Human high molecular weight kininogen. Studies of structure-function relationships and of proteolysis of the molecule occurring during contact activation of plasma.

Human high Mr kininogen was purified from normal plasma in 35% yield. The purified high Mr kininogen appeared homogeneous on polyacrylamide gels in the presence of sodium dodecyl sulfate and mercaptoethanol and gave a single protein band with an apparent Mr = 110,000. Using sedimentation equilibrium techniques, the observed Mr was 108,000 +/- 2,000. Human plasma kallikrein cleaves high Mr kininogen to liberate kinin and give a kinin-free, two-chain, disulfide-linked molecule containing a heavy chain of apparent Mr = 65,000 and a light chain of apparent Mr = 44,000. The light chain is histidine-rich and exhibits a high affinity for negatively charged materials. The isolated alkylated light chain quantitatively retains the procoagulant activity of the single-chain parent molecule. 125I-Human high Mr kininogen undergoes cleavage in plasma during contact activation initiated by addition of kaolin. This cleavage, which liberates kinin and gives a two-chain, disulfide-linked molecule, is dependent upon the presence of prekallikrein and Factor XII (Hageman factor) in plasma. Addition of purified plasma kallikrein to normal plasma or to plasmas deficient in prekallikrein or Factor XII in the presence or absence of kaolin results in cleavage of high Mr kininogen and kinin formation.     

Autoactivatability of human Hageman factor (factor XII)

Purified Hageman factor was found to autodigest upon binding to a negatively charged surface such as kaolin. Assessment by incorporation of tritiated diisopropylfluorophosphate indicated that this cleavage was accompanied by activation and that the two known forms of activated Hageman factor result. Cleavage within a critical disulfide bridge generated activated Hageman factor, a two-chain enzyme of molecular weight 80,000 as well as the active Hageman factor fragment, a 28,000 molecular weight cleavage product. The autocleavage seen was dependent upon the percentage of activated Hageman factor in the starting material and was independent of HMW-kininogen. This result suggest that initiation of the intrinsic coagulation cascade may, in part, depend upon the autoactivatability of Hageman factor described herein. This observation may in turn, account for the ability of prekallikrein deficient plasma to gradually autoactivate as a function of the time of contact with initiating surfaces.     

Rat plasma high-molecular-weight kininogen. A simple method for purification and its characterization

High-molecular-weight kininogen has been isolated from rat plasma in three steps in a relatively high yield. The purified preparation gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the absence and presence of 2-mercaptoethanol, and the apparent Mr was estimated as 100,000. On incubation with rat plasma kallikrein, rat high Mr kininogen yielded a kinin-free protein consisting of a heavy chain (Mr = 64,000) and a light chain (Mr = 46,000), liberating bradykinin. The kinin-free protein was S-alkylated, and its heavy and light chains were separated by a zinc-chelating Sepharose 6B column. The amino acid compositions of rat high Mr kininogen and its heavy and light chains were very similar to those of bovine high Mr kininogen and its heavy and fragment 1.2-light chains, respectively. A high histidine content in the light chain of rat high Mr kininogen indicated the presence of a histidine-rich region in this protein as in bovine high Mr kininogen, although this region was not cleaved by rat plasma kallikrein. Rat high Mr kininogen corrected to normal values the prolonged activated partial thromboplastin time of Brown-Norway Katholiek rat plasma known to be deficient in high Mr kininogen and of Fitzgerald trait plasma. The kinin-free protein had the same correcting activity as intact high Mr kininogen. Rat high Mr kininogen also accelerated approximately 10-fold the surface-dependent activation of rat factor XII and prekallikrein, which was mediated with kaolin, amylose sulfate, and sulfatide. These results indicate that rat high Mr kininogen is quite similar to human and bovine high Mr kininogens in terms of biochemical and functional properties.     

Activation mechanism of human Hageman factor-plasma kallikrein-kinin system by Vibrio vulnificus metalloprotease.

Vivrio vulnificus, an opportunistic human pathogen, secretes a metalloprotease (VVP). The VVP inoculated into a guinea pig is known to generate bradykinin through activation of the Hageman factor-plasma kallikrein-kinin system. VVP was shown to possess the ability to activate the human system through the same mechanism as that clarified in the guinea pig system, namely, VVP converted both human zymogens (Hageman factor and plasma prekallikrein) to active enzymes (activated Hageman factor and plasma kallikrein), and the then generated kallikrein liberated bradykinin from high-molecular-weight kininogen. However, in the presence of plasma alpha 2-macroglobulin (alpha 2M), the VVP action was drastically decreased. This finding suggests that the human system might be activated only at the interstitial-tissue space which contains negligible amounts of alpha 2M or in the bloodstream of the individuals whose plasma alpha 2M level is extremely reduced.     

Activation of prekallikrein from human blood serum by different activators]

It was shown that the activated Hageman factor and its active fragment convert a greater amount of prekallikrein into kallikrein than is observed under the effects of trypsin and kallikrein. The latter two enzymes convert from 30 to 60% of the Hageman factor-activated prekallikrein and its active fragment. A possible existence of two prekallikrein forms is discussed. A mechanism of interaction between individual components of the kininogenase system and their activators is discussed.     

Pathogenesis of serratial infection: activation of the Hageman factor-prekallikrein cascade by serratial protease

A serratial protease with an apparent molecular weight of 56,000 (56K protease), which had been purified from the culture supernatant of a strain of Serratia marcescens isolated from a corneal lesion of a human eye [Matsumoto, K. et al. (1984) J. Bacteriol. 157, 225-232], greatly enhanced vascular permeability when injected into guinea pig skin. The 56K protease, which requires zinc ion for activity, was found to possess plasma kallikrein-like properties in vitro as judged by (i) preferential amidolysis of carbobenzoxy-Phe-Arg-4-methylcoumaryl-7-amide and Pro-Phe-Arg-4-methylcoumaryl-7-amide, which are known substrates for plasma kallikrein; (ii) release of kinin from high-molecular-weight kininogen; and (iii) prompt activation of Hageman factor followed by generation of kallikrein from plasma prekallikrein. These results suggest that the 56K protease enhances vascular permeability through activation of a Hageman factor-kallikrein-kinin pathway in vivo, and this molecular process appears to be a rational mechanism of enhancement of permeability and serratial pathogenesis.     

Purification and characterization of multiple forms of human plasma prekallikrein

Prekallikrein was purified 1,200-fold in 20% yield from human plasma by DEAE-cellulose, arginyl-triazinyl-aminododecyl-agarose, Cm-Sephadex C-50, and Sephadex G-150 chromatography. Isoelectric focusing of the purified proenzyme gave seven peaks, four major ones at pH 8.6, 8.8, 9.1, and 9.3; and three others at pH 7.9, 8.3, and 9.5. The same IEF profile was obtained from plasma of four individuals of three races and both sexes and from three plasma pools, and was not altered by using diisopropyl fluorophosphate, benzamidine, or EDTA during fractionation. Each major IEF form contained Mr = 88,000 (prekallikrein I) and Mr = 85,000 (prekallikrein II) species, in increasing ratios of I:II from about 20:1 in prekallikrein 8.6 (prekallikrein with pI 8.6) to 1:1 in prekallikrein 9.3. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the four zymogens after activation by Hageman factor fragment and reduction gave an Mr = 53,000 H-chain and two L-chains, LI (Mr = 40,000) and LII (Mr = 37,000). Scanning the gels gave LI:LII ratios of 19:1, 5:1, 2:1, and 1:1 for prekallikreins 8.6, 8.8, 9.1, and 9.3, respectively, corresponding to the prekallikrein I:II ratios. The H-chain in turn was split into Mr = 33,000 and 20,000 chains, presumably by autolysis, because the cleavage was prevented by soybean trypsin inhibitor. Each major kallikrein had a pI 0.1-0.2 lower than its zymogen, but the same LI:LII ratio. The four kallikreins were indistinguishable kinetically with human plasma high-molecular weight kininogen and 15 synthetic substrates, and in correcting the activated partial thromboplastin time of prekallikrein-deficient (Fletcher) plasma.     

Interaction of Hageman factor,prekallikrein activator and plasmin

When plasmin was incubated with active Hageman factor prepared from acetone-activated human plasma by adsorption and elution from pre-treated supercel, no change in prekallikrein activator (PKA) activity was found, even though by polyacrylamide gel electrophoresis and by supercel adsorption it was shown that the active Hageman factor had been converted to the 30, 000 molecular weight fragment. These data are in agreement with the concept that PKA is derived from Hageman factor, but do not support the concept that the conversion of plasminogen to plasmin is necessary for maximal generation of PKA activity in human plasma. Also reported is a radiochemical method for the measurement of PKA (active Hageman factor) activity which is 300 times more sensitive than the guinea pig ileum bioassay and 10 times more sensitive than the clotting tests.     

The inhibition of activated factor XII (Hageman factor) by antithrombin III: the effect of other plasma proteinase inhibitors.

Human factor XII was activated by adsorption onto kaolin in the presence of high molecular weight kininogen. The washed kaolin-containing precipitates activate prekallikrein to kallikrein. When antithrombin III was added to the reaction mixture, the conversion of prekallikrein to kallikrein was inhibited, the degree of inhibition depending on the concentration of antithrombin and the time of incubation. Heparin had a slight enhancing effect with low concentrations of antithrombin and short incubation times. However, the inhibition of the generated kallikrein by antithrombin III was markedly enhanced by heparin. Antithrombin III inhibited also the effect of activated factor XII on the partial thromboplastin time, using factor XII-deficient plasma. Of other plasma proteinase inhibitors used (α₁-antitrypsin, α₂-macroglobulin, C$\text{l}$-inactivator) only C$\text{l}$-inactivator inhibited activated factor XII.     

Activation of hageman factor and prekallikrein and generation of kinin by various microbial proteinases

Activation of the Hageman factor-kallikrein-kinin system by serratial 56-kDa proteinase was previously demonstrated (Matsumoto, K., Yamamoto, T., Kamata, T., and Maeda, H. (1984) J. Biochem. (Tokyo) 96, 739-749; Kamata, R., Yamamoto, T., Matsumoto, K., and Maeda, H. (1985) Infect. Immun. 48, 747-753). To investigate whether the activation of the system is specific for 56-kDa proteinase or is found similarly with other microbial proteinases, 11 proteinases of microbial origins were studied; the 56-kDa proteinase was the control. For in vitro studies, activation of guinea pig Hageman factor and prekallikrein was examined in purified systems as well as in plasma as a zymogen source. Specific antibodies and inhibitors confirmed the activation steps of the cascade. In the in vivo study the enhancement of vascular permeability in guinea pig skin and its sensitivity to inhibitors of activated Hageman factor, plasma kallikrein, or a kininase were examined. The results from the in vivo experiments were consistent with those in vitro. Taking all the data together, we classified the 11 microbial proteinases into three groups as follows: 1) Serratia marcescens 56-, 60-, and 73-kDa proteinases, Pseudomonas aeruginosa alkaline proteinase and elastase, and Aspergillus melleus proteinase (this group activated Hageman factor but not prekallikrein); 2) Vibrio vulnificus proteinase, subtilisin from Bacillus subtilis, and thermolysin from Bacillus stearothermophilus (this group activated both Hageman factor and prekallikrein); 3) Streptomyces caespitosus proteinase and V8 proteinase from Staphylococcus aureus (this group activated neither Hageman factor nor prekallikrein, but generated kinin from high molecular weight kininogen directly).     

High molecular weight kininogen-binding site of prekallikrein probed by monoclonal antibodies.

A panel of monoclonal antibodies against human prekallikrein was raised in mice and characterized with respect to the major antigenic epitopes. Of 18 antibodies, nine were directed against the light chain portion performing the proteolytic function of activated kallikrein, and nine recognized the heavy chain mediating the binding of prekallikrein to high molecular weight (H-)kininogen. Among the anti-heavy chain antibodies, one (PK6) interfered with the procoagulant activity of prekallikrein, and prolonged in a concentration-dependent manner the activated partial thromboplastin time of reconstituted prekallikrein-deficient plasma (Fletcher type). Antibody PK6 was subtyped IgG1,k and had an apparent Kass of 6.8 +/- 0.44.10(8) M-1 for prekallikrein. Functional analyses revealed that PK6 does not interfere with prekallikrein activation by activated Hageman factor (beta-F XIIa), and has no effect on the kininogenase function of activated kallikrein. Monoclonal antibody PK6 but none of the other anti-heavy chain antibodies completely prevented complex formation of prekallikrein with H-kininogen, and readily dissociated preformed complexes of prekallikrein and H-kininogen. Likewise, Fab' and F(ab')2 fragments of PK6 blocked H-kininogen binding to prekallikrein. A synthetic peptide of 31 amino acid residues encompassing the entire prekallikrein binding region of H-kininogen effectively competed with PK6 for prekallikrein binding indicating that the target epitope of PK6 is juxtaposed to, if not incorporated in the H-kininogen-binding site of prekallikrein. Extensive cross-reactivity of PK6 with another H-kininogen-binding protein of human plasma, i.e. factor XI, suggested that the structure of the target epitope of PK6 is well conserved among prekallikrein and factor XI, as would be expected for the kininogen-binding site shared by the two proteins. It is anticipated that monoclonal antibody PK6 will be an important tool for the precise mapping of the hitherto unknown kininogen-binding site of prekallikrein.     

Pumpkin seed inhibitor of human factor XIIa (activated Hageman factor) and bovine trypsin

A strong inhibitor of human Hageman factor fragment (HFf, beta-factor XIIa) and bovine trypsin was isolated from pumpkin (Cucurbita maxima) seed extracts by acetone fractionation, by chromatography on columns of diethyl-aminoethylcellulose and carboxylmethyl-Sephadex C-25, and by Sephadex G-50 gel filtration. Pumpkin seed Hageman factor inhibitor (PHFI) is unusual in its lack of inhibition of several other serine proteinases tested--human plasma, human urinary, and porcine pancreatic kallikreins, human alpha-thrombin, and bovine alpha-chymotrypsin. Human plasmin and bovine factor Xa are only weakly inhibited. PHFI also inhibits the HFf-dependent activation of plasma prekallikrein and clotting of plasma. Other properties of PHFI are a pI of 8.3, 29 amino acid residues, amino-terminal arginine, carboxyl-terminal glycine, 3 cystine residues, undetectable sulfhydryl groups and carbohydrate, and arginine at the reactive site. The minimum molecular weight of PHFI is 3268 by amino acid analysis. PHFI may be the smallest protein inhibitor of trypsin known.     

Contact activation of prekallikrein and plasminogen in plasma

Activation of the Hageman factor-prekallikrein system in the whole human blood plasma is studied as affected by organic silica (aerosils) with anionic and cationic properties. Positive- and negative-charged aerosils are shown to possess the same ability to activate prekallikrein. Activity of prekallikrein was manifested in hydrolysis of the chromogenic substrate--Benz-Pro-Phen-Arg-paranitroanilide . HCl, kininogen and protamine sulphate formed by kallikrein. The data permit supposing that optimal activation of the Hageman factor requires the polar (but not ionic) groups with hydrophilic properties on activating surfaces. Plasminogen under contact activation, in contrast to prekallikrein is activated only in the diluted plasma (pH 4.8), and not completely. Possible mechanisms of the contact activation and interaction of the Hageman factor, prekallikrein and high-molecular kininogen in this process are discussed.     

Hageman factor-dependent pathways: mechanism of initiation and bradykinin formation

The concentration of bradykinin in human plasma depends on its relative rates of formation and destruction. Bradykinin is destroyed by two enzymes: a plasma carboxypeptidase (anaphylatoxin inactivator) removes the COOH-terminal arginine to yield an inactive octapeptide, and a dipeptidase (identical to the angiotensin-converting enzyme) removes the COOH-terminal Phe-Arg to yield a fragment of seven amino acids that is further fragmented to an end product of five amino acids. Formation of bradykinin is initiated on binding of Hageman factor (HF) to certain negatively charged surfaces on which it autoactivates by an autodigestion mechanism. Initiation appears to depend on a trace of intrinsic activity present in HF that is at most 1/4000 that of activated HF (HFa); alternatively traces of circulating HFa could subserve the same function. HFa then converts coagulation factor XI to activated factor XI (XIa) and prekallikrein to kallikrein. Kallikrein then digests high-molecular-weight kininogen (HMW-kininogen) to form bradykinin. Prekallikrein and factor XI circulate bound to HMW-kininogen and surface binding of these complexes is mediated via this kininogen. In the absence of HMW-kininogen, activation of prekallikrein and factor XI is much diminished; thus HMW-kininogen has a cofactor function in kinin formation and coagulation. Once a trace of kallikrein is generated, a positive feedback reaction occurs in which kallikrein rapidly activates HF. This is much faster than the HF autoactivation rate; thus most HFa is formed by a kallikrein-dependent mechanism. HMW-kininogen is also therefore a cofactor for HF activation, but its effect on HF activation is indirect because it occurs via kallikrein formation. HFa can be further digested by kallikrein to form an active fragment (HFf), which is not surface bound and acts in the fluid phase. The activity of HFf on factor XI is minimal, but it is a potent prekallikrein activator and can therefore perpetuate fluid phase bradykinin formation until it is inactivated by the C1 inhibitor. In the absence of C1 inhibitor (hereditary angioedema) HFf may also interact with C1 and activate it enzymatically. The resultant augmented bradykinin formation and complement activation may account for the pathogenesis of the swelling characteristic of hereditary angioedema and the serologic changes observed during acute attacks.     

Activation of the contact system of coagulation by a monoclonal antibody directed against a neodeterminant in the heavy chain region of human coagulation factor XII (Hageman factor)

We studied the characteristics of two monoclonal antibodies (mAbs), F1 and F3, against human coagulation factor XII (Hageman factor). Experiments with trypsin-digested 125I-factor XII revealed that the epitope for mAb F1 is located in the NH2-terminal Mr 40,100 portion of factor XII, whereas that for mAb F3 resides in the COOH-terminal Mr 30,000 portion of this protein. Factor XII in fresh plasma (single-chain factor XII) bound approximately 190 times less to mAb F1 than factor XII in dextran sulfate-activated plasma (cleaved factor XII). However, no difference in accessibility of the epitope for mAb F1 was observed between cleaved and single-chain factor XII when bound to glass. mAb F3 appeared to bind to both single-chain and cleaved factor XII in plasma as well as when bound to glass. Neither mAb F1, nor F3 affected the amidolytic activity of factor XIIa, whereas both mAb F1 and F3 inhibited factor XII-coagulant activity to about 15 and 70%, respectively, at a molar ratio of mAb to factor XII of 20 to 1. mAb F1, as well as F(ab')2 and F(ab') fragments of this antibody induced activation of the contact system in plasma, as reflected by the generation of factor XIIa. C1 inhibitor and kallikrein. C1 inhibitor complexes. Activation was induced neither upon incubation with mAb F3, nor with that of control mAbs. mAb F1-induced contact activation required the presence of factor XII, prekallikrein, and high molecular weight kininogen and, in contrast to activation by negatively charged surfaces, was not inhibited by the presence of Polybrene. Based on these results we propose that a conformational change in factor XII is a key event in the activation process of this molecule. This conformational change can be induced by binding of factor XII to a surface as well as by proteolytic cleavage. As mAb F1 can also induce this conformational change, this antibody may provide a unique tool in studies of the activation of factor XII.     

Studies of C1 inactivator-plasma kallikrein complexes in purified systems and in plasma

An enzyme-linked immunosorbent assay (ELISA) has been developed for the quantification of C1 inactivator-kallikrein (C1In-K) complexes. The formation of complexes assayed by this method parallelled the inhibition of plasma kallikrein esterase activity by C1 inactivator in purified systems. C1In-K complexes were detected when a final concentration of 5.7 nM plasma kallikrein was added to plasma, equivalent to the activation of 1% of the plasma prekallikrein. Exogenous Hageman factor fragment added to plasma induced the rapid formation of C1In-K complexes, whereas there was an appreciable delay when the plasma contact system was activated by the addition of kaolin. In both systems, the rate of formation and final amount of complex generated were directly related to the concentration of Hageman factor fragment or of kaolin added, indicating that this proteolytic pathway is tightly regulated. C1In-K complexes were not generated by kaolin in plasma congenitally deficient in Hageman factor or prekallikrein or by kallikrein in hereditary angioedema plasma deficient in C1 inactivator, thus confirming the specificity of the assay. Sucrose gradient ultracentrifugation studies showed plasma C1In-K complexes to have a molecular weight consistent with a 1:1 molar complex. In contrast, the complex displayed an anomalously high molecular weight on gel filtration chromatography. These data demonstrate that a sensitive and specific probe has been developed for documenting plasma kallikrein activation.     

The Hageman factor-dependent system in the vascular permeability reaction

The mechanism by which the Hageman factor-dependent system induces vascular permeability has been analyzed. The Mr-28,000 active fragment of guinea pig Hageman factor (beta-HFa), injected intradermally, induces an increase in local vascular permeability. Inhibition of vascular permeability resulted from pretreatment of the beta-HFa with immunopurified anti-Hageman factor F(ab')2 antibody at concentrations of 10(-6)-10(-7) M as well as by incubation with corn and pumpkin seed inhibitors of beta-HFa. To determine whether prekallikrein and kallikrein participated in the permeability induced by beta-HFa, circulating prekallikrein was depleted by intra-arterial injections of anti-prekallikrein F(ab')2 antibody. This resulted in about 80% diminution of the vascular permeability response to beta-HFa, without affecting the permeability reaction to bradykinin. Soybean trypsin inhibitor (10(-6) M), injected at the same cutaneous site as the beta-HFa, inhibited the vascular permeability response to beta-HFa by more than 90%. This concentration of soybean inhibitor blocked more than 90% of the activity of guinea pig plasma kallikrein, but did not inhibit the amidolytic capacity of beta-HFa. The permeability activity of beta-HFa (but not its amidolytic activity) was augmented 10-fold by simultaneous injection of a synthetic kinin potentiator, SQ 20,881 (Glu-Tyr-Pro-Arg-Pro-Gln-Ile-Pro-Pro-OH), and was almost completely inhibited by the simultaneous injection of a kinin-destroying enzyme, carboxypeptidase B. These results support the hypothesis that the greatest proportion of vascular permeability induced by beta-HFa is produced by the activation of prekallikrein followed by the release of kinin in the cutaneous tissue. These data offer the first in vivo evidence that the Hageman factor-dependent system by itself can induce inflammatory changes.     

Use of chromogenic substrates in the clarification of disorders in the early stages of blood coagulation

The kallikrein specific chromogenic peptide substrates S-2302 (KABI) and Chromozym PK (Boehringer) were used in the first analysis of a familial defect in the early stage of clotting. Slight to extensive prolongation of the activated partial thromboplastin time was seen in the affected persons. Using dextransulfate for activation of plasma marked deficiency in kallikrein activity was found in 3 persons. Using factor XIIa (activated Hageman factor) for activation normal prekallikrein levels were found in 2 of them whereas factor XII levels, however, were below normal. The third had a prekallikrein deficiency presumably caused by oral contraceptives. In a fourth member of the family factor XII deficiency was found with normal kallikrein activity. The application of chromogenic peptide substrates for analysing the early stage of clotting has to take into account the special mechanisms of activation.     

A new protein inhibitor of trypsin and activated Hageman factor from pumpkin (Cucurbita maxima) seeds

A protein inhibitor (CMTI-V; Mr 7106) of trypsin and activated Hageman factor (Factor XIIa), a serine protease involved in blood coagulation, has been isolated for the first time from pumpkin (Cucurbita maxima) seeds by means of trypsin-affinity chromatography and reverse phase high performance liquid chromatography (HPLC). The dissociation constants of the inhibitor complexes with trypsin and Factor XIIa have been determined to be 1.6 x 10(-8) and 4.1 x 10(-8) M, respectively. The primary structure of CMTI-V is reported. The protein has 68 amino acid residues and one disulfide bridge and shows a high level of sequence homology to the Potato I inhibitor family. Furthermore, its amino terminus consists of an N-acetylates Ser. The reactive site has been established to be the peptide bond between Lys44-Asp45. The modified inhibitor which has the reactive site peptide bond hydrolyzed inhibits trypsin but not the Hageman factor.     

Activation of plasma Hageman factor and kallikrein in ongoing allergic reactions in the skin

Ten atopic subjects, sensitive to intradermal injection of less than or equal to 10 protein nitrogen units of ragweed or grass pollen antigen, underwent paired antigen and buffer skin chamber incubation over the base of denuded skin blisters. The chamber fluids were sampled over a 6-hr period for histamine and activated Hageman factor and plasma kallikrein which were complexed to C1 inhibitor. In 9 of 10 subjects significantly (p less than 0.01) increased histamine levels (74 +/- 11 ng/ml vs 1.5 +/- 0.55 ng/ml) and kallikrein-C1 inhibitor complexes (2.15 +/- 0.78 ng/ml/hr vs 0.51 +/- 0.09 ng/ml/hr, p less than 0.25) were detected at antigen sites compared with buffer sites, respectively. Increased levels of activated Hageman factor (ng/ml/hr) were detected at antigen sites (1.35 +/- 0.60) compared with buffer sites (0.11 +/- 0.05), (p less than 0.01), in 8 of 10 subjects. Whereas peak levels of histamine were obtained after 1 hr of challenge, both Hageman factor and kallikrein activation, as assessed by complex formation, tended to peak later from the 2nd to the 5th hr. This represents the first demonstration that cutaneous IgE-mediated allergic responses are associated with local activation of the intrinsic plasma coagulation-kinin pathways.