HMW Kininogen Research Articles

A method of determination of human plasma HMW and LMW kininogen levels by Bradykinin enzyme immunoassay

A method of determination of human plasma HMW and LMW kininogen levels by Bradykinin enzyme immunoassay

T-kinin and T-kininogen--an historical overview.

It is well known that bradykinin liberation of trypsin from mammalian plasmas reflects the levels of high (HMW) and low molecular weight (LMW) kininogens of the plasma. Consequently the addition of trypsin to plasma is classically used to measure the total kininogen content of plasma of all species1,2 by measuring the liberation of kinin. Kinin is generally measured by bioassay or by radioimmunoassay. It is important to understand that until 1983, when our laboratory on HPLC separation of kinins,3 investigators could not differentiate clearly between bradykinin and its analogs by the bioassay or radioimmunoassay techniques. Many assumptions were made particularly in terms that HMW and LMW kininogens were the important proteins liberating kinins in all species. One piece of evidence that seemed to indicate that a third kininogen other than LMW or HMW was present in some fluids was the isolation of leukokininogen from human ascites fluid by Roffman and Greenbaum.4 Leukokininogen was a poor substrate for kallikreins but did liberate a kinin (termed leukokinin by our laboratory) by either cathepsin D or trypsin. Hiroshi Okamoto joined our laboratory in 1982 and began a study on the relative concentration of leukokininogen compared to HMW and LMW kininogens in human, rabbit and rat plasmas. In order to obtain the total kininogen content, the classic concentration of 0.1 mg trypsin added to 1 ml of plasma was initially used.

Individual reaction steps in the release of kallidin from kininogen by tissue kallikrein.

At low pH values (around 6), porcine pancreatic beta-kallikrein B attacks at first the C-terminal ARg bond of the kinin moiety in bovine HMW kininogen. Arg-cleaved kininogen accumulates as an intermediate in the solution. Kallidin is released by cleavage of the aminoterminal Met-Lys bond in a second step. At pH values between 7.6 and 9, however, Arg-cleaved kininogen does not occur as a free intermediate. The participation as a (free, not only enzyme-bound) intermediate of Arg-cleaved kininogen in a short-lived especially reactive conformation or of Met-cleaved kininogen is also unlikely. Probably, both the Met and the Arg bonds are hydrolyzed in one enzyme-substrate complex which does not dissociate between these two events. Kinetic constants for the release of kallidin from native single-chain HMW kininogen and from Arg-cleaved kininogen (even if this Arg residue is removed) remarkably have the same values. Evidently, the rate of the reaction is determined by steps leading to the hydrolysis of the Met bond. As the state of the C-terminal Arg residue has no influence, the efficient cleavage of the Met bond by tissue kallikrein is probably not due to some strain in the kininogen molecule in the region of this bond. As modification of Arg residues of kininogen prevents cleavage also of the Met bond, some Arg residue(s) appear(s) to play a crucial role in this process. kcat/Km (1.4 x 10(6) M-1 sex-1 at pH 9, 25 degrees C) is very high for a proteolytic reaction, mainly because of the low value of Km (0.6 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

910 EVIDENCE FOR REDUCTION OF CONTACT FACTORS IN SICKLE CELL DISEASE

Evidence supporting the existence of intravascular coagulation in homozygous sickle cell (HbSS) disease has been reported. In this study, surface-mediated reactions of clotting were compared in 21 black children with HbSS disease and 12 age-matched controls. Both the coagulant and antigen titers of Hageman factor were decreased (mean coagulant titer 0.49 ± S.D. 0.29 u/ml; mean antigen titer 0.77 ± 0.27 u/ml) in asymptomatic HbSS patients compared to the control group (mean coagulant titer. 1.01 ± 0.39 u/ml; mean antigen titer 0.94 ± 0.25 u/ml). A disparate relationship between the Hageman factor coagulant and antigen titers were observed in HbSS patients. These findings were associated with a slight decrease in the plasma titers of prekallikrein and HMW kininogen (mean 0.78 ± 0.14 and 0.75 ± 0.15 u/ml respectively in HbSS patients compared to 1.05 ± 0.22 and 0.86 ± 0.09 u/ml respectively in the control group). A further reduction from the initially low titers of these contact factors was observed during vaso-occlusive crises (mean Hageman factor titer 0.32 ± 0.15 u/ml; mean prekallikrein titer 0.38 ± 0.18 u/ml; mean HMW kininogen titer 0.56 ± 0.17 u/ml). These data indicate that the initial participants of the intrinsic pathway of coagulation are consumed in patients with HbSS disease. Further, these processes appear to accelerate during vaso-occlusive crises.

Activation of factor XII and prekallikrein with cholesterol sulfate.

Cholesterol sulfate was found to display a strong ability to trigger the activation of Factor XII and prekallikrein in the presence of HMW kininogen. Other sulfate ester derivatives of testosterone, estrone, pregnenolone and dehydroepiandrosterone and cholesterol tested did not show any effect on the activation of Factor XII and prekallikrein. The activity of cholesterol acetate and sulfodeoxycholic acid was very weak. Cholesterol sulfate markedly shortened the partial thromboplastin time of normal human plasma, but not plasmas deficient in Factor XII, Factor XI and HMW kininogen. Upon prolonged incubation, the partial thromboplastin time of prekallikrein-deficient plasma was also shortened. Moreover, as well as kaolin and sulfatide, cholesterol sulfate shortened the partial thromboplastin time of plasmas from monkey, dog, rat, guinea pig, sheep, cow, hog and horse, but not from duck and chicken. Since cholesterol sulfate is distributed in erythrocytes, various organs and body fluids, it may play an important role in the activation of the intrinsic blood coagulation system.

Prolonged activated partial thromboplastin time and deficiency of high molecular weight kininogen in brown norway rat mutant (Katholiek strain)

Activated partial thromboplastin time (APTT) was examined in Brown Norway (B/N) Katholiek rat, which was previously reported as high molecular weight kininogen deficient. APTT of B/N Katholiek was prolonged to 35 sec in comparison with B/N Kitasato and SD rat, showing APTT of 22–24 sec. The mixture of B/N Katholiek plasma and B/N Kitasato plasma (1:1) showed normal APTT value. B/N Katholiek plasma corrected the abnormally prolonged human coagulation factor deficient plasmas, such as XI, XII and prekallikrein deficient plasmas, while it did not correct the APTT of HMW kininogen deficient, Fitzgerald plasma. Intravenous injection of bromelain, which was previously reported to produce prolonged hypotension through the activation of factor XII to release bradykinin, induced slight effect in Katholiek rat, while in Kitasato rat it showed prolonged hypotension in similar degree as SD rat. Contents of coagulation factors in B/N Katholiek thus measured as well as the values of prekallikrein and HMW kininogen previously reported were summarized and suggested that B/N Katholiek rat could be similar deficiency as Fitzgerald trait.

Plasma Contact Activation and Decrease of Factor V Activity on Negatively-Charged Polyelectrolytes

The effect of negatively-charged polymers, used in some artificial devices, on plasma clotting and kinin systems was studied in vitro using polyelectrolyte complexes. Contact activation was observed as an immediate, transient and surface-dependent phenomenon. After incubation of the plasma with the polymer a small decrease of factor XII activity was noticed, which corresponded to a greater reduction of prekallikrein activity and to a marked kinin release. No significant decrease of factor XII, prekallikrein, HMW kininogen could be detected immunologically. Only the initial contact of the plasma with the polyelectrolyte lead to activation, subsequently the surface became inert. Beside contact activation, factor V activity also decreased in the plasma. The decrease was surface and time-dependent. It was independent of contact factor activation, and appeared to be related to the sulfonated groups of the polymer. If purified factor V was used instead of plasma factor V, inactivation was immediate and not time-dependent suggesting a direct adsorption on the surface. A second incubation of the plasma-contacted polymer with fresh plasma resulted in a further loss of Factor V activity.

Kininogen substrates for trypsin and cathepsin D in human, rabbit and rat plasmas

Studies have compared “total”, HMW kininogen and leukokininogen levels in human, rabbit and rat plasmas using trypsin, glass powder and cathepsin D as kininogenases or activators of kininogenases. Rat plasma was found to have about 10 fold more leukokininogen than the other plasmas assayed. When trypsin was used to estimate total kininogen, rat plasma liberated maximal amounts of kinin only in the presence of high concentrations of trypsin (1 mg/ml incubation mixture). In addition, it was found that trypsin in these concentrations liberated from rat plasma both bradykinin and a previously unidentified kinin which we have termed “T-kinin”. The results overall indicate that in the case of rat and rabbit plasma, currently used methods for estimations of total kininogen may not be accurate. T-kinin may represent a leukokininogen or a hitherto undescribed kininogen.

Cellular and Subcellular Mediators of Acute Inflammation

The interactions of the complement system and the contact-activation system (Hageman factor, HMW kininogen, factor XI, and prekallikrein) are discussed, and the role of neutrophils and their complement-mediated activation in acute inflammation are explained.

Characterization of human salivary kallikrein: reactivities to human plasma kininogens and proteinase inhibitors.

In order to clarify the enzymatic properties of human salivary kallikrein, we investigated its actions on human plasma kininogens and response to proteinase inhibitors. Salivary kallikrein released kinin from both LMW and HMW kininogens, but acted preferentially on LMW kininogen. Salivary kallikrein did not completely release the kinin from HMW kininogen even after prolonged incubation (up to 60 min). The kinin releasing activity of salivary kallikrein was not inhibited by plasma proteinase inhibitors such as alpha 2M, alpha 2PI, AT III, and C1 INA after preincubation for 60 min at 37 degrees C, but was inhibited progressively by alpha 1AT as a function of preincubation time. SDS-PAGE revealed that concomitantly with the progressive inhibition a new band of 92,000 daltons, which seemed to be a complex of salivary kallikrein and alpha 1AT, was produced. The kininogen-depleted plasma inhibited the salivary kallikrein to the same degree as alpha 1AT. Therefore, it is suggested that alpha 1AT is the only inhibitor of salivary kallikrein in human plasma.

The kinin-forming enzyme system in pregnancy and obstetrical DIC.

Levels of prekallikrein and HMW kininogen that had increased during pregnancy decreased with start of labor. The role of the kinin-forming system with oxytocin in the mechanism of labor was suggested from the results of decreased prekallikrein and HMW kininogen, appearance of a free kallikrein-like enzyme during labor, and from the case of arrested labor in which both prekallikrein and HMW kininogen were markedly decreased. Prekallikrein was markedly decreased in patients with acute obstetrical DIC and severe toxemia of pregnancy. The excessive activation of prekallikrein in DIC seemed to be of help for understanding such clinical signs as shock, abnormal labor, and increased permeability in obstetrical DIC.

Human tissue kallikrein. I. Isolation and characterization of human pancreatic kallikrein from duodenal juice.

Human pancreatic kallikrein was purified from duodenal juice by ion exchange chromatography on DEAE-Sepharose and immunoaffinity chromatography. Thus, an enzyme preparation with a specific activity (using Ac-Phe-Arg-OEt as substrate) of 1 000 U/mg protein was obtained. A specific biological activity of 1310 KE/mg protein was measured in the dog blood pressure assay and of 0.361 HMW kininogen-U/mg, corresponding to the liberation of 383 micrograms bradykinin-equivalents per mg enzyme per min from HMW kininogen in the rat uterus assay. In dodecyl sulfate gel electrophoresis one protein band corresponding to a molecular mass of 27 kDa was obtained. Using gel filtration on Ultrogel AcA-44 a molecular mass of 40 kDa was measured. The amino-acid composition was determined and isoleucine and alanine were identified as the only N-terminal amino-acid residues. On isoelectric focusing four protein bands with isoelectric points of 5.60, 5.65, 5.70 and 5.85 were separated. The bimolecular velocity constant for the inhibition by diisopropyl fluoro phosphate was determined as 10.5 l x mol-1 x min-1. The dissociation constant Ki of the human pancreatic kallikrein-aprotinin complex was calculated to be 1.5 x 10(-10)M. The kinetic constants for the kallikrein-catalysed hydrolysis of Ac-Phe-Arg-OEt and D Val-Leu-Arg-Nan were determined. Immunological studies showed a close relationship between the human pancreatic kallikrein and other human tissue kallikreins, especially with human urinary kallikrein. Detergents such as Triton X-100, Tween 20 and lysolecithin, as well as human serum albumin, activated the human pancreatic kallikrein preparation.

Inactivation of Factor XIa by Plasma Protease Inhibitors

Factor XIa is a plasma protease that, by activating Factor IX, plays an important role in the early phase of the intrinsic pathway of blood coagulation. Four plasma protease inhibitors, alpha(1)-protease inhibitor, antithrombin III, C1-inhibitor, and alpha(2)-plasmin inhibitor, have been reported to inactivate human Factor XIa, but their quantitative contribution to the inactivation of Factor XIa in plasma has not been fully assessed. Using purified systems, we observed that the second-order rate constants for the reaction of Factor XIa with alpha(1)-protease inhibitor, antithrombin III, and CI-inhibitor were 4.08, 10, and 14.6 M(-1) min(-1) x 10(3), respectively. The pseudo-first-order rate constants, at plasma concentration of the inhibitors, were 1.86 x 10(-1), 4.68 x 10(-2), and 2.4 x 10(-2) min(-1), respectively. These kinetic data predict that alpha(1)-protease inhibitor should account for 68%, antithrombin III for 16%, and C1-inhibitor and the equipotent alpha(2)-plasmin inhibitor each for 8% of the total inhibitory activity of plasma against Factor XIa. The rate of inactivation of Factor XIa in various plasma samples specifically deficient in inhibitors was consistent with these predictions. Factor XI, the zymogen form of Factor XIa, circulates in plasma associated with the contact system cofactor, high molecular weight kininogen (HMW kininogen). Kinetic analysis indicated the existence of a reversible bimolecular Factor XIa-HMW kininogen complex with a dissociation constant (K(d)) = 0.17 muM. The light chain derived from HMW kininogen decreased the inactivation rate of Factor XIa by C1-inhibitor with a K(d) of 0.08 muM for a complex of Factor XIa and the light chain derived from HMW kininogen. The protective effect of HMW kininogen was confirmed by the finding that the inactivation rate of Factor XIa in kininogen-deficient plasma was increased over normal plasma. The present study confirms that alpha(1)-protease inhibitor is the major inhibitor of Factor XIa in plasma, and that the formation of a reversible complex between Factor XIa and HMW kininogen decreases the rate of inactivation of the enzyme by its inhibitors.

Determination of Prekallikrein in Plasma by Means of a Chromogenic Tripeptide Substrate for Plasma Kallikrein

A method for plasma prekallikrein determination utilizing a chromogenic tripeptide substrate is presented. The method has a good re-producibility and can easily be automized. Several parameters have been optimized. By using mixtures of deficient plasmas and pooled normal plasma or purified factors it was proved that prekallikrein was the factor determined and that more than 1O% (of normal plasma concentration) of FXII and HMW kininogen were essential for the activation of prekallikrein in our method. Further experiments showed that the method was fairly selective and was not influenced by inhibitors present in normal plasma. The later finding was attributed to the high dilution of plasma made possible by using a potent activator and a sensitive substrate.

The accelerating effect of bovine plasma HMW kininogen on the surface-mediated activation of factor XII: generation of a derivative form (active kininogen) with maximal cofactor activity by limited proteolysis.

<h2>Abstract</h2> The accelerating effect of HMW kininogen in the kaolin-mediated activation of bovine Factor XII was markedly enhanced after the brief treatment with urinary kallikrein or plasma kallikrein (but not with plasmin). At the early stage of the incubation, the cleavage of a single chain HMW kininogen and the enhancement of the accelerating effect preceded the kinin liberation, indicating that a nicked form of HMW kininogen is more active as a cofactor than the intact form. Thus, in the initial phase of intrinsic blood coagulation, plasma kallikrein seems to play an important role not only in the activation of Factor XII but also in the transformation of HMW kininogen to a nicked form with higher cofactor activity, an "active kininogen".

Mechanisms for Hageman factor activation and role of HMW kininogen as a coagulation cofactor.

Our present concept of the initiating reactions of the intrinsic coagulation pathway is outlined in Figure 5. Although we remain unsure of the etiology shown in Figures 3 and 4, the major function of HMW kininogen is to bind prekallikrein and factor XI in plasma and attach them to surfaces in a conformation that allows activation by HFa. The HMW kininogen--dependent augmentation of the binding of prekallikrein and factor XI to the surface that is seen in plasma (but not buffer systems) would appear to be of lesser importance. Once activated, however, dissociation of kallikrein from the surface allows it to attack adjacent Hageman factor molecules on the same or other particles; this reaction appears to be more rapid than the rate of Hageman factor autoactivation. Thus, the rapid burst of HFa formation seen in normal plasma is kallikrein dependent. It is also dependent upon HMW kininogen, but this appears to be an indirect relationship. The HMW kininogen augments the amount of prekallikrein bound, allows activation to kallikrein, and is needed for kallikrein dissociation from the surface. These three effects all yield a marked increase in the effective ratio of kallikrein/Hageman factor at the surface-fluid interface, and this may be the condition required for rapid HFa formation.

Platelet-coagulant protein interactions in contact activation.

Previous studies have suggested that human platelets can promote the activation of factor XI by two different mechanisms, one requiring factor XII and ADP-treated platelets and the other requiring collagen-treated platelets in the apparent absence of factor XII. To investigate these hypotheses, isolated platelets were tested for their capacity to promote the activation and cleavage of purified factors XII and XI in various mixtures of purified factor XII, kallikrein, high molecular weight kininogen, and factor XI. That ADP- or collagen-treated platelets can promote the proteolytic activation of factor XII in mixtures containing kallikrein and HMW kininogen was shown by (1) the proteolytic cleavage of factor XII, (2) the development of factor XIIa coagulant activity, and (3) the proteolytic cleavage of 125I-labeled factor XII. Platelets treated with collagen or thrombin were shown to both coagulant assays and cleavage studies to participate with HMW kininogen and kallikrein in the proteolytic activation of factor XI by mechanisms that are partially dependent upon and partially independent of factor XII. These studies demonstrate that platelets can promote the proteolytic activation of factor XII by kallikrein and of factor XI by both factor XII-dependent and factor XII-independent mechanisms.

15] HMW and LMW kininogens

Publisher Summary This chapter presents the procedure for purification and assaying of kininogens. In general, purification of LMW kininogen requires many steps and the recovery from plasma is very low. Steps of purification include ion-exchange column chromatography on DEAE-Sephadex or QAE-Sephadex, CM-Sephadex or SP-Sephadex and gel filtration. However, a difficulty in the purification of HMW kininogen is the susceptibility of the kininogen to plasma kallikrein. Kininogen can be estimated by measuring kinin biologically or immunologically following incubation with kinin-releasing enzymes. Differential assay for HMW kininogen and LMW kininogen is obtained by utilizing the specificity of kinin-releasing enzymes. Plasma kallikrein liberates bradykinin from HMW kininogen, whereas glandular kallikrein liberates kallidin from both of high molecular weight and low molecular weight kininogens. Trypsin and snake venom kininogenase liberate bradykinin from both kininogens. For the bioassay of kininogens, attention should be paid to the contamination of kininase or inhibitors for enzymes in kininogen preparation, especially when partially purified kininogen or plasma is used.

Isolation of an enzymatically active glandular kallikrein from human plasma by immunoaffinity chromatography.

A glandular kallikrein from human plasma was isolated by immunoaffinity chromatography and characterized. The molecular weight was determined to 40,000 by gel filtration. The enzyme preparation liberates kinins from human HMW kininogen (specific activity: 0.328 HMW kininogen-U/A280 unit), lowers the blood pressure of dogs after intravenous injection (specific activity: 1090 KE/A280 unit), is inhibited by diisopropyl fluorophosphate and aprotinin, but not by SBTI. In the radioimmunoassay for human urinary kallikrein parallel binding curves were obtained. AcPheArgOEt, DVal-LeuArgOEt and ZTyrONp are cleaved with identical rates by the kallikrein preparation and human urinary kallikrein.

Activation of Human Factor VII in Plasma and in Purified Systems

Factor VII can be activated, to a molecule giving shorter clotting times with tissue factor, by incubating plasma with kaolin or by clotting plasma. The mechanisms of activation differ. With kaolin, activated Factor XII (XII(a)) was the apparent principal activator. Thus, Factor VII was not activated in Factor XII-deficient plasma, was partially activated in prekallikrein and high-molecular weight kininogen (HMW kininogen)-deficient plasmas, but was activated in other deficient plasmas. After clotting, activated Factor IX (IX(a)) was the apparent principal activator. Thus, Factor VII was not activated in Factor XII-,HMW kininogen-, XI-, and IX-deficient plasmas, but was activated in Factor VIII-, X-, and V-deficient plasmas. In further studies, purified small-fragment Factor XII(a) (beta-XII(a)), kallikrein, and Factor IX(a) were added to partially purified Factor VII and to plasma. High concentrations of beta-XII(a) activated Factor VII in a purified system; much lower concentrations of beta-XII(a) activated Factor VII in normal plasma but not in prekallikrein or HWM kininogen-deficient plasmas. Kallikrein alone failed to activate partially purified Factor VII but did so when purified Factor IX was added. Kallikrein also activated Factor VII in normal, Factor XII-, and Factor IX-deficient plasmas. Purified Factor IX(a) activated partially purified Factor VII and had no additional indirect activating effect in the presence of plasma. These results demonstrate that both Factor XII(a) and Factor IX(a) directly activate human Factor VII, whereas kallikrein, through generation of Factor XII(a) and Factor IX(a), functions as an indirect activator of Factor VII.