ADHESIVE PROPERTIES OF OSTEOPONTIN - REGULATION BY A NATURALLY-OCCURRING THROMBIN-CLEAVAGE IN CLOSE PROXIMITY TO THE GRGDS CELL-BINDING DOMAIN

Osteopontin (OPN) is a secreted adhesive glycoprotein with a functiona l glycine-arginine-glycine-aspartate-serine (GRGDS) cell-binding domai n. An interesting feature of OPN structure is the presence of a thromb in-cleavage site in close proximity to the GRGDS region. Cleavage of O PN by thrombin is likely to be of physiological importance, because cl eavage of blood plasma OPN occurs naturally after activation of the bl ood coagulation pathway. To investigate functional consequences of OPN cleavage by thrombin, cell attachment and spreading assays were perfo rmed with uncleaved and cleaved forms of OPN. For all cell lines exami ned, thrombin-cleaved OPN promoted markedly greater cell attachment an d spreading than uncleaved OPN. Cell attachment and spreading on throm bin-cleaved OPN was inhibited both by the soluble GRGDS peptides and a n OPN-specific antibody raised to the GRGDS domain of OPN, thus implic ating the GRGDS region in mediating the increased cell attachment and spreading observed on thrombin-cleaved OPN. Because the GRGDS sequence in OPN is only six residues from the thrombin-cleavage site, the data suggest the possibility that thrombin cleavage allows greater accessi bility of the GRGDS domain to cell surface receptors. To investigate r eceptors that recognize uncleaved and thrombin-cleaved OPN, affinity c hromatography was performed on placental extracts; the cell surface in tegrin alpha(v) beta(3) bound to columns constructed either with nativ e or thrombin-cleaved OPN and was selectively eluted from each with so luble GRGDS peptide and EDTA. Moreover, adhesion assays performed in t he presence of alpha(v) beta(3) blocking monoclonal antibody LM609 ide ntified alpha(v) beta(3) as a major functional receptor for thrombin-c leaved OPN. Several lines of evidence suggest that cleavage of OPN by thrombin occurs in vivo, such as in tumors and at sites of tissue inju ry, and adhesion assay data presented here indicate that such cleavage is important in the regulation of OPN function.