26 research outputs found
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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
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Cobimetinib and trametinib inhibit platelet MEK but do not cause platelet dysfunction
The MEK inhibitors cobimetinib and trametinib are used in combination with BRAF inhibitors to treat metastatic melanoma but increase rates of hemorrhage relative to BRAF inhibitors alone. Platelets express several members of the MAPK signalling cascade including MEK1 and MEK2 and ERK1 and ERK2 but their role in platelet function and haemostasis is ambiguous as previous reports have been contradictory. It is therefore unclear if MEK inhibitors might be causing platelet dysfunction and contributing to increased hemorrhage. In the present study we performed pharmacological characterisation of cobimetinib and trametinib in vitro to investigate potential for MEK inhibitors to cause platelet dysfunction.
We report that whilst both cobimetinib and trametinib are potent inhibitors of platelet MEK activity, treatment with trametinib did not alter platelet function. Treatment with cobimetinib results in inhibition of platelet aggregation, integrin activation, alpha-granule secretion and adhesion but only at suprapharmacological concentrations. We identified that the inhibitory effects of high concentrations of cobimetinib are associated with off-target inhibition on Akt and PKC. Neither inhibitor caused any alteration in thrombus formation on collagen under flow conditions in vitro.
Our findings demonstrate that platelets are able to function normally when MEK activity is fully inhibited, indicating MEK activity is dispensable for normal platelet function. We conclude that the MEK inhibitors cobimetinib and trametinib do not induce platelet dysfunction and are therefore unlikely to contribute to increased incidence of bleeding reported during MEK inhibitor therapy
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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
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RXR ligands negatively regulate thrombosis and hemostasis
OBJECTIVE: Platelets have been found to express intracellular nuclear receptors including the Retinoid X receptors (RXRα and RXRβ). Treatment of platelets with ligands of RXR has been shown to inhibit platelet responses to ADP and thromboxane A2, however the effects on responses to other platelet agonists as well as the underlying mechanism has not been fully characterised.
APPROACH AND RESULTS: The effect of 9-cis-retinoic acid (9-cis-RA), docosahexaenoic acid and synthetic ligand for RXR, methoprene acid on collagen receptor (GPVI) agonists and Thrombin stimulated platelet function; including aggregation, granule secretion, integrin activation, calcium mobilisation, integrin αIIbβ3 outside-in signalling and thrombus formation in vitro and in vivo were determined. Treatment of platelets with RXR ligands resulted in attenuation of platelet functional responses following stimulation by GPVI agonists and thrombin and inhibition of integrin αIIbβ3 outside-in signalling. Treatment with 9-cis-RA caused inhibition of thrombus formation in vitro and an impairment of thrombosis and haemostasis in vivo. Both RXR ligands stimulated protein kinase A activation, measured by VASP S157 phosphorylation, that was found to be dependent on both cAMP and NFκB activity.
CONCLUSIONS: This study identifies a widespread, negative regulatory role for RXR in the regulation of platelet functional responses and thrombus formation and describes novel events that lead to the upregulation of PKA, a known negative regulator of many aspects of platelet function. This mechanism may offer a possible explanation for the cardioprotective effects described in vivo following treatment with RXR ligands
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The metabolites of the dietary flavonoid quercetin possess potent antithrombotic activity, and interact with aspirin to enhance antiplatelet effects
Quercetin, a dietary flavonoid, has been reported to possess antiplatelet activity. However, its extensive metabolism following ingestion has resulted in difficulty elucidating precise mechanisms of action. In this study, we aimed to characterize the antiplatelet mechanisms of two methylated metabolites of quercetin—isorhamnetin and tamarixetin—and explore potential interactions with aspirin. Isorhamnetin and tamarixetin inhibited human platelet aggregation, and suppressed activatory processes including granule secretion, integrin αIIbβ3 function, calcium mobilization, and spleen tyrosine kinase (Syk)/linker for activation of T cells (LAT) phosphorylation downstream of glycoprotein VI with similar potency to quercetin. All three flavonoids attenuated thrombus formation in an in vitro microfluidic model, and isoquercetin, a 3-O-glucoside of quercetin, inhibited thrombosis in a murine laser injury model. Isorhamnetin, tamarixetin, and quercetin enhanced the antiplatelet effects of aspirin more-than-additively in a plate-based aggregometry assay, reducing aspirin IC50 values by an order of magnitude, with this synergy maintained in a whole blood test of platelet function. Our data provide mechanistic evidence for the antiplatelet activity of two quercetin metabolites, isorhamnetin and tamarixetin, and suggest a potential antithrombotic role for these flavonoids. In combination with their interactions with aspirin, this may represent a novel avenue of investigation for the development of new antithrombotic strategies and management of current therapies
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Human platelet protein ubiquitylation and changes following GPVI activation
Platelet activators stimulate post-translational modification of signalling proteins to change their
activity or their molecular interactions leading to signal propagation. One covalent modification is attachment of the small protein ubiquitin to lysine residues in target proteins. Modification by ubiquitin can either target proteins for degradation by the proteasome or act as a scaffold for other proteins. Pharmacological inhibition of deubiquitylases or the proteasome inhibits platelet
activation by collagen, demonstrating a role for ubiquitylation, but relatively few substrates for
ubiquitin have been identified and the molecular basis of inhibition is not established. Here we report the ubiquitome of human platelets and changes in ubiquitylated proteins following stimulation by collagen related peptide (CRP-XL). Using platelets from six individuals over three
independent experiments, we identified 1634 ubiquitylated peptides derived from 691 proteins, revealing extensive ubiquitylation in resting platelets. 925 of these peptides show an increase of more than 2-fold following stimulation with CRP-XL. Multiple sites of ubiquitylation were 16 identified on a number of proteins including Syk, filamin and integrin heterodimer subunits. This work reveals extensive protein ubiquitylation during activation of human platelets and opens the possibility of novel therapeutic interventions targeting the ubiquitin machinery
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Severe platelet dysfunction in NHL patients receiving ibrutinib is absent in patients receiving acalabrutinib
The Bruton’s tyrosine kinase (Btk) inhibitor ibrutinib induces platelet dysfunction and causes increased risk of bleeding. Off-target inhibition of Tec is believed to contribute to platelet dysfunction and other side-effects of ibrutinib. The second generation Btk inhibitor acalabrutinib was developed with improved specificity for Btk over Tec. We investigated platelet function in patients with Non-Hodgkin Lymphoma (NHL) receiving ibrutinib or acalabrutinib by aggregometry and by measuring thrombus formation on collagen under arterial shear. Both patient groups had similarly dysfunctional aggregation responses to collagen and collagen-related peptide (CRP-XL) and comparison with mechanistic experiments in which platelets from healthy donors were treated with the Btk inhibitors suggested that both drugs inhibit platelet Btk and Tec at physiological concentrations. Only ibrutinib caused dysfunctional thrombus formation, while size and morphology of thrombi following acalabrutinib treatment were of normal size and morphology. We found that ibrutinib but not acalabrutinib inhibited SFKs and that SFKs have a critical role in platelet adhesion to collagen that is likely to underpin unstable thrombus formation observed in ibrutinib patients. We found that platelet function was enhanced by increasing levels of vWF and FVIII ex vivo by addition of intermediate purity FVIII (haemate P) to blood from patients, resulting in consistently larger thrombi. We conclude that acalabrutinib avoids major platelet dysfunction associated with ibrutinib therapy, and platelet function may be enhanced in patients with B-cell NHL by increasing plasma vWF and FVIII
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Interspecies differences in protein expression do not impact the spatiotemporal regulation of glycoprotein VI mediated activation
Background
Accurate protein quantification is a vital prerequisite for generating meaningful predictions when using systems biology approaches, a method that is increasingly being used to unravel the complexities of sub cellular interactions and as part of the drug discovery process. Quantitative proteomics, flow cytometry and western blotting have been extensively used to define human platelet protein copy numbers, yet for mouse platelets, a model widely used for platelet research, evidence is largely limited to a single proteomic dataset in which the total amount of proteins were generally comparatively higher than those found in human platelets.
Objectives
To investigate the functional implications of discrepancies between levels of mouse and human proteins in the GPVI signalling pathway using a systems pharmacology model of GPVI
Methods
The protein copy number of mouse platelet receptors was determined using flow cytometry. The Virtual Platelet, a mathematical model of Glycoprotein VI (GPVI) signalling, was used to determine the consequences of protein copy number differences observed between human and mouse platelets.
Results and conclusion
Despite the small size of mouse platelets compared to human platelets they possessed a greater density of surface receptors alongside a higher concentration of intracellular signalling proteins. Surprisingly the predicted temporal profile of Syk activity was similar in both species with predictions supported experimentally. Super resolution microscopy demonstrates that the spatial distribution of Syk is similar between species, suggesting that the spatial distribution of receptors and signalling molecules in activated platelets, rather than their copy number, is important for signalling pathway regulation
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Farnesoid X Receptor and its ligands inhibit the function of platelets
Objective - While initially seemingly paradoxical due to the lack of nucleus, platelets possess a number of transcription factors that regulate their function through DNA-independent mechanisms. These include the Farnesoid X Receptor (FXR), a member of the superfamily of ligand-activated transcription factors that has been identified as a bile acid receptor. In this study, we show that FXR is present in human platelets and FXR ligands, GW4064 and 6-ECDCA, modulate platelet activation nongenomically.
Approach and Results - FXR ligands inhibited the activation of platelets in response to stimulation of collagen or thrombin receptors, resulting in diminished intracellular calcium mobilization and secretion, fibrinogen binding and aggregation. Exposure to FXR ligands also reduced integrin alphaIIbbeta3 outside-in signaling and thereby reduced the ability of platelets to spread and to stimulate clot retraction. FXR function in platelets was found to be associated with the modulation of cGMP levels in platelets and associated downstream inhibitory signaling. Platelets from FXR-deficient mice were refractory to the actions of FXR agonists on platelet function and cyclic nucleotide signaling, firmly linking the non-genomic actions of these ligands to the FXR receptor.
Conclusion – This study provides support for the ability of FXR ligands to modulate platelet activation. The athero-protective effects of GW4064, with its novel antiplatelet effects, indicate FXR as a potential target for prevention of athero-thrombotic disease
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Farnesoid X receptor and liver X receptor ligands initiate formation of coated platelets
The liver X receptors (LXRs) and farnesoid X receptor (FXR) have been identified in human platelets. Ligands of these receptors have been shown to have nongenomic inhibitory effects on platelet activation by platelet agonists. This, however, seems contradictory with the platelet hyper-reactivity that is associated with several pathological conditions that are associated with increased circulating levels of molecules that are LXR and FXR ligands, such as hyperlipidemia, type 2 diabetes mellitus, and obesity. We, therefore, investigated whether ligands for the LXR and FXR receptors were capable of priming platelets to the activated state without stimulation by platelet agonists. Treatment of platelets with ligands for LXR and FXR converted platelets to the procoagulant state, with increases in phosphatidylserine exposure, platelet swelling, reduced membrane integrity, depolarization of the mitochondrial membrane, and microparticle release observed. Additionally, platelets also displayed features associated with coated platelets such as P-selectin exposure, fibrinogen binding, fibrin generation that is supported by increased serine protease activity, and inhibition of integrin αIIbβ3. LXR and FXR ligand-induced formation of coated platelets was found to be dependent on both reactive oxygen species and intracellular calcium mobilization, and for FXR ligands, this process was found to be dependent on cyclophilin D. We conclude that treatment with LXR and FXR ligands initiates coated platelet formation, which is thought to support coagulation but results in desensitization to platelet stimuli through inhibition of αIIbβ3 consistent with their ability to inhibit platelet function and stable thrombus formation in vivo