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.     

Plasma Kallikrein Activation and Inhibition during Typhoid Fever

As an ancillary part of a typhoid fever vaccine study, 10 healthy adult male volunteers (nonimmunized controls) were serially bled 6 days before to 30 days after ingesting 10(5)Salmonella typhi organisms. Five persons developed typhoid fever 6-10 days after challenge, while five remained well. During the febrile illness, significant changes (P < 0.05) in the following hematological parameters were measured: a rise in alpha(1)-antitrypsin antigen concentration and high molecular weight kininogen clotting activity; a progressive decrease of platelet count (to 60% of the predisease state), functional prekallikrein (55%) and kallikrein inhibitor (47%) with a nadir reached on day 5 of the fever and a subsequent overshoot during convalescence. Despite the drop in functional prekallikrein and kallikrein inhibitor, there was no change in factor XII clotting activity or antigenic concentrations of prekallikrein and the kallikrein inhibitors, C1 esterase inhibitor (C1-INH) and alpha(2)-macroglobulin. Plasma from febrile patients subjected to immunoelectrophoresis and crossed immunoelectrophoresis contained a new complex displaying antigenic characteristics of both prekallikrein and C1-INH; the alpha(2)-macroglobulin, antithrombin III, and alpha(1)-antitrypsin immunoprecipitates were unchanged. Plasma drawn from infected-well subjects showed no significant change in these components of the kinin generating system. The finding of a reduction in functional prekallikrein and kallikrein inhibitor (C1-INH) and the formation of a kallikrein C1-INH complex is consistent with prekallikrein activation in typhoid fever. The correlation of these changes with the drop in platelet count suggests that a common mechanism may be responsible.     

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.     

A PREALBUMIN ACTIVATOR OF PREKALLIKREIN : III. APPEARANCE OF CHEMOTACTIC ACTIVITY FOR HUMAN NEUTROPHILS BY THE CONVERSION OF HUMAN PREKALLIKREIN TO KALLIKREIN

Human plasma kallikrein has been shown to directly and selectively attract human neutrophils from a mixed leukocyte population. The capacity of plasma kallikrein to be chemotactic and to generate the nonapeptide bradykinin was maintained during progressive purification. While neither highly purified prekallikrein nor the prealbumin Hageman factor fragments were chemotactic alone, their interaction so as to convert prekallikrein to kallikrein yielded both chemotactic and kinin-generating activity. Both functions of kallikrein were inhibited by treatment with diisopropyl fluorophosphate, indicating an essential role for the active site of the enzyme in the expression of its chemotactic activity.     

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.     

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.     

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.     

Several aspects of the pathogenesis of disturbances of hemostasis in patients with diffuse toxic goiter

Components of the kallikrein-kinin system of the blood and some hemostatic indices were studied in parallel in 32 patients with diffuse toxic goiter. A decrease in the levels of prekallikrein, kininogen, the activity of kallikrein and kininase inhibitors in the blood plasma of the examinees resulted in raised activity of the kallikrein-kinin system in decompensated thyrotoxicosis. Hemostatic changes were characterized by signs of chronic disseminated intravascular coagulation. It was shown that kinin formation and blood coagulation were correlated. A certain parallelism in the increment of the activity of the kinin system and disorders of hemocoagulation was noted with an increase in a degree of severity of thyrotoxicosis. A tendency to improved indices was also noted after thyrostatic therapy.     

Vascular permeability, microcirculation and biologically active substances in patients with hemorrhagic fever with renal syndrome

As many as 56 patients with associated hemorrhagic fever and the renal syndrome were examined for vascular permeability, microcirculation and blood plasma histamine, serotonin, kallikrein and prekallikrein. In all the disease periods, the majority of the patients (n-52) demonstrated a rise of vascular permeability for protein and liquid part of the plasma, with this being marked in particular in the oligoanuric period. Besides, significant perivascular and intravascular changes in microcirculation were noted in the oligoanuric and polyuric disease periods. It was established during examination of biologically active substances that the greatest shifts were experienced by the kinin system. Sometimes the content of kallikrein surpassed the control values more than 7-fold. Less pronounced changes were seen in blood plasma serotonin and histamine. A direct correlation was established between vascular permeability, microcirculation and the content of all biologically active substances explored by the authors. The strongest correlation was found to exist between the above parameters and the kinin system of blood plasma.     

Hepatosplanchnic circulatory dysfunction in acute hepatic infection: the case of dengue hemorrhagic fever

The mechanism of shock in patients with dengue hemorrhagic fever (DHF) has not yet been fully understood. In this study, we investigated the possibility of splanchnic venous pooling as a contributor for circulatory dysfunction in these patients. Ultrasonographic studies of portal vein and inferior vena cava were done in 45 patients with serologically or PCR-confirmed diagnosis of dengue virus infection. The size of portal vein and inferior vena cava, mean blood flow velocity in the right portal vein, and modified portal vein congestion index were compared between patients with dengue fever (DF, n = 20), DHF without shock (n = 14), and dengue shock syndrome (DSS, n = 11) during the toxic stage, convalescent stage, and at follow-up. The portal vein was significantly more dilated in patients with shock (DSS) than DHF without shock and than DF during the toxic and convalescent stages (P < 0.05), but not at follow-up. The change in the size of inferior vena cava followed the opposite trend (not statistically significant). Portal vein blood flow velocity was lower and congestion index was higher in shock cases (DSS) than DHF without shock and than DF at toxic and convalescent stages (P < 0.01). The differences disappeared at follow-up. Hepatosplanchnic venous pooling and/or dysfunction occur and correlate with the severity of circulatory derangement and shock in patients with DHF. The cause(s) and significance of hepatosplanchnic circulatory dysfunction in DHF and possibly other viral hepatic diseases deserve further study.     

Activation of T lymphocytes in dengue virus infections. High levels of soluble interleukin 2 receptor, soluble CD4, soluble CD8, interleukin 2, and interferon-gamma in sera of children with dengue.

It has been reported that the severe complication of dengue virus infection, dengue hemorrhagic fever (DHF) is much more commonly observed during secondary dengue virus infections than primary infections. In order to elucidate the role of T lymphocytes in the pathogenesis of DHF, we attempted to determine whether T lymphocytes are activated in vivo during dengue virus infections, by examining the levels of soluble IL-2 receptor (sIL-2R), soluble CD4 (sCD4), soluble CD8 (sCD8), interleukin-2 (IL-2) and interferon-gamma (IFN gamma) in the sera of 59 patients with DHF and 41 patients with dengue fever (DF). The levels of sIL-2R, sCD4, sCD8, IL-2, and IFN gamma were significantly higher in the acute sera of patients with DHF than in the sera of healthy children (P less than 0.001 for all markers). The acute sera of patients with DF contained higher levels of sIL-2R, sCD4, IL-2, and IFN gamma than the sera of healthy children (P less than 0.001 for sIL-2R, IL-2, and IFN gamma; P less than 0.05 for sCD4), but did not have elevated levels of sCD8. The levels of sIL-2R (P less than 0.05), sCD4 (P less than 0.001), and sCD8 (P less than 0.001) were higher in DHF than in DF on days 3-4 after the onset of fever. The levels of IL-2 and IFN gamma in patients with DHF were highest 1 d before defervescence. There were no significant differences in the levels of sIL-2R, sCD4, sCD8, IL-2, and IFN gamma among grades 1, 2, and 3 of DHF. These results indicate (a) T lymphocytes are activated and produce IL-2 and IFN gamma in vivo during DHF and DF, (b) CD4+ T lymphocytes are activated in DHF and DF, and the level of activation is higher in DHF than in DF, and (c) activation of CD8+ T lymphocytes is evident in DHF, but not in DF.     

The plasma kallikrein kinin system in severely ill and traumatised patients.

Serial estimations of plasma prekallikrein in four critically ill patients in intensive care showed reduced values in each case, suggesting activation of the plasma kallikrein kinin system. In contrast, samples taken early from 15 patients attending an accident and emergency department with multiple trauma showed significantly elevated plasma prekallikrein concentrations; the significance of this observation is at present unclear.     

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.     

Indicators of the blood kinin system in patients with bronchial asthma during health resort treatment

Indicators of the blood kinin system were studied in 57 persons including 42 patients with asthma and 15 healthy persons (control group) in the Kislovodsk health resort area. Activation of the blood kinin system (a decrease in the content of prekallikrein, fast- and slowly reacting kallikrein inhibitors) was observed in patients with infectious-allergic and mixed forms of asthma. Positive shifts of the kinin system indicators pointing to a decrease in the "production" of free kinins were observed in the patients under the influence of multimodality therapy at the health resort.     

Determination of prekallikrein in human plasma: optimal conditions for activating prekallikrein

A method for the assay of human plasma prekallikrein in which a chromogenic synthetic tripeptide, PPAN, is used as a substrate for kallikrein is described. The conversion of prekallikrein to kallikrein is achieved by cold activation (0 degrees C) with water-soluble dextran sulfate. Conditions for obtaining optimal amounts of free kallikrein with respect to concentration of dextran sulfate, activation time, inhibitors (C-1-inactivator), and requirement of factor XII have been determined. The activation procedure is compared to other known procedures. The assay system was worked out for pooled normal plasma and is applicable to any plasma sample not liable to unwanted preactivation or incomplete inactivation, as revealed by control experiments. A survey in 15 apparently health individuals showed a mean activity of 476 +/- 58 (S.D.) mU/ml with a range of 385 to 586 mU/ml.     

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.     

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.     

ADULT DENGUE HAEMORRHAGIC FEVER AT KUALA LUMPUR HOSPITAL: RETROSPECTIVE STUDY OF 102 CASES

SUMMARY A retrospective study involving 102 adults with dengue haemorrhagic fever (DHF) was conducted to investigate the demographic aspect, clinical presenting features, laboratory investigations, complications, and mortality associated with the disease. The clinical diagnosis of DHF was in accordance with WHO recommendations. Epistaxis, gingivitis, haematemesis and gastritis were among the common complications. Platelet levels tended to decline from a higher value on admisssion (mean 67 000/mm³) to lower levels on subsequent days, with the lowest (mean 61 000/mm³) being on day 6 of the fever. Hyponatraemia (46.8%) was commonly observed. Morbidity of DHF was significant (29.4%) but the case fatality rate remained low (2.0%) in our adults, suggesting that adults are less likely than children to suffer from shock syndrome.     

Surface and fluid phase activities of two forms of activated Hageman factor produced during contact activation of plasma

The ability of the two forms of activated Hageman factor (HFa) produced during contact activation of plasma to activate prekallikrein and factor XI was studied. alpha-HFa, defined as an 80,000 mol wt two-chain enzyme which remains bound to the surface was capable of cleaving surface-bound prekallikrein and factor XI. beta-HFa, a 28,000 mol wt single chain molecule, released from the surface during contact activation was able to cleave prekallikrein but showed no activity on factor XI. Cleavage of prekallikrein by beta-HFa occurred irrespective of whether the substrate was surface-bound or in solution. Cleavage of factor XI occurred only when it was surface bound and only the alpha- form of HFa was capable of this proteolytic action. Factor XI was found to remain bound to the surface while prekallikrein and kallikrein rapidly dissociated from the surface into the supernate. These findings suggest that the initiation of intrinsic coagulation through the activation factor XI is a localized event occurring at the site of contact activation and is the result of the action of alpha-HFa. By contrast, kinin generation and fibrinolysis resulting from the formation of kallikrein can be initiated either at the site of contact activation, by alpha-HFa action, or throughout the plasma, by beta-HFa; further dissemination of these activities is assured by the rapid dissociation of kallikrein itself from the surface.     

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.