Contribution of Plasma Protease Inhibitors to the Inactivation of Kallikrein in Plasma

Although l-inhibitor (l-INH) and α₂-macroglobulin (α₂M) have been reported as the major inhibitors of plasma kallikrein in normal plasma, there is little quantitative support for this conclusion. Thus, we studied the inactivation of purified kallikrein in normal plasma, as well as in plasma congenitally deficient in l-INH, or artificially depleted of α₂M by chemical modification of the inhibitor with methylamine. Under pseudo-first-order conditions, the inactivation rate constant of kallikrein in normal plasma was 0.60 min⁻¹. This rate constant was reduced to 0.35, 0.30, and 0.06 min⁻¹, in plasma deficient respectively in l-INH, α₂M, or both inhibitors. Thus l-INH (42%) and α₂M (50%) were found to be the major inhibitors of kallikrein in normal plasma. Moreover all the other protease inhibitors present in normal plasma contributed only for 8% to the inactivation of the enzyme. To confirm these kinetic results, ¹²⁵I-kallikrein (M_r 85,000) was completely inactivated by various plasma samples, and the resulting mixtures were analyzed by gel filtration on Sepharose 6B CL for the appearance of ¹²⁵I-kallikrein-inhibitor complexes. After inactivation by normal plasma, 52% of the active enzyme were found to form a complex (M_r 370,000) with l-INH, while 48% formed a complex (M_r 850,000) with α₂M. After inactivation by l-INH-deficient plasma, >90% of the active ¹²⁵I-kallikrein was associated with α₂M. A similar proportion of the label was associated with l-INH in plasma deficient in α₂M. After inactivation by plasma deficient in both l-INH and α₂M, ¹²⁵I-kallikrein was found to form a complex of M_r 185,000. This latter complex, which may involve antithrombin III, α₁-protease inhibitor, and/or α₁-plasmin inhibitor, was not detectable in appreciable concentrations in the presence of either l-INH or α₂M, even after the addition of heparin (2 U/ml). These observations demonstrate that l-INH and α₂M are the only significant inhibitors of kallikrein in normal plasma confirming previous predictions based on experiments in purified systems. Moreover, in the absence of either l-INH or α₂M, the inactivation of kallikrein becomes almost entirely dependent on the other major inhibitor.     

Recombinant C1 inhibitor P5/P3 variants display resistance to catalytic inactivation by stimulated neutrophils.

Proteolytic inactivation of serine protease inhibitors (serpins) by neutrophil elastase (HNE) is presumed to contribute to the deregulation of plasma cascade systems in septic shock. Here, we report a supplementary approach to construct serpins, in our case C1 inhibitor, that are resistant to catalytic inactivation by HNE. Instead of shifting the specificity of alpha 1-antitrypsin towards the proteases of the contact activation and complement systems, we attempted to obtain a C1 inhibitor species which resists proteolytic inactivation by HNE. 12 recombinant C1 inhibitor variants were produced with mainly conservative substitutions at the cleavage sites for HNE, 440-Ile and/or 442-Val. Three variants significantly resisted proteolytic inactivation, both by purified HNE, as well as by activated neutrophils. The increase in functional half-life in the presence of FMLP-stimulated cells was found to be 18-fold for the 440-Leu/442-Ala variant. Inhibitory function of these variants was relatively unimpaired, as examined by the formation of stable complexes with C1s, beta-Factor XIIa, kallikrein, and plasmin, and as determined by kinetic analysis. The calculated association rate constants (k(on)) were reduced twofold at most for C1s, and appeared unaffected for beta-Factor XIIa. The effect on the k(on) with kallikrein was more pronounced, ranging from a significant ninefold reduction to an unmodified rate. The results show that the reactive centre loop of C1 inhibitor can be modified towards decreased sensitivity for nontarget proteases without loss of specificity for target proteases. We conclude that this approach extends the possibilities of applying recombinant serpin variants for therapeutic use in inflammatory diseases.     

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.     

Inactivation of factor XII active fragment in normal plasma. Predominant role of C-1-inhibitor

To define the factors responsible for the inactivation of the active fragment derived from Factor XII (Factor XIIf ) in plasma, we studied the inactivation kinetics of Factor XIIf in various purified and plasma mixtures. We also analyzed the formation of 125I-Factor XIIf -inhibitor complexes by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). In purified systems, the bimolecular rate constants for the reactions of Factor XIIf with C-1-inhibitor, alpha 2-antiplasmin, and antithrombin III were 18.5, 0.91, and 0.32 X 10(4) M-1 min-1, respectively. Furthermore, SDS-PAGE analysis revealed that 1:1 stoichiometric complexes were formed between 125I-Factor XIIf and each of these three inhibitors. In contrast, kinetic and SDS-PAGE studies indicated that Factor XIIf did not react with alpha 1-antitrypsin or alpha 2-macroglobulin. The inactivation rate constant of Factor XIIf by prekallikrein-deficient plasma was 14.4 X 10(-2) min-1, a value that was essentially identical to the value predicted from the studies in purified systems (15.5 X 10(-2) min-1). This constant was reduced to 1.8 X 10(-2) min-1 when Factor XIIf was inactivated by prekallikrein-deficient plasma that had been immunodepleted (less than 5%) of C-1-inhibitor. In addition, after inactivation in normal plasma, 74% of the active 125I-Factor XIIf was found to form a complex with C-1-inhibitor, whereas 26% of the enzyme formed complexes with alpha 2-antiplasmin and antithrombin III. Furthermore, 42% of the labeled enzyme was still complexed with C-1-inhibitor when 125I-Factor XII was inactivated in hereditary angioedema plasma that contained 32% of functional C-1-inhibitor. This study quantitatively demonstrates the dominant role of C-1-inhibitor in the inactivation of Factor XIIf in the plasma milieu.     

An alpha 2-macroglobulin receptor-dependent mechanism for the plasma clearance of transforming growth factor-beta 1 in mice.

Radioiodinated transforming growth factor-beta 1 (TGF-beta 1) bound to the plasma proteinase inhibitor, alpha 2-macroglobulin (alpha 2M), as determined by chromatography on Superose-6 and native polyacrylamide gel electrophoresis. When alpha 2M conformational change was induced with methylamine, 125I-TGF-beta 1 binding significantly increased. Intravenously injected 125I-TGF-beta 1 cleared from the circulation of mice rapidly at first; however, intravascular radioactivity stabilized near 20% of the initial level. At necropsy, radioactivity was recovered predominantly in the liver (65%); however, the density of radioactivity (disintegrations per minute/g organ wt) was highest in the lungs. Markedly different results were obtained with purified 125I-TGF-beta 1-alpha 2M-methylamine complex. Clearance of the complex occurred as a first-order process with a t1/2 of 4 min. Greater than 90% of the radioactivity was recovered in the liver. The clearance and distribution of 125I-TGF-beta 1-alpha 2M-methylamine were equivalent to those observed with 125I-alpha 2M-methylamine and 125I-alpha 2M-trypsin. The latter two radioligands clear via specific alpha 2M receptors in the liver. Large molar excesses of alpha 2M-trypsin or alpha 2M-methylamine competed with 125I-TGF-beta 1-alpha 2M-methylamine for plasma clearance. Native alpha 2M, which does not bind to the alpha 2M receptor, did not compete. The receptor binding domain of alpha 2M-methylamine was blocked by chemical modification or enzyme treatment. The resulting alpha 2M preparations still bound 125I-TGF-beta 1; however, the complexes did not clear when injected intravenously in mice. The studies presented here demonstrate that alpha 2M can mediate the plasma clearance of a growth factor via the alpha 2M receptor system. We propose that alpha 2M, the alpha 2M receptor, and proteinases may function as a concerted system to regulate TGF-beta 1 activity and the activity of related factors in vivo.     

Z-type alpha 1-antitrypsin is less competent than M1-type alpha 1-antitrypsin as an inhibitor of neutrophil elastase.

Alpha 1-antitrypsin (alpha 1AT) deficiency resulting from homozygous inheritance of the Z-type alpha 1AT gene is associated with serum alpha 1AT levels of less than 50 mg/dl and the development of emphysema in the third to fourth decades. Despite the overwhelming evidence that the emphysema of PiZZ individuals develops because of a "deficiency" of alpha 1AT and hence an insufficient antineutrophil elastase defense of the lung, epidemiologic evidence has shown that levels of alpha 1AT of only 80 mg/dl protect the lung from an increased risk of emphysema. With this background, we hypothesized that homozygous inheritance of the Z-type may confer an added risk beyond a simple deficiency of alpha 1AT by virtue of an inability of the Z-type alpha 1AT molecule to inhibit neutrophil elastase as effectively as the common M1-type molecule. To evaluate this hypothesis, the functional status of alpha 1AT from PiZZ individuals (n = 10) was compared with that of alpha 1AT from PiM1M1 individuals (n = 7) for its ability to inhibit neutrophil elastase (percent inhibition) as well as its association rate constant for neutrophil elastase (K association). Plasma alpha 1AT concentration, measured by radial immunodiffusion, was 34 +/- 1 mg/dl in PiZZ patients vs. 237 +/- 14 mg/dl for PiM1M1 plasma, a sevenfold difference. When titrated against neutrophil elastase, the present inhibition of PiZZ plasma was significantly less than Pi M1M1 plasma (ZZ 78 +/- 1% vs. M1M1 95 +/- 1%, P less than 0.001) as was purified Z type alpha 1AT (ZZ, 63 +/- 2% vs. M1M1 86 +/- 2%, P less than 0.001). Sodium dodecyl sulfate (SDS) gel comparisons of the complexes formed with M1-type alpha 1AT and Z-type alpha 1AT with elastase demonstrated the Z alpha 1AT-elastase complexes were less stable than the M1 alpha 1AT-elastase complexes, thus releasing some of the enzyme to continue to function as a protease. Consistent with these observations, the K association of purified Z-type alpha 1AT for neutrophil elastase was lower than that of M1-type alpha 1AT (ZZ 4.5 +/- 0.3 X 10(6) M-1s-1 vs. M1M1 9.7 +/- 0.4 X 10(6) M-1s-1, P less than 0.001), suggesting that for the population of alpha 1AT molecules, the active Z-type molecules take more than twice as long as the active M1-type alpha 1AT to inhibit neutrophil elastase. Consequently, not only is there less alpha1AT in PiZZ individuals, but the population of Z-type alpha1AT molecules is less competent as an inhibitor of neutrophil elastase than M1-type alpha1AT molecules. This combination of defects suggests that PiZZ individuals have far less functional antielastase protection than suggested by the reduced concentrations of alpha1AT alone, further explaining their profound risk for development of emphysema.     

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.     

Turnover of 125I-labelled tissue kallikrein following intraduodenal or intravenous administration

Tissue kallikrein is released in the body both physiologically and in many inflammatory disorders. Little is, however, known about the turnover of released tissue kallikrein in humans. Approximately 1 mg of tissue kallikrein (mol wt 43,000 Da) was purified from 85 L human urine by: (1) ultracentrifugation, (2) filtration through an aprotinin-coupled Sepharose 4B column, followed by (3) gel filtration over a Sephadex G-75 column. The elimination, after intraduodenal or intravenous administration of purified tissue kallikrein radiolabelled with 125I, was followed by collecting serial samples of plasma, urine and faeces from three volunteers. Within 72 h, about 96% of the intraduodenally administered radioactivity had been excreted in urine, and approximately 5.4% in faeces, mainly as 125I. No intact 125I-tissue kallikrein was found in plasma, urine or faeces after the intraduodenal instillation of the protein. The plasma half-life of 125I-tissue kallikrein up to 3 h after intravenous injection was 9 min and, thereafter, 20 h. The 125I-tissue kallikrein was quickly bound to a plasma protein with a mol wt of about 67 kDa, but some of the radioiodinated tissue kallikrein was still unbound 15 min after injection, judged by gel filtration on Sephadex G-200 columns. Most of the radioactivity was excreted in the urine as 125I, but about 4-6% was recovered as free 125I-tissue kallikrein. CONCLUSION: The use of tissue kallikrein as an oral drug appears, therefore, to be useless. Tissue kallikrein released into plasma seems to be quickly bound to a protein with a mol wt of 67 kDa, probably kallistatin or Protein C inhibitor, but some tissue kallikrein seems to be unbound and may have some physiological or pathophysiological action. The unbound tissue kallikrein is, at least partly, cleared from the circulation by the kidneys, and tissue kallikrein in the urine may partly be derived from plasma.     

Demonstration of modified inactive first component of complement (C1) inhibitor in the plasmas of C1 inhibitor-deficient patients

The first component of complement (C1) inhibitor plays a critical role in the regulation of the classical complement pathway and the contact system, and the deficiency of C1 inhibitor protein or function is associated with recurrent angioedema. In this study we evaluated the size of the C1 inhibitor antigens present in the plasmas of C1 inhibitor-deficient patients. We found that the C1 inhibitor in the plasmas existed in three forms: high molecular weight forms in complex with proteases, native 110-kD C1 inhibitor, and a modified inactive 94-kD form. The proportion of the total C1 inhibitor in the 94-kD form was 28% in nine hereditary angioedema patients, 92% in five acquired C1 inhibitor-deficiency patients, and 1.2% in five normal controls. In vitro activation of normal plasma with kaolin, but not heat-aggregated gamma-globulin generated 94-kD C1 inhibitor from 110-kD C1 inhibitor. Neither kaolin activation nor heat-aggregated gamma-globulin activation generated 94-kD C1 inhibitor in Hageman factor-deficient plasma. These results suggest that 94-kD C1 inhibitor is generated in vitro by activation of the contact system. The in vivo mechanism of 94-kD C1 inhibitor generation in C1 inhibitor-deficient patients is not known.     

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.     

Turnover of 125I-labelled tissue kallikrein following intraduodenal or intravenous administration

Tissue kallikrein is released in the body both physiologically and in many inflammatory disorders. Little is, however, known about the turnover of released tissue kallikrein in humans. Approximately 1 mg of tissue kallikrein (mol wt 43 000 Da) was purified from 85 L human urine by: (1) ultracentrifugation, (2) filtration through an aprotinin-coupled Sepharose 4B column, followed by (3) gel filtration over a Sephadex G-75 column. The elimination, after intraduodenal or intravenous administration of purified tissue kallikrein radiolabelled with ¹²⁵I, was followed by collecting serial samples of plasma, urine and faeces from three volunteers. Within 72 h, about 96% of the intraduodenally administered radioactivity had been excreted in urine, and approximately 5.4% in faeces, mainly as ¹²⁵I. No intact ¹²⁵I-tissue kallikrein was found in plasma, urine or faeces after the intraduodenal instillation of the protein. The plasma half-life of ¹²⁵I-tissue kallikrein up to 3 h after intravenous injection was 9 min and, thereafter, 20 h. The ¹²⁵I-tissue kallikrein was quickly bound to a plasma protein with a mol wt of about 67 kDa, but some of the radioiodinated tissue kallikrein was still unbound 15 min after injection, judged by gel filtration on Sephadex G-200 columns. Most of the radioactivity was excreted in the urine as ¹²⁵I, but about 4- 6% was recovered as free ¹²⁵I-tissue kallikrein. Conclusion: The use of tissue kallikrein as an oral drug appears, therefore, to be useless. Tissue kallikrein released into plasma seems to be quickly bound to a protein with a mol wt of 67 kDa, probably kallistatin or Protein C inhibitor, but some tissue kallikrein seems to be unbound and may have some physiological or pathophysiological action. The unbound tissue kallikrein is, at least partly, cleared from the circulation by the kidneys, and tissue kallikrein in the urine may partly be derived from plasma.     

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.     

Studies on the renin-angiotensin system in a kininogen-deficient individual

The physiological responses of the renin-angiotensin system were studied in an individual with kininogen deficiency (patient 1) with absent plasma bradykinin and markedly impaired pre-kallikrein conversion into kallikrein. After sodium depletion, patient 1 had a low plasma renin activity (1.4 pmol of ANG I h-1 ml-1) and a low angiotensin II concentration (36 pg/ml) compared with values in 11 normal individuals (4.0 +/- 0.94 pmol of ANG I h-1 ml-1) and 63 +/- 6 pg/ml respectively). Unlike normal individuals, in the kininogen-deficient subject there was no significant fall of renin activity or angiotensin II after dietary sodium repletion. Intravenous sodium repletion also failed to further decrease plasma renin activity or angiotensin II. The usual two- to three-fold rise in plasma renin activity and angiotensin II observed in normal subjects on assumption of the upright posture after ingestion of 200 mg of sodium/day failed to occur in the kininogen-deficient individual. These data in vivo are in agreement with observations in vitro that once plasma kallikrein forms it may be important in converting prorenin into renin. In the absence of kininogen, activation of prekallikrein to kallikrein is grossly defective, which may in part account for the diminished response of the renin-angiotensin system to changes in sodium balance and posture.     

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.     

Complex formation between human kallikrein 13 and serum protease inhibitors

BACKGROUND: The kallikrein family is a group of 15 serine protease genes clustered on chromosome 19q13.4. Human kallikrein gene 13 (KLK13) is a member of this family and encodes for a trypsin-like, secreted serine protease (hK13). Given that other kallikreins are sequestered by serum protease inhibitors, we hypothesized that hK13 may also interact with similar inhibitors. Our objective was to identify serum protease inhibitors that interact with human hK13. METHODS: Recombinant hK13 produced in yeast was added to male and female sera and various biological fluids and the spiked samples were analyzed with an hK13 ELISA assay. Enzymatically active hK13 was 125I-labeled and used in in vitro reactions with candidate protease inhibitors and serum samples. The mixtures were then subjected to gel filtration and SDS-PAGE analysis. Candidate inhibitors were also tested in enzymatic assays of hK13 activity. RESULTS: The recovery of recombinant hK13 from male and female sera, measured by three versions of the hK13-ELISA, ranged from 5% to 10%. The same recovery was obtained when serum samples from males and females were spiked with hK13 from amniotic fluid and seminal plasma. However, when hK13 was added to other biological fluids, such as amniotic fluid and breast milk, recovery ranged from 70% to 98%. In vitro analysis indicated that enzymatically active 125I-labeled hK13 forms SDS-stable complexes with alpha2-antiplasmin, alpha2-macroglobulin and alpha1-antichymotrypsin. When added to serum, active hK13 formed stable complexes with molecular masses corresponding to hK13 and the inhibitors mentioned above. CONCLUSIONS: hK13 interacts and forms complexes with serum protease inhibitors, including alpha2-macroglobulin, alpha1-antichymotrypsin and alpha2-antiplasmin.     

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 Inhibitors of the Components of the Fibrinolytic Pathway in Man

The effect of highly purified inhibitor of the first component of complement (CāINH), alpha2 macroglobulin (alpha2M), and alpha1 antitrypsin on the components of the fibrinolytic pathway in human plasma has been examined. CāINH was the only factor active upon the Hageman factor fragments functioning at the initial step of the fibrinolytic pathway, alpha2M was the only factor active against the plasminogen activator and the most active inhibitor of plasmin. The inhibition of plasmin by alpha2M appeared stoichiometric with one molecule of alpha2M inhibiting two molecules of plasmin. All three plasma inhibitors were active against plasmin.     

Recombinant alpha 1-antitrypsin Pittsburgh (Met 358----Arg) is a potent inhibitor of plasma kallikrein and activated factor XII fragment.

In normal plasma, the serine protease inhibitor alpha 1-antitrypsin (alpha 1-AT) plays little or no role in the control of plasma kallikrein or activated Factor XII fragment (Factor XIIf), this function being performed by Cl-inhibitor. Recently, an alpha 1-AT variant was described with a Met----Arg mutation at the reactive center P1 residue (position 358) which altered the specificity of inhibition from the Met- or Val-specific protease neutrophil elastase to thrombin, an Arg-specific protease. We have now examined the inhibition of plasma kallikrein and Factor XIIf, both Arg-specific enzymes, with recombinant alpha 1-AT(Met358----Arg) produced by an Escherichia coli strain carrying a mutated human alpha 1-AT gene. The engineered protein was a very efficient inhibitor of both enzymes. It was more effective than Cl-inhibitor by a factor of 4.1 for kallikrein and 11.5 for Factor XIIf. These results suggest that recombinant alpha 1-AT(Met358----Arg) has therapeutic potential for disease states where activation of the plasma kinin-forming system is observed, for example in hereditary angioedema or septic shock.     

HUMAN PLASMA ALPHA 2-MACROGLOBULIN : AN INHIBITOR OF PLASMA KALLIKREIN

Activation of plasma kallikrein arginine esterase activity by kaolin resulted in peak activity at 1 min of incubation and a 50% reduction in activity at 5 min in normal plasma, and 30% reduction in the plasma of patients with hereditary angioneurotic edema who lacked the C1 inactivator. The peak esterolytic activity was inhibited by soybean trypsin inhibitor whereas the 5 min activity was resistant to this inhibitor. Acid treatment of normal and hereditary angioneurotic edema plasma destroyed the factor responsible for the fall in esterase activity at 5 min and the factor which rendered the esterase resistant to soybean trypsin inhibitor. Purified α₂-macroglobulin inhibited approximately 50% of the TAMe esterase activity of purified plasma kallikrein without changing its activity toward basic amino acid esters. The interaction between the α₂-macroglobulin and kallikrein resulted in alterations in the gel filtration chromatographic pattern of the TAMe esterase and biologic activity of kallikrein, indicating that kallikrein was bound to the α₂-macroglobulin. The TAMe esterase activity of this complex, isolated by column chromatography, was resistant to C1 inactivator and SBTI. Studies of incubation mixtures of kallikrein, α₂-macroglobulin and C1 inactivator suggested that these inhibitors compete for the enzyme, and that the α₂-macroglobulin partially protects the esterase activity of kallikrein from C1 inactivator. The α₂-macroglobulin isolated from kaolin-activated plasma possessed 240 times the esterolytic activity of the α₂-macroglobulin purified from plasma treated with inhibitors of kallikrein and of its activation. The α₂-macroglobulin blocked the uterine-containing activity and vascular permeability-inducing effects of plasma kallikrein. These studies suggest that the α₂-macroglobulin is a major plasma inhibitor of kallikrein and provide a new example of an interrelationship between the coagulation, fibrinolytic, and kallikrein enzyme systems.     

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.