THE CARBOXYL-TERMINUS OF BRADYKININ AND AMINO-TERMINUS OF THE LIGHT-CHAIN OF KININOGENS COMPRISE AN ENDOTHELIAL-CELL BINDING DOMAIN

Bradykinin (BK), a potent vasoactive nonapeptide formed by proteolytic cleavage of kininogen, mediates its activity by binding to specific c ell surface receptors, Delivery of BK to these receptors is limited by cell-bound and plasma kininases that degrade BR to inactive fragments , Binding of its parent compound, kininogen, to specific endothelial c ell receptors may provide an environment in which the degradation of B H by kininases is restricted, The determinants that mediate kininogen binding to endothelial cells have not been fully elucidated, The prese nt studies suggest that part of BK and the amino-terminal amino acids of kininogens' common light chain constitute part of this recognition sequence, Human umbilical vein endothelial cells (HUVEC) in culture at 37 degrees C express 2-3-fold more binding sites for biotinylated hig h molecular weight kininogen (biotin-HK) containing BK than for biotin -HK from which BH: has been liberated by plasma kallikrein, Binding of BK-free biotin-HK was not restored by preincubating HUVEC with BR, ar guing against the possibility that BK released from biotin-HB: stimula ted expression of additional HH receptors, The extent of biotin-HH bin ding to HUVEC at 37 degrees C directly correlated with the amount of B K retained within the protein, Four Lines of evidence suggest that par t of BK and the amino terminus of the light chain of kininogens are pa rt of the sequence recognized by the endothelial cell kininogen recept or, First, monoclonal antibodies to the carboxyl terminus of BK (MBK3) and the common light chains of the kininogens (HKL6, HKL8) inhibited biotin-HK binding to HUVEC, Second, a synthetic, dimeric bradykinin re ceptor antagonist blocked biotin-HK (IC50 = 9 mu M) binding to HUVEC a s did two synthetic tissue kallikrein inhibitors modeled after the car boxyl-terminal sequence of BK. Third, synthetic peptides containing th e carboxyl-terminal portion of BH and the amino terminus of kininogens ' common light chain, MKRPPGFSPFRSSRIG and GFSPFRSSRIG, blocked bindin g of biotin-HK (IC50 = 2-3 mM), whereas an overlapping peptide, SPFRSS RIGEIKEETT, at 5 mM and a scrambled peptide, FSGPKRSPIMGRPSFR, did not . Fourth, biotinylated GMKRPPGFSPFRSSRIG specifically bound to HUVEC, and its binding was blocked by HK. Since the presence of the nonapepti de BE in HK contributes to maximal binding of HK to HUVEC, there is a novel type of BK receptor in which part of the nonapeptide is recogniz ed within the context of its parent molecule, Optimal binding of HK to HUVEC under physiologic conditions requires contact between the kinin ogen receptor and three regions on HK: domain 3 and the light chain of HK and BK. The presentation of HH in its putative receptor may be imp ortant to protect the nonapeptide from kininases thereby modulating it s bioavailability at sites of inflammation.