Studies on human kininogens. I. Isolation, characterization, and cleavage by plasma kallikrein of high molecular weight (HMW)-kininogen.

1. Human high molecular weight (HMW)-kininogen was highly purified from human plasma by chromatographies on QAE-Sephadex A-50 and CM-Sephadex C-50. Human HMW-kininogen thus purified was a mixture of a single chain and a disulfide-linked pair of chains. Human HMW-kininogen is an acidic glycoprotein having a molecular weight of 120,000. The amino acid composition of human HMW-kininogen is quite similar to that of bovine HMW-kininogen. 2. We investigated whether the liberation of kinin from human HMW-kininogen by human plasma kallikrein was accompanied by liberation of histidine-rich fragments, as observed with bovine HMW-kininogen (Han et al. (1975) J. Biochem. 77, 55--68). After prolonged incubation of human HMW-kininogen and human plasma kallikrein followed by gel-filtration on Sephadex G-50, a fragment of molecular weight 8,000 was isolated together with bradykinin. However, the histidine content of the fragment was not as high as that in the bovine fragments. Most of the histidine in human HMW-kininogen was recovered in the kinin-free protein, and the light chain of kinin-free protein was found to be rich in histidine compared with the heavy chain. These results suggest that the histidine-rich sequence in human HMW-kininogen is not released by the action of human plasma kallikrein, but remains bound to the light chain of kinin-free protein.     

Inactivation of human high molecular weight kininogen by human mast cell tryptase

Tryptase, the major neutral protease of human pulmonary mast cell secretory granules, rapidly inactivates human high m.w. kininogen (HMWK) in vitro. HMWK (5600 nM) lost 50% of its capacity to release kinin in response to kallikrein after a 5-min incubation with tryptase (31 nM), even though kinin activity was neither generated nor, when bradykinin was incubated with tryptase, destroyed by tryptase. The procoagulant activity of HMWK (51 nM) and the purified procoagulant chain (40 nM) that is derived from HMWK were each 72% inactivated after 7 min of incubation with tryptase (0.04 nM and 0.02 nM, respectively). Human urinary and pancreatic kallikrein did not inactivate this procoagulant activity under conditions in which kinin generation occurs. Complete cleavage of native single-chain HMWK by tryptase occurred in less than 10 min as analyzed by electrophoresis in sodium dodecyl sulfate polyacrylamide slab gels. The major products formed during the initial 2 min were proteins of 100,000 and 95,000 apparent m.w., and by 10 to 30 min were fragments of 74,000 and 67,000 apparent m.w. Reduction of these cleavage products yielded two major fragments of 67,000 and 66,000 apparent m.w. that were both present by 0.17 min. The presence of lower m.w. products, thought to be primarily from the carboxy-terminal procoagulant region of HMWK, were also detected with and without reduction. The capacity of tryptase to inactivate HMWK is consistent with the ability of other mast cell-derived mediators, such as heparin proteoglycan and prostaglandin D2, to suppress blood coagulation and thrombosis, and may play an important role in the biology of mast cell-dependent events in vivo.     

Studies on porcine high molecular weight kininogen. An improved method for the purification of porcine high molecular weight kininogen and cleavage of the kininogen by the action of porcine plasma kallikrein

By introduction of stepwise DEAE Sephadex A-50 and copper-Chelating Sepharose 6B column chromatographies, about 18.5 mg of high molecular weight kininogen (HK) composed of a single polypeptide chain was obtained from 500 ml of porcine plasma. Molecular weights of reduced or non-reduced preparation were estimated to be 110 kDa and 116 kDa, respectively, by SDS–PAGE. Using the preparation, cleavage of HK by porcine plasma kallikrein (KK) was investigated. A single polypeptide HK was cleaved into two chains cross-linked by disulfide bond(s), accompanying the release of kinin. Further degradation was not observed. Molecular weights of heavy-chain (H-chain) and light-chain (L-chain) were estimated to be 61 kDa and 56 kDa, respectively, by SDS–PAGE. The amino- (N-) terminal sequences of intact HK, reduced and carboxymethylated- (RCM-) H-chain, RCM-L-chain and the peptide around the kinin moiety obtained by BrCN digestion were determined. Their sequences were highly homologous with those of bovine or human HK. These results indicate that plasma KK first cleaved the Arg-Ser bond of HK, and formed nicked HK. The second cleavage yielded bradykinin (BK) and kinin-free protein, which was apparently of equal size to the nicked HK. The structure of HK was from the N-terminus to the carboxy- (C-) terminus, H-chain-BK-L-chain.     

Binding of heparin to human high molecular weight kininogen

The binding of heparin to high molecular weight kininogen (H-kininogen) was analyzed by the effect of kininogen in decreasing the heparin-induced enhancement of the rate of inactivation of thrombin by antithrombin. The conditions were arranged so that the heparin-catalyzed antithrombin-thrombin reaction, monitored in the presence of the reversible thrombin inhibitor p-aminobenzamidine, followed pseudo-first-order kinetics and the observed rate constant (kappa obsd) varied linearly with the heparin concentration. In the absence of metal ions, H-kininogen minimally affected kappa obsd, measured at a constant concentration of heparin with high affinity for antithrombin (30 nM), at I = 0.15, pH 7.4 and 25 degrees C. However, at a saturating concentration of Zn2+ (10 microM), kappa obsd was reduced to 50% at approximately 20 nM H-kininogen and to that of the uncatalyzed reaction at greater than or equal to approximately 0.2 microM H-kininogen. Conversely, at a saturating concentration of H-kininogen (0.5 microM), kappa obsd was decreased to 50% at approximately 0.6 microM Zn2+ and to the kappa obsd of the uncatalyzed reaction at greater than or equal to 10 microM Zn2+. Other metal ions were effective in the order Zn2+ approximately Ni2+ greater than Cu2+ approximately Co2+ approximately Cd2+. The single-chain and two-chain forms of H-kininogen and the H-kininogen light chain reduced the heparin enhancement in the presence of Zn2+ to the same extent, whereas low molecular weight kininogen had no influence.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformation of high molecular weight kininogen: effects of kallikrein and factor XIa cleavage

The effect of kallikrein and factor XIa proteolysis of high molecular weight kininogen (HK) was investigated. Circular dichroism (CD) spectroscopy showed that cleavage of HK by plasma kallikrein or urinary kallikrein, both of which result in an active cofactor (HKa), results in conformational change that is characterized by increase in CD ellipticity at 222 nm. This suggests an increase in organized secondary structures. By contrast, cleavage of HK by factor XIa which results in an inactive cofactor (HKi) is characterized by a dramatic decrease in CD ellipticity at 222 nm suggesting an entirely different type of conformational change. The intrinsic fluorescence of HK is enhanced after cleavage by all three proteases. These conformational changes may play a role in determining the structure and function of HKa and HKi.     

Receptors for high molecular weight kininogen on stimulated washed human platelets

Binding of human high molecular weight kininogen to washed human platelets was studied by measuring platelet-associated radiolabeled ligand in pellets of centrifuged platelets. High molecular weight kininogen was bound to stimulated platelets in the presence of ZnCl2 in a specific and saturable manner. Calcium ions potentiated ligand binding but did not substitute for zinc ions. Optimal binding of high molecular weight kininogen occurred near the plasma concentrations of both zinc and calcium ions. Scatchard analysis yielded 24 200 binding sites for high molecular weight kininogen with an apparent dissociation constant of 20 nM. These studies show that stimulated human platelets can bind many high molecular weight kininogen molecules with high affinity and suggest that the platelet surface may potentially serve as an important site for localizing the initial reactions of the plasma kinin-forming and intrinsic coagulation pathways.     

Study of the effect of high molecular weight kininogen upon the fluid-phase inactivation of kallikrein by C1 inhibitor

Plasma kallikrein and factor XIa circulate bound to high molecular weight kininogen, and such binding has been reported to protect these enzymes from inactivation by their respective inhibitors. However, this observation is controversial, and the effect of high molecular weight kininogen upon the interaction between kallikrein and C1 inhibitor (C1-INH) has been questioned. We have re-evaluated this reaction and studied the rate of inhibition of kallikrein by C1-INH in the presence and absence of high molecular weight kininogen. The second-order rate constant of inhibition of kallikrein by C1-INH was unaffected by saturating concentrations of high molecular weight kininogen. Our results suggest that although high molecular weight kininogen clearly augments the rate of formation of kallikrein and other enzymes of the contact activation pathway, it has no effect on the rate of enzyme inhibition by C1-INH.     

Inhibition of cell adhesion by high molecular weight kininogen

An anti-cell adhesion globulin was purified from human plasma by heparin-affinity chromatography. The purified globulin inhibited spreading of osteosarcoma and melanoma cells on vitronectin, and of endothelial cells, platelets, and mononuclear blood cells on vitronectin or fibrinogen. It did not inhibit cell spreading on fibronectin. The protein had the strongest antiadhesive effect when preadsorbed onto the otherwise adhesive surfaces. Amino acid sequence analysis revealed that the globulin is cleaved (kinin-free) high molecular weight kininogen (HKa). Globulin fractions from normal plasma immunodepleted of high molecular weight kininogen (HK) or from an individual deficient of HK lacked adhesive activity. Uncleaved single-chain HK preadsorbed at neutral pH, HKa preadsorbed at pH greater than 8.0, and HKa degraded further to release its histidine-rich domain had little anti-adhesive activity. These results indicate that the cationic histidine-rich domain is critical for anti-adhesive activity and is somehow mobilized upon cleavage. Vitronectin was not displaced from the surface by HKa. Thus, cleavage of HK by kallikrein results in both release of bradykinin, a potent vasoactive and growth-promoting peptide, and formation of a potent anti-adhesive protein.     

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.     

The expression of high molecular weight kininogen on human umbilical vein endothelial cells

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.     

Cleavage of human high molecular weight kininogen by factor XIa in vitro. Effect on structure and function

We have recently demonstrated that human high molecular weight kininogen (HMWK) is a pro-cofactor that is cleaved by kallikrein to yield a two-chain cofactor (HMWKa) and the nanopeptide bradykinin. This proteolysis enhances its association with an activating surface, an event necessary for expression of its cofactor activity. We now report that factor XIa is capable of hydrolyzing HMWK and releasing bradykinin in a purified system as well as cleaving and inactivating HMWK in a plasma environment during the contact-activation process. The profile of proteolysis differs from that produced by kallikrein and by factor XIIa in that the first cleavage by factor XIa yields 75- and 45-kDa polypeptides, whereas both factor XIIa and kallikrein initially produce 65- and 56-kDa species. Further proteolysis by all three enzymes eventually produces similar heavy chains (Mr = 65,000) and light chains (Mr = 45,000). However, the amount of factor XIa generated in plasma during contact activation further degrades the light chain of HMWK, eventually destroying its coagulant activity. Furthermore, in a purified system, enhancement of the degradation of HMWK coagulant activity by factor XIa was achieved when kallikrein was included in the incubation mixture, suggesting that the preferred substrate for factor XIa is the active form of HMWK (HMWKa), and not the pro-cofactor. These data suggest that factor XIa has the potential to act as a regulator of contact-activated coagulation by virtue of its ability to destroy the cofactor function of HMWK after its generation by either kallikrein, factor XIIa, or to a lesser extent, factor XIa, itself.     

Primary structure requirements for the binding of human high molecular weight kininogen to plasma prekallikrein and factor XI

We recently identified residues 185-224 of the light chain of human high molecular weight kininogen (HMWK) as the binding site for plasma prekallikrein (Tait, J.F., and Fujikawa, K. (1986) J. Biol. Chem. 261, 15396-15401). In the present study, we have further defined the primary structure requirements for binding of HMWK to factor XI and prekallikrein. In a competitive fluorescence polarization binding assay, a 31-residue synthetic peptide (residues 194-224 of the HMWK light chain) bound to prekallikrein with a Kd of 20 +/- 6 nM, indistinguishable from the previously determined value of 18 +/- 5 nM for the light chain. We also prepared three shorter synthetic peptides corresponding to different portions of the 31-residue peptide (residues 205-224, 212-224, and 194-211), but these peptides bound to prekallikrein more than 100-fold more weakly. Factor XI also bound to the same region of the HMWK light chain, but at least 58 residues (185-242) were required for optimal binding (Kd = 69 +/- 4 nM for the light chain; Kd = 130 +/- 50 nM for residues 185-242). The four synthetic peptides inhibited kaolin-activated clotting of blood plasma with potencies paralleling their affinities for prekallikrein and factor XI. Peptide 194-224 can also be used for rapid affinity purification of prekallikrein and factor XI from plasma.     

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.     

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.     

Various properties and kinetics of interaction of high and low molecular weight human kininogens with human plasma kallikrein

A relatively simple procedure for isolation and purification of human blood plasma kallikrein (HPK) by QAE-Sephadex A-50 SP-Sephadex C-50 and affinity chromatography on Sepharose 4B with immobilized soybean trypsin inhibitor with the activity yield of about 40% has been developed. The method allows for simultaneous isolation of low (LMW) and high molecular weight (HMW) kininogens from the same HPK sample. HPK preparations are homogeneous upon 7.5% polyacrylamide gel electrophoresis in the presence of 0.1% SDS; its Mr is 90,000. After treatment with beta-mercaptoethanol, HPK dissociates into two fragments with Mr of 43,000 and 37,000. HPK preparations have high specific activities of esterase (31 microM/min), amidase (78 microM/min), and kininogenase (420 micrograms equiv. bradikinin/min). The high degree of protein purification was demonstrated by titration of active centers with 4-methylumbelliferylguanidine benzoate. The values of equilibrium dissociation constants for the HPK complex with aprotinin (Ki) equal to 1 X 10(-8) M (ethyl ester of N-alpha-benzoyl-L-arginine) and 1,5 X 10(-9) M (HMW) were determined. The kinetics of HPK-induced liberation of bradikinin from purified preparations of HMW and LMW was studied. The kinetic parameters (Km, kcat and kcat/Km) of this reaction suggest a high affinity of HPK for HMW, but not for LMW. LMW does not compete with HMW for the enzyme active center. It is assumed that LMW is not a physiological substrate for HPK.