This research proposal is a response to NHLBI RFA HL-95-007 (Thrombotic Thrombocytopenic Purpura (TTP) and HIV). The long term objectives are to further elucidate the pathogenesis of TTP using in vitro and in vivo experimental models to study factors that may be involved in the pathogenesis of thrombotic microangiopathy (TMA), to determine predisposing factors, early diagnosis and insights into treatment. There are three specific aims: 1) To determine in vitro inflammatory and coagulation responses of the cells most likely to be involved in the pathogenesis of TMA (endothelial cells, platelets and leukocytes) to known inciting agents such as shiga-like toxins (SLTs) and sialidase. The use of micro and macro vascular ECs and comparison of endothelial cells grown under static and shear stress conditions will add significantly to these experiments. Cells and plasma from normal subjects, HIV and TTP patients will be used to elucidate mechanisms in the in vitro system; 2) To determine in vivo responses to SLTs and sialidase using animals (mice and rabbits) known to be susceptible to SLTs. Specific gene (P-selectin, E- selectin, P- and E-selectin, and vWF) knockout mice will be used to determine the specificity of our observations; 3) To prospectively evaluate host factors potentially predisposing to TMA syndromes in HIV infected patients. The research will utilize cell and molecular biological techniques to study toxin and sialidase effects on the various cells of interest. The design of the study will allow us to initiate the experiments with an examination of a broad range of factors using in vitro techniques and then focus the experiments by degree as we find the relevant interactive cells and molecules. The knowledge gained from the in vitro studies will culminate in a series of targeted in vivo experiments looking at specific molecular interactions in whole animals utilizing physiological methods of SLT delivery. At the completion of the project we will know significantly more about the factors and cellular interactions which are critical in the development of TMA in both HIV infected and non-infected patients. This will provide insights for the development of diagnostic, therapeutic and preventative regimens.
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