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.     

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.     

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.     

Sequential changes in the concentration of specific serum proteins during typhoid fever infection in man.

An automated immunoprecipitin system has been utilized to quantitate the concentration of 10 specific proteins in the plasma of man. Values obtained by this technique are in agreement with the published concentrations for these specific plasma proteins. This technique was utilized to determine the sequential change s in 10 individual plasma proteins of volunteers exposed to Salmonella typhi. In those volunteers who developed typical typhoid fever, plasma concentrations of the acute phase proteins, alpha1-acid glycoprotein, alpha1-antitrypsin, and haptoglobin, as well as C3 complement were significantly increased with the onset of febrile illness. In contrast, the concentration of plasma albumin and tranferrin were depressed while plasma IgM became elevated during early convalescence from this infection. No significant changes were observed in the plasma concentrations of alpha2-macroglobulin, IgG, or IgA. In the exposed volunteers who did not become ill, the only significant change was a brief depression of alpha1-antitrypsin. During typhoid fever the patterns of change for individual plasma acute-phase globulins were different from those reported for patients with hepatitis, myocaridal infarction, or surgery.     

Separation of Plasma Thromboplastin Antecedent from Kallikrein by the Plasma α2-Macroglobulin, Kallikrein Inhibitor

Plasma thromboplastin antecedent (PTA, factor XI) is an important intermediate in the intrinsic coagulation system, and plasma kallikrein has been implicated as a mediator of the inflammatory process. Whereas their biologic activities are functionally distinct, their identity as separate entities in plasma has not been fully established, and the nature of their plasma inhibitors has not been completely characterized. A partially purified preparation containing the clotting, tosyl arginine methyl ester (TAMe) esterase and kinin-producing activities of these substances has been prepared by DEAE-cellulose chromatography of a Celite eluate obtained from acid-treated human plasma. These activities were not separable by acrylamide gel electrophoresis nor by isoelectric focusing, their pI being approximately 8.7. Human plasma alpha(2)-macroglobulin has been shown to inhibit the proteolytic activity of kallikrein and to inhibit partially its TAMe esterase activity. An alpha(2)-macroglobulin, PTA, kallikrein incubation mixture was separated by gel filtration chromatography. The alpha(2)-macroglobulin formed a high molecular weight complex with kallikrein and appeared in early chromatographic fractions. The PTA-clotting activity was not inhibited by the alpha(2)-macroglobulin; 64% of the initial PTA activity was isolated in later fractions free of kallikrein-induced kinin-like activity. In contrast, clotting, TAMe esterase, and kinin-forming activities were inhibited after gel filtration chromatography of an incubation mixture of these activities and partially purified C1 inactivator (C1 esterase inhibitor). Electrofocusing of an incubation mixture of an activated PTA, kallikrein preparation, and alpha(2)-macroglobulin resulted in the isolation of a PTA fraction free of kallikrein proteolytic activity, and with 4% of the original TAMe esterase activity. In this manner, activated PTA and plasma kallikrein have been shown to be distinct substances, and methods have been introduced for the further purification of active coagulation factor XI.     

Inhibitors of Kallikrein in Human Plasma

Human plasma was fractionated by ammonium sulfate precipitation, DEAE-cellulose chromatography, and Sephadex G-200 gel filtration to determine which method would give the greatest number of clearly separable kallikrein inhibitory peaks. With G-200 gel filtration three peaks could be separated which were demonstrated to contain alpha(2)-macroglobulin, C1 inactivator, and alpha(1)-antitrypsin. No other kallikrein inhibitors could be identified. The fractions containing C1 inactivator and alpha(2)-macroglobulin appeared to be more effective against kallikrein than that containing alpha(1)-antitrypsin. A patient with hereditary angioneurotic edema was shown to have an abnormal C1 inactivator protein capable of interfering with kallikrein's biologic, but not its esterolytic activity. Heat-treated human plasma, a commonly used source of kininogen for experiments with kallikrein, was shown to have kallikrein inhibitory activity.     

Alpha-1-antitrypsin-Pittsburgh. A potent inhibitor of human plasma factor XIa, kallikrein, and factor XIIf.

Alpha-1-antitrypsin-Pittsburgh is a human variant that resulted from a point mutation in the plasma protease inhibitor, alpha 1-antitrypsin (358 Met----Arg). This defect in the alpha 1-antitrypsin molecule causes it to have greatly diminished anti-elastase activity but markedly increased antithrombin activity. In this report, we demonstrate that this variant protein also has greatly increased inhibitory activity towards the arginine-specific enzymes of the contact system of plasma proteolysis (Factor XIa, kallikrein, and Factor XIIf), in contrast to normal alpha 1-antitrypsin, which has modest to no inhibitory activity towards these enzymes. We determined the second-order-inactivation rate constant (k') of purified, human Factor XIa by purified alpha 1-antitrypsin-Pittsburgh and found it to be 5.1 X 10(5) M-1 s-1 (23 degrees C), which is a 7,700-fold increase over the k' for Factor XIa by its major inhibitor, normal purified alpha 1-antitrypsin (i.e., 6.6 X 10(1) M-1 s-1). Human plasma kallikrein, which is poorly inhibited by alpha 1-antitrypsin (k' = 4.2 M-1 s-1), exhibited a k' for alpha 1-antitrypsin-Pittsburgh of 8.9 X 10(4) M-1 s-1 (a 21,000-fold increase), making it a more efficient inhibitor than either of the naturally occurring major inhibitors of kallikrein (C-1-inhibitor and alpha 2-macroglobulin). Factor XIIf, which is not inhibited by normal alpha 1-antitrypsin, displayed a k' for alpha 1-antitrypsin-Pittsburgh of 2.5 X 10(4) M-1 s-1. This enhanced inhibitory activity is similar to the effect of alpha 1-antitrypsin-Pittsburgh that has been reported for thrombin. In addition to its potential as an anticoagulant, this recently cloned protein may prove to be clinically valuable in the management of septic shock, hereditary angioedema, or other syndromes involving activation of the surface-mediated plasma proteolytic system.     

Evaluation of the plasma kinin system in dengue hemorrhagic fever.

The role of the plasma kinin system in the pathogenesis of dengue hemorrhagic fever (DHF) ws explored by simultaneously measuring factor XLL (Hageman), prekallikrein, kallikrein inhibitors, bradykinin, and complement (C3) in the blood of Thai children with DHF and acute febrile illnesses other than dengue. Prekallikrein, factor XII, and C3 levels were significantly lower in DHF patients compared to fever control patients with the lowest mean levels found in dengue patients with shock. However, bradykinin concentrations were not elevated and mean activity levels of kallikrein inhibitors were not depressed in dengue patients. Two dengue patients first studies at least 2 days before onset of shock had falling C3 levels which were more closely related temporally to the onset of shock than were their rising levels of prekallikrein. The results fail to provide convincing evidence ofr activation of the plasma kinin system leading to free bradykinin or a significant role for bradykinin in the immunopathogenesis of DHF. By contrast, the results refocus attention on complement as a potentially important humoral mediator of the dengue shock syndrome.     

Protection by recombinant alpha 1-antitrypsin Ala357 Arg358 against arterial hypotension induced by factor XII fragment.

The specificity of serpin superfamily protease inhibitors such as alpha 1-antitrypsin or C1 inhibitor is determined by the amino acid residues of the inhibitor reactive center. To obtain an inhibitor that would be specific for the plasma kallikrein-kinin system enzymes, we have constructed an antitrypsin mutant having Arg at the reactive center P1 residue (position 358) and Ala at residue P2 (position 357). These modifications were made because C1 inhibitor, the major natural inhibitor of kallikrein and Factor XIIa, contains Arg at P1 and Ala at P2. In vitro, the novel inhibitor, alpha 1-antitrypsin Ala357 Arg358, was more efficient than C1 inhibitor for inhibiting kallikrein. Furthermore, Wistar rats pretreated with alpha 1-antitrypsin Ala357 Arg358 were partially protected from the circulatory collapse caused by the administration of beta-Factor XIIa.     

The use of the kallikrein-kininogen system indices in the prognosis of the development of hemorrhagic erysipelas]

A number of the kallikrein-kinin system parameters (kallikrein, prekallikrein, total arginine esterase activity, alpha 1 protease inhibitor, and alpha 2 macroglobulin) were measured in 59 patients with erythematous erysipelas and in 51 ones with hemorrhagic erysipelas over the course of the disease. Marked activation of the blood kallikrein-kinin system was seen in all the patients during the initial period of the disease, manifesting by elevated levels of kallikrein, total arginine esterase activity, alpha 1 protease inhibitor, alpha 2 macroglobulin, and a lowered prekallikrein concentration. In erythematous erysipelas the peak of activation was recorded in the first days of the disease, whereas in hemorrhagic condition it was observed during the second week of erysipelatous inflammation. Different patterns of changes in the kallikrein-kinin system over the course of the disease permit using one of its parameters, kallikrein activity, for the prediction of the development of local hemorrhagic syndrome in erysipelas patients already during the earliest (prehemorrhagic) stage of the condition.     

High concentrations of free kinins and kinin system components in abdominal transudate of a patient with nephrotic syndrome.

High levels of bradykinin (60--80 ng/ml) were found in abdominal transudate from a patient with nephrotic syndrome caused by chronic glomerulonephritis. The abdominal transudate contained neutral kininogenase and its precursor, identified with plasma kallikrein and prekallikrein, respectively, as well as both forms of kininogen, the low-molecular-weight form predominating. The abdominal transudate was characterized also by very low kininase activity and low levels of alpha 1-antitrypsin (0.46 g/l) and alpha 2-macroglobulin. Large amounts of very low density lipoproteins were present in the transudate. Despite the difference in total protein content between the abdominal transudate and the patient's serum (4.3 g/l and 48 g/l, respectively) their protein fraction composition was similar. The data obtained suggest that bradykinin is important in maintenance of long-lasting blood vessel hyperpermeability, which, in turn, is a driving force in the pathogenesis of refractory nephrotic edema.     

Amidolytic and immuno-nephelometric determination of alpha 1-proteinase inhibitor and alpha 2-macroglobulin in serum with calculation of specific inhibitor activities in health and disease

In sera of healthy persons (n = 50) and patients with a variety of diseases (n = 197) the two major proteinase inhibitors, alpha 1-proteinase inhibitor (alpha 1-antitrypsin) and alpha 2-macroglobulin, were measured by two methods: a chromogenic (amidolytic) substrate assay to assess the functional activities, and a laser nephelometric method to determine the immunoreactive concentrations of the respective proteins. The specific proteinase inhibitor activities defined as the number of inhibitor units per g inhibitor protein were calculated. The precision and accuracy of both assays were found to be similar, showing a satisfactory correlation of results for the sera of healthy persons (r = 0.916 for alpha 2-macroglobulin and 0.988 for alpha 1-proteinase inhibitor). In diseased individuals the correlation was lower than in normal persons (0.862 for alpha 2-macroglobulin and 0.907 for alpha 1-proteinase inhibitor). A poor correlation was obtained in patients with liver diseases (r = 0.586 for alpha 1-proteinase inhibitor and 0.852 for alpha 2-macroglobulin). Reference ranges were established for functional and immunological concentrations and for specific inhibitor activities, respectively. Normal values followed a Gaussian distribution. In patients with various diseases including those with acute phase response, the specific inhibitor activities of alpha 1-proteinase inhibitor are reduced significantly; this is because inhibitor activity shows a smaller relative increase than immunoreactivity. Among the various diseases, no significant differences were noted. The specific inhibitor activity of alpha 2-macroglobulin changed significantly only in patients with carcinoma, liver diseases and trauma. Follow up of some patients shows also intraindividual variation of specific proteinase inhibitor activities.     

The role of the kallikrein-kinin and renin-angiotensin-aldosterone systems in the development of hypertension in glomerulonephritis

A total of 74 patients with various clinicomorphological variants of glomerulonephritis (GN) were examined. Only a high activity of the enzyme kinase-1 that destroys kinins and the kallikrein inhibitors alpha 1-antitrypsin, alpha 2-macroglobulin is a contribution of the kallikrein-kinin system made to the general antihypertensive "armoury" of the body, as shown by the study. The correlation between the kallikrein activity and the active renin/total renin ratio predetermines that kallikrein may participate in endogenous plasma renin activation in GN patients. In this case, the vasoconstrictive effect of renin may limit the antihypertensive action of kallikrein and kinins by a feedback mechanism.     

Variability of plasma proteins according to molecular size. Long-term and short-term intra-individual variation

The intra-individual variations in serum concentrations of alpha 1-antitrypsin, albumin and alpha 2-macroglobulin were determined using high precision analytical methods. The long-term (3 months) variations were 8.2% for alpha 1-antitrypsin and 2.9% for alpha 2-macroglobulin in five males and five females. The coefficients of variation for albumin were 1.5 and 3.4% for males and females, respectively. In males the long-term variations of albumin and alpha 2-macroglobulin were highly correlated. The short-term (2 days) intra-individual variations in six males were 2.5, 3.8 and 3.4% for alpha 1-antitrypsin, albumin and alpha 2-macroglobulin respectively (coefficients of variation). A diurnal variation was found for albumin with maximal concentrations at 18.00 hours. At 6.00 and 10.00 hours the fractional concentrations of alpha 1-antitrypsin and albumin were lower than for alpha 2-macroglobulin. The variations of the three proteins were positively correlated.     

Neutral proteases of human granulocytes. IV. Interaction between human granulocyte collagenase and plasma protease inhibitors.

Purified human granulocyte collagenase was inactivated by serum through the formation of complexes with alpha 1-antitrypsin and alpha 2-macroglobulin. A molar combining ratio of 1:1 was observed for each inhibitor. The affinity of alpha 2-macroglobulin was about 10 times that of alpha 1-antitrypsin for granulocyte collagenase. The molar concentration of alpha 1-antitrypsin in the blood exceeds that of alpha 2-macroglobulin by about 12 times, so that the inhibitors may be equally important for defence against granulocyte collagenase.     

Determination of functional activity of alpha 1-protease inhibitor and alpha 2-macroglobulin in human plasma using elastase

The competitive binding of human alpha 1-antitrypsin and human alpha 2-macroglobulin to porcine pancreatic elastase was studied. Mixtures of these two protease inhibitors, when titrated against elastase give inhibition curves analogous to those obtained with human plasma. This is however not the case when the individual inhibitors are used. A theoretical treatment enabled us to devise an assay method to determine the amounts of functional activity of alpha 1-protease inhibitor and alpha 2-macroglobulin respectively in human 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.     

Alteration of factor VII activity by activated Fletcher factor (a plasma kallikrein): a potential link between the intrinsic and extrinsic blood-clotting systems.

Although surface contact is known to accelerate the one-stage prothrombin time of human plasma through the participation of Hageman factor (factor XII) and factor VII, it has not been clear whether Hageman factor interacts with factor VII directly or indirectly. Recently, Gj?nnaess reported experiments suggesting that plasma kallikrein was an intermediate between Hageman factor and factor VII. The present study was undertaken to elucidate the interaction of plasma kallikrein and factor VII. Incubation of Fletcher-trait plasma (deficient in a plasma prekallikrein) with kaolin at 0 degrees C. did not induce shortening of the Thrombotest time or enhancement of factor VII activity, in contrast to studies of normal plasma. Monospecific rabbit antiserum against plasma kallikrein blocked the shortening of the Thrombotest time of normal plasma brought about by kaolin. Purified Hageman factor fragments (prekallikrein activator) induced an increase in factor VII activity in normal or Hageman-trait plasma, but not in Fletcher-trait plasma. A purified plasma kallikrein preparation enhanced factor VII activity in all plasmas, including that of Fletcher-trait plasma. The effect of the kallikrein preparation was blocked by soybean trypsin inhibitor, Trasylol, or rabbit antiserum against kallikrein, but not by lima bean trypsin inhibitor or antiserum against Hageman factor. The activity of partially purified factor VII was enhanced by purified kallikrein in the presence, but not in the absence of factor VII-deficient plasma. These results further support the idea that the enhancement of factor VII activity by surface contact is via Hageman factor and plasma kallikrein, suggesting a possible link between the intrinsic and extrinsic pathway of blood clotting. The significance of this phenomenon in hemostasis in vivo remains to be elucidated.     

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.     

Cationic proteins of human granulocytes. V. Interaction with plasma protease inhibitors.

Several cationic proteins of human granulocytes possess chymotrypsin-like and bactericidal activities. The heat-labile chymotrypsin-like activity is inhibited by serum, owing to complex formation with alpha2-macroglobulin and alpha1-antitrypsin. The molar affinity of the cationic proteins for alpha2-macroglobulin is much higher than that for alpha1-antitrypsin. The results indicate that the molar combining ratios are 1:1 for cationic protein to alpha1-antitrypsin and 2:1 for cationic protein to alpha2-macroglobulin. The proteolytic activity against fibrinogen and casein is inhibited by both alpha2-macroglobulin and alpha1-antitrypsin, whereas the activity against small molecular synthetic substrates is inhibited by alpha1-antitrypsin but not alpha2-macroglobulin. The heat-stable bactericidal action of the cationic proteins against Staphylococcus was also inhibited by serum, probably owing to complex formation with alpha2-macroglobulin and alpha1-antitrypsin.