Studies of the mechanisms of bradykinin generation in hereditary angioedema plasma

Studies of the mechanisms of bradykinin generation in hereditary angioedema plasma Kusumam Joseph, PhD*; Thomson B. Tuscano, MD*; and Allen P. Kaplan, MD†

Background: Factor XII– dependent bradykinin formation is thought to be responsible for the swelling associated with the various forms of C1 inhibitor deficiency, and complement activation is augmented during attacks of swelling. Objectives: To further elucidate the interactions of the kinin-forming cascade that lead to complement activation during attacks of swelling and to determine whether fibrinolysis is augmented as well. Methods: We compared spontaneous and kaolin-induced activation of normal plasma with the plasma of patients with hereditary angioedema. Results: Hereditary angioedema plasma demonstrated augmented factor XII activation, production of factor XIIf, prekallikrein activation, and high-molecular-weight kininogen cleavage, and, as a result, bradykinin formation was markedly increased. Baseline levels of C4a and plasmin-␣2 antiplasmin complexes increased, and, on activation with kaolin, levels increased further. Conclusions: All parameters indicative of activation of the bradykinin-forming cascade are activated in hereditary angioedema plasma vs normal plasma. Production of factor XIIf, demonstrated for the first time in whole plasma, may be responsible for C1 activation based on C4a production. The factor XII– dependent fibrinolytic cascade is also activated. Ann Allergy Asthma Immunol. 2008;101:279–286.

INTRODUCTION The swelling that results from C1 inhibitor (C1-INH) deficiency is thought to be dependent on the production of bradykinin1–3 via a plasma cascade that involves activation of factor XII, conversion of prekallikrein to kallikrein, and digestion of high-molecular-weight kininogen (HK) to release bradykinin.4 This can occur whether the C1-INH deficiency is hereditary (as in hereditary angioedema [HAE]) or acquired. C1-INH is known to inhibit all forms of activated factor XII (factor XIIa and factor XIIf) as well as plasma kallikrein5–7; thus, deficiency of C1-INH leads to marked augmentation of bradykinin formation once the cascade is activated. C1-INH also inhibits the fibrinolytic enzyme plasmin, serine proteases associated with the lectin complement pathway (mannosebinding lectin-associated serine proteases),8,9 and the activated C1r and C1s subcomponents of the first component of complement. Although neither plasmin nor C1 is requisite for bradykinin formation, the antifibrinolytic agents ␧-aminocaproic acid and tranexamic acid10 are effective therapeutic agents, and the complement cascade is increasingly activated during attacks of swelling.11 Although activation of factor XII, the initiating enzyme requisite for bradykinin formation, is known to also initiate a

Affiliations: * Department of Medicine, Division of Rheumatology and Clinical Immunology, Medical University of South Carolina, Charleston, South Carolina; † Department of Medicine, Division of Pulmonary, Critical Care, Allergy, and Immunology, Medical University of South Carolina, Charleston, South Carolina. Disclosures: Authors have nothing to disclose. Funding Sources: This study was funded by Lev Pharmaceuticals Inc, New York, New York. Received for publication January 24, 2008; Received in revised form April 11, 2008; Accepted for publication April 14, 2008.

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fibrinolytic cascade,12 the mechanism by which plasminogen is converted to plasmin in HAE plasma is not clear. Nevertheless, excessive fibrinolytic activity in HAE plasma has been clearly demonstrated by assay of plasmin-␣2 antiplasmin (PAP) complexes,13 but the role of plasmin in the production of swelling is unknown. Furthermore, factor XIIf, the active fragment derived from factor XII activation, has been shown to activate the C1r subcomponent of C1 in vitro14; however, factor XIIf formation on activation of human plasma and/or a resultant boost in complement activation in HAE plasma have not been previously demonstrated. The studies described herein address some of these issues and demonstrate, for the first time to our knowledge, formation of factor XIIf in plasma, C1 activation (as evaluated by C4a release) even in an unactivated sample, and excessive fibrinolytic activity in HAE plasma, compared with normal plasma. METHODS Collection of Plasma Normal plasma and C1-INH– deficient plasma obtained from 5 patients with type I HAE were collected, using 0.4% citrate as anticoagulant. Patients were asymptomatic and were not taking prophylactic medication when blood was drawn. The samples were control specimens obtained before a study using infused C1-INH as prophylactic therapy. All 5 patients were from different families and none had acquired angioedema. The protocol for the study was approved by the institutional review board of the National Allergy, Asthma, and Urticaria Centers of Charleston, and written informed consent was obtained from the participants. After collection, the plasma samples were aliquoted and stored at ⫺80°C until use.

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Prekallikrein Activation Assay Prekallikrein activation assays were performed as described previously,15 in assay buffer (10-mmol/L HEPES, 137mmol/L sodium chloride, 4-mmol/L potassium chloride, 11mmol/L D-glucose, 1-mg/mL of radioimmunoassay [RIA] grade bovine serum albumin), with a kallikrein-specific substrate (0.6-mmol/L S2302, Chromogenix, DiaPharma Group, West Chester, Ohio) in 96-well disposable polystyrene microtiter plates (Dynatech Laboratories, Chantilly, Virginia). These microtiter plates were pretreated with 1% polyethylene glycol (Aquacide III; Calbiochem, La Jolla, California) in HEPES-buffered saline (HBS) (10-mmol/L HEPES, 137mmol/L sodium chloride, 4-mmol/L potassium chloride, 11mmol/L D-glucose) for 2 hours to prevent adsorption of the proteins used for the assay. Just before assay, all the proteins were treated with 2.0-mmol/L APMSF (p-amidinophenylmethylsulfonylfluoride; Calbiochem, San Diego, California) for 90 minutes at pH 5.5, after which they were diluted 1:100 with assay buffer and incubated for 30 minutes to allow for the decomposition of any unreacted APMSF at neutral pH. Assay buffer was also pretreated with 0.4␮M APMSF to inactivate any serine protease activity present in the RIAgrade bovine serum albumin. HK and prekallikrein were incubated in the assay buffer in the presence of S2302, and the kallikrein activity was determined by color development. The absorbance (OD at 405 nm) was monitored at room temperature on a Molecular Devices (Sunnyvale, California) THERMOmax microplate reader. Factor XII Activation Assay Factor XII activation assay procedure16 is similar to the prekallikrein assay except that the samples were incubated in assay buffer with a factor XIIa–specific chromogenic substrate (S2222; Chromogenix, DiaPharma Group). Bradykinin Assay For quantification of bradykinin, samples were prepared by incubating plasma or purified proteins at room temperature and withdrawing aliquots at specific intervals. The proteins in the aliquots were precipitated with ice-cold ethanol and centrifuged for 1 hour at 10,000 rpm in a microcentrifuge at 4°C; the supernatant containing free bradykinin was then collected. The supernatant was then evaporated to dryness using a centrifugal concentrator and resuspended in the enzyme immunoassay buffer. Bradykinin enzyme immunoassay was performed using an assay kit supplied by Peninsula Laboratories (San Carlos, California). Briefly, bradykinin antibody was first bound to a 96-well plate. Biotinylated bradykinin and samples or standards then were added, mixed, and incubated for 2 hours at room temperature. After incubation, unbound peptides were removed by washing, and horseradish peroxidase– conjugated streptavidin was added and allowed to bind to the immobilized biotinylated peptide. The wells were again washed and the peroxidase substrate B (3,3⬘,5,5⬘tetramethyl benzidine dihydrochloride) was added to react with the bound horseradish peroxidase. The color intensity is

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inversely proportional to the amount of peptide present in the sample. Concentration of the peptide present in samples was calculated using a standard curve. Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis and Western Blot Samples were prepared by incubating desired protein mixtures at room temperature and withdrawing aliquots at specific intervals. The reaction was stopped by the addition of sodium dodecyl sulfate (SDS) sample buffer containing 5% 2-mercaptoethanol. SDS–polyacrylamide gel electrophoresis was performed using the buffer system of Laemmli.17 Gradient gels (4%–15%) were used for separation of proteins. After electrophoresis, the proteins on the gels were transferred to nitrocellulose membranes overnight. The membranes were then incubated with blocking buffer (1% bovine serum albumin in phosphate-buffered saline) for 1 hour. After blocking, the membranes were probed with monoclonal antibodies for 1 additional hour. Bound probes were visualized by incubating the membranes with alkaline phosphatase– conjugated secondary antibodies followed by color development in 5-bromo-4-chloroindolyl phosphate/nitroblue tetrazolium. Plasmin-␣2 Antiplasmin Assay The quantitative determination of PAP complex in control and HAE plasma samples was performed with the use of the PAP Micro ELISA assay kit from ALPCO Diagnostics. Samples were incubated in a microtiter plate coated with monoclonal antibodies to PAP. The unbound fragments were removed by washing, and peroxidase-conjugated antibodies to plasminogen (which cross-react with plasmin) were added and incubated for 15 minutes. The unbound conjugates were removed by washing, and the substrate was added for color development. The color intensity is proportional to the concentration of PAP, which was calculated using a standard curve. Complement C4a Assay The complement C4a was quantified on the basis of its conversion to C4a des Arg by plasma carboxypeptidases. A competitive immunoassay kit obtained from Assay Designs (Ann Arbor, Michigan) was used. In this method, a polyclonal antibody to human C4a des Arg is used to bind in a competitive manner the human C4a des Arg in the sample or standard or an alkaline phosphatase molecule that has human C4a des Arg attached to it. After incubation at room temperature, the excess reagents were washed and the substrate was added for color development. The intensity of the color is inversely proportional to the concentration of human C4a des Arg in either standards or samples. The measured OD was used to calculate the concentration of human C4a des Arg. Statistical Analysis Statistical analysis was performed with the Student t test. P ⬍ .05 was considered statistically significant.

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Factor XII Activation Figure 4 depicts activation of factor XII using a factor XIIa– specific synthetic substrate (S2222). HAE and control plasma were incubated in the presence of factor XIIa substrate at room temperature, and the conversion of factor XII to activated factor XII was monitored. Slowly increasing levels of activated factor XII were observed in HAE plasma, whereas there was no activity in the control plasma (Fig 4A). When purified proteins were used to demonstrate conversion of factor XII to factor XIIa, the activity was reduced in the presence of C1-INH (Fig 4B). Figure 4C is a Western blot analysis of the activation of factor XII using polyclonal antibody to factor XII. There was activation of factor XII in HAE plasma with the formation of factor XIIf after 3 hours of incubation. Fibrinolysis Fibrinolytic activity in HAE plasma was evaluated by measuring the level of the PAP complex after incubating the

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Prekallikrein Activation Normal plasma and C1-INH– deficient plasma samples were collected using 0.4% citrate as anticoagulant. EDTA was not used because it retards activation of the contact activation pathway by chelating zinc ions, although quantification of bradykinin may be underestimated in citrate because EDTA also inhibits kininases and prevents bradykinin degradation. The rate of conversion of prekallikrein to kallikrein was evaluated using a specific synthetic chromogenic substrate for kallikrein (S2302). Prekallikrein was activated to kallikrein when C1-INH– deficient plasma (Fig 1A) was incubated at room temperature for 1 hour in the absence of any exogenous activator. No activation of prekallikrein occurred in normal plasma during this interval. Figure 1B demonstrates the activation of prekallikrein when purified proteins (HK, prekallikrein, and factor XII) were incubated in the presence or absence of C1-INH at room temperature. Prekallikrein activation was slower in the presence of C1-INH compared with the absence of C1-INH and was barely detectable in normal plasma. Activation of prekallikrein in plasma or in a mixture of purified proteins was also evaluated by checking the cleavage of HK (Fig 2) and the release of bradykinin (Fig 3). Incubation of HAE plasma led to complete cleavage of HK within 15 minutes, whereas there was no HK cleavage in the control plasma at the 2-hour time point (Fig 2A). When purified proteins were used to compare the cleavage of HK, there was faster cleavage and degradation of HK in the absence of C1-INH (cleavage complete by 30 minutes) compared with when C1-INH was present at a concentration of 100-␮g/mL (Fig 2B) (comparable cleavage in 1 hour). The baseline levels of bradykinin were markedly elevated in HAE plasma, which was degraded rapidly (Fig 3), whereas bradykinin levels were not elevated in normal plasma.

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Time in Minutes Figure 1. Prekallikrein (PK) activation in hereditary angioedema (HAE) plasma. A, HAE plasma was prepared by collecting blood in 0.4% sodium citrate. Kallikrein activity was determined by color development. Absorbance (OD at 405 nm) was monitored at room temperature on a microplate reader. The assay was repeated by using 5 different plasma samples; a representative chromatogram is given. B, PK activation assay was performed with the use of purified proteins. High-molecular-weight kininogen (HK), PK, and factor XII (FXII) (with or without C1 inhibitor [INH]) were incubated in the assay buffer (10-mmol/L HEPES, 137-mmol/L sodium chloride, 4-mmol/L potassium chloride, 11-mmol/L D-glucose, 1-mg/mL radioimmunoassay grade bovine serum albumin) in the presence of S2302 and the kallikrein activity was determined by color development. The assay was repeated 3 times. A representative chromatogram is given.

plasma in the presence or absence of kaolin. The baseline fibrinolytic activity was higher in HAE plasma compared with control, but the activity did not increase during incubation in the absence of kaolin. When kaolin was included in the incubation (Fig 5), there was an increase in fibrinolytic activity of both HAE and control plasma, demonstrating that factor XII– dependent contact activation leads to increased fibrinolytic activity. With time, the rate of fibrinolysis was

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Figure 2. High-molecular-weight kininogen (HK) cleavage in hereditary angioedema (HAE) plasma. A, Plasma was incubated at room temperature, and samples were withdrawn at desired time points. The reaction was stopped by the addition of sodium dodecyl sulfate (SDS) sample buffer containing 5% 2-mercaptoethanol. Lanes 1 to 5, normal plasma at different time points (0, 0.25, 0.5, 1, and 2 hours); lanes 6 to 10, HAE plasma at different time points (0, 0.25, 0.5, 1, and 2 hours). B, SDS–polyacrylamide gel electrophoresis and Western blot, using purified proteins. Lanes 1 to 6, HK, prekallikrein (PK), and factor XII (FXII) incubated for different periods (0, 0.25, 0.5 (C1-INH), 1, 2, and 3 hours); lanes 7 to 12, HK, PK, factor XII, and C1 inhibitor incubated for different periods (0, 0.25, 0.5, 1, 2, and 3 hours).

more rapid in HAE plasma, hence the divergence of the lines after 40 minutes. Kaolin-Induced Contact Activation Contact activation can also lead to activation of the complement system (Fig 6). Complement activation was evaluated by measuring the levels of C4a in plasma after incubation with or without kaolin. The baseline levels of C4a were much higher in HAE plasma compared with normal plasma, and the levels increased slowly on further incubation (Fig 6A). In the presence of kaolin there was increased activity up to 1 hour (Fig 6B), and the increment in HAE plasma was more prominent. To test which protein of the contact system (HK, prekallikrein, or factor XII) is involved in the activation of the complement system, we activated normal plasma and plasmas that are deficient in HK, prekallikrein, or factor XII with kaolin and assayed for C4a formation. As shown in

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Figure 3. Bradykinin levels in plasma. Plasmas were incubated at room temperature and aliquots were withdrawn at desired time points. The proteins in the aliquots were precipitated with ice-cold ethanol and centrifuged for 1 hour at 10,000 rpm in a microcentrifuge at 4°C, and the supernatant containing free bradykinin was collected. The supernatant was then evaporated using a centrifugal concentrator to dryness and resuspended in the enzyme immunoassay buffer. The bradykinin enzyme immunoassay was performed using an assay kit supplied by Peninsula laboratories. Data represent mean ⫾ SD of 3 separate assays. *P ⬍.001. HAE indicates hereditary angioedema.

Figure 6C, C4a levels were lower in HK-deficient plasma compared with other plasmas tested. During the first 40 minutes, C4a levels increased significantly compared with the starting value in normal plasma and prekallikrein-deficient plasma but not in plasma deficient in factor XII or HK. This is consistent with an absolute requirement for factor XII and HK for contact activation and subsequent complement activation, whereas prekallikrein-deficient plasma can autoactivate and approximate normal.18 DISCUSSION We confirm that the rate of activation of the factor XII– dependent pathways4,19 in plasma that is deficient in C1-INH is markedly increased, culminating in rapid and excessive generation of bradykinin. The parameters examined include digestion of factor XIIa to produce factor XIIf, an increased rate of conversion of prekallikrein to kallikrein, a marked increase in the rate of HK digestion, and augmented bradykinin formation. Our data for bradykinin are an underestimate because kininases were not inhibited, but this was necessary to accurately evaluate activation of the enzymes in plasma and to observe effects on the complement cascade. Nevertheless, HAE plasma is unstable; elevated bradykinin levels are seen simply from handling the plasma at the earliest time point measured (Fig 3). Similarly, activation of prekallikrein and HK can be discerned on incubation of HAE plasma in a test tube without any “activator” being added, whereas normal plasma requires addition of an exogenous negatively charged macromolecular particle (such as kaolin) to initiate the cascade.

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Figure 5. Plasmin-␣2 antiplasmin (PAP) assay. Quantification of the amount of PAP complex was performed using an enzyme immunoassay kit supplied by ALPCO Diagnostics. Samples were prepared according to the manufacturer’s recommendations. Plasma samples were incubated at room temperature in the presence of kaolin, and samples were withdrawn at desired time points. The reaction was stopped by the addition of 0.05M ␧-aminocaproic acid. Data represent mean ⫾ SD of 4 separate assays. *P ⬍.0001. HAE indicates hereditary angioedema.

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Figure 4. A, Factor XII (FXII) activation in hereditary angioedema (HAE) plasma. HAE plasma was prepared by collecting blood in 0.4% sodium citrate. Plasma was incubated in the presence of a factor XIIa–specific substrate (0.6mmol/L S2222) in 96-well disposable polystyrene microtiter plates. Factor XIIa activity was determined by color development. B, Factor XII activation using purified proteins. High-molecular-weight kininogen (HK), prekallikrein (PK), and factor XII (with or without C1 inhibitor [C1-INH]) were incubated in the assay buffer (10-mmol/L HEPES, 137-mmol/L sodium chloride, 4-mmol/L potassium chloride, 11-mmol/L D-glucose, 1-mg/mL radioimmunoassay-grade bovine serum albumin) in the presence of S2222, and factor XIIa activity was determined by color development. C, Factor XII cleavage in HAE plasma. Plasma was incubated at room temperature and samples were withdrawn at desired time points. The reaction was stopped by the addition of sodium dodecyl sulfate sample buffer. Lanes 1 to 5, HAE plasma incubated for different time points (0, 0.25, 0.5, 1, and 2 hours); lanes 6 to 10, normal plasma incubated for different time points (0, 0.25, 0.5, 1, and 2 hours).

The effect of C1-INH is less prominent when purified proteins are tested (Figs 1B, 2B, and 4B) because there are always traces of active enzyme in purified factor XII20 and

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prekallikrein preparations, and the rate of enzyme interactions far exceeds the rate of inhibition by C1-INH. Also, the final concentration of C1-INH in the assay mixture is about half-normal. Of particular note is the conversion of factor XII to factor XII fragment shown in Figure 5, because factor XIIf is the product of the contact activation cascade that can activate the classic complement cascade.14 Factor XIIa is inactive, and kallikrein destroys C1r but does not inactivate it. Plasmin, however, might contribute to C1 activation.21 Although factor XIIf production is readily demonstrable by using purified proteins,22 its formation in whole plasma has not been previously demonstrated. In fact, absence of C1INH may be requisite for significant levels of factor XIIf to be produced. The marked elevation of C4a levels in HAE plasma at baseline (but not in normal plasma) indicates either in vivo activation in the patient or in vitro activation even as frozen plasma. However, the values may reflect the in vivo situation because C4 levels are depressed even when patients are asymptomatic.23 There is a gradual increase in C4a levels of both normal and HAE plasma with time (Fig 6A); however, addition of kaolin causes an augmented burst of C4a formation in HAE plasma compared with normal plasma that is probably due to factor XIIf formation. Although we assume that the origin of this C4a is cleavage of C4 by activated C1s, the enzymes of the lectin complement pathway (mannosebinding lectin-associated serine proteases) also cleave C4 and are inhibited by C1-INH. Any contribution of this pathway to complement activation has not been addressed, but the boost in C4 cleavage and depressed C4 levels with HAE attacks are dependent on contact activation24 rather than the lectin-dependent complement pathway.8 Neutrophil-derived enzymes can also cleave C4, but these are not inhibited by C1-INH,

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Time in Minutes Figure 6. Human complement C4a measurement in hereditary angioedema (HAE) plasma. Complement C4a in the plasma was quantified using a competitive immunoassay developed by Assay Designs. Plasmas were incubated at room temperature in the absence (A) and presence (B) of kaolin (Ka), and samples were withdrawn at desired time points. Assay was performed according to manufacturer’s recommendations. Data represent mean ⫾ SD of 3 separate assays each in duplicate. Complement C4a measurement in coagulation factor– deficient plasmas (C). Factordeficient plasmas were obtained from George King Biomedical Inc (Overland Park, Kansas). Complement C4a in the plasma was quantified using a competitive immunoassay developed by Assay Designs. Plasmas were incubated at room temperature in the presence of kaolin, and samples were withdrawn at desired time points. Assay was performed according to the manufacturer’s recommendations. Data represent mean ⫾ SD of 3 separate assays. FXII, factor XII; HK, high-molecular weight kininogen; PK, prekallikrein.

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and their contribution to any C4 cleavage seen should not be different in normal vs deficient plasma. With regard to the pathogenesis of swelling, we consider the activation of complement to be an epiphenomenon. C4 levels are decreased in 95% of patients with HAE25 as the result of contact activation as well as autoactivation of C1r when C1-INH is absent.26 The extent of C1r autoactivation in a plasma milieu is considerably slower than is observed with purified C1r.26 Levels of C2 and C3 are generally normal. During an episode of angioedema, however, C2 levels decrease and C4 levels decrease further, often approaching zero, whereas C3 levels (and other complement components) are normal. This increment in complement activation may be due to factor XIIf formation during episodes of swelling with further activation of C1r and C1s.14 If C3 is excessively activated during this circumstance, synthesis clearly compensates so that diminished levels are not seen. Although C4a is an anaphylatoxin (with 10-fold less activity than is seen with C3a or C5a),27 antihistaminics have no effect on the swelling and cannot be used to prevent this form of angioedema. Likewise, the idea that a kininlike fragment is produced on cleavage of C228 has never been reproducible and probably represents an artifact.29 We also noted an increase in baseline PAP complex30 levels in HAE plasma compared with normal plasma, and activation with kaolin raised that level in both HAE plasma and normal plasma, although the levels reached in HAE plasma were greater. Conversion of plasminogen to plasmin in a strictly plasma milieu may be dependent on kallikrein activation of the small amount of prourokinase present,31 although kallikrein can slowly activate plasminogen as well. Plasmin is an enzyme that can cleave and activate factor XII (as does kallikrein)32 to produce factor XIIf, and plasmin can digest both HK33 and C1-INH,34 effects that may lead to augmented bradykinin formation. Plasmin does not directly convert prekallikrein to kallikrein. Nevertheless, the efficacy of antifibrinolytic agents might be due to inhibition of plasmin-dependent functions that have not yet been elucidated. For example, new data regarding the formation of bradykinin at the endothelial cell level reveal activation along the cell surface that depends on binding of HK and factor XII to cell surface ligands such as gC1qR,35,36 cytokeratin 1,37–39 and the urokinase plasminogen activator receptor.40 Activation can be dependent on factor XII as the initiator16 but can occur even in the absence of factor XII by activation of the HK-prekallikrein complex by either heat shock protein 9015,41 or prolyl carboxypeptidase.42 Release of heat shock protein 90 would be a candidate for the observation that stress can induce angioedema episodes in patients with HAE.15 In this scenario, the presence of factor XII augments the reaction as a result of factor XII cleavage by kallikrein,43 but factor XII does not need to serve as the initiator. Conversion of plasminogen to plasmin can also occur at the cell surface.44 Release of tissue plasminogen activator as a result of interactions at the cell surface including bradykinin stimulation of endothelial cells is another consideration. The role of C1-INH (or its absence)

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3. 4. 5. 6. 7.

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Figure 7. Activation of normal vs C1 inhibitor– deficient plasma depicting excessive bradykinin formation and significant activation of complement when C1 inhibitor is absent. HK indicates high-molecular-weight kininogen; PK, prekallikrein.

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at these cell surface reactions has not been examined and might provide insight to aspects of the swelling that we do not understand, such as the seemingly random occurrences of some episodes, the way trauma increases the likelihood of angioedema occurring, the role of stress, and the role of antifibrinolytic agents in effectively inhibiting swelling. It is clear that the absence of C1-INH leads to destabilization of plasma as is seen in patients with HAE with markedly augmented activation of the bradykinin-forming cascade even in the absence of addition of an exogenous initiator with activation of fibrinolysis, complement activation, and augmented bradykinin generation via factor XIIf. This is depicted in Figure 7 for bradykinin formation and C4 cleavage. Although androgens and antifibrinolytic agents are the mainstay of prophylactic treatment in the United States, it is clear that C1-INH replacement therapy should be efficacious for prophylaxis and a good alternative to fresh-frozen plasma for acute episodes of angioedema. Finally, new agents that inhibit plasma kallikrein or inhibit the effects of bradykinin at the B2 receptor level are effective for treatment of acute episodes of swelling and lend further support for the critical role of bradykinin as the mediator of the swelling.45– 47 REFERENCES 1. Fields T, Ghebrehiwet B, Kaplan AP. Kinin formation in hereditary angioedema plasma: evidence against kinin derivation from C2 and in support of “spontaneous” formation of bradykinin. J Allergy Clin Immunol. 1983;72:54 – 60. 2. Shoemaker LR, Schurman SJ, Donaldson VH, Davis AE III. Hereditary

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Requests for reprints should be addressed to: Kusumam Joseph, PhD Department of Medicine Division of Rheumatology and Clinical Immunology Medical University of South Carolina Charleston, SC 29425 E-mail: [email protected]

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