Kininogens, the precursor proteins of the vasoactive kinins, bind specifically, reversibly, and saturably to platelets, neutrophils, and endothelial cells. Two domains of the kininogens expose major cell binding sites: domain D3 that is shared by H- and L-kininogen and domain D5(H) that is exclusively present in H-kininogen. Previously we have mapped the kininogen cell binding sites to 27 residues of D3 (''LDC27'') and 20 residues of D5(H) (''HKH20''), respectively (Herwald, H., Hasan, A. A. K., Godovae-Zimmermann, J., Schmaier, A. H., and Muller-Esterl, W. (1995) J. Biol. Chem. 270, 14634-14642; Hasan, A. A. K., Cines, D. B., Herwald, H., Schmaier, A. H., and Muller-Esterl, W. (1995) J. Biol. Chen. 270, 19256-19261). The corresponding kininogen acceptor site(s) exposed by the cell surfaces are still poorly defined. Using a non-ionic detergent, Nonidet P-40, we have been able to solubilize kininogen binding sites from an endothelial cell line, EA.hy926, in their functionally active form. Affinity chromatography of the solubilized kininogen binding sites on HKH20, a synthetic peptide representing the D5(H) cell binding site, allowed us to isolate a 33-kDa protein (''p33'') that binds specifically and reversibly to H-kininogen with a K-D (apparent dissociation constant) of 9 +/- 2 nM. Preparative SDS electrophoresis followed by NH2-terminal amino acid sequence analysis identified the kininogen-binding protein p33 as the gC1q receptor (''gC1qR''), an extrinsic membrane protein that interacts with the globular domains of the complement component C1q. The purified p33 binds C1q with moderate affinity, K-D = 240 +/- 10 nM. Recombinant expression of the corresponding cDNA in Escherichia coli demonstrated that p33 binds H-kininogen, but not L-kininogen. Peptide HKH20 but not peptide LDC27 inhibited binding of H-kininogen to the recombinant p33 in a concentration-dependent manner, indicating that H-kininogen binds to p33 via domain D5(H). Recombinant p33 efficiently inhibited the binding of H-kininogen to EA.hy926 cells. Factor XII, but not prekallikrein, competed with H-kininogen binding to p33. These findings suggest that an endothelial binding protein mediates the assembly of critical components of the kinin-generating pathway on the surface of endothelial cells, thereby linking the early events of kinin formation and complement activation.
