OPPOSING EFFECTS OF LOW AND HIGH-MOLECULAR-WEIGHT KININOGENS ON CELL-ADHESION

High molecular weight kininogen (HK) blocks cell spreading but not cel l attachment to surfaces coated with vitronectin and other ligands of beta 3 integrins, We sought to learn the structural basis of this phen omenon. Monoclonal antibodies against the histidine-rich D5 domain in the light chain of a-chain HK abolished the inhibitory effect of a-cha in HK on spreading of MG-63 osteosarcoma cells on vitronectin-coated t issue-culture plastic. The antibodies were effective only if incubated with a-chain HK in solution and did not abolish the anti-cell-spreadi ng effect of a-chain HK that was pre-adsorbed to tissue-culture plasti c. Exposure of an epitope in the histidine-rich domain was less when H K was adsorbed to tissue-culture plastic (oxidized polystyrene) than w hen it was adsorbed to ELISA plastic (untreated polystyrene), Loss of the epitope correlated with increased anti-cell-spreading activity of HK on tissue-culture plastic. The light chain of 2-chain HK containing D5 and that containing recombinant D5 both had anti-cell-spreading ac tivity, but only when present in solution during adhesion assays. Pre- adsorption of recombinant D5 to tissue-culture plastic resulted in a s urface on which adsorbed a-chain HK had little anti-cell-spreading act ivity. Binding study revealed that HKa bound to immobilized vitronecti n, The histidine-rich D5 domain of light chain of HK was identified as one of the binding sites of vitronectin, suggesting that the masking of the RGD cell-binding site of immobilized vitronectin is the molecul ar mechanism of anti-cell-spreading effect of HKa. In contrast, low mo lecular weight kininogen (LK), which lacks D5, augmented cell spreadin g on vitronectin-coated tissue-culture plastic, Thus, HK and LK have o pposing effects on VN-dependent cell adhesion. The augmenting effect o f LK was greater if LK was preincubated with cells or adsorbed to the surface at pH>7.0. Analysis of fragments of LK and antibody inhibition studies localized the cell-adhesion activity to the D3 domain that is common to LK and HK, These findings indicate that the D5 domain media tes the adsorption of HK or 2-chain HK to vitronectin substratum in an ti-adhesive conformations, i.e., masking of the RGD cell-binding site of vitronectin. Such conformers inhibit cell spreading on vitronectin even though a cell-adhesion site is present in D3.