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Welcome to my professional homepage. You will find here general information about me and my research, as well as background information concerning the topic of my thesis. I sincerely hope you enjoy it as much as I do...
Lipopolysaccharides-induced liver injury in vivo is characterized by a predominantly mid-zonal multifocal hepatocellular necrosis with neutrophil accumulation in the sinusoid, and formation of fibrin and platelet clumps (1). A marked hyperbilirubinemia and an elevation of the plasma concentration of liver specific enzymes reflects the increased inability of the organ to perform its normal functions.
The administration of anticoagulants like warfarin and heparin (2) protects against the onset of liver damage, despite the fact that other markers of LPS activity, mainly increased TNF-alpha in the plasma and accumulation of neutrophils and platelets in the sinusoids, are still present. The observation of the protective effect of anticoagulants as well as the decrease in plasma fibrinogen and the accumulation of platelets in the liver that normally follow LPS administration suggested that the coagulation system was intricately linked to the hepatotoxicity (3).
The mechanism usually suggested to explain the involvement of the coagulation cascade in the onset of liver injury is the formation of insoluble fibrin clots in the microvasculature of the organ (4, 5). By blocking blood flow to certain areas of the liver, the platelet-fibrin clots create ischemic conditions, and the resulting death of hepatocytes account for the LPS hepatotoxicity.
However, this hypothesis was contradicted by the recent observation that ancrod is not able to prevent liver injury resulting from LPS administration (6). Ancrod is a thrombin-like enzyme extracted from the venom of the malayan pit viper (Agkistrodon rhodostoma) that cleaves fibrinopeptide A but not fibrinopeptide B from fibrinogen. Because ancrod only partially cleaves fibrinogen and does not activate coagulation factor XIII, it does not allow the formation of normal insoluble fibrin clots. The cleavage products are rapidly cleared from the vasculature, effectively preventing further clot formation without disturbing other components of the coagulation system. Pretreatment with ancrod resulted in an effective prevention of platelet-fibrin clot formation, and yet did not protect against LPS-induced liver necrosis, suggesting the involvement of a different mechanism of toxicity.
Recently, the observation that Hirudin, a peptide with specific antithrombin activity could prevent liver damages after intravenous LPS administration pointed toward a critical activity of thrombin in the mechanism of toxicity (8). Heparin and Warfarin do inhibit thrombin, but this occurs along with numerous other elements of the coagulation cascade. Hirudin on the other hand, has been shown to bind specifically to the active site of thrombin to prevent its cleaving activity (9). Thus the demonstration of the ability of Hirudin to protect against LPS induced liver injury pointed strongly toward an involvement of thrombin that would be independent of its ability to cleave fibrinogen and form insoluble clots.
In order to confirm the importance of thrombin activity for the development of liver lesions following LPS administration, we designed a series of experiments using the isolated liver model perfused with different perfusion media. We were able to reproduce the type of liver injury observed in vivo by perfusing livers from LPS-treated animals with ancrod-treated blood. In contrast, we did not observe any toxicity when the perfusion solution contained blood mixed with heparin. In both sets of experiments, the treatment of the perfusion solution prevented increase in intrahepatic pressure, suggesting the absence of fibrin clot formation in the vasculature. Since we were able to reproduce toxicity without the formation of fibrin clots, we concluded that the mechanism required thrombin activity, but that the cleavage of fibrinogen into fibrin was merely a marker of thrombin activation and not the main cause of the observed hepatic injury.
To evaluate the requirement for other coagulation factors beside thrombin in the genesis of injury, livers from LPS-treated animals were perfused with a buffered salt solution with or without bovine a-thrombin. The addition of active thrombin to the perfusate resulted in significant liver injury, as measured by the release of alanine aminotransferase (ALT) into the medium. To examine if thrombin itself, at the dose used in the experiment, could be hepatotoxic, livers from saline-treated animals were perfused with the same thrombin buffer solution. No difference in liver injury was observed as compared to the controls.
We also treated hepatocyte cultures from LPS- or saline-treated animals with increasing doses of thrombin and studied the percent increase of ALT activity in the medium.Thrombin up to 4 U/ml had no effect on ALT release, confirming that thrombin alone was not sufficient to kill hepatocytes.
It therefore appears that thrombin is a critical mediator of LPS-induced liver injury and acts independently of its ability to form obstructive clots in the hepatic vascular beds, since injury can occur in the absence of any other circulating coagulation factor. However, thrombin alone, at the concentration of 4U/ml, is not sufficient to cause damage in naive livers or primary hepatocyte cultures, suggesting more complex interactions in the mechanism of LPS-induced liver toxicity than simple proteolytic damage of the liver parenchymal cells.
In view of these preliminary results, the overall objective of this proposal
is to understand the mechanisms by which thrombin leads to liver injury
following exposure to lipopolysaccharide. Our hypothesis is that thrombin
acts on inflammatory cells present in the liver after LPS administration
through the activation of a specific thrombin receptor, and initiates a
cascade of cellular interactions ultimately resulting in the death of liver
parenchymal cells.
The specific aims are:
The general goal of our research is to clarify the role of thrombin and the thrombin receptor in the mechanisms of liver injury following LPS exposure. In further studies, we will try to understand which cell interactions ultimately result in the death of the hepatocytes following thrombin activation. It is our hope that this information will contribute to the development of new therapeutic treatments for liver toxicity following exposure to gram negative bacteria. Thrombin involvement in the pathogenesis of respiratory distress following endotoxin has also been reported, and anti-thrombin agents have been shown to prevent the lethal consequences of Gram-negative bacterial infections in rats (10) and other animal models (11). Thus, despite the limitation of our research to the study of hepatotoxicity, it is reasonable to envision a broader implication of thrombin in the mechanism of endotoxin-induced multi-organ failure.
Last Updated 01/01/97 Please notify Frederic
Moulin for additions, corrections, or changes.