Isolation and Characterization of Shiga Toxin-Associated Microvesicles.

Microvesicles are shed from cell surfaces during infectious or inflammatory conditions and may contribute to the pathogenesis of disease. During Shiga toxin-producing Escherichia coli (STEC) infection, microvesicles are released from blood cells. These microvesicles play a part in inflammation, thrombosis, hemolysis, and the transfer of the main virulence factor of STEC strains, Shiga toxin, to target organ cells. This chapter describes how to isolate blood cell- and cell culture-derived microvesicles from plasma or cell culture medium, respectively, and how to characterize these microvesicles by various methods, with special focus on Shiga toxin-associated microvesicles.

Shiga Toxin Uptake and Sequestration in Extracellular Vesicles Is Mediated by Its B-Subunit.

Shiga toxin (Stx)-stimulated blood cells shed extracellular vesicles (EVs) which can transfer the toxin to the kidneys and lead to hemolytic uremic syndrome. The toxin can be taken up by renal cells within EVs wherein the toxin is released, ultimately leading to cell death. The mechanism by which Stx is taken up, translocated, and sequestered in EVs was addressed in this study utilizing the B-subunit that binds to the globotriaosylceramide (Gb3) receptor. We found that Stx1B was released in EVs within minutes after stimulation of HeLa cells or red blood cells, detected by live cell imaging and flow cytometry. In the presence of Retro-2.1, an inhibitor of intracellular retrograde trafficking, a continuous release of Stx-positive EVs occurred. EVs from HeLa cells possess the Gb3 receptor on their membrane, and EVs from cells that were treated with a glycosylceramide synthase inhibitor, to reduce Gb3, bound significantly less Stx1B. Stx1B was detected both on the membrane and within the shed EVs. Stx1B was incubated with EVs derived from blood cells, in the absence of cells, and was shown to bind to, and be taken up by, these EVs, as demonstrated by electron microscopy. Using a membrane translocation assay we demonstrated that Stx1B was taken up by blood cell- and HeLa-derived EVs, an effect enhanced by chloropromazine or methyl-ß-cyclodextrin, suggesting toxin transfer within the membrane. This is a novel mechanism by which EVs derived from blood cells can sequester their toxic content, possibly to evade the host response.

Shiga Toxin-Bearing Microvesicles Exert a Cytotoxic Effect on Recipient Cells Only When the Cells Express the Toxin Receptor.

Shiga toxin is the main virulence factor of non-invasive enterohemorrhagic Escherichia coli strains capable of causing hemolytic uremic syndrome. Our group has previously shown that the toxin can reach the kidney within microvesicles where it is taken up by renal cells and the vesicles release their cargo intracellularly, leading to toxin-mediated inhibition of protein synthesis and cell death. The aim of this study was to examine if recipient cells must express the globotriaosylceramide (Gb3) toxin receptor for this to occur, or if Gb3-negative cells are also susceptible after uptake of Gb3-positive and toxin-positive microvesicles. To this end we generated Gb3-positive A4GALT-transfected CHO cells, and a vector control lacking Gb3 (CHO-control cells), and decreased Gb3 synthesis in native HeLa cells by exposing them to the glycosylceramide synthase inhibitor PPMP. We used these cells, and human intestinal DLD-1 cells lacking Gb3, and exposed them to Shiga toxin 2-bearing Gb3-positive microvesicles derived from human blood cells. Results showed that only recipient cells that possessed endogenous Gb3 (CHO-Gb3 transfected and native HeLa cells) exhibited cellular injury, reduced cell metabolism and protein synthesis, after uptake of toxin-positive microvesicles. In Gb3-positive cells the toxin introduced via vesicles followed the retrograde pathway and was inhibited by the retrograde transport blocker Retro-2.1. CHO-control cells, HeLa cells treated with PPMP and DLD-1 cells remained unaffected by toxin-positive microvesicles. We conclude that Shiga toxin-containing microvesicles can be taken up by Gb3-negative cells but the recipient cell must express endogenous Gb3 for the cell to be susceptible to the toxin.

Shiga toxin signals via ATP and its effect is blocked by purinergic receptor antagonism.

Shiga toxin (Stx) is the main virulence factor of enterohemorrhagic Escherichia coli (EHEC), that cause gastrointestinal infection leading to hemolytic uremic syndrome. The aim of this study was to investigate if Stx signals via ATP and if blockade of purinergic receptors could be protective. Stx induced ATP release from HeLa cells and in a mouse model. Toxin induced rapid calcium influx into HeLa cells, as well as platelets, and a P2X1 receptor antagonist, NF449, abolished this effect. Likewise, the P2X antagonist suramin blocked calcium influx in Hela cells. NF449 did not affect toxin intracellular retrograde transport, however, cells pre-treated with NF449 exhibited significantly higher viability after exposure to Stx for 24 hours, compared to untreated cells. NF449 protected HeLa cells from protein synthesis inhibition and from Stx-induced apoptosis, assayed by caspase 3/7 activity. The latter effect was confirmed by P2X1 receptor silencing. Stx induced the release of toxin-positive HeLa cell- and platelet-derived microvesicles, detected by flow cytometry, an effect significantly reduced by NF449 or suramin. Suramin decreased microvesicle levels in mice injected with Stx or inoculated with Stx-producing EHEC. Taken together, we describe a novel mechanism of Stx-mediated cellular injury associated with ATP signaling and inhibited by P2X receptor blockade.

Blockade of the kallikrein-kinin system reduces endothelial complement activation in vascular inflammation.

BACKGROUND: The complement and kallikrein-kinin systems (KKS) are activated during vascular inflammation. The aim of this study was to investigate if blockade of the KKS can affect complement activation on the endothelium during inflammation. METHODS: Complement deposition on endothelial microvesicles was assayed in vasculitis patient plasma samples and controls. Plasma was perfused over glomerular endothelial cells and complement deposition assayed by flow cytometry. The effect of the kinin system was assessed using kinin receptor antagonists and C1-inhibitor. The in vivo effect was assessed in kidney sections from mice with nephrotoxic serum-induced glomerulonephritis treated with a kinin receptor antagonist. FINDINGS: Vasculitis patient plasma had significantly more C3- and C9-positive endothelial microvesicles than controls. Perfusion of patient acute-phase plasma samples over glomerular endothelial cells induced the release of significantly more complement-positive microvesicles, in comparison to remission or control plasma. Complement activation on endothelial microvesicles was reduced by kinin B1- and B2-receptor antagonists or by C1-inhibitor (the main inhibitor of the classical pathway and the KKS). Likewise, perfusion of glomerular endothelial cells with C1-inhibitor-depleted plasma induced the release of complement-positive microvesicles, which was significantly reduced by kinin-receptor antagonists or C1-inhibitor. Mice with nephrotoxic serum-induced glomerulonephritis exhibited significantly reduced glomerular C3 deposition when treated with a B1-receptor antagonist. INTERPRETATION: Excessive complement deposition on the endothelium will promote endothelial injury and the release of endothelial microvesicles. This study demonstrates that blockade of the KKS can reduce complement activation and thereby the inflammatory response on the endothelium. FUNDING: Full details are provided in the Acknowledgements/Funding section.

Exosomes and microvesicles in normal physiology, pathophysiology, and renal diseases.

Extracellular vesicles are cell-derived membrane particles ranging from 30 to 5,000 nm in size, including exosomes, microvesicles, and apoptotic bodies. They are released under physiological conditions, but also upon cellular activation, senescence, and apoptosis. They play an important role in intercellular communication. Their release may also maintain cellular integrity by ridding the cell of damaging substances. This review describes the biogenesis, uptake, and detection of extracellular vesicles in addition to the impact that they have on recipient cells, focusing on mechanisms important in the pathophysiology of kidney diseases, such as thrombosis, angiogenesis, tissue regeneration, immune modulation, and inflammation. In kidney diseases, extracellular vesicles may be utilized as biomarkers, as they are detected in both blood and urine. Furthermore, they may contribute to the pathophysiology of renal disease while also having beneficial effects associated with tissue repair. Because of their role in the promotion of thrombosis, inflammation, and immune-mediated disease, they could be the target of drug therapy, whereas their favorable effects could be utilized therapeutically in acute and chronic kidney injury.

Extracellular vesicles in renal disease.

Extracellular vesicles, such as exosomes and microvesicles, are host cell-derived packages of information that allow cell-cell communication and enable cells to rid themselves of unwanted substances. The release and uptake of extracellular vesicles has important physiological functions and may also contribute to the development and propagation of inflammatory, vascular, malignant, infectious and neurodegenerative diseases. This Review describes the different types of extracellular vesicles, how they are detected and the mechanisms by which they communicate with cells and transfer information. We also describe their physiological functions in cellular interactions, such as in thrombosis, immune modulation, cell proliferation, tissue regeneration and matrix modulation, with an emphasis on renal processes. We discuss how the detection of extracellular vesicles could be utilized as biomarkers of renal disease and how they might contribute to disease processes in the kidney, such as in acute kidney injury, chronic kidney disease, renal transplantation, thrombotic microangiopathies, vasculitides, IgA nephropathy, nephrotic syndrome, urinary tract infection, cystic kidney disease and tubulopathies. Finally, we consider how the release or uptake of extracellular vesicles can be blocked, as well as the associated benefits and risks, and how extracellular vesicles might be used to treat renal diseases by delivering therapeutics to specific cells.

C1-Inhibitor Decreases the Release of Vasculitis-Like Chemotactic Endothelial Microvesicles.

The kinin system is activated during vasculitis and may contribute to chronic inflammation. C1-inhibitor is the main inhibitor of the kinin system. In this study, we investigated the presence of the kinin B1 receptor on endothelial microvesicles and its contribution to the inflammatory process. Compared with controls (n=15), patients with acute vasculitis (n=12) had markedly higher levels of circulating endothelial microvesicles, identified by flow cytometry analysis, and significantly more microvesicles that were positive for the kinin B1 receptor (P<0.001). Compared with microvesicles from wild-type cells, B1 receptor-positive microvesicles derived from transfected human embryonic kidney cells induced a significant neutrophil chemotactic effect, and a B1 receptor antagonist blocked this effect. Likewise, patient plasma induced neutrophil chemotaxis, an effect decreased by reduction of microvesicle levels and by blocking the B1 receptor. We used a perfusion system to study the effect of patient plasma (n=6) and control plasma (n=6) on the release of microvesicles from glomerular endothelial cells. Patient samples induced the release of significantly more B1 receptor-positive endothelial microvesicles than control samples, an effect abrogated by reduction of the microvesicles in the perfused samples. Perfusion of C1-inhibitor-depleted plasma over glomerular endothelial cells promoted excessive release of B1 receptor-positive endothelial microvesicles compared with normal plasma, an effect significantly decreased by addition of C1-inhibitor or B1 receptor-antagonist. Thus, B1 receptor-positive endothelial microvesicles may contribute to chronic inflammation by inducing neutrophil chemotaxis, and the reduction of these microvesicles by C1-inhibitor should be explored as a potential treatment for neutrophil-induced inflammation.

Microvesicle transfer of kinin B1-receptors is a novel inflammatory mechanism in vasculitis.

During vasculitis, activation of the kinin system induces inflammation, whereby the kinin B1-receptor is expressed and activated after ligand binding. Additionally, activated blood cells release microvesicles into the circulation. Here we determined whether leukocyte-derived microvesicles bear B1-kinin receptors during vasculitis, and if microvesicles transfer functional B1-receptors to recipient cells, thus promoting inflammation. By flow cytometry, plasma from patients with vasculitis were found to contain high levels of leukocyte-derived microvesicles bearing B1-receptors. Importantly, renal biopsies from two patients with vasculitis showed leukocyte-derived microvesicles bearing B1-receptors docking on glomerular endothelial cells providing in vivo relevance. Microvesicles derived from B1-receptor-transfected human embryonic kidney cells transferred B1-receptors to wild-type human embryonic kidney cells, lacking the receptor, and to glomerular endothelial cells. The transferred B1-receptors induced calcium influx after B1-receptor agonist stimulation: a response abrogated by a specific B1-receptor antagonist. Microvesicles derived from neutrophils also transferred B1-receptors to wild-type human embryonic kidney cells and induced calcium influx after stimulation. Thus, we found a novel mechanism by which microvesicles transfer functional receptors and promote kinin-associated inflammation.

Complement Interactions with Blood Cells, Endothelial Cells and Microvesicles in Thrombotic and Inflammatory Conditions.

The complement system is activated in the vasculature during thrombotic and inflammatory conditions. Activation may be associated with chronic inflammation on the endothelial surface leading to complement deposition. Complement mutations allow uninhibited complement activation to occur on platelets, neutrophils, monocytes, and aggregates thereof, as well as on red blood cells and endothelial cells. Furthermore, complement activation on the cells leads to the shedding of cell derived-microvesicles that may express complement and tissue factor thus promoting inflammation and thrombosis. Complement deposition on red blood cells triggers hemolysis and the release of red blood cell-derived microvesicles that are prothrombotic. Microvesicles are small membrane vesicles ranging from 0.1 to 1 µm, shed by cells during activation, injury and/or apoptosis that express components of the parent cell. Microvesicles are released during inflammatory and vascular conditions. The repertoire of inflammatory markers on endothelial cell-derived microvesicles shed during inflammation is large and includes complement. These circulating microvesicles may reflect the ongoing inflammatory process but may also contribute to its propagation. This overview will describe complement activation on blood and endothelial cells and the release of microvesicles from these cells during hemolytic uremic syndrome, thrombotic thrombocytopenic purpura and vasculitis, clinical conditions associated with enhanced thrombosis and inflammation.

A novel mechanism of bacterial toxin transfer within host blood cell-derived microvesicles.

Shiga toxin (Stx) is the main virulence factor of enterohemorrhagic Escherichia coli, which are non-invasive strains that can lead to hemolytic uremic syndrome (HUS), associated with renal failure and death. Although bacteremia does not occur, bacterial virulence factors gain access to the circulation and are thereafter presumed to cause target organ damage. Stx was previously shown to circulate bound to blood cells but the mechanism by which it would potentially transfer to target organ cells has not been elucidated. Here we show that blood cell-derived microvesicles, shed during HUS, contain Stx and are found within patient renal cortical cells. The finding was reproduced in mice infected with Stx-producing Escherichia coli exhibiting Stx-containing blood cell-derived microvesicles in the circulation that reached the kidney where they were transferred into glomerular and peritubular capillary endothelial cells and further through their basement membranes followed by podocytes and tubular epithelial cells, respectively. In vitro studies demonstrated that blood cell-derived microvesicles containing Stx undergo endocytosis in glomerular endothelial cells leading to cell death secondary to inhibited protein synthesis. This study demonstrates a novel virulence mechanism whereby bacterial toxin is transferred within host blood cell-derived microvesicles in which it may evade the host immune system.

Shiga toxin-induced complement-mediated hemolysis and release of complement-coated red blood cell-derived microvesicles in hemolytic uremic syndrome.

Shiga toxin (Stx)-producing Escherichia coli (STEC) cause hemolytic uremic syndrome (HUS). This study investigated whether Stx2 induces hemolysis and whether complement is involved in the hemolytic process. RBCs and/or RBC-derived microvesicles from patients with STEC-HUS (n = 25) were investigated for the presence of C3 and C9 by flow cytometry. Patients exhibited increased C3 deposition on RBCs compared with controls (p < 0.001), as well as high levels of C3- and C9-bearing RBC-derived microvesicles during the acute phase, which decreased after recovery. Stx2 bound to P1 (k) and P2 (k) phenotype RBCs, expressing high levels of the P(k) Ag (globotriaosylceramide), the known Stx receptor. Stx2 induced the release of hemoglobin and lactate dehydrogenase in whole blood, indicating hemolysis. Stx2-induced hemolysis was not demonstrated in the absence of plasma and was inhibited by heat inactivation, as well as by the terminal complement pathway Ab eculizumab, the purinergic P2 receptor antagonist suramin, and EDTA. In the presence of whole blood or plasma/serum, Stx2 induced the release of RBC-derived microvesicles coated with C5b-9, a process that was inhibited by EDTA, in the absence of factor B, and by purinergic P2 receptor antagonists. Thus, complement-coated RBC-derived microvesicles are elevated in HUS patients and induced in vitro by incubation of RBCs with Stx2, which also induced hemolysis. The role of complement in Stx2-mediated hemolysis was demonstrated by its occurrence only in the presence of plasma and its abrogation by heat inactivation, EDTA, and eculizumab. Complement activation on RBCs could play a role in the hemolytic process occurring during STEC-HUS.

The combined role of galactose-deficient IgA1 and streptococcal IgA-binding M Protein in inducing IL-6 and C3 secretion from human mesangial cells: implications for IgA nephropathy.

IgA nephropathy (IgAN) is characterized by mesangial cell proliferation and extracellular matrix expansion associated with immune deposits consisting of galactose-deficient polymeric IgA1 and C3. We have previously shown that IgA-binding regions of streptococcal M proteins colocalize with IgA in mesangial immune deposits in patients with IgAN. In the present study, the IgA-binding M4 protein from group A Streptococcus was found to bind to galactose-deficient polymeric IgA1 with higher affinity than to other forms of IgA1, as shown by surface plasmon resonance and solid-phase immunoassay. The M4 protein was demonstrated to bind to mesangial cells not via the IgA-binding region but rather via the C-terminal region, as demonstrated by flow cytometry. IgA1 enhanced binding of M4 to mesangial cells, but not vice versa. Costimulation of human mesangial cells with M4 and galactose-deficient polymeric IgA1 resulted in a significant increase in IL-6 secretion compared with each stimulant alone. Galactose-deficient polymeric IgA1 alone, but not M4, induced C3 secretion from the cells, and costimulation enhanced this effect. Additionally, costimulation enhanced mesangial cell proliferation compared with each stimulant alone. These results indicate that IgA-binding M4 protein binds preferentially to galactose-deficient polymeric IgA1 and that these proteins together induce excessive proinflammatory responses and proliferation of human mesangial cells. Thus, tissue deposition of streptococcal IgA-binding M proteins may contribute to the pathogenesis of IgAN.

Enterohemorrhagic Escherichia coli Pathogenesis and the Host Response.

Enterohemorrhagic Escherichia coli (EHEC) is a highly pathogenic bacterial strain capable of causing watery or bloody diarrhea, the latter termed hemorrhagic colitis, and hemolytic-uremic syndrome (HUS). HUS is defined as the simultaneous development of non-immune hemolytic anemia, thrombocytopenia, and acute renal failure. The mechanism by which EHEC bacteria colonize and cause severe colitis, followed by renal failure with activated blood cells, as well as neurological symptoms, involves the interaction of bacterial virulence factors and specific pathogen-associated molecular patterns with host cells as well as the host response. The innate immune host response comprises the release of antimicrobial peptides as well as cytokines and chemokines in addition to activation and/or injury to leukocytes, platelets, and erythrocytes and activation of the complement system. Some of the bacterial interactions with the host may be protective in nature, but, when excessive, contribute to extensive tissue injury, inflammation, and thrombosis, effects that may worsen the clinical outcome of EHEC infection. This article describes aspects of the host response occurring during EHEC infection and their effects on specific organs.

Complement activation associated with ADAMTS13 deficiency in human and murine thrombotic microangiopathy.

This study addressed the contribution of ADAMTS13 deficiency to complement activation in thrombotic thrombocytopenic purpura (TTP). Renal tissue and blood samples were available from 12 TTP patients. C3 and C5b-9 deposition were demonstrated in the renal cortex of two TTP patients, by immunofluorescence and immunohistochemistry, respectively. C3 was also demonstrated in the glomeruli of Shiga toxin-2-treated Adamts13(-/-) mice (n = 6 of 7), but less in mice that were not Shiga toxin-2 treated (n = 1 of 8, p < 0.05) or wild-type mice (n = 0 of 7). TTP patient plasma (n = 9) contained significantly higher levels of complement-coated endothelial microparticles than control plasma (n = 13), as detected by flow cytometry. Exposure of histamine-stimulated primary glomerular endothelial cells to platelet-rich plasma from patients, or patient platelet-poor plasma combined with normal platelets, in a perfusion system, under shear, induced C3 deposition on von Willebrand factor-platelet strings (on both von Willebrand factor and platelets) and on endothelial cells. Complement activation occurred via the alternative pathway. No C3 was detected when cells were exposed to TTP plasma that was preincubated with EDTA or heat-inactivated, or to control plasma. In the perfusion system, patient plasma induced more release of C3- and C9-coated endothelial microparticles compared with control plasma. The results indicate that the microvascular process induced by ADAMTS13 deficiency triggers complement activation on platelets and the endothelium, which may contribute to formation of thrombotic microangiopathy.

A novel C3 mutation causing increased formation of the C3 convertase in familial atypical hemolytic uremic syndrome.

Atypical hemolytic uremic syndrome has been associated with dysregulation of the alternative complement pathway. In this study, a novel heterozygous C3 mutation was identified in a factor B-binding region in exon 41, V1636A (4973 T > C). The mutation was found in three family members affected with late-onset atypical hemolytic uremic syndrome and symptoms of glomerulonephritis. All three patients exhibited increased complement activation detected by decreased C3 levels and glomerular C3 deposits. Platelets from two of the patients had C3 and C9 deposits on the cell surface. Patient sera exhibited more C3 cleavage and higher levels of C3a. The C3 mutation resulted in increased C3 binding to factor B and increased net formation of the C3 convertase, even after decay induced by decay-accelerating factor and factor H, as assayed by surface plasmon resonance. Patient sera incubated with washed human platelets induced more C3 and C9 deposition on the cell surface in comparison with normal sera. More C3a was released into serum over time when washed platelets were exposed to patient sera. Results regarding C3 and C9 deposition on washed platelets were confirmed using purified patient C3 in C3-depleted serum. The results indicated enhanced convertase formation leading to increased complement activation on cell surfaces. Previously described C3 mutations showed loss of function with regard to C3 binding to complement regulators. To our knowledge, this study presents the first known C3 mutation inducing increased formation of the C3 convertase, thus explaining enhanced activation of the alternative pathway of complement.

Phenotypic expression of ADAMTS13 in glomerular endothelial cells.

BACKGROUND: ADAMTS13 is the physiological von Willebrand factor (VWF)-cleaving protease. The aim of this study was to examine ADAMTS13 expression in kidneys from ADAMTS13 wild-type (Adamts13⁺/⁺) and deficient (Adamts13⁻/⁻) mice and to investigate the expression pattern and bioactivity in human glomerular endothelial cells. METHODOLOGY/PRINCIPAL FINDINGS: Immunohistochemistry was performed on kidney sections from ADAMTS13 wild-type and ADAMTS13-deficient mice. Phenotypic differences were examined by ultramorphology. ADAMTS13 expression in human glomerular endothelial cells and dermal microvascular endothelial cells was investigated by real-time PCR, flow cytometry, immunofluorescence and immunoblotting. VWF cleavage was demonstrated by multimer structure analysis and immunoblotting. ADAMTS13 was demonstrated in glomerular endothelial cells in Adamts13⁺/⁺ mice but no staining was visible in tissue from Adamts13⁻/⁻ mice. Thickening of glomerular capillaries with platelet deposition on the vessel wall was detected in Adamts13⁻/⁻ mice. ADAMTS13 mRNA and protein were detected in both human endothelial cells and the protease was secreted. ADAMTS13 activity was demonstrated in glomerular endothelial cells as cleavage of VWF. CONCLUSIONS/SIGNIFICANCE: Glomerular endothelial cells express and secrete ADAMTS13. The proteolytic activity could have a protective effect preventing deposition of platelets along capillary lumina under the conditions of high shear stress present in glomerular capillaries.

Complement activation on platelet-leukocyte complexes and microparticles in enterohemorrhagic Escherichia coli-induced hemolytic uremic syndrome.

Hemolytic uremic syndrome (HUS) is commonly associated with Shiga toxin (Stx)-producing Escherichia coli O157:H7 infection. This study examined patient samples for complement activation on leukocyte-platelet complexes and microparticles, as well as donor samples for Stx and lipopolysaccharide (O157LPS)-induced complement activation on platelet-leukocyte complexes and microparticles. Results, analyzed by flow cytometry, showed that whole blood from a child with HUS had surface-bound C3 on 30% of platelet-monocyte complexes compared with 14% after recovery and 12% in pediatric controls. Plasma samples from 12 HUS patients were analyzed for the presence of microparticles derived from platelets, monocytes, and neutrophils. Acute-phase samples exhibited high levels of platelet microparticles and, to a lesser extent, monocyte microparticles, both bearing C3 and C9. Levels decreased significantly at recovery. Stx or O157LPS incubated with donor whole blood increased the population of platelet-monocyte and platelet-neutrophil complexes with surface-bound C3 and C9, an effect enhanced by costimulation with Stx and O157LPS. Both Stx and O157LPS induced the release of C3- and C9-bearing microparticles from platelets and monocytes. Released microparticles were phagocytosed by neutrophils. The presence of complement on platelet-leukocyte complexes and microparticles derived from these cells suggests a role in the inflammatory and thrombogenic events that occur during HUS.

Shiga toxin and lipopolysaccharide induce platelet-leukocyte aggregates and tissue factor release, a thrombotic mechanism in hemolytic uremic syndrome.

BACKGROUND: Aggregates formed between leukocytes and platelets in the circulation lead to release of tissue factor (TF)-bearing microparticles contributing to a prothrombotic state. As enterohemorrhagic Escherichia coli (EHEC) may cause hemolytic uremic syndrome (HUS), in which microthrombi cause tissue damage, this study investigated whether the interaction between blood cells and EHEC virulence factors Shiga toxin (Stx) and lipopolysaccharide (LPS) led to release of TF. METHODOLOGY/PRINCIPAL FINDINGS: The interaction between Stx or LPS and blood cells induced platelet-leukocyte aggregate formation and tissue factor (TF) release, as detected by flow cytometry in whole blood. O157LPS was more potent than other LPS serotypes. Aggregates formed mainly between monocytes and platelets and less so between neutrophils and platelets. Stimulated blood cells in complex expressed activation markers, and microparticles were released. Microparticles originated mainly from platelets and monocytes and expressed TF. TF-expressing microparticles, and functional TF in plasma, increased when blood cells were simultaneously exposed to the EHEC virulence factors and high shear stress. Stx and LPS in combination had a more pronounced effect on platelet-monocyte aggregate formation, and TF expression on these aggregates, than each virulence factor alone. Whole blood and plasma from HUS patients (n = 4) were analyzed. All patients had an increase in leukocyte-platelet aggregates, mainly between monocytes and platelets, on which TF was expressed during the acute phase of disease. Patients also exhibited an increase in microparticles, mainly originating from platelets and monocytes, bearing surface-bound TF, and functional TF was detected in their plasma. Blood cell aggregates, microparticles, and TF decreased upon recovery. CONCLUSIONS/SIGNIFICANCE: By triggering TF release in the circulation, Stx and LPS can induce a prothrombotic state contributing to the pathogenesis of HUS.

A novel mutation in the complement regulator clusterin in recurrent hemolytic uremic syndrome.

A novel heterozygous mutation in the clusterin gene, nucleotide position A1298C (glutamine>proline Q433P), was detected in exon 7 of a child with recurrent hemolytic uremic syndrome (HUS). The same mutation was found in the child's two siblings and mother but not in 120 controls. In addition, a previously described heterozygous mutation was detected in the gene encoding membrane cofactor protein (MCP) causing a 6 base-pair deletion 811-816delGACAGT in exon 6. It was found in the patient, both siblings and the father. One sibling had recovered from post-streptococcal glomerulonephritis. Clusterin levels in the patient, siblings and parents were normal as was the migration pattern in a gel. Patient serum induced C3 and C9 deposition on normal washed platelets, and platelet activation, as detected by flow cytometry. The same phenomenon was found in serum taken from the siblings and the mother but not in the sample from the father and controls. Addition of clusterin to patient serum did not inhibit complement activation on platelets. The Q433P mutant, in isolated form, was further studied by binding to the components of the terminal complement complex. The mutant did not bind to C5b-7 that was immobilized onto a BIAcore chip, whereas wild-type clusterin did, indicating that the mutation could lead to defective inhibition of formation of the membrane attack complex under these conditions. Hemolysis of rabbit erythrocytes was inhibited by wild-type clusterin but not by the mutant. Mutated clusterin could thus not prevent assembly of the membrane attack complex on platelets and erythrocytes.