High Mr Kininogen Research Articles

Activation of Purified Human Plasma Prekallikrein Triggered by Cell Wall Fractions of Escherichia coli and Staphylococcus aureus

Whether Escherichia coli and Staphylococcus aureus cell wall fractions can trigger the activation of prekallikrein was investigated in a mixture of purified human factor XII, prekallikrein, and high-relative-molecular-weight (Mr) kininogen. After exposure for 30 min to bacterial preparations (0.02-5 mg/ml) at 0 C, lallikrein amidolytic activity was expressed as a percentage of the optimal activation of prekallikrein induced by dextran sulfate. Lipopolysaccharide (LPS) fractions of five E coli strains and lipid A of E coli O111B4 induced 50%-90% optimal activity. However, the polysaccharide fraction induced less than 5% activity. Peptidoglycan and teichoic acid of S aureus induced 70%-100% optimal activity at 5 mg/ml, but protein A did not generate activity. No activation of prekallikrein occurred in the absence of factor XII. Thus, LPS and lipid A of E coli and peptidoglycan and teichoic acid of S aureus can generate kallikrein amidolytic activity in a mixture of purified factor XII, prekallikrein, and high-Mr kininogen.

Isolation and functional characterization of the active light chain of activated human blood coagulation factor XI.

Human blood coagulation Factor XIa was reduced and alkylated under mild conditions. The mixture containing alkylated heavy and light chains was subjected to affinity chromatography on high Mr kininogen-Sepharose. Alkylation experiments using [14C]iodoacetamide showed that a single disulfide bridge between the light and heavy chains was broken to release the light chain. The alkylated light chain (Mr = 35,000) did not bind to high Mr kininogen-Sepharose while the heavy chain (Mr = 48,000), like Factors XI and XIa, bound with high affinity. The isolated light chain retained the specific amidolytic activity of native Factor XIa against the oligopeptide substrate, pyroGlu-Pro-Arg-p-nitroanilide. Km and kcat values for this substrate were 0.56 mM and 350 s-1 for both Factor XIa and its light chain, and the amidolytic assay was not affected by CaCl2. However, in clotting assays using Factor XI-deficient plasma in the presence of kaolin, the light chain was only 1% as active as native Factor XIa. Human coagulation Factor IX was purified and labeled with sodium [3H]borohydride on its carbohydrate moieties. When this radiolabeled Factor IX was mixed with Factor XIa, an excellent correlation was observed between the appearance of Factor IXa clotting activity and tritiated activation peptide that was soluble in cold trichloroacetic acid. Factor XIa in the presence of 5 mM CaCl2 activated 3H-Factor IX 600 times faster than Factor XIa in the presence of EDTA. In the absence of calcium, Factor XIa and its light chain were equally active in activating 3H-Factor IX. In contrast to Factor XIa, the light chain in this reaction was inhibited by calcium ions such that, in the presence of 5 mM CaCl2, Factor XIa was 2000 times more effective than its light chain. Neither phospholipid nor high Mr kininogen and kaolin affected the activity of Factor XIa or its light chain in the activation of 3H-Factor IX. These observations show that the light chain region of Factor XIa contains the entire enzymatic active site. The heavy chain region contains the high affinity binding site for high Mr kininogen. Furthermore the heavy chain region of Factor XIa plays a major role in the calcium-dependent mechanisms that contribute to the activation of Factor IX.

Kinin release from high molecular weight kininogen by the action of Hageman factor in the absence of kallikrein.

Proteolysis of 125I-high molecular weight (Mr) kininogen occurred in kaolin-activated plasma which was deficient in prekallikrein, but not in plasma which lacked both prekallikrein and Hageman factor (HF) activity. The implication of this observation is that HF itself might be capable of releasing kinin from high Mr kininogen. This concept was further supported by the following studies. In a purified protein system rabbit (Mr = 80,000) two-chain activated HF (alpha-HFa) incubated with human 125I-high Mr kininogen caused rapid proteolysis of the kininogen in the presence, but not in the absence, of kaolin. This was in contrast to the effect of kallikrein on proteolysis of high Mr kininogen which was inhibited 10-fold by the presence of kaolin. The possibilities that the alpha-HFa preparation was contaminated by rabbit kallikrein or that the human high Mr kininogen preparation was contaminated by human prekallikrein were excluded by using specific antibodies (IgG) against these proteins. The proteolytic fragments of 125I-high Mr kininogen generated by both alpha-HFa and kallikrein were indistinguishable by sodium dodecyl sulfate-polyacrylamide gel analysis. Bioassayable kinin was released from high Mr kininogen following incubation with alpha-HFa in the presence of kaolin. The amount of kinin released in proportion to the extent of proteolysis was the same for both kallikrein and alpha-HFa. The data show that activated Hageman factor may cause release of kinin by proteolytic cleavage of high Mr kininogen. This phenomenon occurs not only in a purified system of proteins but also in kaolin-activated plasma.

Inactivation of kallikrein in human plasma.

Human plasma kallikrein is inactivated by plasma protease inhibitors. This study was designed to determine the nature of these protease inhibitors and to assess their relative importance in the inactivation of kallikrein. Therefore, the kinetics of kallikrein inactivation and the formation of kallikrein inhibitor complexes were studied in normal plasma and in plasma depleted of either alpha 2-macroglobulin (alpha 2M), C1 inhibitor, or antithrombin (AT III). Prekallikrein was activated by incubation of plasma with dextran sulfate at 4 degrees C. After maximal activation, kallikrein was inactivated at 37 degrees C. Inhibition of kallikrein amidolytic activity in AT III-deficient plasma closely paralleled the inactivation rate of kallikrein in normal plasma. The inactivation rate of kallikrein in alpha 2M-deficient plasma was slightly decreased compared with normal plasma, but in contrast to normal, C1 inhibitor-deficient, and AT III-deficient plasma, no kallikrein amidolytic activity remained after inactivation that was resistant to inhibition by soybean trypsin inhibitor. Suppression of kallikrein activity in C1 inhibitor-deficient plasma was markedly decreased, and this was even more pronounced in plasma deficient in both C1 inhibitor and alpha 2M. The pseudo first-order rate constants for kallikrein inactivation in normal, AT III-deficient, alpha 2M-deficient, C1 inhibitor-deficient plasma, and plasma deficient in both alpha 2M and C1 inhibitor, were 0.68, 0.60, 0.43, 0.07, and 0.016 min-1, respectively. Sodium dodecyl sulfate gradient polyacrylamide slab gel electrophoresis showed that during inactivation of kallikrein in plasma, high-Mr complexes were formed with Mr at 400,000-1,000,000, 185,000, and 125,000-135,000, which were identified as complexes of 125I-kallikrein with alpha 2M, C1 inhibitor, and AT III, respectively. In addition, the presence of an unidentified kallikrein-inhibitor complex was observed in AT III-deficient plasma. 52% of the 125I-kallikrein was associated with C1-inhibitor, 35% with alpha 2M, and 13% with AT III and another protease inhibitor. A similar distribution of 125I-kallikrein was observed when the 125I-kallikrein inhibitor complexes were removed from plasma by immunoadsorption with insolubilized anti-C1 inhibitor, anti-alpha 2M, or anti-AT III antibodies. These results suggest that only covalent complexes are formed between kallikrein and its inhibitors in plasma. As a function of time, 125I-kallikrein formed complexes with C1 inhibitor at a higher rate than with alpha 2M. No difference was observed between the inactivation rate of kallikrein in high-Mr kininogen-deficient plasma and that in high-Mr kininogen-deficient plasma reconstituted with high-Mr kininogen; this suggests that high-Mr kininogen does not protect kallikrein from inactivation in the plasma milieu. These results have quantitatively demonstrated the major roles of C1 inhibitor and alpha 2M in the inactivation of kallikrein in plasma.

Protection of human plasma kallikrein from inactivation by C1 inhibitor and other protease inhibitors. The role of high molecular weight kininogen.

High Mr kininogen increases the activation rate of prekallikrein by activated factor XII on a surface. The resulting serine protease, plasma kallikrein, Mr 88 000, is inhibited in plasma by C1 inhibitor, Mr 105 000. Since prekallikrein circulates in plasma with high Mr kininogen as a complex and a kallikrein-high Mr kininogen complex can be formed in purified systems, we studied whether the inhibition of kallikrein by C1 inhibitor was influenced by high Mr kininogen. With C1 inhibitor in excess, the inactivation of kallikrein followed pseudo-first-order kinetics. The second-order rate constant for the reaction was 1.7 X 10(4) M-1 s-1, and a kallikrein-C1 inhibitor complex, Mr 190 000 was identified on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Kallikrein and C1 inhibitor formed an irreversible complex without measurable prior equilibrium. The rate of this reaction was decreased by 50% in the presence of high Mr kininogen (1 unit/mL or 0.73 muM). Kinetic analysis indicated that this protection was the result of the formation of a reversible complex between kallikrein and high Mr kininogen, which had a dissociation constant of 0.75 muM. However, low Mr kininogen did not protect kallikrein from inactivation by C1 inhibitor. High Mr kininogen also protected kallikrein from inactivation by diisopropyl fluorophosphate. These findings suggest that the kallikrein-high Mr kininogen complex was formed by noncovalent interactions between the light chains of both kallikrein and high Mr kininogen.

Immunochemical studies of human high molecular weight kininogen and of its complexes with plasma prekallikrein or kallikrein.

Human plasm contains at least two distinct kininogens, designated high Mr and low Mr kininogens, that differ in their Mr and susceptibility to different kallikreins. By limited proteolysis, plasma kallikrein cleaves high Mr kininogen to liberate kinin and gives a molecule containing two disulfide-linked polypeptide chains. Following reduction and alkylation of the two-chain molecule, the heavy chain and the light chain were isolated, and antisera were raised in goats against the alkylated heavy and light chains. The anti-heavy chain antiserum immunoprecipitated both high Mr and low Mr kininogen, whereas the anti-light chain antiserum was specific for high Mr kininogen. Thus, the heavy chain of kinin-free high Mr kininogen and low Mr kininogen extensively share identical immunologic determinants. In contrast, the immunologic determinants of the isolated light chain are unique to high Mr kininogen. Using immunochemical techniques, it was shown that high Mr kininogen or the light chain derived from it forms a complex either with prekallikrein or with kallikrein. Titrations of prekallikrein or kallikrein with increasing amounts of either high Mr kininogen or its alkylated light chain indicated that the complexes contain equimolar amounts of each molecule. These results show that a single site for prekallikrein or kallikrein binding to high Mr kininogen resides in the light chain region of the molecule.

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.

Similarity between a Kininogenase (Kallikrein) from Human Large Intestine and Human Urinary Kallikrein

A human colon kininogenase (kallikrein) was isolated by gel filtration on Sephacryl S-200 and affinity chromatography on Trasylolbound Sepharose, yielding a material with a specific activity of 1.3 U/mg (substrate: AcPheArgOEt). The molecular weight of the enzyme as estimated by gel filtration is approximately 70 000. After reduction with mercaptoethanol two bands were obtained in dodecyl sulfate eletrophoresis with molecular weights of 27 000 and 70 000. The bimolecular velocity constant for the inhibition by diisopropyl fluorophosphate was determined as 4 l x mol-1 x min-1. The preparation was characterized by immunological and enzymatic methods. Using the radioimmumoassay for human urinary kallikrein cross-reactivity and parallel binding curves were obtained. Kinin liberation from human high Mr-kininogen was totally inhibited by antibodies directed against human urinary kallikrein. Trasylol and diisopropyl fluorophosphate, but not by antibodies directed against human trypsin and plasma kallikrein. The effect on dog blood pressure was comparable to that obtained with human urinary kallikrein. The amino acid composition of human large intestine kallikrein is very similar to that of human urinary kallikrein.