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

Aliskiren inhibits renin-mediated complement activation

Certain kidney diseases are associated with complement activation although a renal triggering factor has not been identified. Here we demonstrated that renin, a kidney-specific enzyme, cleaves C3 into C3b and C3a, in a manner identical to the C3 convertase. Cleavage was specifically blocked by the renin inhibitor aliskiren. Renin-mediated C3 cleavage and its inhibition by aliskiren also occurred in serum. Generation of C3 cleavage products was demonstrated by immunoblotting, detecting the cleavage product C3b, by N-terminal sequencing of the cleavage product, and by ELISA for C3a release. Functional assays showed mast cell chemotaxis towards the cleavage product C3a and release of factor Ba when the cleavage product C3b was combined with factor B and factor D. The renin-mediated C3 cleavage product bound to factor B. In the presence of aliskiren this did not occur, and less C3 deposited on renin-producing cells. The effect of aliskiren was studied in three patients with dense deposit disease and this demonstrated decreased systemic and renal complement activation (increased C3, decreased C3a and C5a, decreased renal C3 and C5b-9 deposition and/or decreased glomerular basement membrane thickness) over a follow-up period of four to seven years. Thus, renin can trigger complement activation, an effect inhibited by aliskiren. Since renin concentrations are higher in renal tissue than systemically, this may explain the renal propensity of complement-mediated disease in the presence of complement mutations or auto-antibodies

Numbers and cellular origin of circulating microparticles and TF–expressing microparticles in plasma from HUS patients.

<p>Numbers and cellular origin of microparticles and microparticles with surface-bound tissue factor/mL of plasma shown as median and range values.</p

TF expression on blood cells in complex and free cells induced by incubation with Stx2 and/or LPS.

<p>(A) Incubation of whole blood with Stx2 or O157:H7LPS (0.5 µg/mL) induced TF expression mostly on platelet-monocyte aggregates and to a lesser degree on unbound monocytes as determined by flow cytometry. (B) Likewise, in the neutrophil population TF expression was mostly seen on neutrophils in complex with platelets. Data are expressed as percentage of the platelet-monocyte or platelet-neutrophil population or percentage of unbound monocytes or neutrophils that were positive for the TF antibody±standard deviation (n = 10). ** denotes <i>P</i><0.01 and * <i>P</i><0.05, when comparing TF expression on aggregates in whole blood incubated with Stx2, LPS or the Stx2/O157LPS combination with unstimulated PBS-treated whole blood (except when comparisons are delineated). NS; indicates not significant.</p

Platelet-monocyte and platelet-neutrophil aggregate formation and TF expression induced by shear and Stx2 and/or LPS.

<p>Stx2, O157:H7LPS, O111:B4LPS (both LPS serogroups at 1 µg/mL) or a combination of Stx2 and LPS were added to whole blood immediately before perfusion of whole blood through a flow chamber system and aggregate formation and tissue factor expression determined by flow cytometry. (A) Stx2 and/or LPS induced platelet-monocyte aggregate formation particularly at shear rates between 1000–2000 s⁻¹ (light bars). (B) Aggregate formation between platelets and neutrophils increased minimally by addition of Stx2 or LPS at both low (100–340 s⁻¹) and high shear rates as compared to PBS-treated samples. (C) Stx2 or LPS induced TF expression on platelet-monocyte aggregates at low shear rates which increased even more at high shear rates. Expression increased markedly in Stx2/LPS stimulated samples at higher shear rates. (D) Stx2 and/or LPS induced only a weak increase in TF expression on platelet-neutrophil aggregates. Higher shear rates did not induce more TF expression. Data are expressed as mean±standard deviation (n = 3). Statistical comparisons were not carried out as only three experiments were performed due to the large amounts of blood required.</p

Platelet-monocyte and platelet-neutrophil aggregates induced by incubation with Stx2 and/or LPS determined by flow cytometry.

<p>(A) Incubation of PBS-treated whole blood at 37°C for 4 h induced a slight increase in platelet-monocyte (A) and platelet-neutrophil (B) aggregate formation compared to baseline levels. Baseline levels of platelet-monocyte aggregates were 10% (range 5–17%, 7 experiments) and platelet-neutrophil aggregates 6% (range 2–13%, 7 experiments). Incubation of whole blood with Stx2 and/or O157LPS (0.5 µg/mL) induced predominantly the formation of platelet-monocyte aggregates. (C) Incubation of whole blood with O157LPS (1 µg/mL) at 37°C for 4 h induced significantly more aggregate formation between platelets and monocytes in comparison to the other LPS serotypes tested while no significant differences, between LPS serotypes, were observed in platelet-neutrophil (D) aggregate formation. Results are expressed as the percentage of the monocyte or neutrophil population that was positive for CD38:FITC or CD66:FITC as well as the platelet specific marker CD42b:RPE-Cy5. Data are expressed as mean±standard deviation (n = 10 experiments), ** denotes <i>P</i><0.01 when comparing aggregate formation in whole blood incubated with a stimulant and PBS-treated whole blood and * denotes <i>P</i><0.05, comparing aggregate formation in whole blood incubated with O157LPS with those incubated with O103, O111, O121, or O111:B4LPS. NS; indicates not significant.</p

Numbers and cellular origin of microparticles released from blood cells as determined by flow cytometry.

<p>Data are expressed as median and range of microparticles positive for each surface marker/mL of plasma from four different experiments. *; denotes <i>P value</i> <0.05 comparing microparticle generation in whole blood stimulated with Stx2, O157:H7LPS (1 µg/mL), O111:B4LPS or Stx2/LPS with unstimulated whole blood. The number of microparticles per mL plasma was calculated as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006990#pone.0006990-Sthl1" target="_blank">[18]</a>.</p

Circulating platelet-leukocyte aggregates and TF expression in patients with HUS.

<p>(A) Patients with HUS had increased circulating levels of platelet-monocyte and (B) platelet-neutrophil aggregates during the acute phase of disease compared to levels obtained after recovery. TF expression on platelet-monocyte (C) and platelet-neutrophil (D) aggregates were increased during the acute phase of HUS and decreased at recovery. Samples were available from four patients, thus precluding statistical comparisons.</p

Detection of platelet-monocyte or platelet-neutrophil aggregates and tissue factor expression by flow cytometry.

<p>(A) The neutrophil and monocyte population were identified in whole blood by their characteristic size and granularity. In the monocyte gate 98% of the cells were positive for the monocyte marker CD38:FITC and 99% of the cells in the neutrophil gate were CD66:FITC positive showing the accuracy of the identification of cells by forward and side scatter. (B) Monocytes or neutrophils in complex with platelets (gate 2), were identified by binding of CD38:FITC or CD66:FITC (FL1) and the platelet specific antibody CD42b:RPE-Cy5 (FL3). Cells in gate 3 represent platelet-free monocytes or neutrophils. (C) Surface bound tissue factor was identified by binding of CD142:RPE and CD38:FITC or CD66:FITC (FL2 vs. FL1). (D) Percentage of positive cells was calculated by subtraction of the negative control antibody.</p