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Activation of plasma prekallikein and generation of bradykinin are responsible for the angioedema attacks observed with C1-inhibitor deficiency. Heterozygous individuals with <50% levels of active C1-inhibitor are susceptible to angioedema attacks indicating a critical need for C1-inhibitor to be present at maximum levels to prevent unwanted prekallikrein activation. Studies with purified proteins do not adequately explain this observation. Therefore to investigate why reduction of C1-inhibitor to levels seen in angioedema patients results in excessive kallikrein generation we examined the effect of endothelial cells on the inhibition of kallikrein by C1-inhibitor. Surprisingly, it was found that a C1-inhibitor concentration of greater than 1 microM was needed to inhibit 3 nM kallikrein. We propose that this apparent protection from inhibition was mediated by kallikrein binding to the cells via the heavy chain in a high molecular weight kininogen and zinc independent manner. Protection of kallikrein from inhibition was not observed when C1-inhibitor truncated in the amino-terminal domain by the StcE metalloproteinase was used, which suggests a novel function for this unique domain. The requirement for high concentrations of C1-inhibitor to fully inhibit kallikrein is consistent with the fact that reduced levels of C1-inhibitor result in the kallikrein activation seen in angioedema.

Increased extracellular proteolysis because of unregulated activation of blood coagulation, complement, and fibrinolysis is observed in thrombosis, shock, and inflammation. In the present study, we have examined whether the plasma kallikrein-kinin system, the classical pathway of complement, and the fibrinolytic system could be inhibited by alpha 1-antitrypsin reactive site mutants. Wild-type alpha 1-antitrypsin contains a Met residue at P1 (position 358), the central position of the reactive center. It did not inhibit plasma kallikrein, beta-factor XIIa, plasmin, tissue-type plasminogen activator (t-PA), or urokinase. In contrast, these serine proteases were inhibited by alpha 1-antitrypsin Arg358. For the inhibition of C1s, a double mutant having Arg358 and a Pro----Ala mutation at P2 (position 357) was required. This double modification was made because C1-inhibitor, the natural inhibitor of C1s, has Arg and Ala residues at positions P1 and P2. Plasminogen activator inhibitor 1, the natural inhibitor of t-PA, also has Arg and Ala residues at positions P1 and P2. In a purified system, alpha 1-antitrypsin Ala357-Arg358 was 150-fold less efficient against C1s than C1-inhibitor and 27,000-fold less efficient against t-PA than plasminogen activator inhibitor-1. In plasma, 2.3 microM alpha 1-antitrypsin Ala357-Arg358 reduced by 65% the formation of a complex between kallikrein and C1-inhibitor following activation of the intrinsic pathway of blood coagulation by kaolin. Furthermore, after supplementation by 2.0 microM alpha 1-antitrypsin Ala357-Arg358, zymographic analysis showed that the majority of the free t-PA of normal plasma formed a bimolecular complex with the double mutant. In contrast, 3.4 microM alpha 1-antitrypsin Ala357-Arg358 did not prevent the activation of the classical pathway of complement observed when normal serum is supplemented with anti-C1-inhibitor F(ab')2 fragment. These results demonstrate that alpha 1-antitrypsin Ala357-Arg358 has therapeutic potential for disorders with unregulated activation of the intrinsic pathway of blood coagulation and the fibrinolytic system; however, the double mutant is not an efficient inhibitor for the classical pathway of complement.

Although Cl-inhibitor (Cl-INH) and alpha(2)-macroglobulin (alpha(2)M) 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 Cl-INH, or artificially depleted of alpha(2)M 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 Cl-INH, alpha(2)M, or both inhibitors. Thus Cl-INH (42%) and alpha(2)M (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, (125)I-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 (125)I-kallikrein-inhibitor complexes. After inactivation by normal plasma, 52% of the active enzyme were found to form a complex (M(r) 370,000) with Cl-INH, while 48% formed a complex (M(r) 850,000) with alpha(2)M. After inactivation by Cl-INH-deficient plasma, >90% of the active (125)I-kallikrein was associated with alpha(2)M. A similar proportion of the label was associated with Cl-INH in plasma deficient in alpha(2)M. After inactivation by plasma deficient in both Cl-INH and alpha(2)M, (125)I-kallikrein was found to form a complex of M(r) 185,000. This latter complex, which may involve antithrombin III, alpha(1)-protease inhibitor, and/or alpha(1)-plasmin inhibitor, was not detectable in appreciable concentrations in the presence of either Cl-INH or alpha(2)M, even after the addition of heparin (2 U/ml). These observations demonstrate that Cl-INH and alpha(2)M 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 Cl-INH or alpha(2)M, the inactivation of kallikrein becomes almost entirely dependent on the other major inhibitor.

The inactivation of human plasma kallikrein (EC 3.4.21.8) by the inhibitor of activated complement component 1 (C1 inhibitor) induces the formation of a 1:1 stoichiometric kallikrein-C1 inhibitor complex and a proteolytically modified form of C1 inhibitor. We have produced a monoclonal antibody that recognizes the kallikrein-C1 inhibitor complex as well as modified C1 inhibitor but fails to react with virgin C1 inhibitor or native plasma kallikrein. This observation constitutes an unequivocal demonstration that the reaction between plasma kallikrein and C1 inhibitor leads to the emergence of an epitope that is undetectable on the parent enzyme and inhibitor molecules.

To test whether endotoxin decreases blood pressure acutely in rats by activating the plasma kinin-forming system, plasma kallikrein activity was determined in different experimental settings of endotoxemia. Conscious normotensive rats were infused for 45 min with endotoxin (LPS E. coli 0111:B4) at a dose (0.01 mg/min) which had no effect on blood pressure. Additional rats were infused with the vehicle of endotoxin. Plasma prekallikrein activity was measured at the end of the 45 min infusions. In other rats, a bolus intravenous injection of endotoxin (2 mg) was administered following the 45 min infusion of endotoxin or its vehicle. In these two latter groups of rats, plasma prekallikrein activity was determined 15 min after administration of the bolus dose of endotoxin. In rats pretreated with the endotoxin infusion, the bolus dose of endotoxin had no significant effect on blood pressure, whereas rats infused with the vehicle became and remained hypotensive up to the end of the experiment. There was however no significant difference in plasma prekallikrein activity within the different groups of rats. In another group of rats, dextran sulfate (0.25 mg i.v.), which activates factor XII and thereby the conversion of prekallikrein to kallikrein, induced a short-lasting fall in blood pressure. 15 min after administration of dextran sulfate, plasma prekallikrein activity was almost completely suppressed. These results obtained in unanesthetized rats strongly suggest that the blood pressure fall induced by E. coli endotoxin is not due to activation of prekallikrein and consequently of the kinin-forming system.

Patients with hereditary angioedema lack C-1 inhibitor, a plasma alpha 2-glycoprotein that inhibits both the proteolytic action of C1, the activated first component of the complement system, and the activity of components of the contact phase of coagulation: kallikrein, factor XIa, and factor XIIa. Such patients have been shown to have low levels of C4 and C2, the natural substrates for C-1, but the levels were not correlated with the presence of symptoms. We studied three patients with angioedema for evidence of activation of the contact system and found that during a symptomatic period they had decreased levels of prekallikrein, a substrate for the activated forms of factor XII, and reductions in high-molecular-weight kininogen, a substrate for plasma kallikrein. These observations suggest that zymogens of the contact system are activated during attacks of hereditary angioedema and that some of the clinical manifestations may be mediated through products of this pathway, such as kinins.

Prekallikrein is the zymogen form of plasma kallikrein, a proteolytic enzyme of the contact system of blood coagulation, fibrinolysis and kinin formation. To assess whether prekallikrein was activated in the adult respiratory distress syndrome (ARDS), we examined plasma samples from 12 critically ill patients including five individuals with ARDS. Using the ratio between functional prekallikrein and prekallikrein-kallikrein antigen as an index for prekallikrein activation, we found that prekallikrein was extensively activated in patients with ARDS, while this was significantly less in the case of critically ill patients without ARDS. Following activation of prekallikrein in plasma, kallikrein reacts with its substrates and with protease inhibitors, among which the alpha 2-glycoprotein C1-inhibitor. Thus, we examined whether C1-inhibitor of critically ill individuals was modified in a way suggesting that it had reacted with kallikrein. Such a modification was found in the five patients with ARDS, while it was not detectable in the seven patients without ARDS. This observation further confirmed the activation of prekallikrein in patients with ARDS. We suggest that plasma kallikrein-mediated reactions, which include bradykinin release and neutrophil activation, may contribute to the pathogenesis of ARDS.

High Mr kininogen increases the activation rate of prekallikrein by activated factor XII on a surface. The resulting serine protease, plasma kallikrein, Mr 88 000, is inhibited in plasma by C1 inhibitor, Mr 105 000. Since prekallikrein circulates in plasma with high Mr kininogen as a complex and a kallikrein-high Mr kininogen complex can be formed in purified systems, we studied whether the inhibition of kallikrein by C1 inhibitor was influenced by high Mr kininogen. With C1 inhibitor in excess, the inactivation of kallikrein followed pseudo-first-order kinetics. The second-order rate constant for the reaction was 1.7 X 10(4) M-1 s-1, and a kallikrein-C1 inhibitor complex, Mr 190 000 was identified on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Kallikrein and C1 inhibitor formed an irreversible complex without measurable prior equilibrium. The rate of this reaction was decreased by 50% in the presence of high Mr kininogen (1 unit/mL or 0.73 muM). Kinetic analysis indicated that this protection was the result of the formation of a reversible complex between kallikrein and high Mr kininogen, which had a dissociation constant of 0.75 muM. However, low Mr kininogen did not protect kallikrein from inactivation by C1 inhibitor. High Mr kininogen also protected kallikrein from inactivation by diisopropyl fluorophosphate. These findings suggest that the kallikrein-high Mr kininogen complex was formed by noncovalent interactions between the light chains of both kallikrein and high Mr kininogen.

Plasma protein fraction (PPF) contaminated by factor XII active fragment (XIIf) may cause hypotensive reactions when infused to patients. This study was planned to assess in conscious normotensive rats whether the blood pressure response to the factor XIIf is mediated by an activation of the plasma kallikrein-kinin system or by stimulation of prostaglandin synthesis. To test whether the factor XIIf-induced blood pressure fall is due partially to an enhanced generation of vasodilating prostaglandins, the blood pressure effect of XIIf (1 microgram i.v.) was investigated 15 min after treatment with indomethacin (5 mg i.v.), an inhibitor of cyclo-oxygenase. Factor XIIf reduced mean blood pressure similarly in indomethacin- and vehicle-treated rats (-23 +/- 4 mmHg, n = 5, and -23 +/- 5 mmHg, n = 4, respectively). Other rats received factor XIIf 15 min after depletion of circulating prekallikrein by the administration of dextran sulfate. Thirty minutes after a 0.25 mg i.v. dose of this agent, plasma prekallikrein activity averaged 0.12 +/- 0.015 mumol/min/ml (n = 6) as compared to 2.48 +/- 0.31 mumol/min/ml in control rats (n = 4, P less than .001). Factor XIIf decreased mean blood pressure by only 4 +/- 2 mm Hg in rats pretreated with dextran sulfate. Thus, it was possible to blunt the acute hypotensive effect of factor XIIf by depleting circulating prekallikrein, but not by inhibiting prostaglandin production. This strongly suggests that the blood pressure effects of factor XIIf is mediated by a stimulation of the plasma kallikrein-kinin system.

The reactive center of C1-inhibitor, a plasma protease inhibitor that belongs to the serpin superfamily, is located on a peptide loop which is highly susceptible to proteolytic cleavage. With plasma kallikrein, C1s and beta-Factor XIIa, this cleavage occurs at the reactive site residue P1 (Arg444); with neutrophil elastase, it takes place near P1, probably at residue P3 (Val442). After these cleavages, C1-inhibitor is inactivated and its conformation is modified. Moreover, in vivo, cleaved C1-inhibitor is removed from the blood stream more rapidly than the intact serpin, which suggests that proteolysis unmasks sites responsible for cellular recognition and the uptake of the cleaved inhibitor. In the study reported here, we show, using an MAb, that an identical neoepitope is created on C1-inhibitor after the cleavage of its exposed loop by plasma kallikrein, C1s, beta-Factor XIIa, and by neutrophil elastase.

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

Recent evidence indicates that plasma kallikrein is activated during acute attacks of hereditary angioedema. Plasma kallikrein is known to convert inactive renin, or prorenin, into an active proteolytic enzyme in plasma exposed to acid or low temperatures as well as in purified systems. To establish whether plasma kallikrein could activate prorenin under physiologic or pathologic conditions, prorenin to renin conversion was assessed at neutral pH in plasma deficient in C1 inhibitor (hereditary angioedema). In these plasma samples lacking the two major inhibitors of kallikrein and possessing less than 10% of the inhibitory activity of normal plasma, prorenin was not converted to an active enzyme despite conditions under which prekallikrein was completely activated to plasma kallikrein and despite normal prorenin concentrations and activability.

Human plasma kallikrein consists of an N-terminal heavy chain of molecular weight (mol wt) 52,000, linked by disulfide bonds to two light chain variants (mol wt 36,000 or 33,000). Although the active catalytic site of kallikrein resides on the C-terminal light chain, the role of the N-terminal heavy chain is less clear. We therefore studied an enzyme designated beta-kallikrein, containing a single cleavage in the heavy chain (mol wt 28,000 + 18,000) and compared it to the enzyme, alpha-kallikrein, with an intact heavy chain. The rates of inactivation by C1 inhibitor of plasma alpha- and beta-kallikreins were kinetically identical, as measured by residual amidolytic activity, after various times of incubation with the inhibitor. Both enzymes reacted completely with C1 inhibitor after 18 hours and formed identical C1 inhibitor-kallikrein complexes of mol wt 195,000. The rate of activation of factor XII by alpha-kallikrein and beta-kallikrein was similar. In contrast, the rate of cleavage of high molecular weight kininogen (HMWK) by alpha-kallikrein was at least fivefold faster and the ratio of coagulant activity to amidolytic activity was fourfold greater than for beta-kallikrein. Plasma alpha-kallikrein, at concentrations potentially achievable in plasma, induced aggregation of neutrophils, but beta-kallikrein failed to elicit this response. In addition, human neutrophils pretreated with cytochalasin B released 2.46 +/- 0.10 microgram/10(7) cells of elastase antigen, but beta-kallikrein released only 0.25 +/- 0.10 micrograms/10(7) cells. These observations suggest that cleavage of the heavy chain influences the rate of cleavage of HMWK and decreases its coagulant activity. Moreover, an intact heavy chain appears to be requisite to support the ability of kallikrein to aggregate neutrophils and release elastase.

1. Studies were performed in normal subjects and in rats to assess the effect of angiotensin converting enzyme (ACE) inhibition on the kallikrein-kinin system. As ACE is identical to kininase II, one of the enzymes physiologically involved in bradykinin degradation, bradykinin may be expected to accumulate during ACE inhibition. 2. A competitive antagonist of bradykinin was used to explore in unanaesthetized rats the contribution of circulating bradykinin to blood pressure control under ACE inhibition. 3. No evidence was found for a role of this vasodilating peptide in the blood pressure lowering effect of acute ACE inhibition. 4. The plasma activity of carboxypeptidase N (= kininase I), another pathway of bradykinin degradation, remained intact during a 1 week course of treatment with an ACE inhibitor in normal subjects. This therefore indicates that bradykinin formed during ACE inhibition can still be metabolized.

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.

Prekallikrein was purified from human plasma with a final yield of 76% using as the principal step adsorption to immobilized chicken antikallikrein IgY. When purified prekallikrein (3.4 microM) was incubated in the presence of beta-Factor XIIa (0.068 microM) for 5 min at 37 degrees C and pH 7.5, alpha-kallikrein was obtained. Upon prolonged incubation (0.5-28 h), the Mr 52,000 heavy chain of alpha-kallikrein was progressively cleaved, resulting in the formation of beta-kallikrein. The formation of beta-kallikrein was characterized as an autolytic process because it was prevented by specific inhibitors of kallikrein, including aprotinin and antikallikrein antibody but not by corn trypsin inhibitor, an inhibitor specific for beta-Factor XIIa.

alpha-Kallikrein was prepared using an improved purification protocol (Burger, D., Schleuning, W.-D. and Schapira, M. (1986) J. Biol. Chem. 261, 324-327) and was employed to reevaluate our previous observations indicating that kallikrein activates blood neutrophils by a mechanism requiring an uncleaved Mr 52,000 NH2-terminal heavy chain. Cellular activation was evaluated by measuring neutrophil aggregation, release of both vitamin B12 binding capacity and myeloperoxidase, and generation of superoxide anion. Whereas all these indicators were evoked by exposing neutrophils to f-Met-Leu-Phe, phorbol myristate acetate, zymosan activated serum, or the calcium ionophore A 23187, we show here that neutrophils incubated with alpha-kallikrein remained unactivated. Moreover, we demonstrate that this lack of activation is accompanied by an inability of neutrophils to specifically bind alpha-kallikrein.

Exposure of human blood polymorphonuclear leukocytes (PMN) to purified active plasma kallikrein resulted in PMN aggregation when kallikrein was present at concentrations ranging from 0.4 to 0.6 U/ml (0.18-0.27 microM). Kallikrein-induced PMN aggregation was not mediated through C5-derived peptides, because identical responses were observed whether or not kallikrein had been preincubated with an antibody to C5. Moreover, kallikrein was specific for aggregating PMN, because no aggregation was observed with Factor XII active fragments (23 nM), Factor XIa (0.6 U/ml or 15nM), thrombin (1.6 microM), plasmin (2 microM), porcine pancreatic elastase (2 microM), bovine pancreatic chymotrypsin (2 microM), or bradykinin (1 microM). Bovine pancreatic trypsin (2 microM) aggregated PMN, but to a lesser extent than kallikrein (0.18 microM). Kallikrein was a potent aggregant agent for PMN because similar responses were observed with kallikrein (0.5 U/ml or 0.23 microM) and an optimal dose (0.2 microM) of N-formyl-methionyl-leucyl-phenylalanine. In addition, PMN incubation with kallikrein resulted in stimulation of their oxidative metabolism as assessed by an increased oxygen uptake. Neutropenia and leukostasis observed in diseases associated with activation of the contact phase system may be the result of PMN aggregation by plasma kallikrein.