PROTEIN S AND C4BBP IN THREE COAGULANT MODELS

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The main objective of these studies is to continue study of the influence of protein S and C4b-binding protein on the disseminated intravascular coagulant response in the lethal E.coli model of sepsis. Since these two factors also influence microvascular thrombotic and deep vein thrombotic responses to inflammatory stimuli, we plan to study the role of these two factors in these models as well. The central hypotheses is that protein S has two protective functions. The first is as an anticoagulant cofactor for activated protein C and the second is as an anticomplement cofactor for C4b-binding protein. Conversely, insufficiency of protein S relative to C4bBP can lead to an amplification of both the inflammatory and coagulant responses because they are linked and drive each other. Thus reduction of protein S by consumption or by neutralization by excess C4bBP can leave pro-inflammatory as well as procoagulant activity unchecked unless there is sufficient protein S to serve both its anticomplement and anticoagulant functions. Certain aspects of this hypothesis can be tested in vivo. Using the model of disseminated intravascular coagulant response to lethal E.coli we will intervene with C4bBP/protein S complex or protein S and monitor their effects on physiologic and laboratory parameters. These include C5b-9 and C4bc markers of complement system activation, thrombin-antithrombin complexes and fibrinogen markers of coagulant activation as well as cytokine and components of the protein C pathway (ie. free and bound protein S, protein C and C4bBP). We will determine if there is a pool of C4bBP/protein S complex bound to cell membranes with antibodies to the Gla domain of protein S by which these complexes might be anchored. We will monitor this by assaying for increases of the complex in plasma following infusion of antibodies. We also will study recruitment of radiolabeled complexes to these membranes before and after an inflammatory stimulus (ie. E.coli, TNF). We will study which domains of protein S participate with the appropriate antibodies. We will intervene with anti-C3b antibody and compare its anti -complement effects with those of the C4bBP/protein S complex. Using the models of the microvascular thrombotic response to sublethal E.coli and C4bBP and the deep vein thrombotic response to TNF and C4bBP, we will probe some of the same systems described above as well as features unique to each model. We will examine the role of tissue factor and platelets in the former and that of tissue factor and leukocytes in the latter. To give these studies more perspective, the pathologic responses of these two models will be divided into stages. This includes correlation of laboratory markers with light and electron microscopic studies of tissue. These studies should provide insights into the relationship between inflammation and coagulation in E.coli sepsis and offer new perspectives on diagnosis and treatment.