Understanding hemostasis mechanisms in the brain ultimately requires delineation of the hemostatic functions of the blood-brain barrier (BBB). The balance between clot-forming and clot-dissolving pathways in the brain is closely controlled, but the mechanisms responsible for maintaining this balance are not known. In vitro studies suggest that vascular endothelium of non-BBB origin controls coagulation by regulating expression of binding sites for anticoagulant and procoagulant factors on the cell surface. In the quiescent state, endothelial cells maintain blood fluidity through the thrombomodulin (TM)-protein C anticoagulant system, and the tissue plasminogen activator (tPA) fibrinolytic pathway. In contrast, the procoagulant tissue factor (TF) is normally absent from vascular endothelium. Our preliminary work has demonstrated that a similar scenario may be responsible for the anticoagulant function of the BBB in the basal state, i.e., in the absence of provocative stimuli which may lead to thrombosis. The major stroke risk factors including smoking (e.g., nicotine), diabetes and hypertension, as well as endotoxin, may all produce procoagulant alterations of endothelial cells in vitro, and acting either directly, and/or indirectly via cytokines such as interleukin-1B (IL-1B) and tumor necrosis factor-a (TNF-a). However, the evidence supporting a similar situation in the brain is lacking, except for our preliminary data demonstrating downregulation of BBB tPA expression in diabetes, and complete depletion of BBB tPA following nicotine treatment. The focus of this proposal is directed at BBB hemostatic mechanisms and their transformation by stroke risk factors and endotoxin. Towards this end, we propose to test the following specific hypotheses: 1. Exposure to stroke risk factors results in transformation of the BBB predisposed for thrombogenesis by stroke risk factors. III. IL-1B and TNF-a mediate procoagulant transformation of the BBB induced by endotoxin and stroke risk factors. IV. The effects of middle cerebral artery occlusion (MCAO) are enhanced by the presence of stroke risk factors (nicotine, diabetes, hypertension), and are further enhanced by the presence of endotoxin. Molecular techniques will be used to study expression of tPA, TM, TF, IL-1B and TNF-a at the BBB and in brain parenchyma. MCAO will be used to determine the pathophysiological consequences of exposure to stroke risk factors and endotoxin. We will use well established models of diabetes, hypertension, nicotine, endotoxin and MCAO in rats. The proposed studies will contribute to an understanding of BBB mechanisms in brain hemostasis under normal and pathological conditions related to focal ischemia stroke. Defining the role of anticoagulant, procoagulant and fibrinolytic pathways at the BBB in the presence of provocative stimuli such as major stroke risk factors, endotoxin and MCAO, may ultimately be important in designing therapeutic strategies to counteract thrombogenic processes in the cerebral microvasculature.

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