WO2009007724A2

WO2009007724A2 - Diagnosis and treatment of abnormal blood conditions

Info

Publication number: WO2009007724A2
Application number: PCT/GB2008/002364
Authority: WO
Inventors: Willem Ouwehand; Alison Helena Goodall
Original assignee: EUROPEAN CARDIOVASCULAR GENETICS FOUNDATION
Current assignee: EUROPEAN CARDIOVASCULAR GENETICS FOUNDATION
Priority date: 2007-07-10
Filing date: 2008-07-10
Publication date: 2009-01-15
Anticipated expiration: 2010-01-10
Also published as: GB0713363D0; WO2009007724A3

Abstract

Methods for diagnosing whether a subject has, or is at risk of, an abnormal blood condition are described. The methods comprise determining the expression level of a gene specifically associated with adenosine-di-phosphate (ADP)-stimulated platelet activation, collagen-related peptide (CRP)-stimulated platelet activation, or ADP- and CRP-stimulated platelet activation in a biological sample obtained from the subject. Methods for identifying potential therapeutic agents for the treatment of abnormal blood conditions are also described.

Description

Diagnosis and Treatment of Abnormal Blood Conditions

This invention relates to the diagnosis of abnormal blood conditions, in particular to diagnosis of an increased risk of thrombosis, especially atherothrombosis (AT), or bleeding. The invention also relates to methods for identifying potential therapeutic agents for the treatment of abnormal blood conditions.

AT is the main cause of premature death in Western Society. Myocardial infarction, stroke and peripheral artery thrombosis are three clinical conditions in which activation of platelets is critical to the clinical event of formation of an occlusive thrombus. Collagen is the main extracellular matrix protein which is released upon atherosclerotic plaque rupture and is the prime activator of platelets under this pathological condition ^!"3. Adenosine-di-phosphate (ADP) is contained in platelet granules which are released upon stimulation of platelets by physiological agonists like collagen. ADP release further amplifies platelet activation. The importance of GPVI, the platelet collagen signalling receptor and P2RY1 and P2RY12, the ADP signalling receptor has been convincingly defined in humans because lack of the receptor leads to a bleeding phenotype ⁴'⁵.

We have recently described the extent of interindividual variation in the response of human platelets to the agonists adenosine-di-phosphate (ADP) and cross-linked collagen-related peptide (CRP) ⁶. Signalling of platelets by ADP is via the P2RY1 and P2RY12 G protein coupled receptors (GPCRs) and cross-linked CRP (CRP-XL) signals via the glycoprotein (GP)VI/Fc receptor γ chain. In this platelet function study (PFS) in 506 healthy individuals flow cytometry was used to measure fibrinogen binding and P-selectin expression in response to a single, intermediate dose of ADP or CRP. Pathway specificity was ensured by blocking the main subsidiary signalling pathways. We observed the existence of substantial variation between individuals and between agonists in the level of cellular response. Whilst exogenous factors such as age, hypertension, diabetes mellitus, hypercholesterolemia and cigarette smoking have an effect, it is clear that the platelet functional response is substantially genetically controlled. Studies in siblings ⁷ and twins ⁸ suggest the level of heritability is as high as 50%. Polymorphisms in platelet membrane receptors have been linked to variation response, however the effect is often relatively modest - usually of the order of <5% - and not always reproducible in subsequent studies. This is likely to be because complex functional traits are polygenic, with multiple alleles contributing incrementally to phenotype. Transcriptome studies from our laboratory and others have shown that an estimated 9,000 genes are transcribed in the megakaryocyte, of which ~ 5,000 can be detected in platelets^9"12. To date, efforts to study the relationship between sequence variation and platelet function ^13~15 have been limited by a biased selection of candidate genes based on a priori knowledge, absence of high resolution information on sequence variation resulting in inadequate selection of tagSNPs ¹⁶, often by small cohort sizes, and, importantly, by lack of critical evaluation and standardisation of methods to analyse platelet function.

We have applied a post-genome platforms to study the association between sequence variation and the platelet cellular response to ADP and CRP. A whole genome expression study by micorarray of platelet_. RNA samples from 37 individuals of the PFS cohort selected on their unique functional phenotypes was carried out. By use of this innovative platform, hitherto not applied in platelet biology comparative studies we have found that the transcript level of 69 genes is correlated with function.

According to the invention there is provided an in vitro method of diagnosing whether a subject has, or is at risk of, an abnormal blood condition, which comprises determining the expression level of a gene specifically associated with adenosine-diphosphate (ADP)-stimulated platelet activation, collagen-related peptide (CRP)- stimulated platelet activation, or ADP- and CRP-stimulated platelet activation in a biological sample obtained from the subject.

Preferably the biological sample comprises platelets. The subject is preferably a human subject. A gene specifically associated with ADP-stimulated platelet activation is a gene associated with ADP-stimulated platelet activation, but not with CRP-stimulated platelet activation. Similarly, a gene specifically associated with CRP-stimulated platelet activation is a gene associated with CRP-stimulated platelet activation, but not with ADP-stimulated platelet activation.

The gene specifically associated with ADP-stimulated platelet activation may be any of the following genes: PAIP2; PRKACB; TMSL3; SORD; HBXIP; GTF2A2; LRRFIP1; ST3GAL3; SEPT2; RPS26; PRKACB; FUT6; ADD3; GTF3A; SPINT2; TMAM9B; SNTB1; PRDX6; LOC255783; CMTM1; SYK; NTAN1; GRB2; LMO2.

The gene specifically associated with CRP-stimulated platelet activation may be any of the following genes: COMMD7; TACC3; PFKL; HLA-DPA1; GPR23; Hs.519979-S; CDC16; PNMA1; HIST1H2BN; RGS18; SERPINB1; LYPLAL1 ; CBX3; HIP2; RIOK3; PDCD 10; DNAJB6; COMMD7; RBM38; LOC150928; DNM3; BTK; GTF2A2; NMI; EIF1B; TACC3; SART2; MAD2L1BP; KIAA1191; ATP6V1F; NAP1L1; PGK1 ; ARPC4; WBP1; TPH; TAPBP; ACSBG1; NPC2; TREML1; TSPAN32; ARL2; FAM50A; MPL; TPPl

The gene associated with ADP- and CRP-stimulated platelet activation may be any of the following genes: GTF2A2; TBPL1 ; PPP2CA; NFE2L2; PFKL; ALDOA; PPIA.

If the expression level of the gene specifically associated with ADP-stimulated platelet activation is positively correlated with platelet response (i.e. over expression of the gene is linked to an enhanced platelet response compared with a normal platelet response), the abnormal blood condition may be associated with an increased risk of thrombosis. The gene may be any of the following genes: PAIP2; PRKACB; TMSL3; SORD; HBXIP; GTF2A2; LRRFIP1; RPS26; PRKACB; FUT6; ADD3; GTF3A.

An enhanced platelet response means that the platelet response is enhanced compared to an average response of a population of normal (healthy) individuals (for example the 506 individuals of the PFS).

If the expression level of the gene specifically associated with ADP-stimulated platelet activation is negatively correlated with platelet response (i.e. over expression of the gene is linked to a reduced platelet response compared with a normal platelet response), the abnormal blood condition may be associated with an increased risk of bleeding. The gene may be any of the following genes: PFKL, ST3GAL3; SEPT2; SPINT2; TMAM9B; SNTB1; PRDX6; LOC255783; CMTM1; SYK; NTAN1; GRB2; LMO2.

A reduced platelet response means that the platelet response is reduced compared to an average response of a population of normal (healthy) individuals (for example the 506 individuals of the PFS).

If the expression level of the gene specifically associated with CRP-stimulated platelet activation is positively correlated with platelet response (i.e. over expression of the gene is linked to an enhanced platelet response compared with a normal platelet response), the abnormal blood condition may be associated with an increased risk of thrombosis. The gene may be any of the following genes: COMMD7; TACC3; CDC16; PNMA1; HIST1H2BN; RGS18; SERPINB1; LYPLAL1; CBX3; HIP2; RIOK3; PDCD10; DNAJB6; COMMD7; RBM38; LOC150928; DNM3; BTK; GTF2A2; NMI; EIF1 B; TACC3; SART2; MAD2L1BP.

If the expression level of the gene specifically associated with CRP-stimulated platelet activation is negatively correlated with platelet response (i.e. over expression of the gene is linked to a reduced platelet response compared with a normal platelet response), the abnormal blood condition may be associated with an increased risk of bleeding. The gene may be any of the following genes: PFKL; HLA-DPA1 ; GPR23; HS.519979-S; KIAA1191; ATP6V1F; NAP1L1 ; PGK1; ARPC4; WBP1; TPH; TAPBP; ACSBG1; NPC2; TREML1; TSPAN32; ARL2; FAM50A; MPL; TPP1.

If the expression level of the gene associated with ADP- and CRP-stimulated platelet activation is positively correlated with platelet activation (i.e. over expression of the gene is linked to an enhanced platelet response compared with a normal platelet response), the abnormal blood condition may be associated with increased risk of thrombosis. The gene may be any of the following genes: GTF2A2; TBPL1 ; PPP2CA; NFE2L2.

If the expression level of the gene associated with ADP- and CRP-stimulated platelet activation is negatively correlated with platelet activation (i.e. over expression of the gene is linked to a reduced platelet response compared with a normal platelet response), the abnormal blood condition may be associated with an increased risk of bleeding. The gene may be any of the following genes: PFKL; ALDOA; PPIA.

Platelet response may be measured by any suitable method, for example mobilization of intracellular calcium, platelet aggregometry, or flow cytometric measurement of fibrinogen binding and/or P-selectin expression, in response to adenosine 5'-diphosphate (ADP) or the glycoprotein (GP) Vl-specific crosslinked collagen-related peptide (CRP). Such methods are described, for example, by Jones et al., 2007 (Journal of Thrombosis and Haemostasis, 5: 1756-1765).

A suitable size population of normal individuals may be, for example, 500 or more individuals, preferably Caucasoid individuals. An individual is regarded to be normal if they exhibit a platelet response within a range determined for healthy subjects as described by Jones et al., 2007 (Journal of Thrombosis and Haemostasis, 5: 1756-

1765). In particular, platelet response is preferably determined by flow cytometric measurement of the percentage of platelets positive for fibrinogen binding and/or P- selectin expression in response to a single dose of ADP (10^'7M) or cross-linked CRP

(CRP-XL) (0.1 microg mL^"1) as described by Jones et al. By such methods, a platelet response is considered to be within the range for a healthy subject if: fibrinogen binding to CRP-XL stimulated platelets is within the range 3.1% to 84.4% positive; fibrinogen binding to ADP stimulated platelets is within the range 3.5% to 78.8% positive;

CRP-XL induced P-selectin expression is within the range 6.2% to 90.3% positive; or

ADP induced P-selectin expression is within the range 2.8% to 54.1% positive.

There is also provided according to the invention an in vitro method of diagnosing whether a subject has, or is at risk of, an abnormal blood condition, which comprises determining the expression level of any of the following genes in a biological sample obtained from the subject: PAIP2; PRKACB; TMSL3; SORD; HBXIP; LRRFIP1; ST3GAL3; SEPT2; RPS26; NFE2L2; TBPL1 ; FUT6; PPP2CA; ADD3; GTF3A; SPINT2; TMAM9B; SNTB1; PRDX6; CMTM1; SYK; ALDOA; NTAN1; GRB2; PPIA; LMO2; COMMD7; TACC3; HLA-DPA1; GPR23; CDC16; PNMA1; HIST1H2BN; RGS18; SERPINB1; LYPLAL1 ; CBX3; HIP2; R1OK3; PDCD10; DNAJB6; RBM38; LOC150928; DNM3; BTK; GTF2A2; NMI; EIF1B; SART2; MAD2L1BP; K1AA1191 ; ATP6V1F; NAP1 L1 ; PFKL; PGK1; ARPC4; WBP1 ; TPH; TAPBP; ACSBG1; NPC2; TREML1 ; TSPAN32; ARL2; FAM50A; MPL; TPP1. The expression level of the following genes has been determined to be positively correlated with platelet response: PAIP2; PRKACB; TMSL3; SORD; HBXIP; GTF2A2; LRRFIP1; RPS26; NFE2L2; TBPL1 ; PRKACB; FUT6; PPP2CA; ADD3; GTF3A; COMMD7; TACC3; CDC16; PNMA1; HIST1 H2BN; RGS18; SERPINB1; NFE2L2; LYPLAL1; CBX3; TBPL1; HIP2; RIOK3; PDCD10; DNAJB6; RBM38; LOC150928; DNM3; BTK; GTF2A2; NMI; EIF1B; SART2; MAD2L1B. Accordingly, determination of the expression level of any of these genes may be used to diagnose an increased risk of thrombosis.

The expression level of the following genes has been determined to be negatively correlated with platelet response: PFKL; ST3GAL3; SEPT2; SP1NT2; TMAM9B; SNTB1; PRDX6; CMTM1 ; SYK; ALDOA; NTAN1 ; GRB2; PPIA; LMO2; HLA-DPA1; GPR23; KIAA1191; ATP6V1F; NAP1L1; PGK1; ARPC4; WBP1; TPH; TAPBP; ACSBG1; NPC2; TREML1; TSPAN32; ARL2; FAM50A; MPL; TPP1. Accordingly, determination of the expression level of any of these genes may be used to diagnose an increased risk of bleeding.

Preferably the expression level of at least three (or at least 4, 5, 6, 7, 8, 9, 10, or the majority, or all) of the genes is determined.

There is also provided according to the invention a method of identifying an agent for use in the treatment of an abnormal blood condition, the method comprising screening for a compound that alters the expression level of a gene specifically associated with adenosine-di-phosphate (ADP)-stimulated platelet activation, collagen-related peptide (CRP)-stimulated platelet activation, or ADP- and CRP- stimulated platelet activation.

Agents that decrease the expression level of genes positively correlated with platelet response may be of use in the treatment of thrombosis.

Agents that decrease the expression level of genes negatively correlated with platelet response may be of use in the treatment of a bleeding disorder.

There is also provided according to the invention use of an agent that alters the expression level of a gene specifically associated with adenosine-di-phosphate (ADP)-stimulated platelet activation, collagen-related peptide (CRP)-stimulated platelet activation, or ADP- and CRP-stimulated platelet activation for the treatment of an abnormal blood condition.

There is also provided according to the invention a method for identifying a potential therapeutic agent for the treatment of an abnormal blood condition, which comprises screening for a modulator of expression of any of the following genes: PAIP2; PRKACB; TMSL3; SORD; HBXIP; LRjRFIPl; ST3GAL3; SEPT2; RPS26; NFE2L2; TBPLl; FUT6; PPP2CA; ADD3; GTF3A; SPINT2; TMAM9B; SNTBl; PRDX6; CMTMl; SYK; ALDOA; NTANl; GRB2; PPIA; LM02; C0MMD7; TACC3; HLA- DPAl; GPR23; CDC16; PNMAl; HIST1H2BN; RGS18; SERPINBl; LYPLALl; CBX3; HIP2; RI0K3; PDCDlO; DNAJB6; RBM38; LOC150928; DNM3; BTK; GTF2A2; NMI; EIFlB; SART2; MAD2L1BP; KIAAl 191; ATP6V1F; NAPlLl; PFKL; PGKl; ARPC4; WBPl; TPIl; TAPBP; ACSBGl; NPC2; TREMLl; TSPAN32; ARL2; FAM50A; MPL; TPPl.

There is also provided according to the invention a screening assay to identify a potential therapeutic agent for the treatment of an abnormal blood condition, which comprises screening for a regulator of the activity of any of the proteins encoded by the following genes specified above by: contacting the protein with a candidate regulator and determining the activity of the protein in the presence and absence of the candidate regulator.

Agents that inhibit the activity of a protein encoded by a gene positively correlated with platelet response may be of use in the treatment of thrombosis.

Agents that inhibit the activity of a protein encoded by a gene negatively correlated with platelet response may be of use in the treatment of a bleeding disorder.

There is also provided according to the invention a screening assay to identify a potential therapeutic agent for the treatment of an abnormal blood condition which comprises screening for a regulator of the interaction of any of the proteins encoded by the genes specified above with a binding partner required for the biological effect of the protein by: contacting the protein with the binding partner in the presence of a candidate regulator, and determining binding of the protein to its binding partner in the presence and absence of the candidate regulator. There is also provided according to the invention a screening assay to identify a potential therapeutic agent for the treatment of an abnormal blood condition which comprises screening for a binding partner of any of the proteins encoded by the genes specified above by: contacting the protein with a sample comprising a candidate binding partner, and determining whether the candidate binding partner binds to the protein.

There is also provided according to the invention use of any of the following proteins or nucleic acids in a screening assay to identify a potential therapeutic agent for the treatment of an abnormal blood condition:

(i) proteins encoded by any of the following genes: PAIP2; PRKACB; TMSL3; SORD; HBXP; LRRFIPl; ST3GAL3; SEPT2; RPS26; NFE2L2; TBPLl; FUT6; PPP2CA; ADD3; GTF3A; SPINT2; TMAM9B; SNTBl; PRDX6; CMTMl; SYK; ALDOA; NTANl; GRB2; PPIA; LM02; C0MMD7; TACC3; HLA-DPAl; GPR23; CDC16; PNMAl; HIST1H2BN; RGS18; SERPINBl; LYPLALl; CBX3; HIP2; RI0K3; PDCDlO; DNAJB6; RBM38; LOC150928; DNM3; BTK; GTF2A2; NMI; EIFlB; SART2; MAD2L1BP; KIAAl 191; ATP6V1F; NAPlLl; PFKL; PGKl; ARPC4; WBPl; TPIl; TAPBP; ACSBGl; NPC2; TREMLl; TSPAN32; ARL2; FAM50A; MPL; TPPl ;or ii) nucleic acid encoding any of the proteins of (i) above.

There is also provided according to the invention use of a regulator of expression of any of (i) above in a screening assay to identify a potential therapeutic agent for the treatment of an abnormal blood condition.

There is further provided according to the invention use of a binding partner of any of (i) or (ii) above in a screening assay to identify a potential therapeutic agent for the treatment of an abnormal blood condition.

There is also provided according to the invention a microarray for use in a method of diagnosis of the invention, or in a method or assay of the invention for identifying a potential therapeutic agent for the treatment of an abnormal blood condition.

There is further provided according to the invention a kit for use in a method of diagnosis of the invention, or in a method or assay of the invention for identifying a potential therapeutic agent for the treatment of an abnormal blood condition, the kit comprising means for detecting expression products, or nucleic acids derived from nucleic acid expression products, of a plurality of the following genes: PAIP2; PRKACB; TMSL3; SORD; HBXIP; LRRFIPl; ST3GAL3; SEPT2; RPS26; NFE2L2; TBPLl; FUT6; PPP2CA; ADD3; GTF3A; SPINT2; TMAM9B; SNTBl; PRDX6; CMTMl; SYK; ALDOA; NTANl; GRB2; PPIA; LMO2; C0MMD7; TACC3; HLA- DPAl; GPR23; CDC16; PNMAl; HIST1H2BN; RGS18; SERPINBl; LYPLALl; CBX3; HIP2; RIOK3; PDCDlO; DNAJB6; RBM38; LOCI 50928; DNM3; BTK; GTF2A2; NMI; EIFlB; SART2; MAD2L1BP; KIAAl 191; ATP6V1F; NAPlLl; PFKL; PGKl; ARPC4; WBPl; TPIl; TAPBP; ACSBGl; NPC2; TREMLl; TSPAN32; ARL2; FAM50A; MPL; TPPl.

The kit may further comprise one or more of the following: i) instructions for using the detecting means for diagnosis, prognosis, or therapeutic monitoring; ii) a labelled moiety for detecting the detecting means; iii) a solid phase to which the detecting means is immobilised; iv) a predetermined amount of an isolated expression product of one or more of the genes for use as a standard, or control; v) a label or insert indicating regulatory approval for diagnostic, prognostic or therapeutic use as appropriate.

There is also provided according to the invention a method for identifying a patient having an abnormal blood condition, or a patient suspected of having an abnormal blood condition, who is likely to respond to a therapeutic treatment that alters the level or activity of any of the proteins encoded by the following genes: PAIP2; PRKACB; TMSL3; SORD; HBXIP; LRRFIPl; ST3GAL3; SEPT2; RPS26; NFE2L2; TBPLl; FUT6; PPP2CA; ADD3; GTF3A; SPINT2; TMAM9B; SNTBl; PRDX6; CMTMl; SYK; ALDOA; NTANl; GRB2; PPIA; LM02; C0MMD7; TACC3; HLA- DPAl; GPR23; CDC16; PNMAl; HIST1H2BN; RGS18; SERPINBl; LYPLALl; CBX3; HIP2; RI0K3; PDCDlO; DNAJB6; RBM38; LOC150928; DNM3; BTK; GTF2A2; NMI; EIFlB; SART2; MAD2L1BP; KIAAl 191; ATP6V1F; NAPlLl; PFKL; PGKl; ARPC4; WBPl; TPIl; TAPBP; ACSBGl; NPC2; TREMLl; TSPAN32; ARL2; FAM50A; MPL; TPPl , the method comprising: determining the level of expression of any of the genes encoding the proteins in a patient, or in a biological sample obtained from the patient; and identifying the patient as being likely to respond to the therapeutic treatment if the level of expression of the gene or genes is altered compared to a normal subject.

There is also provided according to the invention a method for selecting a participant in a clinical trial to determine the effectiveness of a potential therapeutic agent for the treatment of an abnormal blood condition, the method comprising: determining the level of expression of any of the following genes: PAIP2; PRXACB; TMSL3; SORD; HBXIP; LRRFIPl; ST3GAL3; SEPT2; RPS26; NFE2L2; TBPLl; FUT6; PPP2CA; ADD3; GTF3A; SPINT2; TMAM9B; SNTBl; PRDX6; CMTMl; SYK; ALDOA; NTANl; GRB2; PPIA; LMO2; COMMD7; TACC3; HLA-DPAl; GPR23; CDC 16; PNMAl; HIST1H2BN; RGS18; SERPINBl; LYPLALl; CBX3; HIP2; RI0K3; PDCDlO; DNAJB6; RBM38; LOCI 50928; DNM3; BTK; GTF2A2; NMI; EIFlB; SART2; MAD2L1BP; KIAAl 191; ATP6V1F; NAPlLl; PFKL; PGKl; ARPC4; WBPl; TPIl; TAPBP; ACSBGl; NPC2; TREMLl; TSPAN32; ARL2; FAM50A; MPL; TPPl in a candidate participant, or in a biological sample obtained from the candidate participant; and selecting the candidate for the clinical trial if the level of expression of the gene or genes is altered compared to a normal subject.

Embodiments of the invention are described in the Example below, with reference to the accompanying drawings and tables, in which:

Figure IA: Example of a correlation plot between the transcript level and the level of P-selectin expression after stimulation of platelets with CRP.

This figure shows the level of transcript as measured on the Illumina microarray platform for the 37 samples. A Pearson's correlation test shows in this case a negative correlation between the level of transcript of the MPL gene and the expression of P-selectin after activation of platelets with CRP. Y axis, microarray intensity signal and X axis, normalised P-selectin expression on CRP. At the right of the graph information is given on the number of transcripts detected in platelets, the number which showed correlation with function and the effect of a stringency filter on the number of transcripts which show positive or negative correlation with function. Figure IB: Overlap of the correlations between transcript level and the four functional measurements.

A total of 69 gene transcripts were identified which either positively or negatively correlated with platelet function. These correlation were observed in a single measurement channel (e.g. P-selectin expression after CRP, see Table 2B) but for five transcripts association with multiple measurements were observed. The number of correlations per functional measurement (FA, FC, PA, PC, see Table 2A and 2B) are presented in the four circles and the number of times that there is overlap between measurements are indicated.

Figure 1C: A heatmap showing the pattern of expression of the 69 platelet transcripts which show an association between transcript level and platelet function.

The heatmap shows expression in megakaryocytes, erythroblasts and in six mature blood cell elements (CD4-T cells, CD8-T cells, CD14-monocytes, CD19-B cells, CD56-NK cells, CD65-granulocytes). The inset shows details of the expression pattern of xx genes which are uniquely transcribed in megakaryocytes and platelets but not in the other 7 blood cell elements.

Figure 2: A protein-protein interaction network visualised by Cytoscope using first- order protein-protein interaction data from Reactome and IntAct using the proteins encoded by the 69 genes identified in the microarray study and 98 candidate genes as drivers.

The protein-protein interaction network was constructed using the 69 genes/proteins uncovered in the microarray experiment and another 100 candidate genes/proteins, which are known to be of importance to the ADP and CRP signalling pathways, as drivers. Source information on first-order protein-protein interaction data are from Reactome and IntAct. A total of 409 nodes (proteins) and 424 edges (interactions) are visualised. The proteins encoded by the candidate genes are triangles and squares are the 69 genes/proteins from the microarray experiment. Diamonds are on both lists. The grey colour scale of the nodes from light to dark indicates the relative level of the transcript for the node/protein in megakaryocytes, with blank symbols indicating the lack of a probe on the Hlumina microarray Version 2 platform.

Table IA: This table provides a listing of the genes for which the transcript level in platelets is correlated with the response of platelets to ADP. For each of the genes listed, the type of correlation is given (positive or negative) and the measurement channel has been indicated. Further information is provided on the characteristics of the transcript level and the strength of statistical correlation.

Table IB: This table provides a listing of the genes for which the transcript level in platelets is correlated with the response of platelets to CRP. For each of the genes listed, the type of correlation is given (positive or negative) and the measurement channel has been indicated. Further information is provided on the characteristics of the transcript level and the strength of statistical correlation.

Table 1C: This table list the transcripts for which platelet transcript level shows association with more than one functional measurement.

Table ID: Categorisation of the 69 genes which showed a correlation between cellular function and RNA level by Gene Ontology.

Example

Microarray study of platelet RNA samples

Differentially expressed genes

Expression studies with genome- wide expression (GWE) arrays were performed with the RNA from highly purified platelets of 37 individuals from the platelet function study cohort. Two groups of 16 individuals, each at the extreme ends of the platelet cellular response for both ADP and CRP and a group of 15 with normal ADP and CRP response pheno types. Analysis of the microarray results indicates that transcripts of 5325 genes can be detected in platelets (Figure IA). This number is lower than in megakaryocytes and is best explained by the platelet being anuclear and by the degradation of RNA post platelet formation. For 69 of these transcripts a correlation between microarray signal level and platelet function was observed (Tables IA and IB for ADP and CRP and Table 1C for overlap).

Expression of the 69 transcripts in other haematopoietic cells Interestingly the analysis of expression of the 69 features in six different blood cell lineages (CD4-T cells, CD8-T cells, CD19-B cells, CD14-monocytes, CD56-NK cells, CD65-granulocytes) and the red cell and platelet precursor (CD235a⁺- erythroblasts and CD61 ^megakaryocytes) suggests megakaryocyte lineage-specific gene transcription for four of the 69 transcripts. Even more intriguing Gene Ontology (GO) analysis of the 69 transcripts indicated that approximately halve of the 69 observed associations between RNA level and platelet function concern transcripts which encode proteins implicated in gene transcription (Table ID). The other GO categories are shown in Table ID.

The signalling cascades

The signalling cascades for ADP and CRP are assumed to be distinct for the receptor proximal segments of the pathways. There is therefore minimal cross-talk between the two pathways for the molecular events before the release of Ca²⁺ from the intracellular stores. After Ca²⁺ release the ADP and CRP signalling events are common but too diverge for a large extent distinct for the change in the configuration of αllbβ3 (Glycoprotein [GP] HbIIIa, allowing fibrinogen binding to occur which was one of the functional read-outs) and for the expulsion of α granules leading to the expression of P-selectin (the second read-out).

The 'first order' interaction of the proteins encoded by 97 genes which were on an a priori candidate gene list was determined by using Reactome and IntAct protein- protein information and a programme application called Cytoscape. Information on connectivity and interdependency between the pathways were obtained from the two databases for ~ 50% of the proteins. The results from the network analysis are depicted in Figure 2. In this figure the lines indicate 'first order' connectivity between proteins (nodes) and the 'grey' intensity of the node indicates the transcript level of the corresponding genes in megakaryocytes.

Addition of the genes/proteins from the microarray experiment allowed expansion of the network to one of 409 hubs and 424 edges. The nodes identified in the microarray experiment or which were present in the a priori candidate gene list have been visualised as squares and as triangles respectively. Diamond shaped nodes are present in both lists. Grey intensity shows the transcript level of the nodes in megakaryocytes. One highly connected node or so called hub is EGFR, a gene known to not be transcribed hi megakaryocytes or other haematological lineages. Absence of this hub is incompatible with network stability as shown here and we postulate on basis of structural analysis that in megakaryocytes the EGFR protein is replaced by PEARl, an EGF domain containing platelet membrane protein which was in the a priori candidate gene list. Proteomics studies and immunoblots are required to validate the suggested EGFR replacements.

Discussion

We have observed that the level of transcript of 69 of the 5325 genes, which are transcribed in megakaryocytes and can be detected in platelets, is either positively or negatively correlated with platelet function. The excess of transcripts encoding proteins involved in gene transcription strongly suggests that the functional phenotype of the platelet is too a large extent determined by transcriptional and molecular events during megakaryopoiesis. The mechanism by which the level of transcript for a group of proteins which is involved in the gene transcription modifies platelet function requires further studies of gene silencing and over-expression in model organisms (e.g. zebrafish and mice) and in human haematopoietic stem cells by RNAi.

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Claims

1. An in vitro method of diagnosing whether a subject has, or is at risk of, an abnormal blood condition, which comprises determining the expression level of a gene specifically associated with adenosine-di-phosphate (ADP)-stimulated platelet activation, collagen-related peptide (CRP)-stimulated platelet activation, or ADP- and CRP-stimulated platelet activation in a biological sample obtained from the subject.

2. A method according to claim 1 , wherein the gene specifically associated with ADP-stimulated platelet activation is any of the following genes: PAIP2; PRKACB; TMSL3; SORD; HBXIP; GTF2A2; LRRFIP1; ST3GAL3; SEPT2; RPS26; PRKACB; FUT6; ADD3; GTF3A; SPINT2; TMAM9B; SNTB1 ; PRDX6; LOC255783; CMTM1; SYK; NTAN1; GRB2; LMO2.

3. A method according to claim 1 , wherein the gene specifically associated with CRP-stimulated platelet activation is any of the following genes: COMMD7; TACC3; PFKL; HLA-DPA1; GPR23; Hs.519979-S; CDC16; PNMA1; HIST1H2BN; RGS18; SERPINB1; LYPLAL1 ; CBX3; HIP2; RIOK3; PDCD10; DNAJB6; COMMD7; RBM38; LOC150928; DNM3; BTK; GTF2A2; NMI; EIF1B; TACC3; SART2; MAD2L1BP; KIAA1191; ATP6V1F; NAP1L1; PGK1; ARPC4; WBP1; TPH; TAPBP; ACSBG1 ; NPC2; TREML1 ; TSPAN32; ARL2; FAM50A; MPL; TPP1.

4. A method according to claim 1 , wherein the gene associated with ADP- and CRP-stimulated platelet activation is any of the following genes: GTF2A2; TBPL1; PPP2CA; NFE2L2; PFKL; ALDOA; PPIA.

5. A method according to claim 1, wherein the abnormal blood condition is associated with an increased risk of thrombosis, and the expression level of the gene specifically associated with ADP-stimulated platelet activation is positively correlated with platelet response.

6. A method according to claim 5, wherein the gene is any of the following genes: PAIP2; PRKACB; TMSL3; SORD; HBXIP; GTF2A2; LRRFIP1 ; RPS26; PRKACB; FUT6; ADD3; GTF3A.

7. A method according to claim 1 , wherein the abnormal blood condition is associated with an increased risk of bleeding, and the expression level of the gene specifically associated with ADP-stimulated platelet activation is negatively correlated with platelet response.

8. A method according to claim 7, wherein the gene is any of the following genes: ST3GAL3; SEPT2; SPINT2; TMAM9B; SNTB1; PRDX6; LOC255783; CMTM1; SYK; NTAN1; GRB2; LMO2.

9. A method according to claim 1, wherein the abnormal blood condition is associated with an increased risk of thrombosis, and the expression level of the gene specifically associated with CRP-stimulated platelet activation is positively correlated with platelet response.

10. A method according to claim 9, wherein the gene is any of the following genes: COMMD7; TACC3; CDC16; PNMA1; HIST1H2BN; RGS18; SERPINB1; LYPLAL1; CBX3; HIP2; RIOK3; PDCD10; DNAJB6; COMMD7; RBM38; LOC150928; DNM3; BTK; GTF2A2; NMI; EIF1B; TACC3; SART2; MAD2L1BP.

11. A method according to claim 1, wherein the abnormal blood condition is associated with an increased risk of bleeding, and the expression level of the gene specifically associated with CRP-stimulated platelet activation is negatively correlated with platelet response.

12. A method according to claim 11, wherein the gene is any of the following genes: PFKL; HLA-DPA1; GPR23; Hs.519979-S; KIAA1191; ATP6V1F; NAP1L1; PGK1; ARPC4; WBP1 ; TPH; TAPBP; ACSBG1; NPC2; TREML1 ; TSPAN32; ARL2; FAM50A; MPL; TPP1.

13. A method according to claim 1, wherein the abnormal blood condition is associated with increased risk of thrombosis, and the expression level of the gene associated with ADP- and CRP-stimulated platelet activation is positively correlated with platelet activation.

14. A method according to claim 13, wherein the gene is any of the following genes: GTF2A2; TBPL1; PPP2CA; NFE2L2.

15. A method according to claim 1 , wherein the abnormal blood condition is associated with an increased risk of bleeding, and the expression level of the gene associated with ADP- and CRP-stimulated platelet activation is negatively correlated with platelet activation.

16. A method according to claim 15, wherein the gene is any of the following genes: PFKL; ALDOA; PPIA.

17. An in vitro method of diagnosing whether a subject has, or is at risk of, an abnormal blood condition, which comprises determining the expression level of any of the following genes in a biological sample obtained from the subject: PAIP2; PRKACB; TMSL3; SORD; HBXIP; LRRFIP1 ; ST3GAL3; SEPT2; RPS26; NFE2L2; TBPL1; FUT6; PPP2CA; ADD3; GTF3A; SPINT2; TMAM9B; SNTB1; PRDX6; CMTM1; SYK; ALDOA; NTAN1; GRB2; PPIA; LMO2; COMMD7; TACC3; HLA- DPA1; GPR23; CDC16; PNMA1 ; HIST1H2BN; RGS18; SERPINB1; LYPLAL1; CBX3; HIP2; RIOK3; PDCD10; DNAJB6; RBM38; LOC150928; DNM3; BTK; GTF2A2; NMI; EIF1B; SART2; MAD2L1BP; KIAA1191; ATP6V1F; NAP1L1; PFKL; PGK1 ; ARPC4; WBP1; TPM; TAPBP; ACSBG1; NPC2; TREML1; TSPAN32; ARL2; FAM50A; MPL; TPP1.

18. A method according to claim 17, wherein the abnormal blood condition is associated with increased risk of thrombosis, and the expression level of any of the following genes is determined: PAIP2; PRKACB; TMSL3; SORD; HBXIP; GTF2A2; LRRFIP1 ; RPS26; NFE2L2; TBPL1; PRKACB; FUT6; PPP2CA; ADD3; GTF3A; COMMD7; TACC3; CDC16; PNMA1; HIST1H2BN; RGS18; SERPINB1; NFE2L2; LYPLAL1; CBX3; TBPL1 ; HIP2; RIOK3; PDCD10; DNAJB6; RBM38; LOC150928; DNM3; BTK; GTF2A2; NMI; EIF1 B; SART2; MAD2L1B.

19. A method according to claim 17, wherein the abnormal blood condition is associated with an increased risk of bleeding, and the expression level of any of the following genes is determined: PFKL; ST3GAL3; SEPT2; SPINT2; TMAM9B; SNTB1; PRDX6; CMTM1; SYK; ALDOA; NTAN1; GRB2; PPIA; LMO2; HLA-DPA1 ; GPR23; KIAA1191; ATP6V1F; NAP1L1; PGK1; ARPC4; WBP1 ; TPH; TAPBP; ACSBG1; NPC2; TREML1 ; TSPAN32; ARL2; FAM50A; MPL; TPP1.

20. A method according to any preceding claim, wherein the expression level of at least three of the genes is determined.

21. A method according to any preceding claim, wherein the biological sample comprises platelets.

22. A method of identifying an agent for use in the treatment of an abnormal blood condition, the method comprising screening for a compound that alters the expression level of a gene specifically associated with adenosine-di-phosphate (ADP)-stimulated platelet activation, collagen-related peptide (CRP)-stimulated platelet activation, or ADP- and CRP-stimulated platelet activation.

23. Use of an agent that alters the expression level of a gene specifically associated with adenosine-di-phosphate (ADP)-stimulated platelet activation, collagen-related peptide (CRP)-stimulated platelet activation, or ADP- and CRP- stimulated platelet activation for the treatment of an abnormal blood condition.

24. A method for identifying a potential therapeutic agent for the treatment of an abnormal blood condition, which comprises screening for a modulator of expression of any of the following genes: PAIP2; PRKACB; TMSL3; SORD; HBXIP; LRRFEPl; ST3GAL3; SEPT2; RPS26; NFE2L2; TBPLl; FUT6; PPP2CA; ADD3; GTF3A; SPINT2; TMAM9B; SNTBl; PRDX6; CMTMl; SYK; ALDOA; NTANl; GRB2; PPIA; LM02; COMMD7; TACC3; HLA-DPAl; GPR23; CDC16; PNMAl; HIST1H2BN; RGS18; SERPINBl; LYPLALl; CBX3; HIP2; RI0K3; PDCDlO; DNAJB6; RBM38; LOC150928; DNM3; BTK; GTF2A2; NMI; EIFlB; SART2; MAD2L1BP; KIAAl 191; ATP6V1F; NAPlLl; PFKL; PGKl; ARPC4; WBPl; TPIl; TAPBP; ACSBGl; NPC2; TREMLl; TSPAN32; ARL2; FAM50A; MPL; TPPl.

25. A screening assay to identify a potential therapeutic agent for the treatment of an abnormal blood condition, which comprises screening for a regulator of the activity of any of the proteins encoded by the genes specified in claim 24 by: contacting the protein with a candidate regulator and determining the activity of the protein in the presence and absence of the candidate regulator.

26. A screening assay to identify a potential therapeutic agent for the treatment of an abnormal blood condition which comprises screening for a regulator of the interaction of any of the proteins encoded by the genes specified in claim 24 with a binding partner required for the biological effect of the protein by: contacting the protein with the binding partner in the presence of a candidate regulator, and determining binding of the protein to its binding partner in the presence and absence of the candidate regulator.

27. A screening assay to identify a potential therapeutic agent for the treatment of an abnormal blood condition which comprises screening for a binding partner of any of the proteins encoded by the genes specified in claim 24 by: contacting the protein with a sample comprising a candidate binding partner, and determining whether the candidate binding partner binds to the protein.

28. Use of any of the following proteins or nucleic acids in a screening assay to identify a potential therapeutic agent for the treatment of an abnormal blood condition:

(i) proteins encoded by the genes specified in claim 24: or ii) nucleic acid encoding any of the proteins of (i) above.

29. Use of a regulator of expression of any of (i) of claim 28 in a screening assay to identify a potential therapeutic agent for the treatment of an abnormal blood condition.

30. Use of a binding partner of any of (i) or (ii) of claim 28 in a screening assay to identify a potential therapeutic agent for the treatment of an abnormal blood condition.

31. A microarray for use in a method of diagnosis according to any of claims 1 to 21 , or in a method or assay for identifying a potential therapeutic agent for the treatment of an abnormal blood condition according to any of claims 22, or 24 to 27.

32. A kit for use in a method of diagnosis according to any of claims 1 to 21, or in a method or assay for identifying a potential therapeutic agent for the treatment of an abnormal blood condition according to any of claims 22, or 24 to 27, the kit comprising means for detecting expression products, or nucleic acids derived from nucleic acid expression products, of a plurality of the genes specified in claim 24.

33. A kit according to claim 32, which further comprises one or more of the following: vi) instructions for using the detecting means for diagnosis, prognosis, or therapeutic monitoring; vii) a labelled moiety for detecting the detecting means; viii) a solid phase to which the detecting means is immobilised; ix) a predetermined amount of an isolated expression product of one or more of the genes for use as a standard, or control; x) a label or insert indicating regulatory approval for diagnostic, prognostic or therapeutic use as appropriate.

34. A method for identifying a patient having an abnormal blood condition, or a patient suspected of having an abnormal blood condition, who is likely to respond to a therapeutic treatment that alters the level or activity of any of the proteins specified in (i) of claim 28, the method comprising: determining the level of expression of any of the genes encoding the proteins specified in (i) of claim 28 in a patient, or in a biological sample obtained from the patient; and identifying the patient as being likely to respond to the therapeutic treatment if the level of expression of the gene or genes is altered compared to a normal subject.

35. A method for selecting a participant in a clinical trial to determine the effectiveness of a potential therapeutic agent for the treatment of an abnormal blood condition, the method comprising: determining the level of expression of any of the genes encoding the proteins specified in (i) of claim 28 in a candidate participant, or in a biological sample obtained from the candidate participant; and selecting the candidate for the clinical trial if the level of expression of the gene or genes is altered compared to a normal subject.