The applicant proposes to study the fibrinolytic system by in vitro and in vivo techniques in three areas: 1) Previous in vivo and in vitro studies by the applicant and others have identified a murine liver receptor which binds fibrinogen and fibrin degradation products containing the D domain. These studies indicate that the receptor is present on reticuloendothelial cells. In vitro binding studies will be performed with cultured rat and mouse macrophages/reticuloendothelial cells. Receptor number and Kd will be determined with each type of cell employing 125 I-labeled fibrinogen fragments X, Y, D and fibrin fragment D1 dimer. Previous work by others (and recently confirmed in the laboratory of the applicant) has shown that fibrinogen fragment D interacts with macrophages with elaboration of a protein which stimulates fibrinogen synthesis by hepatocytes. Studies will be designed to determine whether binding of each of the above fragments to macrophages triggers release of the factor which stimulates rat hepatocytes to produce fibrinogen. If degradation fragments containing the D domain bind to macrophages/reticuloendothelial cells, the appropriate cell fraction will be employed to prepare membranes for binding studies. A long term goal of this work is purification of the putative fragment D receptor. 2) Streptokinase is an important drug for therapy of thromboembolic disease. Its use is limited by its short half life and its antigenicity and immunogenicity. Polyethylene glycol (PEG) can be coupled to proteins increasing their half lives and dramatically decreasing immune recognition. The applicant has recently described a new and very gentle procedure for coupling PEG to proteins. It is proposed to PEG modify streptokinase and study the in vitro effects on plasminogen interaction and on the activator potential of the modified complex. In vivo studies in the mouse model will define the effect of the modification on clearance. 3) Deficiency of vascular plasminogen activator (PA) in humans result in a significantly increased thrombolic risk. Utilizing a quantitative PA assay developed in this laboratory, and a recently developed immunoradiometric assay it is proposed to study the relationship between low levels of vascular PA and myocardial infarction and stroke. Other studies will examine whether lower levels of vascular PA increase the post operative risk for thrombosis in a group of patients with gynecologic malignancies.
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