Tissue Factor-Driven Enhanced Platelet Aggregation in Flowing Blood in a Rabbit Model of Childhood Hemolytic Uremic Syndrome.

The Dutch belted rabbit constitutes a promising animal model of the childhood hemolytic uremic syndrome (HUS; A. Garcia, J. Infect. Dis. 2002). When Shiga toxin (Stx)-producing E. coli are administered orally, these rabbits develop HUS. Platelet-fibrin thrombus formation in this model was examined on unfixed renal tissue (5 μm sections) after mounting on 150-μm glass slides in test animal-control animal pairs. Normal donor blood, collected into 4U/ml low MW heparin and 10 μM quinacrine dihydrochoride, was drawn at shear rates of 270–650 sec-1 through a parallel-plate flow chamber for which one surface was one of the above slides. Such shear rates give rise to shear stresses which approach the 20–25 dynes/cm² estimated to exist in glomerular arterioles. Platelets depositing on the sections were imaged in real time using epifluorescence digital videomicroscopy, with quantitation of adherent platelets via image processing. For 16 pairs of test and control sections, the percent of image pixels occupied by adherent platelets after 6–9 min of blood flow was 17.7 ± 3.06 (mean ± SD) for test sections vs 5.71 ± 5.18 for controls (P<0.001). This 3.1-fold increase for sections from Stx-treated rabbits is all the more striking considering that individual platelet aggregates on these sections were also larger and thicker, as judged from greater local pixel intensities. In 4 additional experiments, preincubation of sections with 300 nM of a monoclonal antibody (courtesy of Y. Nemerson, Mt. Sinai Medical Center, NY, NY) directed against human tissue factor (TF) with known cross-reactivity to rabbit TF markedly reduced platelet adhesion/aggregation on both test and control sections. Residual platelet deposition, which only slightly favored test sections, may be due to either a non-TF-dependent mechanism or incomplete neutralization of TF activity by the amount of antibody used. Studies of parallel fibrin deposition are in progress. Thus, renal tissue from the Dutch belted HUS model is reactive to platelets in large part via expression of TF, an expression augmented on test sections. These observations support the hypothesis that the TF pathway of coagulation plays a major role in childhood HUS.