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Scand. J. Immunol. 44, 185­192, 1996

Activation of the Complement, Coagulation, Fibrinolytic and Kallikrein­Kinin Systems During Attacks of Hereditary Angioedema

° E. WAAGE NIELSEN*, H. THIDEMANN JOHANSEN, K. HØGA SEN, W. WUILLEMIN, C. E. HACK & T. E. MOLLNES

*Department of Anaesthesiology, Nordland Central Hospital, Bodø, and University of Tromsø, Tromsø, Norway; Institute of Pharmacy, University of Oslo, Oslo; Institute of Immunology and Rheumatology, University of Oslo, Oslo, Norway; §Central Laboratory of the Netherlands Red Cross Blood Transfusion Service and Laboratory for Experimental and Clinical Immunology, University of Amsterdam, Amsterdam, the Netherlands; and Department of Immunology and Transfusion Medicine, Nordland Central Hospital, Bodø, and University of Tromsø, Tromsø, Norway

(Received 12 April 1996; Accepted in revised form 2 May 1996)

° Waage Nielsen E, Thidemann Johansen H, Høgasen K, Wuillemin W, Hack CE, Mollnes TE. Activation of the Complement, Coagulation, Fibrinolytic and Kallikrein­Kinin Systems During Attacks of Hereditary Angioedema. Scand J Immunol 1996;44:185­192 Five patients with hereditary angioedema (HAE) were studied during attacks and remission as were healthy controls. The high levels of C1/C1-INH complexes, low C4 and high ratio C4 activation products (C4bc)/C4 also differed significantly during remission compared to controls.During attacks C4bc/C4 increased (922­ 2007; P 0 022, remission versus attacks, median values throughout), C2 and CH50 dropped (111­31%; P 0 043 and 110­36%; P 0 016, respectively), TCC (C5b-9) increased (0.88­1.23 AU/ml; P 0 028). Cleavage of HK increased to be almost complete during attacks (20­90%; P 0 009). While factor XIa/serpin-complexes did not increase, a more than twofold rise in thrombin/antithrombincomplexes (0.20­0.50 g/l; P 0 009) and in plasmin/alpha-2-antiplasmin-complexes (7.3­17 nmol/l; P 0 028) was observed. For the first time cascade activation in HAE was studied simultaneously, and corroborates that attacks lead to activation of the kallikrein-kinin system, fibrinolysis and early part of the classical complement pathway. In addition, the authors present novel data of terminal complement and coagulation activation, the latter apparently not via FXIa. Erik Waage Nielsen, Department of Anaesthesiology, Nordland Central Hospital, 8017 Bodø, Norway

kininogen (HK) into smaller fragments, compatible with liberation of bradykinin, the major mediator of edema formation. A kinin-like peptide cleaved off from C2b by plasmin [8], and C4a [9] are probably less important mediators. Fibrinolysis may be enhanced as shown by elevated plasmin-alpha-2-antiplasmin (PAP) complexes [10, 11]. The critical inhibition by C1-INH of kallikrein, factor XII [12], factor XI [12, 13], and to some extent plasmin [14] and tissue-type plasminogen activator (t-PA) [15], underlines the widespread function of this protein and indicates that C1-INH is probably essential [16] in preventing massive activation of the complement and kallikrein-kinin systems. Although studies such as those mentioned above have clearly demonstrated activation of complement, the kallikrein-kinin and fibrinolytic systems, during attacks of HAE, no single study has documented these phenomena in the same patient group. Therefore, the aim of the present study 185

INTRODUCTION Hereditary angioedema (HAE) is inherited as an autosomal dominant trait. It is caused by lack of functional C1-inhibitor (C1-INH), a key regulator of plasma cascade systems, particularly the kallikrein-kinin system and the classical complement pathway. Affected members experience various degrees of swellings in the subcutaneous tissue, the mucosa of the upper respiratory tract and the gastrointestinal tract. A substantial amount of fluid can pass through leaky endothelial cells in the capillaries [1], and the haematocrit may reach 60­75% in extreme cases [2]. Complexes between C1 and C1-INH are increased in HAE patients, while C4 and C2 are depressed [3, 4]. Functional- [5] and immunoblot assays [6, 7] of plasma from HAE patients show degradation of high molecular weight

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186 E. Waage Nielsen et al. was to thoroughly examine the extent of complement activation, as well as activation in the coagulation, fibrinolytic and the kallikrein-kinin systems at the same time in patients suffering from hereditary angioedema outside and during acute attacks.

with heat aggregated IgG and defined to contain 1000 arbitrary units (AU)/ml. This antibody does not react with native C4 and thus accurately reflects the amount of C4 activation products. Due to the very low total amounts of C4 relative to the C4bc, the ratio C4bc/total C4 was calculated to give a more correct impression of the degree of C4 activation. C3 activation products (C3bc). ACtivation of C3 was quantified using the neoepitope specific MoAb bH6 does not react with the native protein and thus accurately reflects the amount of C3 activation products. In this, and the following complement activation assays, zymosan-activated serum defined to contain 1000 AU/ml, was used as a standard. Activation was obtained by incubating 10 mg zymosan per ml serum for 1 h at 37 C followed by centrifugation at 27,000 g for 30 min. C3a. Using a neoepitope specific MoAb, C3a was quantified in a double antibody EIA principally as described by Zilow et al. [22]. The MoAbs were kindly provided by Progen Biotechnik, Heidelberg, Germany. The standard was as described for C3bc. C5a and TCC. Activation of C5 was evaluated by quantification of C5a/C5a desArg using the neoepitope-specific MoAb C17/5 as capture antibody and biotinylated MoAb G25/2 as detection antibody. These antibodies were a generous gift from Otto Gotze. Gottingen, Germany. ¨ ¨ The assay has been described in detail by Oppermann et al. [23]. Formation of the terminal sC5b-9 complement complex (TCC) was quantified using the C9 neoepitope specific MoAb aE11 essentially as described previously [24], and later modified [25]. CH50. Total haemolytic complement activity was evaluated using microtitre techniques based on the standard detection system of lysis of sensitized sheep red blood cells. The red cells (2% v/v final concentration) were suspended in veronal buffered saline (VBS) containing 0.2 mM CaCl2 and 0.8 mM MgCl2 (Veronal Calcium Magnesium, Biomerieux, Marcy l'Etoile, France). Normal human serum was used as ´ standard and defined to contain 100% lytic activity. Standard curves were made 3/2-fold from 150%. Serum samples were diluted 1/150. The incubation was undertaken for 1 h at 37 C, the plates were centrifuged immediately and the supernatants transferred to another plate and measured spectrophotometrically at 410 nm. Factor XI, complexes between factor XIa and its inhibitors C1-INH, alpha-1-antitrypsin, alpha-2-antiplasmin and antithrombin. Factor XI antigen levels were measured using EIA with an anti-factor XI MoAb (XI-5) as capturing antibody and another biotinylated anti-factor XI MoAb (XI-3) as detecting antibody. Serial dilutions of pooled (n 15) normal human plasma were used as standards, Results were expressed as percentage of the amount of factor XI present in the pooled plasma. Complexes between factor XIa and its inhibitors were determined using an EIA as described [13]. Thrombin/antithrombin complexes (TAT). These were measured using a novel IEA. Briefly, MoAb CLB-TR3, which is directed against (pro-) thrombin was used as capture antibody, and MoAb CLB-AT III-0, raised against antithrombin III as detecting antibody. The latter antibody was biotinylated according to standard procedures. Bound TAT complexes were finally visualized by incubation with streptavidin-polymerized horseradish peroxidase (CLB). Results obtained with this assay correlated significantly with TAT levels measured with a commercial assay (Behringwerke AG, Marburg, Germany; Spearman rank correlation coefficient 0 94 for 23 samples with TAT levels ranging from normal to 1000 g/l. In this separate methodology study normal TAT levels were less than 4 g/l, the detection limit of the assay was 0.05 g/l). t-PA antigen. These were measured by sandwich EIA which used MoAbs against recombinant t-PA [26].

1996 Blackwell Science Ltd, Scandinavian Journal of Immunology, 44, 185­192

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MATERIALS AND METHODS

Samples were taken from four patients suffering from HAE type 1 (reduced function and C1-INH antigen) and one patient with type 2 (reduced function but normal C1-INH antigen) during typical attacks of angiodema and in remission. Five age- and sex-matched normals plus, in some cases, six healthy blood donors, served as controls. Blood for analysis of complement activation products was collected into Vacutainer tubes containing ethylene diamine tetraacetic-acid (EDTA) and kept on ice until frozen. Siliconized tubes containing polybrene-EDTA were used to collect blood for analyses of the coagulation, fibrinolytic and kallikrein-kinin system, and these samples were kept at room temperature until frozen. Serum was used for quantitation of complement factors. Aliquots were frozen without delay at 70 C and so kept until analysed in one batch. C1-INH complexes. Radioimmunoassay methods were utilized as described previously [17, 18]. A MoAb recognizing complexed C1INH was used as capture antibody on Sepharose beads (Pharmacia AB, Uppsala, Sweden), 125 I labelled polyclonal antibodies towards human C1s, factor XII, or kallikrein to detect bound complexes, respectively. Results were expressed in molar concentrations, referring to in-house standards consisting of normal human serum, in which maximal amounts of C1/C1-INH were generated by incubation with heat-aggregated immunoglobulins [19], or of normal human plasma in which maximal amounts of factor XIIa/C1-INH and kallikrein/C1-INH complexes were generated by incubation with dextran sulfate MW 500,000, as described [17]. Total C3 and C4. The total antigenic amounts of the complement components C3 and C4 were quantified by nephelometry (Behringwerke AG, Marbug, Germany) in the routine laboratory at the Institute of Immunology and Rheumatology, Oslo according to the manufacturer's instructions. Total C2. The total antigenic amount of the complement component C2 was quantified in an enzyme immunoassay (EIA) using monoclonal (MoAb) and polyclonal (poAb) antibodies kindly provided by Peter Garred and Claus Koch. Microtitre wells (NUNC Maxisorp, NUNC, Copenhagen, Denmark) were coated with mouse MoAb dilute 1/1000 in phosphate-buffered saline (PBS) at 4 C overnight. Standard (normal human serum diluted two-fold from 1/25) and samples diluted 1/100 in PBS containing 0.2% Tween 20 (PBS-T) were incubated for 45 min at 37 C. The rabbit pAb to C2 and a peroxidase-linked donkey anti-rabbit Ig antibody (Amersham, Buckinghamshire, UK), both diluted 1/1000 in PBS-T, were subsequently incubated for 45 min at 37 C. The plates were washed with PBS containing 0.05% Tween in a Dynawasher (Dynatech Laboratories, Alexandria, Virginia, USA) four times between each incubation. Susbtrate was 2,2 azino-di(3-ethyl)benzthiazoline sulfonate (0.3 mM) and H2 O2 (0.7 mM) in 0.1 M sodium acetate buffer, pH 4.0. Colour formation was measured spectrophotometrically at 410 nm using 490 nm as reference (Dynatech Model MR 7000). Values were referred to the standard defined to contain 100% C2. C4 activation products (C4bc). Activation of C4 was quantified using a neoepitope specific MoAb reacting with C4b, iC4b and C4c (C4bc) as described by Wolbink et al. [20]. Standard was a serum pool activated

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Table 1. Median values (range in parentheses) of 23 analyses covering the complement, coagulation, fibrinolytic and kallikrein kinin systems performed in samples from HAE patients during attacks and in remission, as well as in samples from healthy controls. P values are reported exact

C1/C1-INH complex (NM) C4 (g/l) C4bc (AU*.ml) Ratio C4bc/C4 C2 (%) Classical pathway CH50 (%) C3 (g/l) C3a (ng/ml) C3bc (AU/ml) C5a (ng/ml) TCC (AU/ml) FXI* antigen (%) FXIa/C1-INH* complex (pM) FXIa/ -1-AT* complex (pM) FXIa/ -2-AP* complex (pM) FXIa/ATIII* complex (pM) TAT* complex ( g/l) t-PA*( g/l) t-PA/C1-INH complex (pM) PAP* complex (nM) Kal/C1-INH* complex (nM) Cleaved HK* (% of total) HNE/ -1-AT* complex (ng/ml) 49 0.03 84 2007 31 36 0.77 23 11 12 1.23 110 11 0* 0* 0* 0.5 2.3 24 17.0 0.2 90 52 (34­167) (0.03­0.06) (54­141) (1810­2807) (7­53) (0­114) (0.51­1.01) (13­95) (8­157) (9­23) (0.93­1.72) (74­149) (0*­56) (0­19) ­ ­ (0.3­1.7) (2.0­2.9) (11­47) (11.0­24.0) (0.03­0.6) (24­100) (37­99) 32 0.11 50 922 111 110 0.75 26 21 8 0.88 107 9 0* 0* 0* 0.2 2.1 12 7.3 0.1 20 38 (24­35) (0.03­0.18) (33­115) (251­1089) (40­400) (2­127) (0.53­0.98) (16­39) (6­32) (7­14) (0.54­1.15) (76­146) (0*­56) ­ ­ ­ (0.05­02) (2.0­3.4) (11­54) (5.2­15.0_ (0.01­2.4) (17­24) (27­51) 20 0.25 38 131 106 114 0.75 20 7 13 0.60 116 0* 0* 0* 0* 0.2 2.0 34 6.6 0.2 19 35 (15­23) (0.19­0.29) (10­155) (39­816) (79­309) (98­155) (0.55­0.85) (8­28) (5­10) (1­16) (0.40­1.42) (78­178) (0*­90) ­ ­ ­ (0.05­23.0) (2.0­3.0) (11­282) (5.6­30.0) (0.04­2.4) (15­22) (20­44)

Cascade Activation During Attacks of HAE

* Abbreviations: / denotes complexes. AU, arbitrary units; C1-INH, C1-inhibitor; TCC, terminal complement complex; FXIa, activated factor XI; -1-AT, alpha-1-antitrypsin; -2-AP, alpha-2-antiplasmin; ATIII, antithrombin III; TAT, thrombin/antithrombin complex; PAP, plasmin complexed with alpha-2-antiplasmin; Kal/C1-INH, kallikrein complexed with C1-inhibitor; HNE, human neutrophil elastase. # Explications: 0 is not detected; ­ is range where values were not detectable.

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1996 Blackwell Science Ltd, Scandinavian Journal of Immunology, 44, 185­192

Analysis 0.0216 N.S. N.S. 0.0090 0.0433 0.0163 N.S. N.S. N.S. N.S. 0.0283 N.S. N.S. N.S. N.S. N.S. 0.0090 N.S. N.S. 0.0283 N.S. 0.0090 N.S.

Attack

Remission

Controls

P (Attack vs. Remission)

P (Remission vs. Controls) 0.0090 0.0090 N.S. 0.0163 N.S. N.S. N.S. N.S. 0.0290 N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S.

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Plasmin/alpha-2-antiplasmin-complexes (PAP). PAP complexes were measured by radioimmunoassay as described previously. A MoAb against complexed alpha-2-antiplasmin was used as capture- and an iodine 125 labelled MoAb against PAP as detecting antibody. Results were referred to standard curves of normal human plasma, in which maximal amounts of PAP complexes were obtained by adding urokinase [27]. High molecular weight kininogen (HK). Proteins from plasma containing polybrene-EDTA were immunoblotted as previously described for C1-INH [28], except that rabbit antiserum against human HK was used. The developed immunoblots were scanned with a Shimadzu CS9000 scanner used in reflection mode at 600 nm. The areas obtained form bands were used as a measure of the relative amounts of the native kininogen and degradation products. Complexes between alpha-1-antitrypsin and neutrophil elastase/ These were measured with a radioimmunoassay described previously [29]. Statistics. Differences between the three groups were examined with non-parametric Kruskal­Wallis one-way analysis of variance, followed by Mann­Whitney U-test for comparison of two groups. Analyses and box plot presentation were done in Statview statistical software for MacIntosh (Abacus, CA, USA). Exact P values are reported, with values below 0.05 are considered significant.

Fig. 2. Terminal complement complex (TCC) (AU/ml) in samples from five patients during attacks and in remission, as well as 11 controls.

RESULTS Table 1 shows the median and range values of all analyses. Also presented are P values for the comparison of values between normal controls and those of HAE patients in remission, and for the comparison of attack and remission values. Complexes between C1 and C1-INH were higher during remission than in controls, and increased further during attacks. Total C4 in remission was lower than in controls, whereas the difference between remission and attack samples did not reach statistical significance. The ratio between C4 activation products C4bc (C4b, iC4b, C4c) and total C4 was higher during remission than

in controls, and increased further during attacks (Fig. 1). C2 in remission samples and controls were similar. During attacks, however, C2 and the classical pathway CH50 values dropped significantly. CH50 was virtually zero in two patients during attacks. C3a values were not significantly different. The activation products C3bc (C3b, iC3b, C3c) were higher during remission than in controls, whereas the difference between attack and remission samples did not reach statistical significance. The rise in C5a during attacks was not statistically significant, but TCC rose significantly (Fig. 2). No significant differences were observed for tissue plasminogen activator (t-PA) or t-PA complexed with C1-INH. Complexes between plasmin and alpha-2antiplasmin (PAP) in remission samples and controls were similar. During attacks, however, PAP increased significantly

Fig. 1. Ratio C4bc (AU) to C4 (g/l) in samples from five patients during attacks and in remission, as well as five controls. The horizontal lines in the boxplot present from below the 10th, 25th, 50th (median), 75th and 90th percentiles, respectively. P values are reported exact.

Fig. 3. Plasmin/antiplasmin (PAP) complexes (nmol/l) in samples from five patients during attacks and in remission, as well as five controls.

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Fig. 5. Cleaved high molecular weight kininogen (HK) in percentage of total HK in samples from five patients during attacks and in remission, as well as five controls.

Fig. 4. Increase in thrombin-antithrombin (TAT) complexes ( g/l) during attacks in five HAE patients.

(Fig. 3). Although no evidence for factor XI activation was found, a more than twofold increase in complexes between thrombin and antithrombin was observed during attacks (Fig. 4). The cleavage of high molecular weight kininogen (HK) in remission samples and controls were similar, but almost all HK was cleaved during attacks (Fig. 5). Elastase/alpha-1-antitrypsin complexes was higher during attacks, but only the difference between attacks and controls was significant. DISCUSSION Elevated levels of C1/C1-INH complexes in HAE patients demonstrate the importance of C1-INH as a regulator of the early part of the classical complement pathway. In contrast to, e.g. the kallikrein-kinin system (referred to below), no other known inhibitor can replace C1-INH in the early part of the classical complement pathway, and complexes between C1 and C1-INH are thus a sensitive marker of early

complement activation in HAE patients. This is illustrated by the additional rise of these complexes during attacks and corresponds to the findings of Cugno et al. also in five patients [11], and by the demonstration of increased levels during physical stress in HAE patients but not in normals [30]. The increased generation of C1/C1-INH complexes in patients with HAE at first glance may seem contradictory since these patients have a genetic deficiency of C1-INH. However, this deficiency is heterozygous and therefore some residual C1INH complexes in patients with HAE at first glance may seem contradictory since these patients have a genetic deficiency of C1-INH. However, this deficiency is heterozygous and therefore some residual C1-INH activity (10­30% of normal levels) is always present in blood samples from these patients. Levels of C1 are normal in HAE patients [31]. This condition favours autoactivation of C1 [32], which explains the increase of C1/ C1-INH complexes. It also implicates that a significant proportion of the tiny functional C1-INH pool in HAE patients is consumed, mainly by complexing to C1, yielding residual levels much lower than 50%, which would be expected from the heterozygous deficiency. Previous studies have shown low concentrations of total haemolytic activity (CH50), C4 and to a lesser degree C2 in HAE patients, with a further drop during attacks [4]. This is consistent with out findings and suggests further complement activation during these attacks. Enhanced complement activation was indeed evident from the observed increase of the ratio between C4 activation products and native C4 (Fig. 1). This ratio has previously successfully applied to remission samples as an aid in making the diagnosis of HAE in newborns [33]. The use of ratios between complement activation products and their native proteins as such is not novel; it has been proven valuable in other studies also [34]. Previous studies have reported C3 to be within normal limits

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190 E. Waage Nielsen et al. in HAE during remission as well as during attacks [2, 35]. Complement activation is believed to stop at the level of C3 in this condition, which can be explained by pointing to the susceptibility of C4b in the fluid phase for inactivation by factor I, and thereby the classical C3 convertase [36]. Moreover, Gronsky et al. [37] showed a six- to eightfold increase in the ratio between C4b-binding protein (C4BP) and C4 in HAE patients and considered this as evidence for efficient inhibition at the level of C4. Two previous reports, however, could indicate an activation beyond C2, apparently with increased catabolic rate of C3 [38, 39]. Our finding of increased C3bc in HAE patients support this latter observation and, for the first time, we show a modest but significant activation of the terminal part of the complement system in HAE patients. These increased values in the TCC assay, which previously was shown to have a higher sensitivity in detecting complement activation than the C3bc assay [4], also indicate a significantly enhanced activation of C5 and later components during attacks. The lack of increased levels of C5a is not surprising since this activation product is cleared rather rapidly from the circulation. The half-life of C5a is about 3 min [41], compared to approximately 1 h for TCC [42]. Indirect support for the activation of C5 during attacks of HAE was also the observation that levels of elastase/alpha-1-antitrypsin increased, since C5a is capable of mediating the release of elastase from neutrophils. Activation of the later components of complement raises the possibility that other vasocactive split products, such as C5a, play in concert with bradykinin in the pathogenesis of the oedema. We demonstrate for the first time that coagulation is activated during attacks of HAE, represented by a twofold increase of complexes between thrombin and antithrombin (TAT). However, we did not detect activation of factor XI using very sensitive assays for the complexes of this factor with its inhibitors, that allows detection of 0.1% activation of factors in plasma [13]. This finding is against the theory of a downstream activation of the intrinsic pathway of coagulation via activation of factor XI by factor XII a and also failed to detect any effect of thrombin on FXI [43]. Another possibility for thrombin generation in HAE patients could be via the direct activation of factor VII by Factor XIIa [44, 45]. This may be in accordance with the recent findings of increased factor VIIa levels in remission HAE patients, examined in a novel assay in which only the pre-existing content of factor VIIa is measured [46]. Finally, it has recently been demonstrated that relatively low concentrations of plasmin, insufficient to deplete alpha-2-antiplasmin, induced marked increases in thrombin activity in plasma [47]. Plasmin activation obviously takes place during acute attacks of HAE, in this study represented by more than twofold increase of circulating plasmin/alpha-2-antiplasmin complexes, but to what extent this mechanism explains the thrombin generation during attacks of HAE is unclear. The novel observation of activation of the coagulation system in HAE represents a counterweight to the previously found increased fibrinolysis and may, in part, explain why neither bleeding nor thromboenic episodes are experienced by patients suffering from hereditary angioedema. The normal antigenic values of t-PA, both in remission and during attacks, are consistent with previous studies on HAE attacks [11], in which urokinase-type plasminogen activator (uPA) was also found to be normal. Since neither t-PA nor u-PA are released it has been postulated that, in HAE patients, plasmin, as shown by increased PAP complexes [11], is at least partly generated by other mechanisms. It is possible that kallikrein and FXIIa, poorly inhibited in HAE patients, can participate in this matter as they directly activate plasminogen [48, 49]. Complexes between kallikrein and C1-INH did not differ from normal values nor did they rise during attacks. In the kallikreinkinin system, alpha-2-macroglobulin may to a certain extent compensate for lack of C1-INH. Even if HAE patients have enough C1-INH to complex to kallikrein, activated kallikrein tends to prefer alpha-2-macroglobulin, and complexes between kallikrein and alpha-2-macroglobulin may therefore b more sensitive in detecting activation of the kallikrein-kinin system in HAE patients [50, 51]. Although we did not measure alpha2-macroglobulin complexes in our patients, we found significant activation of the kallikrein-kinin system, since a significant increase in HK conversion during attacks was observed. These results are in accordance with the findings of Cugno et al. [11], and increase the few cases with this observation in HAE patients. The increase of complexes between neutrophil elastase and alpha-1-antitrypsin indicate that neutrophils are degranulated during attacks of HAE. On the one hand this degranulation may have resulted from exposure to complement activation products during attacks as discussed above. On the other hand, factor XIIa, largely unopposed in HAE, and kallikrein could also co-operate in attracting and degranulating neutrophils [52, 53]. In conclusion, this study shows that attacks of HAE are accompanied by significant activation of the terminal part of the complement cascade, as well as by activation of the coagulation system, neutrophil cells and kallikrein-kinin and fibrinolytic systems. These observations raise the possibility that in addition to bradykinin, other vasocative peptides may also contribute to the pathogenesis of angioedema in this condition.

ACKNOWLEDGMENTS We thank Grethe Bergseth, Tone Reitstad, Bente Falang Hoaas, Adam Babinski and Anke J. M. Eerenberg for excellent technical support. The study was financially supported by Behringwerke, Anders Jahre's Fund for the Promotion of Science, and the Research Council of Norway. REFERENCES

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