This investigation seeks to identify and selectively inhibit the reactions that occur in blood during cardiopulmonary bypass (CPB) and long-term extracorporeal life support (ECLS). Extracorporeal perfusion activates four plasma protein systems: coagulation, contact, complement and fibrinolysis, and four blood cells: neutrophils, monocytes, platelets and endothelial cells. Inhibition of these reactions should prevent the bleeding, thrombotic and inflammatory complications associated with CPB and ECLS and is the strategy of this project. The investigators have recently found that monocytes express tissue factor (TF) during prolonged extracorporeal perfusion.They intend to pursue this discovery by examining the assembly of coagulation factors on the surface of monocytes and also platelets during extracorporeal perfusion. Because monocyte expression of TF is delayed, they will study the timing of monocyte mRNA transcription for TF and other coagulant proteins. They will test the hypothesis that cell-bound thrombin, which is shielded from heparin during CPB and ECLS, is the agonist for activation of procoagulant mechanisms. They will study specific inhibitors of monocyte coagulant protein expression using leukospecific antibodies, factor Xa inhibitors and peptides of high molecular weight kininogen. They will study platelet coagulation protein assembly using iloprost, a disintegrin, and specific antibody fragments. Lastly, they will study direct thrombin inhibitors (r-hirudin, small peptide derivatives of hirudin, a boroarginine peptide and a chloromethyl-ketone for their ability to inhibit cell-bound thrombin. Studies of blood activation during ECLS and the possible role of surface- bound heparin are severely compromised by the lack of an appropriate animal model. The investigators propose to """"""""complete the sheep model"""""""" by developing additional assays of plasma proteases and blood cell activation. They plan to study the mechanism of platelet adhesion during CPB and ECLS and the mechanisms by which single dose disintegrins and surface-bound heparin reduce adhesion and apparently preserve platelet function. In the final year, the investigators plan to devise an inhibitory """"""""cocktail"""""""" to block blood activation during CPB and ECLS by first inhibiting cell-bound thrombin and platelet activation and then adding a protease inhibitor to block contact, complement and neutrophil activation.
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