Stimulation of platelet P2Y1 receptors by different endogenous nucleotides leads to functional selectivity via biased signalling.

BACKGROUND AND PURPOSE: Platelet function during inflammation is dependent on activation by endogenous nucleotides. Non-canonical signalling via the P2Y1 receptor is important for these non-thrombotic functions of platelets. However, apart from ADP, the role of other endogenous nucleotides acting as agonists at P2Y1 receptors is unknown. This study compared the effects of ADP, Ap3A, NAD+ , ADP-ribose, and Up4A on platelet functions contributing to inflammation or haemostasis. EXPERIMENTAL APPROACH: Platelets obtained from healthy human volunteers were incubated with ADP, Ap3A, NAD+ , ADP-ribose, or Up4A, with aggregation and fibrinogen binding measured (examples of function during haemostasis) or before exposure to fMLP to measure platelet chemotaxis (an inflammatory function). In silico molecular docking of these nucleotides to the binding pocket of P2Y1 receptors was then assessed. KEY RESULTS: Platelet aggregation and binding to fibrinogen induced by ADP was not mimicked by NAD+ , ADP-ribose, and Up4A. However, these endogenous nucleotides induced P2Y1 -dependent platelet chemotaxis, an effect that required RhoA and Rac-1 activity, but not canonical PLC activity. Analysis of molecular docking of the P2Y1 receptor revealed distinct differences of amino acid interactions and depth of fit within the binding pocket for Ap3A, NAD+ , ADP-ribose, or Up4A compared with ADP. CONCLUSION AND IMPLICATIONS: Platelet function (aggregation vs motility) can be differentially modulated by biased-agonist activation of P2Y1 receptors. This may be due to the character of the ligand-binding pocket interaction. This has implications for future therapeutic strategies aimed to suppress platelet activation during inflammation without affecting haemostasis as is the requirement of current ant-platelet drugs. LINKED ARTICLES: This article is part of a themed issue on Platelet purinergic receptor and non-thrombotic disease. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v181.4/issuetoc.

Novel pharmacological therapies for the treatment of bronchial asthma.

Asthma has long been recognized as a chronic inflammatory disease of the airways, often in response to inhaled allergens prompting inappropriate activation of the immune response involving a range of cells including mast cells, Th2 lymphocytes and eosinophils alongside a wide range of inflammatory mediators. First-line therapy for treatment of persistent asthma involves the use of inhaled corticosteroids (ICS) in combination with inhaled ß2-agonists enabling both the control of the underlying airways inflammation and a reduction of airway hyperresponsiveness. However, many patients remain symptomatic despite high-dose therapy. Therefore, there is a continued unmet clinical need to develop specifically new anti-inflammatory therapies for patients with asthma, either as an add-on therapy to ICS or as replacement monotherapies. The success of fixed dose combination inhalers containing both a bronchodilator and an anti-inflammatory drug has also led to the development of "bifunctional" drugs which are molecules specifically designed to have two distinct pharmacological actions based on distinct pharmacophores. In this review we will discuss these different pharmacological approaches under development for the treatment of bronchial asthma and the available preclinical and clinical data.

Red Blood Cells Elicit Platelet-Dependent Neutrophil Recruitment Into Lung Airspaces.

ABSTRACT: Hemolysis that occurs in intravascular hemolytic disorders, such as sickle cell disease and malaria, is associated with inflammation and platelet activation. Alveolar hemorrhage, for example following primary blast lung injury or acute respiratory distress syndrome, results in the escape of erythrocytes (RBCs) into alveolar spaces, where they subsequently lyse and release their intracellular contents. However, the inflammatory effects of RBCs in the airways are not fully understood. We hypothesized that RBCs in the airway induce an inflammatory response, associated with platelet activation. By instilling whole RBCs or lysed RBCs into the airways of mice, we have demonstrated that whole RBCs elicit macrophage accumulation in the lung. On the other hand, lysed RBCs induce significant inflammatory cell recruitment, particularly neutrophils and this was associated with a 50% increase in circulating platelet neutrophil complexes. Platelet depletion prior to lysed RBC exposure in the lung resulted in reduced neutrophil recruitment, suggesting that the presence of intracellular RBC components in the airways can elicit inflammation that is platelet dependent. To identify specific platelet-dependent signaling pathways involved in neutrophil recruitment, anti-P-selectin ligand and anti-PSGL1 blocking antibodies were tested; however, neither affected neutrophil recruitment. These findings implicate an involvement for other, as yet unidentified platelet-dependent signaling and adhesion mechanisms. Further understanding of how platelets contribute to lung inflammation induced by the presence of RBCs could offer novel therapeutic approaches to attenuate inflammation that occurs in conditions associated with alveolar hemorrhage.

A pleurocidin analogue with greater conformational flexibility, enhanced antimicrobial potency and in vivo therapeutic efficacy.

Antimicrobial peptides (AMPs) are a potential alternative to classical antibiotics that are yet to achieve a therapeutic breakthrough for treatment of systemic infections. The antibacterial potency of pleurocidin, an AMP from Winter Flounder, is linked to its ability to cross bacterial plasma membranes and seek intracellular targets while also causing membrane damage. Here we describe modification strategies that generate pleurocidin analogues with substantially improved, broad spectrum, antibacterial properties, which are effective in murine models of bacterial lung infection. Increasing peptide-lipid intermolecular hydrogen bonding capabilities enhances conformational flexibility, associated with membrane translocation, but also membrane damage and potency, most notably against Gram-positive bacteria. This negates their ability to metabolically adapt to the AMP threat. An analogue comprising D-amino acids was well tolerated at an intravenous dose of 15 mg/kg and similarly effective as vancomycin in reducing EMRSA-15 lung CFU. This highlights the therapeutic potential of systemically delivered, bactericidal AMPs.

Animal models of mechanisms of SARS-CoV-2 infection and COVID-19 pathology.

The coronavirus disease 2019 (COVID-19) pandemic caused by SARS-CoV-2 infections has led to a substantial unmet need for treatments, many of which will require testing in appropriate animal models of this disease. Vaccine trials are already underway, but there remains an urgent need to find other therapeutic approaches to either target SARS-CoV-2 or the complications arising from viral infection, particularly the dysregulated immune response and systemic complications which have been associated with progression to severe COVID-19. At the time of writing, in vivo studies of SARS-CoV-2 infection have been described using macaques, cats, ferrets, hamsters, and transgenic mice expressing human angiotensin I converting enzyme 2 (ACE2). These infection models have already been useful for studies of transmission and immunity, but to date only partly model the mechanisms involved in human severe COVID-19. There is therefore an urgent need for development of animal models for improved evaluation of efficacy of drugs identified as having potential in the treatment of severe COVID-19. These models need to reproduce the key mechanisms of COVID-19 severe acute respiratory distress syndrome and the immunopathology and systemic sequelae associated with this disease. Here, we review the current models of SARS-CoV-2 infection and COVID-19-related disease mechanisms and suggest ways in which animal models can be adapted to increase their usefulness in research into COVID-19 pathogenesis and for assessing potential treatments. LINKED ARTICLES: This article is part of a themed issue on The Pharmacology of COVID-19. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.21/issuetoc.

LPS-induced Lung Platelet Recruitment Occurs Independently from Neutrophils, PSGL-1, and P-Selectin.

Platelets are recruited to inflammatory foci and contribute to host defense and inflammatory responses. Compared with platelet recruitment in hemostasis and thrombosis, the mechanisms of platelet recruitment in inflammation and host defense are poorly understood. Neutrophil recruitment to lung airspaces after inhalation of bacterial LPS requires platelets and PSGL-1 in mice. Given this association between platelets and neutrophils, we investigated whether recruitment of platelets to lungs of mice after LPS inhalation was dependent on PSGL-1, P-selectin, or interaction with neutrophils. BALB/c mice were administered intranasal LPS (O55:B5, 5 mg/kg) and, 48 hours later, lungs were collected and platelets and neutrophils quantified in tissue sections by immunohistochemistry. The effects of functional blocking antibody treatments targeting the platelet-neutrophil adhesion molecules, P-selectin or PSGL-1, or treatment with a neutrophil-depleting antibody targeting Ly6G, were tested on the extent of LPS-induced lung platelet recruitment. Separately in Pf4-Cre × mTmG mice, two-photon intravital microscopy was used to image platelet adhesion in live lungs. Inhalation of LPS caused both platelet and neutrophil recruitment to the lung vasculature. However, decreasing lung neutrophil recruitment by blocking PSGL-1, P-selectin, or depleting blood neutrophils had no effect on lung platelet recruitment. Lung intravital imaging revealed increased adhesion of platelets in the lung microvasculature which was not associated with thrombus formation. In conclusion, platelet recruitment to lungs in response to LPS occurs through mechanisms distinct from those mediating neutrophil recruitment, or the occurrence of pulmonary emboli.

A dichotomy in platelet activation: Evidence of different functional platelet responses to inflammatory versus haemostatic stimuli.

INTRODUCTION: Platelets participate in inflammatory disorders through a variety of different functional responses, including chemotaxis, platelet-leukocyte complex formation and facilitation of leukocyte recruitment that are thought to be distinct from platelet aggregation. This may account for why classical anti-platelet drugs have failed to ameliorate inflammatory disorders where platelets are known to participate, suggesting that distinct pathways may control inflammatory and haemostatic functions of platelets. In the present study, we have therefore investigated the effect of different stimuli on several different functions of platelets preferentially involved either in haemostasis or in inflammation. MATERIALS AND METHODS: Human platelets were stimulated with either inflammatory (fMLP, histamine, IL-1ß, LPS, MDC/CCL22, SDF-1α/CXCL12 and 5-HT) or haemostatic (ADP, collagen, convulxin, epinephrine, TRAP-6 and U46619) stimuli. Aggregation, platelet-leukocyte complex formation, platelet migration and platelet protein phosphorylation were assessed. RESULTS: Haemostatic stimuli induced platelet aggregation, whilst inflammatory agonists induced platelet migration. The haemostatic stimuli, with the exception of epinephrine, and some of the inflammatory stimuli induced platelet-leukocyte complex formation, even if to a different extent. Furthermore, inflammatory stimuli induced a shorter lasting profile of platelet protein phosphorylation compared with haemostatic stimuli. CONCLUSIONS: Stimulation of platelets with inflammatory stimuli triggers the activation of non haemostatic functions different from those induced by haemostatic stimuli, supporting the existence of alternative platelet responses depending on the stimulus (haemostatic or inflammatory). A deeper understanding of the biochemical pathways behind these functional differences may lead to the development of novel therapeutic options targeting the inflammatory actions of platelets, without affecting their critical role in haemostasis.

Diverse signalling of the platelet P2Y1 receptor leads to a dichotomy in platelet function.

Platelet P2Y1 receptor signalling via RhoGTPases is necessary for platelet-dependent leukocyte recruitment, where no platelet aggregation is observed. We investigated signalling cascades involved in distinct P2Y1-dependent platelet activities in vitro, using specific inhibitors for phospholipase C (PLC) (U73122, to inhibit the canonical pathway), and RhoGTPases: Rac1 (NSC23766) and RhoA (ROCK inhibitor GSK429286). Human platelet rich plasma (for platelet aggregation) or isolated washed platelets (for chemotaxis assays) was treated with U73122, GSK429286 or NSC23766 prior to stimulation with adenosine diphosphate (ADP) or the P2Y1 specific agonist MRS2365. Aggregation, chemotaxis (towards f-MLP), or platelet-induced human neutrophil chemotaxis (PINC) towards macrophage derived chemokine (MDC) was assessed. Molecular docking of ADP and MRS2365 to P2Y1 was analysed using AutoDock Smina followed by GOLD molecular docking in the Accelrys Discovery Studio software. Inhibition of PLC, but not Rac1 or RhoA, suppressed platelet aggregation induced by ADP and MRS2365. In contrast, platelet chemotaxis and PINC, were significantly attenuated by inhibition of platelet Rac1 or RhoA, but not PLC. MRS2365, compared to ADP had a less pronounced effect on P2Y1-induced aggregation, but a similar efficacy to stimulate platelet chemotaxis and PINC, which might be explained by differences in molecular interaction of ADP compared to MRS2365 with the P2Y1 receptor. Platelet P2Y1 receptor activation during inflammation signals through alternate pathways involving Rho GTPases in contrast to canonical P2Y1 receptor induced PLC signalling. This might be explained by selective molecular interactions of ligands within the orthosteric site of the P2Y1 receptor.

Platelets Play a Central Role in Sensitization to Allergen.

Platelet activation occurs in patients with allergic inflammation, and platelets can be activated directly by allergen via an IgE-dependent process. Platelets have been shown to activate APCs such as CD11c+ dendritic cells in vitro. Although CD11c+ dendritic cells are a requisite for allergen sensitization, the role of platelets in this process is unknown. In this study, we investigated whether platelets were necessary for allergen sensitization. Balb/c mice sensitized to ovalbumin were exposed to subsequent aerosolized allergen (ovalbumin challenge). We analyzed lung CD11c+ cell activation, colocalization with platelets, and some other indices of inflammation. The role of platelets at the time of allergen sensitization was assessed through platelet depletion experiments restricted to the period of sensitization. Platelets colocalized with airway CD11c+ cells, and this association increased after allergen sensitization as well as after subsequent allergen exposure. Temporary platelet depletion (>95%) at the time of allergen sensitization led to a suppression of IgE and IL-4 synthesis and to a decrease in the pulmonary recruitment of eosinophils, macrophages, and lymphocytes after subsequent allergen exposure. Furthermore, in mice previously depleted of platelets at the time of sensitization, the recovered platelet population was shown to have reduced expression of FcεRI. Pulmonary CD11c+ cell recruitment was suppressed in these mice after allergen challenge, suggesting that the migration of CD11c+ cells in vivo may be dependent on direct platelet recognition of allergen. We conclude that platelets are necessary for efficient host sensitization to allergen. This propagates the subsequent inflammatory response during secondary allergen exposure and increases platelet association with airway CD11c+ cells.

Platelet Depletion Impairs Host Defense to Pulmonary Infection with Pseudomonas aeruginosa in Mice.

Platelets have been implicated in pulmonary inflammatory cell recruitment after exposure to allergic and nonallergic stimuli, but little is known about the role of platelets in response to pulmonary infection with Pseudomonas aeruginosa. In this study, we have investigated the impact of the experimental depletion of circulating platelets on a range of inflammatory and bacterial parameters, and their subsequent impact on mortality in a murine model of pulmonary infection with P. aeruginosa. P. aeruginosa infection in mice induced a mild, but significant, state of peripheral thrombocytopenia in addition to pulmonary platelet accumulation. Increased platelet activation was detected in infected mice through increased levels of the platelet-derived mediators, platelet factor-4 and ß-thromboglobulin, in BAL fluid and blood plasma. In mice depleted of circulating platelets, pulmonary neutrophil recruitment was significantly reduced 24 hours after infection, whereas the incidence of systemic dissemination of bacteria was significantly increased compared with non-platelet-depleted control mice. Furthermore, mortality rates were increased in bacterial-infected mice depleted of circulating platelets. This work demonstrates a role for platelets in the host response toward a gram-negative bacterial respiratory infection.

RhoA signaling through platelet P2Y1 receptor controls leukocyte recruitment in allergic mice.

BACKGROUND: Clinical studies reveal platelet activation in patients with asthma, allergic rhinitis, and eczema. This is distinct from platelet aggregation, which is critical for the maintenance of hemostasis and in which a role for platelet purinergic receptors is well documented. However, purines are also essential for inflammatory cell trafficking in animal models of allergic lung inflammation, which are known to be platelet dependent, yet the role of purines in the platelet activation accompanying inflammation is unknown. OBJECTIVES: We investigated whether the involvement of purine activation of platelets during allergic inflammation is distinct from purine involvement in platelet aggregation. METHODS: BALB/c mice were sensitized to ovalbumin and subsequent airway ovalbumin challenge. Bronchoalveolar lavage fluid was analyzed for inflammatory cells, and blood samples were assessed for platelet activation. The role of platelet purinergic receptors and associated signaling mechanisms (RhoA) were assessed. RESULTS: P2Y1, but not P2Y12 or P2X1, antagonism inhibited pulmonary leukocyte recruitment. The formation of platelet-leukocyte complexes in vivo and platelet/P-selectin-dependent polymorphonuclear cell migration in vitro were exclusively platelet P2Y1 receptor dependent. Furthermore, platelet P2Y1 activation resulted in RhoA activity in vivo after allergen challenge, and RhoA signaling in platelets through P2Y1 stimulation was required for platelet-dependent leukocyte chemotaxis in vitro. Leukocyte recruitment in thrombocytopenic mice remained suppressed after reinfusion of platelets pretreated with a P2Y1 antagonist or a Rho-associated kinase 1 inhibitor, confirming the crucial role of platelet P2Y1 receptor and subsequent activation of RhoA. CONCLUSION: RhoA signaling downstream of platelet P2Y1, but not P2Y12, represents a clear dichotomy in platelet activation during allergic inflammation versus hemostasis.

P-Rex and Vav Rac-GEFs in platelets control leukocyte recruitment to sites of inflammation.

The small GTPase Rac is required for neutrophil recruitment during inflammation, but its guanine-nucleotide exchange factor (GEF) activators seem dispensable for this process, which led us to investigate the possibility of cooperation between Rac-GEF families. Thioglycollate-induced neutrophil recruitment into the peritoneum was more severely impaired in P-Rex1(-/-) Vav1(-/-) (P1V1) or P-Rex1(-/-) Vav3(-/-) (P1V3) mice than in P-Rex null or Vav null mice, suggesting cooperation between P-Rex and Vav Rac-GEFs in this process. Neutrophil transmigration and airway infiltration were all but lost in P1V1 and P1V3 mice during lipopolysaccharide (LPS)-induced pulmonary inflammation, with altered intercellular adhesion molecule 1-dependent slow neutrophil rolling and strongly reduced L- and E-selectin-dependent adhesion in airway postcapillary venules. Analysis of adhesion molecule expression, neutrophil adhesion, spreading, and migration suggested that these defects were only partially neutrophil-intrinsic and were not obviously involving vascular endothelial cells. Instead, P1V1 and P1V3 platelets recapitulated the impairment of LPS-induced intravascular neutrophil adhesion and recruitment, showing P-Rex and Vav expression in platelets to be crucial. Similarly, during ovalbumin-induced allergic inflammation, pulmonary recruitment of P1V1 and P1V3 eosinophils, monocytes, and lymphocytes was compromised in a platelet-dependent manner, and airway inflammation was essentially abolished, resulting in improved airway responsiveness. Therefore, platelet P-Rex and Vav family Rac-GEFs play important proinflammatory roles in leukocyte recruitment.