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We lead multidisciplinary applied research and training to rethink the way health care is delivered in general practice and across the community.
Integrin-linked kinase regulates the rate of platelet activation and is essential for the formation of stable thrombi
Background: Integrin-linked kinase (ILK) and its associated complex of proteins are involved in many cellular activation processes, including cell adhesion and integrin signaling. We have previously demonstrated that mice with induced platelet ILK deficiency show reduced platelet activation and aggregation, but only a minor bleeding defect. Here, we explore this apparent disparity between the cellular and hemostatic phenotypes. Methods: The impact of ILK inhibition on integrin αIIbβ3 activation and degranulation was assessed with the ILK-specific inhibitor QLT0267, and a conditional ILK-deficient mouse model was used to assess the impact of ILK deficiency on in vivo platelet aggregation and thrombus formation. Results: Inhibition of ILK reduced the rate of both fibrinogen binding and α-granule secretion, but was accompanied by only a moderate reduction in the maximum extent of platelet activation or aggregation in vitro. The reduction in the rate of fibrinogen binding occurred prior to degranulation or translocation of αIIbβ3 to the platelet surface. The change in the rate of platelet activation in the absence of functional ILK led to a reduction in platelet aggregation in vivo, but did not change the size of thrombi formed following laser injury of the cremaster arteriole wall in ILK-deficient mice. It did, however, result in a marked decrease in the stability of thrombi formed in ILK-deficient mice. Conclusion: Taken together, the findings of this study indicate that, although ILK is not essential for platelet activation, it plays a critical role in facilitating rapid platelet activation, which is essential for stable thrombus formation. © 2014 The Authors Journal of Thrombosis and Haemostasis.
Platelet endothelial cell adhesion molecule-1 inhibits platelet response to thrombin and von willebrand factor by regulating the internalization of glycoprotein Ib via AKT/glycogen synthase kinase-3/dynamin and integrin αiIbβ3
OBJECTIVE - Platelet endothelial cell adhesion molecule-1 (PECAM-1) regulates platelet response to multiple agonists. How this immunoreceptor tyrosine-based inhibitory motif-containing receptor inhibits G protein-coupled receptor-mediated thrombin-induced activation of platelets is unknown. APPROACH AND RESULTS - Here, we show that the activation of PECAM-1 inhibits fibrinogen binding to integrin αIIbβ3 and P-selectin surface expression in response to thrombin (0.1-3 U/mL) but not thrombin receptor-activating peptides SFLLRN (3×10-1×10 mol/L) and GYPGQV (3×10-1×10 mol/L). We hypothesized a role for PECAM-1 in reducing the tethering of thrombin to glycoprotein Ibα (GPIbα) on the platelet surface. We show that PECAM-1 signaling regulates the binding of fluorescein isothiocyanate-labeled thrombin to the platelet surface and reduces the levels of cell surface GPIbα by promoting its internalization, while concomitantly reducing the binding of platelets to von Willebrand factor under flow in vitro. PECAM-1-mediated internalization of GPIbα was reduced in the presence of both EGTA and cytochalasin D or latrunculin, but not either individually, and was reduced in mice in which tyrosines 747 and 759 of the cytoplasmic tail of β3 integrin were mutated to phenylalanine. Furthermore, PECAM-1 cross-linking led to a significant reduction in the phosphorylation of glycogen synthase kinase-3β Ser, but interestingly an increase in glycogen synthase kinase-3α pSer. PECAM-1-mediated internalization of GPIbα was reduced by inhibitors of dynamin (Dynasore) and glycogen synthase kinase-3 (CHIR99021), an effect that was enhanced in the presence of EGTA. CONCLUSIONS - PECAM-1 mediates internalization of GPIbα in platelets through dual AKT/protein kinase B/glycogen synthase kinase-3/dynamin-dependent and αIIbβ3-dependent mechanisms. These findings expand our understanding of how PECAM-1 regulates nonimmunoreceptor signaling pathways and helps to explains how PECAM-1 regulates thrombosis. © 2014 American Heart Association, Inc.
Gap junctions and connexin hemichannels underpin hemostasis and thrombosis
Background-Connexins are a widespread family of membrane proteins that assemble into hexameric hemichannels, also known as connexons. Connexons regulate membrane permeability in individual cells or couple between adjacent cells to form gap junctions and thereby provide a pathway for regulated intercellular communication. We have examined the role of connexins in platelets, blood cells that circulate in isolation but on tissue injury adhere to each other and the vessel wall to prevent blood loss and to facilitate wound repair. Methods and Results-We report the presence of connexins in platelets, notably connexin37, and that the formation of gap junctions within platelet thrombi is required for the control of clot retraction. Inhibition of connexin function modulated a range of platelet functional responses before platelet-platelet contact and reduced laser-induced thrombosis in vivo in mice. Deletion of the Cx37 gene (Gja4) in transgenic mice reduced platelet aggregation, fibrinogen binding, granule secretion, and clot retraction, indicating an important role for connexin37 hemichannels and gap junctions in platelet thrombus function. Conclusions-Together, these data demonstrate that platelet gap junctions and hemichannels underpin the control of hemostasis and thrombosis and represent potential therapeutic targets. © 2012 American Heart Association, Inc.
Clot retraction
The study of clot retraction in vitro has been adopted as a simple and reproducible approach to assess platelet function. Plasma clots should retract away from the sides of a glass tube within a few hours allowing the rapid characterization of outside-in signaling through platelet integrin α IIbβ 3. In this chapter, we describe the role of platelets in fibrin clot retraction and provide a detailed description of the methods used to assess this process. © 2012 Springer Science+Business Media, LLC.
A functional proteomic method for the enrichment of peripheral membrane proteins reveals the collagen binding protein Hsp47 is exposed on the surface of activated human platelets
Platelets are small blood cells vital for hemostasis. Following vascular damage, platelets adhere to collagens and activate, forming a thrombus that plugs the wound and prevents blood loss. Stimulation of the platelet collagen receptor glycoprotein VI (GPVI) allows recruitment of proteins to receptorproximal signaling complexes on the inner-leaflet of the plasma membrane. These proteins are often present at low concentrations; therefore, signaling-complex characterization using mass spectrometry is limited due to high sample complexity. We describe a method that facilitates detection of signaling proteins concentrated on membranes. Peripheral membrane proteins (reversibly associated with membranes) were eluted from human platelets with alkaline sodium carbonate. Liquid-phase isoelectric focusing and gel electrophoresis were used to identify proteins that changed in levels on membranes from GPVI-stimulated platelets. Immunoblot analysis verified protein recruitment to platelet membranes and subsequent protein phosphorylation was preserved. Hsp47, a collagen binding protein, was among the proteins identified and found to be exposed on the surface of GPVI-activated platelets. Inhibition of Hsp47 abolished platelet aggregation in response to collagen, while only partially reducing aggregation in response to other platelet agonists. We propose that Hsp47 may therefore play a role in hemostasis and thrombosis. © 2009 American Chemical Society.
Proteomic analysis of resting and thrombin-stimulated platelets reveals the translocation and functional relevance of HIP-55 in platelets
The platelet surface is a dynamic interface that changes rapidly in response to stimuli to coordinate the formation of thrombi at sites of vascular injury. Tight control is essential as loss of organisation may result in the inappropriate formation of thrombi (thrombosis) or excessive bleeding. In this paper we describe the comparative analysis of resting and thrombin-stimulated platelet membrane proteomes and associated proteins to identify proteins important to platelet function. Surface proteins were labelled using a biotin tag and isolated by NeurtrAvidin affinity chromatography. Liquid phase IEF and SDS-PAGE were used to separate proteins, and bands of increased intensity in the stimulated platelet fractions were digested and identified by FT-ICR mass spectrometry. Novel proteins were identified along with proteins known to be translocated to the platelet surface. Furthermore, many platelet proteins revealed changes in location associated with function, including G6B and Hip-55. HIP-55 is an SH3-binding protein important in T-cell receptor signalling. Further analysis of HIP-55 revealed that this adaptor protein becomes increasingly associated with both Syk and integrin β3 upon platelet activation. Analysis of HIP-55 deficient platelets revealed reduced fibrinogen binding upon thrombin stimulation, suggesting HIP-55 to be an important regulator of platelet function. © 2009 Wiley-VCH Verlag GmbH & Co. KGaA.
Peripheral tachykinins and the neurokinin receptor NK1 are required for platelet thrombus formation
Platelets play an important role in hemostasis, with inappropriate platelet activation being a major contributor to debilitating and often fatal thrombosis by causing myocardial infarction and stroke. Although current antithrombotic treatment is generally well tolerated and effective, many patients still experience cardiovascular problems, which may reflect the existence of alternative underlying regulatory mechanisms in platelets to those targeted by existing drugs. In this study, we define a role for peripherally distributed members of the tachykinin family of peptides, namely substance P and the newly discovered endokinins A and B that are present in platelets, in the activation of platelet function and thrombus formation. We have reported previously that the preferred pharmacologically characterized receptor for these peptides, the NK1 receptor, is present on platelets. Inhibition or deficiency of the NK1 receptor, or SP agonist activity, resulted in substantially reduced thrombus formation in vitro under arterial flow conditions, increased bleeding time in mice, and a decrease in experimentally induced thromboembolism. Inhibition of the NK1 receptor may therefore provide benefit in patients vulnerable to thrombosis and may offer an alternative therapeutic target. © 2008 by The American Society of Hematology.
Future innovations in anti-platelet therapies
Platelets have long been recognized to be of central importance in haemostasis, but their participation in pathological conditions such as thrombosis, atherosclerosis and inflammation is now also well established. The platelet has therefore become a key target in therapies to combat cardiovascular disease. Anti-platelet therapies are used widely, but current approaches lack efficacy in a proportion of patients, and are associated with side effects including problem bleeding. In the last decade, substantial progress has been made in understanding the regulation of platelet function, including the characterization of new ligands, platelet-specific receptors and cell signalling pathways. It is anticipated this progress will impact positively on the future innovations towards more effective and safer anti-platelet agents. In this review, the mechanisms of platelet regulation and current anti-platelet therapies are introduced, and strong, and some more speculative, potential candidate target molecules for future anti-platelet drug development are discussed. © 2008 Nature Publishing Group All rights reserved.
A dual role for integrin-linked kinase in platelets: Regulating integrin function and ά-granule secretion
Integrin-linked kinase (ILK) has been implicated in the regulation of a range of fundamental biological processes such as cell survival, growth, differentiation, and adhesion. In platelets ILK associates with β1- and β3-containing integrins, which are of paramount importance for the function of platelets. Upon stimulation of platelets this association with the integrins is increased and ILK kinase activity is up-regulated, suggesting that ILK may be important for the coordination of platelet responses. In this study a conditional knockout mouse model was developed to examine the role of ILK in platelets. The ILK-deficient mice showed an increased bleeding time and volume, and despite normal ultrastructure the function of ILK-deficient platelets was decreased significantly. This included reduced aggregation, fibrinogen binding, and thrombus formation under arterial flow conditions. Furthermore, although early collagen stimulated signaling such as PLCγ2 phosphorylation and calcium mobilization were unaffected in ILK-deficient platelets, a selective defect in α-granule, but not dense-granule, secretion was observed. These results indicate that as well as involvement in the control of integrin affinity, ILK is required for (v-granule secretion and therefore may play a central role in the regulation of platelet function. © 2008 by The American Society of Hematology.
Understanding the physiology of pre-fertilisation events in the human spermatozoa--a necessary prerequisite to developing rational therapy.
Sperm dysfunction is the single most common defined cause of infertility. One in 15 men is sub-fertile and the condition is increasing in frequency. However, the diagnosis is poor and, excluding assisted conception, there is no treatment. The reason for this is our limited understanding of the biochemical, molecular and genetic functions of the spermatozoon. The underlying premise of our research programme is to establish a rudimentary understanding of the processes necessary for successful fertilisation. In this manuscript, we detail advances in our understanding of calcium signalling in the cell and outline genetic and proteomic technologies that are being used to improve the diagnosis of the condition.
Platelet PECAM-1 inhibits thrombus formation in vivo
Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a cell surface glycoprotein receptor expressed on a range of blood cells, including platelets, and on vascular endothelial cells. PECAM-1 possesses adhesive and signaling properties, the latter being mediated by immunoreceptor tyrosine-based inhibitory motifs present on the cytoplasmic tail of the protein. Recent studies in vitro have demonstrated that PECAM-1 signaling inhibits the aggregation of platelets. In the present study we have used PECAM-1-deficient mice and radiation chimeras to investigate the function of this receptor in the regulation of thrombus formation. Using intravital microscopy and laser-induced injury to cremaster muscle arterioles, we show that thrombi formed in PECAM-1-deficient mice were larger, formed more rapidly than in control mice, and were more stable. Larger thrombi were also formed in control mice that received transplants of PECAM-1-deficient bone marrow, in comparison to mice that received control transplants. A ferric chloride model of thrombosis was used to investigate thrombus formation in carotid arteries. In PECAM-1-deficient mice the time to 75% vessel occlusion was significantly shorter than in control mice. These data provide evidence for the involvement of platelet PECAM-1 in the negative regulation of thrombus formation. © 2006 by The American Society of Hematology.
Is if functional?
This report describes the first scientific meeting of the British Society for Proteome Research (BSPR), which was organised jointly with the European Bioinformatics Institute (EBI) and held in July 2004. The focus of the conference was functional proteomics with an emphasis on possible clinical application. The main subjects described here are: the need to simplify samples, the use of biological fluids verses tissue, consideration of biological and experimental variation and the creation of databases to achieve menaingful functional analysis.
Physiological and proteomic approaches to studying prefertilization events in the human
This research aims firstly to understand, in cellular and molecular terms, how a mature human spermatozoon is prepared for fertilization, and secondly, to identify what factors are involved in the initial signalling interactions between the egg and spermatozoon. In order to achieve these objectives, a combination of approaches is being used, including single-cell imaging, patch clamping and proteomics. Single-cell imaging reveals hidden complexity and heterogeneity in signalling responses in spermatozoa. Characterization of cell physiology at the single-cell level must be a future aim, including the study of ion channel expression and function by patch clamping. Proteomic experiments are aimed at identifying defects in protein expression in specific subgroups of men, e.g. those with globozoospermia. A better understanding of prefertilization events will allow the development of non-assisted reproductive therapy, drug-based treatments for male infertility.
Sperm proteome mapping of a patient who experiencd failed fertilization at IVF reveals altered expression of at least 20 proteins compared with fertile donors: Case report
The aim of this study was to compare the sperm protein expression profile (proteome map) from a patient who experienced failed fertilization at IVF with fertile controls. One patient and three fertile donor sperm samples were characterized using two-dimensional electrophoresis. Differences in protein expression were established using gel analysis software before attempted protein identification. Gel analysis of the fertile donor proteome maps revealed excellent reproducibility as well as very low intra-donor and inter-donor variability in the presence of protein spots. In the patient samples, we have noted 20 consistent differences in protein expression (six spots missing, three additional spots, four less abundant, seven more abundant) compared with the controls. Two proteins that were more intense in the patient have been conclusively identified as secretory actin-binding protein and outer dense fibre protein 2/2. In conclusion proteome variation between different fertile donors was very low. In contrast, the patient proteome exhibited 20 differences compared with controls, which we believe is an underestimate. These proteins merit further investigation to determine whether failed fertilization at IVF might be caused by abnormalities in their expression. This case report represents a proof of principle that proteomics may be useful to study defects in sperm function. © European Society of Human Reproduction and Embryology 2004; all rights reserved.
Platelet Proteomics
Features of this book include: Coverage of non-gel based separation methods (MudPit), analysis of the general platelet proteome and signaling cascades by gel ...
The distribution and expression of a biotrophy-related gene, CIH1, within the genus Colletotrichum
During the biotrophic phase of the infection process of the hemibiotrophic anthracnose fungus Colletotrichum lindemuthianum, an intracellular hypha develops within epidermal cells of its host, Phaseolus vulgaris. This is followed by the formation of secondary hyphae during the necrotrophic phase. Previous work using a monoclonal antibody, UB25, has identified a glycoprotein that is specific to the interfacial matrix that forms between the wall of the intracellular hypha and the invaginated host plasma membrane. The gene encoding the protein identified by UB25 was cloned by immunoscreening and designated CIH1.The predicted amino acid sequence revealed a proline-rich glycoprotein, and biochemical evidence suggested that it formed a cross-linked structure at the biotrophic interface. Although CIH1 is a fungal gene, its product has several similarities to plant cell wall proteins. In this paper, we have surveyed the distribution and expression of CIH1 within the genus Colletotrichum, encompassing both necrotrophic and hemibiotrophic species. The results show that homologues of the CIH1 gene are present in all the Colletotrichum species tested. Northern blot studies of the time course of the infection process in planta have shown that CIH1 is expressed by both C. lindemuthianum in bean and C. trifolii in alfalfa during the biotrophic phase of fungal development, Immunofluorescence labelling of infected epidermal strips with UB25 revealed that the intracellular hyphae formed by C. destructivum as it infects alfalfa were specifically labelled in a similar way to those formed by C. lindemuthianum in bean. Northern and Western analysis showed that CIH1 was also expressed by C. lindemuthianum in vitro, though not constitutively. Overall, the evidence supports a role for CIH1 in biotrophy within the genus Colletotrichum.
NAADP regulates human platelet function
Platelets play a vital role in maintaining haemostasis. Human platelet activation depends on Ca release, leading to cell activation, granule secretion and aggregation. NAADP (nicotinic acid-adenine dinucleotide phosphate) is a Ca -releasing second messenger that acts on acidic Ca stores and is used by a number of mammalian systems. In human platelets, NAADP has been shown to release Ca in permeabilized human platelets and contribute to thrombin-mediated platelet activation. In the present study, we have further characterized NAADP-mediated Ca release in human platelets in response to both thrombin and the GPVI (glycoprotein VI)-specific agonist CRP (collagen-related peptide). Using a radioligand-binding assay, we reveal an NAADP-binding site in human platelets, indicative of a platelet NAADP receptor. We also found that NAADP releases loaded Ca from intracellular stores and that total platelet Ca release is inhibited by the proton ionophore nigericin. Ned-19, a novel cell-permeant NAADP receptor antagonist, competes for the NAADP-binding site in platelets and can inhibit both thrombin- and CRP-induced Ca release in human platelets. Ned-19 has an inhibitory effect on platelet aggregation, secretion and spreading. In addition, Ned-19 extends the clotting time in whole-blood samples. We conclude that NAADP plays an important role in human platelet function. Furthermore, the development of Ned-19 as an NAADP receptor antagonist provides a potential avenue for platelet-targeted therapy and the regulation of thrombosis. © The Authors Journal compilation © 2012 Biochemical Society. 2+ 2+ 2+ 2+ 2+ 45 2+ 2+ 2+