In studies of the role of C4b binding protein (C4bBP) in the baboon response to E. coli, we observed a microvascular thrombotic (MVT) variant which mimics the hematolytic uremic syndrome (HUS). We identified the key inflammatory and coagulant mediators (ie. tumor necrosis factor, tissue factor, etc.) and anticoagulant regulators (ie. protein C and S, etc.). We established that inflammatory mediators initiated the coagulant response. We did not, however, establish how the inflammatory, coagulant, and anticoagulant systems interacted. In this proposal, we hypothesize that coagulant mediators can amplify inflammatory activity, and that as a consequence, anticoagulants also act as anti-inflammatory regulators. Conversely, we hypothesize that these inflammatory mediators decrease anticoagulant activity. This is a circle in which the regulator systems are influenced by disturbances in the two mediator systems and visa versa. We will ask the following three questions: First, does C4bBP neutralization of anticoagulant protein S affect platelet/coagulant status, and is the stage set for an increased inflammatory (MVT) response to TNF, I1-6, etc.? Second, conversely do these cytokines disable the normal regulatory protein C anticoagulant/fibrinolytic response to factor Xa phospholipid (XaPCPS) and """"""""tip the balance"""""""" in favor of MVT? Third, do these or similar events involving these mediator/regulator systems occur in a baboon model of MVT/HUS induced by Shiga-like toxin? The coagulant, anticoagulant, and inflammatory (neutrophil), responses to C4bBP alone will be studied. These studies will include determination of neutrophil opsonin/receptor expression by blood luminescence analysis, and appearance of enzyme/inhibitor complexes composed of components of regulator systems. Platelet receptor, and, neutrophil receptor participation will be assessed using appropriate Fab monoclonal antibodies. Both the C4bBP and XaPCPS models will be perturbed with TNF and I1-6, etc., to determine the effect of the absence of protein C and S on the response to these mediators and their effect on a stressed, but compensated, anticoagulant/fibrinolytic response to XaPCPS. We will determine whether the compensated response to XaPCPS can be converted into a response by these cytokines and/or by coinfusion of antibodies to both protein C and tissue plasminogen activator. The experience and techniques used in the above intervention and reconstitution studies will be applied to mechanistic, diagnostic and treatment studies of the fully expressed Shiga-like toxin model of HUS/MVT.
Showing the most recent 10 out of 42 publications
