The plasma contact system in atopic asthma

The plasma

contact

system

in atopic

asthma

Elliott C. Lasser, M.D., Joseph H. Lang, Ph.D., John G. Curd, M.D.,’ Charles G. Cochrane, M.D.,2 Sandra G. Lyon, B.A., Mitzi M. Howard, A. Elizabeth Hamblin, B.S., and Susan D. Revak, B.A.2 San Diego

and La Jolla,

B.A.,

Calif.

An in vitro test for the rate of’ appearance qf kallikrein in plasma due to contact system activation by dextran sulfate at 0” C was applied to plasmas of 19 atopic asthma patients and 19 age- and sex-matched controls without atopy. The average prekallikrein activation rate was markedly higher in the plasmas of the atopic patients. Mean endogenous heparin levels were also elevated. (JALLERGYCLINIMMUNOL 72:83-88,1983.)

In studying sensitivity to x-ray contrast media in humans, we observed that the rate of dextran sulfate-catalyzed conversion of plasmaprekallikrein to kallikrein in vitro was elevated in the plasma of individuals with documented contrast media reactions. ’ Since sensitivity to radiocontrast media occurs more frequently in atopic asthma,’ we questioned whether prekallikrein-to-kallikrein conversion rates might similarly be elevated in atopic asthmaplasmas. We now report that under the conditions of our test, the mean rate of conversion of prekallikrein to kallikrein was markedly elevated in the plasma of these atopic individuals. Such plasmasalso showedsignificant elevations in their mean endogenousheparin levels. Exogenous hepatin resembledthe endogenous heparin of theseatopic patients in its relative concentration of heparin active against thrombin and against factor Xa but differed from normal values in this respect. MATERIALS Subjects

AND METHODS

Nineteenpatients(8 male, 11female)with severeallergic asthmawere studied.The patients’agesrangedfrom 3

to 57 yr, with 11beinglessthan 16. Thediagnosis of atopic asthmawasmadein eachcaseby a characteristichistoryof episodicwheezing,and two or moreof the following: diminishedPEFR, positive skin test to dustsand molds, eczema,persistentrhinitis, seasonal asthma,elevatedIgE levels,or a positiveRAST test.Themajority of patientshad threeor moreof these associated signs or symptoms. Nine of the 19patientshadreceivedsystemicsteroidswithin the 2 weeks before the study. Eight individuals

were receiving

nonsteroidaltherapy for asthma(bronchodilators,antihistamines,expectorants)at the timeof samplecollection.The durationof knownasthmain the grouprangedfrom 1 to 36 yr (averageabout 1 I). Bloodspecimens wereobtainedfrom thepatientsand 19 age- sex-matched controls (volunteers and patients without demonstrable atopic disease) during outpatient evaluations, in accordance with procedures approved by the Institutional Human Use Committee. Blood from these subjects was sampled by vein puncture with plastic syringes and transferred to siliconized tubes containing sodium citrate (Becton, Dickinson & Co., Orangeburg, N. J.) The samples were centrifuged (3000 x g) and stored in plastic cuvettes at -70” C in small aliquots until analyzed. All assays were performed with plasma frozen and thawed only once.

Prekallikrein-to-kallikrein

conversion

rates

Measurement of the rateof conversionof prekallikreinto From the Department of Radiology, University of California, San Diego, School of Medicine, and the Departments of ‘Molecular Immunology and Clinical Research and of 21mmunopathology, Scripps Clinic and Research Foundation, La Jolla, Calif. Supported by P.H.S. grants GM 22794, AM 00543, and AI 10386 from the National Institutes of Health, Bethesda, Md. Received for publication Oct. 11, 1982. Accepted for publication Jan. 27, 1983. Reprint requests: Elliott C. Lasser, M.D., Department of Radiology, UCSD Medical Center, University Hospital, 225 Dickinson Street, San Diego, CA 92103.

kallikrein was carried out by a modification of Kluft’s method” for the assay of total prekallikrein and was previously reportedin detail.’ Briefly, 40 ~1 of test plasmawas

incubatedat 0” C with 10 ~1 of 1% zwitterionic detergent (Zwittergent 3-08; Calbiochem-Behring Corp., La Jolla, Calif.) and 50 ~1 of 1.5 mg/L dextran sulfate (mol. wt.

500,000; Sigma ChemicalCo., St. Louis, MO.). Aliquots were removed at 10, 20, and 30 min. The 15 ~1 aliquots were added to cuvettes containing1 ml of PPAN (Vega Biochemicals, Tucson, Ariz.), 1 mM, in Tris-NaCl buffer, pH 7.9, and the change of O.D. at 405 nm was 83

84

Lasser

1 ALLERGY

et al

Ahhrcviuti0tr.s

PEFR: PPAN: NHP: CIINH: HF: HMWK: SDS-PAGE: AT Ill: EDTA: O.D.: FEV,: APTT:

(‘:!N

IMMU.\O~ ,I,[ \ iCj8.G

rrseri

Peak exploratory flow rate ,Y-benzoyl-L-prolyl-t~-phenylalanylI.-arginine-/7-nitroanilide Normal human plasma CT-e\terase inhibitor Hageman factor High-molecular-weight kininogen Sodium dodecyl sulfatepolyacrylamide gel electrophoresis Antithrombin III Ethylenediamine tetraacetic acid Optical density Forced expiratory volume in 1 set Activated partial thromboplastin time

recorded at 37” for about 3 min. Under conditions of the assay, hydrolysis of PPAN was shown to be specific for kallikrein.’ Rates of appearance of kallikrein activity were determined for the plasma of atopic asthma patients and controls. The effects of heparin were measured by addition of IO ~1 of commercial porcine heparin or of normal saline to 10 ~1 of Zwittergent. 40 ~1 of control plasma, and 60 ~1 of 1.5 mg/L dextran sulfate. The control plasma for heparin addition studies was obtained from a single individual with no history of atopy or contrast material reactivity (single NHP). The assay of total prekallikrein was similar, except that the dextran sulfate solution was 25 mg/L and aliquots were withdrawn after 7 and 10 min at 0”. The higher of the two rates was recorded as “total prekallikrein. ”

Hageman factor cleavage Under essentially the same conditions as those used for total prekallikrein assay (I 2.5 mg/L dextran sulfate final concentration), a trace amount of ‘“Y-HF labeled by the chloramine T method’ was included. Aliquots were removed at I, 2, 3, 4, 6, and IO min and were assayed for kallikrein activity and for HF cleavage. HF cleavage was detected by subjecting timed aliquots to slab gel SDS-PAGE under reducing conditions. Autoradiography of dried gels showed the locations of the intact (unactivated) HF and the two fragments resulting from cleavage,” which parts were carefully cut out and counted for quantitation of cleavage.

Other assays Functional CiINH was determined by the effect of the test plasma on the esterase activity of a Cis preparation as previously described.” Quantitative antigenic determinations of HMWK were done by radial immunodiffusion assay.’ Qualitative assays were by double diffusion.’ Coagulation assays for HF were performed by the standard single-stage method utilizing HF-deficient plasma (George King Biomedical, inc., Overland Park, Kans.). The anti-factor Xa (anti-Xa) heparin assay was a mod-

MINUTES INCUBATION AT 0°C FIG. 1. Time course of average (2 S.E.M.) prekaliikrein tivation under test conditions in plasma samples asthma patients and controls. Ordinate, Kallikrein expressed as rate of change of O.D. of chromogenic strate.

acof 19

activity sub-

ification of the method of Teien and Lie.!’ A 100 ~1 aliquot of a mixture of 25 ~1 of test plasma, 25 g1 of AT 111 (1 U/ml; AB Kabi, Stockholm, Sweden), and 200 11 of buffer (0.2M Tris plus EDTA, pH 8.4) was incubated 3 min at 37” in a plastic semimicro cuvette. A 50 ~1 volume of factor Xa (7 pkatiml; Kabi) was added and incubated 3 min at 37”. followed by 100 ~1 of Tris buffer and 100 ~1 of the chromogenic substrate S-2222 (I .5 mg/mI: Kabi). Tne O.D. at 405 nm was recorded for about 3 min and the rate was calculated from the slope of the tracing. The anti-thrombin assay was that of Larsen et al .I” A 100 ~1 aliquot of a mixture of 20 yl of test plasma, 20 ~1 of AT 111 (I U/ml), and 460 ~1 of the Tris buffer was placed in a plastic semimicro cuvette. After 2 min at 37”. 20 ~1 of thombin (4 U/ml; Calbiochem) was added. and after an additional 2 min at 37”, 180 ~1 of Tris buffer and 100 ~1 of chromogenic substrate S-2238 (0.75 mM; Kabi) was added. The O.D. at 405 nm was recorded for about 3 min and the rate was calculated from the slope of the tracing. In both assays, excess AT III is added to minimize any pre-existing differences in the concentration uf this substance in a given plasma. Heparin values are expressed as the differences in rates of hydrolysis of their respective chromogenic substrates by factor Xa or thrombin, in the presence and absence of test plasma, in units of d0.D 405imin. This avoids the implication that endogenous heparin is identical with the commercial exogenous heparin that is commonly utilized for standardization of heparin assays.

statistics Statistical evaluations were done by the two-tailed t test. All values are cited in terms of mean f S.E.M. Significance is attached to values where p < 0.05.

RESULTS Prekatlirein-ta-keMkr&n

eonversi~n r&es

The average rate of dextran sulfate-catalyzed conversion of prekallikrein to kallikrein in plasmaof the asthmaticpatientswas significantly higher than that of

VOLUME NUMBER

TABLE

Plasma contact system in atopic asthma

72 1

I. Contact

system

proteins Total

Asthma patients Controls

and CilNH

in asthma

prekallikrei#

>o.

110 t 5 (17) 109 k 7 (18)

II.

Correlation

Subject

I, asthma

2. asthma

3. control

4. control

of HF cleavage Incubation (min)

HMWKC

and kallikrein

CilNHD

83 k 5 (17) 75 2 4 (18)

>O.l

1

HMWK by antigen determination: all others by functional determination. All values are means ? S.E.M.; number of subjects in parentheses. ’ Factors participating in contact system activity that did not differ significantly “Expressed as 1O.D. 405/min X I@. ’ Expressed as percent of normal pooled plasmas. “Expressed as mg/dl.

TABLE

plasmasA

IiF

784 k 45 (19) 779 + 49 (19)

p value

and control

85

>o.

in asthmatics

1

16.2 k 0.8 (18) 15.2 k 0.9 (18) x.

1

and controls.

activity

time

1 2 3 4 6 10 1 2 3 4 6 10 1 2 3 4 6 IO 1 2 3 4 6 10

r = correlation coefficient. Slab gel SDS-PAGE analysis of “’ l-factor of kallikrein activity at timed intervals. the time frame utilized.

% factor

XII activated

20 16 25 40 60 62 17 23 35 48 68 73 19 14 16 17 30 46 18 19 22 55 64 66

Kallikrein

(mu/ml)

36 103 473 750 991 913 119 370 926 945 1017 1356 0 34 70 298 750 822 36 114 645 848 1020 971

r value

0.942 (p < 0.005)

0.911 (p < 0.01)

0.890

(p < 0.02)

0.912 (p < 0.01)

XII activation on incubation with 12.5 mg/L dextran sulfate at 0” C, correlated with appearance This concentration of dextran sulfate produced total, or near total, activation of prekallikrein in

the normal controls (Fig. 1). The greatestdifferences between the asthmatic and normal plasmasoccurred after 10 min of incubation. There wassomeoverlap of the asthmaticplasmavalues with thoseof the normals at each time point. In contrast to the rates of conversion of prekallikrein to kallikrein, the average total prekallikrein level in the plasmaof asthmaticpatients was not significantly different from that of the normal controls (Table I). Measurementsof the antigen concentrations of HMWK in the plasmasrevealed no significant differences (p > 0.1) between the asthma

and control groups. Similarly, HF procoagulant activity and functional CiINH activity were not significantly different in the two groups (Table I). The experiments using ‘251-labeledHF were performed with plasmasamplesfrom two asthmapatients and two controls. HF wascleaved in a time-dependent fashion, and all four plasmasdemonstrateda good correlation between the rates of cleavage and the appearanceof kallikrein activity (Table II). The overall mean correlation coefficient was 0.913; the p value was p < 0.01.

86

Lasser

et al

1

I 20

I

I

30 40 0’ C INCUBATION TIME (minutes) FK;. 2. Time course of prekallikrein normal human plasma by 1.5 mg/L Effect of added heparin.

Effects

of added

activation dextran

of a single sulfate at 0”.

heparin

The addition of increasing concentrations of commercial heparin to NHP incubated with dextran sulfate at 0” C produced a stepwise increment in the rate of prekallikrein conversion (Fig. 2). Heparin concentrations asthmatics and controls

in plasma

of atopic

Seventeen of the 19 test and control plasmas were assayed for heparin content by both anti-thrombin and anti-Xa methods. In terms of relative concentration units, the mean concentration of heparin active against thrombin in asthmatics significantly exceeded that of the controls (1401 + 340 vs. 210 ? 34; p < 0.02). The anti-Xa mean value in the asthmatic group was also higher than that of the controls but was not significantly different (107 1 t 260 vs. 479 rt 38; p < 0.1). The ratio of relative heparin active against thrombin to relative heparin active against Xa differed significantly in asthmatics and controls (I .09 + 0.15 vs. 0.50 ? 0.10; p < 0.002). In the asthmatics, there was a high correlation between individual antithrombin heparin values and anti-Xa values (r = 0.952; p < O.OOOS), whereas in controls, no such correlation existed (r = -0.252; p > 0.3). The relative concentration of ‘ ‘anti-thrombin heparin ” to “antiXa heparin” in the porcine heparin preparation used in these studies was identical to the mean ratio found in the asthmatics (1.09). DISCUSSK)fU The primary results reported in this study are that (1) the dextran sulfate-catalyzed prekallikrein activation rate is, on the average, elevated in plasma obtained from patients with allergic asthma and (2) the

plasma of atopic individuals with clinical asthma contains, on the average, significant elevaritrnr IP, anti. thrombin heparin. The demonstration that HF IS cleaved under thehi. test conditions confirms on a molecular ievci that the contact system is indeed involved. The correlation oi the rate of HF cleavage with the rate oC kallikrein activity appearance implies that the difference between atopics and normals manifests itself at a step preceding the activation of prekallikrein. l,evels oi the protein concentrations and/or activities of total plasma HF, prekallikrein, HMWK. dnd CilNH showed no significant differences between patient3 and controls. Therefore factors other than the concentrations of these components are responsible for the differences found in the test, The potentiating fattar(s) present in the plasmas of these atopic patients is not known, but it could be heparin-like material o! heparin itself because heparin is found in increased concentrations in these plasmas and heparin added directly to test plasma potentiates the activity Furthermore. heparin alone. in the increased concentrations noted in some of the asthma plasmas, can activate the contact system by itself.* Nevertheless, the increased prekallikrein conversion rate noted in the asthmatic patients may be entirely independent of endogenous heparin levels. Although heparin potrntiates the prekallikrein conversion rate when dextran sulfate is the activator. no potentiation cxcurs when sulfatides are utilized to activate the system.:: Hence, it is possible that heparin functions in the dextran sulfate incubation by neutralizing cations that would otherwise diminish the polyanionic activating potential of dextran sulfate. The significant increase in the ratio of antithrombin heparin relative to anti-Xa heparin in these patients suggests either that greater quantities of antithrombin heparin are mobilized in atopic asthmatics or that these patients catabolize this less effectively than normals. The high correlation between antithrombin heparin and anti-Xa heparin in these patients, the lack of such correlation in controls, and the correspondence of anti-thrombin/anti-Xa ratios to that found in commercial porcine heparin, but not to that found in controls, suggest as an additional possibility that the primary source of heparin in these two groups might differ. In atopy, the mast cell is clearly the primary source of heparin. Heparin is present in high concentrations in the granules of mast cells,” ~‘I and heparin-like material is abundantly present within other cells. ‘I. I5 Activation of prekallikrein to kallikrein depends *Lasser

EC, Lang JH: Unpublished

data.

VOLUME NUMBER

72 1

primarily on the binding of the proenzymes HF and prekallikrein (in complex with HMWK) to a negatively charged surface. rfi-iX The binding is accompanied by activation of HF. Activated HF converts prekallikrein to kallikrein, which then reciprocally activates HF in a feedback loop. The rate of kallikrein generation in the described assay reflects the net rate of temperature-diminished activation of HF vs. temperature-diminished inhibition of this activation. The specific factor (or factors) potentiating the HF activation rate must await further studies, but heparin itself remains as one viable candidate. Several studies suggest that in some circumstances IgE may mediate activation of the contact system. Pinckard et al.‘” showed that specific IgE, raised in young rabbits consequent to inoculation with beef serum albumin, induced consumption of factors XII, XI, and IX during antigen challenge. De Shazo et al.“” pointed out that the IgE-dependent late-phase cutaneous reaction to intradermal antigens in man was characterized by fibrin deposition, signaling activation of the coagulation system. Newball et al.‘] noted that a specific kallikrein activity was generated from human basophils via an IgE-mediated immune reaction. Most recently, Dale and Venge” reported that 16 patients, challenged by inhalation with an allergen to which they had positive skin and RAST tests, responded with a fall in PEFR accompanied by a profound shortening of plasma recalcification time. Half of these patients showed a delayed, as well as an immediate, fall in PEFR, with normal values intervening. This group uniformly demonstrated an increased activation rate of HF, determined by incubating diluted patient plasma with HF-deficient plasma for 60 set before the addition of calcium. In the present study, we elected to evaluate patients with atopic asthma by the prekallikrein conversion rate test because these patients are known to exhibit a relatively high incidence of adverse reactions to x-ray contrast media’ and since we had shown accelerated rates of prekallikrein conversion in an earlier study of contrast material reactors. ’ Many stimuli, including allergen exposure, exercise, irritant aerosols, viral respiratory tract infection, and methacholine, are known to precipitate exacerbations of asthma. The pathogenic mechanisms mediating these exacerbations are not clearly understood. However, release of inflammatory mediators such as histamine, slowreacting substances (including perhaps leukotrienes C, D, and E), and eosinophil chemotactic factor and activation of complement have been implicated in different asthmatic responses. Z-Z Activation of the contact system of coagulation has been infrequently reported in association with spontaneous or provoked

Plasma

contact

system

in atopic

asthma

87

asthma, but some studies have reported that plasma obtained during asthmatic attacks contained elevated levels of bradykinin, measured by bioassay,2fi. 2i and aerosolized bradykinin (in common with many other aerosols) was found to produce bronchospasm in asthmatic (but not healthy) children and in guinea pigs.2H. 29 Additional studies will be necessary to determine whether the mean increase in prekallikrein conversion rates noted in the present report will be found in other forms of allergic disease and whether the activation dynamics occurring in vitro partially reflect those that might occur in asthma in vivo. In vivo activators may share some of the properties of in vitro activators such as dextran sulfate, a substance that resembles heparin in a number of ways, including anticoagulant activity with AT III. Whether or not elevated endogenous heparin levels act in the fashion postulated above to accelerate or initiate in vivo contact system dynamics, the simple recognition that endogenous heparin is elevated in atopy raises the possibility of utilizing this substance as a marker for putative mast cell activity. The halflife of circulating heparin30 is much longer than that of histamine3i or neutrophilic chemotactic factors ,‘12 mediators currently utilized as markers of mast cell release. Furthermore, the assay for heparin employing chromogenic substrates is far simpler than the assays for the other markers. Ultimately, the full value of heparin as a marker will depend on temporally demonstrated concentration changes after specific antigen challenge in hypersensitive individuals. These studies are currently under way. In future studies, it will be important to attempt to identify patients with atopic asthma who have never received medication for this condition. In the present study, the nine patients who had received steroid therapy within the preceding 2 weeks had a higher 30-min mean level of prekallikrein conversion rate than did the 10 patients who had not been so treated (440 vs. 351; AO.D./min x 104). Although this difference may simply reflect more intense disease in the patients who required steroid therapy, other explanations are possible. If the nine patients receiving steroid therapy were eliminated, however, the 30 min prekallikrein conversion rates of the remaining 10 patients would still differ significantly from those of controls (p = 0.02). SUMMARY Atopic asthma patients demonstrated an elevation in the dextran sulfate-catalyzed activation rate of prekallikrein. Mean heparin levels were also elevated in these plasmas. The ratio of heparin active against thrombin to heparin active against factor Xa in these

88

Lasser

et

al

patients corresponded to that of commercial porcine heparin but differed significantly from the mean ratios found in nonatopic, nonasthmatic individuals. The relation of IgE-mediated events to the factor(s) producing the elevated rate in the prekallikrein activation test requires further study, as do the precise role of the contact system in the pathophysiology of atopic asthma and the role of endogenous heparin as a facilitator of HF activity and/or as a marker of mast cell release activity. Note added in proof. Since the original submission of this manuscript, we have had the opportunity to examine 11 patients subjected to oral or inhalational or parenteral challenges by antigens or drugs to which they were hypersensitive. In each of these patients, baseline and sequential plasma samples were obtained over at least a 1 hr period. Each of the patients responded to individual challenges by a demonstrated fall in FEV, of 25% or more. Each patient demonstrated an increase in the prekallikrein conversion rate. The average maximal (20 min incubation) increment was 201% in these 11 patients. To date, seven of these patients have had endogenous heparin concentrations assayed and/or APTT values established. In each instance, the fall in FEV, was accompanied by either an increment in the heparin concentration and/or a prolongation in the APTT value. Thesepatients will be the subjectof a separatereport.

IO.

I I. 12.

I.?.

14. 15. 16.

17.

18. 19.

20.

21. REFERENCES I. Lasser EC, Lang JH, Lyon SO, et al: Prekallikrein-kallikrein conversion: conversion rates as a predictor for contrast catastrophies. Radiology 140: 1 I , 198 1. 2. Ansell G, Tweedie MCK, West CR, Evans P, Couch L: The current status of reactions to intravenous contrast media. Invest Radio1 15(Suppl.):S32, 1980. 3. Kluft C: Determination of prekallikrein in human plasma: optimal conditions for activating prekallikrein. J Lab Clin Med 91:83, 1978. 4. McConahey PJ. Dixon FJ: A method of trace iodination of proteins for immunologic studies. Int Arch Allergy Appl Immunol 29: I 85, 1966. 5. Dunn JT, Silverberg M, Kaplan AP: The cleavage and formation of activated human Hageman Factor by autodigestion and by kallikrein. J Biol Chem 257:1779, 1982. 6. Lasser EC, Lang JH, Lyon SG, et al: Complement and contrast material reactors. J ALLERGY CLIN IMMUNOL 64~102, 1979. 7. Mancini G. Carbonara AO, Heremans JF: Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochemistry 2:235, 1965. 8. Ouchterlony 0, Nilsson LA: Immunodiffusion and immunoelectrophoresis. In Weir DM, editor: Handbook of experimental immunology. Oxford, 1973, Blackwell Scientific Publications, chap. 19. 9. Teien AN, Lie M: Evaluation of an amydolytic heparin assay

22. 23. 24. 25. 26.

27. 28.

29. 30. 31. 32.

method: increased sensitivity by adding purlfred antrrhromh~r III. Thromb Res l&399. lY77. Larsen ML. Abildgaard U. Teien AN. Gjesdal k. .A\\a) 0: plasma heparin using thrombin and the chromogenic substrati: H-D-Phe-Pip-Arg-pNA (S2238). Thromb Reh 13t-188. iY?R Kaliner MA: The mast cell-~ a fascinating &die ‘i I%$ ,i Med 301:498. 1979 (editorial ). Uvas B. Aborg C-H, Bergendorff A Storapc o!‘ histamme !n mast cells. Evidence for an ionic binding of histamine to protein carboxyls In the granule heparin-protein