Thrombin-binding antibody molecules and uses thereof
US10370454B2 (en) 2011-12-14 2019-08-06 Janssen Pharmaceuticals, Inc. Thrombin-binding antibody molecules and uses thereof
US11155637B2 (en) 2011-12-14 2021-10-26 Janssen Pharmaceuticals, Inc. Thrombin-binding antibody molecules and uses thereof
CN108472503A (zh) * 2015-12-23 2018-08-31 朱利叶斯-马克西米利维尔茨堡大学 用于治疗血栓性疾病的可溶性糖蛋白v
WO2019030706A1 (fr) 2017-08-10 2019-02-14 Janssen Pharmaceutica Nv Molécules d'anticorps anti-thrombine et méthodes d'utilisation en chirurgie orthopédique
WO2019035055A1 (fr) 2017-08-16 2019-02-21 Janssen Pharmaceutica Nv Molécules d'anticorps anti-thrombine et procédés d'utilisation avec des agents antiagrégants plaquettaires

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