Identification of a circulating microvesicle protein network involved in ST-elevation myocardial infarction.

Membrane microvesicles (MVs) are released from activated cells, most notably platelets, into the circulation. They represent an important mode of intercellular communication, and their number is increased in patients with acute coronary syndromes. We present here a differential proteomic analysis of plasma MVs from ST-elevation myocardial infarction (STEMI) patients and stable coronary artery disease (SCAD) controls. The objective was the identification of MVs biomarkers/drug targets that could be relevant for the pathogenesis of the acute event. Proteome analysis was based on 2D-DIGE, and mass spectrometry. Validations were by western blotting in an independent cohort of patients and healthy individuals. A systems biology approach was used to predict protein-protein interactions and their relation with disease. Following gel image analysis, we detected 117 protein features that varied between STEMI and SCAD groups (fold change cut-off ≥2; p<0.01). From those, 102 were successfully identified, corresponding to 25 open-reading frames (ORFs). Most of the proteins identified are involved in inflammatory response and cardiovascular disease, with 11 ORFs related to infarction. Among others, we report an up-regulation of α2-macroglobulin isoforms, fibrinogen, and viperin in MVs from STEMI patients. Interestingly, several of the proteins identified are involved in thrombogenesis (e.g. α2-macroglobulin, and fibrinogen). In conclusion, we provide a unique panel of proteins that vary between plasma MVs from STEMI and SCAD patients and that might constitute a promising source of biomarkers/drug targets for myocardial infarction.

A detailed proteomic analysis of rhodocytin-activated platelets reveals novel clues on the CLEC-2 signalosome: implications for CLEC-2 signaling regulation.

C-type lectin-like receptor 2 (CLEC-2) is an essential platelet-activating receptor in hemostasis and thrombosis that is activated by the snake venom rhodocytin. We present here a differential proteomic analysis of basal and rhodocytin-activated platelets with the aim of providing novel clues on CLEC-2 signaling regulation. Proteome analysis was based on 2D-DIGE, phosphotyrosine immunoprecipitations followed by 1D SDS-PAGE and mass spectrometry. Protein-protein interactions were studied by coimmunoprecipitations and a systems biology approach. Overall, we identified 132 proteins differentially regulated after CLEC-2 platelet activation, including most of the major players reported so far in the signaling cascade. In addition, we identified various proteins not previously known to participate in CLEC-2 signaling, such as the adapters Dok-2 and ADAP, tyrosine kinase Fer, and tyrosine phosphatase SHIP-1. We also report an increased association between Dok-2 and SHIP-1 in rhodocytin-stimulated platelets, which might negatively regulate CLEC-2 signaling. Moreover, we also present a comparative analysis of proteomic data for CLEC-2 and glycoprotein VI signaling. We think that our data provide thrombosis-relevant information on CLEC-2 signaling regulation, contributing to a better understanding of this important signaling cascade.

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.

Comparative analysis of platelet-derived microparticles reveals differences in their amount and proteome depending on the platelet stimulus.

Platelet-derived microparticles (PMP) are elevated in a number of disorders (e.g. cardiovascular disease and cancer). In the present study, we have carried out a high-resolution 2-DE-based proteomic analysis of PMP originated following platelet activation with different stimulus. Flow cytometric analysis revealed a higher average number of microparticles when platelets are activated with shear (1800 s(-1)) compared to 0.5 U/mL thrombin. Regarding the proteomic analysis, 30 protein features were found to be differentially regulated between shear and thrombin groups in the pI 4-7 range, from which 28 were successfully identified by MS, corresponding to 26 open-reading frames. Signaling proteins constituted the major functional group, including membrane receptors, and adapters. Ingenuity Pathways Analysis (IPA) software revealed that 21 of the 26 differentially regulated unique proteins identified are part of a common network related to cell assembly and organization and cell morphology. Western blotting analyses confirmed that Dok-2 and the integrin α6 are differentially regulated in PMP depending on the platelet stimulus. Interestingly, both proteins participate in mechanisms regulating angiogenesis so could be part of PMP regulation of endothelial cells function. In conclusion, this study shows that platelets shed microparticles in different amounts and with a different proteome depending on the stimulus.This article is part of a Special Issue entitled: Integrated omics.

Variations in platelet proteins associated with ST-elevation myocardial infarction: novel clues on pathways underlying platelet activation in acute coronary syndromes.

OBJECTIVE: Our aim in this study was to provide novel information on the molecular mechanisms playing a major role in the unwanted platelet activation associated with ST-elevation myocardial infarction (STEMI). METHODS AND RESULTS: We compared the platelet proteome of 11 STEMI patients to a matched control group of 15 stable chronic ischemic cardiopathy patients. In addition, we did a prospective study to follow the STEMI patients over time. Proteins were separated by high-resolution 2D gel electrophoresis, identified by mass spectrometry, and validated by Western blotting. Platelets from STEMI patients on admission displayed 56 protein spot differences (corresponding to 42 unique genes) compared with the control group. The number of differences decreased with time during the patients' follow-up. Interestingly, the adapter protein CrkL and the active form of Src (phosphorylated in Tyr418) were found to be upregulated in platelets from STEMI patients. Major signaling pathways related to the proteins identified include integrin, integrin-linked kinase, and glycoprotein VI (GPVI) signaling. Interestingly, a study on an independent cohort of patients showed a higher degree of activation of GPVI signaling in STEMI patients. CONCLUSIONS: CrkL, the active form of Src, and GPVI signaling are upregulated in platelets from STEMI patients.

High-resolution two-dimensional gel electrophoresis analysis of atrial tissue proteome reveals down-regulation of fibulin-1 in atrial fibrillation.

BACKGROUND: Atrial fibrillation (AF) is the most common cardiac arrhythmia found in clinical practice. We combined high-resolution two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) to compare the atrial proteome of subjects with AF versus controls with sinus rhythm (SR). Our aim was to identify novel differentially regulated proteins that could be related to the development of the arrhythmia. METHODS: Human atrial appendage tissue samples from patients undergoing heart surgery with AF or SR were analyzed by high-resolution 2-DE. Proteins of interest were identified by MS and validated by western blotting and inmunohistochemistry. RESULTS: Our analysis allowed the detection of over 2300 protein spots per gel. Following differential image analysis, we found 22 spot differences between the AF and SR groups in the 4-7 isoelectric point range, leading to the identification of 15 differentially regulated proteins. The main group of proteins identified was that of heat shock proteins (HSPs), including TRAP-1, HspB3, HspΒ6 and AHA1. Some of the differences detected between AF and SR for the above proteins were due to post-translational modifications. In addition, we identified the structural protein fibulin-1 as down-regulated in atrial tissue from AF patients. CONCLUSIONS: High-resolution 2-DE analysis of human atrial tissue revealed that AF is associated with changes in structural proteins and an important number of HSPs. The lower expression of the structural protein fibulin-1 in atrial tissue from AF patients might reflect the myocardial structural changes that take place in the arrhythmia.