Andrés F. Parguiña

Proteins Involved in Platelet Signaling Are Differentially Regulated in Acute Coronary Syndrome: A Proteomic Study

Background Platelets play a fundamental role in pathological events underlying acute coronary syndrome (ACS). Because platelets do not have a nucleus, proteomics constitutes an optimal approach to follow platelet molecular events associated with the onset of the acute episode. Methodology/Principal Findings We performed the first high-resolution two-dimensional gel electrophoresis-based proteome analysis of circulating platelets from patients with non-ST segment elevation ACS (NSTE-ACS). Proteins were identified by mass spectrometry and validations were by western blotting. Forty protein features (corresponding to 22 unique genes) were found to be differentially regulated between NSTE-ACS patients and matched controls with chronic ischemic cardiopathy. The number of differences decreased at day 5 (28) and 6 months after the acute event (5). Interestingly, a systems biology approach demonstrated that 16 of the 22 differentially regulated proteins identified are interconnected as part of a common network related to cell assembly and organization and cell morphology, processes very related to platelet activation. Indeed, 14 of those proteins are either signaling or cytoskeletal, and nine of them are known to participate in platelet activation by αIIbβ3 and/or GPVI receptors. Several of the proteins identified participate in platelet activation through post-translational modifications, as shown here for ILK, Src and Talin. Interestingly, the platelet-secreted glycoprotein SPARC was down-regulated in NSTE-ACS patients compared to stable controls, which is consistent with a secretion process from activated platelets. Conclusions/Significance The present study provides novel information on platelet proteome changes associated with platelet activation in NSTE-ACS, highlighting the presence of proteins involved in platelet signaling. This investigation paves the way for future studies in the search for novel platelet-related biomarkers and drug targets in ACS.

Proteomic analysis of integrin alphaIIbbeta3 outside-in signaling reveals Src-kinase-independent phosphorylation of Dok-1 and Dok-3 leading to SHIP-1 interactions.

Summary.  Background and Objectives: Outside‐in integrin αIIbβ3 signaling involves a series of tyrosine kinase reactions that culminate in platelet spreading on fibrinogen. The aim of this study was to identify novel tyrosine phosphorylated signaling proteins downstream of αIIbβ3, and explore their role in platelet signaling. Methods and Results: Utilizing proteomics to search for novel platelet proteins that contribute to outside‐in signaling by the integrin αIIbβ3, we identified 27 proteins, 17 of which were not previously shown to be part of a tyrosine phosphorylation‐based signaling complex downstream of αIIbβ3. The proteins identified include the novel immunoreceptors G6f and G6b‐B, and two members of the Dok family of adapters, Dok‐1 and Dok‐3, which underwent increased tyrosine phosphorylation following platelet spreading on fibrinogen. Dok‐3 was also inducibly phosphorylated in response to the GPVI‐specific agonist collagen‐related peptide (CRP) and the PAR‐1 and ‐4 agonist thrombin, independently of the integrin αIIbβ3. Tyrosine phosphorylation of Dok‐1 and Dok‐3 was primarily Src kinase‐independent downstream of the integrin, whereas it was Src kinase‐dependent downstream of GPVI. Moreover, both proteins inducibly interacted with Grb‐2 and SHIP‐1 in fibrinogen‐spread platelets. Conclusions: This study provides new insights into the molecular mechanism regulating αIIbβ3‐mediated platelet spreading on fibrinogen. The novel platelet adapter Dok‐3 and the structurally related Dok‐1 are tyrosine phosphorylated in an Src kinase‐independent manner downstream of αIIbβ3 in human platelets, leading to an interaction with Grb2 and SHIP‐1.

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 Alzheimers 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.

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.

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.

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.

Platelet proteomics in transfusion medicine: a reality with a challenging but promising future.

Dear Sir, Platelet proteomics is a young field that took off at the beginning of this century and soon yielded relevant results. The basic concept for platelet proteomic researchers is that, because platelets do not have a nucleus, proteomics is an ideal tool to approach their biochemistry. This concept proved to be true and thus, during the last ten years proteomics allowed the discovery of new platelet receptors and signaling proteins, some of which were shown to play a significant role in platelet activation. Indeed, some of these proteins are being studied as potential anti-thrombotic drug targets1. The initial success was taken with cautious optimism by platelet researchers, some of whom took the challenge of applying platelet proteomics to more clinical orientated studies. The aim was to take advantage of the technology in a clinical environment in order to find novel biomarkers and drug targets for those pathologies where unwanted platelet activation plays a relevant role. One of the fields where platelet clinical proteomics has shown more productive results is transfusion medicine. The storage of platelets is of critical importance in today’s clinical practice. Platelet concentrates (PLCs) are obtained, either by apheresis or pooled buffy coats, and administered routinely as life-saving procedures during surgery or chemotherapy to patients with low numbers of platelets or those who have their function impaired2,3. The problem with PLCs is their short shelf life when stored at 22 °C, which may lead to a deterioration of platelet functionality, a condition known as platelet storage lesion. Even though improvements in storage materials have lengthened the storage time of PCs, it is still restricted to 5 days for the risk of bacterial infection3. In contrast to other blood products, which can be stored for longer periods of time using colder temperatures (e.g. erythrocyte concentrates and plasma), platelets can’t be preserved in cold conditions because they undergo intense modifications in shape and functionality. These limitations create a constant shortage of PLCs in blood transfusion services. Platelet proteomics aims to help to understand the altered morphological, biochemical and functional features that constitute the phenomenon of storage lesion. By filling this gap of knowledge, proteomics will improve the understanding of the loss of function during storage and help to design new strategies and methods to enhance our ability to maintain platelets in optimal conditions during longer periods of time. During the last 5 years a combination of two-dimensional gel electrophoresis (2-DE)- and mass spectrometry (MS)-based proteomic approaches have been used to profile alterations in platelet proteins during storage. Back in 2007, Thiele and coworkers used two-dimensional differential in-gel electrophoresis (2D-DIGE) technology and MS to assess the effects of storage on the global proteome profile of therapeutic PCs and identify proteins that could be used as sensitive markers for storage related changes2. Their results showed that the platelet proteome remains quite stable during the first 5–9 days of storage, in fact, 97% of the cytosolic platelet proteins didn’t suffer any alterations at day 9. Major alterations of the platelet proteome occurred between day 9 and 15 of storage and these changes are most likely caused by degradation2. Proteins that were detected to change in the remaining 3% of cytosolic platelet proteins after 9 days of storage included septin-2, gelsolin and β-actin. Interestingly septin 2 and gelsolin are affected during apoptosis, indicating that apoptosis in PCs may have an impact on platelet storage. Moreover, those altered proteins could also be used as sensitive markers of platelet deterioration during storage. This study - as the authors recognize - suffers from one the most important limitations of 2-DE-based proteomics, the under-representation of membrane proteins due to their high hydrophobicity. To overcome this limitation, and comprehensively analyze changes suffered by PLCs during storage, Thon and co-workers performed a multi-technique study of the platelet proteome in PLCs over a 7-day storage period3. The authors combined protein-centric (2-DE/DIGE) and peptide-centric techniques (isotope-coded affinity tagging, ICAT; and isotope tagging for relative and absolute quantitation, iTRAQ) and could identify a total of 503 differentially expressed proteins. More precisely, 93 proteins were identified by 2-DE /DIGE, 355 by iTRAQ, and 139 by ICAT. Comparative analysis of 2-DE/DIGE, iTRAQ, and ICAT indicated that only five proteins were common to all three proteomic approaches employed3. There was some overlap among studies, especially between those with the same orientation (peptide- or protein-centric). They could also identify the altered proteins detected by Thiele and collaborators2 (septin-2, gelsolin, β-actin), which further consolidate them as possible markers for changes happening in PLCs. Other protein changes, like the ones seen in talin, 14-3-3, tubulin and thrombospondin, were also in excellent agreement between the two studies. Nevertheless, there were also some disagreements, which might be due to differences in instrumentation, variations in protocols from laboratory-to-laboratory, undiscovered changes in post-translational modifications, or the lack of specific protein detection due to low abundance.4 Platelets stored for transfusion produce prothrombotic and pro-inflammatory mediators implicated in adverse transfusion reactions. Correspondingly, these mediators are central players in pathological conditions including cardiovascular disease, the major cause of death in diabetics. In view of this, in 2009 Springer and coworkers performed a MS-based study where they analyzed platelet proteome changes in PLCs from diabetic patients and healthy donors over a 5-day storage period5. They identified 122 proteins that were either up- or down-regulated in type-2 diabetics relative to non-diabetic controls and 117 proteins whose abundances changed during the storage period. They could replicate some of the findings by Thiele and colleagues2 seeing that septin and actin were increased with storage. The study by Springer and colleagues was the first to characterize the proteome of platelets from diabetics before and after storage for transfusion. Their findings allowed the authors to formulate new hypotheses and experimentation to improve clinical outcomes by targeting “high risk platelets” that render platelet transfusion less effective or even unsafe. In conclusion, the application of platelet proteomics to address clinically relevant issues in the transfusion medicine field is already a promising reality. Obviously, there are many challenges ahead that should be addressed before we can have standardized methods that can be routinely introduced in the clinical practice. The proteomic method/s of choice should be robust, reproducible, and relatively rapid. Multi-center studies could bring light into these issues but there is no doubt the future of this young field is as promising as it was its beginning.