cGMP signaling inhibits platelet shape change through regulation of the RhoA-Rho Kinase-MLC phosphatase signaling pathway

Background: Platelet shape change, spreading and thrombus stability require activation of the actin cytoskeleton contractile machinery. The mechanisms controlling actin assembly to prevent unwanted platelet activation are unclear. Objectives: We examined the effects of nitric oxide on the signaling pathways regulating platelet actinmyosin activation. Results: S-nitrosoglutathione (GSNO) inhibited thrombin-induced platelet shape change and myosin phosphorylation of the myosin light chain (MLC). Because thrombin stimulates phospho-MLC through the RhoA/ ROCK dependent inhibition of MLC phosphatase (MLCP) we examined the effects of NO on this pathway. Thrombin caused the GTP loading and activation of RhoA, leading to the ROCK-mediated phosphorylation of MLCP on threonine 853 (thr853), which is known to inhibit phosphatase activity. Treatment of platelets with GSNO blocked ROCK-mediated increases in phosphoMLCPthr853 induced by thrombin. This effect was mimicked by the direct activator of protein kinase G, 8-pCPT-PET-cGMP, and blocked by the inhibition of guanylyl cyclase, but not inhibitors of protein kinase A. Further exploration of the mechanism demonstrated that GSNO stimulated the association of RhoA with protein kinase G (PKG) and the inhibitory phosphorylation (serine188) of RhoA in a cGMP-dependent manner. Consistent with these observations, in vitro experiments revealed that recombinant PKG caused direct phosphorylation of RhoA. The inhibition of RhoA by GSNO prevented ROCK-mediated phosphorylation and inhibition of MLCP activity. Conclusions: These data suggest novel crosstalk between the NO-cGMP-PKG and RhoA/ROCK signaling pathways to control platelet actin remodeling

Alterations in Platelet Alpha-Granule Secretion and Adhesion on Collagen under Flow in Mice Lacking the Atypical Rho GTPase RhoBTB3

Typical Rho GTPases, such as Rac1, Cdc42, and RhoA, act as molecular switches regulating various aspects of platelet cytoskeleton reorganization. The loss of these enzymes results in reduced platelet functionality. Atypical Rho GTPases of the RhoBTB subfamily are characterized by divergent domain architecture. One family member, RhoBTB3, is expressed in platelets, but its function is unclear. In the present study we examined the role of RhoBTB3 in platelet function using a knockout mouse model. We found the platelet count, size, numbers of both alpha and dense granules, and surface receptor profile in these mice were comparable to wild-type mice. Deletion of Rhobtb3 had no effect on aggregation and dense granule secretion in response to a range of agonists including thrombin, collagen, and adenosine diphosphate (ADP). By contrast, alpha-granule secretion increased in mice lacking RhoBTB3 in response to thrombin, collagen related peptide (CRP) and U46619/ADP. Integrin activation and spreading on fibrinogen and collagen under static conditions were also unimpaired; however, we observed reduced platelet accrual on collagen under flow conditions. These defects did not translate into alterations in tail bleeding time. We conclude that genetic deletion of Rhobtb3 leads to subtle alterations in alpha-granule secretion and adhesion to collagen without significant effects on hemostasis in vivo

Biochemical and immunocytochemical characterization of coronins in platelets

Rapid reorganization of the actin cytoskeleton in response to receptor-mediated signaling cascades allows platelets to transition from a discoid shape to a flat spread shape upon adhesion to damaged vessel walls. Coronins are conserved regulators of the actin cytoskeleton turnover but they also participate in signaling events. To gain a better picture of their functions in platelets we have undertaken a biochemical and immunocytochemical investigation with a focus on Coro1. We found that class I coronins Coro1, 2 and 3 are abundant in human and mouse platelets whereas little Coro7 can be detected. Coro1 is mainly cytosolic, but a significant amount associates with membranes in an actin-independent manner and does not translocate from or to the membrane fraction upon exposure to thrombin, collagen or prostacyclin. Coro1 rapidly translocates to the Triton insoluble cytoskeleton upon platelet stimulation with thrombin or collagen. Coro1, 2 and 3 show a diffuse cytoplasmic localization with discontinuous accumulation at the cell cortex and actin nodules of human platelets, where all three coronins colocalize. Our data are consistent with a role of coronins as integrators of extracellular signals with actin remodeling and suggests a high extent of functional overlap among class I coronins in platelets

Pre-analytical conditions for multiparameter platelet flow cytometry

Background Flow cytometry is an important technique for understanding multiple aspects of blood platelet biology. Despite the widespread use of the platform for assessing platelet function, the optimisation and careful consideration of pre-analytical conditions, sample processing techniques and data analysis strategies should be regularly assessed. When set up and designed with optimal conditions it can ensure the acquisition of robust and reproducible flow cytometry data. However, these parameters are rarely described despite their importance. Objectives We aimed to characterise the effects of several pre-analytical variables on the analysis of blood platelets by multiparameter fluorescent flow cytometry. Methods We assessed anticoagulant choice, sample material, sample processing and storage times on four distinct and commonly used markers of platelet activation including fibrinogen binding, expression of CD62P and CD42b, and phosphatidylserine exposure. Results The use of sub-optimal conditions led to increases in basal platelet activity and reduced sensitivities to stimulation, however the use of optimal conditions protected the platelets from artefactual stimulation and preserved basal activity and sensitivity to activation. Summary The optimal pre-analytical conditions identified here for the measurement of platelet phenotype by flow cytometry suggests a framework for future development of multiparameter platelet assays for high quality datasets and advanced analysis

Coronin 1 Is Required for Integrin β2 Translocation in Platelets

Remodeling of the actin cytoskeleton is one of the critical events that allows platelets to undergo morphological and functional changes in response to receptor-mediated signaling cascades. Coronins are a family of evolutionarily conserved proteins implicated in the regulation of the actin cytoskeleton, represented by the abundant coronins 1, 2, and 3 and the less abundant coronin 7 in platelets, but their functions in these cells are poorly understood. A recent report revealed impaired agonist-induced actin polymerization and cofilin phosphoregulation and altered thrombus formation in vivo as salient phenotypes in the absence of an overt hemostasis defect in vivo in a knockout mouse model of coronin 1. Here we show that the absence of coronin 1 is associated with impaired translocation of integrin β2 to the platelet surface upon stimulation with thrombin while morphological and functional alterations, including defects in Arp2/3 complex localization and cAMP-dependent signaling, are absent. Our results suggest a large extent of functional overlap among coronins 1, 2, and 3 in platelets, while aspects like integrin β2 translocation are specifically or predominantly dependent on coronin 1

Atherogenic Lipid Stress Induces Platelet Hyperactivity Through CD36-Mediated Hyposensitivity To Prostacyclin-; The Role Of Phosphodiesterase 3A

Prostacyclin (PGI2) controls platelet activation and thrombosis through a cyclic adenosine monophosphate (cAMP) signalling cascade. However, in patients with cardiovascular diseases this protective mechanism fails for reasons that are unclear. Using both pharmacological and genetic approaches we describe a mechanism by which oxidised low density lipoproteins (oxLDL) associated with dyslipidaemia promote platelet activation through impaired PGI2 sensitivity and diminished cAMP signalling. In functional assays using human platelets, oxLDL modulated the inhibitory effects of PGI2, but not a PDE-insensitive cAMP analogue, on platelet aggregation, granule secretion and in vitro thrombosis. Examination of the mechanism revealed that oxLDL promoted the hydrolysis of cAMP through the phosphorylation and activation of phosphodiesterase 3A (PDE3A), leading to diminished cAMP signalling. PDE3A activation by oxLDL required Src family kinases, Syk and protein kinase C. The effects of oxLDL on platelet function and cAMP signalling were blocked by pharmacological inhibition of CD36, mimicked by CD36-specific oxidised phospholipids and ablated in CD36-/- murine platelets. The injection of oxLDL into wild type mice strongly promoted FeCl3 induced carotid thrombosis in vivo, which was prevented by pharmacological inhibition of PDE3A. Furthermore, blood from dyslipidaemic mice was associated with increased oxidative lipid stress, reduced platelet sensitivity to PGI2 ex vivo and diminished PKA signalling. In contrast, platelet sensitivity to a PDE-resistant cAMP analogue remained normal. Genetic deletion of CD36, protected dyslipidaemic animals from PGI2 hyposensitivity and restored PKA signalling. These data suggest that CD36 can translate atherogenic lipid stress into platelet hyperactivity through modulation of inhibitory cAMP signalling.  

Ergothioneine supplementation in people with metabolic syndrome (ErgMS): protocol for a randomised, double-blind, placebo-controlled pilot study

Background Ergothioneine is a naturally occurring metabolite of histidine found in many foods and in high amounts in mushrooms. In vivo, ergothioneine acts as an antioxidant and is widely distributed in most mammalian tissues. While ergothioneine is sold as a dietary supplement for its antioxidant and anti-inflammatory properties, to date there are no published intervention trials examining its health benefits in humans. The aim of this work was to develop a study protocol for a pilot interventional trial that will establish the primary and secondary outcomes, and the power required, for a definitive randomised controlled trial to test the hypothesis that ergothioneine supplementation is beneficial for people with metabolic syndrome. Methods We have designed the ErgMS study as a single-centre, randomised, double-blind, placebo-controlled, 3-arm parallel, pilot intervention trial, which aims to supplement participants with either placebo, 5 or 30 mg/day ergothioneine for 12 weeks. Measurements of metabolic syndrome risk factors, serum markers of oxidative stress (lipid peroxidation), inflammation, blood platelet function and liver function will take place at baseline, and after 6 weeks and 12 weeks of supplementation. In addition, we will examine if there are any changes in the serum metabolome in response to ergothioneine supplementation. Linear regression and two-way ANOVA will be utilised to analyse the association between ergothioneine and measured variables. Discussion The ErgMS study will be the first study to address the question does ergothioneine supplementation have health benefits for people with metabolic syndrome. Study results will provide preliminary data as to which dose may improve inflammatory markers in adults with metabolic syndrome and will inform dose and primary outcome selection for a definitive randomised controlled trial. Trial registration ISRCTN, ISRCTN25890011 Registered February 10th, 202

Protein Kinase A Regulates Platelet Phosphodiesterase 3A through an A-Kinase Anchoring Protein Dependent Manner

Platelet activation is critical for haemostasis, but if unregulated can lead to pathological thrombosis. Endogenous platelet inhibitory mechanisms are mediated by prostacyclin (PGI2)-stimulated cAMP signalling, which is regulated by phosphodiesterase 3A (PDE3A). However, spatiotemporal regulation of PDE3A activity in platelets is unknown. Here, we report that platelets possess multiple PDE3A isoforms with seemingly identical molecular weights (100 kDa). One isoform contained a unique N-terminal sequence that corresponded to PDE3A1 in nucleated cells but with negligible contribution to overall PDE3A activity. The predominant cytosolic PDE3A isoform did not possess the unique N-terminal sequence and accounted for >99% of basal PDE3A activity. PGI2 treatment induced a dose and time-dependent increase in PDE3A phosphorylation which was PKA-dependent and associated with an increase in phosphodiesterase enzymatic activity. The effects of PGI2 on PDE3A were modulated by A-kinase anchoring protein (AKAP) disruptor peptides, suggesting an AKAP-mediated PDE3A signalosome. We identified AKAP7, AKAP9, AKAP12, AKAP13, and moesin expressed in platelets but focussed on AKAP7 as a potential PDE3A binding partner. Using a combination of immunoprecipitation, proximity ligation techniques, and activity assays, we identified a novel PDE3A/PKA RII/AKAP7 signalosome in platelets that integrates propagation and termination of cAMP signalling through coupling of PKA and PDE3A

Platelet function following induced hypoglycaemia in type 2 diabetes

Aim: Strict glycaemic control has been associated with an increased mortality rate in subjects with type 2 diabetes (T2DM). Here we examined platelet function immediately and 24 hours following induced hypoglycaemia in people with type 2 diabetes compared to healthy age-matched controls. Methods: Hyperinsulinaemic clamps reduced blood glucose to 2.8 mmol/L (50 mg/dl) for 1 hour. Sampling at baseline; euglycaemia 5 mmol/L (90 mg/dl); hypoglycaemia; and at 24 post clamp were undertaken. Platelet function was measured by whole blood flow cytometry. Results: 10 subjects with T2DM and 8 controls were recruited. Platelets from people with T2DM showed reduced sensitivity to prostacyclin (PGI2, 1 nM) following hypoglycaemia. The ability of PGI2 to inhibit platelet activation was significantly impaired at 24 hours compared to baseline in the T2DM group. Here, inhibition of fibrinogen binding was 29.5% (10.3–43.8) compared to 50.8% (36.8–61.1), (P < 0.05), while inhibition of P-selectin expression was 32% (16.1–47.6) vs. 54.4% (42.5–67.5) (P < 0.05). No significant changes in platelet function were noted in controls. Conclusion: Induced hypoglycaemia in T2DM enhances platelet hyperactivity through impaired sensitivity to prostacyclin at 24 hours