Application of Proteomics to the Study of Platelet Regulatory Mechanisms

BRIEF REVIEWS Application of Proteomics to the Study of Platelet Regulatory Mechanisms Patricia B. Maguire,* Niamh Moran Gerard Cagney, and Desmond J. Fitzgerald

Newly developed proteomic technologies now permit the routine identification of hundreds or even thousands of proteins in a single experiment. However, the global study of any proteome has unique challenges that set it apart from comprehensive studies of genes and transcripts. The detection of low-abundance, biologically relevant proteins poses a particular challenge, especially given that the dynamic range of proteins in cells is estimated to be z106. Nevertheless, the incorporation of proteomics into functional biochemical and biologic investigation has proved to be a powerful tool when applied to platelet biology. This review highlights recent proteomic approaches to the characterization of the proteins released from activated platelets and to the identification of integrin-associated regulators of platelet function. Also described are efforts to link platelet-proteomic and platelettranscriptional data. (Trends Cardiovasc Med 2004;14:207–220) D 2004, Elsevier Inc.

Sequencing of the human genome heralded a new era in the investigation of biologic processes, highlighting, in particular, the importance of identifying all human proteins. It became known that

Patricia B. Maguire, Niamh Moran, Gerard Cagney, and Desmond J. Fitzgerald are at the Department of Clinical Pharmacology, Royal College of Surgeon’s in Ireland, Dublin, Ireland. * Address correspondence to: Dr. Patricia B. Maguire, Dept. of Clinical Pharmacology, Royal College of Surgeon’s in Ireland, 123, St. Stephen’s Green, Dublin 2, Ireland. Tel.: (+00) 353-1-716-6957; fax: (+00) 353-1-7166701; e-mail: [email protected]. D 2004, Elsevier Inc. All rights reserved. 1050-1738/04/$-see front matter

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each gene, of an estimated N10,000 expressed at any one time in a mammalian cell, could potentially—through altered transcription, variation in translation, and extensive posttranslational modification—generate hundreds of different proteins with altered structure or function (Miklos and Rubin 1996). Thus, theoretically, a more complete picture of the consequences of gene expression could be achieved through the characterization of proteins. The asyet unrealized aim of global proteomic studies is to provide a comprehensive overview of biologic processes by cataloguing the components of biologic networks and pathways and by defining their interactions, thereby complementing and extending traditional single molecule analysis. Indeed, by providing

a global analysis of proteins in biologic fluids, proteomics is one of the technologies most likely to foster the translation of basic biologic knowledge into clinical applications. Proteomics utilizes a series of interfaced technologies to identify proteins. Mass spectrometry (MS) methods for proteomics normally introduce one peptide or protein to the instrument at a time, and thus a separation step is required. Separation steps include protein separation of complex mixtures by two-dimensional electrophoresis (2-DE) or by multidimensional chromatography. Both of these methods separate peptides or proteins based on their physical or chemical properties. 2-DE uses isoelectric focusing and gel electrophoresis to separate proteins by charge and molecular mass (Gorg et al. 2000), whereas the most common multidimensional chromatographic method separates peptides by charge and hydrophobicity, using ion exchange and reverse-phase chromatography (Link et al. 1999). This method, unlike 2-DE, is amenable to automation and detects proteins not well represented by 2-DE, such as transmembrane and basic proteins. A variety of instrument configurations can be used to obtain mass spectra from the peptides or proteins. Two related, computerized, interpretation procedures are normally used for identification in proteomic experiments because manual identification is very time consuming and requires skilled workers. In effect, bidealizedQ spectra for proteins or peptides found in the sequence databases are generated and the experimentally observed spectra are compared with these until a match is found (Griffin and Aebersold 2001). The ideal spectra may represent the predicted trypsin digest products of single protein species (bpeptide mass spectraQ) or predicted fragmentation patterns resulting from tandem MS experiments using individual peptides. Although a variety

207

of algorithms have been used to match the ideal and experimental spectra (Fenyo 2000), recent efforts have focused on standardized approaches that give a statistical description of the likelihood that a match is correct rather than simply indicating that a particular match is the best one (Von Haller et al. 2003). Some mention here should be made concerning the relative merits of the different methods. Although multidimensional chromatography has resulted in up to a 10-fold increase in the number of proteins identified in yeast studies (Washburn et al. 2001), information concerning the abundance of protein present is lost. Using 2-DE, the intensity of the stained protein bspotsQ gives some indication of the level of protein present, as well as other information such as protein modification and proteolysis. Advanced methods have been described recently that combine multidimensional approaches with procedures that permit identification of protein relative abundance and posttranslational modifications (Mawuenyega et al. 2003). Because the multidimensional chromatography methods use digested proteins, the incidence of proteolysis is difficult or impossible to measure using these methods. These technologies have transformed protein identification and provided the tools for efficient and comprehensive characterization of thousands of proteins. (For a more indepth review of protein separation and identification, mass spectrometric techniques, and instrumentation, please refer to Maguire and Fitzgerald 2003 and Yates 2000). Proteomics—or, more specifically, protein identification by MS—has also contributed to the in vivo analysis of protein–protein interactions, traditionally performed using co-immunoprecipitation. Recently, high-density protein expression arrays have been used for in vitro analysis of protein interactions (Cahill 2001, Phizicky et al. 2003). Other applications of protein expression arrays include the characterization of antibody epitopes and the construction of peptide maps of trypsin-digested proteins for high-throughput mass spectrometric analysis in proteomics (Lueking et al. 2003). Studies such as these hint at the power of high-throughput parallel approaches should analytic techniques

208

for proteins begin to approach those currently available for nucleic acids. 

The Platelet Proteome

The proteome of the platelet is independent of alterations in gene expression, because platelets are anucleate (Fox 1996). That said, valuable information has been obtained from profiling platelet RNA (Gnatenko et al. 2003, McRedmond et al. 2004), and protein synthesis in platelets has been demonstrated (Lindemann et al. 2001). Proteomics offers the opportunity to comprehensively explore the proteins involved in the various pathways of platelet function, from the signals triggered by adhesion to extracellular matrix proteins, to the subsecond events of platelet shape change and aggregation, to the later phases when granule secretion occurs and stable aggregates are formed. Despite the extensive studies of platelet biology over the past 20 years, the number of proteins identified in platelets is relatively low (for a review of these studies, see Maguire and Fitzgerald 2003). Recently, Gevaert et al. (2003) developed a peptide-sorting approach based on the principle of diagonal chromatography called COmbined FRActional DIagonal Chromatography (COFRADIC). Using this technique, they selected the N-terminal peptides of proteins present in the cytosolic and membrane skeleton proteomes of platelets and identified 264 platelet proteins and 78 in vivo acetylated proteins. Additionally, Garcia et al. extended a previously reported analysis of the pI 4–5 region of the human platelet proteome to the pI 5–11 region using 2-DE followed by tandem MS (Garcia et al. 2004a). In the pI 5-11 region (O’Neill et al. 2002) they identified 760 protein features, corresponding to 311 different genes, mainly involved in cytoskeletal regulation and intracellular signaling and regulation. Global characterization of the platelet proteome is very challenging and still does not tell us of the role of those proteins in the signaling pathways and regulatory biology of the platelet. The detection of low-abundance proteins poses a particular challenge, given that the dynamic range of proteins in platelets is at least 106. Furthermore, no amplification method—analogous to the polymerase chain reaction method

for amplifying DNA or RNA—exists for proteins. Therefore, it is necessary when designing experiments to consider approaches that will enrich and capture relevant, lower-abundance proteins that answer specific biologic questions about the platelet. 

Mechanisms of Platelet Activation

Platelet activation is initiated by the binding of adhesive ligands (collagen and von Willebrand factor) to cognate receptors (GPVI, GPIb-V-IX), reinforced by excitatory agonists such as thrombin and adenosine diphosphate (ADP) acting on the G-protein-coupled membrane receptors protease-activated receptor 1 (PAR1) and P2Y1, respectively. Stimuli originating from each of these interactions trigger cascades of signaling events that induce remodeling of the platelet cytoskeleton and activation of the major platelet integrin receptor aIIbh3. Fibrinogen binds to the active conformation of aIIbh3 and initiates another wave of signals into the cell, referred to as outside-in signaling. These late signals regulate several cellular processes, such as secretion by modulating the activity of tyrosine kinases, triggering amplification signals such as the generation of thromboxane, and activating ion transport (Phillips et al. 2001). These signaling events arise from multimolecular complexes formed between integrin cytoplasmic tails, signaling molecules, and structural cytoskeletal proteins and are essential in reinforcing platelet aggregation (Shattil et al. 1998). Complex assembly continues until a bfocal adhesionQ is formed, before the supply of new components is exhausted or inhibitory signals supervene, at which time the complex disassembles. The challenge for researchers is to determine the array of proteins involved in the formation of these multiproteinsignaling complexes regulating platelet activation and aggregation. As inside-out (the signals generated by soluble agonists such as thrombin) and outside-in signaling pathways are regulated both positively and negatively by phosphorylation, proteomics studies have focused on providing an overview of these cascades. Strategies employed include Western blot analysis of whole-platelet proteomes with an antibody to tyrosinephosphorylated residues or radioactive TCM Vol. 14, No. 6, 2004

[32P]-orthophosphate labeling (Immler et al. 1998, Marcus et al. 2000, 2003). Such approaches have been used to monitor phosphorylation of specific proteins. For example, Butt et al. (2001), using 2-DE of radiolabeled platelets in combination with nano-electrospray ionization MS identified the heat shock protein 27 (Hsp27) as a novel substrate of cyclic guanosine monophosphatedependent protein kinase (cGK). Using Hsp27 mutants, they demonstrated that phosphorylation of Hsp27 at different sites by the enzyme’s mitogen-activated protein-kinase-activated protein kinase 2 or cGK could positively or negatively contribute to platelet activation by regulating actin polymerization (Butt et al.

2001). This group also identified the LIM and SH3 domain protein (LASP) as a substrate for cGK serine phosphorylation in platelets, and demonstrated a role for this protein in cytoskeletal organization (Butt et al. 2003). Recently, using 2-DE of control and Thrombin-receptor activating peptide (TRAP)-activated platelets, Garcia et al. (2004b) detected 62 differentially regulated protein spots, 41 of which were identified by tandem MS including the adapter downstream of tyrosine kinases-2 and two regulators of G-protein-signaling (RGS) proteins, RGS 10 and 18. The most comprehensive proteomic studies of tyrosine phosphorylation in platelets to date involved enrichment by

immunoprecipitation—with the use of the monoclonal antibody 4G10—of the dynamic tyrosine phosphorylation events occurring upon thrombin activation of platelets (Maguire et al. 2002, Marcus et al. 2003). By the use of 1-DE in combination with tandem MS, 28 different candidate proteins were identified—some of which were already known to be tyrosine phosphorylated, including actin, filamin, and tubulin (Marcus et al. 2003). With the use of 2DE in combination with MALDI-TOF MS and immunoblotting, 67 differences were highlighted between control and thrombin-activated platelets and several of these proteins were identified, including FAK, Syk, mitogen-activated protein

Table 1. Proteins released from thrombin-activated platelets identified by MALDI-TOF mass spectrometry

Spot no.

Protein identity

1

Apolipopotein A1 fragment Apolipopotein A1 fragment 14-3-3 protein zeta/delta TMP4-ALK fusion oncoprotein type 2 Haptoglobin Haptoglobin Haptoglobin Haptoglobin Actin Osteonectin Osteonectin Thrombospondin Alpha-1 antitrypsin Alpha-1 antitrypsin Alpha-1 antitrypsin Serum albumin Serum albumin Serum albumin Albumin Serum albumin Serum albumin Albumin

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

Accession number

Molecular weight (Mr)

Theoretic isoelectric point (pI)

Theoretic sequence coverage

MOWSE score

CAA00975

28061

5.27

59%

232

CAA00975

28061

5.27

46%

125

1QJBA

26297

4.99

38%

116

Q9HBZ0

27570

4.77

29%

102

AAC27432 AAC27432 AAC27432 AAC27432 CAA27396 O08953 O08953 CAA32889 CAA00206

38722 38722 38722 38722 39446 35129 35129 133261 44291

6.14 6.14 6.14 6.14 5.78 4.81 4.81 4.71 5.36

25% 36% 30% 36% 54% 37% 37% 15% 21%

91 151 137 140 158 127 119 111 120

CAA00206

44291

5.36

21%

109

CAA00206

44291

5.36

21%

120

CAA00298 1AO6A CAA00298 CAA01216 1AO6A 1AO6A AA64922

68588 676090 68588 68425 67690 67690 53416

5.67 5.63 5.67 5.67 5.63 5.63 5.69

30% 27% 27% 29% 25% 17% 33%

194 161 159 154 164 90 150

Protein identifications were generated from the MASCOT database. The MOWSE probability score quantified the validity of the matches, and the percentage of the protein sequence matched by the generated peptides (the sequence coverage) was documented and satisfied the criteria for protein identification by peptide mass fingerprinting as outlined by Lefkovits et al. 2000 (Maguire and Fitzgerald 2003). From: Coppinger JA, Cagney G, Toomey S, et al. Characterization of the proteins released from activated platelets leads to localization of novel platelet proteins in human atherosclerotic lesions. Blood 2004;103:2096–2104. Copyright American Society of Hematology, used with permission.

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Table 2. Summary of 81 proteins from the thrombin-activated platelet releasate identified by MudPIT

Name

Gene name

Known Known to platelet be released/ protein exocytosed

mRNA rank in platelets

Function

Proteins in the platelet releasate known to be released from platelets Thrombospondin 1

TSP1_HUMAN

Yes

From platelet a granules

Fibrinogen-a chain

FIBA_HUMAN

Yes

From platelet a granules

Fibrinogen-g chain

FIBG_HUMAN

Yes

From platelet a granules

Platelet basic protein

SZO7_HUMAN

Yes

From platelet a granules

Platelet factor 4

PLF4_HUMAN

Yes

Serum albumin

ALBU_HUMAN

Yes

From platelet a granules From platelet a granules

Endothelial cell multimerin SPARC (osteonectin) a-actinin 1

ECM_HUMAN

Yes

SPRC_MOUSE

Yes

AAC1_HUMAN

Yes

a-1 antitrypsin

A1AT_HUMAN

Yes

Fibrinogen h chain

FIBB_HUMAN

Yes

Factor V

FA5_HUMAN

Yes

From platelet a granules

Secretory granule proteoglycan core protein Thymosin h 4

PGSG_HUMAN

Yes

From platelet a granules

TYB4_MOUSE

Yes

From platelets a granules

Fructose biphosphate aldolase

ALFA_MOUSE

Yes

From platelets and exosomes from dendritic cells

Clusterin

CLUS_HUMAN

Yes

From platelet a granules

Coagulation factor XIIIA chain

F13A_HUMAN

Yes

From platelet a granules

From platelet a granules From platelet a granules From platelet a granules

From platelet a granules From platelet a granules

Upon secretion, can bind aIIbh3, av h3 , and glycoprotein IV. Can potentiate aggregation by complexing with fibrinogen and becoming incorporated into fibrin clots Cofactor in platelet aggregation Endocytosed into platelets from plasma Cofactor in platelet aggregation Endocytosed into platelets from plasma Proteolytic cleavage yields the chemokines h thromboglobulin and neutophil-activating peptide 2 Platelet-specific chemokine with neutrophil-activating properties Major plasma protein secreted from the liver into the blood. Endocytosed into platelets from plasma Carrier protein for platelet factor V/Va Upon secretion, forms a specific complex with thrombospondin Actin-binding and actinincross-linking protein found in platelet a granules. Interacts with thrombospondin on the platelet surface Acute phase protein, similar to complement, inhibits proteinases Cofactor in platelet aggregation. Endocytosed into platelets from plasma Cofactor that participates with factor Xa to activate prothrombin to thrombin Function not known. Associates and coreleased with inflammatory mediators such as platelet factor 4 G-actin-binding protein. Functions as an antimicrobial peptide when secreted Glycolytic enzymes that converts fructose 1,6-bis phosphate to glyceraldeyde 3-phosphate and dihydroxyacetone phosphate Function not clear. Possibly platelet-derived apolipoprotein J participates in short-term wound repair and chronic pathogenic processes at vascular interface Coagulation protein involved in the formation of the fibrin clots

213

632 — 23 8 —

— 14 171

— — — 343 5 79

1

19

(continued on next page)

210

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Table 2. (continued)

Gene name

Known Known to platelet be released/ protein exocytosed

Metalloproteinase inhibitor 1

TIM1_HUMAN

Yes

From platelet a granules

Platelet glycoprotein V

GPV_HUMAN

Yes

Cleaved from platelet surface

von Willebrand factor Amyloid h-A4 protein (protease nexin II)

VWF_HUMAN

Yes

A4_HUMAN

Yes

From platelet a granules From platelet a granules

Name

Latent transforming LTBS_HUMAN growth factor (TGF)-h-binding protein isoform 1S Alpha-actinin 2 AAC2_MOUSE

Yes

From platelet a granules

Yes

From platelet a granules

Latent TGF-h binding protein 1L Proactivator polypeptide

O88349

Yes

From platelet a granules

SAP_HUMAN

Yes

From lysosomes

Platelet glycoprotein Iba chain

GPBA_HUMAN

Yes

Cleaved from platelet surface (glycocalicin)

Vitamin Kdependent protein S Platelet factor 4 variant Alpha-2 macroglobulin Alpha-actinin 4

PRTS_HUMAN

Yes

From platelet a granules

PF4V_HUMAN

Yes

A2MG_HUMAN

Yes

AAC4_HUMAN

Yes

From platelet a granules From platelet a granules From platelet a granules

mRNA rank in platelets

Function Interacts with metalloproteinases and inactivates them. Stimulates growth and differentiation of erythroid progenitors, dependent on disulfide bonds Part of the GPIb-IX-V complex on the platelet surface. Cleaved by the protease thrombin during thrombin-induced platelet activation Binds GPIb-IX-V Exhibits potent protease inhibitor and growth factor activity. May play a role in coagulation by inhibiting factors XIa and IXa Subunit of the TGF-h1 complex secreted from platelets Actin-binding and actinincross-linking protein found in platelet a granules. Interacts with thrombospondin on the platelet surface Subunit of the TGF beta 1 complex secreted from platelets Activator proteins for sphingolipid hydrolases (saposins) that stimulate the hydrolysis of sphingolipids by lysosomal enzymes Surface membrane protein of platelets that participates in formation of platelet plug by binding A1 domain of von Willebrand factor Cofactor to protein C in the degradation of coagulation factors Va and VIIIA Platelet-specific chemokines with neutrophil activating properties Acute-phase protein, similar to compliment, inhibits proteinases Actin-binding and actinin-crosslinking protein found in platelet a granules. Interacts with thrombospondin on the platelet surface

112

—

2465 379

—

—

617 147

26

1810 346

467

Secretory proteins in the platelet releasate not previously identified in platelets Vitamin D-binding protein

VTDB-HUMAN

No

From liver to plasma

Carries vitamin D sterols. Prevents actin polymerization. Has T-lymphocyte surface association

—

(continued on next page)

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211

Table 2. (continued)

Gene name

Known Known to platelet be released/ protein exocytosed

h2 microglobulin

B2MG_HUMAN

No

Hemoglobin a chain

HBA_HUMAN

No

Plasminogen

PLMN_HUMAN

Yes

Serotransferrin

TRFE_HUMAN

Yes

Pyruvate kinase, M2 isozyme

KPY2_MOUSE

Yes

Actin, aortic smooth muscle

ACTA_HUMAN

Yes

Actin

ACTB_HUMAN

Yes

14-3-3 protein s/y

143Z_MOUSE

Yes

Hemopexin

HEMO_HUMAN

No

Hemoglobin h chain

HBB_HUMAN

No

Peptidyl-prolylcis isomerase A (cyclophilin A) Calumenin

CYPH_MOUSE

No

CALU_MOUSE

No

Adenylyl cyclase-associated protein 1 (CAP 1)

CAP1_MOUSE

No

Tubulin

TBA1_HUMAN

Yes

Apolipoprotein A-1

APA1_HUMAN

Yes

Name

Exosomes from dendritic cells, B cells, enterocytes, tumor cells, and T cells Exosomes from dendritic cells and phagosomes in macrophages From kidney into plasma

mRNA rank in platelets

Function Is the h chain of the major histocompatibility complex class I molecule

3

Oxygen transport. Potentiates platelet aggregation through thromboxane receptor

21

Dissolves fibrin in blood clots, proteolytic factor in tissue remodeling, tumor invasion, and inflammation From Precursor to macromolecular liver into activators of phagocytosis, plasma which enhance leukocyte phagocytosis via the FcgRII receptor B-cell Involved in final stage of exosomes glycolysis. Presented as an autoantigen by dendritic cells Exosomes from B cells, Major cytoskeletal protein dendritic cells, enterocytes, and mastocytes Major cytoskeletal protein Exosomes from External function unknown B cells, dendritic cells, enterocytes and mastocytes External function unknown. Exosomes from Involved intracellularly in signal dendritic cells and transduction; however, may have phagosomes in a role in regulating exocytosis macrophages From liver to plasma Haem-binding protein with metalloproteinase domains From liver to plasma Oxygen transport and phagosomes from macrophages From smooth Cellular protein with isomerase muscle cells activity. Secreted vascular smooth muscle cell growth factor An inhibitor of the vitamin K From many cells, epoxide reductase–warfarin including fibroblasts interaction and COS cells Phagosomes from Contains a WH2 actin-binding macrophages domain (as h-thymosin 4). Known to regulate actin dynamics. May mediate endocytosis Cytoskeletal protein involved Exosomes from in microtubule formation dendritic cells and phagosomes from macrophages From liver to plasma; Role in high-density lipoprotein binding to platelets from monocytes and exosomes of dendritic cells

—

—

61 —

11

63

— 9 116 1816 174

33

—

(continued on next page)

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Table 2. (continued)

Gene name

Known Known to platelet be released/ protein exocytosed

Compliment C3

CO3_HUMAN

No

From liver cells and monocytes

Transthyretin

TTHY_HUMAN

No

From choroid plexus into cerebrospinal fluid (CSF)

Cofilin

COF1_MOUSE

Yes

Profilin

PRO1_MOUSE

Yes

Secretogranin III

SG3_MOUSE

No

Exosome from dendritic cells Exosome from dendritic cells From neuronal cells

Phosphoglycerate kinase

PGK1_MOUSE

Yes

From tumor cells

a-IB glycoprotein

A1BG_HUMAN

No

From many cells including white blood cells

Compliment C4 precursor

CO4_HUMAN

No

From many cells including white blood cells

Prothrombin

THRB_HUMAN

No

From liver to plasma

Glyceraldehyde 3-phosphate dehydrogenase a1-acid glycoprotein

G3P2_HUMAN

Yes

A1AH_HUMAN

No

From B-cell exosomes and phagosomes from macrophages From liver to plasma

GELS_HUMAN

Yes

Name

Gelsolin

Secreted isoform released from liver and adipocytes

mRNA rank in platelets

Function Activator of the compliment system. Cleaved to a, h, and g chains normally prior to secretion, and is a mediator of the local inflammatory response Thyroid hormone-binding protein secreted from the choroid plexus and the liver into CSF and plasma, respectively Actin demolymerization/ regulation in cytoplasm Actin demolymerization/ regulation in cytoplasm Function not clear; possibly involved in secretory granule biogenesis. May be cleaved into active inflammatory peptide-like secretogranin II Glycotic enzyme. Secreted from tumor cells and involved in angiogenesis Found in plasma, function not clear; possibly involved in cell recognition as a new member of the immunoglobulin family Activator of the compliment system. Cleaved normally prior to secretion, its products mediate the local inflammatory response Converts fibrinogen to fibrin and activates coagulation factors including factor V Mitochondrial enzyme involved in glycolysis. May catalyse membrane fusion Modulates activity of the immune system during the acute phase reaction. Binds platelet surface Two isoforms, a cytoplasmic actinmodulating protein and a secreted isoform involved in the inflammatory response

\

—

31 70 \

378 \

—

2143 60 — 438

Proteins in the platelet releasate not previously reported to be released from any cell Calmodulin

CALM_HUMAN

Yes

No evidence

Pleckstrin

PLEK_HUMAN

Yes

No evidence

Nidogen

NIDO_HUMAN

Yes

No evidence

Known to regulate calciumdependent acrosomal exocytosis in neuroendocrine cells A substrate for protein kinase C, its phosphoryation is important for platelet secretion Glycoprotein found in basement membranes, interacts with laminin, collagen, and integrin on neutrophils

55 532 —

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213

Table 2. (continued) mRNA rank in platelets

Gene name

Known Known to platelet be released/ protein exocytosed

Function

Fibrinogen-type protein Rho guanosine diphospate (GDP) dissociation inhibitor 2

Q8VCM7

No

No evidence

Similar to fibrinogen

GDIS_MOUSE

Yes

No evidence

Rho guanosine triphosphatase (GTP) activating protein Transgelin

Q92512

Yes

No evidence

TAG2_HUMAN

No

No evidence

Vinculin WD-repeat protein Superoxide dismutase (SOD)

VINC_HUMAN WDR1_HUMAN SODC_HUMAN

Yes No Yes

No evidence No evidence No evidence

78-kDa glucoserelated protein

GR78_MOUSE

No

No evidence

BRF3_HUMAN Bromodomain and Plant Homology Domain finger-containing protein 3 (fragment) Titin Q8WZ42

No

No evidence

Regulates the GDP/GTP exchange reaction of Rho proteins. Regulates platelet aggregation. Involved in exocytosis in mast cells Promotes the intrinsic GTP hydrolysis activity of Rho family proteins. Involved in regulating myosin phosphorylation in platelets Actin-binding protein. Loss of transgelin expression important in early tumor progression. May serve as a diagnostic marker for breast and colon cancer Actin-binding protein Actin-binding protein Important enzyme in cellular oxygen metabolism, role for SOD-1 in inflammation Chaperone in the endoplasmic reticulum involved in inhibition of secreted coagulation factors, thus reducing prothrombotic potential of cell Function not known

Yes

No evidence

Q99JW3

No

Q9BYX7 Q9UQ35

Name

Similar to hepatocellular carcinomaassociated antigen 59 FKSG30 RNA-binding protein Hypothetic protein Intracellular hyaluronanbinding protein p57 Hypothetic protein Filamin fragment (hypothetic 54( ) kDa protein) Filamin

— 97

\

7

44 127 1730 —

\

—

No evidence

Anchoring protein of actinomyosin filaments. Role in secretion of myostatin Tumor marker

—

No No

No evidence No evidence

Actin-binding protein RNA-binding protein

\ —

Q9BTV9

No

No evidence

Unknown

1744

Q9JKS5

No

No evidence

Unknown

—

Y586_HUMAN

No

No evidence

Unknown

\

Q99KQ2

Yes

No evidence

Unknown

—

FLNA_HUMAN

Yes

No evidence

Actin-binding protein. Essential for GP1b a anchorage at high shear. Substrate for caspase 3

43

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Table 2. (continued)

Gene name

Known Known to platelet be released/ protein exocytosed

Talin

TALI_HUMAN

Yes

No evidence

Zyxin

ZYX_HUMAN

Yes

No evidence

Name

Function Actin-binding protein that binds to integrin-h3 domain Associates with the actin cytoskeleton near adhesion plaques. Binds a actinin and vasodilator-stimulated Phosphoprotein

mRNA rank in platelets 17 145

Eighty-one proteins were identified using MudPIT from the thrombin-stimulated platelet supernatant fraction. Spectra were identified with the SEQUEST program and a composite mouse and human database (National Centre for Biotechnology Information July 2002 release) in three replicate experiments. Information on their functions and whether they are secretory proteins are provided. Also indicated is whether these proteins have a corresponding platelet mRNA. The rank of abundance of the message is denoted numerically in the last column. —, below the threshold for detection on the Affymetrix microarray; \, not present on Affymetrix microarray; MudPIT, multidimensional protein identification technology. From: Coppinger JA, Cagney G, Toomey S, et al. Characterization of the proteins released from activated platelets leads to localization of novel platelet proteins in human atherosclerotic lesions. Blood 2004;103:2096–2104. Copyright American Society of Hematology, used with permission.

kinase, and activin receptor-like kinase 4, which contain the known tyrosine phosphorylation motifs DXXY and EXXY (Maguire et al. 2002). This review highlights additional proteomic approaches used in our laboratory to (a) characterize the proteins released from activated platelets (Coppinger et al. 2004), (b) link proteomic data to transcriptional data (McRedmond et al. 2004), and (c) identify integrin-associated regulators of platelet function (Larkin et al. 2004).

Characterization of the Platelet Releasate When an agonist, such as collagen or thrombin, activates resting platelets, a granules [some containing exosomes of endosomal origin (Heijnen et al. 1999)], dense granules, and lysosomes migrate to the cell surface and exocytose their contents (Fukami et al. 2001). Proteins released in this manner act in an autocrine or paracrine fashion to modulate cell signaling. Several of the proteins (e.g., factor XIII, growth arrest-specific gene 6) are prothrombotic, whereas others (e.g., platelet derived growth factor) regulate cell proliferation (AngelilloScherrer et al. 2001). Others are immune modulators, such as platelet basic protein, whose proteolytic product is neutrophil-activating peptide 2 (Castor et al. 1989). Interestingly, platelet-derived chemokines such as platelet factor 4 (PF4) are found in atherosclerotic plaques, and may contribute to the inflammation TCM Vol. 14, No. 6, 2004

that is a hallmark of the disease (Huo et al. 2003). Thus, the platelet releasate contains factors that conceivably contribute to the development of atherothrombosis, yet the complement of proteins comprising the platelet releasate is largely uncharacterized. We used a multilayered, comprehensive proteomics approach to isolate, separate, and identify the proteins released by platelets upon activation by thrombin (Coppinger et al. 2004). The supernatant fraction of thrombin-activated platelets contained known secreted proteins but no signaling (focal adhesion kinase) or membrane proteins (aIIb), indicating that the preparation was comparatively free of cytosolic fractions or microvesicles. Following two-dimensional (2D) gel electrophoresis of the activated platelet releasate, 22 protein spots were

excised, proteolytically digested, and identified with the use of MALDI-TOF MS, including known platelet-secreted proteins ostenoectin, thrombospondin, a1-antitrypsin, 14-3-3 protein s/y, and actin (Figure 1, Table 1). Certain classes of proteins may be underrepresented on 2D gels—for instance, very large or small, charged, or hydrophobic proteins (Gygi et al. 2000). Multidimensional liquid chromatography approaches are generally less biased, although still not completely representative of the natural proteome (Link et al. 1999). The released protein fraction from thrombin-activated platelets was digested with trypsin and the resulting peptides were separated by tandem strong cation exchange and reverse-phase chromatography (multidimensional protein identification tech-

Figure 1. Two-dimensional electrophoresis (2-DE) of the releasate fraction from thrombin-activated platelets. Four hundred micrograms of the releasate fraction from thrombin-activated platelets was separated by 2-DE and stained with Coomassie Blue dye. Spots were excised and digested with trypsin and the resulting peptides analyzed by matrix-assisted laser desorption ionisationTime of flight Mass Spectrometry (MALDITOF MS). A representative gel is shown and the proteins identified are listed (see Table 1). Molecular weight markers and pI values are indicated. (From: Coppinger JA, Cagney G, Toomey S, et al. Characterization of the proteins released from activated platelets leads to localization of novel platelet proteins in human atherosclerotic lesions. Blood 2004;103:2096–2104. Copyright American Society of Hematology, used with permission.)

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Figure 2. Multidimensional chromatography followed by tandem mass spectrometry (MS) analysis. This figure displays (A) a chromatogram from the third step of a seven-step, multidimensional chromatography run of the releasate fraction. Illustrated in (B) is a characteristic tandem MS spectrum for a peptide (IPESGGDNSVFDIFELTGAARK) that was identified using the SEQUEST program as being from thrombospondin, a known secreted platelet protein. (From: Coppinger JA, Cagney G, Toomey S, et al. Characterization of the proteins released from activated platelets leads to localization of novel platelet proteins in human atherosclerotic lesions. Blood 2004;103:2096–2104. Copyright American Society of Hematology, used with permission.)

nology [MudPIT]). Seven successive salt elutions and high-performance liquid chromatography cycles were used to separate the peptides before introduction into an ion-trap mass spectrometer (Figure 2). Employing this approach to analyze the platelet releasate enabled the discovery of N300 proteins. Of 81 proteins observed in two or three experiments (see Table 2), 37% had been reported previously to be released from platelets, including thrombospondin, PF4, and metalloproteinase inhibitor 1. A further 35% were previously known to be released from other secretory cells, including cofilin, profiling, and 14-3-3s from dendritic cells (Thery et al. 2001) and cyclophilin A from smooth muscle

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cells (Jin et al. 2000). The remaining proteins have not been reported as released from any cell type and include many proteins of unknown function that mapped to expressed sequence tags. Three proteins—secretogranin III (SgIII), a potential monocyte chemoattractant precursor (Kahler et al. 2002); cyclophilin A, a vascular smooth muscle cell growth factor (Jin et al. 2000); and calumenin, an inhibitor of the vitamin K epoxide reductase-warfarin interaction (Wallin et al. 2001)— not previously recognized to be present in or released by platelets, were examined further because they are of potential interest in the pathogenesis of atherosclerosis. These three proteins

were localized to platelets by Western blot (Figure 3), confocal microscopy, and flow cytometry and, for cyclophilin A and calumenin, by microarray analysis of mRNA. Furthermore, whereas absent from normal artery, all three proteins were present in human atherosclerotic plaque, which stained also for the platelet-specific proteins integrin aIIbh3 and PF4. It is possible that these and other proteins released from platelets could contribute to atherosclerosis and to the thrombosis that complicates the disease. Moreover, secreted proteins are suitable therapeutic targets, given their extracellular localization. Thus, neutralization of platelet-derived proinflammatory soluble factors may provide a novel therapeutic TCM Vol. 14, No. 6, 2004

Figure 3. Western blot for calumenin (a), cyclophilin A (b), and secretogranin III (c). The presence of calumenin, cyclophilin, and secretogranin III was confirmed in lysates from control (platelet control [PC]) and thrombin-activated (platelet activated [PA]) platelets, as well as the thrombin-activated releasate (releasate activated [RA]). These proteins were not found in the supernatant from unactivated platelets (releasate control [RC]). In addition, SgIII and calumenin were not detected in a crude leukocyte lysate (WBC), although cyclophilin A was present in low amounts. (From: Coppinger JA, Cagney G, Toomey S, et al. Characterization of the proteins released from activated platelets leads to localization of novel platelet proteins in human atherosclerotic lesions. Blood 2004;103:2096–2104. Copyright American Society of Hematology, used with permission.)

strategy in atherosclerosis without the risk of bleeding that complicates direct inhibition of platelet activation.

Linking Proteomic Data to Transcriptional Data Recently, we have probed Affymetrix oligonucleotide arrays with platelet RNA and identified 2928 distinct messages present in platelets (McRedmond et al. 2004). Hypothesizing that the platelet proteome is reflected in the transcriptome, we used UniGene clusters to compare this transcriptional profile with the proteomic analysis of the platelet releasate, as well as data from two previously published platelet proteomic studies (Marcus et al. 2000, O’Neill et al. 2002). We found that 75 of the 81 released proteins were matched to UniGene clusters, 68 of which had corresponding Affymetrix probesets. Of these 68 array-comparable proteins, messages corresponding to 46 (68%) were detected in platelet mRNA, including messages for several of the secreted proteins not previously attributed to platelets (McRedmond et al. 2004; Table 2). Remarkably, 18 of the 50 most abundant platelet messages were represented in the releasate. Other proteins released that do not have a corresponding mRNA may be endocytosed by platelets; for instance, fibrinogen (Sixma et al. 1977). Protein synthesis in the platelet is limited (Kieffer et al. 1987) and the platelet transcriptome may largely reflect that of the parent megakaryocyte (Shaw et al. 1984). The messages for TCM Vol. 14, No. 6, 2004

many secreted proteins may therefore be transcribed in the megakaryocyte and passed to the daughter platelets. Thus, the profile of platelet mRNA correlated well with protein expression, and transcriptional analysis can be used to predict the presence of novel proteins in the platelet.

Identification of Specific Integrin-Associated Proteins in Platelet The platelet-specific integrin aIIbh3 is one of a family of proteins that acts as

cell membrane receptors of adhesive proteins. Each integrin is a unique heterodimer of an a and h subunit and displays preferential affinity for distinct ligands, depending on their conformation. Platelet aIIbh3 binds fibrinogen preferentially, a critical step for platelet aggregation and secretion. In resting platelets, aIIbh3 displays a low or absent affinity for soluble fibrinogen. Following platelet activation, the integrin undergoes a major change in conformation and expresses a high-affinity binding site permitting fibrinogen binding. Each fibrinogen molecule, a complex of three pairs of identical subunits, has at least two binding sites for aIIbh3 and consequently can bridge adjacent platelets in a growing thrombus. The interaction between fibrinogen and platelet aIIbh3 is insufficient to support complete platelet aggregation (Phillips 2001). Receptor engagement triggers further activation of the cell through so-called outside-in signaling, which includes Ca2+ transients, a wave of protein phosphorylation and dephosphorylation reaction, and the release of several factors that reinforce platelet activation, such as ADP and thromboxane. There is secondary reorganization of the cytoskeleton and, for weak agonists, platelet secretion. aIIbh3 is a large protein complex with the largest component, the aminotermi-

Figure 4. Identification of integrin-binding proteins in a recombinant human protein expression library. Biotin-tagged KVGFFKR, corresponding to the highly conserved a-integrin cytoplasmic motif, bound to multiple Escherichia coli clones expressing recombinant human proteins on a protein array. Proteins were purified by Ni-Agarose affinity chromatography by virtue of their 6-His tag. Dot-blot analysis of these purified proteins (0.1–1 fmol) confirmed that two clones expressed proteins that bound the probe peptide with high affinity (panel A; 100 AM biotin-KVGFFKR) but failed to bind to the control peptide (panel B; 100 AM biotin-KAAAAAR). These clones corresponded to a hypothetic protein (clone B) and a chloride channel protein (clone D). Purified his-tagged chloride channel protein is shown in (C).

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nal, projecting externally. Both subunits have single transmembrane domains and short carboxyl cytoplasmic tails. Proteins interacting with the cytoplasmic tails of the two subunits regulate the activation-dependent change in receptor conformation and the outside-in signaling triggered by aIIbh3. One region that appears to be important is the highly conserved motif, KVGFFKR, which is in the membrane-proximal region of the cytoplasmic tail of aIIb. Mutations of this region result in a constitutively activated receptor, whereas peptides corresponding to the sequence coupled to a lipid trigger platelet signals similar to those following engagement of aIIbh3 (Stephens et al. 1998). Various methods have been used to characterize protein–protein interactions in signaling complexes; for instance, two-hybrid and chemical cross-linking (Uetz et al. 2000). Alternatively, epitope-tagged proteins may be

expressed in a suitable cell line (e.g., one derived from megakaryocytes) and purified along with interacting proteins by a method such as tandem affinity purification (Gavin et al. 2002, Puig et al. 2001). We used a high-density, recombinant human fetal brain expression array of 37,000 robotically arrayed recombinant proteins to identify the proteins that bind to the KVGFFKR peptide (Cahill 2001). Biotin-tagged synthetic peptides corresponding to the known integrin regulatory motif (biotin-KVGFFKR) were reacted with the array and compared with a control peptide. Thirty proteins on the array reacted with the labeled peptide and 19 of these were confirmed in a dot-blot assay of purified protein. Two proteins emerged as potential integrin-associated proteins (Figure 4). One was not expressed in platelets as determined by polymerase chain reaction analysis of platelet mRNA. The

other, a chloride channel protein, was expressed in platelets both at the RNA and protein level. The chloride channel protein co-immunoprecipitated with platelet aIIbh3; and an inhibitor of the channel protein, acyclovir (Gschwentner et al. 1995), dose dependently inhibited platelet aggregation (Larkin et al. 2004). A 6-amino-acid peptide sequence (6-AA) common to the chloride channel and other integrin binding proteins, and consequently representing a potential integrin-binding domain, also inhibited platelet function (Figure 5). The data suggest that the chloride channel provides some of the signaling mediated by aIIbh3, following complex formation between the two proteins (Larkin et al. 2004). The approach illustrates a potential evolution of proteomics technology that overcomes the confounding limitations of protein separation/MS; in particular, the poor detection of lowabundant proteins.

Figure 5. Inhibition of platelet function by a peptide corresponding to the chloride channel integrin-recognition domain. A six-amino-acid sequence (6-AA) was identified by ClustalW alignment of the chloride channel with other putative integrin-binding proteins. Cell-permeable peptides corresponding to this sequence (6-AA; 100 AM), but not control peptides (6-Ctl; 100 AM) inhibited platelet spreading on immobilized fibrinogen. Fibrinogen (10 Ag/mL) was coated onto glass slides and blocked with bovine serum albumin. Gel-filtered platelets were allowed to adhere in the absence or presence of peptides or vehicle for the indicated time points before fixing and staining with Alexa 488-phalloidin (molecular probes). Slides were viewed on a Zeiss LSM501 confocal microscope in differential interference contrast mode or fluorescent mode. 6-AA inhibited cell spreading and actin assembly in adhering platelets. 6-Ctl or vehicle (0.02% methanol) had no effect.

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The Future

The current explosion in proteomics, bioinformatics, and related technologies will have a major impact on cardiovascular biology and on the way cardiovascular disease will be diagnosed, treated, and managed. High-throughput, two-hybrid approaches and analysis of protein complexes using affinity tag purification have yielded valuable protein–protein interaction maps in yeast and drosophila (Giot et al. 2003, Uetz et al. 2000). Application of these technologies to the platelet is challenging, yet possible, if modified strategies are used. Perhaps more exciting—in the context of the platelet releasate and given recent evidence of platelet protein synthesis (Lindemann et al. 2001)—are technologies that allow for relative abundance determination. A suite of enabling technologies have recently been described, and coupling of these methods to high-resolution proteome analysis methods, such as MudPIT, promises the emergence of qualitative studies of whole proteomes that rival gene expression analysis (Cagney and Emili 2002, Gygi et al. 1999, Mirgorodskaya et al. 2000, Oda et al. 1999, Yao et al. 2001). For example, it will be possible to compare platelet proteins in patients with arterial thrombosis and those with stable arterial disease and thus link changes in protein abundance to the risk of the disease. Proteomic analysis may also be applied to monitor the response to drug therapy, possibly resulting in new platelet function assays. As the function of each platelet protein is understood and the mechanisms regulating protein modifications are unraveled, new potential protein drug targets will be discovered, forming the basis for the design of more effective antithrombotic drugs.



Acknowledgments

This work was funded in part by a fellowship to P.B.M. from Enterprise Ireland. P.B.M., N.M., and D.J.F. acknowledge research grants from the Health Research Board of Ireland. D.J.F., N.M., G.C., and P.B.M. are also funded under the Programme for Research in Third Level Institutions, administered by the HEA. G.C. is a recipient of a Science FoundaTCM Vol. 14, No. 6, 2004

tion Ireland awards (foundation grant # 02/IN.1/B117).

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