Annexins are a family of calcium-dependent phospholipid binding proteins with diverse physiological functions that generally involve membrane binding either within or outside the cell. Annexin V is also being developed for clinical use as an imaging agent for detection of organ transplant rejection, response to cancer chemotherapy, and myocardial infarction. Membrane binding is critical for the proposed functions and clinical uses of annexins, yet the structural requirements for membrane binding are still poorly understood. Furthermore, wild-type annexin V has certain properties that limit its utility as a nuclear-medicine imaging agent. This proposal will clarify the mechanism of annexin-membrane binding, and will evaluate the utility of site-directed annexin V mutants as potential imaging agents. The long-term goals are to understand the structure-function relationships of annexins, and to develop clinical applications of annexins as diagnostic and therapeutic agents. By better understanding the structural correlates of membrane binding, we will be able to design improved agents for diagnosis and treatment in thrombosis, organ transplantation, and cancer chemotherapy.
The specific aims of this proposal are: 1) Develop a family of mutant annexin V molecules with a wide range of membrane binding affinities. Using newly developed methods, measure the binding of the mutant annexin V molecules to phospholipid vesicles and cell membranes in vitro under conditions of low membrane occupancy. 2) Develop a family of mutant annexin V molecules with different net charges. Measure in vivo biodistribution of Tc-labeled _mutant proteins in normal mice. Correlate in vitro properties (binding affinity and net charge) with in vivo organ uptake. 3) Develop a set of mutant annexin V proteins with a wide range of dissociation rates. Measure biodistribution and target uptake of selected mutants in mice with hepatic apoptosis. Determine which mutant proteins have lower renal uptake without loss of uptake in apoptotic target tissues. Serially measure target uptake and retention of wild-type annexin V and selected mutants in mice with apoptosis of liver. Determine which mutant proteins have faster removal from the target site, thus allowing for repeated measurement of PS expression over short periods of time (up to one day).
