Increased Tissue Factor Bearing Microparticles in Hemolytic Uremic Syndrome.

Abstract

Abstract Microparticles (MP) are circulating cellular fragments that are increased in thrombotic conditions including TTP, antiphospholipid syndrome, and HIT. Tissue factor (TF)-bearing MP are thought to be procoagulant in these conditions. Hemolytic Uremic Syndrome (HUS) is a thrombotic disease characterized by microangiopathic hemolytic anemia, thrombocytopenia and renal failure secondary to glomerular thrombotic microangiopathy. In children, HUS most often occurs after infection with E. coli O157H7. It is hypothesized that E. coli verotoxin injures renal and other endothelium leading to activation of hemostasis, thrombotic microangiopathy and HUS. We studied 56 children at the time of presentation with bloody diarrhea associated with E. coli O157H7 infection (typically day 4 of illness) prior to developing HUS, and 5 healthy children as controls. Subsequently 14 children (Pre-HUS) went on to develop HUS while 42 (Uncomplicated) resolved their illness without developing HUS. We also studied 15 children that presented with already developed HUS (HUS group). We analyzed the total number of MPs, percent of TF-bearing microparticles and their cellular derivation using a 5-color flow cytometry assay. Endothelial-derived MP are identified as positive for CD144 (Ve-Cad)-PE, monocyte-derived MP are positive for CD14-PE-Cy7, and platelet derived MP are positive for CD41a-PerCP. MPs were collected from plasma by ultracentrifugation (100,000g). MPs were defined based on size (0.5–1 um) and strong annexin V binding. The MP distribution in the control group was similar to reports by others: 204 ±98 ×103 MPs/mL of plasma (55% platelet-, 5% endothelial- and 15% monocyte-derived), only 2.6% of MP express TF. The MP distribution in Uncomplicated was 996 ±766 ×103 MPs/mL (36% plat-, 11% endo- and 15% mono-derived) and 5.4% express TF; while pre-HUS showed 1197±1108 ×103 MPs/mL (21% plat-, 12% endo- and 11% mono-derived) and 8.2% express TF. The MP distribution in the HUS group was 1183±949 ×103 MPs/mL (45% plat-, 15% endo- and 19% mono-derived) and 10.9% express TF. Compared to controls, children infected with E. coli O157H7 showed a higher number of total MPs (p<0.02, Mann Whitney) and a higher number of tissue factor bearing MP (p<0.004). Compared to the Uncomplicated group, the pre-HUS group showed fewer platelet MPs (p<0.04). Progression from Pre-HUS to HUS is characterized by increased number of TF-bearing MP distributed in all cellular populations (p<0.01). In a prior study the pre-HUS group showed increased hemostatic activation as indicated by higher levels of F1.2 and D-dimer. We conclude that E. coli 0157:H7 infection results in increased microparticle production versus controls, and increased tissue factor positive microparticles. Development of HUS is associated with further increases in tissue factor positive microparticles, which correlates with the increased hemostatic activation observed in previous studies, suggesting a pathologic role for the release TF-bearing MP in HUS. Interestingly, Pre-HUS patients show reduced numbers of platelet derived microparticles versus uncomplicated patients that resolve without developing HUS, but when HUS develops the number of TF-bearing MP increases. Whether this reduction of platelet-derived microparticles preceding HUS development results from increased binding of MP to endothelium or reduced production of MP in pre-HUS are ongoing studies that may shed some light into the pathogenesis of HUS.