Tityus discrepans scorpion venom activates platelets through GPVI and a novel Src-dependent signaling pathway

In humans and other mammals, Tityus discrepans (Td) scorpion envenomation produces a variety of systemic effects including respiratory distress, a generalized inflammatory reaction, modulation of blood pressure, fibrin formation, and platelet activation. For many of these effects, the venom components and underlying mechanisms are not known. In the present study, we demonstrate that Td venom (TdV) stimulates integrin αIIbβ3-dependent aggregation of washed human and mouse platelets downstream of Src kinase activation. The pattern of increase in tyrosine phosphorylation induced by TdV in human platelets is similar to that induced by the collagen receptor GPVI, and includes FcR γ-chain, Syk, and PLC γ 2. Confirmation of GPVI activation by TdV was achieved by expression of human GPVI in chicken DT40 B cells and use of a reporter assay. To our surprise, TdV was able to activate mouse platelets deficient in the GPVI-FcR γ-chain complex through a pathway that was also dependent on Src kinases. TdV therefore activates platelets through GPVI and a second, as yet unidentified Src kinase-dependent pathway.

The novel Syk inhibitor R406 reveals mechanistic differences in the initiation of GPVI and CLEC-2 signaling in platelets.

The inhibitory effect of R406 provides direct evidence of a role for Syk in GPVI, CLEC-2 and integrin alphaIIbbeta3 signaling in human platelets. Further, the results demonstrate a critical role for Syk in mediating tyrosine phosphorylation of CLEC-2, suggesting a novel model in which both Src and Syk kinases regulate tyrosine phosphorylation of the C-type lectin receptor leading to platelet activation.

A global proteomics approach identifies novel phosphorylated signaling proteins in GPVI-activated platelets: involvement of G6f, a novel platelet Grb2-binding membrane adapter.

Collagen-related peptide (CRP) stimulates powerful activation of platelets through the glycoprotein VI (GPVI)-FcR gamma-chain complex. We have combined proteomics and traditional biochemistry approaches to study the proteome of CRP-activated platelets, focusing in detail on tyrosine phosphorylation. In two separate approaches, phosphotyrosine immunoprecipitations followed by 1-D-PAGE, and 2-DE, were used for protein separation. Proteins were identified by MS. By following these approaches, 96 proteins were found to undergo PTM in response to CRP in human platelets, including 11 novel platelet proteins such as Dok-1, SPIN90, osteoclast stimulating factor 1, and beta-Pix. Interestingly, the type I transmembrane protein G6f was found to be specifically phosphorylated on Tyr-281 in response to platelet activation by CRP, providing a docking site for the adapter Grb2. G6f tyrosine phoshporylation was also found to take place in response to collagen, although not in response to the G protein-coupled receptor agonists, thrombin and ADP. Further, we also demonstrate for the first time that Grb2 and its homolog Gads are tyrosine-phosphorylated in CRP-stimulated platelets. This study provides new insights into the mechanism of platelet activation through the GPVI collagen receptor, helping to build the basis for the development of new drug targets for thrombotic disease.

Comparison of CLEC-2 and GPVI signaling in platelets: the role of adaptor proteins

GPVI activates platelets through an ITAM pathway by activation of Src and Syk kinases leading to activation of PLCy2. CLEC-2 has been shown to activate platelets using an ITAM-like sequence in its cytoplasmic tail that is also dependent on Src and Syk kinases, but shows a partial rather than an absolute dependence on adapter SLP-76 for activation of PLCy2. The aim of this thesis is to understand some of the key differences in these signalling pathways. GPVI is in complex with FcRwhich contains the ITAM sequence (Yxx(L/I)x6−12Yxx(L/I)). These two tyrosines provide a docking site for the tandem-SH2 domains of Syk. In this thesis I show that CLEC-2 signalling through Syk is mediated by phosphorylation of the CLEC-2 YxxL sequence, receptor dimerisation and cross-linking by the Syk SH2 domains. I also show that the differential requirement for SLP-76 is not mediated by Gads. Both signalling pathways also show partial dependency for LAT. I also show that a novel protein, G6f, is not able to substitute for LAT in this signalling pathway and also exclude the LAT-family proteins PAG, LIME, LAX and NTAL as potential LAT replacements in platelet activation by GPVI. These results extend our understanding of platelet activation by CLEC-2.

A novel interaction between FlnA and Syk regulates platelet ITAM-mediated receptor signaling and function

Filamin A (FlnA) cross-links actin filaments and connects the Von Willebrand factor receptor GPIb-IX-V to the underlying cytoskeleton in platelets. Because FlnA deficiency is embryonic lethal, mice lacking FlnA in platelets were generated by breeding FlnA(loxP/loxP) females with GATA1-Cre males. FlnA(loxP/y) GATA1-Cre males have a macrothrombocytopenia and increased tail bleeding times. FlnA-null platelets have decreased expression and altered surface distribution of GPIbalpha because they lack the normal cytoskeletal linkage of GPIbalpha to underlying actin filaments. This results in approximately 70% less platelet coverage on collagen-coated surfaces at shear rates of 1,500/s, compared with wild-type platelets. Unexpectedly, however, immunoreceptor tyrosine-based activation motif (ITAM)- and ITAM-like-mediated signals are severely compromised in FlnA-null platelets. FlnA-null platelets fail to spread and have decreased alpha-granule secretion, integrin alphaIIbbeta3 activation, and protein tyrosine phosphorylation, particularly that of the protein tyrosine kinase Syk and phospholipase C-gamma2, in response to stimulation through the collagen receptor GPVI and the C-type lectin-like receptor 2. This signaling defect was traced to the loss of a novel FlnA-Syk interaction, as Syk binds to FlnA at immunoglobulin-like repeat 5. Our findings reveal that the interaction between FlnA and Syk regulates ITAM- and ITAM-like-containing receptor signaling and platelet function.

Ibrutinib inhibits platelet integrin αIIbβ3 outside-in signaling and thrombus stability but not adhesion to collagen

OBJECTIVE: Ibrutinib is an irreversible Bruton tyrosine kinase inhibitor approved for treatment of Waldenstrom macroglobulinemia, chronic lymphocytic leukemia, and mantle cell lymphoma that increases the risk of bleeding among patients. Platelets from ibrutinib-treated patients exhibit deficiencies in collagen-evoked signaling in suspension; however, the significance of this observation and how it relates to bleeding risk is unclear, as platelets encounter immobile collagen in vivo. We sought to clarify the effects of ibrutinib on platelet function to better understand the mechanism underlying bleeding risk. APPROACH AND RESULTS: By comparing signaling in suspension and during adhesion to immobilized ligands, we found that the collagen signaling deficiency caused by ibrutinib is milder during adhesion to immobilized collagen. We also found that platelets in whole blood treated with ibrutinib adhered to collagen under arterial shear but formed unstable thrombi, suggesting that the collagen signaling deficiency caused by ibrutinib may not be the predominant cause of bleeding in vivo. However, clot retraction and signaling evoked by platelet adhesion to immobilized fibrinogen were also inhibited by ibrutinib, indicating that integrin αIIbβ3 outside-in signaling is also effected in addition to GPVI signaling. When ibrutinib was combined with the P2Y12 inhibitor, cangrelor, thrombus formation under arterial shear was inhibited additively. CONCLUSIONS: These findings suggest that (1) ibrutinib causes GPVI and integrin αIIbβ3 platelet signaling deficiencies that result in formation of unstable thrombi and may contribute toward bleeding observed in vivo and (2) combining ibrutinib with P2Y12 antagonists, which also inhibit thrombus stability, may have a detrimental effect on hemostasis.

Blockade of ActRIIB signaling triggers muscle fatigability and metabolic myopathy

Myostatin regulates skeletal muscle size via the activin receptor IIB (ActRIIB). However, its effect on muscle energy metabolism and energy dependent muscle function remains largely unexplored. This question needs to be solved urgently since various therapies for neuromuscular diseases based on blockade of ActRIIB signaling are being developed. Here we show in mice that four months of pharmacological abrogation of ActRIIB signaling by treatment with soluble ActRIIB-Fc triggers extreme muscle fatigability. This is associated with elevated serum lactate levels and a severe metabolic myopathy in the mdx mouse, an animal model of Duchenne muscular dystrophy. Blockade of ActRIIB signaling down-regulates Porin, a crucial ADP/ATP shuttle between cytosol and mitochondrial matrix leading to a consecutive deficiency of oxidative phosphorylation as measured by in vivo Phophorus Magnetic Resonance Spectroscopy (31P-MRS). Further, ActRIIB blockade reduces muscle capillarization, which further compounds the metabolic stress. We show that ActRIIB regulates key determinants of muscle metabolism, such as Pparβ, Pgc1α, and Pdk4 thereby optimizing different components of muscle energy metabolism. In conclusion, ActRIIB signaling endows skeletal muscle with high oxidative capacity and low fatigability. The severe metabolic side effects following ActRIIB blockade caution against deploying this strategy, at least in isolation, for treatment of neuromuscular disorders.

Endothelin-1 and fibroblast growth factors stimulate the mitogen-activated protein kinase signaling cascade in cardiac myocytes. The potential role of the cascade in the integration of two signaling pathways leading to myocyte hypertrophy.

Maximally effective concentrations of endothelin-1 (ET-1), acidic FGF (aFGF), or 12-O-tetradecanoylphorbol-13-acetate (TPA) activated mitogen-activated protein kinase (MAPK) by 3-4-fold in crude extracts of myocytes cultured from neonatal rat heart ventricles. Maximal activation was achieved after 5 min. Thereafter, MAPK activity stimulated by ET-1 or aFGF declined to control values within 1-2 h, whereas activation by TPA was more sustained. Two peaks of MAPK activity (a 42- and a 44-kDa MAPK) were resolved in cells exposed to ET-1 or aFGF by fast protein liquid chromatography on a Mono Q column. One major and one minor peak of MAPK kinase (MAPKK) was stimulated by ET-1 or aFGF. Cardiac myocytes expressed protein kinase C (PKC)-alpha, -delta, -epsilon and -zeta as shown immunoblotting. Exposure to 1 microM TPA for 24 h down-regulated PKC-alpha, -delta, and -epsilon, but not PKC-zeta. This maneuver wholly abolished the activation of MAPK on re-exposure to TPA but did not affect the response to aFGF. The effect of ET-1 was partially down-regulated. ET-1 stimulated phospho[3H]inositide hydrolysis 18-fold, whereas aFGF stimulated by only 30%. Agonists which initially utilize dissimilar signaling pathways may therefore converge at the level of MAPKK/MAPK and this may be relevant to the hypertrophic response of the heart.

Signaling through the extracellular signal-regulated kinase 1/2 cascade in cardiac myocytes.

The extracellular signal-regulated kinases 1/2 (ERK1/2) are particularly implicated in the growth response of cardiac myocytes. In these cells, the ERK1/2 pathway is potently activated by Gq protein-coupled receptor agonists (such as endothelin-1 or alpha-adrenergic agonists), which activate protein kinase C isoforms. Here, we review the mechanisms associated with the activation of the ERK1/2 pathway by these agonists with particular emphasis on signal integration into the pathway. Signaling to the nucleus and the regulation of transcription factor activity associated with ERK1/2 activation in cardiac myocytes are also discussed.

Oxidative stress and growth-regulating intracellular signaling pathways in cardiac myocytes

The toxic effects of oxidative stress on cells (including cardiac myocytes, the contractile cells of the heart) are well known. However, an increasing body of evidence has suggested that increased production of reactive oxygen species (ROS) promotes cardiac myocyte growth. Thus, ROS may be 'second messenger' molecules in their own right, and growth-promoting neurohumoral agonists might exert their effects by stimulating production of ROS. The authors review the principal growth-promoting intracellular signaling pathways that are activated by ROS in cardiac myocytes, namely the mitogen-activated protein kinase cascades (extracellular signal-regulated kinases 1/2, c-Jun N-terminal kinases, and p38-mitogen-activated protein kinases) and the phosphoinositide 3-kinase/protein kinase B (Akt) pathway. Possible mechanisms are discussed by which these pathways are activated by ROS, including the oxidation of active site cysteinyl residues of protein and lipid phosphatases with their consequent inactivation, the potential involvement of protein kinase C or the apoptosis signal-regulating kinase 1, and the current models for the activation of the guanine nucleotide binding protein Ras.

Signaling pathways mediating cardiac myocyte gene expression in physiological and stress responses

The contractile cells in the heart (the cardiac myocytes) are terminally differentiated. In response to pathophysiological stresses, cardiac myocytes undergo hypertrophic growth or apoptosis, responses associated with the development of cardiac pathologies. There has been much effort expended in gaining an understanding of the stimuli which promote these responses, and in identifying the intracellular signaling pathways which are activated and potentially involved. These signaling pathways presumably modulate gene and protein expression to elicit the end-stage response. For the regulation of gene expression, the signal may traverse the cytoplasm to modulate nuclear-localized transcription factors as occurs with the mitogen-activated protein kinase or protein kinase B/Akt cascades. Alternatively, the signal may promote translocation of transcription factors from the cytoplasm to the nucleus as is seen with the calcineurin/NFAT and JAK/STAT systems. We present an overview of the principal signaling pathways implicated in the regulation of gene expression in cardiac myocyte pathophysiology, and summarize the current understanding of these pathways, the transcription factors they regulate and the changes in gene expression associated with the development of cardiac pathologies. Finally, we discuss how intracellular signaling and gene expression may be integrated to elicit the overall change in cellular phenotype.

Platelet signaling: a complex interplay between inhibitory and activatory networks

The role of platelets in hemostasis and thrombosis is dependent on a complex balance of activatory and inhibitory signaling pathways. Inhibitory signals released from the healthy vasculature suppress platelet activation in the absence of platelet receptor agonists. Activatory signals present at a site of injury initiate platelet activation and thrombus formation; subsequently, endogenous negative signaling regulators dampen activatory signals to control thrombus growth. Understanding the complex interplay between activatory and inhibitory signaling networks is an emerging challenge in the study of platelet biology and necessitates a systematic approach to utilize experimental data effectively. In this review, we will explore the key points of platelet regulation and signaling that maintain platelets in a resting state, mediate activation to elicit thrombus formation or provide negative feedback. Platelet signaling will be described in terms of key signaling molecules that are common to the pathways activated by platelet agonists and can be described as regulatory nodes for both positive and negative regulators. This article is protected by copyright. All rights reserved.

Cognition and anxiety are regulated by thyroid hormone signaling

Anxiety and cognition are both linked to deficits in thyroid hormone concentrations in humans and in rodent models. Both processes have also been shown to be affected by the loss of the thyroid hormone receptors (TR) or by mutant transgenic TRs. Specifically, the unbalanced action of the unliganded TRα1 is thought to be important in the memory deficit and extreme anxiety seen in transgenic mice. The contribution of TRβ is less well defined and the molecular mechanisms that underlie these deficits are also unknown. We review the literature that demonstrates the importance of the thyroid hormone (TH) and the TR in these processes and focus on the mechanisms, in particular adult hippocampal neurogenesis in the dentate gyrus, that might be important in mediating both state anxiety and cognition by thyroid hormone.