Shiga Toxin and Lipopolysaccharide Induce Platelet-Leukocyte Aggregates and Tissue Factor Release, a Thrombotic Mechanism in Hemolytic Uremic Syndrome

Abstract

BackgroundAggregates 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 FindingsThe 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/SignificanceBy triggering TF release in the circulation, Stx and LPS can induce a prothrombotic state contributing to the pathogenesis of HUS.

Direct in vivo observations of P-selectin glycoprotein ligand-1-mediated leukocyte–endothelial cell interactions in the pulmonary microvasculature in abdominal sepsis in mice

“…Leukocyte-platelet complexes are larger in size and have been suggested to be susceptible for size-restricted microvascular trapping [27]. We observed that the number of leukocyte-platelet aggregates increased in septic mice, which is in line with previous studies showing increased generation of leukocyteplatelet complexes in inflammatory diseases [28][29][30]. Interestingly, we found that immunoneutralization of PSGL-1 completely inhibited sepsis-triggered formation leukocyteplatelet aggregates, which might help to explain the inhibitory effect of anti-PSGL-1 antibody on the accumulation of leukocytes in pulmonary capillaries in abdominal sepsis.…”

The Antibiotic Polymyxin B Impairs the Interactions between Shiga Toxins and Human Neutrophils

“…Briefly, the seminal paper demonstrating for the first time such an interaction (18) was followed by a second paper (20) in which some of the authors reversed their position. However, we and other groups confirmed these findings in vitro and in patients (16,(21)(22)(23)(24)(25).…”

“…It has been shown that Stx2 added to human blood induced the formation of neutrophil/platelet or monocyte/platelet aggregates with activated thrombocytes and leukocytes, leading to the release of microvesicles (24,36). Complement activation on these complexes and on microvesicles should play a definite role in the development of HUS, because patients show higher platelet/leukocyte aggregates in blood with respect to healthy subjects (24,36).…”

“…Various arguments have been put forward as a causative role: 1) free Stx have never been found in sera of patients with HUS (27)(28)(29), and, conversely, the toxins have been detected on the surface of their neutrophils (19,27,30); 2) patients' neutrophils are activated, degranulated, and hyporesponsive to other stimuli (19,31); 3) neutrophilia is a common finding in patients with HUS and is considered a poor prognostic factor (32-34); 4) the greater the amount of toxins in patients' feces, the greater the saturation of neutrophil receptors by Stx (35); 5) the transfer of the toxins from neutrophils to target endothelial cells has been demonstrated in vitro (18) during leukocyte transmigration (22); 6) HUS patients with lower amounts of toxins in their neutrophils had severe renal involvement (19,27,30); 7) HUS with only mild renal involvement developed in patients with a high detectable amount of toxins on neutrophil membrane, although they were at risk for neurologic complications (19,27,30); 8) Stx2 has been found on leukocytes (monocyte or neutrophil)/platelet aggregates circulating in patients with HUS (24); and 9) leukocyte-or platelet-derived microvesicles containing Stx2 have been found in the blood patients with HUS (24,36) and in their renal cortex (37).…”

Soluble CD40 Iigand and Oxidative Response Are Reciprocally Stimulated during Shiga Toxin-Associated Hemolytic Uremic Syndrome

“…This in turn would contribute to endothelial damage and to further platelet activation leading to a positive feedback loop between platelets and monocytes, in which ROS and sCD40L stimulates each other. This represents a new pathogenic pathway mediated by monocyte-platelet interaction during HUS, that adds to the already described monocyte-platelet aggregate formation [51].…”