PHOSPHOLIPID-TARGETED FIBRINOLYTIC AGENTS

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The long-term objective of this research is to develop new approaches for detecting and treating thrombosis. In this project, a new method will be developed for detection and targeting of procoagulant phospholipid exposure. This approach is based on the phospholipid binding protein annexin V, which binds with high affinity to activated platelets and to membranes containing phosphatidylserine. The specific aims are: 1. Prepare phospholipid-targeted fibrinolytic agents by chemical crosslinking of annexin V with single-chain urokinase, streptokinase and plasminogen. The hybrid proteins will be evaluated as to molecular weight, enzyme activity, binding to procoagulant phospholipid membranes, binding to resting and activated platelets, and in vitro and in vivo fibrinolytic activity. 2. Prepare and characterize recombinant hybrid proteins in order to extend the studies described under Aim 1. 3. Evaluate annexin V as an agent for detecting, targeting and imaging thrombi in vivo. 4. Study the structure/function relationships of annexin V by site-directed mutagenesis in order to localize the binding site(s) for phospholipid surfaces. 5. Evaluate annexin V as a potential endogenous regulator in atherosclerosis by studying its expression in normal and atherosclerotic human coronary arteries. These studies represent a novel approach to the problem of detecting and treating thrombosis in the coronary arteries and elsewhere. They will also contribute to basic knowledge about the annexin protein family and its potential involvement in vascular pathology and atherosclerosis.