Plasma prekallikrein: quantitative determination by direct activation with Hageman factor fragment (beta-XIIa)

This report describes a plasma prekallikrein assay which, unlike methods that employ contact activation, is not affected by the factor XII or HMW kininogen content of the plasma analyzed. In this assay beta-XIIa, a potent fluid-phase activator of prekallikrein, is added to diluted plasma in the presence of 20% acetone (to inactivate kallikrein inhibitors) at 30 degrees C and the kallikrein generated is measured with the chromogenic substrate S-2302. Prekallikrein is fully activated under these conditions and the activity remains stable for at least 30 hr. The mean prekallikrein concentration in plasma samples from 24 healthy individuals was 1.50 +/- 0.35 (S.D.) S-2302 U/ml, corresponding to 20.3 +/- 4.7 micrograms/ml prekallikrein (the specific activity of highly purified human prekallikrein was determined to be 74 S-2302 U/mg). In contrast, the mean concentration in five plasma samples from patients deficient in HMW kininogen was 0.38 +/- 0.02 S-2302 U/ml. No activity was generated in prekallikrein-deficient plasma, and essentially normal levels (1.35 +/- 0.18 S-2302 U/ml) were measured in plasmas from three patients with factor XII deficiency. Plasma prekallikrein was also quantitated by radial immunodiffusion, which gave results similar to those obtained by functional assay with beta-XIIa. The determination of plasma prekallikrein by direct activation with beta-XIIa in the presence of acetone offers several advantages over the use of contact activators such as dextran sulfate. These advantages include complete inactivation of kallikrein inhibitors and total activation of prekallikrein (even in plasmas deficient in other contact factors) without simultaneous generation of plasmin.     

Plasma prekallikrein assay: reversible inhibition of C-1 inhibitor by chloroform and its use in measuring prekallikrein in different mammalian species

The assay of plasma prekallikrein requires activation of prekallikrein to kallikrein and sufficient inactivation of the plasma protease inhibitors of kallikrein to accurately measure the generated kallikrein activity. One method of elimination of the plasma protease inhibitors to kallikrein is to chemically pretreat the plasma. Methylamine has previously been employed to selectively inactivate alpha 2-macroglobulin. Our study examines the effect of sequential preincubation of plasma with chloroform and methylamine on the plasma prekallikrein assay. Chloroform was demonstrated to be a chemical inhibitor of purified C-1 inhibitor, but alpha 2-macroglobulin was not. Chloroform inhibition of C-1 inhibitor was not caused by precipitation of the protein into the interface between the water and organic solvent phase. Greater than 95% of C-1 inhibitor antigen was recovered in the supernatant of chloroform-treated purified C-1 inhibitor, and chloroform-saturated buffer inhibited purified C-1 inhibitor. Chloroform did not dissociate a preformed complex of kallikrein and C-1 inhibitor, but its inhibition of C-1 inhibitor was reversible. The addition of methylamine to plasma pretreated with chloroform in the plasma prekallikrein assay allowed for only a slight increase in the amount of kallikrein measured at 1 minute kaolin activation times, but provided for sustained measurement of activated prekallikrein when kaolin activation times were 5 to 7 minutes. Without chemical pretreatment, prekallikrein was not measurable in rabbit plasma. Both rabbit and pig plasma prekallikrein was measurable after exposure of the plasma to chloroform and methylamine, although the peak activation times and the contribution of each animals' protease inhibitors varied with the species. Our results show that chloroform is a reversible inhibitor of C-1 inhibitor, and that the plasma prekallikrein assay in which it is used is useful for the measurement of prekallikrein in nonhuman mammalian plasma samples.     

Evaluation of a microassay for human plasma prekallikrein

Current methods for determining plasma prekallikrein, one of three zymogens of the contact phase of plasma proteolysis, are laborious and impractical for general use in a clinical laboratory. Therefore, we have developed a simple, reliable assay using commercially available reagents. By use of the substrate H-D-Pro-Phe-Arg-p-nitroanilide-HCI (S-2302), a functional assay, performed in a 96-well microplate, was designed to measure prekallikrein in plasma. Measures were taken to destroy the naturally occurring plasma protease inhibitors of kallikrein without affecting the integrity of the plasma prekallikrein, which allowed complete activation of the zymogen to virtually 100% of predicted activity when compared with that of purified kallikrein. Besides permitting full activation, the use of low pH to destroy critical plasma protease inhibitors allowed the conversion of prekallikrein to kallikrein in as many as 44 plasma samples at one time without the tedious individual timing step usually required to activate each sample. An excellent correlation was found (r = 0.92) when this functional microassay was compared with a functional spectrophotometric assay performed in three subject populations: normal individuals, women receiving oral contraceptives (who frequently exhibit high plasma prekallikrein concentrations), and patients with liver disease (who manifest low plasma prekallikrein levels). This plasma prekallikrein microassay should facilitate the increased determination of plasma prekallikrein in pathophysiologic conditions as well as the monitoring of the progression of various diseases in which contact activation occurs.     

Human plasma prekallikrein (Fletcher factor) clotting activity and antigen in health and disease

Human plasma prekallikrein (Fletcher factor) clotting activity and antigen levels have been examined in various clinical conditions. Prekallikrein antigen was measured by a newly developed, specific, and sensitive radioimmunoassay. The assay had no demonstrable cross-reactivity with human urinary kallikrein nor, in the species tested, animal plasma prekallikrein. This assay was able to measure plasma kallikrein after its biological functions had been inactivated by plasma inhibitors. Normal human pooled plasma contained approximately 50 microgram/ml prekallikrein. Quantitative measurement of plasma prekallikrein was possible for concentrations as low as 0.3% of that of normal pooled plasma. A good correlation (correlation coefficient = 0.71) existed between titers of plasma prekallikrein measured by Fletcher factor clotting assays and radioimmunoassays among 40 normal subjects. Both prekallikrein clotting activity and antigen were significantly reduced in plasmas of patients with advanced hepatic cirrhosis or DIC. Prekallikrein activity and antigen were mildly decreased in plasmas or serums of patients with chronic renal failure and nephrotic syndrome but were normal in those of patients under treatment with warfarin or suffering from SLE, rheumatoid arthritis, sarcoidosis, or HANE. Human cord serum contained a lower titer of prekallikrein antigen than adult serum. Strenuous physical exercise did not significantly change plasma prekallikrein levels.     

Enzymes of the contact phase of blood coagulation: kinetics with various chromogenic substrates and a two-substrate assay for the joint estimation of plasma prekallikrein and factor XI

Kinetic constants (Km and kcat) of kallikrein and factor XIa for the chromogenic substrates H-D-L-prolyl-L-phenylanyl-L-arginine-p-nitroanilide (S-2302) and L-pyroglutamyl-L-propyl-L-arginine-p-nitroanilide (S-2366) were determined. The determined constants allow the use of S-2302 and S-2366 in an assay that leads to the joint estimation of factor XI and prekallikrein in activated plasma. The assay reports approximately 3.1 micrograms/ml factor XIa and 34.5 micrograms/ml kallikrein in kaolin-activated plasma (kaolin content 2 mg/ml). The dual-substrate amidolytic assay shows good correlation with the coagulant assay of both factors (0.92 with the prekallikrein assay and 0.98 with the factor XI assay). It is capable, through the joint estimation of factor XI and prekallikrein levels, of differentiating among plasma samples deficient in components of the contact phase of blood coagulation. Kinetic constants of factor beta-XIIa (factor XII fragment) for these substrates and for N-benzol-L-isoleusyl-L-glutamyl-glycyl-L-arginine-P-nitro ani lide (S-2222) were determined, and they allowed the assessment of the contribution of this factor to this assay and its estimation in the activated phase.     

Mechanized assay of plasma prekallikrein by activation with Pseudomonas aeruginosa elastase and amidolysis of chromogenic substrate.

An automated assay of plasma prekallikrein is described. Prekallikrein was converted to kallikrein with Pseudomonas aeruginosa elastase, and the hydrolytic activity of kallikrein to H-D-Pro-Phe-Arg-paranitroanilide subsequently measured.

The conversion was complete within 8 minutes and the amidolytic activity remained stable at least another 10 min at 37 ° C. This method worked in plasma deficient in Hageman factor (blood coagulation factor XII). Using anti-prekallikrein antibody and plasma deficient in prekallikrein, the amidolytic activity generated in normal plasma was identified as due to kallikrein. With plasma samples, the coefficients of variation (CV) for multiple measurements within run (n = 10) and between run (n = 10) were as low as 5.0% and 6.6%, respectively, and the minimum measurable concentration of prekallikrein in plasma was 10% of the normal level.     

Activation and function of human Hageman factor. The role of high molecular weight kininogen and prekallikrein.

The activation and function of surface-bound Hageman factor in human plasma are dependent upon both high molecular weight (HMW) kininogen and prekallikrein. HMW kininogen does not affect the binding of Hageman factor to surfaces, but it enhances the function of surface-bound Hageman factor as assessed by its ability to activate prekallikrein and Factor XI. The initial conversion of prekallikrein to kallikrein by the surface-bound Hageman factor in the presence of HMW kininogen is followed by a rapid enzymatic activation of Hageman factor by kallikrein. The latter interaction is also facilitated by HMW kininogen. Kallikrein therefore functions as an activator of Hageman factor by a positive feedback mechanism and generates most of the activated Hageman factor during brief exposure of plasma to activating surfaces. HMW kininogen is a cofactor in the enzymatic activation of Hageman factor by kallikrein and it also augments the function of the activated Hageman factor generated. The stoichiometry of the Hagman factor interaction with HMW kininogen suggests that it enhances the activity of the active site of Hageman factor. Since HMW kininogen and prekallikrein circulate as a complex, HMW kininogen may also place the prekallikrein in an optimal position for its reciprocal interaction with Hageman factor to proceed. The surface appears to play a passive role upon which bound Hageman factor and the prekallikrein-HMW kininogen complex can interact.     

Mechanized assay of plasma prekallikrein by activation with Pseudomonas aeruginosa elastase and amidolysis of chromogenic substrate

An automated assay of plasma prekallikrein is described. Prekallikrein was converted to kallikrein with Pseudomonas aeruginosa elastase, and the hydrolytic activity of kallikrein to H-D-Pro-Phe-Arg-paranitroanilide subsequently measured.The conversion was complete within 8 minutes and the amidolytic activity remained stable at least another 10 min at 37 ° C. This method worked in plasma deficient in Hageman factor (blood coagulation factor XII). Using anti-prekallikrein antibody and plasma deficient in prekallikrein, the amidolytic activity generated in normal plasma was identified as due to kallikrein. With plasma samples, the coefficients of variation (CV) for multiple measurements within run (n = 10) and between run (n = 10) were as low as 5.0% and 6.6%, respectively, and the minimum measurable concentration of prekallikrein in plasma was 10% of the normal level.     

Human plasma kallikrein releases neutrophil elastase during blood coagulation.

Elastase is released from human neutrophils during the early events of blood coagulation. Human plasma kallikrein has been shown to stimulate neutrophil chemotaxis, aggregation, and oxygen consumption. Therefore, the ability of kallikrein to release neutrophil elastase was investigated. Neutrophils were isolated by dextran sedimentation, and elastase release was measured by both an enzyme-linked immunosorbent assay, and an enzymatic assay using t-butoxy-carbonyl-Ala-Ala-Pro-Val-amino methyl coumarin as the substrate. Kallikrein, 0.1-1.0 U/ml, (0.045-0.45 microM), was incubated with neutrophils that were preincubated with cytochalasin B (5 micrograms/ml). The release of elastase was found to be proportional to the kallikrein concentration. Kallikrein released a maximum of 34% of the total elastase content, as measured by solubilizing the neutrophils in the nonionic detergent Triton X-100. A series of experiments was carried out to determine if kallikrein was a major enzyme involved in neutrophil elastase release during blood coagulation. When 10 million neutrophils were incubated in 1 ml of normal plasma in the presence of 30 mM CaCl2 for 90 min, 2.75 micrograms of elastase was released. In contrast, neutrophils incubated in prekallikrein-deficient or Factor XII-deficient plasma released less than half of the elastase, as compared with normal plasma. The addition of purified prekallikrein to prekallikrein-deficient plasma restored neutrophil elastase release to normal levels. Moreover, release of elastase was enhanced in plasma deficient in C1-inhibitor, the major plasma inhibitor of kallikrein. This release was not dependent upon further steps in the coagulation pathway, or on C5a, since levels of elastase, released in Factor XI- or C5-deficient plasma, were similar to that in normal plasma, and an antibody to C5 failed to inhibit elastase release. These data suggest that kallikrein may be a major enzyme responsible for the release of elastase during blood coagulation.     

Centrifugal analysis for plasma kallikrein activity, with use of the chromogenic substrate S-2302

The amidolytic activity of activated kallikrein in plasma can be measured by use of the chromogenic substrate, S-2302 (H-D-Pro-Phe-Arg-pNA). Plasma prekallikrein was activated to kallikrein by exposure to 50 mg/L dextran sulfate in acetone/water (35/65 by vol) at 0 degrees C for 15 min. The acetone slows anti-kallikrein activity and increases the kallikrein activity by 30%. The 37 degrees C reaction mixture contained 0.54 mmol of S-2302 substrate per liter of Tris buffer (pH 7.5 at 37 degrees C). We monitored the change in absorbance at 405 nm for 60 s. The specificity of the substrate for kallikrein was demonstrated by using plasma deficient in prekallikrein (Fletcher trait) diluted with pooled normal human plasma. We recommend collecting blood specimens with sodium citrate as the anticoagulant and with use of a double-syringe technique and all-plastic containers. Plasma kallikrein activity with Chromozym-PK (Bz-Pro-Phe-Arg-pNA) as substrate (y-axis) compared with S-2302 as substrate (x-axis) gave the relation: y = 0.28x + 0.82 (r = 0.94). Day-to-day analytical variation was 2.4% for a pooled plasma with a mean value of 85.9 mukat/L. The mean (and 2 SD) for 50 healthy adults was 86.4 (32.4) mukat/L.     

Characterization of a variant prekallikrein, prekallikrein Long Beach, from a family with mixed cross-reacting material-positive and cross-reacting material-negative prekallikrein deficiency.

Studies of plasma prekallikrein in a family with prekallikrein deficiency were made. Three children had no clotting activity but approximately 35% antigen levels, and the mother and five children had twice as much prekallikrein antigen as clotting activity, suggesting the presence of a dysfunctional molecule. A nonfunctional variant form of prekallikrein was purified that contained no prekallikrein clotting activity. The variant and normal molecules were both 80,000 mol wt, immunologically indistinguishable and complexed similarly with high molecular weight kininogen. Isoelectric focusing studies suggested a difference of one charged amino acid residue. The variant was cleaved by beta-Factor XIIa 200 times slower than the normal molecule, and no amidolytic activity was detected for the cleaved variant. These data and other observations suggest that an amino acid was substituted in the variant near the NH2-terminal end of the kallikrein light chain resulting in slower cleavage by beta-Factor XIIa and the absence of enzymatic activity.     

Immunochemical Studies of Plasma Kallikrein

A monospecific antibody against human plasma kallikrein has been prepared in rabbits with kallikrein further purified to remove gamma globulins. The antisera produced contained antikallikrein and also anti-IgG, in spite of only 8% contamination of kallikrein preparation with IgG. The latter antibody was removed by adsorption of antisera with either Fletcher factor-deficient plasma or with purified IgG. Both kallikrein and prekallikrein (in plasma) cross-react with the antibody with no apparent difference between the precipitation arcs developed during immunoelectrophoresis and no significant difference in reactivity when quantified by radial immunodiffusion.Kallikrein antibody partially inhibits the esterolytic and fully inhibits the proteolytic activity of kallikrein. In addition, the antibody inhibits the activation of prekallikrein, as measured by esterase or kinin release. The magnitude of the inhibition is related to the molecular weight of the activator used. Thus, for the four activators tested, the greatest inhibition is observed with kaolin and factor XII(A), while large activator and the low molecular weight prekallikrein activators are less inhibited.With the kallikrein antibody, the incubation of kallikrein with either plasma or partially purified C1 esterase inactivator results in a new precipitin arc, as detected by immunoelectrophoresis. This finding provides physical evidence for the interaction of the enzyme and inhibitor. No new arc could be demonstrated between kallikrein and alpha(2)-macroglobulin, or alpha(1)-antitrypsin, although the concentration of free kallikrein antigen decreases after interaction with the former inhibitor.By radial immunodiffusion, plasma from healthy individuals contained 103+/-13 mug/ml prekallikrein antigen. Although in mild liver disease, functional and immunologic kallikrein are proportionally depressed, the levels of prekallikrein antigen in plasma samples from patients with severe liver disease remains 40% of normal, while the functional kallikrein activity was about 8%. These observations suggest that the livers of these patients have synthesized a proenzyme that cannot be converted to active kallikrein.     

Factor XII and prekallikrein-kallikrein-kinin in the cryoactivation of human plasma prorenin

Cryoactivation of human plasma 'prorenin' was followed for 24 h at -4 degrees C. Chromogenic assays were used to determine factor XII (FXII), FXIIa, prekallikrein and kallikrein in relation to the observed cold-induced increase in plasma renin activity (PRA). Bradykinin activity was also determined using the rat uterus bioassay. PRA increased rapidly and became significantly higher after just 6 h of cryoactivation, by which time prekallikrein had almost disappeared, while kallikrein and kinin levels increased. In contrast, FXII did not change notably, but some FXIIa was indeed formed. The bacteriostat neomycin sulphate did not affect the course of cryoactivation, but did block the dextran sulphate- and kaolin-induced activation of prekallikrein and FXII respectively, and was therefore omitted. Thus cryoactivation of prorenin is accompanied by, and may depend upon, the activation of FXII and prekallikrein, supporting other evidence in favour of this hypothesis.     

Prorenin-renin conversion by the contact activation system in human plasma: role of plasma protease inhibitors

Acid-pretreated normal human plasma generates renin activity at 0 degree C and neutral pH by the activation of prorenin. The activation is caused by kallikrein generated from prekallikrein by activated factor XII. Nonacidified plasma also generates renin at 0 degree C, but at a lower rate (cold-promoted activation). In normal plasma, 14% +/- 1% of prorenin (mean +/- SEM, n = 30) was activated during incubation at 0 degree C for 7 days (range 6% to 26%). Cold-promoted activation of prorenin was within the normal range in plasma deficient in factor XI, X, IX, VIIIC, VII, V, prothrombin, or high mol wt kininogen. Cold-promoted activation of prorenin was less than or equal to 1% in plasma deficient in factor XII or prekallikrein. Reconstitution of these plasmas with highly purified factor XII or prekallikrein restored normal prorenin activation. Correction of high mol wt kininogen deficiency had no effect. Thus cold-promoted activation of prorenin depends on the presence of factor XII and prekallikrein, whereas the other clotting factors are not essential. The influence of the inhibitors C1 esterase-inhibitor, alpha 2-macroglobulin, antithrombin III, and alpha 1-antitrypsin on the activation of prorenin was studied in factor XII-deficient plasma from which one or more of these inhibitors had been selectively removed by immunoadsorption. Factor XII was subsequently added, and the generation of renin at 37 degrees C was observed after complete factor XII-high mol wt kininogen-mediated activation of prekallikrein induced by dextran sulfate. No activation of prorenin was observed at 37 degrees C after depletion of C1 esterase inhibitor, alpha 2-macroglobulin, antithrombin III, or alpha 1-antitrypsin. When prekallikrein was activated in plasma depleted of both C1 esterase-inhibitor and alpha 2-macroglobulin, 6% of prorenin was activated in 2 hours at 37 degrees C. After additional depletion of antithrombin III, the activation increased to 47%. These results indicate that the contact activation system is capable of activating prorenin in plasma at physiologic pH and temperature when the three most important kallikrein inhibitors, C1 esterase-inhibitor, alpha 2-macroglobulin, and antithrombin III, are absent.     

Fletcher factor deficiency. A diminished rate of Hageman factor activation caused by absence of prekallikrein with abnormalities of coagulation, fibrinolysis, chemotactic activity, and kinin generation

Fletcher factor-deficient plasma is deficient in prekallikrein and therefore generates no bradykinin upon activation with kaolin. It also possesses a diminished rate of kaolin-activable coagulation and fibrinolysis and possesses a defect in kaolin-activable chemotactic activity. These abnormalities are also corrected by reconstitution with purified prekallikrein. Addition of intact activated Hageman factor corrected the coagulation, fibrinolytic, and chemotactic defects and addition of Hageman factor fragments corrected the fibrinolytic defect and partially corrected the chemotactic defect; neither of these corrected the kinin-generating defect. Although the Hageman factor-dependent pathways appear to be initiated by contact activation of Hageman factor, the kallikrein generated activates more Hageman factor; this feedback is necessary for the Hageman factor-dependent pathways to proceed at a normal rate. It is the absence of this feedback in Fletcher factor-deficient plasma that accounts for the diminished rate of activation of Hageman factor and therefore a diminished rate of activation of the coagulation and fibrinolytic pathways. The ability of prekallikrein to correct the coagulation, fibrinolytic, kinin-generating, and chemotactic defects of Fletcher factor-deficient plasma is consistent with the identity of the Fletcher factor and prekallikrein.     

Anaphylactic release of a prekallikrein activator from human lung in vitro.

We have demonstrated the in vitro IgE-mediated release of a prekallikrein activator from human lung. The lung prekallikrein activator was partially purified by sequential chromatography on sulfopropyl-Sephadex, DEAE-Sephacel, and Sepharose 6B. Purified human prekallikrein was converted to its active form (kallikrein) by the lung protease. The generated kallikrein was shown to be biologically active; that is, it generates bradykinin from purified human high-molecular weight kininogen and also cleaves benzoyl-propyl-phenyl-arginyl-p-nitroanilide, a known synthetic substrate of kallikrein. The lung prekallikrein activator differs from the known physiologic activators of prekallikrein (the activated forms of Hageman factor) with respect to: (a) size (it has a mol wt of approximately 175,000); (b) synthetic substrate specificity (D-propyl/phenyl/arginyl-p-nitroanilide is a substrate for the activated forms of Hageman factor, but not the lung protease); (c) antigenic specificity (an anti-Hageman factor immunoadsorbent column did not remove significant amounts of the lung protease, while it removed most of the activity of activated Hageman factor fragments); and (d) inhibition profile (the lung proteases was not inhibited by corn trypsin inhibitor). This prekallikrein activator provides a physiologic mechanism by which prekallikrein can be directly activated during IgE-mediated reactions of the lung. While the role of this lung prekallikrein activator in immediate hypersensitivity reactions and in other inflammatory processes is not clear, it does represent a first and important interface between IgE-mediated reactions and the Hageman factor-dependent pathways of the inflammatory response.     

Dextran sulfate-dependent fibrinolysis in whole human plasma

This study shows that Flu-beta-Ala can reduce the ability of human plasma to inhibit plasmin. This observation was utilized to develop a method for generating detectable fibrinolytic activity in whole human plasma as assessed on a radiolabeled fibrin plate. Plasma was pretreated with Flu-beta-Ala to remove inhibitors of fibrinolysis: then dextran sulfate was added and the mixture was further incubated at 4 degrees C. When normal plasma was treated in this manner, the rate of generation of fibrinolytic activity after 0.75 hr incubation with radiolabeled fibrin was equivalent to that of 35 ng/ml plasmin. The plasminogen dependence of this activity was tested by pretreating plasma with antibodies against plasminogen. The generation of fibrinolytic activity was totally blocked by this treatment, indicating that the observed fibrinolytic activity was plasminogen-dependent. When plasmas deficient in prekallikrein, factor XII, or high-molecular-weight kininogen were treated with Flu-beta-Ala and dextran sulfate, the initial rate of fibrinolytic activity was less than normal. But after 3 hr incubation with radiolabeled fibrin, the rate of fibrinolytic activity in these deficient plasmas approached that of normal plasma. Thus this dextran sulfate-dependent fibrinolytic activity is dependent on factor XII, prekallikrein, and high-molecular-weight kininogen, but the requirement is not absolute.     

Potentiation of the function of Hageman factor fragments by high molecular weight kininogen.

Patients lacking high molecular weight (HMW) kininogen have profound abnormalities of the Hageman factor-dependent pathways of coagulation, kinin formation, and fibrinolysis. The ability of HMW kininogen to potentiate the Hageman factor fragments (HFf) activation of prekallikrein and Factor XI in plasma was studied. HFf only partially converted Factor XI to XIa and prekallikrein to kallikrein in plasma deficient in HMW kininogen (Williams trait), while enhanced activation of Factor XI and prekallikrein by HFf resulted after reconstitution with HMW kininogen. In a system using highly purified components, HMW kininogen increased the initial rate of prekallikrein activation whether the kallikrein formed was assayed by arginine esterase activity or kininforming ability. The potentiation of prekallikrein activation occurred over a 12-fold range of enzyme (HFf) concentration and was nonhyperbolic with respect to substrate (prekallikrein). HMW kininogen exerted its effect even in the absence of prekallikrein since the hydrolysis of acetylglycyl-lysine methyl ester by HFf was increased by HMW kininogen. These results suggest that one of the functions of HMW kininogen is to augment the catalytic action of HFf.     

Immunoreactive tissue kallikrein in human serum

Human urinary kallikrein and an antiserum to it raised in the rabbit were used to detect and quantitate immunoreactive tissue kallikrein in human serum. Both 125I-labeled kallikrein and the unlabeled purified enzyme appear complexed to higher molecular weight entities in serum, but specific binding between radiolabeled enzyme and antiserum was unaffected by the presence of serum or plasma. Parallelism to standard displacement curves was always seen with radioimmunoassay of normal sera as well as with human mixed saliva or pancreatic extracts. Assay sensitivity is 160 pg/ml of serum, or 16 pg per tube. Purified plasma kallikrein or prekallikrein in concentrations up to 10 micrograms/ml showed no displacement. Acetone-kaolin activation of plasma produced the expected 30-fold increase in Tos-Arg-OMe esterase activity but no change in immunoreactive tissue kallikrein levels. Serum concentrations were 3.8 +/- 0.7 (mean +/- SE) ng/ml in 21 normal volunteers, and were similar in patients with Fletcher trait or Hageman factor deficiency. Significantly increased serum concentrations were seen with long-term low dietary sodium intake or acute forms of pancreatitis. Although the relation of this immunoreactive material to any active tissue kallikrein within the circulation remains to be determined, our studies provide a new parameter for the assessment of a system repeatedly suggested to have some role in regulation of vascular resistance.     

The Kallikrein-Kinin System in Bartter''s Syndrome and Its Response to Prostaglandin Synthetase Inhibition

The kallikrein-kinin system was characterized in seven patients with Bartter's syndrome on constant metabolic regimens before, during, and after treatment with prostaglandin synthetase inhibitors. Patients with Bartter's syndrome had high values for plasma bradykinin, plasma renin activity (PRA), urinary kallikrein, urinary immunoreactive prostaglandin E excretion, and urinary aldosterone; urinary kinins were subnormal and plasma prekallikrein was normal. Treatment with indomethacin or ibuprofen which decreased urinary immunoreactive prostaglandin E excretion by 67%, decreased mean PRA (patients recumbent) from 17.3+/-5.3 (S.E.M.) ng/ml per h to 3.3+/-1.1 ng/ml per h, mean plasma bradykinin (patients recumbent) from 15.4+/-4.4 ng/ml to 3.9+/-0.9 ng/ml, mean urinary kallikrein excretion from 24.8+/-3.2 tosyl-arginine-methyl ester units (TU)/day to 12.4+/-2.0 TU/day, but increased mean urinary kinin excretion from 3.8+/-1.3 mug/day to 8.5+/-2.5 mug/day. Plasma prekallikrein remained unchanged at 1.4 TU/ml. Thus, with prostaglandin synthetase inhibition, values for urinary kallikrein and kinin and plasma bradykinin returned to normal pari passu with changes in PRA, in aldosterone, and in prostaglandin E. The results suggest that, in Bartter's syndrome, prostaglandins mediate the low urinary kinins and the high plasma bradykinin, and that urinary kallikrein, which is aldosterone dependent, does not control kinin excretion. The high plasma bradykinin may be a cause of the pressor hyporesponsiveness to angiotensin II which characterizes the syndrome.