Current Proteomics - Volume 8, Issue 3, 2011

This Special Edition of Current Proteomics is devoted to recent advances in blood platelets that have adapted proteomic or genomic technologies to dissect platelet function. The contents are relevant to the broad audience of biomedical researchers, clinicians, and biotechnology companies interested in novel approaches related to platelet biology. Platelets are fundamentally important in regulating normal hemostasis and pathological thrombus formation, and represent major cellular targets for drug discovery and therapeutics related to cerebro- and cardiovascular disorders. It is fully anticipated that platelet-directed therapies will likely have broader (and unexpected) therapeutic applicability, as highlighted by the recent application of platelet rich plasma as a novel therapeutic modality in sports medicine. From a historical perspective, the emergence of platelet-directed dialogues in the lay press is quite startling for cellular elements that were initially described as “particulate dust” during the early days of microscopy. This Special Edition contains seven original manuscripts from accomplished investigators that summarize basic and applied research that have re-defined the complexity of platelet biology, while developing novel model systems that will re-shape the fluctuating paradigm of platelet function. The lead two articles provide highly informative reviews that survey the range of proteomic and bioinformatic technologies that collectively serve to define the proteins and networks functionally important during quiescent and activated biologic conditions. The article by Premsler et al. presents an overview of the subcellular contents (granules, membrane composition and platelet-derived microparticles) that collectively define the platelet proteome, while the complementary article by Dittrich et al. provides insight into the dynamic networks that accompany intracellular signaling events. Presentation of a unique bioinformatic resource (PlateletWeb) that helps to dissect the functional interactome represents a highlight of this review. The article by Campbell et al. is a forward-looking review that updates the reader on the evolving field of platelet protein synthesis and translational controls, a surprisingly fresh area of investigation that delineates the capacity of blood platelets to synthesize proteins constitutively and upon activation. The related article by Landry et al. summarizes the experimental evidence and advances delineating the unique role of microRNAs as modulators of the platelet proteome. Support for this area of investigation is predicated on data highlighting the abundance and diversity of miRNAs in human platelets that among other functions may regulate expression of the platelet P2Y12 (ADP) receptor. The article by Maguire et al. was specifically included because of its application of proteomic approaches to dissect a novel pathway in platelet function (canonical Wnt pathway), with recent evidence that suppression of platelet activation and adhesion can be effected by a targeted Wnt ligand. The subsequent article by Dr. Klement provides an overview of the health-related implications of identifying plateletrestricted biomarkers as proteomic tools for disease diagnosis and monitoring. The article reviews the capacity of platelets to sequester plasma proteins, and how this provides a unique opportunity for biomarker development, emphasizing not only the interplay between platelets, angiogenesis, and cancer metastases, but also its relevance to other clinical conditions; this article is especially relevant for development of translational technologies. The Special Edition concludes with a comprehensive article on the application of new proteomic tools for identification of novel platelet targets. Building on progress in cancer and other diseases, the application and development of novel activity-based probes coupled with mass spectrometric technologies is likely to provide new insight into platelet signaling pathways and targeted drug development. In concluding, it is important to acknowledge the dedicated input of all the authors who have not only provided articles of the highest caliber, but were instrumental in providing highly thoughtful critiques that have collectively enhanced the overall quality of this Special Edition The successful completion of such a volume requires expert administrative assistance which was provided by Ms. Stacey Hondropulos.

As the clinical use of biologic response modifiers continues to increase, the old way of estimating effective doses in oncology, i.e. reaching dose-limiting toxicities, is becoming obsolete. Biologic response modifiers and targeted therapies are less toxic and their effective dose is often left shifted on the dose response curve. This is why the majority of pharmaceutical companies have opened new programs seeking biomarker of therapeutic response, and why numerous research programs have been announcing Request for Applications in order to find and evaluate biomarkers of disease. Because of the ease of procurement of the clinical specimen, plasma and serum, remain the favorite clinical analytes. However, the sheer numbers of different plasma proteins, the many thousand fold differences in the amounts of the potential protein biomarkers, and the lack of specificity of plasma proteins have hindered the search. The ability to detect changes in the levels of proteins expressed in picomolar quantities are mired by the presence of more abundant nonspecific proteins such as albumin or immunoglobulins. Most meaningful changes, those that occur in the amounts of free proteins, remain very difficult to evaluate. The recent discovery that angiogenesis regulators are actively and selectively sequestered in platelets early in cancer, has led to renewed hopes of finding circulating biomarkers that would help in early disease detection, and improve our ability to detect an early therapeutic response. There are early indicators that this is the case in other diseases as well, and that the focus may be shifting from analyzing plasma and serum, to analyzing platelets.

Blood platelets are key players mainly involved in physiological thrombus formation as well as pathological events, such as atherosclerosis, coronary artery disease, or myocardial infarction. Consequently, numerous studies have focused on gaining insight into the platelet proteome, with biomolecular mass spectrometry providing an efficient technology to cope with this task as it nowadays enables both protein identification and differential quantification of cellular material. To assemble a comprehensive proteomic dataset, platelet-centric studies have to be performed not only on the global scale, but also on the subcellular level, which implies subfractionation techniques and targeted approaches prior to detailed proteomic analyses. In this review we present an overview of advances in platelet subcellular proteomics focussing on the platelet membrane proteome and the protein content of both platelet granules and platelet-derived microparticles. Since posttranslational modifications are furthermore known to decisively participate in platelet function e.g. upon activation, analytical methods for the platelet phospho- as well as the glycoproteome will also be discussed.

MicroRNAs are short 21- to 24-nucleotide (nt) RNA species that act as key regulators of gene expression. Known primarily to modulate mRNA translation through recognition of specific binding sites located in the 3'untranslated region (UTR) of messenger RNA (mRNA) targets, microRNAs may regulate between 30% to 92% of the genes in human, thereby controlling a plethora of biological processes. Although devoid of a nucleus and lacking genomic DNA, platelets may be no exception, as recent experimental evidences indicate that they contain all the protein and RNA components and features required for microRNA-regulated mRNA translation: (i) the platelet transcriptome is astonishingly diverse, representing between 15 and 32% of all human genes, (ii) platelet mRNAs can be translated into proteins, (iii) platelets contain an abundant and diverse array of microRNAs, and (iv) the host Dicer and Argonaute 2 (Ago2) complexes. The latter ones are functional in microRNA biogenesis and function, respectively. In this review article, we will summarize and discuss the experimental evidences as well as the most recent advances supporting a role for microRNAs as modulators of the platelet proteome. Expected to play a central role in health and disease, a dysfunctional microRNA-based regulation of gene expression in platelets may represent an important etiologic factor underlying platelet-related and cardiovascular diseases.

Human platelets are released from the cytoplasm of megakaryocytes and average 2-3 μm in diameter. In response to vascular injury, platelets stick together like glue, seal the wounded area, and dispense their contents into the nearby milieu. Taken at face value, these features and functions seem simplistic and unrefined. As one digs deeper, however, it becomes apparent that platelets are intricately wired and created for multifunctional purposes. One of their duties, which cropped up over the last decade, is to synthesize proteins both continually and on demand. It turns out that platelets possess thousands of template mRNAs, ribosomes, and requisite translational machinery that they use to generate new proteins. Platelets also retain tools that grant them the ability to process precursor mRNAs and microRNAs. In this review, we briefly describe what we currently know about protein synthesis in platelets, its functional significance, and where the field is likely to take us over the next decade.

Unbiased proteomic analyses of cell fractions, interactomes and protein modifications coupled with more targeted approaches are adding to an impressive database of the signalling pathways in platelets. In addition to the well characterised receptors that are known to exist on the platelet surface, platelet proteomic studies continue to expose novel transmembrane proteins including CD148, CLEC-2, Eph kinases and Ephrins, Frizzled-4 and -6, G6b, HIP-55, HSP47, LRP5/6 and PEAR-1. In turn identification of novel platelet receptors has led to the discovery of new platelet signalling pathways such as the collagen/CLEC-2 receptor pathway, as well as the canonical WNT pathway. This review focuses on the canonical WNT pathway, providing background information to WNT ligands, receptors and signalling pathways and then focusing on canonical WNT signalling in anucleate platelets, where the suppression of platelet activity and adhesion by its ligand, Wnt-3a has recently been demonstrated.

Human platelets are thought to express approximately 2000-3000 proteins, but post-translational modifications, alternatively spliced variants and a rich diversity of vertebrate domain architectures likely make this a conservative estimate. Even though rapidly advancing proteomic techniques have catalyzed the identification of roughly one third of the platelet proteome, a combination of abundance-based and activity-based proteomics is needed for elucidation of platelet functional characteristics including the definition of a “core proteome” and recognition of diverse enzyme activity profiles associated with various physiological states. In this review, we describe the latest mass spectrometry-based techniques capable of providing some of these physiological details required for more comprehensive evaluation of the human platelet repertoire.

Human platelets are anucleate cells or rather cellular fragments derived from megakaryocytes. With a life span of just a few days they are nevertheless central components of the haemostatic system and, apart from being the major players in haemostatic processes, they are also involved in the pathophysiology of many cardiovascular diseases and are engaged in inflammatory processes like sepsis. To achieve this, platelets are optimized for rapid adaptation and signalling and are filled with a number of intricate signalling cascades. Their interplay enables a number of different system states, ranging from quiescence, basic activation, rapid and irreversible full activation to more or less strong inhibitory states. Appropriate databases and references are given together with a primer on integrated network analysis of the platelet starting from its proteome and transcriptome. There are different preferences of network states in healthy or ill conditions including sepsis and disseminated coagulopathy. Both clinical and basic researchers have great interest in understanding platelet physiology by untangling the intricate network of cellular signalling. This holds promise for better diagnostics, monitoring and therapy as well as identifying potential novel drug targets and development of novel antithrombotic strategies.

Of systemic type of amyloidosis, transthyretin (TTR)-related familial amyloidotic polyneuropathy (FAP) is a fatal hereditary amyloidosis with autosomal dominant trait. As of today, reports of 121 different points of mutations or a deletion in the TTR gene have been identified. In addition, senile systemic amyloidosis induced by wild type TTR has been focused in the recent attention. Early diagnosis of FAP is absolutely imperative for the treatments, because duration of the disease is one of the most important prognostic factors for patients' survival after treatments including liver transplantation. To make a diagnosis of FAP sooner, we established a novel rapid diagnostic system. It included histopathological, genetic and proteomic techniques used to detect amyloid deposits in tissues, genetic mutations of the TTR gene, and variant TTR in serum. Mass spectrometric analyses can clearly detect TTR variants in the most of FAP cases and can be used for screening and double checking TTR variants with the genetic testing. In this review, we introduce recent research progress in ATTR amyloidosis and our diagnostic system.