Defining the roles of adenosine, anesthetics, and selective integrin targeting during leukocyte and platelet activation -A basis for new therapeutic strategies aiming to treat systemic inflammation, ischemia-reperfusion injury and thrombosis

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The systemic inflammatory response syndrome (SIRS) is a major cause of morbidity and mortality in intensive care medicine. During sepsis, SIRS is caused by infectious microorganisms. SIRS can also result from non-infectious causes such as extracorporeal circulation (ECC) in cardiac surgery or organ ischemia and reperfusion. During SIRS pro-inflammatory mediators induce the activation and interaction of leukocytes, platelets and endothelium. These cellular interactions are of central importance for the maintenance of SIRS and lead to life-threatening inflammatory organ failure. Leukocyte activation is associated with increases in expression and ligand affinity of adhesion molecules, including the Mac-1 integrin receptor. Mac-1 plays a central role in leukocyte adhesion to endothelium and platelets. In contrast, the ecto-apyrase CD39 plays a crucial role in generating the anti-inflammatory molecule adenosine, which inhibits leukocyte and platelet activation. The anesthetics sevoflurane and propofol also carry anti-inflammatory potential. These agents interact with cell surface molecules and probably activate adenosine receptors. In this study we will analyze mechanisms underlying the development of SIRS with the aim to establish new pharmacological treatment strategies. For this purpose, we have identified three promising approaches to increase anti-inflammatory effects, which we will test. Ex vivo we will induce leukocyte and platelet activation using physiological agonists and simulated ECC. In vivo we will employ a murine intravital microscopy model to study leukocyte-platelet-endothelium interaction during SIRS and will investigate leukocyte and platelet activation during ischemia and reperfusion of mouse myocardium and liver. Our three approaches include: Activation-specific blockade of Mac-1, activation of adenosine receptors, and increase of adenosine production. First, to define the role of Mac-1 in leukocyte and platelet interaction we will use a KO mouse strain and analyze the effect of the single chain antibody fragment MAN-1 on leukocyte and platelet interaction. MAN-1 selectively binds to the activated conformation of Mac-1 without affecting the overall function of Mac-1 in the immune system. Second, we will use sevoflurane and propofol to induce adenosine receptor activation and evaluate the effects of these anesthetics on leukocyte and platelet activation and look at different adenosine receptor KO mice. Third, we will employ a bivalent molecule, which carries the ecto-apyrase CD39 and is targeted against the activated platelet GP IIb/IIIa receptor. Using this approach, apyrase activity can be confined to sites of platelet-leukocyte interaction to remove the prothrombotic platelet agonist ADP and to produce the anti-inflammatory agent adenosine. Pharmacological advances to prevent leukocyte and platelet activation represent a novelty in the treatment of SIRS, as it represents a major step towards symptomatic treatment.