In biological fluids, proteins bind to the surface of nanoparticles to form a coating known as the protein corona, which can critically affect the interaction of the nanoparticles with living systems. As physiological systems are highly dynamic, it is important to obtain a time-resolved knowledge of protein-corona formation, development and biological relevancy. Here we show that label-free snapshot proteomics can be used to obtain quantitative time-resolved profiles of human plasma coronas formed on silica and polystyrene nanoparticles of various size and surface functionalization. Complex time- and nanoparticle-specific coronas, which comprise almost 300 different proteins, were found to form rapidly (<0.5 minutes) and, over time, to change significantly in terms of the amount of bound protein, but not in composition. Rapid corona formation is found to affect haemolysis, thrombocyte activation, nanoparticle uptake and endothelial cell death at an early exposure time.

The symbiotic gut microbiota play pivotal roles in host physiology and the development of cardiovascular diseases, but the microbiota-triggered pattern recognition signaling mechanisms that impact thrombosis are poorly defined. In this article, we show that germ-free (GF) and Toll-like receptor-2 ()-deficient mice have reduced thrombus growth after carotid artery injury relative to conventionally raised controls. GF and wild-type (WT) mice were indistinguishable, but colonization with microbiota restored a significant difference in thrombus growth between the genotypes. We identify reduced plasma levels of von Willebrand factor (VWF) and reduced VWF synthesis, specifically in hepatic endothelial cells, as a critical factor that is regulated by gut microbiota and determines thrombus growth in mice. Static platelet aggregate formation on extracellular matrix was similarly reduced in GF WT, , and heterozygous mice that are all characterized by a modest reduction in plasma VWF levels. Defective platelet matrix interaction can be restored by exposure to WT plasma or to purified VWF depending on the VWF integrin binding site. Moreover, administration of VWF rescues defective thrombus growth in mice in vivo. These experiments delineate an unexpected pathway in which microbiota-triggered TLR2 signaling alters the synthesis of proadhesive VWF by the liver endothelium and favors platelet integrin-dependent thrombus growth.

A loop-mediated isothermal amplification (LAMP) procedure for the detection of Cryptosporidium in environmental and fecal samples was developed and evaluated. This is the first demonstration of LAMP applied to detection of Cryptosporidium. Due to its specificity and simplicity, the method could become a useful diagnostic tool for epidemiologic studies of Cryptosporidium presence.

Our results demonstrate a functional role for the commensal microbiota in atherothrombosis. In a ferric chloride injury model of the carotid artery, GF C57BL/6J mice had increased occlusion times compared to colonized controls. Interestingly, in late atherosclerosis, HFD-fed GF Ldlr−/− mice had reduced plaque rupture-induced thrombus growth in the carotid artery and diminished ex vivo thrombus formation under arterial flow conditions.

Objective: Recruitment of neutrophils and formation of neutrophil extracellular traps (NETs) contribute to lethality in acute mesenteric infarction. To study the impact of the gut microbiota in acute mesenteric infarction, we used gnotobiotic mouse models to investigate whether gut commensals prime the reactivity of neutrophils towards formation of neutrophil extracellular traps (NETosis). Approach and Results: We applied a mesenteric ischemia-reperfusion (I/R) injury model to germ-free (GF) and colonized C57BL/6J mice. By intravital imaging, we quantified leukocyte adherence and NET formation in I/R-injured mesenteric venules. Colonization with gut microbiota or monocolonization with Escherichia coli augmented the adhesion of leukocytes, which was dependent on the TLR4 (Toll-like receptor-4)/TRIF (TIR-domain–containing adapter-inducing interferon-β) pathway. Although neutrophil accumulation was decreased in I/R-injured venules of GF mice, NETosis following I/R injury was significantly enhanced compared with conventionally raised mice or mice colonized with the minimal microbial consortium altered Schaedler flora. Also ex vivo, neutrophils from GF and antibiotic-treated mice showed increased LPS (lipopolysaccharide)-induced NETosis. Enhanced TLR4 signaling in GF neutrophils was due to elevated TLR4 expression and augmented IRF3 (interferon regulatory factor-3) phosphorylation. Likewise, neutrophils from antibiotic-treated conventionally raised mice had increased NET formation before and after ischemia. Increased NETosis in I/R injury was abolished in conventionally raised mice deficient in the TLR adaptor TRIF. In support of the desensitizing influence of enteric LPS, treatment of GF mice with LPS via drinking water diminished LPS-induced NETosis in vitro and in the mesenteric I/R injury model. Conclusions: Collectively, our results identified that the gut microbiota suppresses NETing neutrophil hyperreactivity in mesenteric I/R injury, while ensuring immunovigilance by enhancing neutrophil recruitment.

Cofactor‐independent antiphospholipid antibodies (CI‐aPL) are generally considered non‐pathogenic. We analyzed the effects of human monoclonal CI‐aPL in a mouse model of venous thrombosis. As shown in vitro, CI‐aPL induce a procoagulant state in vivo by activation of endosomal NADPH‐oxidase. Contrary to common belief, CI‐aPL induce venous thrombosis in vivo. Summary BackgroundThere is general consensus that the antiphospholipid syndrome (APS) is caused by antiphospholipid antibodies (aPL) with antibodies against β2‐glycoprotein‐I being the most relevant. aPL that bind phospholipids in the absence of protein cofactors are generally considered pathogenetically irrelevant. We showed that cofactor‐independent human monoclonal aPL isolated from APS patients induce proinflammatory and procoagulant cellular responses by activating endosomal NADPH‐oxidase 2 (NOX2). Similar aPL were detected in all IgG fractions from APS patients analyzed. ObjectivesWe aimed to clarify if cofactor‐independent aPL can be thrombogenic in vivo and, if so, whether these effects are mediated via activation of NOX2. MethodsTwo cofactor‐independent human monoclonal aPL, HL5B and RR7F, were tested in a mouse model of venous thrombosis. Genetically modified mice and in vitro assays were used to delineate the mechanisms underlying thrombus induction. ResultsHL5B and RR7F dramatically accelerate thrombus formation in this mouse model. Thrombus formation depends on tissue factor activation. It cannot be induced in NOX2‐deficient mice. Bone marrow chimeras of C57BL/6J mice reconstituted with NOX2‐deficient bone marrow showed that leukocyte activation plays a major role in thrombus formation. Neither TLR4 signaling nor platelet activation by our aPL is required for venous thrombus formation. ConclusionsCofactor‐independent aPL can induce thrombosis in vivo. This effect is mainly mediated by leukocyte activation, which depends on the previously described signal transduction via endosomal NOX2. Because most APS patients have been shown to harbor aPL with similar activity, our data are of general relevance for the APS.

The commensal gut microbiota is an environmental factor that has been implicated in the development of cardiovascular disease. The development of atherosclerotic lesions is largely influenced not only by the microbial-associated molecular patterns of the gut microbiota but also by the meta-organismal trimethylamine N-oxide pathway. Recent studies have described a role for the gut microbiota in platelet activation and arterial thrombosis. This review summarizes the results from gnotobiotic mouse models and clinical data that linked microbiota-induced pattern recognition receptor signalling with atherogenesis. Based on recent insights, we here provide an overview of how the gut microbiota could affect endothelial cell function and platelet activation, to promote arterial thrombosis. LINKED ARTICLES: This article is part of a themed section on When Pharmacology Meets the Microbiome: New Targets for Therapeutics? To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.24/issuetoc.

Under ischemic conditions, tissues are exposed to hypoxia. Although human physiology, to a certain extent, can adapt to hypoxic conditions, the impact of low oxygen levels on platelet function is unresolved. Therefore, we explored how reduction of atmospheric oxygen levels to 1% might affect agonist-induced aggregation and static adhesion of isolated human platelets. We uncovered that isolated, washed human platelets exposed to hypoxic conditions show reduced thrombin receptor-activating peptide-6 (TRAP-6) and convulxin-induced aggregation. Of note, this hypoxia-triggered effect was not observed in platelet-rich plasma. Independent of the agonist used (TRAP-6, ADP), activation of the platelet fibrinogen receptor integrin αIIbβ3 (GPIIbIIIa, CD41/CD61) was strongly reduced at 1% and 8% oxygen. The difference in agonist-induced integrin αIIbβ3 activation was apparent within 5 minutes of stimulation. Following hypoxia, re-oxygenation resulted in the recovery of integrin αIIbβ3 activation. Importantly, platelet secretion was not impaired by hypoxia. Static adhesion experiments revealed decreased platelet deposition to fibrinogen coatings, but not to collagen or vitronectin coatings, indicating that specifically the function of the integrin subunit αIIb is impaired by exposure of platelets to reduced oxygen levels. Our results reveal an unexpected effect of oxygen deprivation on platelet aggregation mediated by the fibrinogen receptor integrin αIIbβ3.