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

Ståhl AL, Sartz L, Nelsson A, Békássy ZD, Karpman D - PLoS ONE (2009)

Bottom Line: 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.Blood cell aggregates, microparticles, and TF decreased upon recovery.By triggering TF release in the circulation, Stx and LPS can induce a prothrombotic state contributing to the pathogenesis of HUS.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Clinical Sciences Lund, Lund University, Lund, Sweden.

ABSTRACT

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.

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Related in: MedlinePlus

Detection of platelet-monocyte or platelet-neutrophil aggregates and tissue factor expression by flow cytometry.(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.
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pone-0006990-g001: Detection of platelet-monocyte or platelet-neutrophil aggregates and tissue factor expression by flow cytometry.(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.

Mentions: Flow cytometry was performed using a FACSCalibur instrument with CELLQuest software (Becton Dickinson Immunocytometry Systems, San Jose, CA). Forward and side scatter measurements were made with gain settings in linear mode for the analysis of platelet-monocyte or platelet-neutrophil interactions. The monocyte and the neutrophil populations were thus easily distinguished (Figure 1A). A minimum of 2000 monocytes and 5000 neutrophils were acquired for each determination. A three-color analysis was used for simultaneous detection of platelet-leukocyte aggregates and platelet-leukocyte activation. Monocytes and neutrophils were further gated into a CD42b-positive platelet-bound population, gate two, and a CD42b-negative platelet-free population, gate three (FL1 versus FL3, Figure 1B).


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

Ståhl AL, Sartz L, Nelsson A, Békássy ZD, Karpman D - PLoS ONE (2009)

Detection of platelet-monocyte or platelet-neutrophil aggregates and tissue factor expression by flow cytometry.(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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2735777&req=5

pone-0006990-g001: Detection of platelet-monocyte or platelet-neutrophil aggregates and tissue factor expression by flow cytometry.(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.
Mentions: Flow cytometry was performed using a FACSCalibur instrument with CELLQuest software (Becton Dickinson Immunocytometry Systems, San Jose, CA). Forward and side scatter measurements were made with gain settings in linear mode for the analysis of platelet-monocyte or platelet-neutrophil interactions. The monocyte and the neutrophil populations were thus easily distinguished (Figure 1A). A minimum of 2000 monocytes and 5000 neutrophils were acquired for each determination. A three-color analysis was used for simultaneous detection of platelet-leukocyte aggregates and platelet-leukocyte activation. Monocytes and neutrophils were further gated into a CD42b-positive platelet-bound population, gate two, and a CD42b-negative platelet-free population, gate three (FL1 versus FL3, Figure 1B).

Bottom Line: 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.Blood cell aggregates, microparticles, and TF decreased upon recovery.By triggering TF release in the circulation, Stx and LPS can induce a prothrombotic state contributing to the pathogenesis of HUS.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Clinical Sciences Lund, Lund University, Lund, Sweden.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus