Integrins aIIbb3 and aVb3 mediate key adhesive responses in platelets and endothelial cells during vascular repair. Ligand binding to b3 integrins is regulated by """"""""inside-out"""""""" signals, but the nature of these signals and their relative effects on integrin affinity and avidity are unclear. We have developed recombinant ligand-mimetic antibodies that discriminate between changes in b3 integrin affinity and avidity in human and murine platelets and endothelial cells. These new reagents and several complementary model cell systems will now be used to address three major unresolved questions pertaining to functional regulation of the b3 integrins. First, how does von Willebrand factor binding to platelet GP Ib-IX-V, a non-integrin receptor, lead to activation of aIIbb3? We hypothesize that it is the lateral clustering of GP Ib-IX-V complexes and associated signaling molecules that triggers activation of aIIbb3. To test this, GP IX will be fused at its cytoplasmic tail to FKBP repeats, which can be clustered by cell-permeable chemical dimerizers. After co-expression with other members of the GP Ib-IX-V complex in CHO cells and in platelets of transgenic mice, the effects of conditional clustering of GP Ib-IX-V on aIIbb3 affinity and avidity will be assessed, and the signaling molecules involved will be identified. Second, how do the cytoskeletal proteins, VASP and actin, regulate ligand binding to aIIbb3? Fibrinogen binding to aIIbb3 is increased in VASP-/- murine platelets. To establish the mechanism, full-length or truncated forms of VASP will be expressed in VASP+/- or VASP-/- megakaryocytes, and the effects on aIIbb3 affinity and avidity will be determined. At low concentrations, compounds that inhibit actin polymerization activate integrins. Therefore, we will determine whether they primarily affect aIIbb3 affinity or avidity and whether they influence VASP regulation of aIIbb3. Third, what is the significance of affinity regulation of aVb3 or the related integrin, aVb5, in endothelial cells? Preliminary studies indicate that growth factors regulate both aVb3 affinity and the selective recruitment of activated aVb3 to lamellipodial edges of endothelial cells. We will now determine whether aVb5 is subject to similar regulation, identify the signaling pathways involved, and establish whether high affinity aVb3 or aVb5 is required for endothelial cell migration and angiogenesis. These studies will lead to a better understanding of the mechanisms and biological consequences of integrin activation in vascular cells.
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