This proposal is complementary to the existing BIOMED programmePL 931685. The purpose of this project is to further characterize structure and function of the aIIbb3 and aVb3 integrin, their interaction with high molecular weight ligands, and their role in binding of fibrinogen. Since platelet-platelet interactions resulting from the receptor occupancy by fibrinogen are critical for thrombus formation, ligand binding to the major platelet receptor for fibrinogen, aIIbb3 is a key event in haemostasis an thrombosis. This project is focused on the following aims: (a) identification of conformational changes in both receptors, aIIbb3 and aVb3, induced by different agonists, antibodies, peptides and disintegrins. This will be analyze with the purified receptors, receptors incorporated into liposomes, and in suspension of platelets and endothelial cells. (b) Characterization of conformational changes in aIIbb3 induced by palmitoylation.
We demonstrated previously that b3 in platelet suspensions is palmitoylated and provided suggestive evidence that this process may lead to the activation of platelet receptors. We propose to compare ligand binding by control and palmitoylated aIIbb3. Control experiments will include palmitoylation of aVb3. Since palmitoylated aIIbb3 appears to be more active than native integrin, we will try to isolate it on an RGD-affinity column. Interaction of palmitoylated and native aIIbb3 with short RGD peptides and disintegrins will be investigated using various approaches. Subsequently we will try to digest palmitoylated aIIb with various proteolytic enzymes to isolate palmitoylated peptides by means of HPLC and to identify amino acids (probably serine and threonine) which form est bonds with palmitic acid. Similar experiments will be done with endothelial cel in order to see whether b3 in aVb3 becomes palmitoylated.
To accomplish these aims we are going to use the following approaches: (a) Conformational changes of aIIbb3 associated with transition from resting to activated state will be analyzed by spectrometric techniques using different fluorescent and spin probes. (b) Conformational changes occurring in different regions of a and b subunits of both cytoadhesins will be monitored by antipepti antibodies reacting with well defined antigenic epitopes. Among them antibodies against peptides b3(90-102) and b3(631-653) and to peptide fragments of aIIb corresponding to sequences 242-255, 296-309, 374-377, 425-438, 842-857 are to b used. (c) We will estimate a range of conformational changes induced upon activation of aIIbb3 and aVb3 by measurements of fluorescence resonance energy transfer. For this purpose we will use antibodies to b3(90-102) and b3(631653). The surface orientation of b subunit in aIIbb3 and aVb3 during activation of platelets by thrombin, ADP, and RGD, the gamma peptide and disintegrins - eristostatin and albolabrin will be evaluated by this approach.