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High molecular weight kininogen (HMWK) functions as a cofactor for activation of plasma serine zymogens and as an inhibitor of tissue cysteine proteases. Cell surfaces to which HMWK binds may provide sites for regulation of these systems. Localization of these HMWK-dependent processes at sites of vascular injury may depend on its binding to specific receptors on endothelial cells. In culture, passaged human umbilical vein endothelial cells (HUVEC) bind anti-HMWK antibody to the cell surface and contain 171 +/- 75 ng of HMWK/10(8) cells. [35S]Methionine-labeled HUVEC in culture synthesize a 120-kDa protein immunoisolated using an anti-kininogen antibody, and a 3500-nucleotide message for human HMWK was detected by Northern blot in RNA extracted from HUVEC. HUVEC also express unoccupied binding sites for HMWK on their surface. 125I-HMWK specifically binds to HUVEC in a reaction requiring Zn2+. 125I-HMWK binding to HUVEC is saturable at 4 degrees C but not at 23 degrees C. 125I-HMWK binds to HUVEC with equal affinity as unlabeled HMWK. Kallikrein, factor XII, fibrinogen, fibronectin, and thrombin do not inhibit 125I-HMWK binding to HUVEC. 125I-HMWK-HUVEC binding remains fully reversible at 60 min following the addition of a 50-fold molar excess HMWK. HUVEC express 9.3 +/- 2.0 X 10(5) (mean +/- S.E.) HMWK binding sites/cell (Kd = 52 +/- 13 nM). Both added and cell-bound 125I-HMWK migrate at 120 kDa on sodium dodecyl sulfate gel electrophoresis, suggesting that the protein remains uncleaved upon binding to the HUVEC surface. These studies indicate that HUVEC synthesize HMWK and the HUVEC surface has a site for its expression. By synthesizing and localizing HMWK to the cell surface, endothelial cells may contribute to the activation of plasma's contact serine zymogens and regulation of tissue cysteine proteases.

The purpose of the present study was to confirm expression of kininogen in human kidneys. As the human kininogen gene is alternatively transcribed to high molecular weight and low molecular weight kininogen messenger (m)RNA, we constructed a probe which can discern these two transcripts by the S1 nuclease mapping analysis. The low molecular weight kininogen mRNA was detected in human kidney cortex and medulla. This finding strongly supports the existence of a local kinin-kallikrein system in human kidneys.

In order to investigate the structures and expressions of the angiotensinogen and kininogen genes, we have isolated cDNA and genomic clones and characterized their structures. The following results were obtained. (a) The primary structures of rat and human angiotensinogens and bovine, human, and rat kininogens were determined, and they exhibit characteristic features. (b) Angiotensinogen and alpha 1-antitrypsin are related in not only protein structures but also gene organizations. (c) Angiotensinogen mRNA is synthesized not only in the liver but also in the brain, kidney, lung, adrenal gland, and ovary. (d) Angiotensinogen mRNA markedly increased in the liver but not in the brain after induction of acute inflammation. (e) Low molecular weight (LMW) and high molecular weight (HMW) kininogen mRNAs are generated from a single gene by alternative RNA processing events. (f) At least four types of kininogen mRNAs exist in rat liver. Two of them are LMW and HMW kininogen mRNAs, while the other two encode additional forms of kininogens (T-kininogens). (g) Two T-kininogen mRNAs but not LMW and HMW kininogen mRNAs dramatically increased after induction of acute inflammation, and T-kininogen turned out to be identical to alpha 1-major acute phase protein. (h) Kininogens have multifunctional domains; an amino-terminal domain for cystein proteinase inhibitor, a bradykinin moiety, and in the case of HMW kininogen a carboxyl-terminal domain for a cofactor of blood coagulation.

cDNA sequences for both human high molecular weight (HMW) and low molecular weight (LMW) prekininogens have been isolated by molecular cloning and determined by sequence analysis. The sequence determination together with the S1 nuclease mapping and RNA blot-hybridization analyses indicate that human HMW and LMW prekininogen mRNAs share an identical sequence throughout the 5'-untranslated region and the protein-coding region up to the sequence encoding the 12 amino acids distal to the bradykinin sequence, and the two mRNAs then completely diverge from each other. The signal peptide, the heavy chain (H chain), and the bradykinin moiety, which are common between the two prekininogens, consist of 18, 362, and 9 amino acids, respectively, while the light chains (L chains) of the HMW and LMW prekininogens are composed of 255 and 38 amino acids, respectively. All 17 cysteine residues present in the human and bovine H chains are located at exactly equivalent positions, indicating that the human H chain, like the bovine counterpart, can form 8 loop structures, each connected by two adjacent cysteine residues. The L chains of human and bovine kininogens differ in the protein lengths as well as in some amino acids crucial for the processing of the kininogens by kallikrein. Based upon this finding, we have discussed the molecular basis for the different modes of processing of human and bovine HMW kininogens and for the different kinetics of contact activation reactions exhibited by the two HMW kininogens.

The primary structures of the angiotensinogen precursor and the low molecular weight (LMW) kininogen precursors have been deduced by determining the nucleotide sequences of cloned DNAs complementary to their mRNAs. The angiotensinogen precursor consists of a mature angiotensinogen of 453 amino acid residues and a putative signal peptide of 24 amino acid residues. An angiotensin moiety is located at the amino-terminal part of angiotensinogen, preceded directly by the signal peptide and followed by a large carboxyl-terminal sequence that contains two internally homologous sequences and three potential glycosylation sites. The LMW kininogen precursors are encoded by two very similar but distinct mRNAs and composed of 436 and 434 amino acid residues. Both kininogens contain two internally homologous sequences in which all amino acid differences between the two kininogens are located. This suggests that these homologous regions may be biologically significant in relation to the existence of two LMW kininogens.

This article reviews most recent developments in the field of the multifunctional kininogens which are involved in major physiologic systems such as the blood coagulation cascade, the inflammatory reaction, the inhibitor defense mechanism, and the acute phase response. Particular attention is given to the interaction of kininogens with endothelial cells and platelets, the post-translational modification of kinin(ogen)s by proline hydroxylase, and the placing of kininogens among structurally and functionally related plasma proteins.

The complete disulfide loop structure of human alpha 2-HS glycoprotein has been elucidated. alpha 2-HS glycoprotein isolated from human plasma was found to be a two-chain protein composed of a heavy and a light chain. The heavy chain comprises the A-chain of alpha 2-HS glycoprotein (Yoshioka, Y., Gejyo, F., Marti, T., Rickli, E. E., Bürgi, W., Offner, G. D., Troxler, R. F., and Schmid, K. (1986) J. Biol. Chem. 261, 1665-1676) and part of the connecting peptide which has been predicted from the corresponding cDNA sequence (Lee, C. C., Bowman, B. H., and Yang, F. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 4403-4407), whereas the light chain corresponds to the beta-chain of alpha 2-HS glycoprotein (Gejyo, F., Chang, J. L., Bürgi, W., Schmid, K., Offner, G. D., Troxler, R. F., Van Halbeek, H., Dorland, L., Gerwig, G. J., Vliegenthart, J. F. G. (1983) J. Biol. Chem. 258, 4966-4971). Twelve half-cystine residues are present in the alpha 2-HS glycoprotein molecule, and 11 of them are positioned in the heavy chain and a single one in the light chain of the molecule; they form six disulfide bridges. The first and the last half-cystine residues of the amino acid sequence of alpha 2-HS glycoprotein are engaged in the formation of a loop spanning the extreme NH2- and COOH-terminal portions of the molecule, thereby connecting the heavy and light chains. The other 10 half-cystines residues are linked consecutively in the heavy chain and form five loops which span 4-19 amino acid residues. Among them are two pairs of loops which are characterized by mutual sequence homology. The particular arrangement of disulfide loops in alpha 2-HS glycoprotein is similar to the patterns of linearly arranged and tandemly repeated disulfide loops of cysteine proteinase inhibitors, i.e. the cystatins and the kininogens. It is concluded that alpha 2-HS glycoprotein represents a structural prototype of a novel family among the cystatin superfamily, characterized by the presence of two cystatin-like building blocks. Extensive similarity among the NH2-terminal sequences of alpha 2-HS glycoprotein and human histidine-rich glycoprotein suggest that the latter protein is another candidate protein of this new family.