Platelet Proteome Research Articles

Proteomic profiling of platelet signalling

Platelets play a crucial role in hemostasis; activating and aggregating to arrest bleeding following vascular injury. Platelet activation is a complex and dynamic process, involving the co-ordination of numerous receptors to initiate shape change and aggregation. Under pathological conditions, alterations in the normal platelet response can favor a prothrombotic state and increase the risk of acute coronary syndromes (ACS). Receptor stimulation and the tyrosine phosphorylation of key signaling molecules underpin platelet activation in both hemostasis and cardiovascular disease. A lack of nucleus and low mRNA levels makes protein function the primary focus of platelet research. Advancements in proteomic technologies now allow for comprehensive analysis of the platelet proteome and its associated post-translational modifications. In this review, recent applications of proteomics in platelet signaling studies are discussed with particular focus on the elucidation of novel phosphorylation events following receptor activation.

Aspirin Exposure Reveals Novel Genes Associated With Platelet Function and Cardiovascular Events

The aim of this study was to develop ribonucleic acid (RNA) profiles that could serve as novel biomarkers for the response to aspirin. Aspirin reduces death and myocardial infarction (MI), suggesting that aspirin interacts with biological pathways that may underlie these events. Aspirin was administered, followed by whole-blood RNA microarray profiling, in a discovery cohort of healthy volunteers (HV1) (n = 50) and 2 validation cohorts of healthy volunteers (HV2) (n = 53) and outpatient cardiology patients (OPC) (n = 25). Platelet function was assessed using the platelet function score (PFS) in HV1 and HV2 and the VerifyNow Aspirin Test (Accumetrics, Inc., San Diego, California) in OPC. Bayesian sparse factor analysis identified sets of coexpressed transcripts, which were examined for associations with PFS in HV1 and validated in HV2 and OPC. Proteomic analysis confirmed the association of validated transcripts in platelet proteins. Validated gene sets were tested for association with death or MI in 2 patient cohorts (n = 587 total) from RNA samples collected at cardiac catheterization. A set of 60 coexpressed genes named the "aspirin response signature" (ARS) was associated with PFS in HV1 (r = -0.31, p = 0.03), HV2 (r = -0.34, Bonferroni p = 0.03), and OPC (p = 0.046). Corresponding proteins for the 17 ARS genes were identified in the platelet proteome, of which 6 were associated with PFS. The ARS was associated with death or MI in both patient cohorts (odds ratio: 1.2 [p = 0.01]; hazard ratio: 1.5 [p = 0.001]), independent of cardiovascular risk factors. Compared with traditional risk factors, reclassification (net reclassification index = 31% to 37%, p ≤ 0.0002) was improved by including the ARS or 1 of its genes, ITGA2B. RNA profiles of platelet-specific genes are novel biomarkers for identifying patients who do not respond adequately to aspirin and who are at risk for death or MI.

Exploring the potential of the platelet membrane proteome as a source of peripheral biomarkers for Alzheimer's disease

IntroductionPeripheral biomarkers to diagnose Alzheimer's disease (AD) have not been established. Given parallels between neuron and platelet biology, we hypothesized platelet membrane-associated protein changes may differentiate patients clinically defined with probable AD from noncognitive impaired controls.MethodsPurified platelets, confirmed by flow cytometry were obtained from individuals before fractionation by ultracentrifugation. Following a comparison of individual membrane fractions by SDS-PAGE for general proteome uniformity, equal protein weight from the membrane fractions for five representative samples from AD and five samples from controls were pooled. AD and control protein pools were further divided into molecular weight regions by one-dimensional SDS-PAGE, prior to digestion in gel. Tryptic peptides were analyzed by reverse-phase liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Ionized peptide intensities were averaged for each identified protein in the two pools, thereby measuring relative protein abundance between the two membrane protein pools. Log2-transformed ratio (AD/control) of protein abundances fit a normal distribution, thereby permitting determination of significantly changed protein abundances in the AD pool.ResultsWe report a comparative analysis of the membrane-enriched platelet proteome between patients with mild to moderate AD and cognitively normal, healthy subjects. A total of 144 proteins were determined significantly altered in the platelet membrane proteome from patients with probable AD. In particular, secretory (alpha) granule proteins were dramatically reduced in AD. Of these, we confirmed significant reduction of thrombospondin-1 (THBS1) in the AD platelet membrane proteome by immunoblotting. There was a high protein-protein connectivity of proteins in other pathways implicated by proteomic changes to the proteins that define secretory granules.ConclusionsDepletion of secretory granule proteins is consistent with a preponderance of post-activated platelets in circulation in AD. Significantly changed pathways implicate additional AD-related defects in platelet glycoprotein synthesis, lipid homeostasis, amyloidogenic proteins, and regulators of protease activity, many of which may be useful plasma membrane-expressed markers for AD. This study highlights the utility of LC-MS/MS to quantify human platelet membrane proteins and suggests that platelets may serve as a source of blood-based biomarkers in neurodegenerative disease.

Platelet Proteome Analysis Reveals Integrin-dependent Aggregation Defects in Patients with Myelodysplastic Syndromes

Bleeding complications are a significant clinical problem in patients with myelodysplastic syndromes even at sufficient platelet counts (>50,000/μl). However, the underlying pathology of this hemorrhagic diathesis is still unknown. Here, we analyzed the platelet proteome of patients with myelodysplastic syndromes by quantitative two-dimensional difference gel electrophoresis followed by mass spectrometric protein identification. Proteins identified with lower concentrations, such as Talin-1, Vinculin, Myosin-9, Filmain-A, and Actin play critical roles in integrin αIIbβ3 signaling and thus platelet aggregation. Despite normal agonist receptor expression, calcium flux, and granule release upon activation, the activation capacity of integrin αIIbβ3 was diminished in myelodysplastic syndrome platelets. Förster resonance energy transfer analysis showed a reduced co-localization of Talin-1 to the integrin's β3-subunit, which is required for receptor activation and fibrinogen binding. In addition, platelet spreading on immobilized fibrinogen was incomplete, and platelet aggregation assays confirmed a general defect in integrin-dependent platelet aggregation in patients with myelodysplastic syndromes. Our data provide novel aspects on the molecular pathology of impaired platelet function in myelodysplastic syndromes and suggest a mechanism of defective integrin αIIbβ3 signaling that may contribute to the hemorrhagic diathesis observed in these patients.

Proteomic analysis of platelets treated with gamma irradiation versus a commercial photochemical pathogen reduction technology

Several strategies are currently being tested to reduce the risk of pathogen transmission associated with platelet (PLT) transfusion. Within the framework of the Italian Platelet Technology Assessment Study, we investigated the variations of the protein profiles (proteomics) of apheresis PLT concentrates (PCs) upon treatment with riboflavin and ultraviolet (UV) light (Mirasol; 6.24 J/mL; 280-400 nm). Control, gamma-irradiated, and Mirasol-treated apheresis PCs were assayed on Days 1 and 5 of storage by means of gel-based analytical approaches (two-dimensional gel electrophoresis) and mass spectrometry-based identification of significant (p < 0.05 analysis of variance) differential proteins. Supernatants were then assayed for metabolism and oxidative stress-related metabolites through multiple reaction monitoring mass spectrometry. Only a handful of modifications could be observed in the PLT proteome profiles in response to the Mirasol treatment, which included proteins involved in oxidative stress responses, PLT metabolism, and activation. Results confirmed increased metabolic rate and oxidative stress in the supernatants of treated PLTs (both gamma irradiated and Mirasol treated). From this investigation, it emerges that, from a proteomics standpoint, gamma irradiation results in the acceleration of PLT storage lesions and the Mirasol treatment only moderately exacerbates these phenomena.

The integration of proteomics and systems approaches to map regulatory mechanisms underpinning platelet function

Platelets in the circulation are triggered by vascular damage to activate, aggregate and form a thrombus that prevents excessive blood loss. Platelet activation is stringently regulated by intracellular signalling cascades, which when activated inappropriately lead to myocardial infarction and stroke. Strategies to address platelet dysfunction have included proteomics approaches which have lead to the discovery of a number of novel regulatory proteins of potential therapeutic value. Global analysis of platelet proteomes may enhance the outcome of these studies by arranging this information in a contextual manner that recapitulates established signalling complexes and predicts novel regulatory processes. Platelet signalling networks have already begun to be exploited with interrogation of protein datasets using in silico methodologies that locate functionally feasible protein clusters for subsequent biochemical validation. Characterization of these biological systems through analysis of spatial and temporal organization of component proteins is developing alongside advances in the proteomics field. This focused review highlights advances in platelet proteomics data mining approaches that complement the emerging systems biology field. We have also highlighted nucleated cell types as key examples that can inform platelet research. Therapeutic translation of these modern approaches to understanding platelet regulatory mechanisms will enable the development of novel anti-thrombotic strategies.

Identification of AADACL1 As a Novel Regulator of Human Platelets Via Chemoproteomics

AbstractAbstract 381A comprehensive knowledge of the platelet proteome is necessary for understanding important vascular pathologies such as myocardial infarction and stroke. To discover new signaling pathways in human platelets that may represent potential therapeutic targets, we took a combinatorial approach utilizing small library screening coupled with an innovative proteomics technique known as competitive activity based protein profiling (ABPP), a highly sensitive mass spectrometry (MS) technique that queries active enzymes from endogenous biological sources. Human platelets were pretreated with a novel carbamate library designed to interrogate enzymatically active proteins belonging to the serine hydrolase superfamily, which is estimated to represent 1% of the human proteome. Carbamate screening resulted in the identification of several novel inhibitors that reduced fibrinogen binding and P-selectin exposure on agonist-stimulated platelets. An endogenous protein target of the most selective inhibitor was identified by competitive ABPP as arylacetamide deacetylase-like 1 (AADACL1, also known as NCEH1), a lipid deacetylase implicated in cancer growth and atherosclerosis. Using highly selective second generation inhibitors of AADACL1 and MS-based metabolomics, we show that AADACL1 regulates platelet aggregation, Rap1 and PKC activation, lipid metabolism and fibrinogen binding. Elimination of AADACL1 protein levels via RNA interference in a megakaryocytic cell line that responds to platelet agonists also reduces inside-out signaling to αllbβ3. Furthermore, inhibition of AADACL1 reduces collagen-dependent thrombus growth under arterial shear conditions, suggesting that AADACL1 may be a relevant therapeutic target. Collectively, these data identify AADACL1 as a novel regulator of important signaling pathways in platelets and megakaryocytes and validate activity-based proteomics as a means to identify new classes of anti-platelet targets for thrombotic diseases. Disclosures:Parise:BD: Consultancy; Biogen-Idec: Consultancy; NIH: Research Funding; AHA: Research Funding; SCDAC-NIH: Membership on an entity's Board of Directors or advisory committees; BRI Milwaukee: Membership on an entity's Board of Directors or advisory committees. Off Label Use: Eptifibatide, a glycoprotein IIb/IIIa inhibitor, was evaluated as treatment for acute pain episodes in patients with sickle cell disease.

The first comprehensive and quantitative analysis of human platelet protein composition allows the comparative analysis of structural and functional pathways

AbstractAntiplatelet treatment is of fundamental importance in combatting functions/dysfunction of platelets in the pathogenesis of cardiovascular and inflammatory diseases. Dysfunction of anucleate platelets is likely to be completely attributable to alterations in posttranslational modifications and protein expression. We therefore examined the proteome of platelets highly purified from fresh blood donations, using elaborate protocols to ensure negligible contamination by leukocytes, erythrocytes, and plasma. Using quantitative mass spectrometry, we created the first comprehensive and quantitative human platelet proteome, comprising almost 4000 unique proteins, estimated copy numbers for ∼ 3700 of those, and assessed intersubject (4 donors) as well as intrasubject (3 different blood samples from 1 donor) variations of the proteome. For the first time, our data allow for a systematic and weighted appraisal of protein networks and pathways in human platelets, and indicate the feasibility of differential and comprehensive proteome analyses from small blood donations. Because 85% of the platelet proteome shows no variation between healthy donors, this study represents the starting point for disease-oriented platelet proteomics. In the near future, comprehensive and quantitative comparisons between normal and well-defined dysfunctional platelets, or between platelets obtained from donors at various stages of chronic cardiovascular and inflammatory diseases will be feasible.

Chronic estradiol treatment reduces platelet responses and protects mice from thromboembolism through the hematopoietic estrogen receptor α

AbstractAlthough estrogens are known to have a deleterious effect on the venous thrombosis risk and a preventive action on the development of arterial atheroma, their effect on platelet function in vivo remains unclear. Here, we demonstrate that a chronic high physiologic level of estradiol (E2) in mice leads to a marked decrease in platelet responsiveness ex vivo and in vivo compared with ovariectomized controls. E2 treatment led to increased bleeding time and a resistance to thromboembolism. Hematopoietic chimera mice harboring a selective deletion of estrogen receptors (ERs) α or β were used to demonstrate that the effects of E2 were exclusively because of hematopoietic ERα. Within ERα the activation function-1 domain was not required for resistance to thromboembolism, as was previously shown for atheroprotection. This domain is mandatory for E2-mediated reproductive function and suggests that this role is controlled independently. Differential proteomics indicated that E2 treatment modulated the expression of platelet proteins including β1 tubulin and a few other proteins that may impact platelet production and activation. Overall, these data demonstrate a previously unrecognized role for E2 in regulating the platelet proteome and platelet function, and point to new potential antithrombotic and vasculoprotective therapeutic strategies.

A combined proteomic and genetic analysis of the highly variable platelet proteome: From plasmatic proteins and SNPs

High biological variation in protein expression represents a major challenge in clinical proteomics. In a study based on 2D-DIGE, we found that the standardised abundance of only a few proteins varied by more than 50%. While some of the highest variable proteins in platelets of 52 healthy elderly were of plasmatic origin, such as albumin or haptoglobin, absence of several other high-abundant plasma proteins strongly suggests that plasma-derived proteins represent an integral part of the platelet proteome. Amongst the highly variable platelet-derived proteins, two spots were both identified as GSTO1 and assigned to either the wild-type or mutant isoform of SNP A140D. Remarkably, when the spots were considered within the respective genotype groups, their CV decreased to about the median variation. Albeit 2D-DIGE allowed correct genotyping, two individuals seemed to be GSTO1*A140 deficient. Probing 2D-Western blots with novel mAb, however, detected A140 protein as additional spot at pH 8.1, caused by the SNPs E155del and E208K. In contrast to previous studies, we show that GSTO1 protein is expressed in vivo, despite the deletion E155. Our data indicate that incorporation of exogenous proteins and genetic polymorphisms of endogenous proteins represent the main source of extreme biological variation in the platelet proteome.

Recent advances in platelet proteomics

Platelets are the fundamental players in primary hemostasis, but are also involved in several pathological conditions. The remarkable advances in proteomic methodologies have allowed a better understanding of the basic physiological pathways underlying platelet biology. In addition, recent platelet proteomics focused on disease conditions, helping to elucidate the molecular mechanisms of complex and/or unknown human disorders and to find novel biomarkers for early diagnosis and drug targets. The most common and innovative proteomic techniques, both gel-based and gel-free, used in platelet proteomics will be reviewed here. A particular focus will be given to studies that used a subproteomic strategy to analyze specific platelet conditions (resting or activated), compartments (membrane, granules and microparticles) or fractions (phosphoproteome or glycoproteome). The thousands of platelet proteins and interactions discovered so far by these different powerful proteomic approaches represent a precious source of information for both basic science and clinical applications in the field of platelet biology.

Impact of Clopidogrel and Aspirin Treatment on the Expression of Proteins in Platelets from Type-2 Diabetic Patients with Stable Coronary Ischemia

The purpose of this study was to compare the effect of dual antiplatelet therapy [clopidogrel + aspirin (ASA)] with respect to ASA on the protein expression of platelets from controlled type-2 diabetic patients with stable coronary ischemia. Patients had been taking ASA (100 mg day) and they were randomized to receive (n = 29) or not (n = 28) 75 mg day clopidogrel for 12 ± 2 weeks in a blind form. Protein expression was analyzed by two-dimensional electrophoresis and mass spectrometry. The protein expression of a limited number of proteins such as actin-binding protein isotypes 2 and 5, lactate dehydrogenase, serotransferrin isotype 4, protein disulfide isomerase-A3 isotype 1, fibrinogen beta chain isotype 5, Ras-related protein Rab-7b isotypes 1 and 6, and immunoglobulin heavy chain was changed after dual antiplatelet therapy. Plasma level of platelet factor 4 (PF4), an in vivo marker of platelet activity, was not different between both groups. These changes suggest lower platelet reactivity after dual antiplatelet therapy in the studied patients. However, the variation in platelet proteome was lower than it would be initially expected, taking into account the apparent clinical beneficial effects of dual antiplatelet therapy. PF4 plasma level was not further decreased in the platelets treated for a longer time than 9-12 months with ASA + clopidogrel, as compared with ASA alone.

Simplified platelet sample preparation for SDS‐PAGE‐based proteomic studies

The goal of this study was to design an easy and simple protocol for platelet isolation and sample preparation for proteomic studies based on 2DE (IEF-SDS-PAGE) followed by Coomassie blue staining. Blood was collected by venipuncture into tubes coated with EDTA and platelet-rich plasma (PRP) was immediately obtained by centrifugation. PRP was stored refrigerated in closed Falcon tubes for 0, 1, 2, 3, 5, and 7 days and platelets were isolated by centrifugation. 2DE gels were stained with colloidal Coomassie blue stain and evaluated using the Progenesis SameSpots software. Spots that differed significantly in the gels of fresh and stored platelet samples were excised, digested with trypsin, and further analyzed using nanoLC-MS/MS. During the 7-day follow-up period, we found 20 spots that differed significantly (ANOVA p <0.05). During the first 2 days of PRP storage in test tubes, however, only nine spots significantly differed in all donors. In these spots, we identified 14 different proteins. In conclusion, for proteome investigations, whenever it is not feasible to prepare washed platelets immediately after blood collection, the EDTA-anticoagulated PRP can be stored in test tubes at 4°C for up to 2 days for the platelet proteome investigation.

Calpain inhibition stabilizes the platelet proteome and reactivity in diabetes

AbstractPlatelets from patients with diabetes are hyperreactive and demonstrate increased adhesiveness, aggregation, degranulation, and thrombus formation, processes that contribute to the accelerated development of vascular disease. Part of the problem seems to be dysregulated platelet Ca2+ signaling and the activation of calpains, which are Ca2+-activated proteases that result in the limited proteolysis of substrate proteins and subsequent alterations in signaling. In the present study, we report that the activation of μ- and m-calpain in patients with type 2 diabetes has profound effects on the platelet proteome and have identified septin-5 and the integrin-linked kinase (ILK) as novel calpain substrates. The calpain-dependent cleavage of septin-5 disturbed its association with syntaxin-4 and promoted the secretion of α-granule contents, including TGF-β and CCL5. Calpain was also released by platelets and cleaved CCL5 to generate a variant with enhanced activity. Calpain activation also disrupted the ILK-PINCH-Parvin complex and altered platelet adhesion and spreading. In diabetic mice, calpain inhibition reversed the effects of diabetes on platelet protein cleavage, decreased circulating CCL5 levels, reduced platelet-leukocyte aggregate formation, and improved platelet function. The results of the present study indicate that diabetes-induced platelet dysfunction is mediated largely by calpain activation and suggest that calpain inhibition may be an effective way of preserving platelet function and eventually decelerating atherothrombosis development.

Effect of supplementation with an 80:20 cis9,trans11 conjugated linoleic acid blend on the human platelet proteome

The dietary fatty acid cis9,trans11 conjugated linoleic acid (cis9,trans11 CLA) has been shown to modify the function of endothelial cells, monocytes, and platelets, all of which are involved in the development of atherosclerosis. Potential mechanisms for the platelet effects have not been assessed previously. In this study, we assessed how supplementation of the diet with an 80:20 cis9,trans11 CLA blend affects the platelet proteome. In a double-blind, randomized, placebo-controlled, parallel-group trial, 40 overweight but apparently healthy adults received either 4 g per day of cis9,trans11 CLA-enriched oil or placebo oil, consisting of palm oil and soybean oil, for 3 months. Total platelet proteins were extracted from washed platelets, separated using two-dimensional gel electrophoresis and differentially regulated protein spots were identified by LC-ESI-MS/MS. Supplementation with the CLA blend, compared with placebo, resulted in significant alterations in levels of 46 spots (p < 0.05), of which 40 were identified. Network analysis revealed that the majority of these proteins participate in regulation of the cytoskeleton and platelet structure, as well as receptor action, signaling, and focal adhesion. The platelet proteomics approach revealed novel insights into regulation of cellular biomarkers of atherogenic and thrombotic pathways by an 80:20 cis9,trans11 CLA blend.

Proteomics applied to the study of platelet-related diseases: Aiding the discovery of novel platelet biomarkers and drug targets

Platelets play a fundamental role in hemostasis. Because they do not have a nucleus, proteomics is an ideal way to approach their biochemistry. Platelet proteomics is still a young field that emerged a decade ago. Initial platelet proteomic research focused on general proteome mapping followed by the exploration of sub-cellular compartments, the membrane proteome, and signaling pathways. The initial studies were later completed with the analysis of the platelet releasate and microparticle proteome. The success of these studies led to the application of platelet proteomics to the study of several pathologies where platelets play a fundamental role. Those include platelet-related disorders, such as storage pool disease, gray platelet syndrome, and Quebec platelet disorder; diseases where unwanted platelet activation is highly relevant, such as thrombosis and cardiovascular disease; and other diseases, such as cystic fibrosis, uremia, or Alzheimer's disease. In the present review article, we revise the most relevant proteomic studies on platelet-related diseases carried out to date, paying special attention to sample preparation requirements for platelet clinical proteomic studies. This article is part of a Special Issue entitled: Integrated omics.

Shaking hands with the future through omics application in transfusion medicine and clinical biochemistry.

Shaking hands with the future through omics application in transfusion medicine and clinical biochemistry.

Profiling alterations in platelets induced by Amotosalen/UVA pathogen reduction and gamma irradiation--a LC-ESI-MS/MS-based proteomics approach.

Pathogen reduction in platelet concentrates (PC) using Amotosalen/UVA-light reduces the risk of transfusion transmitted infections but also decreases the post-transfusion platelet count increment. Little is known about potential platelet lesions caused by Amotosalen/UVA-light, which may reduce therapeutic efficacy of PCs. Platelets from buffy coat (n=15) derived PCs were pooled and split into three equal PC-units. PC-1 was left untreated (control). PC-2 was Amotosalen/UVA treated using the INTERCEPT Blood System(™), PC-3 was gamma irradiated (30 Gy). Samples were prepared one and five days after PC-production for LC-ESI-MS/MS analysis. Proteins displaying treatment-dependent changes in intensity were classified according to Gene Ontology. In total, 948 proteins were identified, 721 with ≥2 peptides. At day 1, Amotosalen/UVA-treatment triggered alteration of 23 proteins, and gamma irradiation of 49 proteins (overlap: 11 proteins). Five days storage revealed 58 (Amotosalen/UVA treated), 50 (gamma irradiated), and 36 (controls) changes in the platelet proteome compared to control platelets at day 1. Gene Ontology analysis revealed that many affected proteins were displaying specific catalytic activities and/or protein/nucleic acid binding capacity. We identified platelet endothelial aggregation receptor 1 precursor, chloride intracellular channel protein 4, and protein-tyrosine sulfotransferase 2 as proteins uniquely and consistently altered after treatment and storage of Amotosalen/UVA treated platelets. While Amotosalen/UVA-treatment causes less pronounced proteome changes than gamma irradiation at day 1, our data indicate an increase in storage lesions at day 5 caused by this pathogen reduction treatment.

Effect of Uremia and Hemodialysis on Proteome Profile of Blood Platelets and Plasma

The aim of the present research was to assess the differences in blood platelet and plasma proteome profiles of patients with uremia in comparison with healthy participants. It was found that 23 peptides in the platelet proteome profiles of hemodialyzed patients and only 6 peptides in nondialyzed patients were upregulated. On the other hand, 18 peptides with reduced expression in nondialyzed patients and only 1 peptide in hemodialyzed patients were found. For serum, only 6 upregulated peptides in patients undergoing hemodialysis and 15 peptides in nondialyzed patients were found, most of these were about 10 kDa. A decrease in serum peptide expression was not observed. In conclusion, it should be noted that the process of hemodialysis modifies the platelet proteome to a greater extent than uremia alone, however the sera of nondialyzed patients have much larger amounts of low-molecular-weight peptides than those of hemodialyzed patients.

Platelet proteome and clopidogrel response in patients with stable angina undergoing percutaneous coronary intervention

We analyzed the platelet proteome of circulating platelets during the onset of clopidogrel therapy in patients with stable angina underwent percutaneous coronary intervention in order to investigate the mechanisms that control platelet reactivity and clopidogrel response in this context. Twenty patients were enrolled in this study. Blood samples were collected before coronary angiography (T0), 12 h after 600 mg of clopidogrel (T1) and 24 h after percutaneous coronary intervention (PCI) (T2). Platelet reactivity, Clopidogrel response and proteomic analysis were examined. Clopidogrel loading dose produced a significant inhibition in all markers of platelet activation in both flow cytometry and aggregation tests. Among the proteins found differentially expressed, eighteen were identified by MS/MS analysis and they resulted involved in the cytoskeleton rearrangement (profilin-1, calpain, α-soluble NSF attachment protein, thrombospondin), in the energetic metabolism (ubiquitin-like modifier-activating enzyme 1, protein-L-isoaspartate-(D-aspartate) O-methyltransferase and nucleoside diphosphate kinase B) and in the oxidative stress (heat shock 70 kDa protein 5 and anti-stress induced phosphoprotein 1. The present study provides novel information on platelet proteome changes associated with platelet activation and clopidogrel response. This investigation supports the development of further proteomic studies for the identification of novel platelet biomarkers.