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Platelets and their role in thrombotic and cardiovascular disease: the impact of proteomic analysis
No full textThis chapter provides an overview of how proteomics research impacts on our understanding of platelets. In addition to their role in hemostasis, inappropriate platelet activation is strongly related to the leading cause of death in Western societies: thrombotic cardiovascular disease. The known processes of platelet activation and the signaling mechanisms that regulate these are detailed here, but this knowledge is incomplete. Mass-spectrometry-based proteomics has already contributed to a growth in the understanding of platelets and presents itself as a tool that can unravel the details of the control of platelet function in health and disease through continuing refinements in technology and experimental design toward the development of diagnostic tools and antithrombotic drugs. © 2011 John Wiley and Sons, Inc
Isolation and characterization of cotiaractivase, a novel low molecular weight prothrombin activator from the venom of Bothrops cotiara.
No full textIn this study, we isolated a novel prothrombin activator from the venom of Bothrops cotiara, a Brazilian lance-headed pit viper (Cotiara, Jararaca preta, Biocotiara), which we have designated "cotiaractivase" (prefix: cotiar- from B. cotiara; suffix: -activase, from prothrombin activating activity). Cotiaractivase was purified using a phenyl-Superose hydrophobic interaction column followed by a Mono-Q anion exchange column. It is a single-chain polypeptide with a molecular weight of 22,931 Da as measured by mass spectroscopy. Cotiaractivase generated active alpha-thrombin from purified human prothrombin in a Ca2+-dependent manner as assessed by S2238 chromogenic substrate assay and SDS-PAGE. Cotiaractivase cleaved prothrombin at positions Arg271-Thr272 and Arg320-Ile321, which are also cleaved by factor Xa. However, the rate of thrombin generation by cotiaractivase was approximately 60-fold less than factor Xa alone and 17 x 10(6)-fold less than the prothrombinase complex. The enzymatic activity of cotiaractivase was inhibited by the chelating agent EDTA, whereas the serine protease inhibitor PMSF had no effect on its activity, suggesting that it is a metalloproteinase. Interestingly, S2238 inhibited cotiaractivase activity non-competitively, suggesting that this toxin contains an exosite that allows it to bind prothrombin independently of its active site. Tandem mass spectrometry and N-terminal sequencing of purified cotiaractivase identified peptides that were identical to regions of the cysteine-rich and disintegrin-like domains of known snake venom metalloproteinases. Cotiaractivase is a unique low molecular weight snake venom prothrombin activator that likely belongs to the metalloproteinase family of proteins
Platelet proteomics: state of the art and future perspective.
No full textPlatelets pose unique challenges to cell biologists due to their lack of nucleus and low levels of messenger RNA. Platelets cannot be cultured in great abundance or manipulated using common recombinant DNA technologies. As a result, platelet research has lagged behind that of nucleated cells. The advent of mass spectrometry and its application to protein biochemistry brought with it great hopes for the platelet community that are now being realized. This technology is ideally suited for identifying low-abundance proteins, protein-protein interactions, and post-translational modifications in complex protein mixtures. Over the past 10 years, proteomics has delivered in many ways, providing platelet biologists with a comprehensive list of proteins expressed in platelets, information on post-translational modifications, protein interactions and sub-cellular localization. Several novel and important platelet membrane proteins, including CLEC-2, CD148, G6b-B, G6f, and Hsp47, have been identified using proteomics-based approaches. New, more sensitive instrumentation and novel approaches are making it increasingly possible to identify ever lower amounts of proteins. In this chapter we highlight some of the major achievements of platelet proteomics to date, discussing challenges and how they were overcome. We also discuss new frontiers and applications of proteomics to platelets and microparticles in health and disease, as we strive to better understand the molecular mechanisms underlying the platelet response to vascular injury
Targeting Receptor-Type Protein Tyrosine Phosphatases with Biotherapeutics: Is Outside-in Better than Inside-Out?
No full textProtein tyrosine phosphatases (PTPs), of the receptor and non-receptor classes, are key signaling molecules that play critical roles in cellular regulation underlying diverse physiological events. Aberrant signaling as a result of genetic mutation or altered expression levels has been associated with several diseases and treatment via pharmacological intervention at the level of PTPs has been widely explored; however, the challenges associated with development of small molecule phosphatase inhibitors targeting the intracellular phosphatase domain (the “inside-out” approach) have been well documented and as yet there are no clinically approved drugs targeting these enzymes. The alternative approach of targeting receptor PTPs with biotherapeutic agents (such as monoclonal antibodies or engineered fusion proteins; the “outside-in” approach) that interact with the extracellular ectodomain offers many advantages, and there have been a number of exciting recent developments in this field. Here we provide a brief overview of the receptor PTP family and an update on the emerging area of receptor PTP-targeted biotherapeutics for CD148, vascular endothelial-protein tyrosine phosphatase (VE-PTP), receptor-type PTPs σ, γ, ζ (RPTPσ, RPTPγ, RPTPζ) and CD45, and discussion of future potential in this area
Faculty Opinions recommendation of A dominant gain-of-function mutation in universal tyrosine kinase SRC causes thrombocytopenia, myelofibrosis, bleeding, and bone pathologies.
No full textSignalling Pathways Regulating Platelet Biogenesis
No full textPlatelet biogenesis is a complex process controlled by a combination of cell-extrinsic and cell-intrinsic factors. Cell-extrinsic factors include cytokines and growth factors, chemokines, extracellular matrix proteins, cell-cell interactions and shear forces within the bone marrow milieu that act on megakaryocytes. Cell-intrinsic factors include receptors and associated signalling pathways, cytoskeletal structures, lipid and cation concentrations found within megakaryocytes. Collectively, these two sets of factors control the differentiation, proliferation and survival of megakaryocytes and produce platelets. Recent discoveries have greatly increased our understanding of the molecular mechanisms controlling platelet biogenesis; however, major gaps remain in our knowledge regarding signalling events regulating the transition from megakaryopoiesis to thrombopoiesis, the triggering of proplatelet formation and platelet release and the inhibition of activation signals within megakaryocytes during the course of platelet biogenesis. A major step will be to map all of the signalling networks within megakaryocytes and elucidate their interconnectedness. In this chapter, we describe some of the major signalling pathways that regulate platelet biogenesis, novel modes of regulation and inhibitory mechanisms that prevent uncontrolled megakaryocyte and platelet activation. We also highlight key questions that remain to be addressed and propose potential mechanisms. It is only through a comprehensive understanding of how platelet biogenesis is regulated that we will be able to identify key signalling nodes that can be targeted to modulate platelet homeostasis in health and disease
Proplatelets slip slidin' away
No full textIn this issue of Blood, Bender et al provide compelling evidence that the motor protein cytoplasmic dynein provides the necessary force for microtubule sliding and proplatelet elongation from megakaryocytes.</p
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