Human platelet activation by Escherichia coli: roles for FcγRIIA and integrin αIIbβ3

Gram-negative Escherichia coli cause diseases such as sepsis and hemolytic uremic syndrome in which thrombotic disorders can be found. Direct platelet–bacterium interactions might contribute to some of these conditions; however, mechanisms of human platelet activation by E. coli leading to thrombus formation are poorly understood. While the IgG receptor FcγRIIA has a key role in platelet response to various Gram-positive species, its role in activation to Gram-negative bacteria is poorly defined. This study aimed to investigate the molecular mechanisms of human platelet activation by E. coli, including the potential role of FcγRIIA. Using light-transmission aggregometry, measurements of ATP release and tyrosine-phosphorylation, we investigated the ability of two E. coli clinical isolates to activate platelets in plasma, in the presence or absence of specific receptors and signaling inhibitors. Aggregation assays with washed platelets supplemented with IgGs were performed to evaluate the requirement of this plasma component in activation. We found a critical role for the immune receptor FcγRIIA, αIIbβ3, and Src and Syk tyrosine kinases in platelet activation in response to E. coli. IgG and αIIbβ3 engagement was required for FcγRIIA activation. Moreover, feedback mediators adenosine 5’-diphosphate (ADP) and thromboxane A₂ (TxA₂) were essential for platelet aggregation. These findings suggest that human platelet responses to E. coli isolates are similar to those induced by Gram-positive organisms. Our observations support the existence of a central FcγRIIA-mediated pathway by which human platelets respond to both Gram-negative and Gram-positive bacteria

Neutrophil extracellular traps (NETS): role in disease

Neutrophil extracellular traps (NETs) are web-like structures composed by a chromatin backbone, histones and antimicrobial proteins. NETs constitute yet another mechanism deployed by neutrophils to immobilise and kill microorganisms, thus contributing to the host innate immunity. Neutrophils cast NETs upon stimulation by a variety of stimuli, including bacteria, protozoa, fungi, viruses, their products and also host factors like chemokines, complement and activated platelets. NETs production or NETosis occurs as a result of activation of neutrophil PKC, Raf-MEK-ERK and NADPH oxydase signalling pathways. Driven mostly by peptidylarginine deiminase 4 (PAD4) citrullination of histones, the hallmark of NETosis is chromatin decondensation, rupture of nuclear membrane and release of nuclear and granular contents into the cytoplasm, prior their release into the extracellular space. NETs control propagation of pathogens by entrapping them within the lose chromatin web and kills them with the antimicrobial molecules –granule proteins and histones- present in high concentrations within the chromatin network. Despite contributing to host defence, aberrant NET formation may damage tissues and activate inflammatory cells, contributing to several pathologies, including sepsis, systemic inflammatory response syndrome, autoimmune diseases and thrombosis. This review presents an overview of our current knowledge of NETs physiology and their role in fighting and propagating disease.Sociedad Argentina de FisiologíaFacultad de Ciencias Médica

Towards 3D in vitro models for the study of cardiovascular tissues and disease

The field of tissue engineering is developing biomimetic biomaterial scaffolds which are showing increasing therapeutic potential for the repair of cardiovascular tissues. However, a major opportunity exists to use them as 3D in vitro models for the study of cardiovascular tissues and disease in addition to drug development and testing. These in vitro models can span the gap between 2D culture and in vivo testing thus reducing cost, time and the ethical burden of current approaches. This review outlines the progress to date and requirements for the development of ideal in vitro 3D models for blood vessels, heart valves and myocardial tissue

Amplification of bacteria-induced platelet activation is triggered by FcγRIIA, integrin αiIbβ3, and platelet factor 4

Bacterial adhesion to platelets is mediated via a range of strain-specific bacterial surface proteins that bind to a variety of platelet receptors. It is unclear how these interactions lead to platelet activation. We demonstrate a critical role for the immune receptor FcγRIIA, αIIbβ3, and Src and Syk tyrosine kinases in platelet activation by Staphylococcus aureus, Streptococcus sanguinis, Streptococcus gordonii, Streptococcus oralis, and Streptococcus pneumoniae. FcγRIIA activation is dependent on immunoglobulin G (IgG) and αIIbβ3 engagement. Moreover, feedback agonists adenosine 59-diphosphate and thromboxane A2 aremandatory for platelet aggregation. Additionally, platelet factor 4 (PF4) binds to bacteria and reduces the lag time for aggregation, and gray platelet syndromea-granule-deficient platelets do not aggregate to 4 of 5 bacterial strains. We propose that FcγRIIA-mediated activation is a common response mechanism used against a wide range of bacteria, and that release of secondary mediators and PF4 serve as a positive feedback mechanism for activation through an IgG-dependent pathway. © 2014 by The American Society of Hematology

Full scale pedestrian impact testing with PMHS: A pilot study

The complexity of vehicle-pedestrian collisions necessitates extensive validation of pedestrian computational models. While body components can be individually simulated, overall validation of human pedestrian models requires full-scale testing with post mortem human surrogates (PMHS). This paper presents the development of a full-scale pedestrian impact test plan and experimental design, and some results from a pilot experiment. The test plan and experimental design are developed based on the analysis of a combination of literature review, multi-body modeling, and epidemiologic studies. The proposed system has proven effective in testing an anthropometrically correct rescue dummy in multiple instances, and in one test with a PMHS.Postprint (published version

Molecular Cross-Talk between Integrins and Cadherins Leads to a Loss of Vascular Barrier Integrity during SARS-CoV-2 Infection

The vascular barrier is heavily injured following SARS-CoV-2 infection and contributes enormously to life-threatening complications in COVID-19. This endothelial dysfunction is associated with the phlogistic phenomenon of cytokine storms, thrombotic complications, abnormal coagulation, hypoxemia, and multiple organ failure. The mechanisms surrounding COVID-19 associated endotheliitis have been widely attributed to ACE2-mediated pathways. However, integrins are emerging as possible receptor candidates for SARS-CoV-2, and their complex intracellular signaling events are essential for maintaining endothelial homeostasis. Here, we showed that the spike protein of SARS-CoV-2 depends on its RGD motif to drive barrier dysregulation by hijacking integrin αVβ3, expressed on human endothelial cells. This triggers the redistribution and internalization of major junction protein VE-Cadherin which leads to the barrier disruption phenotype. Both extracellular and intracellular inhibitors of integrin αVβ3 prevented these effects, similarly to the RGD-cyclic peptide compound Cilengitide, which suggests that the spike protein—through its RGD motif—binds to αVβ3 and elicits vascular leakage events. These findings support integrins as an additional receptor for SARS-CoV-2, particularly as integrin engagement can elucidate many of the adverse endothelial dysfunction events that stem from COVID-19

Staphylococcus aureus protein A causes osteoblasts to hyper-mineralise in a 3D extra-cellular matrix environment.

Osteomyelitis is an inflammatory bone infection that is caused most commonly by the opportunistic pathogen Staphylococcus aureus. Research into staphylococcal induced bone infection is typically conducted using traditional 2D in vitro culture settings, which is not fully representative of the dynamic in vivo environment. In this study we utilised a collagen glycosaminoglycan scaffold, previously developed for bone tissue engineering, as a representative 3D model of infection. The scaffold resisted degradation and retained its pore structure, which is important for cellular function and survival, when seeded with both cells and bacteria. Using this model, we showed that in the presence of S. aureus, osteoblast proliferation was reduced over 21 days. Interestingly however these cells were more metabolically active compared to the uninfected cells and demonstrated increased mineralisation. Protein A (SpA) is a virulence factor found on the surface of S. aureus and has been shown to interact with osteoblasts. When SpA was removed from the surface of S. aureus, the osteoblasts show comparable activity with the uninfected cells-demonstrating the importance of SpA in the interaction between bone cells and S. aureus. Our results suggest that infected osteoblasts are capable of over-compensating for bone loss and bone destruction by increasing mineralisation in a 3D environment, key elements required for ensuring bone strength. It also reinforces our previously established result that S. aureus SpA is a critical mediator in osteomyelitis and might be a potential novel drug target to treat osteomyelitis by preventing the interaction between S. aureus and osteoblasts

Platelets and the innate immune system: Mechanisms of bacterial-induced platelet activation.

It has become clear that platelets are not simply cell fragments that can plug the leak in a damaged blood vessel, they are in fact key components in the innate immune system which is supported by the presence of Toll-like receptors (TLRs) on platelets. As the first responding cell to a site of injury they are well placed to direct the immune response to deal with any resulting exposure to pathogens. The response is triggered by bacteria binding to platelets which usually triggers platelet activation and the secretion of anti-microbial peptides. The main platelet receptors that mediate these interactions are GPIIb/IIIa, GPIbα, FcνRIIa, complement receptors and TLRs. This may involve direct interactions between bacterial proteins and the receptors or can be mediated by plasma proteins such as fibrinogen, von Willebrand factor, complement and IgG. Here we review the variety of interactions between platelets and bacteria and look at the potential for inhibiting these interactions in diseases such as infective endocarditis and sepsis.</p

Pre-emptive and therapeutic value of blocking bacterial attachment to the endothelial alphaVbeta3 integrin with cilengitide in sepsis

Pre-emptive and therapeutic value of blocking bacterial attachment to the endothelial alphaVbeta3 integrin with cilengitide in sepsis