Human high molecular weight kininogen. Studies of structure-function relationships and of proteolysis of the molecule occurring during contact activation of plasma.

Human high Mr kininogen was purified from normal plasma in 35% yield. The purified high Mr kininogen appeared homogeneous on polyacrylamide gels in the presence of sodium dodecyl sulfate and mercaptoethanol and gave a single protein band with an apparent Mr = 110,000. Using sedimentation equilibrium techniques, the observed Mr was 108,000 +/- 2,000. Human plasma kallikrein cleaves high Mr kininogen to liberate kinin and give a kinin-free, two-chain, disulfide-linked molecule containing a heavy chain of apparent Mr = 65,000 and a light chain of apparent Mr = 44,000. The light chain is histidine-rich and exhibits a high affinity for negatively charged materials. The isolated alkylated light chain quantitatively retains the procoagulant activity of the single-chain parent molecule. 125I-Human high Mr kininogen undergoes cleavage in plasma during contact activation initiated by addition of kaolin. This cleavage, which liberates kinin and gives a two-chain, disulfide-linked molecule, is dependent upon the presence of prekallikrein and Factor XII (Hageman factor) in plasma. Addition of purified plasma kallikrein to normal plasma or to plasmas deficient in prekallikrein or Factor XII in the presence or absence of kaolin results in cleavage of high Mr kininogen and kinin formation.     

Rat plasma high-molecular-weight kininogen. A simple method for purification and its characterization

High-molecular-weight kininogen has been isolated from rat plasma in three steps in a relatively high yield. The purified preparation gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the absence and presence of 2-mercaptoethanol, and the apparent Mr was estimated as 100,000. On incubation with rat plasma kallikrein, rat high Mr kininogen yielded a kinin-free protein consisting of a heavy chain (Mr = 64,000) and a light chain (Mr = 46,000), liberating bradykinin. The kinin-free protein was S-alkylated, and its heavy and light chains were separated by a zinc-chelating Sepharose 6B column. The amino acid compositions of rat high Mr kininogen and its heavy and light chains were very similar to those of bovine high Mr kininogen and its heavy and fragment 1.2-light chains, respectively. A high histidine content in the light chain of rat high Mr kininogen indicated the presence of a histidine-rich region in this protein as in bovine high Mr kininogen, although this region was not cleaved by rat plasma kallikrein. Rat high Mr kininogen corrected to normal values the prolonged activated partial thromboplastin time of Brown-Norway Katholiek rat plasma known to be deficient in high Mr kininogen and of Fitzgerald trait plasma. The kinin-free protein had the same correcting activity as intact high Mr kininogen. Rat high Mr kininogen also accelerated approximately 10-fold the surface-dependent activation of rat factor XII and prekallikrein, which was mediated with kaolin, amylose sulfate, and sulfatide. These results indicate that rat high Mr kininogen is quite similar to human and bovine high Mr kininogens in terms of biochemical and functional properties.     

Interaction of human plasma kallikrein and its light chain with alpha 2-macroglobulin

Human plasma kallikrein participates in the contact activation system of plasma. The light chain of kallikrein contains the enzymatic active site; the heavy chain is required for binding to high molecular weight kininogen and for surface-dependent activation of coagulation. This study has examined the functional contributions of the heavy chain of kallikrein and of high molecular weight kininogen in the inactivation of kallikrein and of its isolated light chain by alpha 2-macroglobulin (alpha 2M). Irreversible inhibition was observed for both kallikrein and its light chain, with the initial formation of a reversible enzyme-inhibitor complex. The second-order rate constants for these reactions were 3.5 X 10(5) and 4.8 X 10(5) M-1 min-1 for kallikrein and its light chain, respectively. When present in excess, high molecular weight kininogen decreased the rate of kallikrein inactivation by alpha 2M, whereas the rate of inactivation of the light chain was unaffected by high molecular weight kininogen. Although at a drastically reduced rate, high molecular weight kininogen was cleaved by alpha 2M-bound kallikrein. Sodium dodecyl sulfate gradient polyacrylamide gel electrophoresis was used to study complex formation between alpha 2M and kallikrein or its light chain. Under reducing conditions, four kallikrein-alpha 2M complexes were observed. Three of these complexes consisted of alpha 2M and the light chain of kallikrein (Mr 123 000, 235 000, and 330 000). Two alpha 2M-kallikrein light chain complexes incorporated [3H]diisopropyl fluorophosphate ( [3H]DFP) whereas the Mr 330 000 complex did not react with [3H]DFP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biochemical characterization and substrate specificity of rat prostate kallikrein (S3): comparison with tissue kallikrein, tonin and T-kininogenase.

A tissue kallikrein-like enzyme encoded by S3 mRNA was purified to homogeneity from rat prostate gland. The apparent molecular mass of the prostate enzyme is 32 kDa as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The intact 32 kDa enzyme is split into two bands of lower molecular mass, 18 and 14 kDa, under reducing conditions on SDS-PAGE. NH2-terminal amino acid sequence analyses of the intact enzyme and heavy and light chains revealed the identity to the translated sequence of a prostate kallikrein cDNA (S3). Isoelectric focusing indicated that the prostate enzyme is a basic protein with pI of 7.30-7.45. Specific activities of the prostate kallikrein toward angiotensin I, angiotensinogen and rat low M(r) kininogen as well as tripeptide chromogenic substrates were compared with those of tissue kallikrein, tonin and T-kininogenase. The kinin-releasing activity is inhibited by leupeptin, antipain, benzamidine and soybean trypsin inhibitor. A sensitive and specific radioimmunoassay for the rat prostate kallikrein shows that the immunoreactive kallikrein levels in prostate and submandibular gland were 23.78 +/- 2.62 micrograms/mg protein (n = 5) and 12.29 +/- 2.25 micrograms/mg protein (n = 5), respectively. The results indicate that the prostate kallikrein S3 is expressed at high levels in both prostate and submandibular glands.     

Studies on human high molecular weight (HMW) kininogen. II. Structural change of HMW kininogen by the action of human plasma kallikrein

We have investigated in detail the cleavage of human high molecular weight (HMW) kininogen by human plasma kallikrein and revealed the formation of a nicked kininogen and a novel kinin-free protein (KFP) as intermediate cleavage products. The cleavage of a single chain HMW kininogen (Mr=120,000) by plasma kallikrein was a three-step reaction. The first cleavage yielded a nicked kininogen composed of two disulfide-linked 62,000 and 56,000 daltons chains. The second cleavage yielded kinin and an intermediate kinin-free protein, KFP-I, which was apparently of equal size to the nicked kininogen. The third cleavage yielded a stable kinin-free protein, KFP-II, composed of two disulfide-linked 62,000 and 45,000 daltons chains. The liberation of an 8,000 daltons fragment was identified when the 56,000 daltons chain isolated by SP-Sephadex C-50 chromatography of reduced and alkylated KFP-I was cleaved by plasma kallikrein into the 45,000 daltons chain. Although the antiserum against HMW kininogen cross-reacted with low molecular weight (LMW) kininogen, the antiserum against the 45,000 daltons chain was specific for HMW kininogen. These results suggest that the antigenic determinant groups common to HMW and LMW kininogens are located in the 62,000 daltons heavy chain, while those specific for HMW kininogen are located in the 45,000 daltons light chain, which is known to retain blood coagulation activity.     

Single-chain, low molecular weight kininogen in the blood plasma of rabbits: isolation and fragmentation by porcine pancreatic kallikrein

A highly purified preparation of low molecular weight kininogen (LMrK) was isolated from the plasminogen-free rabbit blood plasma, using chromatography on DEAE-Sepharose CL-6B, gel filtration on Ultrogel AcA 34 and Sephadex G-100 as well as gradient chromatography on a hydroxylapatite column. The yield of the 320-fold purified LMrK was 16%. Trypsin released 13-14 micrograms-eq. of bradykinin (BK) from 1 mg of LMrK or 0.85-0,95 mol of BK per mol of kininogen. Rabbit LMrK consists of one polypeptide chain of Mr 69 000 and pI 4.63. Porcine pancreatic kallikrein splits off kinin from the LMrK polypeptide chain by disrupting two peptide bonds resulting in the formation of S-S-bound two chain molecule. After reduction of the S-S bonds by dithioerithritol the latter is separated into a heavy (Mr 61 000) and light (Mr 6 800) chains. A biologically active peptide was isolated from the products of CNBr cleavage of LMrK. This peptide consists of Lys-BK elongated from the C-terminal with several amino acid residues. Rabbit LMrK closely resembles human LMrK in terms of Mr, pI and location of the kinin fragment in the protein molecule.     

Interaction of human plasma kallikrein and its light chain with C1 inhibitor

The light chain of human plasma kallikrein contains the enzymatic active site. The inactivation of kallikrein and of its isolated light chain by C1 inhibitor was investigated to assess the functional contributions of the heavy-chain region of kallikrein and of high molecular weight kininogen to this reaction. The second-order rate constants for the inactivation of kallikrein or its light chain were respectively 2.7 X 10(6) and 4.0 X 10(6) M -1 min -1. High molecular weight kininogen did not influence the rate of kallikrein inactivation. The nature of the complexes formed between kallikrein or its light chain and C1 inhibitor was studied by using sodium dodecyl sulfate (SDS) gradient polyacrylamide slab gel electrophoresis. Kallikrein as well as its light chain combined with C1 inhibitor to form stable stoichiometric complexes that were not dissociated by SDS and that exhibited apparent molecular weights (Mr's) of 185 000 and 135 000, respectively, on nonreduced SDS gels. Reduction of the kallikrein-C1 inhibitor complex gave a band at Mr 135 000 that comigrated with the complex seen for the light chain-C1 inhibitor complex. During the inactivation of both kallikrein and its light chain, a Mr 94 000 fragment of C1 inhibitor was formed which was unable to inactivate or bind kallikrein or its light chain. Kallikrein inactivated by diisopropyl phosphofluoridate did not form SDS-stable complexes with C1 inhibitor. These results demonstrate that the functional binding site for C1 inhibitor is localized in the light chain of kallikrein.(ABSTRACT TRUNCATED AT 250 WORDS)     

High-molecular-weight kininogen from horse plasma. Isolation, characterization and comparison with bovine high-Mr kininogen

High-molecular-weight (high-Mr) kininogen was purified from horse plasma by chromatography on columns of DEAE-Sephadex A-50, CM-Sephadex C-50, p-chlorobenzylamine-Sepharose and Sephadex G-150. The yield was about 150 mg from 81 of fresh plasma. The purified material gave a single band on sodium dodecylsulfate/polyacrylamide gel electrophoresis and a single precipitin line on immunodiffusion and immunoelectrophoresis. The molecular weight of horse high-Mr kininogen was estimated to be 78000 by dodecylsulfate gel electrophoresis using the Ferguson plot. Its polypeptide content was determined to be 86% by amino acid analysis and there was a total of 581 amino acid residues/molecule of protein. The kininogen contained a total of 13.9% carbohydrates, consisting of hexoses (7.8%), glucosamine (1.9%), galactosamine (0.6%) and sialic acid (3.6%). On incubation of horse high-Mr kininogen with bovine and horse plasma kallikreins, several fragments which contained extremely high levels of histidine, were liberated, in addition to kinin. After the liberation of kinin and histidine-rich fragments, a protein free of kinin and its fragments was isolated. This protein consisted of two polypeptide chains, heavy chain and light chain, which are bridged by disulfide bonds. The molecular weight and amino acid composition of the heavy chain and the light chain from horse high-Mr kininogen were very similar to those of the heavy and light chains from bovine high-Mr kininogen, respectively. From these results, it was revealed that horse high-Mr kininogen is quite similar to bovine high-Mr kininogen in terms of their physicochemical and chemical properties, although they are immunologically distinguishable.     

Proteinase-sensitive regions in the heavy chain of low molecular weight kininogen map to the inter-domain junctions

Low molecular weight kininogen from human plasma was subjected to limited proteolysis with trypsin, chymotrypsin, elastase, and bromelain, and the resulting fragments of 20,000 or 40,000 Da were isolated. Amino-terminal sequence analysis of the fragments disclosed for the various proteinases eight independent cleavage sites distinct from the typical kallikrein cleavage sites flanking the kinin region. All the identified cleavage sites cluster in two stretches of 11-12 residues of the kininogen heavy chain. These short segments represent the primary attack sites for proteinases ("proteinase-sensitive regions") in the heavy chain portion of human low molecular weight kininogen. The amino acid sequences of the two proteinase-sensitive regions are mutually homologous; they are further characterized by the presence of a single copy each of the consensus tetrapeptide Cys-X-Gly-Cys known to form a narrow disulfide loop (Kellermann, J., Thelen, C., Lottspeich, F., Henschen, A., Vogel, R., and Müller-Esterl, W. (1987) Biochem. J. 247, 15-21). The proteinase-sensitive regions are located at the junctions of the three cystatin-like domains constituting the kininogen heavy chain. Proteolytic cleavage at the sensitive regions dissects the kininogen heavy chain and releases single domains of 20,000 Da and combined domains of 40,000 Da which can function as cysteine proteinase inhibitors. The presence of kininogen heavy chain domains in plasma samples under pathologic conditions suggests that cleavage of the proteinase-sensitive regions might also occur in vivo.     

The role of bovine high-molecular-weight (HMW) kininogen in contact-mediated activation of bovine factor XII: interaction of HMW kininogen with kaolin and plasma prekallikrein

Previous studies from our laboratories (Sugo et al. (1980) Biochemistry 19, 3215-3220) have shown that bovine high-molecular-weight (HMW) kininogen remarkably accelerates the kaolin-mediated activation of Factor XII in the presence of prekallikrein, and that both fragment 1.2 and the light chain regions located in the COOH terminal half of the kininogen molecule are essential for the activation. In the present study, we demonstrate that the accelerating effect of HMW kininogen is mediated through its adsorption on the kaolin surface through the fragment 1.2 region and its complex formation with prekallikrein through the light chain region. The evidence is as follows: 1. HMW kininogen radio-labeled with 125I was adsorbed on kaolin and the adsorption was inhibited by the prior treatment of kaolin with fragment 1.2, fragment 1.2-light chain, kinin-free protein or HMW kininogen, but not with kinin- and fragment 1.2-free protein, light chain or low molecular-weight (LMW) kininogen. 2. The complex formation of HMW kininogen with prekallikrein in bovine plasma or in the purified system was examined by gel-filtration on a column of Sephacryl S-200 In bovine plasma, prekallikrein was eluted in the same fraction as HMW kininogen, showing an apparent molecular weight of 250,000, whereas purified prekallikrein was eluted in the fraction corresponding to an apparent molecular weight of 100,000. When purified prekallikrein was mixed with purified HMW kininogen in a mol ratio of 1 to 2, all prekallikrein was found to be associated with HMW kininogen. Furthermore, purified prekallikrein mixed with kininogen derivatives, such as kinin- and fragment 1.2-free protein, fragment 1.2-light chain or light chain, was eluted in the higher molecular weight fraction. HMW kininogen did not form a complex with prekallikrein. Using the same technique, it was shown that kinin- and fragment 1.2-free protein forms a complex not only with prekallikrein but also with kallikrein.     

Isolation and characterization of ornitho-kininogen

Ornitho-kininogen was purified from chicken blood plasma by a two-stage method using chromatography on columns of S-alkylated papain-Cellulofine and DEAE-5PW. The yield was 1.7 mg from 44 ml plasma. The isolated preparation gave a single band on sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) with or without 2-mercaptoethanol and on disc/polyacrylamide gel electrophoresis. The relative molecular mass, Mr, of ornitho-kininogen was estimated as 74,000 on SDS-PAGE using the Ferguson plot method. Ornitho-kininogen was found to have the similar properties to those of mammalian high-Mr kininogen, in terms of the amino acid composition, molecular mass, and susceptibility to plasma kallikrein. No kininogen corresponding to mammalian low-Mr kininogen and rat T-kininogen could be detected in chicken plasma. In fact, ornitho-kininogen was degraded rapidly by bovine plasma kallikrein, liberating a kinin. This kinin was isolated from the digest by reversed-phase HPLC. The primary structure of the isolated kinin was determined as Arg1-Pro2-Pro3-Gly4-Phe5-Thr6-Pro7-Leu8-Arg9. The sequence of this peptide, named ornitho-kinin, was similar to that of bradykinin except for the substitution of Thr6 and Leu8 for Ser6 and Phe8. The isolated ornitho-kinin induced a contraction of chicken smooth muscle and had a strong hypotensive effect in the chicken. However, it did not contract the isolated rat uterus. It is suggested that this specificity difference is due to the replacement of Phe8 by Leu8. The sequence of residues 1-30 of ornitho-kininogen exhibited 43% identity with that of bovine kininogen.     

Hypothesis. The molecular 'double-pivot' mechanism for water oxidation

High-molecular-weight (HMW) kininogen was purified from guinea-pig plasma by measuring its ability to correct the prolonged clotting time in human HMW kininogen deficient plasma (Fitzgerald trait). The purified HMW kininogen demonstrated a homogeneous band in disc gel electrophoresis in the presence of sodium dodecyl sulfate under reducing or non-reducing conditions with an apparent molecular weight of 100,000. Kinin released from HMW kininogen by treatment with guinea-pig plasma kallikrein was identified as bradykinin by reverse-phase HPLC and amino-acid analysis. The capacity of HMW kininogen as a thiol-proteinase inhibitor was realized by its dose-dependent inhibitory activity to papain. The Ki value for papain was estimated to be 42 pM. The kinin-free HMW kininogen maintained the inhibitor and clotting-factor activities with similar capacities to those of the HMW kininogen molecule. Heavy chain (H-chain) and light chain (L-chain) of HMW kininogen were prepared from reduced and alkylated kinin-free HMW kininogen by HPLC. The S-alkylated H-chain, but not L-chain, demonstrated the inhibitor activity with the Ki value 6.9 nM for papain, whereas the S-alkylated L-chain, but not H-chain, maintained the clotting activity one-third of the capacity of HMW kininogen. Specific antibodies recognized HMW kininogen, but also a probable low-molecular-weight kininogen(s) with an apparent molecular weight of 60,000 in the guinea-pig plasma. All of these properties are consistent with the reports on human, bovine and rat HMW kininogen.     

Protein-protein interactions in contact activation of blood coagulation. Binding of high molecular weight kininogen and the 5-(iodoacetamido) fluorescein-labeled kininogen light chain to prekallikrein, kallikrein, and the separated kallikrein heavy and light chains

Binding of the 5-(iodoacetamido)fluorescein (IAF)-labeled high molecular weight (HMW) kininogen light chain to prekallikrein and D-Phe-Phe-Arg-CH2Cl-inactivated kallikrein was monitored by a 0.040 +/- 0.002 increase in fluorescence anisotropy. Indistinguishable average dissociation constants and stoichiometries of 14 +/- 3 nM and 1.1 +/- 0.1 mol of prekallikrein/mol of IAF-light chain and 17 +/- 3 nM and 0.9 +/- 0.1 mol of kallikrein/mol of IAF-light chain were determined for these interactions at pH 7.4, mu 0.14 and 22 degrees C. Prekallikrein which had been reduced and alkylated in 6 M guanidine HCl lost the ability to increase the fluorescence anisotropy of the IAF-kininogen light chain, suggesting that the native tertiary structure was required for tight binding. The kallikrein heavy and light chains were separated on the basis of the affinity of the heavy chain for HMW-kininogen-Sepharose, after mild reduction and alkylation of kallikrein under nondenaturing conditions. Under these conditions, alkylation with iodo [14C]acetamide demonstrated that only limited chemical modification had occurred. Binding of the IAF-kininogen light chain to the isolated alkylated kallikrein heavy chain, when compared to prekallikrein and kallikrein, was characterized by an indistinguishable increase in fluorescence anisotropy, average dissociation constant of 14 +/- 3 nM, and stoichiometry of 1.2 +/- 0.1 mol of kallikrein heavy chain/mol of IAF-light chain. In contrast, no binding of the D-Phe-Phe-Arg-CH2Cl-inactivated kallikrein light chain was detected at concentrations up to 500 nM. Furthermore, 300 nM kallikrein light chain did not affect IAF-kininogen light chain binding to prekallikrein, kallikrein, or the kallikrein heavy chain. The binding of monomeric single chain HMW-kininogen to prekallikrein, kallikrein, and the kallikrein heavy and light chains was studied using the IAF-kininogen light chain as a probe. Analysis of the competitive binding of HMW-kininogen gave average dissociation constants and stoichiometries of 12 +/- 2 nM and 1.2 +/- 0.1 mol of prekallikrein/mol of HMW-kininogen, 15 +/- 2 nM and 1.3 +/- 0.1 mol of kallikrein/mol of HMW-kininogen, 14 +/- 3 nM and 1.4 +/- 0.2 mol of kallikrein heavy chain/mol of HMW-kininogen, and no detectable effect of 300 nM kallikrein light chain on these interactions. We conclude that a specific, nonenzymatic interaction between sites located exclusively on the light chain of HMW-kininogen and the heavy chain of kallikrein or prekallikrein is responsible for the formation of 1:1 noncovalent complexes between these proteins.     

Purification and characterization of alpha 1-thiol proteinase inhibitor and its identity with kinin- and fragment 1.2-free high molecular weight kininogen

1. alpha 1-Thiol proteinase inhibitor (alpha 1 TPI) purified from outdated human plasma was a glycoprotein with Mr 83,000 and was composed of heavy and light chains held together with a disulfide bond. 2. The data on amino acid composition, amino terminal sequence of the light chain and carboxyl terminal sequences of the heavy and light chains indicate that alpha 1 TPI is identical with kinin- and fragment 1.2-free HMW kininogen. 3. Purified human plasmin generated a derivative having the same molecular weight (Mr 83,000), same subunit structure (heavy and light chains) and same inhibitory capacity as alpha 1 TPI from HMW kininogen and kinin-free HMW kininogen. This indicated the possibility that alpha 1 TPI is derived from HMW kininogen by plasmin.     

Localization of distinct functional domains on prekallikrein for interaction with both high molecular weight kininogen and activated factor XII in a 28-kDa fragment (amino acids 141-371).

The predominant autolytic form of human kallikrein, beta-kallikrein, was used to localize the high molecular weight kininogen (HK) binding site on kallikrein as well as the substrate recognition site for activated factor XII on prekallikrein. beta-Kallikrein is formed by autolysis of the kallikrein heavy chain to give two fragments of approximately 18 and 28 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A ligand binding technique established that the HK binding site on kallikrein residues on the 28-kDa fragment of the heavy chain. Limited NH2-terminal sequencing of this portion of beta-kallikrein showed that this fragment of the heavy chain consists of the COOH-terminal 231 amino acids of the heavy chain. A panel of five murine monoclonal antibodies to human prekallikrein (PK) were found to have epitopes on this same fragment of the heavy chain. None of the monoclonal antibodies were able to block binding of HK to PK. Three of the monoclonal antibodies (13G11, 13H11, and 6A6) were able to inhibit the activation of PK to kallikrein in both a plasma system and a purified system. The 28-kDa fragment of the PK heavy chain was purified and was able to compete with HK for binding to PK. The HK binding site and the site of recognition of factor XII are separate and distinct on PK, and both are contained in the COOH-terminal 231 amino acids of the PK heavy chain.     

Role of the light chain of high molecular weight kininogen in adhesion, cell-associated proteolysis and angiogenesis

Cleavage of high molecular weight kininogen (HK) by plasma kallikrein results in a light chain and a heavy chain (HK). The light chain has two domains: D6, which binds (pre)kallikrein, and D5, which binds to anionic surfaces, including heparin as well as zinc. Initially, HK was thought to be important for surface-activated coagulation. HKa or D5 binds to the urokinase receptor on endothelial cells, thereby enhancing the conversion of prourokinase to urokinase by kallikrein, and, thus, cell-associated fibrinolysis. HKa or D5 is antiadhesive by competing with vitronectin binding to the urokinase receptor and/or forming a complex with vitronectin. D5 inhibits endothelial cell migration, proliferation, tube formation and angiogenesis, thus modulating inflammation and neovascularization.     

Human plasma prekallikrein, a zymogen to a serine protease that contains four tandem repeats

The amino acid sequence of human plasma prekallikrein was determined by a combination of automated Edman degradation and cDNA sequencing techniques. Human plasma prekallikrein was fragmented with cyanogen bromide, and 13 homogeneous peptides were isolated and sequenced. Cyanogen bromide peptides containing carbohydrate were further digested with trypsin, and the peptides containing carbohydrate were isolated and sequenced. Five asparagine-linked carbohydrate attachment sites were identified. The sequence determined by Edman degradation was aligned with the amino acid sequence predicted from cDNAs isolated from a lambda gt11 expression library. This library contained cDNA inserts prepared from human liver poly(A) RNA. Analysis of the cDNA indicated that human plasma prekallikrein is synthesized as a precursor with a signal peptide of 19 amino acids. The mature form of the protein that circulates in blood is a single-chain polypeptide of 619 amino acids. Plasma prekallikrein is converted to plasma kallikrein by factor XIIa by the cleavage of an internal Arg-Ile bond. Plasma kallikrein is composed of a heavy chain (371 amino acids) and a light chain (248 amino acids), and these 2 chains are held together by a disulfide bond. The heavy chain of plasma kallikrein originates from the amino-terminal end of the zymogen and is composed of 4 tandem repeats that are 90 or 91 amino acid residues in length. These repeat sequences are also homologous to those in human factor XI. The light chain of plasma kallikrein contains the catalytic portion of the enzyme and is homologous to the trypsin family of serine proteases.     

The hyaluronan-binding serine protease from human plasma cleaves HMW and LMW kininogen and releases bradykinin

The influence of the hyaluronan-binding protease (PHBSP), a plasma enzyme with FVII- and pro-urokinase-activating potency, on components of the contact phase (kallikrein/kinin) system was investigated. No activation or cleavage of the proenzymes involved in the contact phase system was observed. The pro-cofactor high molecular weight kininogen (HK), however, was cleaved in vitro by PHBSP in the absence of any charged surface, releasing the activated cofactor and the vasoactive nonapeptide bradykinin. Glycosoaminoglycans strongly enhanced the reaction. The cleavage was comparable to that of plasma kallikrein, but clearly different from that of coagulation factor FXIa. Upon extended incubation with PHBSP, the light chain was further processed, partially removing about 60 amino acid residues from the N-terminus of domain D5 of the light chain. These cleavage site(s) were distinct from plasma kallikrein or FXIa cleavage sites. PHBSP and, more interestingly, also plasma kallikrein could cleave low molecular weight kininogen in vitro, indicating that domains D5H and D6H are no prerequisite for kininogen cleavage. PHBSP was also able to release bradykinin from HK in plasma where the pro-cofactor circulates predominantly in complex with plasma kallikrein or FXI. In conclusion, PHBSP represents a novel kininogen-cleaving and bradykinin-releasing enzyme in plasma that shares significant catalytic similarities with plasma kallikrein. Since they are structurally unrelated in their heavy chains (propeptide), their similar in vivo catalytic activities might be directed at distinct sites where PHBSP could induce processes that are related to the kallikrein/kinin system.     

Inhibition of human blood coagulation factor XIa by C-1 inhibitor

The inactivation of activated factor XI (factor XIa) and of its isolated light chain by C-1 inhibitor was studied. Irreversible inhibition was observed in a reaction in which no reversible enzyme-inhibitor complex was formed. The second-order rate constants for the inactivation of factor XIa or its light chain by C-1 inhibitor were 2.3 X 10(3) and 2.7 X 10(3) M-1 s-1, respectively. High molecular weight kininogen did not affect the rate of inactivation. The nature of the complexes formed between factor XIa or its light chain and C-1 inhibitor was studied by using sodium dodecyl sulfate gradient polyacrylamide slab gel electrophoresis. Under nonreducing conditions, two factor XIa-C-1 inhibitor complexes were observed with apparent molecular weights of 230,000 and 300,000. Reduction of these complexes resulted in the formation of a single band with a molecular weight of 130,000. This band is also formed in the reaction of the isolated light chain of factor XIa with C-1 inhibitor. These results demonstrate that two C-1 inhibitor molecules can become bound to the light chains of a factor XIa molecule. In addition, the mechanism of interaction of factor XIa or its isolated light chain with C-1 inhibitor appears identical, and the rate of inactivation of the enzyme by C-1 inhibitor is very similar. Neither the heavy chain of factor XIa nor high molecular weight kininogen is significantly involved in the inactivation of factor XIa by C-1 inhibitor.     

Bradykinin and kininogens in bovine milk

Two peptides exhibiting kinin activity in an isolated rat uterus assay were purified from pasteurized skim bovine milk. The amino acid sequence of the more prominent peptide was found to be that of bradykinin. Partially purified kinin preparations were also obtained from N-tosyl-L-phenylalanyl chloromethyl ketone-treated trypsin digests of non-fat dry milk and insoluble lactalbumin. The application of fast atom bombardment/mass spectrometry permitted detection of the bradykinin protonated molecular ion in each of these samples. Collision-activated decomposition of the ion of m/z 1061 confirmed it to be the protonated molecular ion of bradykinin. Fast atom bombardment/mass spectrometry analysis further confirmed the occurrence of bradykinin in a pancreatic kallikrein digest of a partially purified bovine milk kininogen preparation. In apparent contrast with bovine plasma kininogens, the forms of kininogen which occur in milk include a high Mr kininogen (Mr greater than 68,000) and a low Mr kininogen (Mr 16,000-17,000). Kinin formation from the high Mr kininogen is catalyzed by porcine pancreatic kallikrein or trypsin.