Adam, Jacqueline Michelle; (2000) Modulation of tissue factor activity from a human monocytic cell line. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The contribution of oxidised low-density lipoprotein (oxLDL) in the pathogenesis of atherosclerosis is well established, as is the presence of oxLDL within atherosclerotic plaques. An important concomitant of atherosclerosis is the increased tendency for thrombosis in the region of the plaque. This may be explained by an interaction between oxLDL and the constituent cells of the plaque as in vitro studies have demonstrated tissue factor (the initiator of the coagulation cascade) is expressed from vascular cells following incubation with oxLDL. The exact mechanism by which LDL is oxidised in vivo has yet to be elucidated however, one candidate for the oxidising species is peroxynitrite, which forms via a reaction between nitric oxide and the superoxide anion (both released from macrophages during inflammatory periods). In addition to its ability to oxidise LDL, peroxynitrite has been shown to nitrate tyrosine to 3-nitrotyrosine, an effect that influences the biological activity of certain proteins. In the present study, the effect of native LDL and LDL oxidatively modified by cupric ions and peroxynitrite were studied with respect to their ability to influence maximal cell surface tissue factor activity and to induce tissue activity expression in THP-1 macrophages. Native LDL inhibited maximal cell surface tissue factor activity and did not induce tissue factor expression in the macrophages. Conversely, LDL modified by both cupric ions and peroxynitrite enhanced maximal cell surface tissue factor activity and induced tissue factor activity expression, above basal levels. These results indicate peroxynitrite-oxidised LDL modulates the tendency for blood to coagulate contributing to the hypercoagulable-state associated with atherosclerosis. The direct effect of peroxynitrite on tissue factor was investigated as 11 of its 12-tyrosine residues are in its active domain. 8 of these are exposed and thus susceptible to attack by peroxynitrite. Following exposure of tissue factor to peroxynitrite, there was an inhibition of procoagulant activity, accompanied by an increase in the nitrotyrosine content of the tissue factor, determined by ELISA. Furthermore, exposure of LPS to peroxynitrite was shown to diminish its ability to induce tissue factor expression in THP-1 monocytes. These results may indicate a protective mechanism afforded by peroxynitrite under inflammatory conditions as an anticoagulant, to minimise the extent of the coagulation response, but also indicate procoagulant properties through oxidation of LDL.

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