Physiological Agonists Research Articles

Attenuation of Hypertrophy in Human MSCs via Treatment with a Retinoic Acid Receptor Inverse Agonist.

In vitro chondrogenically differentiated mesenchymal stem cells (MSCs) have a tendency to undergo hypertrophy, mirroring the fate of transient “chondrocytes” in the growth plate. As hypertrophy would result in ossification, this fact limits their use in cartilage tissue engineering applications. During limb development, retinoic acid receptor (RAR) signaling exerts an important influence on cell fate of mesenchymal progenitors. While retinoids foster hypertrophy, suppression of RAR signaling seems to be required for chondrogenic differentiation. Therefore, we hypothesized that treatment of chondrogenically differentiating hMSCs with the RAR inverse agonist, BMS204,493 (further named BMS), would attenuate hypertrophy. We induced hypertrophy in chondrogenic precultured MSC pellets by the addition of bone morphogenetic protein 4. Direct activation of the RAR pathway by application of the physiological RAR agonist retinoic acid (RA) further enhanced the hypertrophic phenotype. However, BMS treatment reduced hypertrophic conversion in hMSCs, shown by decreased cell size, number of hypertrophic cells, and collagen type X deposition in histological analyses. BMS effects were dependent on the time point of application and strongest after early treatment during chondrogenic precultivation. The possibility of modifing hypertrophic cartilage via attenuation of RAR signaling by BMS could be helpful in producing stable engineered tissue for cartilage regeneration.

In vitro Induction and Detection of Acrosomal Exocytosis in Human Spermatozoa.

The acrosome reaction is a highly regulated exocytotic event that primes spermatozoa for successful fertilization. Upon induction, acrosomal exocytosis proceeds via a wave of vesiculation that radiates across the sperm head, destabilizing the acrosomal vesicle and resulting in the release of the acrosomal contents. Having shed their acrosome, spermatozoa are then capable of penetrating the outer vestments of the oocyte and initiating fertilization. Accordingly, the failure of spermatozoa to complete an acrosome reaction represents a relatively common etiology in male infertility patients, and the ability to induce acrosomal exocytosis has found clinical utility in the evaluation of sperm fertilizing capacity. Here, we firstly describe protocols for driving the capacitation of human spermatozoa in vitro using chemically defined media in order to prime the cells for completion of acrosomal exocytosis. We then describe methodology routinely used for the induction of acrosomal exocytosis incorporating either a physiological agonist (i.e., the steroidal hormone, progesterone) or pharmacological reagent (i.e., the divalent cation ionophore, A23187). Finally, we describe the application of histochemical and immunofluorescence techniques that can be applied to study the completion of the acrosome reaction. Such protocols have important diagnostic utility for sperm function testing in both clinical and andrological research laboratories.

Evaluation of cytokine concentrations in a trehalose-stabilised lyophilised canine platelet product: a preliminary study.

BackgroundPlatelet transfusion is indicated for haemorrhage due to severe thrombocytopenia and for trauma associated coagulopathy. Febrile non-haemolytic transfusion reactions are a common complication of platelet transfusions in people and may be due to accumulated inflammatory cytokines. The present study aimed to determine the cytokine profile of a novel canine lyophilised platelet product following reconstitution, to assess the lyophilised platelets’ activation response to physiological platelet agonists and to compare the cytokine profiles of basal and stimulated canine lyophilised platelets.MethodsCell counts and biochemical analyses were conducted following reconstitution. Cytokine concentrations were measured with a canine-specific multiplex immunocapture assay and with an electrochemiluminescent ELISA. Aliquots of reconstituted product from three separate vials were activated for 10 minutes under non-stirred conditions using adenosine diphosphate, thrombin or convulxin and their cytokine concentrations compared with unactivated samples. Flow cytometry and light-transmission aggregometry were used to evaluate the product’s ability to express a procoagulant surface, degranulate and aggregate. Fresh platelet-rich plasma was used as a positive control.ResultsThe product had a mean±SD particle count of 1.23±0.2×109/ml, contained platelets that expressed surface phosphatidylserine before agonist stimulation and was capable of aggregation in response to thrombin stimulation suggesting that the product may have haemostatic potential following in vivo administration. Cytokine concentrations measured by the immunocapture assay were generally low, while twofold to threefold increases relative to published intervals were noted for several cytokines using the ELISA. Concentrations of chemokine (C-X-C) motif ligand 8 and tumour necrosis factor-α were significantly increased as measured by the ELISA, but not by the immunocapture assay, while concentrations of KC-like were significantly increased as measured by the immunocapture assay. Stimulation with platelet agonists did not affect measured cytokine concentrations.ConclusionFurther study of the effects of administration of this lyophilised platelet product is warranted.

Detection of Intact Transcription Factors in Human Neutrophils.

The crucial contribution of neutrophils to innate immunity extends well beyond their traditional role as professional phagocytes. Indeed, it is now well established that neutrophils generate a plethora of inflammatory cytokines and chemokines that are profoundly involved in the onset and evolution of the inflammatory reaction. Several recent studies have shown that neutrophils can represent an important source of inflammatory cytokines in pathophysiological settings. The inflammatory and immunomodulatory cytokines produced by neutrophils are generally encoded by immediate-early response genes, which in turn depend on the activation of transcription factors such as those belonging to the nuclear factor κB (NF-κB) and signal transducers and activators of transcription (STAT) families. We have shown in the past that the expression of such factors is induced in neutrophils stimulated by physiological agonists. However, the detection of intact (i.e., undegraded) transcription factors in neutrophils requires special precautions and a specially designed protocol, due to the huge amounts of endogenous proteases present in these cells. This protocol is the focus of this chapter.

Detection and Analysis of Gene Mutations in Patients with Glanzmann's Thrombasthenia

Background: Glanzmann thrombasthenia (GT) is a rare inherited disorder of bleeding, and it is characterized by the impaired or absent platelet aggregation to multiple physiologic agonists such as collagen, adenosine diphosphate (ADP), arachidonic acid(AA), but normal reaction to ristocetin. There is qualitative or quantitative defect in platelet integrin αIIbβ3(GPIIb/IIIa). Pathogenic variants of either αIIb or β3 unit could cause GT. The database of gene mutations is continuously updated on the Internet (http://www.hgmd.org); it totally lists 236 variants of ITGA2B gene and 170 variants of ITGB3 gene. Aim: To characterize the clinical manifestation and molecular basis of GT patients in China, and update the pathogenic variants database. Method: Clinical features are evaluated in 104 patients with GT. New generation sequencing was performed with a custom designed panel for the bleeding and platelet disease involving 76 genes, while ITGA2B and ITGB3 were enrolled. Result: The initial bleeding occurred before 1 age in most patients. Incidence of consanguinity is 12.5%. Symptoms lessened with age in about 30% patients. Female patients suffered more severe bleeding than male patients. Fifty different mutations were detected, among which 15 were novel. Most patients were compound heterozygotes and most mutations detected were missense mutations. Among 15 novel mutations, there were 7 missense mutations, 2 nonsense mutations, 2 splicing mutations, 4 frameshift mutations. Pathogenicity of all novel mutations were evaluated according to the standards and guidelines of ACMG. All variants detected were pathogenic or likely pathogenic. Furthermore, c.1750C>T [p.R584X] and c.2333A>C [p.Q778P] in ITGA2B were detected in 10 and 16 unrelated families, strongly suggesting a founder effect. Conclusion: Our study reports the largest cohort of GT in China, describing the clinical, laboratory and genetic characteristics of 104 patients. We found 15 novel pathogenic mutations in ITGA2B and ITGB3 causing GT. Theses novel findings expand the GT mutation spectrum. Disclosures No relevant conflicts of interest to declare.

Acute β-adrenoceptor mediated glucose clearance in brown adipose tissue; a distinct pathway independent of functional insulin signaling

Objectiveβ-adrenoceptor mediated activation of brown adipose tissue (BAT) has been associated with improvements in metabolic health in models of type 2 diabetes and obesity due to its unique ability to increase whole body energy expenditure, and rate of glucose and free fatty acid disposal. While the thermogenic arm of this phenomenon has been studied in great detail, the underlying mechanisms involved in β-adrenoceptor mediated glucose uptake in BAT are relatively understudied. As β-adrenoceptor agonist administration results in increased hepatic gluconeogenesis that can consequently result in secondary pancreatic insulin release, there is uncertainty regarding the importance of insulin and the subsequent activation of its downstream effectors in mediating β-adrenoceptor stimulated glucose uptake in BAT. Therefore, in this study, we made an effort to discriminate between the two pathways and address whether the insulin signaling pathway is dispensable for the effects of β-adrenoceptor activation on glucose uptake in BAT. MethodsUsing a specific inhibitor of phosphoinositide 3-kinase α (PI3Kα), which effectively inhibits the insulin signaling pathway, we examined the effects of various β-adrenoceptor agonists, including the physiological endogenous agonist norepinephrine on glucose uptake and respiration in mouse brown adipocytes in vitro and on glucose clearance in mice in vivo. ResultsPI3Kα inhibition in mouse primary brown adipocytes in vitro, did not inhibit β-adrenoceptor stimulated glucose uptake, GLUT1 synthesis, GLUT1 translocation or respiration. Furthermore, β-adrenoceptor mediated glucose clearance in vivo did not require insulin or Akt activation but was attenuated upon administration of a β3-adrenoceptor antagonist. ConclusionsWe conclude that the β-adrenergic pathway is still functionally intact upon the inhibition of PI3Kα, showing that the activation of downstream insulin effectors is not required for the acute effects of β-adrenoceptor agonists on glucose homeostasis or thermogenesis.

JAK2/STAT3 Pathway is Required for α7nAChR-Dependent Expression of POMC and AGRP Neuropeptides in Male Mice.

Cholinergic signalling mediated by the activation of muscarinic and nicotinic receptors has been described in the literature as a classic and important signalling pathway in the regulation of the inflammatory response. Recent research has investigated the role of acetylcholine, the physiological agonist of these receptors, in the control of energy homeostasis at the central level. Studies have shown that mice that do not express acetylcholine in brain regions regulating energy homeostasis present with excessive weight gain and hyperphagia. However, it has not yet been well-described in the literature which cholinergic receptor subunits are involved in this response; moreover, the signalling pathways responsible for the observed effects are not fully delineated. The hypothalamus is the regulating centre of energy homeostasis, and the α7 subunit of the nicotinic acetylcholine receptor (α7nAChR) is highly expressed in this region. When active, α7nAChR recruits proteins such as JAK2/STAT3 to mediate its signalling; the same intracellular components are required by leptin, an anorexigenic hormone. The aim of the present study was to evaluate the role of the hypothalamic α7nAChR in the control of energy homeostasis. The work was performed on Swiss male mice. Initially, using immunofluorescent staining on brain sections, the presence of α7nAChR in hypothalamic cells regulating energy homeostasis was evaluated. Animals were submitted to stereotaxis in the lateral ventricle and intracerebroventricular stimulation (ICV) was used for the administration of an agonist (PNU) or antagonist (α-bungarotoxin) of α7nAChR. Metabolic parameters were evaluated and the expression of neuropeptides was evaluated in the hypothalamus by real-time PCR and western blot. The expression of hypothalamic neuropeptides was evaluated in mice treated with siRNA or inhibitors of JAK2/STAT3 (AG490 and STATTIC) proteins. We also evaluated food intake in α7nAChR knockout animals (α7KO). Additionally, in mouse hypothalamic cell culture (the mypHoA-POMC/GFP lineage), we evaluated the expression of neuropeptides and pSTAT3 after stimulation with PNU. Our results indicate co-localisation of α7nAChR with α-MSH, AgRP and NPY in hypothalamic cells. Pharmacological activation of α7nAChR reduced food intake and increased hypothalamic POMC expression and decreased NPY and AgRP mRNA levels and the protein content of pAMPK. Inhibition of α7nAChR with an antagonist increased the mRNA content of NPY and AgRP. Inhibition of α7nAChR with siRNA led to the suppression of POMC expression and an increase in AgRP mRNA levels. α7KO mice showed no changes in food intake. Inhibition of proteins involved in the JAK2/STAT3 signalling pathway reversed the effects observed after PNU stimulation. POMC-GFP cells, when treated with PNU, showed increased POMC expression and nuclear translocation of pSTAT3. Thus, selective activation of α7nAChR is able to modulate important markers of the response to food intake, suggesting that α7nAChR activation can suppress the expression of orexigenic markers and favour the expression of anorexics using the intracellular JAK2/STAT3 machinery.

Platelet Oxidative Stress and its Relationship with Cardiovascular Diseases in Type 2 Diabetes Mellitus Patients.

Enhanced platelet activation and thrombosis are linked to various cardiovascular diseases (CVD). Among other mechanisms, oxidative stress seems to play a pivotal role in platelet hyperactivity. Indeed, upon stimulation by physiological agonists, human platelets generate and release several types of reactive oxygen species (ROS) such as O2 -, H2O2 or OH-, further amplifying the platelet activation response via various signalling pathways, including, formation of isoprostanes, Ca2+ mobilization and NO inactivation. Furthermore, excessive platelet ROS generation, incorporation of free radicals from environment and/or depletion of antioxidants induce pro-oxidant, pro-inflammatory and platelet hyperaggregability effects, leading to the incidence of cardiovascular events. Here, we review the current knowledge regarding the effect of oxidative stress on platelet signaling pathways and its implication in CVD such as type 2 diabetes mellitus. We also summarize the role of natural antioxidants included in vegetables, fruits and medicinal herbs in reducing platelet function via an oxidative stress-mediated mechanism.

1I. Vitamin D receptor-like receptors (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

Vitamin D (VDR), Pregnane X (PXR) and Constitutive Androstane (CAR) receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Nuclear Hormone Receptors [47]) are members of the NR1I family of nuclear receptors, which form heterodimers with members of the retinoid X receptor family. PXR and CAR are activated by a range of exogenous compounds, with no established endogenous physiological agonists, although high concentrations of bile acids and bile pigments activate PXR and CAR [47].

A novel missense variant in the RASGRP2 gene in patients with moderate to severe bleeding disorder

Inherited platelet function disorder-18 (IPD-18) is a relatively new non-syndromic autosomal recessive bleeding disorder. It is characterized by deficient or dysfunctional CalDAG-GEFI protein. The distinctive feature of the disease is impaired platelet aggregation in response to multiple physiologic agonists. We here report a family with a platelet-type bleeding disorder and a novel mutation in the RASGRP2 gene. The overall bleeding score for the affected individuals was 15 and 12. Based on the initial diagnosis of Glanzmann thrombasthenia, targeted sequencing of integrin subunit alpha 2b and integrin subunit beta 3 encoding genes ITGA2B and ITGB3 were carried out in both affected members of a family. Sequence alignment failed to identify the disease-causing variant(s) in both genes. Therefore, whole exome sequencing in one affected individual was performed. Data analysis detected a novel homozygous missense variant (c.956C>T; p.Pro319Leu) in the exon 9 of the RASGRP2 gene. Five additional individuals of a family including both parents, an affected individual and two asymptomatic individuals were Sanger sequenced for the variant (c.956C>T). The variant segregates in the family in an autosomal recessive manner. Several in silico tools predicted the variant as pathogenic. Protein modeling studies suggest that the mutation (p.Pro319Leu) cause a conformational change in the loop structure of the RasGEF domain of the CalDAG-GEFI protein. Reported variants in the RasGEF domain impair expression and/or nucleotide exchange activity of CalDAG-GEFI protein and thus inhibit the activation of Rap1 protein required for platelet adhesion and hemostatic plug formation.

Structure of Heteropentameric GABAA Receptors and Receptor-Anchoring Properties of Gephyrin.

γ-Aminobutyric acid type A receptors (GABAARs) mediate the majority of fast synaptic inhibition in the central nervous system (CNS). GABAARs belong to the Cys-loop superfamily of pentameric ligand-gated ion channels (pLGIC) and are assembled from 19 different subunits. As dysfunctional GABAergic neurotransmission manifests itself in neurodevelopmental disorders including epilepsy and anxiety, GABAARs are key drug targets. The majority of synaptic GABAARs are anchored at the inhibitory postsynaptic membrane by the principal scaffolding protein gephyrin, which acts as the central organizer in maintaining the architecture of the inhibitory postsynaptic density (iPSD). This interaction is mediated by the long intracellular loop located in between transmembrane helices 3 and 4 (M3–M4 loop) of the receptors and a universal receptor-binding pocket residing in the C-terminal domain of gephyrin. In 2014, the crystal structure of the β3-homopentameric GABAAR provided crucial information regarding the architecture of the receptor; however, an understanding of the structure and assembly of heteropentameric receptors at the atomic level was lacking. This review article will highlight recent advances in understanding the structure of heteropentameric synaptic GABAARs and how these structures have provided fundamental insights into the assembly of these multi-subunit receptors as well as their modulation by diverse ligands including the physiological agonist GABA. We will further discuss the role of gephyrin in the anchoring of synaptic GABAARs and glycine receptors (GlyRs), which are crucial for maintaining the architecture of the iPSD. Finally, we will also summarize how anti-malarial artemisinin drugs modulate gephyrin-mediated inhibitory neurotransmission.

The physiological hypertrophic agonist, norepinephrine, is able to induce PLC‐mediated PI4P hydrolysis in cardiac myocytes via a pool of internal β‐adrenergic receptors

Previously, we have shown that PLC∑‐mediated phosphatidylinositol‐4‐phosphate (PI4P) can be activated by raising cAMP in cardiac myocytes, however, activation by Isoproterenol was unable to induce PI4P hydrolysis. In the accompanying abstract by Wei et al, we have demonstrated that the cell permeant β‐agonist, Dobutamine is able to induce PI4P hydrolysis. Here, we seek to determine if the physiological agonist, norepinephrine is able to induce PI4P hydrolysis, despite being membrane impermeant. In contrast to Iso, norepinephrine induced sustained PI4P hydrolysis that was blocked by the cell permeant β‐adrenergic antagonist, metoprolol (100μM). The cell impermeant βAR antagonist, sotalol (5mM) could not block norepinephrine effects, suggesting that a pool of internal β‐adrenergic receptors is responsible for NE‐mediated PI4P hydrolysis. To directly determine if internal β1‐ARs in NRVMs can be activated by norepinephrine, we monitored translocation of a YFP‐tagged mini Gs protein in β1AR transfected NRVMs2. Norepinephrine induced robust and rapid recruitment of mini Gs to both the plasma membrane, and the perinuclear region corresponding to the Golgi apparatus. Golgi localization could be reversed by Metoprolol but not Sotalol. Isoproterenol, on the other hand, induced a strong recruitment to the plasma membrane but not to the Golgi. NE‐mediated PI4P hydrolysis is also abrogated by an inhibitor of a cation transporter Oct3, Corticosterone (100μM). Oct3 has been recently shown to be required for epinephrine‐mediated β1‐AR stimulation at the Golgi envelope in HELA cells3. Taken together, these data suggest that norepinephrine can activate β‐adrenergic receptors in internal membranes, potentially at the Golgi apparatus, stimulating PI4P hydrolysis. These internal GPCRs may be responsible for cardiac hypertrophy and heart failure and targeting them may allow for the development of selective therapies which can treat heart failure with little to no effect on other cardiac functions.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Solvent isotope effect on leukocytes disintegration after large mechanical deformations

The objective of this study was to explore the mechanisms through which water may contribute to the structural integration of the cells and to their mechanical failure at large cellular deformations. To this end, we performed acute substitution of H2O with D2O in THP-1 myeloid leukocytes, then forced them at constant flow rates through filter pores smaller than their average resting diameter and analyzed the resulting fragments by flow cytometry and confocal microscopy. We found that this solvent replacement in culture media maintained the THP-1 cells viability, as shown by maintained plasma membrane integrity and a preserved cell response to a physiological agonist, while increasing their volume and actin polymerization. However, D2O exposure dramatically reduced filterability of cells and amplified their overall fragmentation. At the same time, size distribution of cytoplasmic fragments remained larger in D2O than in water, unlike those derived from the nuclei, which remained unchanged. Our data suggests that water as biological solvent is ‘gluing’ not only on the plasma membrane through hydrophobic effects, but also the cytoplasm, via the reciprocal influence between actin polymerization and the solvent’s molecular network.

Complement Activation Occurs at the Surface of Platelets Activated by Streptococcal M1 Protein and This Results in Phagocytosis of Platelets.

Platelets circulate the bloodstream and principally maintain hemostasis. Disturbed hemostasis, a dysregulated inflammatory state, and a decreased platelet count are all hallmarks of severe invasive Streptococcus pyogenes infection, sepsis. We have previously demonstrated that the released M1 protein from S. pyogenes activates platelets, and this activation is dependent on the binding of M1 protein, fibrinogen, and M1-specific IgG to platelets in susceptible donors. In this study, we characterize the M1-associated protein interactions in human plasma and investigate the acquisition of proteins to the surface of activated platelets and the consequences for platelet immune function. Using quantitative mass spectrometry, M1 protein was determined to form a protein complex in plasma with statistically significant enrichment of fibrinogen, IgG3, and complement components, especially C1q. Using flow cytometry, these plasma proteins were also confirmed to be acquired to the platelet surface, resulting in complement activation on M1-activated human platelets. Furthermore, we demonstrated an increased phagocytosis of M1-activated platelets by monocytes, which was not observed with other physiological platelet agonists. This reveals a novel mechanism of complement activation during streptococcal sepsis, which contributes to the platelet consumption that occurs in sepsis.

Effects of β3-adrenergic receptor stimulation on the resting holding current of medial prefrontal cortex pyramidal neurons in young rats

While the expression of β3-adrenergic receptors is firmly established in adipose, kidney and heart tissue, their expression and function in the brain remains unclear despite their potential role in depression and stress-related disorders. This study aimed to investigate the expression of β3-adrenoreceptors and their involvement in the mechanism controlling the resting holding current in layer V medial prefrontal cortex (mPFC) pyramidal neurons in young rats. Applications of the selective β3-adrenergic receptor agonists BRL 37344 and SR 58611 A evoked inward currents in the tested neurons. The inward currents evoked by BRL 37344 or noradrenaline (a nonselective physiological adrenergic receptor agonist) were prevented or decreased, respectively, by the selective β3-receptor antagonist L-748,337. Western blot and fluorescence immunohistochemistry analyses revealed β3-adrenergic receptor protein expression in the mPFC. Thus, based on the results obtained here, functional β3-adrenergic receptors are expressed in layer V mPFC pyramidal neurons and their activation evokes inward currents.

Histamine causes influx via T-type voltage-gated calcium channels in an enterochromaffin tumor cell line: potential therapeutic target in adverse food reactions.

The P-STS human ileal neuroendocrine tumor cells, as a model for gut enterochromaffin cells, are strongly and synergistically activated by histamine plus acetylcholine (ACh), presumably via histamine 4 receptors, and weakly activated by histamine alone. Sensing these signals, enterochromaffin cells could participate in intestinal intolerance or allergic reactions to food constituents associated with elevated histamine levels. In this study we aimed to analyze the underlying molecular mechanisms. Inhibition by mepyramine and mibefradil indicated that histamine alone caused a rise in intracellular calcium concentration ([Ca2+]i) via histamine 1 receptors involving T-type voltage-gated calcium channels (VGCCs). Sensitivity to histamine was enhanced by pretreatment with the inflammatory cytokine tumor necrosis factor-α (TNF-α). In accordance with the relief it offers some inflammatory bowel disease patients, otilonium bromide, a gut-impermeable inhibitor of T-type (and L-type) VGCCs and muscarinic ACh receptors, efficiently inhibited the [Ca2+]i responses induced by histamine plus ACh or by histamine alone in P-STS cells. It will take clinical studies to show whether otilonium bromide has promise for the treatment of adverse food reactions. The cells did not react to the nutrient constituents glutamate, capsaicin, cinnamaldehyde, or amylase-trypsin inhibitors and the transient receptor potential channel vanilloid 4 agonist GSK-1016790A. The bacterial product butyrate evoked a rise in [Ca2+]i only when added together with ACh. Lipopolysaccharide had no effect on [Ca2+]i despite the presence of Toll-like receptor 4 protein. Our results indicate that inflammatory conditions with elevated levels of TNF-α might enhance histamine-induced serotonin release from intestinal neuroendocrine cells. NEW & NOTEWORTHY We show that histamine synergistically enhances the intracellular calcium response to the physiological agonist acetylcholine in human ileal enterochromaffin tumor cells. This synergistic activation and cell activation by histamine alone largely depend on T-type voltage-gated calcium channels and are inhibited by the antispasmodic otilonium bromide. The cells showed no response to wheat amylase-trypsin inhibitors, suggesting that enterochromaffin cells are not directly involved in nongluten wheat sensitivity.

The protease‐activated receptor 4 Ala120Thr variant alters platelet responsiveness to low‐dose thrombin and protease‐activated receptor 4 desensitization, and is blocked by non‐competitive P2Y12 inhibition

Essentials The rs773902 SNP results in differences in platelet protease-activated receptor (PAR4) function. The functional consequences of rs773902 were analyzed in human platelets and stroke patients. rs773902 affects thrombin-induced platelet function, PAR4 desensitization, stroke association. Enhanced PAR4 Thr120 effects on platelet function are blocked by ticagrelor. SUMMARY: Background F2RL3 encodes protease-activated receptor (PAR)4 and harbors an A/G single-nucleotide polymorphism (SNP) (rs773902) with racially dimorphic allelic frequencies. This SNP mediates an alanine to threonine substitution at residue120 that alters platelet PAR4 activation by the artificial PAR4-activation peptide (PAR4-AP) AYPGKF. Objectives To determine the functional effects of rs773902 on stimulation by a physiological agonist, thrombin, and on antiplatelet antagonist activity. Methods Healthy human donors were screened and genotyped for rs773902. Platelet function in response to thrombin was assessed without and with antiplatelet antagonists. The association of rs773902 alleles with stroke was assessed in the Stroke Genetics Network study. Results As compared with rs773902 GG donors, platelets from rs773902 AA donors had increased aggregation in response to subnanomolar concentrations of thrombin, increased granule secretion, and decreased sensitivity to PAR4 desensitization. In the presence of PAR1 blockade, this genotype effect was abolished by higher concentrations of or longer exposure to thrombin. We were unable to detect a genotype effect on thrombin-induced PAR4 cleavage, dimerization, and lipid raft localization; however, rs773902 AA platelets required a three-fold higher level of PAR4-AP for receptor desensitization. Ticagrelor, but not vorapaxar, abolished the PAR4 variant effect on thrombin-induced platelet aggregation. A significant association of modest effect was detected between the rs773902 A allele and stroke. Conclusion The F2RL3 rs773902 SNP alters platelet reactivity to thrombin; the allelic effect requires P2Y12 , and is not affected by gender. Ticagrelor blocks the enhanced reactivity of rs773902 A platelets. PAR4 encoded by the rs773902 A allele is relatively resistant to desensitization and may contribute to stroke risk.

Comparing a New Bovine Source Heparin to the Clinically Used Porcine Heparin for Platelet Function Effects and Heparin-Induced Thrombocytopenia Potential

BackgroundHeparin is a sulfated polysaccharide obtained from intestinal mucosa with anticoagulant properties that is widely used as a standard clinical therapeutic agent to treat and prevent thrombosis. Heparin is known to affect platelet function, and among its side effects is heparin-induced thrombocytopenia (HIT) that can occur in about 1% of patients exposed to heparin. Presently, only porcine source heparin is approved for use in the United States. The aims of this study were to determine if platelet activation by physiological agonists and platelet aggregation induced by HIT antibodies would be equivalent in the presence of bovine source heparin and porcine source heparin.Materials and MethodsSeven lots of bovine heparin from Eurofarma and 3 lots of commercial clinical grade porcine heparin (Pfizer/Hospira) were evaluated. The USP Reference Standard for porcine heparin was used to determine anti-Xa and anti-IIa potencies of the bovine heparins. For each study, blood was collected from healthy volunteers (n=5 per test group), anticoagulated with sodium citrate, and centrifuged to obtain platelet rich plasma (PRP). Platelet aggregation responses were assessed using the BioData PAP-8 platelet aggregometer. For the first aim to evaluate platelet function, PRP was combined with heparin at final concentrations of 10.0, 1.0, and 0.1 µg/mL, covering both therapeutic and prophylactic ranges. Platelet agonists included adenosine diphosphate (ADP), collagen, epinephrine, arachidonic acid, and thrombin receptor agonist peptide (TRAP). The aggregation response was quantitated in terms of primary slope (PS), area under the curve (AUC), maximum aggregation (MA), and final aggregation (FA). For the second aim to evaluate the HIT potential, antibodies to the complex of heparin-platelet factor 4 (H-PF4) from banked HIT patient apheresis fluid were combined with donor PRP and heparin. Heparins were tested at final concentrations of 0.1, 0.4, 0.8, 1, and 100 U/mL. PS and FA results were recorded. For all data, comparisons were analyzed with 2-Way ANOVA using SigmaPlot software.ResultsIn the presence of either bovine (BMH) or porcine heparin (PMH), the normal platelet aggregation response of all donors was not altered from that obtained with saline (see representative aggregation tracing in the image below). All heparin concentrations produced the same response. There were no significant differences between the bovine and porcine heparins for each of the 4 platelet aggregation parameters for ADP, arachidonic acid, collagen, epinephrine, and TRAP. Variation in the PS for arachidonic acid and collagen need to be assessed in a larger pool of donors to assure the lack of significant difference. Platelet activation to H-PF4 antibodies was strong at 0.1 to 1 U/mL concentrations with the expected inhibition observed when using 100 U/mL heparin. The HIT potential between bovine heparin and porcine heparin demonstrated no significant difference between the heparins (see MA responses in the image below). There were no lot to lot differences for the bovine heparins or the porcine heparins in either the platelet aggregation studies or the assessment for HIT.ConclusionIn these studies of platelet function, the bovine and porcine source heparins were comparable with regards to their effects on platelet aggregation induced by multiple different agonists and their HIT potential. [Display omitted] DisclosuresWalenga:Eurofarma: Research Funding.

HMGB1 As a Novel Platelet Agonist That Acts Synergistically with ADP to Activate Platelets in Sickle Cell Disease

Background: Sickle cell disease (SCD) is a proinflammatory and prothrombotic disorder that exhibits increased platelet activation. High mobility group box 1 (HMGB1) is a nuclear protein that can mediate inflammation when released from inflammatory or ischemic cells. HMGB1 is increased in many inflammatory disease states including SCD. Recent data suggests HMGB1 activates platelets and may work synergistically with potent platelet agonists such as collagen and thrombin, but little is known regarding HMGB1-platelet interactions in combination with weaker agonists like ADP, or in isolated platelets. Moreover, the effect of HMGB1 on platelet activation has not been evaluated in SCD. We hypothesized that the in vitro addition of low-dose recombinant HMGBI (rHMBG1) to isolated platelets will lower the threshold dose of physiologic agonists required to achieve platelet activation, and that this effect is exaggerated in SCD.Methods: Platelets were isolated from healthy controls (n=4) and patients with hemoglobin SS disease (SCD; n=5). The level of platelet activation was assessed after treatment with ADP at concentrations of 0 μM, 0.5 μM, 2 μM, and 5 μM with the addition of either low-dose rHMGB1 (10 μg/mL) or the same volume of vehicle. Percent platelet activation was measured via flow cytometry using PE antibody to GPIIb (CD41) to select for platelets, and PAC1 to detect the activation-dependent conformational change in integrin αIIbβ3 (GP IIb-IIIa). Platelet activation was interpreted as percent of platelets that bound PAC1. Data was analyzed using FlowJo software and nonparametric statistical tests.Results: Mean baseline platelet activation was 1.5% (range 0.4-3.3%) for control platelets and 7.3% (1.4-17.7%) for SCD platelets (p=0.19). In the SCD group, the addition of low-dose rHMGB1 (10 μg/mL) increased the mean percent of activated platelets from 7.3% to 26.5% (10.9-43%) (p=0.01). In comparison, mean activation of control platelets increased from only 1.5% to 19.5% (8.3-42.7%) after addition of rHMGB1 (p=0.12).Having illustrated that rHMGB1 can activate washed SCD platelets, we then compared the synergistic effect of rHMGB1 with ADP. There was increased platelet activation observed when ADP was added to rHMGB1 in SCD platelets: ADP 0.5 μM increased mean platelet activation from 13.8% (range 0.3-25.3%) to 54.4% (6.7-84.9%) with the addition of rHMGB1 (p=0.02); ADP 2 μM increased platelet activation from 14.1% (2.8-23.8%) to 56.2% (22.2-88.6%) with rHMGB1 (p=0.006); and ADP 5 μM increased platelet activation from 21.4% (2.5-30.1%) to 65.3% (31.7-85.9%) after adding rHMGB1 (p=0.004) (Fig 1; * and **, diamonds vs stars). We did not find a similar statistically significant synergistic effect in the control samples treated with ADP compared to combined HMGB1 + ADP, except at ADP dose 2 μM where platelet activation increased from 14.94% (4.6-28.6%) to 39.1% (19.5-56.0%) after the addition of rHMGB1 (p=0.04; Fig 1; #, square vs circle).Activation of platelets with just ADP was not different comparing control with SCD platelets (Fig 1; circles vs stars). Similarly, activation of platelets with both ADP and rHMGB1 was not significantly different comparing control with SCD platelets except for a trend at 0.5 μM ADP + rHMGB1 10 μg/mL with 19.29% (6.6-38.7) in controls vs 54.44% (6.7-84.9) in the SCD group (p=0.07) (Fig 1; diamonds vs squares).Summary: We found that rHMGB1 acts both independently and synergistically with ADP to increase platelet activation in SCD platelets. In our small cohort, SCD platelets had increased responsiveness to low dose-rHMGB1 compared to control platelets. Moreover, combining rHMGB1 with ADP greatly enhanced platelet activation in SCD but not control platelets. Our data suggest that SCD platelets are sensitized to HMGB1 in the presence of weaker agonists such as ADP. This heightened responsiveness of SCD platelets to HMGB1 may explain the enhanced platelet activation and inflammation associated with SCD in vivo. With further study, HMGB1 could be a target of clinical drug-directed therapy in SCD patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Novel Insights into the Crosstalk between Mineralocorticoid Receptor and G Protein-Coupled Receptors in Heart Adverse Remodeling and Disease

The mineralocorticoid hormone aldosterone regulates sodium and potassium homeostasis but also adversely modulates the maladaptive process of cardiac adverse remodeling post-myocardial infarction. Through activation of its mineralocorticoid receptor (MR), a classic steroid hormone receptor/transcription factor, aldosterone promotes inflammation and fibrosis of the heart, the vasculature, and the kidneys. This is why MR antagonists reduce morbidity and mortality of heart disease patients and are part of the mainstay pharmacotherapy of advanced human heart failure. A plethora of animal studies using cell type–specific targeting of the MR gene have established the importance of MR signaling and function in cardiac myocytes, vascular endothelial and smooth muscle cells, renal cells, and macrophages. In terms of its signaling properties, the MR is distinct from nuclear receptors in that it has, in reality, two physiological hormonal agonists: not only aldosterone but also cortisol. In fact, in several tissues, including in the myocardium, cortisol is the primary hormone activating the MR. There is a considerable amount of evidence indicating that the effects of the MR in each tissue expressing it depend on tissue- and ligand-specific engagement of molecular co-regulators that either activate or suppress its transcriptional activity. Identification of these co-regulators for every ligand that interacts with the MR in the heart (and in other tissues) is of utmost importance therapeutically, since it can not only help elucidate fully the pathophysiological ramifications of the cardiac MR’s actions, but also help design and develop novel better MR antagonist drugs for heart disease therapy. Among the various proteins the MR interacts with are molecules involved in cardiac G protein-coupled receptor (GPCR) signaling. This results in a significant amount of crosstalk between GPCRs and the MR, which can affect the latter’s activity dramatically in the heart and in other cardiovascular tissues. This review summarizes the current experimental evidence for this GPCR-MR crosstalk in the heart and discusses its pathophysiological implications for cardiac adverse remodeling as well as for heart disease therapy. Novel findings revealing non-conventional roles of GPCR signaling molecules, specifically of GPCR-kinase (GRK)-5, in cardiac MR regulation are also highlighted.