Shiga toxin enhances functional tissue factor on human glomerular endothelial cells: implications for the pathophysiology of hemolytic uremic syndrome.
BACKGROUND
The pathogenesis of Shiga toxin (Stx)-mediated childhood hemolytic uremic syndrome (HUS) is not fully delineated, although current evidence implicates a prothrombotic state. We hypothesized that the tissue factor (TF) pathway plays a major role in the pathophysiology of HUS.


METHODS
We measured cell surface TF activity in response to tumor necrosis factor-alpha (TNF-alpha) (20 ng mL(-1), 2-144 h), Stx-1 (10(-11) mol L(-1), 4-144 h), or their combination (TNF-alpha 22 h and Stx-1 for the last 0.5-4 h of TNF-alpha incubation) on human glomerular (microvascular) endothelial cells (HGECs) and human umbilical vein (macrovascular) endothelial cells (HUVECs).


CONCLUSIONS
We observed that while TNF-alpha caused an increase in cell surface TF activity on both cell types, the combination of TNF-alpha and Stx-1 differentially affected HGECs. On these cells, TF activity was increased further by 2.67 +/- 0.38-fold (n = 38, P < 0.001), consistent with our parallel observation that Stx-1 binds to HGECs but not to HUVECs. Anti-TF antibody abolished functional TF while anti-tissue factor pathway inhibitor antibody enhanced TF activity. Stx-1 alone did not induce TF activity on either cell type. Measurement of TF antigen levels and quantitative real-time polymerase chain reaction demonstrated that exposure to TNF-alpha markedly increased TF protein and TF mRNA for HGECs, but the exposure to the combination of TNF-alpha and Stx-1 did not increase further the amount of either TF protein or TF mRNA. We conclude that cytokine-activated HGECs, but not HUVECs, undergo a significant augmentation of cell surface TF activity following exposure to Stx, suggesting an important role for TF in the coagulopathy observed in HUS.
Nestoridi
E
E
Cardiovascular Thrombosis Laboratory, MassGeneral Hospital for Children, Harvard Medical School, Boston, MA, USA.
Tsukurov
O
O
Kushak
R I
RI
Ingelfinger
J R
JR
Grabowski
E F
EF
D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002863:Chromogenic Compounds / Q000737:chemistry; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism*; D015951:Factor Xa / Q000737:chemistry; D005353:Fibronectins / Q000378:metabolism; D006463:Hemolytic-Uremic Syndrome / Q000503:physiopathology*; D006801:Humans; D007678:Kidney Glomerulus / Q000166:cytology* / Q000378:metabolism*; D008074:Lipoproteins / Q000378:metabolism; D008833:Microcirculation; D008856:Microscopy, Fluorescence; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D022622:Shiga Toxin 1 / Q000378:metabolism / Q000494:pharmacology*; D013925:Thromboplastin / Q000096:biosynthesis* / Q000378:metabolism; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D014471:Umbilical Veins / Q000166:cytology; D015854:Up-Regulation
D002863:Chromogenic Compounds; D005353:Fibronectins; D008074:Lipoproteins; D012333:RNA, Messenger; D022622:Shiga Toxin 1; D014409:Tumor Necrosis Factor-alpha; C051928:lipoprotein-associated coagulation inhibitor; D012313:RNA; D013925:Thromboplastin; D015951:Factor Xa