Amplification and propagation of local thrombosis is likely to be dependent upon the local microenvironment. Activated factors Xa and IXa produced by upstream, stimulated endothelial cells (EC) under prevailing shear stress conditions, as well as any blood-borne tissue factor (TF) and tissue factor pathway inhibitor (TFPI) secreted by these same ECs may induce thrombosis. The investigators plant to test the hypothesis that TF-expressing EC promote local mural thrombi in arteries and arterioles, and that reductions in shear stress from baseline levels determine the extent to which stimulated EC become functionally procoagulant. The investigators will focus on Shiga toxin injury to the vessel wall in the hemolytic uremic syndrome (HUS) as a paradigm for general endothelial injury.
Specific Aim 1 is designed to show that for simultaneous exposure to shear stress and TNF-a, the endothelium limits its expression of functional TF by immediately increasing its secretion of TFPI, only after several hours being able to downregulate the gene for TF. Conversely, reduction of shear stress to near-stasis levels allows such endothelium to become procoagulant. The importance of the transcription factors Egr-1, Sp1, NFKb, and AP-1 in upregulating TF mRNA after endothelial cell exposure to shear stress and TNF-a, individually and together, will be examined.
Aim 2 is to assess platelet adhesion / aggregation to glomerular endothelial cells injured by Shiga toxin, and exposed to flowing blood, and to determine the mechanism by which Shiga toxin, following exposure of endothelium to TNF-a, further augments functional TF, as demonstrated previously by the investigators. A candidate mechanism is de-encryption of preformed TF.
Aim 3 is to demonstrate in real time and under controlled blood flow conditions that TF on endothelial and/or leukocyte origin co-localizes with growing platelet aggregates, and is present on platelet aggregates adherent to Shiga toxin-injured glomerular endothelium in kidney segments derived from a primate model of HUS.
These aims i ncorporate measurement of factor Xa and TFPI production, immunogold labeling to TF and TFPI antigen, RT-PCR amplification production of TF and TFPI mRNAs, and nuclear runoff studies and electrophoretic mobility shift assays. The investigators will also employ immunoglold lableling for fibrin on the surface of platelet aggregates on fibrinogen-coated glass and glomerular endothelium. Real-time imaging of platelet and TF co-localization will be achieved by means of dual-label epiflourescence videomicroscopy. A clearer understanding of the manner in which shear stress regulates the expression of TF by activated endothelium is important to the development of strategies to interrupt the TF pathway in HUS and conceptually related disorders of arterial / arteriolar thrombosis.
| Nestoridi, Eirini; Kushak, Rafail I; Tsukurov, Olga et al. (2008) Role of the renin angiotensin system in TNF-alpha and Shiga-toxin-induced tissue factor expression. Pediatr Nephrol 23:221-31 |
