Klytaimnistra Kiouptsi

Rapid formation of plasma protein corona critically affects nanoparticle pathophysiology

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.

Cofactor Independent Human Antiphospholipid Antibodies Induce Venous Thrombosis in Mice

Essentials 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.

Gut microbiota regulate hepatic von Willebrand Factor synthesis and arterial thrombus formation via Toll-like receptor-2

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 (Tlr2)-deficient mice have reduced thrombus growth after carotid artery injury relative to conventionally raised controls. GF Tlr2-/- 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 Tlr2-/- mice. Static platelet aggregate formation on extracellular matrix was similarly reduced in GF WT, Tlr2-/- , and heterozygous Vwf+/- 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 Tlr2-/- 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.

Impact of the commensal microbiota in atherosclerosis and arterial thrombosis

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.

Hypoxia impairs agonist-induced integrin α IIb β 3 activation and platelet aggregation

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.

Mice deficient in the anti-haemophilic coagulation factor VIII show increased von Willebrand factor plasma levels

Von Willebrand factor (VWF) is the carrier protein of the anti-haemophilic Factor VIII (FVIII) in plasma. It has been reported that the infusion of FVIII concentrate in haemophilia A patients results in lowered VWF plasma levels. However, the impact of F8-deficiency on VWF plasma levels in F8-/y mice is unresolved. In order to avoid confounding variables, we back-crossed F8-deficient mice onto a pure C57BL/6J background and analysed VWF plasma concentrations relative to C57BL/6J WT (F8+/y) littermate controls. F8-/y mice showed strongly elevated VWF plasma concentrations and signs of hepatic inflammation, as indicated by increased TNF-α, CD45, and TLR4 transcripts and by elevated macrophage counts in the liver. Furthermore, immunohistochemistry showed that expression of VWF antigen was significantly enhanced in the hepatic endothelium of F8-/y mice, most likely resulting from increased macrophage recruitment. There were no signs of liver damage, as judged by glutamate-pyruvate-transaminase (GPT) and glutamate-oxalacetate-transaminase (GOT) in the plasma and no signs of systemic inflammation, as white blood cell subsets were unchanged. As expected, impaired haemostasis was reflected by joint bleeding, prolonged in vitro clotting time and decreased platelet-dependent thrombin generation. Our results point towards a novel role of FVIII, synthesized by the liver endothelium, in the control of hepatic low-grade inflammation and VWF plasma levels.

Contribution of the commensal microbiota to atherosclerosis and arterial thrombosis

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.

Contribution of the commensal microbiota to atherosclerosis and arterial thrombosis: Microbiota and thrombosis

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.

Protein disulfide-isomerase – a trigger of tissue factor-dependent thrombosis

The plasmatic coagulation system is tightly controlled by natural anticoagulant mechanisms, preventing thrombosis to ensure the supply of tissues with oxygen and nutrients without provoking susceptibility to bleeding diatheses. The membrane receptor tissue factor (TF) is the principal initiator of coagulation, forming a high affinity complex with coagulation factor VIIa, thus catalyzing quantitative formation of the coagulation factor Xa. While it has been well-established that the TF-VIIa complex is efficiently blocked by factor Xa associated with tissue factor pathway inhibitor (TFPI), it was uncovered during the last decade that TF contains an intramolecular allosteric disulfide, which is prone to reduction and is crucial for TFs procoagulant and prothrombotic function. The compromised integrity of the allosteric TF disulfide pair Cys186/Cys209 was demonstrated to be responsible for the cryptic nature of TF procoagulant activity on monocytes and other cell types as well as in mouse thrombosis models. Cell surface associated protein disulfide-isomerases (PDI) were identified as activators of cryptic tissue factor. Here, we review this current concept of TF de-encryption by PDI oxidoreductases that have become interesting targets for the development of new antithrombotic treatment strategies.