WO2002017711A2 - Therapeutics and diagnostics based on a novel signal transduction system in platelets - Google Patents

Abstract

The present invention relates to therapeutics and diagnostics that take advantage of a novel signal transduction pathway in platelets. In addition, the present invention provides methods for identifying agents that modulate activities mediated by the novel transduction pathway including platelet activation and thrombosis.

Description

THERAPEUTICS AND DIAGNOSTICS BASED ON A NOVEL SIGNAL TRANSDUCTION SYSTEM IN PLATELETS

Inventors: Vanitha Ramakrishnan and David Phillips

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to United States provisional patent application serial number 60/229,047, filed August 31, 2000, and to United States provisional patent application serial number 60/230,566, filed August 31, 2000, the disclosures of which are specifically incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to the field of hematology. In particular, the present invention relates to a novel signal transduction system in platelets and to therapeutic and diagnostic methods based thereon.

BACKGROUND OF THE INVENTION

Glycoproteins GP V and the GP Ib-IX-V Complex The GP Ib-IX-N complex is a large multimeric protein complex on the platelet surface which consists of 4 different subunits GP Ibα, GP Ibβ, 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 Ibα subunit contains the high affinity binding site for thrombin and the binding site for vWf. This complex has been implicated in the initial events associated with arterial thrombosis (Savage et al. (1996) Cell 84:289-297 and Weiss (1995) Thrombosis and Haemostasis 74(1):117-122).

It is known that 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). Following adhesion, binding of other agonists such as thrombin, adenosine diphosphate (ADP), and collagen, induce signaling events that ultimately activate the receptor function of αllbβ3 for soluble fibrinogen, leading to platelet aggregation (Clemetson et al, 1995). While 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).

The importance of the 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). 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.

Several studies indicate functional activities for the GP V subunit of the GP Ib- LX-V complex. In one example, 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). In another example, 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. Finally, 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.

SUMMARY OF THE INVENTION

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. Examples of 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. In some embodiments, the tlrrombin may be inactive. Examples of preferred inactivated thrombins include-but are not limited to-PPACK- inactivated thrombin, S205A thrombin, and DlP-thrombin. h other embodiments, 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. Examples of agents that bind GP Ib-LX include-but are not limited to-antibodies that bind specifically to GP Ib-LX. h some embodiments, 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. The 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. hi some embodiments, 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. In some preferred embodiments, the determining may be performed by fluorescence activated cell sorting (FACS). In other preferred embodiments, determining may be performed by Western blot. hi another embodiment of the present invention, 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 deteπnining 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. Examples of preferred inactivated thrombins include-but are not limited to-PPACK-inactivated thrombin, S205A tlrrombin, and DLP- thrombin. hi other embodiments, the thrombin may be active. In some embodiments, the signal transduction level is determined by measuring platelet aggregation.

In another aspect, 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.

BRIEF DESCRIPTION OF THE DRAWINGS

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-Hmdiπ was cloned into the corresponding sites around the Neo^r cassette, as shown.

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 FΪTC~vWf and botrocetin and analyzed by flow cytometry. The data is representative of 3 experiments done in duplicate.

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.

Figures 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. EC₅₀ 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. Inset. 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). C. GP V null platelets were aggregated with S205A- thrombin (lμM) in the absence (line 1) or presence (line 2) of a αllbβ3 inhibitor. The tracings represent experiments done at least 3 times with pooled platelets from 4 mice each. Higher concentrations of the CHO-expressed proteins were required to elicit a response in the GP V null platelets (l-2μM) than of the plasma derived thrombin (K_d range=100-400nM, Fig 10A inset), a requirement based on CHO-expressed thrombin having 70% less clotting activity than the plasma-derived thrombin (see Methods).

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 (2μM, line 2) or control rabbit IgG (2μM, line 1) for 10 min at room temp. A. Aggregation was initiated by the addition of DIP-thrombin (lOOnM). B. Aggregation was initiated by the addition of thrombin (lOnM). C. Glycocalicin-purified Ab 3584 IgG (1.89μM) 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 (2μM, line 1) or Ab 3584 (2μM, line 2) for 10 min and aggregation was initiated by the addition of lμM 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 ) (4.45μM, Panels E-H), and aggregation responses were measured compared to that obtained in the absence of heparin or PGI₂. GP V null platelets aggregated with 500nM DIP-thrombin (A, E) or lOnM thrombin (B, F) and thrombin-pre- treated wt platelets aggregated with 500nM DIP-thrombin (C, G) or wt platelets aggregated with lOnM thrombin (D, H). 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), 0.46μM CHO-expressed wt DIP-thrombin (Panel D), 0.75μM CHO-expressed DIP-

R89/R93/E94 thrombin (Exosite II mutant, Panel E) or 0.75μM CHO-expressed DIP-R98A thrombin (Exosite JJ mutant, Panel F). The number of animals used and the statistical significance is shown in each graph.

Figure 14 shows thrombin-induced platelet activation. Two pathways exist on the platelet for activation by thrombin. 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 Ibα by thrombin results in a signalling response which leads to αllbβ3 activation. Additionally, thrombin can cleave the PARs on platelets that will then also stimulate platelet aggregation.

DETAILED DESCRIPTION The present invention relates to the production and use of nonhuman transgenic animals, preferably mammals, that contain or comprise a modified GP V gene. Specifically, 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. Thus, 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. In one aspect of the present invention, 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. Once generated, such a transgenic or genetically-engineered animal, for example, a "knock-out mouse", can be used to 1) identify biological and pathological processes mediated by GP V; 2) identify proteins and other genes that interact with the GP V protein; 3) identify agents that can be exogenously supplied to overcome the absence or reduction in GP V protein function; and 4) serve as an appropriate screen for identifying agents that modulate {i.e., increase or decrease) the activity of the transgenic cells of knock-out mice or other animals so modified. In general, a transgenic nonhuman animal according to the present invention 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. For example, in the production of a transgenic mouse according to the present invention, 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). Through conventional breeding techniques thereafter, 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. For example, 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. h other types of assays, 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.

In a preferred embodiment of the present invention, 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. Specifically, 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. Unless defined otherwise, all technical and scientific terms used in this application have the same meaning as would commonly be understood by a person having an ordinary level of skill in the field to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.

As used herein "thrombin-induced activation" is activitation mediated by the GP Ib-LX signal transduction system. Activities induced by thrombin include-but are not limited to- thrombosis and platelet activation.

For example, 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.

The term "knock-out" generally refers to mutant organisms, usually mice, which contain a null or non-functional allele of a specific gene. The term "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. Such mutations include insertions of heterologous sequences, deletions, frame shift mutations and any other mutations that prevent, disrupt or alter GP V expression. The transgenic mammals of the present invention therefore may display non-normal platelet aggregation and/or other effects. By comparing the physiological and morphological characteristics between the transformed and non-transformed animals, one skilled in the art can thereby determine the effect of the presence or absence of the GP V gene and its expression product on the corresponding animal.

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. 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.

The cells, platelets, tissues and whole organisms of the disclosed transgenic animals specifically have utility in testing the effect of various agents for their ability to reduce or increase GP Ib-LX-V complex mediated processes. Agents that can be tested include various anticoagulant, thrombolytic and antiplatelet therapeutics and drugs. Examples of such agents include glycosaminoglycans such as heparin; oral anticoagulants such as dicumarol, anisindione, and bromadkiolone; tissue plasminogen activator (t-PA); urokinase; aspirin; dipyridamole; and ticlopidine. See, Majerus, et al, Anticoagulant, Thrombolytic, and Antiplatelet Drugs, in Goodman & Gihnan's The Pharmacological Basis of Therapeutics, Ninth Edition, Chapter 54 (1996) for a more complete list of such agents and their pharmacology.

The cells and whole organisms of the transgenic animals of the present invention, quite apart from their uses in veterinary and human medicine, may also be used to investigate gene regulation, expression and organization in animals. In general, for further examples of diagnostic and research uses of transgenic mammals, especially transgenic mice, see U.S. Patent No. 5,569,824.

Homologous Recombination Techniques

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. Similarly, 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.

Homologous recombination permits site-specific modifications in endogenous genes and thus inherited or acquired mutations may be corrected, and/or novel alterations may be engineered into the target animal's genome. To prepare cells for homologous recombination in the generation of transgenic animals, embryonic stem cells or a stem cell line maybe 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.

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. In contrast to the conventional microinjection technique, the homologous respective endogenous gene can be removed from a chromosome by homologous recombination with the transgene. By subsequent cross-breeding experiments, 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.

Production of Transgenic Animals

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.

The term "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.

If such offspring in fact possess some or all of that alteration or genetic information, then they, too, are transgenic animals.

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.

The development of 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. For example, 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.

5,602,307; Mullins et al., 1993 Hypertension 22, 630-633; Brenin βt al., 1997 Surg. Oncol. 6, 99-110; Tuan (ed.), 1997 Recombinant Gene Expression Protocols, Methods in Molecular

Biology No. 62, Humana Press).

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

40 T-antigen (U.S. Patent No. 5,728,915); lack the expression of interferon regulatory factor 1 (DRF-l) (U.S. Patent No. 5,731,490); exhibit dopaminergic dysfunction (U.S. Patent No.

5,723,719); express at least one human gene which participates in blood pressure control (U.S. Patent No. 5,731 ,489); display greater similarity to the conditions existing in naturally occurring Alzheimer's disease (U.S. Patent No. 5,720,936); have a reduced capacity to mediate cellular adhesion (U.S. Patent No. 5,602,307); possess a bovine growth hormone gene (Clutter et al. 1996 Genetics 143, 1753-1760); and, are capable of generating a fully human antibody response (Zou et al. , 1993 Science 262, 1271-1274).

While 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

Reprod. Nutr. Dev. 35, 609-617; Petters, 1994 Reprod. Fertil. Dev. 6,643-645; Schnieke et al. 1997 Science 278, 2130-2133; and Amoah, 1997 J. Animal Science 75, 578-585).

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. Thus, 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). This aggregation is physiologically relevant since aggregation occurs by a GP IJb-IJJa mediated process. Furthermore, we have shown that 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. Thus this mechanism is relevant because it is the first evidence that 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. Thus 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. Additionally, diagnostic screens based on the measurement of cleaved GP V (GP Vfl) can be used as indicators of ongoing thrombotic events and/or a predisposition to thrombotic events. Likewise, platelets lacking the GP Vfl portion can be used to assay for ongoing thrombotic events and/or a predisposition to such events. Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compositions of the present invention and practice the claimed methods. The following working examples therefore, specifically point out preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure. Other generic configurations will be apparent to one skilled in the art.

EXAMPLES METHODS Proteins and Antibodies. Rabbit antibodies against peptides based on mouse GP V generated by standard methods (Harlow et al, 1988) include Ab #810 (against mouse GP V residues C⁴⁷²-A⁴⁹⁰) and Ab#808 (against mouse GP V residues L⁴³²-R⁴⁵⁰ ). Anti-GP Ib-IX (rabbit polyclonal Ab #3584) was kindly provided by Dr. Beat Steiner, Hoffinan-LaRoche (Poujol et al, 1998). Anti- αllbβ3 (Ab #41) was described previously (Law et al, 1999). Human fibrinogen (Enzyme Research Labs) or human vWf (Haematologic technologies) were FITC-labeled in 0.1M NaHCO₃ (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-Hindiπ fragment from the EcoRI plasmid generating the 3' HR upstream of the Neo^r cassette. Thus in the recombinant, the coding region of mouse GP V (including the putative initiator Met) to Leu³⁸⁹ was replaced by a 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. Confirmation of recombination and germline transmission were performed using a probe designed to show linkage following Sphl digestion of genomic DNA (10μg) isolated as described (Laird et al, 1991) and Southern analysis. Heterozygote (+/-) animals identified by Southern blotting were bred to homozygosity.

Mouse Platelet Preparation.

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, 0.4μM prostaglandin El (PGE1)) for platelet-rich plasma (PRP) or (b) Acid-Citrate-Dextrose (ACD, 85mM sodium citrate, 0.11 IM glucose, 714mM citric acid, 0.4μM PGE1), for washed platelets (WP). Diluted blood was centrifuged at 82xg for 10 min.

The supernatant from (a) normalized to 2xl0⁸platelets/mL and ImM Mg²⁺ (final) was PRP. For WP, 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, ρH7) and resuspended in calcium-free Tyrodes-Hepes buffer (CFTH;10mM Hepes, 5.56mM glucose, 137mM NaCl, 12mM NaHCO₃, 2.7mM KC1, 0.36mM NaH₂PO₄, ImM MgCl₂, pH7.4). Platelets were normalized to 2xl0⁸/ml. PRP or WP were incubated at room temp for 30 min prior to use.

Glycoprotein Expression.

Flow cytometry was carried out as follows: PRP (lOμl) 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

30 min at 4°C in the dark. Samples were diluted to 400μL in PBS containing 0.1%) BSA and analyzed on a FACSORT (Becton-Dickinson).

Western analysis was carried out as follows: WP (5x10⁷) were solubilized in reducing sample buffer (Laemmli, 1970), electrophoresed, transferred to membranes and probed with primary rabbit antibody overnight at 4 ° C . Blots were incubated with peroxidase conjugated mouse anti-rabbit secondary antibody (1:5000; Jackson ImmunoResearch) for 1 hour at 4°C and developed by ECL (Amersham).

FITC~Ligand Binding Assay. For the determination of solution-phase vWf binding, pooled PRP was incubated with

FITC~vWf and botrocetin (4μM-40μM) for lOmin. Samples were diluted in CFTH buffer prior to analysis. For FITC~fibrinogen binding, WP were isolated from individual mice and incubated in duplicate (lxlO⁶) α-thrombin for 10 min. The reaction was terminated with PPACK (phenylalaninylprolylargininylchloromethylketone; 50μM final). The platelets were incubated with FITC-labeled fibrinogen (lOOμgs/ml) for 30 min, fixed with ^-formaldehyde (10%)) for 20 min and diluted into P/o^-formaldehyde in CFTH and analyzed by flow cytometry.

Binding Assays.

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 Mg²⁺-free CFTH buffer (1.2xl0⁸/mL) containing botrocetin (4μM), and incubated immobilized human vWf for lhour at room temp. Following two PBS washes, bound platelets were lysed and intracellular acid phosphatase activity was quantitated colorimetrically using the substrate pNPP (Sigma).

Platelet Aggregation.

Each experiment used pooled WP from 6-10 littermate mice of each genotype. Platelet aggregation initiated by thrombin was measured in a lumi-aggregometer (Chrono-Log) with stirring (1000-1200rpm) at 37°C.

Determination of Bleeding Time.

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.

Briefly, anaesthetized 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.

Example 1. Generation of the Targeting Vector.

1. Obtaining the murine GP V genomic DNA

The sequence of murine GP V was unknown at the start of this project. We therefore generated degenerate primers based on the human GP V sequence which had been published (Lanza et al, 1993, U.S. Patent Application No. 08/089,455, filed July 9, 1993, which is incorporated by reference herein and Figure 3) . These primers had the following sequences:

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' GA(CT)AA(AG)ATGGTG(CT)TC(CT)TGGA(GA)CA 3' (SEQ ID NO: 3) corresponds to human GP V residues DKMVLLEQ (SEQ ID NO: 4).

5' CC(CT)GG(CGA)AC(AC)TT(TC)AG(CT)GA(CT)(CT)TGAT(GC)AA 3' (SEQ ID NO: 5) corresponds to human GP V residues PGTFSDLIK (SEQ ID NO: 6).

Complements of Portions of the Coding Sequence: 5' (AG)TT(TGC)C(TG)(AG)AA(AG)GC(AG)GC(AG)GC(AG)GG 3'(SEQ ED NO: 7) corresponds to the complement of the human GP V sequence encoding PAAAFRN (SEQ ED NO: 8)

5'GGCCCCA(AG)(TG)CC(AG)CA(AG)TC(AG)CAGA(AG)CCA(AG)GA 3' (SEQ ID NO: 9) 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). Several clones were expanded for sequencing. Sequence analysis showed that clone Bl-12 was homologous to the human GP V gene, and was the rat homologue of the GP V gene. The insert from this clone was isolated and used to screen the mouse 129 BAG library (Genome Systems) by hybridization (see Shizuya et al. 1992). 2 clones 11487 and 11488 were positive. Genomic DNA was isolated from these clones. Approximately ~22Kb of the insert was mapped using Southern blotting with the Bl-12 insert. The mouse genomic DNA for GP V was identified by homology to the published human GP V DNA sequence (Figures 1 and 2).

2. Construction of the Targeting Vector.

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 (Neo¹) 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

Acc651(blunt)-BamHl sites in the polylinker which was at the 3' end of the Neo^r cassette between the Neo^r and TK cassettes , such that its orientation was opposite to that of the Neo gene. 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.

Example 3. Generation of GP V Knock-out mice.

To evaluate the specific role of GP V in both platelet function and the GP Ib-LX-V complex expression, we generated a mouse strain that lacked the GP V gene using homologous recombination techniques (Koller et al, 1992). Since rat GP V RNA would have greater homology to mouse GP V than the cloned human gene and was easier to isolate than mouse platelet RNA, rat platelet RNA for GP V was isolated and used as a probe for isolating genomic mouse GP V which was mapped and cloned from a BAC 129/Sv library.

Complete sequencing of 3 separate clones showed the mouse 129/Sv GP V gene to have 99.9%> homology in the coding region to the published mouse C57BL/6J mouse sequence (Ravanat et al, 1997) at both the DNA and protein levels (not shown). Sequencing also proved that the mouse and rat coding sequences were more homologous (DNA=92%, protein=87%), than human and mouse GP V (DNA=78%, protein=70%).

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.

Analysis of whole blood from GP V-deficient animals showed the platelets were normal in both number and size. Platelet counts in whole blood were within the normal range {wt males =6.52xl0⁸/ml (n=17) and females =5.6 xl0⁸/ml (n=8); +/- males =7.56xl0⁸/ml (n=15) and females =5.12xl0⁸/ml (n=12); -/- males =7.02xl0⁸/ml (n=21) and females =5.36xl0⁸/ml (n=9)}. There was a statistically significant difference in platelet recovery from whole blood from wt animals (males =74±22%>; females =82.4± 13%) and -/- animals (males =62± 19.5%, p =0.05; females =67 ± 14%», p=0.03). +/- animals showed intermediate recovery numbers, which were not statistically different {males =71 ± 19%, p^ _{t0 +}/-) =0.55 and p(+/_ _t0 -/-) =0.14; and females =78± 12%, p(_{Wt t0 +/}-) =0.46 and p(_+/-_t0-_/-) =0.065 }.

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.

Example 4. Effect of GP V gene deletion on GP Ib-IX expression and function.

GP V is usually expressed in platelets as a complex with GP Ib-LX (Meyer et al, 1995; Modderman et al. , 1992).

We used two techniques to determine whether absence of GP V from the platelet surface affected the expression of the other subunits of the GP Ib-LX-V complex. FACS analysis using an antibody specific for GP Ib-IX showed similar levels of GP Ib-LX in all three genotypes (Mean relative fluorescence units (RFU) ± sd for wt = 547+ 106; +/- = 391 ± 65; -/- = 483+56 with (wtto-/-)^ 0.42, p (wtto+/-)⁼ 0.11 and (+/-to-/-)⁼ 0.14). Western blot analysis confirmed that similar levels of the GP lb and GP EX were also present in platelet lysates from all three genotypes.

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. Under these conditions, the binding of vWf to platelets is mediated entirely by GP lb, since purified human glycocalicin (a soluble, extracellular fragment of GP Ibα that contains the vWf binding domain), inhibited botrocetin-induced binding of platelets to vWf in a concentration- dependent manner (not shown). We also found that soluble, activated vWf bound identically to platelets from all three genotypes in PRP (Figure 7). Again, botrocetin-induced vWf binding could be completely inhibited by purified human glycocalicin (not shown). Furthermore, stimulation of αllbβ3 on platelets by ADP and epinephrine did not induce soluble vWf binding (Figure 7). Thus GP Ib-LX expressed in the GP V-/- platelets was functional.

Example 5. Effect of GP V gene deletion on thrombin-induced platelet function. As shown in Figure 8, thrombin at low concentrations (0.5nM) induced significantly increased binding of FITC-fibrinogen in GP V -/- platelets compared to wt (Mean RFU + sd wt=7+1.2, +/-=13.2±0.9 and -/- = 22±0.8). This difference persisted at lnM thrombin (Mean RFU ± sd wt =11.5±6.8, +/- = 27.9±9.45 and -/- = 42± 1.8). However, platelets from all genotypes were able to bind FITC-fibrinogen equivalently at high (20nM) thrombin concentrations (Mean RFU± sd wt= 66.5 ±8.6, +/- =76.1 + 11.3 and -/- =72 ±0.2). The apparent EC₅₀ values for thrombin were approximately 2nM for wt platelets and 0.7nM for the -/- platelets. P-Selectin expression was also greater in the GP V-/- platelets at low thrombin concentrations, compared to wt (not shown). Tins 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 being contacted with 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.

Consistent with the FITC~fibrinogen results, platelets lacking GP V exhibited an increased aggregation response to thrombin compared to wt platelets. Indeed, 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. In addition to thrombin, other GP Ib-IX signaling activators may be used in place of thrombin. Examples include-but are not limited to-proteolytically inactive thrombin. As observed in the fibrinogen binding studies, 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 αllbβ3, using an antibody specific for the mouse fibrinogen receptor. The levels of αllbβ3 were comparable on platelets from animals of all three genotypes by flow cytometry (Mean RFU wt =1554±386; +/- =1246±202; and -/- = 1435±77; p _(wtto-/-r °-⁶⁵ _> P ( tto+/-)= 0.31 andp ₍+/-to-/-)= 0.24), and were also similar by Western blotting. Example 6. Determination of bleeding time.

To determine the consequences of enhanced platelet function in GP V -/- mice, bleeding time measurements were performed using a tail cut model, which was previously shown to be platelet dependent (Hodivala-Dilke et al, 1999; Tsakiris et al, (1999).

Consistent with the in vitro data, GP V -/- mice had a statistically shorter bleeding time (Mean ± sem=178±21sec) than wt littermate control mice (276 ±35 sec, Student's T-test p=0.016). The bleeding time in the +/- animals was intermediate (224±25 sec) but not statistically different from either wt or -/- mice. Furthermore, 70% of the -/- mice had bleeding times less than 120 sec, compared to 50%> of the wt and +/- mice. Conversely, 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. The difference in bleeding time is also statistically significant using non-parametric analysis (Maim- Whitney test p=0.046). Thus the increased aggregability of the platelets from GP V-/- mice observed in in vitro assays translates into a shorter bleeding time in vivo. This assay can be readily adapted to screen for agents that modulate clotting time.

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.

Discussion of Examples 1-6 Two conclusions on the role of GP V in platelet function can be made from the present study. First, 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. Second, although GP V is a subunit of the GP lb- EX-V complex, GP V is not required for the expression or function of GP Ib-IX. 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). Gene targeting of PARs reduces responsiveness of platelets to thrombin (Kahn et al, 1998; Kahn et al, 1999). 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). Additionally, the GP V subunit is a thrombin substrate and is cleaved during thrombin-induced platelet aggregation. Three observations have supported a role for GP V in GP Ib-LX-V complex expression.

First, subunits of a complex usually signify a mutual requirement for surface expression (Modderman et al, 1992). Second, all three subunits are missing in the platelets of patients with BSS, an autosomal recessive bleeding disorder (Lopez et al, 1998) linked to defects in GP lb or GP IX. Third, co-transfection of the GP V gene into heterologous cells expressing GP lb- EX resulted in increased expression (Calverley et al, 1995; Meyer et al, 1995; Lopez et al, 1992). These data taken together have led to the hypothesis that GP V is important for the expression of the vWf receptor (Calverley et al, 1995). Surprisingly, examination of platelets from the GP V-deficient mice by flow cytometry and Western analysis using a GP Ib-EX- specific antisera, revealed that lack of GP V did not compromise the expression of GP Ib-EX on the platelet surface. Thus, unlike observations in heterologous cells, GP V is not required for optimal expression of the GP Ib-EX complex on the platelet surface. Platelets from GP V-/- mice were normal in size in contrast to the giant size and low numbers of platelets observed in both BSS patients that lack the GP Ib-LX-V complex (Lopez et al, 1998), and in mice deficient in GP lb (Ware et al, 1998). The loss of the actin binding protein anchoring is believed to cause the large platelets in BSS. The present data suggest that GP V is not required for this interaction. These observations suggest that GP V does not regulate expression of GP Ib-IX, but that thrombin cleavage of GP V may be involved in vascular pathologies. It is of interest to note that we performed bleeding times on a large number of animals, in order to confirm that the phenotype was consistent with the in vitro data. Furthermore, the absence of GP V may occur and yet be undetected in the human population. Recently, it has been shown that platelets from the 12-lipoxygenase-deficient mouse have increased sensitivity to ADP (Johnson et al, 1998), which resulted in a prothombotic phenotype. Thus modifiers of platelet function which upregulate responses to agonists provide additional means of modifying platelet function. Arterial 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 .

Example 7. A Novel Thrombin Receptor Function for Platelet GP lb-IX Unmasked by Cleavage of GP V.

Generation of CHO-Expressed Thrombins.

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 lOμM 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 Chromozyme™ 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 Ibα (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.

Aggregation. 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.

Detection of Cleaved GP V (GP V fl) in Supematants of Wt Platelets Treated with Thrombin. Washed wt mouse platelets were isolated from 20 mice, and incubated with or without

50-1 OOpM thrombin for 30 min without stirring at 37°C. Platelets were centrifuged at 100,000 g to remove microparticles, and the supernatant was lyophilised, reconstituted in reducing REPA buffer and boiled for 5 min. Reduced samples were buffer-exchanged by dialysis into non-reducing REPA buffer and immunoprecipitations (EPs) were carried out with Ab 808 (previously shown to recognise an epitope not available in native GP V (Ramakrishnan et al, 1999), or control IgG. Samples were electrophoresed by reducing SDS-PAGE, and Western analysis was done with Ab 808.

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 . Optionally, 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.

Thrombosis Model in Mice. This model is a modification of that described by Leon et al (Leon et al. 1999). Briefly, mice were anaesthetised and the jugular vein was exposed surgically. A retro-orbital bleed (125μL) was taken using heparinised tubes, which was transferred into tubes containing 0.9%> saline/50mM EDTA (125μL), to determine the baseline platelet counts. Then, various agonists were injected into the jugular vein in a volume of lOOμl, 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

Discussion of Example 7

Cellular responses to thrombin are mediated, in part, by the proteolysis of one or more of 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 Ibα can bind thrombin (De Marco et al, 1991) and could therefore be important for this effect. To explore this possibility, we examined the effect of proteolytically-inactive thrombin on the aggregation of platelets that lack GP V and thus express a mutant GP Ib-EX-V complex (Ramakrishnan, V. et al, 1999). Surprisingly, di- isopropylphospho (DEP)-thrombin induced platelet activation in GP V null platelets even without the addition of any active thrombin (Fig 10A). Aggregation of GP V null platelets required about 10-fold more DEP-thrombin (lOOnM) than untreated thrombin (lOnM), confirming the Kd values determined in recent studies (De Cristofaro et al, 2000). In contrast, platelets from wild-type (wt) mice failed to aggregate in response to inactive thrombin, unless they had been pre-treated with sub-optimal doses (~50pM) of active thrombin (Fig 10B). Western analysis showed that 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). En other experiments, we also found that GP V null platelets aggregated in response to either phenylalaninylprolylargininylchloromethyl ketone (PPACK)-inactivated thrombin (data not shown), or to recombinant tlrrombin carrying a mutation (S205A) that inactivates its proteolytic capacity (Fig 10C). CHO-expressed wt thrombin inactivated by DFP also caused aggregation in GP V null platelets. Aggregation of mutant and wt platelets in response to all forms of thrombin was sensitive to inhibition by an antagonist of the αllbβ3 integrin, indicating that the aggregation reactions involved the characteristic agonist-induced pathway (not shown) .

Antibody 3584 (Ab 3584) recognises GP Ib-EX on human and mouse platelets (Ramakrishnan, V. et al., 1999). Because GP Ibα 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 Ibα 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). Further, MAb LJ-1B10, which inhibits thrombin binding to GP Ibα (De Marco et al, 1994), inhibits the aggregation in human platelets rendered GP V deficient by thrombin pre-treatment (not shown). These data implicate thrombin-binding to GP Ibα in the aggregation of platelets induced by proteolytically inactive thrombin.

The exosite II of thrombin binds heparin and may also be involved in the interaction with GP Ibα (De Cristofaro et al., 2000). We therefore examined whether aggregation induced by DEP-thrombin could be inhibited by heparin. Within the standard therapeutic dose range (0.3U/ml; standard range 0.2-0.7U/ml (Barrow et al, 1994)) 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). Thus, the data suggest that binding between exosite Et of thrombin and GP Ibα on platelets is essential for platelet activation in response to proteolytically-inactive thrombin. Since heparin blocks this interaction at concentrations used therapeutically, this mechanism may also be involved in the anti-thrombotic activity of heparin.

To evaluate the physiological significance of the ligand binding function of thrombin, we examined the effect of a systemic infusion of DEP-thrombin into mice. Infusion of a platelet agonist induces a decrease in platelet counts due to (ongoing) thrombosis (Leon, C. et al. 1999). As can be seen in Fig 13, there was significant platelet loss following the infusion of lOnM thrombin in both GP V null and wt mice (Fig 13C), with no statistical difference obse ved between the 2 groups. Injection of lnM thrombin had a marginal effect on platelet counts in either GP V null mice or wt mice (Fig 13B). hi contrast, 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. As additional controls we used 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. As can be seen in Fig 13E and 13F, there was no major loss in platelets in either group, h contrast, 460nM CHO-expressed thrombin inactivated with DFP (Fig 13D) caused a significant platelet loss in GP V null mice, with little effect in wt mice. These results show that binding of inactive forms of thrombin to GP V-deficient platelets can occur in vivo, and that the binding results in platelet activation and thrombosis. Our data further substantiate the binding site for GP Ibα being via exosite Et of thrombin. These results support a novel role for GP Ibα in thrombin-induced signalling in platelets which consequently supports aggregation and thrombosis.

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 Ibα that is phosphorylated at Ser609 (Bodnar et al, 1999). Other studies have shown that agents like PGI₂ and VGEi negatively regulate GP Ib-EX signalling. The mechanism of inhibition by the adenylyl cyclase activators appears to be through the activation of the cAMP-dependent kinase and phosphorylation of the cytoplasmic domain of GP Ibα (Fox et al, 1987; Fox et al, 1989). To determine whether the thrombin signalling function of GP Ib-EX-V is similarly regulated, we evaluated the role of the cAMP-dependent pathway in DEP-thrombin induced signalling. Fig. 12E shows that PGI₂ completely inhibited DEP -thrombin-induced shape change and aggregation of GP V -/- platelets (IC₅₀∞ 70nM). PGE_ls another activator of adenylyl cyclase, also inhibited DEP-thrombin induced aggregation in GP V -/- platelets (IC₅₀«300nM). PGI₂ inhibited DEP -thrombin-induced aggregation in both mouse (Fig 12G) and human platelets pre- treated with sub-optimal doses of active thrombin. hi contrast, thrombin-induced aggregation was not affected significantly by PGI₂ even at high concentrations (4.45 μM, Fig 12F and H). h fact, PG-₂-treated platelets that fail to respond to DEP-thrombin were fully responsive to lOnM α-thrombin (data not shown). Thus, the 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 PGI₂ or PGE₁ 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 PGE₁ during platelet isolation and desensitisation studies described therein may have inhibited signalling through GP Ib-EX-V. Our data support a model (Fig 14) for thrombin-induced platelet activation that 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. Following the loss of GP V upon cleavage by thrombin, thrombin binding to GP Ibα results in activation of αllbβ3 and consequently in aggregation. As shown here, the GP Ibα-bound thrombin need not be catalytically functional for this response to occur. The data show not only a novel functional role for thrombin, but also a novel mechanism by which this pathway can mediate thrombosis independent of proteolytic activity. It has been calculated that clot-bound thrombin concentrations can be as high as 1.4μM, which encompasses the range described in these studies (van 't Veer et al, 1997; Mann et al, 1999). The binding of thrombin to GP Ibα 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 Ibα-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. Suitable 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. As used herein, 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. One class of 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. As used herein, 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. As used herein, 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. As described above, in some embodiments there are two sites of action for agents that block the thromin-GP Ib-LX interaction: the binding site on GP Ib-LX and exosite JJ of thrombin. 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. For example, 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.

One class of 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. During the immunization schedule, titers of antibodies are taken to determine adequacy of antibody formation.

While the polyclonal antisera produced in this way may be satisfactory for some applications, for pharmaceutical compositions, use of monoclonal preparations is preferred. 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. When the appropriate immortalized cell culture secreting the desired antibody is identified, 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.

The agents of the present invention can be provided alone, or in combination with other agents that modulate a particular pathological process. For example, 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. As used herein, 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 μg/kg body wt. The preferred dosages comprise 0.1 to 10 μg/kg body wt. The most preferred dosages comprise 0.1 to 1 μg/kg body wt. In addition to the pharmacologically active agent, 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. In addition, 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. Optionally, 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. h practicing the methods of this invention, 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, cattle, pigs, dogs, cats, rats and mice, or in vitro.

Although the present invention has been described in detail with reference to examples above, it is understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims. All cited patents, applications and publications referred to in the application are hereby incorporated by reference in their entirety.

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Claims

WHAT IS CLAIMED IS:

1. A method of identifying an agent that inhibits thrombin-induced platelet activation, wherein the activation is modulated by GP V, comprising a) administering a test agent and proteolytically inactive thrombin to a GP V null non-human transgenic animal; and b) monitoring aggregation of platelets of the animal to identify inhibition.

2. The method of claim 1 , wherein the administered thrombin is selected from the group consisting of PPACK-inactivated thrombin, S205A thrombin, and DEP-thrombin.

3. The method of claim 1 , wherein the test agent binds GP Ib-EX.

4. The method of claim 1 , wherein the test agent is selected from the group consisting of antibodies that bind specifically to GP Ib-EX and agents that bind exocite Et of thrombin.

5. A method of identifying an agent that inliibits thrombin-induced activity, wherein the activity is inhibited by GP V, comprising a) administering a test agent and thrombin to a platelet isolated from a GP V null non-human transgenic animal; and b) monitoring aggregation of platelets to identify modulation of the activity.

6. The method of claim 5, wherein the administered thrombin is proteolytically inactive.

7. The method of claim 6, wherein the thrombin is selected from the group consisting of PPACK-inactivated thrombin, S205A thrombin, and DEP-thrombin.

8. The method of claim 5, wherein the thrombin is active.

9. The method of claim 5, wherein the test agent binds GP Ib-IX.

10. The method of claim 5, wherein the test agent is selected from the group consisting of antibodies that bind specifically to GP Ib-EX, peptides and small molecules.

11. A method of identifying an agent that modulates thrombin activity, wherein the activity is inhibited by GP V, comprising: a) administering a test agent and thrombin to a platelet isolated from a GP V null non-human transgenic animal; and b) monitoring aggregation of the platelets for modulation of the activity.

12. The method of claim 11 , wherein the thrombin is inactive.

13. The method of claim 12, wherein the thrombin is selected from the group consisting of PPACK-inactivated thrombin, S205A thrombin, and DEP-thrombin.

14. The method of claim 11 , wherein the thrombin is active.

15. The method of claim 11 , wherein the test agent binds GP Ib-EX.

16. The method of claim 11 , wherein the test agent is selected from the group consisting of antibodies that bind specifically to GP Ib-EX, peptides and small molecules.

17. A composition, comprising: a platelet that does not display GP V on its surface; and a GP Ib-EX signaling activator.

18. A method according to 17, wherein the signaling activator is a proteolytically inactive thrombin.

19. A composition according to claim 17, further comprising an agent that modulates an activity of the platelet induced by thrombin.

20. A composition according to claim 17, wherein the thrombin is selected from the group consisting of PPACK-inactivated thrombin, S205A thrombin, and DEP-thrombin.

21. 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.

22. A method according to claim 21, wherein the determining is performed using an antibody specific for GP Vfl .

23. A method of 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.

24. A method according to claim 23, wherein the determining is performed by fluorescence activated cell sorting (FACS).

25. A method according to claim 23, wherein the determining is performed by Western blot.

26. A method of inhibiting thrombosis in a subject, comprising: administering to the subject an inhibitor of GP Ib-IX signal transduction, with the proviso that the agent is not heparin.

27. A method according to claim 26, wherein the agent binds to exosite II of thrombin.

28. A method of screening for antithrombotic agents, comprising: contacting a platelet that does not display GP V on its surface with an agent; contacting the platelet with a GP Ib-IX signaling activator; and determining GP Ib-EX mediated signal transduction level, wherein a reduced level is indicative of an antithrombotic agent.

29. A method according to claim 28, wherein the GP Ib-EX activator is thrombin.

30. A method according to claim 28, wherein the tlirombin is inactive.

31. A method according to claim 30, wherein the thrombin is selected from a group consisting of PPACK-inactivated thrombin, S205A thrombin, and DEP-thrombin.

32. A method according to claim 28, wherein signal transduction level is determined by measuring platelet aggregation.

33. A method of preventing platelet activation in a subject, comprising: administering to the subject an agent, wherein the agent prevents interaction of thrombin exosite IE with GP Ib-EX, with the proviso that the agent is not heparin.

34. An agent identified by the method of claim 1.

35. A pharmaceutical composition comprising the agent of claim 34 in an amount suitable to ameliorate a condition characterized by platelet activation.