WO2010060918A1 - Methods and kits for monitoring anti-platelet therapy - Google Patents

Abstract

Methods and kits are provided for assessing the effect of anti-platelet therapy, in particular for assessing the efficacy of anti-platelet agents that target platelet surface receptors. The methods of the present invention include determining the activity of a surface receptor targeted by an anti-platelet drug by measuring the phosphorylation state of Akt in platelets obtained from a patient receiving the anti-platelet drug. The methods of the invention are useful for adjusting or optimizing the dose of anti-platelet agent administered to a patient to achieve therapeutic efficacy and/or to reduce side effects, and for detecting biological resistance to anti-platelet treatment. More generally, the ability to monitor the responsiveness to anti-platelet therapy allows individualization of treatment.

Description

Methods and Kits for Monitoring Anti-Platelet Therapy

Related Applications

The present application claims priority to European Patent Application No. EP 08 305 859.4 filed on November 27, 2008. The European patent application is incorporated herein by reference in its entirety.

Background of the Invention

The main function of platelets is the maintenance of hemostasis, the body's normal physiological process that prevents excessive blood loss. Hemostasis is primarily achieved by the formation of a thrombus (or blood clot) following damage to the endothelium of blood vessels. In addition to their beneficial role in the prevention of hemorrhage, platelets are also implicated in a number of pathological conditions such as thrombosis, atherosclerosis and inflammation. Platelets have therefore become a major target for therapeutic intervention in cardiovascular diseases. In particular, anti-platelet therapy has emerged as a critical element in the management of patients with acute coronary syndromes (ACS) and coronary artery disease (CAD), and patients undergoing percutaneous coronary intervention (PCI).

Currently approved anti-platelet agents include aspirin, thienopyridines such as clopidogrel, and glycoprotein Ilb/IIIa receptor antagonists. Although anti-platelet therapy provides significant clinical benefits in patients with a variety of thrombotic or ischemic diseases, available anti-platelet agents have less than satisfactory safety and efficacy. Limitations to current therapeutic options include modest levels of platelet inhibition, variability in response, slow rates of onset, side effects such as problem bleeding, and continued thrombotic and atherosclerotic events despite treatment. For example, while numerous studies have demonstrated the benefit of clopidogrel used, in combination with aspirin, to prevent thrombosis after coronary stent implantation, research also indicates that 5 to 30% of patients do not respond adequately to the drug (Gurbel et al, Circulation, 2003, 107: 2908-2913; Jaremo et al, J. Intern. Med., 2002, 252: 233-238; Muller et al, Thromb. Haemostasis, 2003, 89: 783-787; Grossmann et al, Thromb. Haemost., 2004, 92: 1201-1206). The medical consequences can be dramatic: patients who are non-responsive to clopidogrel are at much greater risk for a serious cardiovascular event including not only heart attack or stroke but also blockage of coronary stents. Because of the limitations and potential risks of current anti-platelet drugs, the medical and research community has been focusing more aggressively on how this clinical problem could be solved. In the last decade or so, substantial progress has been made in understanding the regulation of platelet function, including the characterization of new ligands, platelet- specific receptors and cell signaling pathways. This progress is expected to lead to the development of more effective and safer anti-platelet drugs; and several pharmaceutical companies already have, in their pipelines, anti-platelets at different stages of development. The need for novel antiplatelet agents is all the more important that the clinical burden of cardiovascular diseases is set to increase with an ageing population and increasing levels of obesity. In addition to these epidemiological factors, the forthcoming patent expiry faced by clopidogrel is another incentive to the development of novel anti-platelet drugs.

The clinical importance of non-responsiveness to anti-platelet therapy has also led to an increased interest in the use of platelet function tests to assess patients' individual response to anti-platelet agents. A conventional test of platelet function is light transmission aggregometry (LTA), which measures platelet aggregation in plasma after exposure to substrates such as arachidonic acid, epinephrine, collagen or adenosine diphosphate (ADP). Other techniques for assessing platelet function include the platelet function analyzer PFA- 100^®, which determines high shear stress- dependent platelet aggregation in whole blood; the Thrombelastograph^® (TEG) PlateletMapping™ assay, which evaluates clot strength and provides a quantitative analysis of platelet function; the VerifyNow™ (aspirin, P2Yi₂ and Ilb/IIIa) assays, which measure platelet function based upon the ability of activated platelets to bind to fibrinogen-coated beads; and flow cytometry techniques used to assess the state of platelet activation through determination of conformation changes in membrane glycoproteins and surface expression of P-selectin and phosphotidylserine.

However, most of these platelet function tests have well established limitations for monitoring anti-platelet therapy. In particular, it has been suggested that methods to evaluate the effects of anti-platelet agents should be designed to measure the direct pharmacodynamic effect of the drug, rather than the consequences for global platelet function. Accordingly, a more specific test measuring the extent of phosphorylation of the cytoplasmic vasodilator-stimulated phosphoprotein (VASP) by flow cytometry has recently been developed and is now commercially available (Biocytex/Diagnostic Stago, France). VASP phosphorylation is regulated by the cytoplasmic levels of cyclic adenosine monophosphate (cAMP). cAMP levels are reduced following inhibition of adenylyl cyclase which results from stimulation of the Gi-coupled P2Yi₂ receptor, the target of thienopyridines such as clopidogrel. To the Applicant's knowledge, this test is the first to specifically address the target of anti-platelet agents.

Clearly, there is a need in the art for simple, rapid and reliable monitoring assays for the determination of anti-platelet therapy efficacy. In particular, assays that provide information about the effects of anti-platelet agents on their primary targets are highly desirable.

Summary of the Invention

The present invention is directed to a novel biomarker for the monitoring of antiplatelet therapies. The present Applicants have shown that the therapeutic efficacy of clopidogrel, the precursor of a potent inhibitor of the purinergic adenosine diphosphate (ADP) receptor P2Yi₂ could be monitored by following the phosphorylation of the serine-threonine kinase Akt in platelets (see Example 1). Activation of P2Yi₂ results in activation of phosphoinositide 3-kinase (PDK) followed by activation of several kinases, including Akt. The Applicants have also demonstrated that the effects of wortmannine, a PDK inhibitor (see Example 2) and of SCH79797, a selective inhibitor of the thrombin receptor PAR-I (see Example 3) could be assessed by measurements of Akt phosphorylation by flow cytometry.

Accordingly, the present invention provides for the use of Akt phosphorylation as a biomarker for the monitoring of anti-platelet therapies, in particular for the monitoring of anti-platelet agents that target platelet surface receptors, preferably for the monitoring of anti-platelet agents that are inhibitors or antagonists of a platelet surface receptor.

In certain preferred embodiments, the anti-platelet agents that target platelet surface receptors are inhibitors or antagonists of a platelet surface receptor, in particular of a platelet surface receptor selected from the group consisting of an ADP receptor, preferably P2Yi₂; a thrombin receptor, preferably PAR-I; a collagen receptor, preferably GPVI; a von Willebrand factor receptor, preferably GPIb-IX-V; a thromboxane A2 receptor, preferably TP; an integrin receptor, preferably GPIIb/IIIa; or a C-type lectin receptor, preferably CLEC -2. In one aspect, the present invention provides methods for characterizing the effects of an anti-platelet therapy on a subject. More specifically, the present invention provides an in vitro method for monitoring a subject's response to an antiplatelet agent which targets a platelet surface receptor, the method comprising a step of: determining the phosphorylation state of Akt in a sample containing platelets from the subject.

In certain embodiments, the anti-platelet agent is an antagonist of the platelet surface receptor. For example, the anti-platelet agent may be an antagonist of a platelet surface receptor selected from the group consisting of an ADP receptor, preferably P2Yi₂; a thrombin receptor, preferably PAR-I; a collagen receptor, preferably GPVI; a von Willebrand factor receptor, preferably GPIb-IX-V; a thromboxane A2 receptor, preferably TP; an integrin receptor, preferably GPIIb/IIIa; or a C-type lectin receptor, preferably CLEC -2.

In certain embodiments, the sample containing platelets is selected from the group consisting of whole blood, platelet rich plasma, and isolated platelets.

In certain embodiments, the in vitro methods of monitoring of the present invention further comprise a step of: stimulating the platelets with an agonist of the platelet surface receptor targeted by the anti-platelet agent prior to determining the phosphorylation state of Akt. In certain embodiments, the agonist of the platelet surface receptor targeted by the anti-platelet agent is selected from the group consisting of ADP, thrombin, thrombin receptor activating peptide (TRAP), collagen, collagen related peptide (CRP), convulxin, von Willebrand factor under flow conditions, thromboxane A2 (TxA₂), TxA₂ analogue U46619, fibrinogen under adhesion conditions, and any combination thereof.

In certain embodiments, determining the phosphorylation state of Akt comprises using at least one detectable probe selective (or specific) for phosphorylated Akt, and optionally, at least one detectable probe selective (or specific) for non-phosphorylated Akt. The detectable probe selective for phosphorylated Akt may comprise a labeled phospho- specific antibody selective for phosphorylated Akt, or alternatively a phospho- specific antibody selective for phosphorylated Akt and a labeled secondary antibody against the phospho-specific antibody. The detectable probe selective for non-phosphorylated Akt may comprise a labeled antibody selective for non- phosphorylated Akt or alternatively an antibody selective for non-phosphorylated Akt and a labeled secondary antibody against the antibody selective for non- phosphorylated Akt.

In certain embodiments, the phospho-specific antibody is a phospho-specific S473 antibody or a phospho-specific T308 antibody.

In certain embodiments, determining the phosphorylation state of Akt comprises using a method selected from the group consisting of Western blot, immunoblot, enzyme-linked immunosorbant assay (ELISA), radioimmunoassay (RIA), immunoprecipitation, surface Plasmon resonance, immunohistochemistry (IHC), matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, microcytometry, microscopy, fluorescence activated cell sorting (FACS), and flow cytometry (FC). In certain preferred embodiments, determining the phosphorylation state of Akt comprises using flow cytometry.

In another aspect, the present invention provides a kit for monitoring a subject's response to an anti-platelet agent comprising at least one detectable probe selective for phosphorylated Akt, and, optionally, at least one detectable probe selective for non- phosphorylated Akt.

The detectable probe selective for phosphorylated Akt included in the kit may comprise a labeled phospho-specific antibody selective for phosphorylated Akt, or a phospho-specific antibody selective for phosphorylated Akt and a labeled secondary antibody against the phospho-specific antibody. For example, the phospho-specific antibody may be a phospho-specific S473 antibody or a phospho-specific T308 antibody.

The detectable probe selective for non-phosphorylated Akt optionally included in the kit may comprise a labeled antibody selective for non-phosphorylated Akt, or an antibody selective for non-phosphorylated Akt and a labeled secondary antibody against the antibody selective for non-phosphorylated Akt.

In certain embodiments, the kit further comprises one or more of: a platelet activation inhibitor selected from the group consisting of aprotinin, theophylline, apyrase, prostaglandin El, and any combination thereof; and a platelet stimulator selected from the group consisting of ADP, thrombin, thrombin receptor activating peptide (TRAP), collagen, collagen related peptide (CRP), convulxin, von Willebrand factor, thromboxane A2 (Tx A2), TxA2 analogue U46619, fibrinogen, and any combination thereof.

These and other objects, advantages and features of the present invention will become apparent to those of ordinary skill in the art having read the following detailed description of the preferred embodiments.

Brief Description of the Drawing Figure 1 is a set of pictures of polyacrylamide gels showing P2Yi₂-mediated phosphorylation of Akt on Ser473 in ADP- stimulated platelets. (A) and (B): Platelets pre-incubated in the absence and presence of AR-C69931 MX (10 μM) or MRS-2179 (100 μM) were stimulated by ADP (10 μM) at 37⁰C for 10 minutes in (A) and for a time as indicated on the figure in (B), without stirring. (C): Platelets were stimulated by ADP (10 μM) and PGEl (10 μM) at 37⁰C for 10 minutes. Platelets were separated by SDS-PAGE, Western blotted and probed with anti-P-AKt (Ser473) and anti-Akt antibodies. Results are representative of 2 to 4 experiments.

Figure 2 is a set of pictures of polyacrylamide gels showing typical patterns of Akt-phosphorylation in ADP- stimulated platelets in patients treated with clopidogrel. (A): no phosphorylation of Akt, suggestive of P2Yi₂ inhibition by clopidogrel in vivo (found in 26 out of 55 patients). (B): Akt-phosphorylation, prevented by addition of ARC-C69931 MX in vitro, suggestive of a poor inhibition of P2Yi₂ by clopidogrel in vivo (found in 25 out of 55 patients). (C): undetermined profile (Akt-phosphorylation not inhibited by clopidogrel in vivo or by ARC-C69931 MX in vitro) (found in 4 out of 55 patients).

Figure 3 is a graph showing the distribution of VASP-PRI values according to P-Akt index. Boxes span the interquartile range. Horizontal lines bisecting the boxes indicate median values. The dotted line indicates the commonly used cut-off.

Figure 4 is a graph showing the correlation between VerifyNow^® P2Y_i2 inhibition index and peak aggregation induced by 10 μM ADP. Closed circles denote patients with low P-Akt index (group A); open circles denote patients with high P-Akt index (group B).

Figure 5 is a set of two graphs showing platelet aggregation according to P2Yi₂ signaling measured by ADP-induced Akt-phosphorylation (A) or VASP-PRI (B). Platelet aggregation was expressed as peak aggregation induced by 10 μM in PRP

(LTA) and VerifyNow^® P2Yi₂ inhibition index. Boxes span the interquartile range.

Horizontal lines bisecting the boxes indicate median values.

Figure 6 is a graph showing the phosphorylation of Akt measured by flow cytometry in human platelets in a resting state or stimulated by TRAP (thrombin receptor activating peptide - 50 μM) in the absence and in the presence of wortmannine, a PI3-kinase inhibitor, which activates Akt.

Figure 7 is a graph showing the phosphorylation of Akt measured by flow cytometry in human platelets in resting state or stimulated by TRAP (thrombin receptor activating peptide - 50 μM) in the absence and in the presence of SCH79797, a selective PAR-I inhibitor.

Definitions

Throughout the specification, several terms are employed that are defined in the following paragraphs.

The term "thrombotic disorder" refers to any disorder relating to, or associated with, the formation or presence of a blood clot within a blood vessel. Thrombotic disorders include, but are not limited to, myocardial infarction, unstable angina, abrupt closure following angioplasty, or stent placement, and thrombosis as a result of peripheral vascular surgery.

The term "antithrombotic agent", as used herein, refers to any compound, agent or factor that belongs to a family of drugs useful in the treatment of thrombotic disorders. There are three main classes of antithrombotic drugs: anticoagulant drugs, anti-platelet drugs and thrombolytic or fibrinolytic drugs. Anticoagulant and antiplatelet drugs inhibit the coagulation process and can therefore be administered acutely to prevent the initial formation of blood clots, or thrombi, in patients with recognized risk factors. Thrombolytic or fibrinolytic drugs act by dissolving existing thrombi or emboli and therefore play a role in the acute treatment of thrombosis. As used herein, the term "anti-platelet agent" refers to any agent which, irrespective of its specific mechanism of action, blocks, reduces or inhibits the formation of blood clots.

As used herein, the term "subject" refers to a human or another mammal (e.g., primate, dog, cat, rat, rabbit, and the like) that can be affected by a thrombotic disorder but may or may not be affected by the disease. Non-human subjects may be transgenic or otherwise modified animals. In many embodiments of the present invention, the subject is a human being. In such embodiments, the subject is often referred to as an "individual", or to a "patient" if the individual is receiving a treatment (e.g., an anti-platelet agent) or has been diagnosed with a thrombotic disorder. The terms "individual" and "patient" do not denote a particular age.

The term "treatment" is used herein to characterize a method or process that is aimed at (1) delaying or preventing the onset of a disease or condition (e.g., a thrombotic disorder); (2) slowing down or stopping the progression, aggravation, or deterioration of the symptoms of the disease or condition; (3) bringing about amelioration of the symptoms of the disease or condition; and/or (4) curing the disease or condition. A treatment may be administered prior to the onset of the disease or condition, for a prophylactic or preventing action. Alternatively or additionally, a treatment may be administered after initiation of the disease or condition, for a therapeutic action.

The term "platelets" has herein its art understood meaning and refers to small cytoplasmic bodies derived from cells. Like red blood cells, platelets have no nucleus. They circulate in the blood and help mediate blood clotting at sites of damage. They also release various factors that stimulate healing. One of the major functions of platelets is to initiate blood clotting. Some characteristics of platelet function include, but are not limited to, platelet aggregation, ATP secretion and platelet cell shape change.

The terms "inhibitor of a platelet surface receptor" and "antagonist of a platelet surface receptor" are used herein interchangeably. They refer to a receptor ligand, compound or drug that does not provoke a biological response itself upon binding to the receptor, but blocks or inhibits agonist-mediated responses. The term "Akt" refers to protein kinase B (also known as PKB), an enzyme that is activated via phosphatidylinositol lipids such as PIP3 or PI(3,4)P₂ and is capable of phosphorylating glycogen synthase kinase-3 (GSK3). In humans, there are three genes in the Akt family: AKTl (Accession Number: NM_005163), AKT2 (Accession Number: NM_001626), and AKT3 (Accession Number: NM_181690). These genes code for enzymes that are members of the serine/threonine- specific protein kinase family. Akt denotes multiple enzymes, including Aktl (also known as PKBα), Akt2 (also known as PKBβ) and Akt3 (also known as PKBγ). hi many embodiments of the invention, Akt refers to human Aktl (Accession number: NP_005154), which can be phosphorylated at threonine 308 (Thr³⁰⁸) and/or serine 473 (Ser⁴⁷³). Protein kinase B, and isoforms thereof, are reviewed by PJ. Coffer et ah, Biochem. J., 1998, 335: 1-13, which is incorporated herein by reference in its entirety.

As used herein, the term "phosphorylation" refers to the addition of a phosphate group to an amino acid, e.g., to a serine or a threonine in the case of Akt. The terms "phosphorylation state" and "phosphorylation level" are used herein interchangeably.

A compound or agent is herein said to be "selective" or "specific" for a particular cell or substance if it reacts or associates more frequently, more rapidly, for a greater duration and/or with greater affinity with that particular cell or substance than it does with alternative cells or substances. Thus, as used herein, the term "probe selective for phosphorylated Akt" refers to any molecule, compound, agent or moiety that reacts or associates more frequently, more rapidly, for a greater duration and/or with greater affinity with phosphorylated Akt than it does with non-target molecules {e.g., non-phosphorylated Akt, phosphorylated kinases or proteins other than phosphorylated Akt, and the like). A selective probe for phosphorylated Akt selectively recognizes and binds to phosphorylated Akt {e.g., under conditions of a binding assay). The term "recognizes and binds to" is meant to include detectable biochemical interactions between the probe and phosphorylated Akt, such as protein- protein interactions, protein-nucleic acid interactions, and protein-organic or inorganic compound interactions. Selective probes suitable for use in the methods of the present invention include, but are not limited to, biomolecules such as proteins, phospholipids, and DNA-hybridizing probes. In certain embodiments, selective probes are phospho- specific antibodies. The term "antibody" , as used herein, refers to any immunoglobulin, including antibodies {i.e., intact immunoglobulin molecules) and fragments thereof {i.e., active portions of immunoglobulin molecules), that binds to a specific epitope. The term encompasses monoclonal antibodies and antibody compositions with polyepitopic specificity {i.e., polyclonal antibodies). All derivatives and fragments thereof, which maintain specific binding ability, are also included in the term {e.g. , humanized antibodies, antibody binding fragments, recombinant antibodies, and the like). The term also covers any protein having a binding domain, which is homologous or largely homologous to an immunoglobulin-binding domain. These proteins may be derived from natural sources, or partly or wholly synthetically produced.

The term "phospho-specific antibody" (or "phosphorylation-speciflc antibody") refers to an antibody which selectively/specifically recognizes and binds to at least one phosphorylated residue of a protein of interest {e.g., Akt). Preferred phospho- specific antibodies selectively/specifically recognize and bind to one particular phosphorylated amino acid residue {e.g., phosphorylated serine at position 473 or phosphorylated threonine at position 308 in Akt). Phospho-specific antibodies and their methods of preparation are known in the art. Phospho-specific antibodies are also commercially available, for example, from New England Biolabs, Inc. (Beverly, MA, USA), BD Biosciences/Pharmingen (San Diego, CA, USA), Sigma-Genosys (The Woodlands, TX, USA), and Upstate Biologicals (Lake Placid, NY, USA).

The terms "labeled", "labeled with a detectable agent" and "labeled with a detectable moiety" are used herein interchangeably. These terms are used to specify that an entity {e.g., phosphorylated Akt) can be visualized, for example, following binding to another entity {e.g., a probe selective for phosphorylated Akt). Preferably, a detectable agent or moiety is selected such that it generates a signal which can be measured and whose intensity is related to the amount of bound entity. In array-based methods, a detectable agent or moiety is also preferably selected such that it generates a localized signal, thereby allowing spatial resolution of the signal from each spot of the array. Methods for labeling probes {e.g., proteins or polypeptides) are well known in the art. Labeled probes can be prepared by incorporation of or conjugation to a label that is directly or indirectly detectable by spectroscopic, chemical, biochemical, immunochemical, electrical, optical or chemical means, or any other suitable means. Suitable detectable agents include, but are not limited to, various ligands, radionuclides, fluorescent dyes, chemiluminescent agents, microparticles, enzymes, colorimetric labels, magnetic labels, and haptens.

The term "fluorescently detectable" , when applied to a probe, is used herein to specify (1) that the probe is conjugated or linked to a fluorescent label (for example, the probe may be a phospho-specific antibody attached to a fluorescent molecule) or (2) that the probe may be specifically recognized by a secondary probe that is conjugated or linked to a fluorescent label (for example, the probe may be a phospho- specific antibody which is specifically recognized by a secondary antibody that is attached to a fluorescent molecule). The terms "approximately" and "about', as used herein in reference to a number, generally include numbers that fall within a range of 10% in either direction of the number (greater than or less than the number) unless otherwise stated or otherwise evident from the concept (except where such number would exceed 100% of a possible value).

Detailed Description of Certain Preferred Embodiments

As mentioned above, the present invention relates to improved systems and strategies for the monitoring of anti-platelet therapies, in particular for the monitoring of anti-platelet agents that target platelet surface receptors.

I - Phosphorylated Akt as Biomarker for Monitoring Anti-Platelet Therapy The in vitro methods provided by the present invention comprise determining the activity of a surface receptor targeted by an anti-platelet agent by measuring the phosphorylation state of Akt in a sample comprising platelets obtained from a patient receiving the anti-platelet agent, as treatment.

Anti-Platelet Agents The methods of the invention may be used to monitor the effects of currently approved anti-platelet agents as well as of drugs that are being (or will be) developed and/or that are (or will be) under clinical investigation. The anti-platelet agent may be a naturally occurring or non-naturally occurring {i.e., synthetic or recombinant) molecule, such as a biological molecule (e.g., nucleic acid, polypeptide, protein, antibody), an organic or inorganic molecule {e.g., small molecule), or an extract made from biological materials, such as bacteria, plants, fungi, or animal (particularly mammalian, including human) cells or tissues. The anti-platelet agent may be a single molecule, a mixture of two or more molecules, or a complex of at least two molecules.

Prior to obtaining platelets from a patient, the anti-platelet agent may be administered to the patient by any suitable method known in the art. Examples of suitable routes include oral administration; parenteral administration, including intravenous, intramuscular, intraperitoneal and subcutaneous injections; transdermal administration; enteral administration, and the like. The dose of anti-platelet agent administered to the patient is generally determined by a physician. The methods of the present invention may be used to monitor the therapeutic efficacy of a wide variety of anti-platelet agents that interfere with thrombus formation. In particular, the methods described herein are useful for monitoring the efficacy of anti-platelet agents which target platelet surface receptors that play a role in platelet activation, platelet adhesion and/or platelet aggregation. Preferred anti- platelet agents are inhibitors or antagonists of platelet surface receptors whose signaling involves activation and phosphorylation of Akt.

In certain embodiments, the methods of the invention are used to characterize the response of a patient to an anti-platelet agent that acts as an inhibitor or antagonist of a platelet ADP receptor. Platelet activation by ADP is mediated by two G protein- coupled receptors, P2Yi and P2Yi₂. Since it is mainly present on blood platelets and does not significantly occur in other tissues, the P2Yi₂ receptor is considered as an ideal candidate for pharmacological approaches. Therefore, in certain preferred embodiments, the methods of the invention are used to characterize the response of a patient to an anti-platelet agent that acts as an inhibitor or antagonist of P2Yi₂. Known P2Yi₂ inhibitors and antagonists include, but are not limited to, thienopyridines such as ticlopidine (which is indicated to reduce the risk of thrombotic stroke in patients who have had a stroke or stroke precursors), clopidogrel (which is currently the treatment of choice, in combination with aspirin, for the prevention of stent thrombosis) and prasugrel (CS 747 - LY 640315, developed by Daiichi Sankyo Co. and currently under clinical development in cooperation with Eli Lilly and Co. for acute coronary syndromes planned for percutaneous coronary intervention). Other known P2Yi₂ inhibitors and antagonists include non-thienopyridines such as ticagrelor (AZD6140, produced by AstraZeneca, currently under clinical investigation for the prevention of thromboemolism) and cangrelor (AR C69931MX, from AstraZeneca), which, in contrast to clopidogrel and prasugel, both block P2Yi₂ in a reversible manner. In other embodiments, the methods of the invention are used to monitor the therapeutic efficacy of an anti-platelet agent that acts as an inhibitor or antagonist of platelet thrombin receptors. Thrombin is the main effector protease of the coagulation system and is among the most effective activators of platelets. Activation of platelets by thrombin is mediated by protease-activated receptors (PARs). Of the four identified PARs, PAR-I and PAR-4 are present on human platelets, with PAR-I proposed as the principal thrombin receptor. Therefore, in certain preferred embodiments, the methods of the invention are used to characterize the response of a patient to an anti-platelet agent that acts as an inhibitor or antagonist of PAR-I.

Two oral PAR-I antagonists, or thrombin receptor antagonists (TRAs), are currently in Phase II or Phase III investigation as anti-platelet agents: SCH 530348 (a compound based on the natural product himbacine and under development by

Schering Plough for the treatment and prevention of atherothrombotic events in patients) and E55555 (developed by Eisai Medical Research Inc. as a potential treatment for critical care acute coronary syndrome). Another specific PAR-I inhibitor is SCH 19191 , which was developed by Schering-Plough.

In still other embodiments, the methods of the invention are used to characterize the response of a patient to an anti-platelet agent that acts as an inhibitor or antagonist of platelet collagen receptors. A number of proteins have been implicated as binding collagen or acting as receptors on platelets. Among these, glycoprotein VI (GPVI) plays a critical role in the platelet response to collagen. GPVI is a member of the Ig superfamily. It is constitutively associated and expressed with the Fc receptor γ chain (FcRγ), an immunoreceptor tyrosine-based motif-bearing (ITAM) receptor. Upon GPVI engagement, phosphorylation of the ITAM motif of FcRγ initiates a signaling cascade, leading to platelet activation. Therefore, in certain preferred embodiments, the methods of the present invention are used to monitor the therapeutic effects of an anti-platelet agent that acts as an inhibitor or antagonist of GPVI. Examples of GPVI inhibitors or antagonists include, but are not limited to, GPVI-neutralizing human antibodies derived from a combinatorial phage display library of single-chain antibodies developed by Millennium Pharmaceuticals, Inc (Qian et al, Hum. Antibodies, 2002, 11: 97-105). In other embodiments, the methods of the invention are used to monitor the effects in a patient of an anti-platelet agent that acts as an inhibitor or antagonist of platelet receptors of the von Willebrand factor. The GPIb complex plays an important role in the first steps of platelet adhesion and arterial thrombus formation. GPIb-IX-V ligands also include alpha-thrombin, clotting factors XI/XIIa, and high-molecular- weight kininogen. Interactions involving GPIb-IX-V are therefore central to vascular processes of thrombosis and inflammation. Recent animal studies have supported inhibition of PGIb as a good candidate for anti-thrombotic drug development (Clemetson et al, Thromb. Haemost, 2008, 99: 473-479). Therefore, in certain preferred embodiments, the methods of the present invention are used to characterize the response of a patient to an anti-platelet agent that acts as an inhibitor or antagonist of GPIb-IX-V.

Examples of GPIb-IX-V inhibitors or antagonists include, but are not limited to, human monoclonal antibodies that compete with von Willebrand factor for binding to human platelets (Hagay et al, MoI. Immunol, 2006, 43: 443-453; Perrault et al, Thromb. Haemost., 2001, 86: 1238-1248); peptide antagonists to GPIb (Benard et al, Biochemistry, 2008, 47: 4674-4682); and snake venom proteins (Kanaji et al, J. Biol. Chem., 2003, 278: 39452-39460).

In other embodiments, the methods of the invention are used to characterize the response of a patient to an anti-platelet agent that acts as an inhibitor or antagonist of platelet thromboxane A2 (TxA₂) receptors. TxA₂, which is the major COX-I (cyclooxygenase) product of arachidonic acid metabolism in platelets, is considered as one of the most powerful agonists for platelet activation and thus, thrombus formation. The TxA₂ receptor (TP) is also activated by prostaglandin endoperoxides and isoprostanes, prostaglandin-like compounds. Thus, in certain preferred embodiments, the methods of the invention are used to monitor the effects of an antiplatelet agent that acts as an inhibitor or antagonist of TP. Examples of known TP inhibitors or antagonists include, but are not limited to, terutroban also known as S 18886 developed by Servier Laboratories (Husted, Eur. Heart J. Suppl., 2007, 9: D20-27). Terutroban is currently in Phase II clinical trial for the secondary prevention of acute thrombotic complications. Other examples of known TP inhibitors or antagonists that inhibit platelet aggregation include prostaglandin analogues such as beraprost, prostacyclin, iloprost and treprostinil.

In still other embodiments, the methods of the invention are used to monitor the effects in a patient of an anti-platelet agent that acts as an inhibitor or antagonist of platelet integrin receptors. A major downstream consequence of engagement of primary platelet adhesive receptors such as GPIb-IX-V or GPVI is the rapid activation of platelet integrins. Platelet integrins include, in particular, GPIIb/IIIa (or αllbβ3), which binds fibrinogen and von Willebrand factor and is critically involved in stable thrombus formation at high shear stress. Thus, in certain preferred embodiments, the methods of the present invention are used to characterize the response of a patient to an anti-platelet agent that acts as an inhibitor or antagonist of GPIIb/IIIa.

Examples of known GPIIb/IIIa inhibitors or antagonists include, but are not limited to, humanized blocking antibody abciximab (manufactured by Centocor and

(S) distributed by Eli Lilly under the trade name ReoPro ); eptifibatide (a cyclic heptapeptide developed and commercialized by Millennium Pharmaceuticals, Inc. under the trade name Integrilin^®); and non-peptide tirofiban (Aggrastat^®, Medicure Pharma).

In yet other embodiments, the methods of the invention are used to characterize the response of a patient to an anti-platelet agent that acts as an inhibitor or antagonist of platelet lectin receptors, in particular C-type lectin receptors, such as CLEC-2. CLEC-2 has recently been shown to function as a receptor for the snake venom toxin rhodocytin (also known as aggretin), which elicits powerful platelet activation (Fuller et al, J. Biol. Chem., 2007, 282: 12397-12409).

Preparation of Samples containing Platelets

Samples comprising platelets to be used in the methods of the invention can be obtained directly or indirectly {e.g., by a healthcare provider) and can be prepared by any method suitable for the particular sample {e.g., whole blood, platelet rich plasma, isolated platelets) and assay format selected. Most commonly, platelets are obtained either as a component of a whole blood unit or via plateletpheresis (i.e., by withdrawing only platelets from a donor and re-infusing the remaining of the blood back into the donor).

In certain embodiments, the sample comprising platelets used in the methods of the invention is whole blood. Whole blood can be collected by any suitable method known in the art, for example, by venipuncture into a container containing an anticoagulant such as heparin, ACD-A (anticoagulant citrate dextrose), or EDTA, or from an in-dwelling arterial line into such a container. In other embodiments, the sample comprising platelets used in the methods of the invention is platelet-rich plasma (PRP). PRP can be obtained by any suitable method known in the art. For example, a sample of blood may be centrifuged, leading to the formation of three layers: the inferior layer composed of red blood cells, the intermediate layer composed of white cells and the superior layer made up of plasma. The plasma layer is collected and centrifuged to obtain a two-part plasma: the upper part consisting of platelet-poor plasma (PPP) and the lower part consisting of platelet-rich plasma (PRP). Alternatively, the monitoring methods of the present invention may be performed on isolated platelets. As mentioned above, platelets may be obtained by plateletpheresis or isolated from a blood sample. For example, platelets may be obtained by differential centrifugation of a whole blood sample, as described in Example 1. One or more washing steps may be included in the preparation of isolated platelets (see Example 1).

In certain embodiments, the sample to be used in an assay of the present invention comprises a pre-determined number of platelets. Adjusting the number of platelets in a sample can easily be performed by one skilled in the art. In certain embodiments, prior to performing an assay according to the invention, the platelets obtained from the patient are treated so as to retain their endogeneous (in vivo) activation level. Such treatment may include addition of an activation inhibitor well known in the art such as aprotinin, theophylline, apyrase and/or prostaglandin El.

In certain embodiments, the methods of the invention include a step of fixing the platelets. This step is performed to preserve or "freeze" a cell in a certain state, preferably so that an accurate representation of the structure of the cell is maintained.

For example, it is often desirable to maintain the cell's original size and shape, to minimize loss of cellular materials, and/or to retain the reactivity and/or status of its intracellular constituents (for example, the cell's phosphorylation level). Platelets may be fixed by any of a variety of suitable chemical and physical methods. Methods of cell fixation typically rely on crosslinking and/or rapid dehydration agents, such as formaldehyde, paraformaldehyde, glutaraldehyde, acetic acid, methanol, ethanol, and acetone. Platelets are preferably incubated in the presence of the fixing agent at a certain temperature (for example at room temperature, i.e., between 18°C and 25°C) and for a certain period of time (for example between 5 and 10 minutes). Excess fixing agent may be removed after centrifugation by aspiration of the supernatant. In certain embodiments, the step of fixing the platelets is followed by a step of permeabilization of the platelets. Permeabilization is performed to facilitate access to cellular cytoplasm or intracellular molecules, components or structures of a cell. In particular, permeabilization may allow an agent (such as a phospho- selective antibody) to enter into a platelet and reach a concentration within the platelet that is greater than that which would normally penetrate into the platelet in the absence of such permeabilizing treatment. Permeabilization of the platelets may be performed by any suitable method including, but not limited to, exposure to a detergent or to an organic alcohol (such as methanol or ethanol). Selection of an appropriate permeabilizing agent and optimization of the incubation conditions and time can easily be performed by one of ordinary skill in the art.

Stimulation of Platelets

The methods of the present invention generally include a step of stimulating the platelets of the sample prior to measuring the phosphorylation state of Akt. In preferred embodiments, the platelet stimulator used is an agonist of the receptor that is targeted by the anti-platelet agent whose efficacy is being monitored. Thus, if the anti-platelet agent is an inhibitor or antagonist of a platelet ADP receptor (e.g., P2Y₁₂), the platelet stimulator will preferably be ADP. If the anti-platelet agent is an inhibitor or antagonist of a platelet thrombin receptor (e.g., PAR-I), the platelet stimulator will preferably be thrombin or a thrombin receptor activating peptide (TRAP). If the anti-platelet agent is an inhibitor or antagonist of a platelet collagen receptor (e.g., GPVI), the platelet stimulator will preferably be collagen, CRP (collagen related peptide), or convulxin. If the anti-platelet agent is an inhibitor or antagonist of a platelet von Willebrand factor receptor (e.g., GPIb), the platelet stimulator will preferably be the von Willebrand factor under flow conditions. If the anti-platelet agent is an inhibitor or antagonist of a platelet thromboxane A2 receptor (e.g., TP), the platelet stimulator will preferably by TxA₂ or its analogue U46619. If the anti-platelet agent is an inhibitor or antagonist of a platelet integrin receptor such as GPIIb/IIIa, the platelet stimulator will preferably be fibrinogen under adhesion conditions.

The stimulation is preferably carried out under conditions suitable for activation of platelets in the sample and for a period of time effective to efficiently activate said platelets. Conditions include, for example, quantity of platelet stimulator and incubation conditions such as medium, temperature, and the like. One skilled in the art will know (or will know how to determine) such conditions and period of time.

In certain embodiments, for control purposes, the sample containing platelets obtained from the patient is divided into aliquots (or portions), and platelets in one aliquot are stimulated while platelets in another aliquot are not stimulated (referred to as "resting platelets"). Thus, in the second aliquoted sample, platelets are maintained at the endogenous platelet activation level. The phosphorylation state of Akt is then measured in both aliquots and the results obtained may be compared.

If desired, additional controls may be performed. For example, prior to stimulating the platelets of a sample, an aliquot of the sample may be contacted with a positive control, i.e., a compound or agent that is known to be a specific inhibitor or antagonist of the platelet surface receptor targeted by the anti-platelet agent whose efficacy is being monitored. The platelets of this aliquot are then stimulated and the phosphorylation state of Akt is then measured.

Probes Selective for Phosphorylated Akt In the assays of the present invention, measuring the phosphorylation state of

Akt in platelets obtained from a patient receiving an anti-platelet agent may be performed using any suitable method and/or any suitable means. In certain preferred embodiments, measuring the phosphorylation state of Akt involves using a probe selective for phosphorylated Akt. A selective probe may be any molecule, compound, agent or moiety that reacts or associates more frequently, more rapidly, for a greater duration and/or with greater affinity with phosphorylated Akt than it does with non-target molecules. The affinity for phosphorylated Akt may be governed by non-covalent interactions (e.g. , ionic or electrostatic interactions, hydrophobic interactions, hydrogen bonds, Van der Waals forces, dipole-dipole intermolecule forces) or by covalent bonding. A wide variety of selective probes may be used in the practice of the present invention including, but not limited to, biomolecules such as proteins, phospholipids, and nucleic acid probes. Due to their high degree of specificity for binding to a single molecule target in a mixture of molecules as complex as a cell or cell lysate, preferred probes are antibodies, preferably phospho-specific antibodies.

A phospho-specific antibody suitable for use in the practice of the present invention specifically recognizes and binds to an amino acid residue of Akt when this amino acid residue is phosphorylated. In certain preferred embodiments, the phospho- specific antibody specifically recognizes and binds to threonine 308 (Thr³⁰⁸) or to serine 473 (Ser⁴⁷³) of Akt when Thr³⁰⁸ or Ser⁴⁷³ is phosphorylated. Phospho-specific antibodies may be polyclonal or monoclonal. The methods of the invention are not limited to the use of whole antibodies (i.e., intact immunoglobulin molecules), but include fragments thereof (e.g., active portions of immunoglobulin molecules), derivatives thereof, and equivalent molecules, such as protein binding domains or nucleic acid aptamers, which bind, in a phospho-specific manner, to essentially the same phosphorylated epitope to which the particular phospho-specific antibodies bind. Phospho-specific antibodies for use in the practice of the assay methods of the invention may be produced using methods known in the art or, alternatively, may be purchased from different commercial sources (see below). As will be appreciated by one of ordinary skill in the art, any type of antibody can be generated and/or modified to specifically recognize and bind to an epitope of phosphorylated Akt at one or more serine or threonine residues.

Methods for producing custom polyclonal antibodies are well known in the art and include standard procedures such as immunization of rabbits or mice with pure protein or peptide (see, for example, Mage and Lamoyi in "Monoclonal Antibody Production Techniques and Applications" ^', 1987, Marcel Dekker, Inc., pp. 79-97). Monoclonal antibodies that specifically bind to phosphorylated Akt may be prepared using any technique that provides for the production of antibody molecules by continuous cell lines in culture. These techniques include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV- hybridoma technique (see, for example, Kohler and Milstein, Nature, 1975, 256: 495- 497; Kozbor et ai, J. Immunol. Methods, 1985, 81: 31-42; and Cote et al., Proc. Natl. Acad. Sci. 1983, 80: 2026-2030). Monoclonal antibodies may also be made by recombinant DNA methods (see, for example, U.S. Pat. No. 4,816,567). Other methods have been reported and can be employed to produce monoclonal antibodies for use in the practice of the assay methods of the invention (see, for example, Lerner, Nature, 1982, 299: 593-596; Nairn et al, Nature, 1982, 299: 734-736; Czernik et al, Methods Enzymol. 1991, 201: 264-283; Czernik et al, Neuromethods: Regulatory Protein Modification: Techniques & Protocols, 1997, 30: 219-250; Czernik et al, Neuroprotocols, 1995, 6: 56-61; and Zhang et al, J. Biol. Chem. 2002, 277: 39379- 39387).

A biologically active fragment or portion of a phospho- specific monoclonal antibody may be a Fab fragment or portion, a F(ab')₂ fragment or portion, a variable domain, or one or more CDRs (complementary determining regions) of the antibody. Alternatively, a biologically active fragment or portion may be derived from the carboxyl portion or terminus of the antibody protein and may comprise an Fc fragment, an Fd fragment or an Fv fragment. Antibody fragments may be produced by any suitable method known in the art including, but not limited to, enzymatic cleavage (e.g., proteolytic digestion of intact antibodies) or by synthetic or recombinant techniques. F(ab')₂, Fab, Fv and ScFv (single chain Fv) antibody fragments can, for example, be expressed in and secreted from mammalian host cells or from E. coli. Antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons have been introduced upstream of the natural stop site. The various portions of antibodies can be joined together chemically by conventional techniques, or can be prepared as a contiguous protein using genetic engineering techniques.

Techniques developed for the production of chimeric antibodies, the slicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate specificity and biological activity, can, alternatively, be used in the preparation of antibodies (Morrison et al, Proc. Natl. Acad. Sci., 1984, 81: 6851-6855; Neuberger et al, Nature, 1984, 312: 604-608; Takeda et al, Nature, 1985, 314: 452-454). Monoclonal and other antibodies can also be "humanized"; sequence differences between rodent antibodies and human sequences can be minimized by replacing residues which differ from those in the human sequences by site-directed mutagenesis of individual residues or by grafting of entire complementarity determining regions (CFRs). Humanized antibodies can also be produced using recombinant methods (see, for example, GB 2 188 638 B).

Antibodies to be used in the monitoring assays of the invention can be purified by methods well known in the art (see, for example, Minden, "Monoclonal Antibody Purification" , 1996, IBC Biomedical Library Series: Southbridge, MA). For example, antibodies can be affinity-purified by passage over a column to which a phosphorylated Akt molecule is bound. The bound antibodies can then be eluted from the column using a buffer with a high salt concentration.

Instead of being prepared, phospho- specific antibodies may be purchased, for example, from Cell Signaling (Beverly, MA), BD Biosciences/Pharmingen (San Diego, CA); Upstate Biologicals, Inc. (Lake Placid, NY), Bethyl Laboratories, Inc. (Montgomery, TX), Alexis Biochemicals (San Diego, CA), Sigma-Genosys (The Woodlands, TX), Affinity BioReagents, Inc. (Golden, CO), New England Biolabs, Inc. (Beverly, MA), Covance Research Products, Inc. (Berkeley, CA), and Stressgen Biotechnologies Corp. (Victoria, BC, Canada).

In embodiments where a probe selective for phosphorylated Akt is used, the assay methods of the invention comprise a step of contacting the sample comprising platelets with the selective probe under conditions and for a time allowing reaction or association (e.g., binding) of the selective probe with any phosphorylated Akt present in the platelets. One skilled in the art will know the conditions and reaction time to be used or will know how to determine them. The amount of selective probe to be added to the sample will depend on its avidity (affinity) for phosphorylated Akt and on the number of platelets present in the sample. Such amount can easily be determined by one of ordinary skill in the art.

In certain embodiments, a monitoring method of the invention includes the use of more than one probe selective for phosphorylated Akt {e.g., two or more probes selective for phosphorylated Akt). For example, in certain embodiments, an antibody against threnonine 308 of phosphorylated Akt and an antibody against serine 473 of phosphorylated Akt may be used in the same assay. This allows the determination of the ratio of phosphorylation of Akt at two different positions. Alternatively or additionally, a monitoring method of the invention includes the use of one probe selective for Akt (i.e., selective for non-phosphorylated Akt). Thus, in certain embodiments, an antibody against threonine 308 of phosphorylated Akt, an antibody against serine 473 of phosphorylated Akt and an antibody against Akt are used in the same assay. This allows the determination of the ratio of phosphorylated Akt and non-phosphorylated Akt.

Detectable Selective Probes

In preferred embodiments of the present invention, the probe selective for phosphorylated Akt is detectable. The probe may be directly detectable (e.g., it may be attached to a detectable label or moiety) or indirectly detectable (e.g. , it may be specifically recognized by a secondary probe that is attached to a detectable label or moiety).

Detectable labels and moieties suitable for use in the practice of the present invention may be any agents which allow for the detection and visualization of a probe after reaction or association with (e.g., binding to) phosphorylated Akt. Suitable detectable labels and moieties include, but are not limited to, labels and moieties detectable by spectroscopic, photochemical, electrical, optical or chemical means. Useful labels and moieties in the present invention include, for example, biotin staining with labeled streptavidin conjugate, magnetic beads (e.g., Dynabeads™), fluorescent dyes (see below), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc). Methods for labeling a probe (or secondary probe) with a detectable moiety are well known in the art (see, for example, "Affinity Techniques. Enzyme Purification: Part B", Methods in Enzymol., 1974, Vol. 34, Jakoby and Wilneck (Eds.), Academic Press: New York, NY; and Wilchek and Bayer, Anal. Biochem., 1988, 171: 1-32). The association between the probe (or secondary probe) and the detectable moiety can be covalent or non-covalent. Preferably, the association is covalent. More preferably, in order to permit quantitative studies, a defined number of detectable moieties are covalently attached to a molecule of probe (e.g., one detectable moiety per probe). Detectable moieties can be attached to a probe (or secondary probe) either directly or indirectly through a linker. Linkers or spacer arms of various lengths are known in the art and are commercially available. Such linkers can, for example, be selected to reduce steric hindrance. Preferably, attachment of a detectable moiety to a probe (or secondary probe) does not significantly affect the specific affinity of the probe.

In certain embodiments, the selective probe (or secondary probe) is fluorescently labeled, i.e., it is attached to a fluorescent labeling agent. Favorable optical properties of fluorescent labeling agents to be used in the practice of the invention include high molar absorption coefficient, high fluorescence quantum yield, and photostability. In certain embodiments, preferred fluorescent dyes exhibit absorption and emission wavelengths in the visible {i.e., between 400 and 700 nm) or the near infra-red {i.e., between 700 and 950 nm) rather than in the ultraviolet range {i.e., below 400 nm) of the spectrum. Selection of a particular fluorescent label will be governed by the nature and characteristics of the illumination and detection systems of the apparatus used for measuring the phosphorylation state of Akt. For example, if a flow cytometry system is used in the assay, a suitable fluorescent label is one that can be efficiently excited by the light beam of the system and whose emission can be efficiently detected by its detector.

Numerous fluorescent labels of a wide variety of structures and characteristics are suitable for use in the practice of the present invention. Suitable fluorescent labels include, but are not limited to, quantum dots {i.e., fluorescent inorganic semiconductor nanocrystals) and fluorescent dyes such as Texas red, fluorescein isothiocyanate

(FITC), phycoerythrin (PE), rhodamine, fluorescein, carbocyanine, Cy-3 and Cy-5

{i.e., 3- and S-N^N'-diethyltetramethylindodicarbocyanine, respectively), mero- cyanine, styryl dye, oxonol dye, BODIPY dye {i.e., boron dipyrromethene difluoride fluorophore), and analogues or derivatives of these molecules.

Methods for fluorescently-labeling probes {e.g., antibodies) are well-known in the art. Fluorescent dyes are usually commercially available as NHS-esters, maleimides, and hydrazides to make them suitable for labeling via reaction with different chemical groups such as amine, thiol and aldehyde groups, respectively. Fluorescent labeling dyes as well as labeling kits are commercially available from, for example, Amersham Biosciences Inc. (Piscataway, NJ), Molecular Probes Inc. (Eugene, OR), Prozyme, Inc. (San Leandro, CA) and New England Biolabs Inc. (Berverly, MA).

Alternatively, fluorescently-labeled phospho-specific antibodies may be purchased from, for example, BD Biosciences/Pharmingen (San Diego, CA) and AnaSpec (San Jose, CA). Fluorescently-labeled secondary antibodies are also commercially available, for example, from Santa Cruz Biotechnology (Santa Cruz, CA), Jackson ImmunoResearch Labs Inc. (West Grove, PA), and Rockland Immunochemicals Inc. (Gilbertsville, PA).

Selection of a particular fluorescent label and/or labeling technique will depend on the situation and will be governed by several factors, such as the ease and cost of the labeling method, the quality of labeling desired, the effects of the fluorescence label on the affinity of the probe {e.g., on the rate and/or efficiency of the binding process), the nature of the illumination and detection systems to be used, the nature and intensity of the signal generated by the fluorescent label, and the like. When two probes (e.g., one antibody against threonine 308 of phosphorylated

Akt and one antibody against serine 473 of phosphorylated Akt) are used in a monitoring method of the invention, the first probe is attached to a first detectable moiety and the second probe is attached to a second detectable moiety. Alternatively, the first probe is recognized by a first secondary probe that is attached to a first detectable moiety and the second probe is recognized by a second secondary probe that is attached to a second detectable moiety. In such embodiments, the probes (or secondary probes) are preferably differentially labeled, i.e., the first and second detectable moieties exhibit distinct detectable properties and produce distinguishable signals. For example, if the first and second detectable moieties are fluorescent labels, the first and second fluorescent labels preferably produce a dual-color fluorescence upon excitation. Examples of matched pairs of fluorescent dyes include, but are not limited to, Cy-3™ and Cy-5™; Spectrum Red™ and Spectrum Green™; and rhodamine and fluorescein.

Determination of Phosphorylation State of Akt The methods of the present invention comprise a step of determining the phosphorylation state of Akt. Determination of the phosphorylation state of Akt can be carried out using any of a variety of methods well known in the art, including, but not limited to, Western blot, immunoblot, enzyme-linked immunosorbant assay (ELISA), radioimmunoassay (RIA), immunoprecipitation, surface Plasmon resonance, immunohistochemistry (IHC), matrix-assisted laser desorption/ionization time-of- flight (MALDI-TOF) mass spectrometry, microcytometry, microscopy, fluorescence activated cell sorting (FACS), and flow cytometry (FC). The choice of the method will depend on the situation (e.g., extent of phosphorylation of Akt, number of samples to be analyzed, manual or automatic analysis, etc ..) and/or the setting (e.g., clinical laboratory or research laboratory).

Immunoassay formats and variations thereof which may be useful for carrying out the methods disclosed herein are well known in the art (see, for example, Tijssen,

In: Practice and theory of enzyme immunoassays, eds. Burdon and v. Knippenberg,

Elsevier, Amsterdam (1990), pp. 221-278 and various volumes of Methods in

Enzymology, Eds. Colowick et al.., Academic Press, dealing with immunological detection methods). Any of a number of variations of the sandwich assay technique may be used to perform an immunoassay. The most commonly used detectable moieties in immunoassays are enzymes (e.g., horseradish peroxidase, glucose oxidase, beta-galactosidase, alkaline phosphatase) and fluorophores.

Alternatively, the phosphorylation state of Akt may be determined in an ELISA or reverse-phase array format. For the ELISA format, the phospho-specific antibody (or capture antibody) is affixed to a solid substrate such as a plastic ELISA plate, nitrocellulose membrane or bead. The lysate obtained from the patient is incubated with the substrate allowing for the capture of phosphorylated Akt to the substrate via the capture antibody. The substrate is then washed, and the captured phosphorylated Akt is then detected using a second antibody specific for phosphorylated Akt. The bound detection antibody may be detected by a labeled secondary antibody or by labeling (fluorescent or enzyme) the detection antibody.

Alternatively, phospho-specific antibodies may be optimized for use in other clinically- suitable applications, for example, bead-based multiplex-type assays, such as IGEN, Luminex and/or Bioplex assay formats, or otherwise optimized for antibody arrays formats.

Methods like immunohistochemistry and flow cytometry are well-known and accepted clinical procedures, and thus are highly-desirable assay formats for clinical and monitoring assays. They have the ability to rapidly analyze multiple samples in parallel.

Flow cytometry is a sensitive and quantitative technique that analyzes particles (such as cells) in a fluid medium based on the particle's optical characteristics (for background information on flow cytometry, see, for example, Shapiro, "Practical Flow Cytometry", 3^rd Ed., 1995, Alan R. Liss, Inc.; and "Flow Cytometry and Sorting, Second Edition", Melamed et al. (Eds), 1990, Wiley-Liss: New York, which are incorporated herein by reference in their entirety). The fundamental concept of flow cytometry is simple. A flow cytometer hydrodynamically focuses a fluid suspension of particles which have been attached to one or more fluorophores, into a thin stream so that the particles flow down the stream in a substantially single file and pass through an examination or analysis zone. A focused light beam, such as a laser beam, illuminates the particles as they flow through the examination zone. Optical detectors within the flow cytometer measure certain characteristics of the light as it interacts with the particles. Light interaction with the particles is generally measured as light scatter and particle fluorescence at one or more wavelengths.

Fluorescence measurements allow one to determine, with high accuracy, relative quantities of a variety of cell constituents simultaneously. Furthermore, when the measurements are recorded in a list mode, it is possible to attribute each of these features on a cell-by-cell basis. In particular, standard flow cytometry has been shown to provide a rapid and efficient way to measure kinase activity and study kinase cascades in individual cells (see, for example, Krutzik et al., Cytometry, 2003, 55A: 61-70; Hickerson et al, Hematol. Oncol. Clin. North Am. 2002, 16: 421-454; Perez et al, Nature Biotechnology, 2002, 20: 155-162; Chow et al, Cytometry, 2001, 46: 72- 78; Uzel et al, Clin. Immunol. 2001, 100: 270-276; Lund-Johansen et al, Cytometry, 2000, 39: 250-259; Maino et al, Cytometry, 1998, 34: 207-215; Prussia, J. Clin. Immunol. 1997, 17: 195-204; and Hubert et al , Cytometry, 1997, 29: 83-91).

Briefly and by way of example, the following protocol for cytometric analysis (or a variation thereof) may be employed. Cells are fixed with 1% paraformaldehyde for 15 minutes at 37°C followed by permeabilization in 90% methanol for 30 minutes on ice. Cells may then be stained with a phospho- specific antibody or antibodies, washed and labeled with a fluorescently-labeled secondary antibody (or antibodies). Alternatively, the cells may be stained with a fluorescently-labeled primary antibody (or antibodies). The cells may then be analyzed using a flow cytometer (e.g., a Beckman Coulter FC 500) according to the specific protocols of the instrument used.

The results of a monitoring assay of the present invention may be presented under any suitable form, as long as the form selected provides information about the effect or efficacy of the anti-platelet therapy administered to the patient being tested. For example, if flow cytometry is used, the results may be (or may be derived from) fluorescence measurements. In many embodiments, the phosphorylation state of Akt measured in platelets stimulated by an agonist of the platelet surface receptor targeted by the anti-platelet agent administered to the patient from whom the platelets have been obtained is compared to the phosphorylation state of Akt measured in platelets (obtained from the same patient) that were not submitted to stimulation with the agonist. Using these values, the results of a monitoring assay of the invention may be presented as a percentage or an index of Akt phosphorylation (or as a percentage or an index of reduction in Akt phosphorylation). Alternatively or additionally, the phosphorylation state of Akt measured in a sample of stimulated platelets and in a sample of non-stimulated platelets can be normalized to the amount of non- phosphorylated Akt in each of the samples.

In certain embodiments, the methods of the invention further comprise a step of evaluating the efficacy of the anti-platelet agent in the patient based on the level of Akt phosphorylation determined. In general, an inefficient blockade of a platelet surface receptor by an antagonist anti-platelet agent will result in a higher Akt phosphorylation level in stimulated platelets than an efficient blockade of the platelet surface receptor.

II - Utilization of Monitoring Methods As already mentioned above, the methods of the present invention may be employed to monitor the efficacy of any of a variety of anti-platelet agents. Using the methods described herein, skilled physicians may individualize anti-platelet therapy. More specifically, based on the results obtained in a monitoring assay of the invention, a physician may adjust or optimize the dose of anti-platelet agent administered to the patient tested in order to achieve therapeutic efficacy and/or to reduce side effects. Alternatively, based on the results obtained in a monitoring assay of the invention, a physician may decide to stop administration of the anti-platelet agent to which the patient shows resistance.

Accordingly, in certain embodiments, the methods of the invention further comprise a step of increasing the dose of anti-platelet agent administered to the patient if the level of Akt phosphorylation detected is higher than a desirable value. In other embodiments, the methods of the invention further comprise a step of decreasing the dose of anti-platelet agent administered to the patient if the level of Akt phosphorylation detected is lower than a desirable value. In yet other embodiments, the methods of the invention further comprise a step of stopping administration of the anti-platelet agent to the patient if the level of Akt phosphorylation detected is such that it indicates resistance to the anti-platelet agent. As methods of the invention will be used, physicians will be able to quantitatively correlate anti-platelet agent efficiency with Akt phosphorylation levels and therefore identify a "desirable value" of Akt phosphorylation level for a given anti-platelet agent. Similarly, physicians will be able to identify Akt phosphorylation levels that are indicative of resistance to a given anti-platelet agent.

Selection of an appropriate therapeutic regimen for a given patient may be made based solely on the results of a monitoring assay of the invention. Alternatively, the physician may also consider other clinical or pathological parameters and/or take into account results from other tests such as platelet function tests.

Ill - Kits

In another aspect, the present invention provides kits comprising materials useful for monitoring anti-platelet therapy according to the invention. The monitoring procedures described herein may be performed by diagnostic or analysis laboratories, experimental laboratories, or practitioners. The invention provides kits which can be used in these different settings.

Materials and reagents for characterizing biological samples from a patient and/or monitoring the efficiency of an anti-platelet agent in a patient according to the inventive methods may be assembled together in a kit. In certain embodiments, an inventive kit comprises at least one reagent that specifically detects the phosphorylation state of Akt. For example, a kit may comprise at least one probe selective for phosphorylated Akt, preferably a detectable probe selective for phosphorylated Akt as described above (e.g., a detectable phospho-specific antibody against phosphorylated Akt or a phospho-specific antibody against phosphorylated Akt and a secondary labeled antibody specific for the phospho-specific antibody). For example, a kit may comprise an antibody against threnonine 308 of phosphorylated Akt (i.e., phospho-specific T308 antibody) or an antibody against serine 473 of phosphorylated Akt (i.e., phospho-specific S473 antibody). In certain embodiments, a kit may comprise more than one probe selective for phosphorylated Akt. For example, a kit may comprise a phospho-specific T308 antibody and a phospho- specific S473 antibody. In certain embodiments, a kit further comprises a probe selective for Akt

(i.e., non-phosphorylated Akt). For example, a kit may comprise a phospho-specific T308 antibody and an antibody selective against Akt; or a phospho-specific S473 antibody and an antibody selective against Akt; or a phospho-specific T308 antibody, a phospho-specific S473 antibody and an antibody selective against Akt. These phospho-specific antibodies will either be labeled or the kit will also comprise secondary labeled antibodies against the phospho-specific antibodies.

In certain embodiments, a kit according to the invention further comprises a platelet activation inhibitor. For example, a kit may comprise one or more of aprotinin, theophylline, apyrase and prostaglandin El. In certain embodiments, a kit according to the invention further comprises at least one platelet stimulator. Preferably, the platelet stimulator is an agonist of the receptor that is targeted by the anti-platelet agent whose efficacy is to be monitored using the kit. Suitable platelet stimulators include ADP, if the anti-platelet agent to be monitored using the kit is an antagonist of a platelet ADP receptor (e.g., P2Y₁₂); thrombin or a thrombin receptor activating peptide (TRAP) if the anti-platelet agent to be monitored is an antagonist of a platelet thrombin receptor (e.g., PAR-I); collagen, CRP (collagen related peptide or convulxin if the anti-platelet agent to be monitored is an antagonist of a platelet collagen receptor (e.g., GPVI); von Willebrand factor under flow conditions if the anti-platelet agent to be monitored is an antagonist of a platelet von Willebrand factor receptor (e.g., GPIb); TxA₂ or its analogue U46619 if the antiplatelet agent to be monitored is an antagonist of a platelet thromboxane A2 receptor (e.g., TP); and fibrinogen under adhesion conditions if the anti-platelet to be monitored is an antagonist of an integrin receptor such as GPIIb/IIIa. Kits of the present invention may be designed for the monitoring of only one type of anti-platelet agents (e.g., antagonists of platelet ADP receptors, or antagonists of platelet thrombin receptors). In such embodiments, the kit will comprise a platelet stimulator that is an agonist of the receptor that is targeted by the particular type of anti-platelet agents. Alternatively, kits of the present invention may be designed for the monitoring of several types of anti-platelet agents. In such embodiments, the kit will comprise more than one platelet stimulator.

Depending on the procedure, the kit may further comprise one or more of: extraction buffer and/or reagents, fixing buffer and/or reagents, permeabilization buffer and/or reagent, lysis buffer and/or reagent, immunodetection buffer and/or reagents, labeling buffer and/or reagents, and detection means. Protocols for using these buffers and reagents for performing different steps of the procedure may be included in the kit.

Kits of the invention may further comprise negative and/or positive controls, blocking agents, protein stabilizing agents, and the like.

The reagents may be supplied in a solid (e.g., lyophilized) or liquid form. The kits of the present invention may comprise different containers (e.g., vials, ampoules, test tubes, flasks or bottles) for each individual buffer and/or reagent. Each component will generally be suitable as aliquoted in its respective container or provided in a concentrated form. Other containers suitable for conducting certain steps of the disclosed methods may also be provided. The individual containers of the kit are preferably maintained in close confinement for commercial sale.

In certain embodiments, the kit further comprises instructions for using the kit according to a method of the invention. Such instructions may comprise instructions for processing the biological sample obtained from the patient and/or for performing the test, instructions for interpreting the results as well as a notice in the form prescribed by a governmental agency (e.g., FDA) regulating the manufacture, use or sale of pharmaceuticals or biological products.

Examples The following examples describe some of the preferred modes of making and practicing the present invention. However, it should be understood that the examples are for illustrative purposes only and are not meant to limit the scope of the invention. Furthermore, unless the description in an Example is presented in the past tense, the text, like the rest of the specification, is not intended to suggest that experiments were actually performed or data were actually obtained. Some of the results reported below are described in a poster entitled

"Assessment of platelet response to clopidogrel through measurement of ADP- induced Akt phosphorylation" presented at the 5 International Meeting ADP 2008 Platelet P2 Receptors in June 19-21, 2008 in Bornio (Italy) and in a manuscript entitled "Assessment of platelet response to clopidogrel through measurement of ADP-induced Akt phosphorylation" by D. Carrie et al This manuscript has been published in the Journal of Thrombosis and Haemostasis (2009, 7: 1411-1413).

Example 1: Assessment of Platelet Response to Clopidogrel through Measurement of ADP-induced Akt Phosphorylation

Introduction Clopidogrel, the precursor of a potent inhibitor of the purinergic adenosine diphosphate (ADP) receptor P2Yi₂, is currently used, in combination with aspirin, to prevent thrombosis after coronary stent implantation (Mehta et al, Lancet, 2001, 358: 527-533). Variability in the degree of platelet inhibition by clopidogrel is well documented (Gurbel et al, Circulation, 2003, 107: 2908-2913; Jaremo et al, J. Intern. Med., 2002, 252: 233-238; Muller et al, Thromb. Haemostasis, 2003, 89: 783-787; Grossmann et al, Thromb. Haemost, 2004, 92: 1201-1206). A significant proportion of patients with coronary artery disease have high on-treatment residual platelet activity, mainly due to inefficient metabolism of the drug. There is increasing evidence that patients with inadequate response to clopidrogrel are at higher risk for the development of cardiovascular events.

Platelet response to clopidogrel was first defined on the basis of functional tests, such as conventional light transmission aggregometry (LTA), whole blood platelet aggregation, or flow cytometry assessment of in vitro platelet activation in response to ADP. A more specific test measuring the extent of phosphorylation of the cytoplasmic vasodilator- stimulated phosphoprotein (VASP) by flow cytometery is now commercially available. VASP phosphorylation is regulated by the cytoplasmic levels of cyclic adenosine monophosphoate (cAMP). The stimulation of the Gi- coupled P2Yi₂ receptor results in Goc_rmediated inhibition of adenylyl cyclase, followed by a subsequent reduction in cAMP, and thereby VASP de-phosphorylation (Schwartz et al, Thromb. Haemost, 1999, 82: 1145-1152). Although rather indirect, since it requires a pre- stimulation of adenylyl cyclase by prostaglandin El (PGEl), this test specifically addresses the target of thienopyridines including clopidogrel.

The stimulation of P2Yi₂ results in Gβγ_rmediated activation of phosphoinositide 3-kinase (PI3K), followed by an activation of several kinases, including the serine- threonine kinase Akt (Kim et al, J. Biol. Chem., 2004, 279: 4186-419). Through downstream signaling, the reduction in cAMP levels and the activation of PI3K contribute to the stabilization of the integrin (Xπ_bβ₃ in its active conformation, in combination with the stimulation of the second ADP platelet receptor P2Yi (Kahner et al, J. Thromb. Haemost., 2006, 4: 2317-2326). Reduction of ADP-induced Akt- phosphorylation has been demonstrated in vitro, using platelets pretreated with a selective P2Yi₂ antagonist, and in vivo in platelets from mice treated with clopidogrel (Kim et al, J. Biol. Chem., 2004, 279: 4186-419). However, reduction of ADP- induced Akt-phosphorylation has not been demonstrated in a clinical setting.

Although the VASP assay correlates with LTA in situations where P2Yi₂ is strongly inhibited, either in vitro after addition of a receptor antagonist (Pampuch et al, Thromb. Haemost., 2006, 96: 767-773; Aleil et al, J. Thromb. Haemost., 2005, 3: 85-92) or in vivo in healthy subjects after intake of the potent antagonist prasugrel (Jakubowski et al, Thromb. Haemost., 2008, 99: 215-222), the relation is weaker in clopidogrel-treated patients, in whom a wide variety of P2Yi₂ inhibition, overlapping the values in patients not receiving the drag, is observed (Aleil et al, J. Thromb. Haemost., 2005, 3: 85-92; Jakubowski et al, Thromb. Haemost., 2008, 99: 215-222). The first aim of the study presented here was to compare two Gi-dependent events resulting from P2Yi₂ stimulation (i.e., Akt phosphorylation and VASP dephosphorylation) as metabolic end-points of clopidogrel activity, after a loading dose before elective percutaneous coronary intervention (PCI). The second objective was to study the correlation, in this population, between the results of these assays and the inhibition of platelet aggregation, measured by conventional LTA or in whole blood by the VerifyNow^® P2Yi₂ point-of-care assay. Materials and Methods

Study Population. The study was approved by the ethical committee of

Hospital Purpan, Toulouse (France). All the subjects studied gave informed consent.

Between 8 and 12 hours before sampling, consecutive patients (n=59) scheduled for elective PCI (Percutaneous Coronary Intervention) were given a loading dose of

600 mg of clopidogrel, 160 mg aspirin and prophylactic subcutaneous enoxaparin.

Four (4) patients were excluded from analysis because of low platelet count (n=2), or because of a low membrane transfer of total Akt (n=2). Baseline characteristics of evaluated patients (n=55) are shown in Table 1. Coronary stenting was performed in 33 patients (61%). Within the first month of follow-up, no patient died but one presented a recurrence of cardiovascular event.

Table 1: Baseline demographic and clinical characteristics of the study population.

Blood Sampling. The blood samples were obtained before PCI from an antecubital vein, using a 21-guage needle in vacuum tubes containing 0.105 M citrate, and processed in the laboratory within 1 hour.

Analysis of Platelet Akt Phosphorylation. Platelets, isolated from blood by differential centrifugation, were prepared essentially as previously described (Gratacap et al, Blood, 2000, 96: 3439-3446). Briefly, platelets were washed once in Tyrode's-Hepes buffer (140 mM NaCl, 5 mM KCl, 5 mM KH₂PO₄, 1 mM MgSO₄, 1 mM glucose, 10 mM Hepes adjusted to pH 7.3 and containing 2 mg bovine serum albumin). Before each centrifugation and re-suspension, 500 nM PGI2 was added to the buffer. The platelet suspension was finally adjusted at 500 G/L in the same buffer without albumin but with 1 mM CaCl₂ and incubated at 37°C for 30 minutes with 0.02 U/mL of apyrase, an ADP scavenger. The platelets were then stimulated with 10 μM ADP for 5 minutes at 37°C without stirring. For control purposes, platelets were processed in the same way but without ADP (these platelets are referred to as "resting" platelets), or were pre-incubated, before the addition of ADP, with 10 μM of the P2Yi2 selective antagonist, AR-C69931 MX, for 10 minutes at 37°C.

In preliminary experiments, platelets were pre-incubated or not for 10 minutes with 10 μM of AR-C69931 MX or 100 μM of MRS-2179, an antagonist of P2Yi receptor, prior to stimulation for different periods of time with 10 μM ADP, 10 μM PGEl, or both, at 37°C. Platelet stimulation was halted by the addition of electrophoresis sample buffer [100 mM Tris-HCl pH 6.8 containing 3% sodium dodecylsulfate (SDS), 15% (v/v) glycerol, 0.01% bromophenol blue and 15% β- mercaptoethanol] . The samples were boiled for 5 minutes and stored at -20°C until use.

The proteins (50 μg) were separated by electrophoresis in 7.5% polyacrylamide gel and electro-transferred onto nitrocellulose membranes (Millipore, Billerica, USA). After blocking using 5% milk protein in Tris-buffered saline-Tween (TBS-T: 20 mM Tris pH 7.6, 137 mM NaCl, 0.1% Tween 20), the membranes were incubated for 24 hours at 4°C with monoclonal primary antibodies (1 : 1000) against phosphorylated Akt and non-phosphorylated Akt in TBS-T containing 5% milk protein. The anti P-Akt (Ser473) antibody was purchased from Cell Signaling (Beverly, MA, USA) and the anti-Akt antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). After washing in TBS containing 1% milk protein and 1% albumin, the membranes were labeled with a secondary anti-rabbit antibody conjugated to horseradish peroxidase (1:3000) in 1% albumin. The anti-rabbit horseradish peroxidase conjugated secondary antibody was purchased from Cell Signaling Inc. Technology (Boston, MA, USA). The membranes were incubated with SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology, Inc., Rockford, USA), washed with TBS-T, and exposed to X-ray film. Bands were revealed and quantified by densitometry using Gene tools multi-gel (Syngene, Frederick, USA). Unless otherwise specified, all reagents were from Sigma- Aldrich (St. Louis, MI, USA). Analysis of Platelet VASP Phosphorylation. Analyses of platelet VASP phosphorylation were performed using a standardized flow cytometry assay (Platelet VASP^®, Diagnostica Stago/Biocytex, Asnieres, France) according to manufacturer's recommendations, on a Coulter Epics XL-M flow cytometer (Beckman/Coulter, Fullerton, CA, USA). Briefly, the platelets in whole blood were stimulated by PGEl in the absence or the presence of 10 μM ADP, before labeling with a monoclonal antibody against phosphorylated VASP. The results are expressed as platelet reactivity index (PRI), calculated from the median fluorescence intensity (mfi) of the samples incubated with PGElor PGEl + ADP, as follows: PRI= [mfi₍PGEi) - mfi_(PGEi + ADP)]/mfi_(PGEi)

Platelet Aggregation Assays. Platelet function tests were performed within 2 hours after sampling. Platelet rich plasma (PRP) was prepared for light transmission aggregometry (LTA) by centrifugation of whole blood, at 130 g for 10 minutes at room temperature. Platelet aggregation was measured on a Packs-4 optical aggregometer (Helena Laboratories, Beaumont, Texas, USA), as maximal (peak) aggregation induced by 10 μM ADP in stirred (900 rpm) non-adjusted PRP. Results are expressed in %, with PRP used as a reference for 100% aggregation.

The VerifyNow^® assay (Accumetrics, Inc., San Diego, CA, USA) was performed on whole blood using P2Yi₂ test cartridges, according to the manufacturer's recommendations. The assay records platelet agglutination induced in two separate channels, one by 20 μM ADP in the presence of PGEi to reduce the contribution from ADP binding to P2Yi receptor (P2Yi₂ reaction unit or PRU), and one by iso-TRAP (base). Results are expressed as % inhibition, calculated as follows:

% inhibition = (1 - PRUftase) x 100.

Statistical Analysis. Categorical data are presented as frequencies and percentages. The chi-square test was used for dichotomous analysis of categorical data. Results were checked for normality by a Kolmogorov-Smirnov test. Continuous data are presented as median, mean values ± SD and compared using the Mann-Withney U test. The linear regression between continuous variables was determined by Spearman's correlation analysis. Results

P2Yi₂ receptor-mediated Akt phosphorylation in ADP-stimulated platelets.

Akt phosphorylation was first investigated in ADP-stimulated platelets from healthy human volunteers not taking clopidogrel (Figure 1). The level of P- Akt (i.e., phosphorylated Akt) increased during the 10 min-period studied. This increase was efficiently inhibited by AR-C69931MX, as well as by PGEi, indicating that in ADP- stimulated platelets Akt activation is regulated by the balance between the P2Yi₂/Gi signaling and the PGEl/Gs signaling.

Akt phosphorylation was then measured in washed platelets obtained from the studied population of patients (n=55). When not stimulated by ADP, the P- Akt signal was low in all patients. Upon ADP stimulation, 3 different profiles of response were recorded (Figure 2). About half of the patients failed to display detectable phosphorylated Akt, whether or not AR-C69931MX was present. This profile is suggestive of an inhibition of P2Yi₂ by clopidogrel in vivo. About the same proportion of patients displayed phosphorylated Akt with return to the level of resting platelets when the P2Yi₂-receptor antagonist was present. This profile is typical of an impaired response to clopidogrel. Four patients displayed detectable P-Akt in response to ADP, which could not be blunted by AR-C69931MX in vitro. This profile was called "undetermined" and the patients were excluded from the analysis as far as P-Akt was concerned.

After densitometry analysis, an index of phosphorylation was calculated as follows:

Index of phosphorylation = §ADp/[§rest + §_{ADP + AR})/2], where §_ADP, §_rest and §_{ADP + AR} are the signals of P-Akt measured in the respective tracks. The index varied widely among individuals, asymmetrically from 0.4 to 9.9, with a median of 1.5 (at this value, ADP induces a signal of P-Akt 50% above the baseline).

For further analysis, the population was dichotomized using the median as cutoff. Two (2) groups of patients were defined: group A (n=26, 50%) with a low ADP- induced Akt phosphorylation, the expected pharmacological response, (median of index: 1, extremes 0.4-1.5); and group B (poor responders, n=25, 50%) with higher P- Akt index (median: 3.95; extremes 1.8-9.9). Relationship between Akt-phosphorylation and VASP phosphorylation assays. VASP-PRI values were distributed around 45 ± 29 % (m ± SD), with extremes ranging from 0 to 100%, indicating a large inter-individual variability. The correlation between P-Akt index and VASP-PRI was highly significant (p<0.001). As shown in Figure 3, VASP-PRI was significantly lower in group A compared to group B (19, 26 ± 21 vs 74, 65 ± 23 respectively, median, m ± sd, p<0.0001). VASP-PRI cut-off values of 48% (R. Blindt et al, Thromb. Haemost, 2007, 98: 1329-1334) or 50% (F. Bonello et al, J. Thromb. Haemost., 2007, 5: 1630-1636) have recently been proposed to categorize patients according to the risk of stent thrombosis. Patient's allocation using this criterion (50% cut-off) and the P-Akt index as defined above was concordant for 45 patients and discordant for 6 (see Table 2 below).

Table 2. Correlation between patients' allocation according to P-Akt index and VASP-PRI. Patients at high and low risk of stent thrombosis were defined using a cut-off value of VASP-PRI of 50 %, according to Bonello et al (J. Thromb. Haemost., 2007, 5: 1630-1636). (p<0.001, chi-square test).

This good agreement indicates that the two assays, in spite of profound differences in methodology, are equivalent in assessing the pharmacological effect of clopidogrel through its effect on P2Yi₂ Gi-dependent signaling pathways.

VASP-PRI of the 4 patients with "undetermined" P-Akt profiles were 16, 25, 36 and 52% indicating that these patients had a rather good inhibition of P2Yi₂ receptor by clopidogrel in vivo.

Relationship between P2Yi₂ signaling and platelet aggregation. Clopidogrel- evoked platelet aggregation inhibition exhibited considerable individual heterogeneity, without clear cut-offs between "responder" and "non-responder" subjects irrespectively of the method used (LTA and VerifyNow^® P2Yi₂). Correlation of % inhibition assessed by VerifyNow^® P2Y12 assay with ADP-induced peak aggregation yielded a Pearson correlation coefficient (r) of 0.70 (p<0.001, Figure 4). ADP-induced platelet aggregation was significantly lower in group A, with low

P-Akt index, than in group B, with high P-Akt index (6 ± 4 % vs 28 ± 22 % respectively, m±SD, p<0.01) and the inhibition (%) of aggregation measured by VerifyNow^® P2Yi₂ was stronger in group A compared to group B (75 ± 22 % vs 34 ± 27 % respectively, m±SD, p<0.05) (Figure 5(A)).

A very similar figure was obtained using VASP-PRI as a marker of P2Yi₂ signaling (including the 4 patients classified as "undetermined" by P-Akt analysis).

ADP-induced platelet aggregation was significantly lower in patients with PRI <50% than those with a value >50% (6 ± 4 % vs 26 ± 22 % respectively, m±SD, p<0.01) and the inhibition (%) of aggregation measured by VerifyNow^® P2Yi₂ was stronger in the patients with PRI <50% than in those with a value >50% (77 ± 29 % vs 35 ± 26 % respectively, m±SD, p<0.05) (Figure 5(B)).

All patients with "undetermined" P-Akt profile had a complete inhibition of platelet aggregation (<6% peak aggregation by LTA and >84% inhibition by VerifyNow^® P2Y_i2 assay).

Unexpectedly, a significant proportion of patients displayed almost complete inhibition of platelet aggregation without apparent impairment of P2Yπ signaling.

While all patients in group A displayed peak aggregation <20% (empirical cut-off value), this was recorded in 10 out of 25 patients (40%) in group B. And while 24 out of 25 patients in group A (96%, one missing value) displayed >50% inhibition by

VerifyNow^®, this was recorded in 9 out of 23 patients in group B (39%, 2 missing values). Similarly, while all patients in the group with VASP-PRI <50% had peak aggregation <20%, this was recorded in 12 out of 28 patients (43%) in the group with

VASP-PRI >50%. And while all patients of the first group displayed >50% inhibition, this was recorded in 7 out of 24 patients of the second group (29%, 3 missing values). Taken together, this data show that platelet response to ADP assessed by functional assays was still inhibited in 30-40% of patients without detectable impairment of P2Yi₂ signaling.

Discussion

Biological resistance to anti-platelet drugs is a poorly defined condition. It also largely depends on the method used to measure platelet function (Kim et al., Blood, 2006, 107: 947-954). Global tests measuring platelet aggregation are expected to be less specific than pharmacological tests aiming at the molecular targets of the drugs.

Measurement of intra-platelet VASP dephosphorylation induced by ADP is currently the only pharmacological method to assess the effect of P2Yi₂ antagonists. As the activation of Gi-coupled receptor P2Yi₂ activates the Akt-dependent pathway (Kim et al, J. Biol. Chem., 2004, 279: 4186-419; Kim et al, Blood, 2006, 107: 947-954), measurement of Akt phosphorylation induced by ADP could be an alternative to the VASP assay. The results presented in this study confirm the possibility of detecting P-Akt by Western blotting after ADP- stimulation of washed platelets. They also demonstrate the specificity of the assay for P2Yi₂ in the conditions used (see Fig.l).

The first result of this work is that, in spite of the administration of a loading dose of 600 mg of clopidogrel, 8-12 hours before sampling, P2Yi₂-dependent phosphorylation of platelet Akt still occurs in about half of the patients. The Applicants have no explanation for the "undertermined" profile observed in 4 patients who appeared to be responsive to clopidogrel on the basis of VASP and platelet function assays.

The good correlation between the P-Akt index and VASP-PRI confirms that adenylyl-cyclase inhibition and PDK activation, two Gi-dependent events, occur in parallel, downstream of P2Yn activation. Although the Western blot assay for measuring Akt phosphorylation was only semi-quantitative, it allowed patients to be categorized in the same groups as those determined by quantitative flow cytometric assay of VASP, in respect to their response to clopidogrel. This gives indirect support to the value of 50% inhibition proposed as a cut-off for this test. In the present study, one half of the patients were below this cut-off, a proportion similar to that determined by others after the same loading dose of 600 mg: 55% in healthy volunteers (Jakubowski et al, Thromb. Haemost, 2008, 99: 215-222) and 48% in patients undergoing PCI (Bonello et al, J. Am. Coll. Cardiol., 2008, 51: 1404-1411). Platelet aggregation was only partially related to the inhibition of P2Y₁₂ receptor. Although LTA is generally considered as a gold standard for measurement of platelet function, it is poorly standardized: pre-analytical and analytical variables, including the anticoagulant used, conditions of the test, batches, brands and doses of ADP influences the results. Since, in response to ADP, P2Y_i2 is considered as being (mainly) responsible for the stabilization of aggregation, and P2Yi for its extent, it has been claimed that late aggregation would be a better index than maximal (peak) aggregation for measuring the effects of clopidogrel (Labarthe et al, J. Am. Coll. Cardiol., 2005, 46: 638-345). However, direct comparison indicates that the two are practically interchangeable (van Werkum et al, J. Thromb. Haemost, 2006, 4: 2516- 2518; van Werkum et al, J. Thromb. Haemost., 2007, 5: 884-886).

Finally, the mode of expression of the results as a single measurement of platelet aggregation on-treatment, or decrease between pre-and on-treatment is of importance. The latter was not possible in the present study, since the patients may have had received treatment before their admission for PCI. VerifyNow^® is a substitute to conventional platelet aggregometry designed for point-of-care use due to its convenience, rapidity and reproducibility (Malinin et al., Thromb. Res., 2007, 119: 277-284). Several studied have demonstrated a high level of correlation between the % inhibition measured by VerifyNow^® P2Yi₂ and the results of ADP-induced aggregation measured by LTA (van Werkum et al., J. Thromb. Haemost., 2006, 4: 2516-2518; Malinin et al, Thromb. Res., 2007, 119: 277-284; von Beckerath et al, Thromb. Haemost., 2006, 95: 910; Jakubowski et al, Thromb. Haemost., 2008, 99: 409-415). In the present study, the correlation between the two assays was reasonably good. Persistent platelet aggregation (empirical cut-off values: >20% of maximal amplitude by LTA, and/or <30% inhibition by the VerifyNow^® assay) was found in a significant proportion of patients and 13 patients (24%) matched both criteria.

All patients who demonstrated blockade of P2Yi₂ receptor also had low residual platelet aggregation irrespective of the assay used (Figure 5). The reverse was not true since about 30-40% of patients, depending on the test used, displayed strongly reduced aggregation without gross impairment of P2Yi₂ signaling. It is worth noting that this proportion may be underestimated due to the fact that aggregation tests are performed in low, non-physiological, Ca²⁺ concentrations. How can this discrepancy be explained? P2Yi₂ signaling may occur through Gi-independent pathways. One pathway involves the activation of RhoA and Rho-kinase, leading to the reorganization of the cytoskeleton (Soulet et al, J. Thromb. Haemost., 2004, 2: 135- 146). Another pathway involves the activation of G-protein-gated inwardly rectifying potassium channel (Shankar et al, Blood, 2004, 104: 1335-1343). These events are blocked by the active metabolite of clopidogrel in vitro as well. Since the active metabolite disrupts P2Yi₂ receptor oligomers, partitions them out of lipid rafts, and prevents ADP binding (Savi et al, Proc. Natl. Acad. Sci., 2006, 103: 11069-11074), it is expected to affect the signaling functions evenly. A more likely explanation is that the inhibition of platelet aggregation in vitro is obtained with incomplete inhibition of P2Yi₂ signaling. A discrepancy between low platelet aggregation measured by LTA and relatively high VASP-PRI in platelets pre-treated in vitro by the direct agonist cangrelor before ADP challenge has been observed (Pampuch et al., Thromb. Haemost, 2006, 96: 767-773). A similar finding has been shown in a comparative study of clopidogrel (300, 600 mg) and prasugrel (60 mg) in healthy volunteers (Jakubowski et al., Thromb. Haemost., 2008, 99: 215-222). A recent study has shown that, in rat platelets, marginal inhibition of VASP is associated with > 75% reduction in P2Yi₂ binding and late aggregation (Schumacher et al., J. Pharmacol. Exp. Ther., 2007, 322: 369-377). So receptor occupancy, post-receptor signaling and platelet aggregation may be non-proportional.

In summary, the present study demonstrates, for the first time in cardiologic patients, that ADP-induced phosphorylation of platelet Akt is inhibited by clopidogrel in fair parallelism with the phosphorylation of VASP. However, although the inhibition was incomplete in a large proportion of patients, it was sufficient to inhibit platelet aggregation measured by LTA or VerifyNow^® P2Yi₂.

Example 2: Assessment of Platelet Response to Wortannine through Measurement of TRAP-induced Akt Phosphorylation

Platelets, isolated from blood by differential centrifugation, were prepared essentially as previously described (Gratacap et al., Blood, 2000, 96: 3439-3446). Briefly, platelets were washed once in Tyrode's Hepes buffer (140 mM NaCl, 5 mM KCl, 5mM KH₂PO₄, 1 mM MgSO₄, 1 mM glucose, 10 mM Hepes adjusted to pH 7.3 and containing 2 mg bovine serum albumin). Before each centrifugation and re- suspension, 500 mM PGI2 was added to the buffer. The platelet suspension was finally adjusted at 500 G/L in the same buffer without albumin but with ImM CaCl₂ and incubated at 37⁰C for 30 minutes with 0.02 U/mL of apyrase, an ADP scavenger. The platelets were then stimulated with 50 μM TRAP for 5 minutes at 37°C without stirring and in the presence of the GPIIb/IIIa antagonist integrilin (4 μg/mL). For control purposes, platelets were processed in the same way but without TRAP (these platelets are referred to as "resting platelets"), or were pre-incubated before the addition of TRAP, with 100 nM of the PI3K inhibitor, wortmannin for 10 minutes at 37°C. Akt phosphorylation was followed by flow cytometry, as described above. The results of these experiments are presented on Figure 6. Example 3: Assessment of Platelet Response to SCH79797 through Measurement of TRAP-induced Akt Phosphorylation

The platelets were prepared as described in Example 2. The platelets were then stimulated with 50 μM TRAP for 5 minutes at 37°C without stirring and in the presence of the GPIIb/IIIa antagonist integrilin (4 μg/mL). For control purposes, platelets were processed in the same way but without TRAP (these platelets are referred to as "resting platelets"), or were pre-incubated before the addition of TRAP, with 5 μM of the PARI antagonist SCH78787 for 5 minutes at 37⁰C. Akt phosphorylation was followed by flow cytometry, as described above. The results of these experiments are presented on Figure 7. Similar results were obtained when platelets were stimulated in platelet rich plasma in the presence of GPIIb/IIIa antagonists to prevent platelet aggregation.

Other Embodiments

Other embodiments of the invention will be apparent to those skilled in the art from a consideration of the specification or practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope of the invention being indicated by the following claims.

Claims

ClaimsWhat is claimed is:

1. An in vitro method for monitoring a subject's response to an anti-platelet agent which targets a platelet surface receptor, the method comprising a step of: determining the phosphorylation state of Akt in a sample containing platelets from the subject.

2. The method according to claim 1, wherein the anti-platelet agent is an antagonist of the platelet surface receptor.

3. The method according to claim 2, wherein the platelet surface receptor is an ADP receptor, preferably P2Yi₂; a thrombin receptor, preferably PAR-I; a collagen receptor, preferably GPVI; a von Willebrand factor receptor, preferably GPIb-IX-V; a thromboxane A2 receptor, preferably TP; an integrin receptor, preferably GPIIb/IIIa; or a C-type lectin receptor, preferably CLEC- 2.

4. The method according to claim 1 or claim 2, wherein the sample containing platelets is selected from the group consisting of whole blood, platelet rich plasma, and isolated platelets.

5. The method according to any one of claims 1-4, further comprising a step of: stimulating the platelets with an agonist of the platelet surface receptor targeted by the anti-platelet agent prior to determining the phosphorylation state of Akt.

6. The method according to claim 5, wherein the agonist of the platelet surface receptor targeted by the anti-platelet agent is selected from the group consisting of ADP, thrombin, thrombin receptor activating peptide (TRAP), collagen, collagen related peptide (CRP), convulxin, von Willebrand factor under flow conditions, thromboxane A2 (TxA₂), TxA2 analogue U46619, fibrinogen under adhesion conditions, and any combination thereof.

7. The method according to any one of the preceding claims, wherein determining the phosphorylation state of Akt comprises using at least one detectable probe selective for phosphorylated Akt, and, optionally, at least one detectable probe selective for non-phosphorylated Akt.

8. The method according to claim 7, wherein the detectable probe selective for phosphorylated Akt comprises: a labeled phospho-specific antibody selective for phosphorylated Akt, or a phospho-specific antibody selective for phosphorylated Akt and a labeled secondary antibody against the phospho-specific antibody, and wherein the detectable probe selective for non-phosphorylated Akt comprises: a labeled antibody selective for non-phosphorylated Akt, or an antibody selective for non-phosphorylated Akt and a labeled secondary antibody against the antibody selective for non-phosphorylated Akt.

9. The method according to claim 8, wherein the phospho-specific antibody is a phospho-specific S473 antibody or a phospho-specific T308 antibody.

10. The method according to any one of the preceding claims, wherein determining the phosphorylation state of Akt comprises using a method selected from the group consisting of Western blot, immunoblot, enzyme- linked immunosorbant assay (ELISA), radioimmunoassay (RIA), immunoprecipitation, surface Plasmon resonance, immunohistochemistry (IHC), matrix-assisted laser desorption/ionization time-of-flight (MALDI- TOF) mass spectrometry, microcytometry, microscopy, fluorescence activated cell sorting (FACS), and flow cytometry (FC).

11. The method according to claim 10, wherein determining the phosphorylation state of Akt comprises using flow cytometry.

12. A kit for monitoring a subject's response to an anti-platelet agent comprising at least one detectable probe selective for phosphorylated Akt and, optionally, at least one detectable probe selective for non-phosphorylated Akt.

13 The kit according to claim 8, wherein the detectable probe selective for phosphorylated Akt comprises: a labeled phospho-specific antibody selective for phosphorylated Akt, or a phospho-specific antibody selective for phosphorylated Akt and a labeled secondary antibody against the phospho-specific antibody, and wherein the detectable probe selective for non-phosphorylated Akt comprises: a labeled antibody selective for non-phosphorylated Akt, or an antibody selective for non-phosphorylated Akt and a labeled secondary antibody against the antibody selective for non-phosphorylated Akt.

14. The kit according to claim 13, wherein the phospho-specific antibody is a phospho-specific S473 antibody or a phospho-specific T308 antibody.

15. The kit according to any one of claims 12-14 further comprising one or more of: a platelet activation inhibitor selected from the group consisting of aprotinin, theophylline, apyrase, prostaglandin El, and any combination thereof; and a platelet stimulator selected from the group consisting of ADP, thrombin, thrombin receptor activating peptide (TRAP), collagen, collagen related peptide (CRP), convulxin, von Willebrand factor, thromboxane A2 (Tx A2), TxA2 analogue U46619, fibrinogen, and any combination thereof.