The fibrinolytic system is composed of multiple enzymes which promote or inhibit the lysis of fibrin clots. Binding sites for plasminogen, the inactive precursor of plasmin, are present on fibrin and on many cell types including vascular endothelium and hepatocytes. Alpha2-antiplasmin (alpha2-AP), a serine protease inhibitor, inactivates plasmin by forming a stable complex, which is cleared from the circulation by specific hepatic receptors. Clinically, alpha2-AP depletion is associated with a hemorrhagic diathesis due to excess fibrinolysis. Therapeutic inhibition of alpha2-AP function by specific antibodies in patients with thromboemboli promotes fibrinolysis. Therefore, modulation of circulating levels of alpha2-AP may have therapeutic potential in disorders of hemostasis and thrombosis; however, the mechanisms regulating the synthesis and secretion of alpha2-AP are presently unknown. The goals of this project are to characterize the hepatocyte receptor system for the alpha2-AP-plasmin complex, and to study the mechanisms involved in the regulation of hepatic alpha2-AP synthesis and secretion. The initial phase of this project will focus on hepatocyte receptor systems using studies of radiolabeled alpha2-AP-plasmin complex binding to hepatocytes in primary culture. The second phase of this project will focus on hepatic synthesis of alpha2-AP. Fibrinolytic proteins for which specific hepatocyte receptors have been described will be evaluated as potential modulators of alpha2-AP synthesis and secretion. These will include plasminogen, plasmin, alpha2-AP-plasmin complexes, tissue plasminogen activator, fibrinogen, degradation fragments of fibrinogen, IL-6 and TGF-beta. Alterations in fibrinolytic homeostasis result in fluctuations in the circulating levels of these proteins. Changes in the concentrations of these proteins may then act as signals to the hepatocyte to alter the rate of synthesis and secretion of other fibrinolytic proteins, such as alpha2-AP, via interaction with their hepatocyte receptors. alpha2-AP synthesis will be studied at the levels of transcription and translation. Newly synthesized (alpha2-AP will be determined by incorporation of 35S-methionine in pulse-chase experiments. Regulation of hepatic alpha2-AP mRNA levels will be studied by northern blotting. The data produced by these investigations will lead to an increased understanding of fibrinolytic homeostasis and potentially to the development of novel therapeutic interventions in patients with disorders of thrombosis and hemostasis.
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