Numerous studies have documented the activation of the coagulation system either in septic patients (28) or following LPS administration in animal models. In the rat model of LPS-induced liver injury employed in our laboratory, circulating plasma fibrinogen concentration falls more than 90% within 6 hours following intravenous injection of LPS. This is accompanied by the appearance of numerous fibrin clots within the liver sinusoids.
The importance of this activation of the coagulation system in the pathogenesis of liver injury is further emphasized by the ability of anticoagulants to abolish the hepatotoxicity. Pretreatment with Warfarin and heparin prevents the decrease in plasma fibrinogen concentration and offers complete protection against LPS-induced liver injury (2, 6).
However, pretreatment with ancrod, a snake-venom derived enzyme resulted in an 89% decrease in plasma fibrinogen, and yet did not prevent LPS-induced liver injury. This suggests that the role of the coagulation system is independant of the circulating fibrinogen level and the ability to generate insolubles fibrin clots; and that the decrease in plasma fibrinogen concentration is merely a marker of the activation of a system responsible for the onset of liver injury.
The recent demonstration that hirudin, a small peptide with specific antithrombin activity could prevent the onset of injury up to two hours after LPS administration pointed strongly toward the involvment of thrombin as a critical and distal mediator of the toxicity. This was further confirmed by the ability of thrombin to mimic LPS toxicity when infused in the portal vein (5) or when added to the perfusate in the isolated liver model (preliminary results), and by the ability of antithrombin III to reduce the symptoms and improve the survival rate of patients with septic shock (29). Thus, thrombin appears to be an important mediator of the LPS-induced hepatocellular necrosis that acts after the accumulation of inflammatory cells in the liver and the release of certain inflammatory mediators (TNF-a). However, the mechanisms of its action, and how it interacts with other cellular or soluble mediators remain to be elucidated.
Thrombin is not a normal constituant of the circulating blood. It is generated by the catalytic cleavage of its plasma precursor, prothrombin (factor II), by the activated Stuart factor (factor Xa). This is the final step of one or both of the two convergent chains of reactions called the intrinsic and extrinsic pathways of coagulation. The transformation requires the presence of an activated cofactor, factor Va, released from factor V by thrombin itself, and whose binding to prothrombin accelerates the activity of factor Xa in a non-enzymatic manner.
Thrombin is a glycoprotein formed by two peptides chains of 36 and 259 amino-acids linked by disulfure bonds. Three important sites have been identified on the surface of the enzyme: The catalytic site that confers to the molecule its serine protease activity, the exosite one responsible for the binding of the substrate (fibrinogen or thrombin receptor) and the exosite two responsible for the binding of antithrombin III and inactivation of thrombin (30).
The earliest identified function of thrombin is the cleavage of fibrinogen into fibrin monomers and the activation of the fibrin-stabilizing factor (factor XIII) and protein C. Clotted blood is a meshwork of insoluble fibrin threads that traps blood cells and serum. Thrombin has the property of activating factor XIII to act as a transaminase and form covalent links between the carboxyl and amino groups of two different fibrin monomers, enhancing the strength of the clot.
Thrombin is, however, more than a simple plasma enzyme. Its properties to stimulate platelets and cause them to expand aggregate and release components of the alpha and dense granules were recognized earlier on (31, 33). Thrombin also has numerous effects on various cells, some of them being of major importance in the development of LPS-induced liver injury: It alters the synthesis, expression and release of proteins from endothelial cells (34, 35). This results in increased production of the platelet-derived growth factor (PDGF), factor XIII, factor VIII, tPA, PAI, platelet activating factor (PAF), modification of the interactions between endothelial cells and the underlying matrix or between endothelial cells, and the expression of adhesion glycoproteins to the cell surface, thereby increasing the binding of inflamatory cells to the endothelium. It induces chemotaxis in neutrophils (36) and promotes the release of inflammatory components (37, 39). Thrombin is also a potent chemotaxin for macrophages, and can alter their production of cytokines and arachidonic acid metabolites (40). Little is known of thrombin activity in the liver resident macrophages, but high affinity binding sites for thrombin have been identified on the rat kupffer cells (41). The direct effect of thrombin on hepatic parenchymal cells is poorly understood, and no function have been yet associated with the thrombin binding sites described on these cells (42).
The mechanism by which thrombin activates platelets or other cells was until recently unknown, and if traditional binding studies had identified thrombin-binding proteins, the signal transduction pathway remained mysterious. In 1991, a thrombin platelet receptor was cloned (43), revealing a new mechanism of receptor activation. After binding to its receptor, thrombin cleaves the amino-terminal extension to expose a new amino-acid sequence that binds to the third extracellular loop of the receptor and activates it. This sequence capable of self-activation was named the tethered ligand. Various peptides similar to the sequence of the tethered ligand have now been synthetised (44), and their ability to activate the thrombin receptor has been demonstrated (45, 46). In essence, the agonist peptide bypasses the thrombin-mediated receptor proteolysis and activates the receptor independently of thrombin. This mechanism of activation has since been demonstrated on cell types other than platelets (47).
Despite more than thirty years of research, the mechanisms of LPS-induced liver toxicity remain poorly understood. It is now clear that they involve a complex and sequential series of interactions between cellular and humoral mediators.
In the same periode of time, gram-negative systemic sepsis and its sequallae have become a major health concern, and our attempts to use monoclonal antibodies directed against LPS or various inflammatory mediators have only yield therapeutical failures.
One of the reasons currently invoqued to explain the lack of efficiency in the clinic, of technics that were successfull in the laboratory animals, is the delay between the actual exposure to LPS and the time of admission in the critical care unit. From a medical viewpoint, the first exposure to LPS and the rise in TNF-alpha in the patient's circulation are not in close enough temporal apposition to the appearence of the first clinical symptomes to be considered an interesting target for therapeutic intervention.
In contrary, our evidences suggest that thrombin is a critical mediator of liver injury, and that its involvment closely preceeds the onset of hepatotoxicity. Given that thrombin action does not appear to be dependant on the formation of insoluble fibrin clots as previously suggested, this proposal will focuse on explaining the mechanisms by which it contributes to hepatocyte necrosis following LPS administration.
It is our hope that this work will contribute to a better understanding
of the events closely preceeding the onset of organ injury, and by doing
so unmask new directions toward a better treatment of the consequences of
endotoxemia.
