MAPPING THE CELL-BINDING SITE ON HIGH-MOLECULAR-WEIGHT KININOGEN DOMAIN-5

Investigations mapped the region(s) on the light chain of high molecul ar weight kininogen (HK) that participates in cell binding. Sequential and overlapping peptides of domain 5 (D5(H)) were synthesized to dete rmine its cell binding site(s). Three peptides from non-overlapping re gions on D5(H) were found to inhibit biotin-HK binding to endothelial cells. Peptides GKE19 and HNL21 weakly inhibited biotin-HK binding wit h IC50 of 792 and 215 mu M, respectively. Peptide HKH20 inhibited biot in-HK binding with an IC50 of 0.2 mu M. Two peptides, GGH18 and HVL24, which overlapped HKH20, also inhibited biotin HK binding to endotheli al cells with IC50 values of 108 and 0.8 mu M, respectively. Biotinyla ted HKH20 directly bound to endothelial cells. HK and HKH20 bound at o r near the same site on endothelial cells because HK inhibited biotin- HKH20 binding (IC50 0.2 mu M). A polyclonal anti-HKH20 antibody also b locked biotin-HK binding. Peptides HKH20 and HVL24 and anti-HKH20 anti body also inhibited the procoagulant activity of plasma HK. These data indicated that the cell and artificial surface binding sites on D5(H) overlap. The orientation of HK on endothelial cells may be critical f or the assembly and activation of contact system enzymes and the expre ssion of kininogen's anti-thrombin activity.