Endothelium has a central role in the regulation of vascular permeability and the coagulation mechanism. In the prethrombotic state associated with certain malignancies, the coagulation mechanism and vascular permeability appear to be altered. This proposal examines the effects of an apparently novel polypeptide, isolated from murine meth A tumor cells, on barrier function and cell surface coagulant properties of the endothelial cell. Preliminary studies, which resulted in the identification of an isolation procedure for this 24,000 dalton protein from tumor-conditioned medium (meth A factor), will be optimized and scaled-up to produce sufficient material for further biochemical characterization, production of antisera and physiology studies in vitro and in vivo. The human counterpart of the murine factor, which has been partially purified from a melanoma cell line, will be completed. Using purified meth A factor and cultured endothelium (micro- and macrovascular), pilot studies demonstrating modulation of endothelial cell coagulant and barrier function by this mediator will be extended, and the underlying mechanisms identified: partially purified or purified meth A factor suppresses two anticoagulant mechanisms of endothelium (the protein C/protein S and fibrinolytic pathways), enhances induction of the procoagulant cofactor tissue factor in response to TNF, and increases monolayer permeability. In vivo studies have shown a selective procoagulant response in the tumor vasculature of meth A sarcomas following the administration of low concentrations of TNF, which we hypothesize is related to the meth A factor. Furthermore, partially purified meth A factor augments the increase in vascular permeability observed in the presence of TNF in vivo. These observations will be followed up with systematic studies in rats to define the effects of meth A factor on coagulant function and vascular blood flow/permeability. Activation of coagulation will be monitored by assays for fibrinopeptide A and fragment 1+2, and direct examination for the presence of intravascular fibrin. Measurements made by intravital microscopy will define blood flow and transvascular exchange of macromolecules. The presence of tumor factor in malignant tissues will be assessed using our immunological reagents. These specific plans are directed toward our long-term objective: understanding the role of endothelium in the pathogenesis of the prethrombotic state.
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