DEVELOPMENT OF AN ASSAY FOR SCREENING PDGF ANTAGONISTS

VASCULAR SMOOTH MUSCLE CELL PROLIFERATION CAUSING RESTENOSIS FOLLOWING SUCCESSFUL PERCUTANEOUS TRANSLUMINAL CORONARY ANGIOPLASTY IS THE MAJOR LIMITING FACTOR FOR LONG-TERM EFFICACY OF THIS PROCEDURE. PLATELET DERIVED GROWTH FACTOR (PDGF), A POTENT MITOGEN FOR VASCULAR SMOOTH MUSCLE CELLS, IS RELEASED BY PLATELETS AT THE SITE OF VASCULAR INJURY AND IS EXPRESSED BY VASCULAR ENDOTHELIAL CELLS AND SMOOTH MUSCLE CELLS. THEREFORE, IT IS LIKELY THAT PARACRINE AN/OR AUTOCRINE STIMULATION OF SMOOTH MUSCLE CELLS BY PDGF PLAYS A ROLE IN VASCULAR RESTENOSIS. HUMAN PDGF FORMS COMPRISE DISULFIDE-LINKED HETERODIMERS AND HOMODIMERS OF TWO DISTINCT BUT RELATED POLYPEPTIDE CHAINS, A AND B. PDGF MEDIATES ITS BIOLOGICAL EFFECTS THROUGH BINDING AND ACTIVATION OF CELL SURFACE PDGF RECEPTORS OF WHICH THERE ARE TWO SUBTYPES, ALPHA AND BETA. THE MAIN GOAL OF PHASE I IS TO DEVELOP A SENSITIVE, SPECIFIC, EASILY- AUTOMATED MICROTITER SOLID PHASE BINDING ASSAY TO IDENTIFY ANTAGONISTS OF PDGF BINDING. FOR THIS PURPOSE, WE WILL PURIFY BETA PDGF RECEPTOR EXTRACELLULAR DOMAIN WHICH RETAINS PDGF BINDING ACTIVITY. THE EXTRACELLULAR DOMAIN OF THE ALPHA PDGF RECEPTOR WILL ALSO BE EXPRESSED IN PHASE I AND THIS RECOMBINANT PROTEIN WILL BE USED TO DEVELOP A BINDING ASSAY IN PHASE II. THESE ASSAYS WILL BE FURTHER DEVELOPED IN PHASE II WITH BIOTINYLATED PDGF WHICH AVOIDS THE USE OF RADIOISOTOPES AND EXPEDITES ANALYSIS BY UTILIZING AN ELISA PLATE READER. AT THIS POINT LARGE CHEMICAL LIBRARIES WILL BE INTENSIVELY SCREENED TO IDENTIFY COMPOUNDS WITH THERAPEUTIC POTENTIAL FOR THE TREATMENT OF VASCULAR RESTENOSIS.