These studies focus on the suppression of anticoagulant factors by inflammatory mediators. We hypothesize that 1) C4bBP in responding to E. coli binds protein S favoring both coagulation and amplification of inflammation (TNF). 2) TNF/Il-1 in responding to E. coli likewise suppresses thrombomodulin favoring both coagulation and amplification of inflammation. We believe that this suppression of the regulatory activity of the anticoagulant factors is reflected by the fall of proteins C and S concentrations following E. coli infusion and bears directly on survival and on the relative resistance to rescue therapy with activated protein C. We propose to test these hypotheses by specifically modifying the response to E. coli of each of these putative components using monoclonal antibodies against protein S, thrombomodulin, C4bBP, TNF, Il-1 and using regulatory proteins including protein S, C4bBP, and activated protein C. Finally, we propose to use this information to design optimal therapeutic schedules. Using the appropriate monoclonal antibodies or regulatory proteins, we will pursue the following aims:
In Aim 1, we will examine the role of C4bBP and its interactions with protein S in the acute response to LD100 E. coli. We also will examine whether this C4bBP-protein S interaction influences the inflammatory response (TNF).
In Aim 2, we will study whether thrombomodulin as well as protein S is suppressed in response to LD100 E. coli and whether TNF and Il-1 play a role in this phenomena.
In Aim 3, we will study the role of complement and the response of platelets to LD100 E. coli. Anti-TNF and anti-tissue factor antibodies protect but only attenuate the coagulopathic response 50%. Activation of complement therefore may play a role in promoting expression of coagulant activity of both platelets and endothelium. We examine this possibility by analysis of complement (C5b-9) and prothrombinase factor V assembly on activated platelets by flow cytometry using the appropriate fluorescein labeled antibodies.
In Aim 4, we will study whether C4bBP and TNF/Il-1 play a role in the subacute responses to E. coli. This question is addressed using a model of the microangiopathic response recently developed in our laboratory.
In Aim 5 we will apply the information gained in Aims 1-4 to the design of tests for early diagnosis and more effective activated protein C therapy.
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