Variability in Purified Dysfunctional C1-inhibitor Proteinsfrom Patients with Hereditary Angioneurotic EdemaFunctional and Analytical Gel Studies

Virginia H. Donaldson, Richard A. Harrison, Fred S. Rosen, David H. Bing, George Kindness,John Canar, Constance J. Wagner, and Semie AwadDepartments ofPediatrics and Medicine, University of Cincinnati College ofMedicine, Children's Hospital Research Foundation,Cincinnati, Ohio 45229; The Mechanisms in Tumour Immunity Unit, The Medical Research Council Centre, Cambridge, England,CB2 2QH; The Children's Hospital Medical Center and Department of Pediatrics, Harvard Medical School, Boston, Massachusetts;The Beth Israel Hospital, Boston, Massachusetts; The Departments ofMolecular and Life Sciences,College of Technology, Dundee, Scotland

Abstract Introduction

Cl-inhibitor (CI-INH) proteins from normal persons andmembers of eight different kindred with dysfunctional Ci-INHproteins associated with hereditary angioneurotic edema(HANE) were compared with respect to their inhibitory activityagainst purified preparations of Cls, plasma kallikrein, activatedforms of Hageman factor, and plasmin. Each dysfunctionalCi-INH protein showed a unique spectrum of inhibitory activityagainst these enzymes. Although none of the dysfunctional Ci-INH proteins significantly impaired amidolysis by plasmin, allbut one inhibited activated Hageman factor. One purifieddysfunctional Ci-INH (Ta) inhibited purified Cli to a normaldegree. Another Ci-INH (Za) had almost seven times as muchinhibitory activity as normal Ci-INH against activated Hage-man factor, but had decreased activity against Cli and noactivity against plasmin. Analyses of mixtures of plasmin andCi-INH proteins in SDS gel electrophoresis revealed variabilityin the patterns of complex formation and cleavage of dysfunc-tional proteins after exposure to Cli and plasmin. Some boundto plasmin and were cleaved, even though none significantlyimpaired the amidolytic activity of plasmin. Two were cleavedby Cli, whereas neither normal or other dysfunctional C1-INH were cleaved. Dysfunctional Ci-INH proteins from pa-tients with HANE are thus heterogeneous in their inhibitoryproperties and there must be different structural requirementsfor the inhibition of the various plasma enzymes that can beregulated by normal Cl-INH. The data suggest that in additionto common sites of interactions between these proteases andCl-INH, there are also points of contact that are specific foreach protease. Genetic mutations leading to structural changesat some of these sites may have differing effects on theinteraction between individual proteases and abnormal Cl-INH proteins. These alterations may allow these proteins toserve as probes for structural requirements for inhibitoryactions of normal Ci-INH.

Address reprint requests to Dr. Donaldson, Children's Hospital MedicalCenter.

Received for publication 28 Seplember 1983 and in revised form17 July 1984.

Plasmas from persons with hereditary angioneurotic edema(HANE)' are either markedly deficient in Cl-inhibitor (Cl-INH) activity, as well as CI-INH antigens (type I), or theycontain normal or elevated concentrations of dysfunctionalCi-INH protein (type II). Both types of Ci-INH deficiencyare inherited as autosomal dominant traits in kindred withHANE. Plasma from patients with type II HANE do notadequately inhibit Cl, or its Cli subunit, and C4 and C2,both of which are substrates of Cl, were readily inactivated inplasma from patients with type II HANE as well as those withtype I (1-3).

The abnormal CI-INH proteins in plasma and serum ofpatients with type II HANE were electrophoretically andfunctionally heterogeneous (3). At least four structural variantsof Cl-INH have been identified within this group of patients(3). Despite the heterogeneity among this group of patients,both the tendency to bouts of HANE, and the biochemicaldisorder, were inherited as autosomal dominant traits inaffected kindred (3). Because of the heterogeneity of theseproteins, dysfunctional CI-INH was isolated from plasma of12 affected individuals from eight different kindred with typeII HANE. This report describes the capacity of each of theseproteins to inhibit ClI, plasma kallikrein, activated Hagemanfactor (HFa, or Factor XIIa), Hageman factor fragments (HFf),and plasmin; normal CI-INH can inhibit each ofthese proteasesin vitro. The inhibition of purified preparations of each ofthese enzymes by individual dysfunctional Ci-INH proteinsdiffered from one enzyme to another. In addition, the dys-functional proteins appeared to have unique patterns of inhi-bition of each enzyme when compared to one another. Thissuggests that the sites of contact between inhibitor and enzymeare, in part, different and specific to each protease.

Methods

MaterialsResins used in column chromatography included SP-Sephadex C-50,DEAE-Sephadex A-50, DEAE-Sephadex C-50, CM-Sephadex C-50,DEAE, activated Sepharose 4B, Sephacryl S-200 superfine and Sephadex

1. Abbreviations used in this paper: HFa, activated Hageman factor;CT-INH, Cl-inhibitor; HFf, Hageman factor fragments; HANE, hered-itary angioneurotic edema; HTP, hydroxylapatite; ALTEE, N-acetyl-L-tyrosine ethyl ester; PNA, paranitroaniline; PAGE, polyacrylamidegel electrophoresis.

124 Donaldson, Harrison, Rosen, Bing, Kindness, Canar, Wagner, and Awad

J. Clin. Invest.C The American Society for Clinical Investigation, Inc.0021-9738/85/01/0124/09 $ 1.00Volume 75, January 1985, 124-132

G-150, all of which were obtained from Pharmacia Fine Chemicals,Piscataway, NJ. Hydroxylapatite (HTP) was obtained from Bio-RadLaboratories, Richmond, CA; AcA 54 Ultragel from LKB Instruments(Bromma, Sweden or Rockville, MD). Polyacrylamide was obtainedfrom Fisher Chemical Co., Norwood, OH. Synthetic chromogenicamides used as substrates in assays for enzymatic activities includedS-2302 (Pro-Phe-Arg-paranitroaniline [PNA]), S-2251 (Val-Leu-Lys-paranitroaniline) and S-2160 (N-Benz.-Phe-Val-Arg-PNA), which wereobtained from Kabi, Stockholm, Sweden, or Stamford, CT. CBZ-L-tyrosine-O-paranitroaniline was obtained from Sigma Chemical Co.,St. Louis, MO. Na-BZ-Pro-Phe-Arg-PNA hydrochloride was obtainedfrom Pentapharm, Basle, Switzerland. N-acetyl-L-tyrosine ethyl ester(ALTEE) was obtained from the Aldrich Chemical Co., Milwaukee,WI; soybean trypsin inhibitor from Worthington Chemical Corp.,Freehold, NJ; streptokinase (SK) (high purity) from Lederle Laboratories,Pearl River, NY; benzamidine and L-lysine hydrochloride from Mathe-son, Coleman and Bell, Norwood, OH; sodium dodecyl sulfate (SDS)from Fisher Chemical Co., Norwood, OH; and BRIJ-35 from Calbi-ochem-Behring Corp., La Jolla, CA. Lysine-Sepharose was preparedusing the instructions of the manufacturer for conjugating the lysinewith Sepharose. Soybean trypsin inhibitor was similarly complexedwith Sepharose.

Ellagic acid (4,4,5,5',6,6' hexahydroxydiphenic acid 2,6,2',6'-dilac-tone) used to activate Hageman factor was synthesized as described(4). Stock solutions were prepared in barbital saline buffer, pH 7.4, byhomogenization in a mechanical homogenizer and centrifuged at15,000 g for 20 min. These solutions did not contain microscopicallyvisible crystals. The barbital-saline buffer used in coagulation assayscontained 2.06 g of sodium barbital, 2.76 g of barbital, and 7.8 g ofsodium chloride in I liter of distilled water.

Plasmas were obtained from normal individuals and from personswith HANE associated with dysfunctional C1-INH protein previouslycharacterized (3, 5) after informed consent was given by the donors.The plasmas were separated by plasmapheresis, and immediatelymixed with benzamidine and disodium EDTA (see below).

MethodsProtein isolation. CI-INH was isolated from normal plasma by theprocedure of Harrison (6, 7), except that in some instances gel filtrationon Sephacryl S-200 replaced that on Sephadex G-150. This providedCJ-INH, which was usually homogeneous in SDS gel electrophoresis,but in some preparations two closely approximated bands were seen(8) with as much as 70% recovery.

Plasma kallikrein. Plasma kallikrein was isolated by the methodof Nagase and Barrett (9), modified in that a second affinity step onSBTI-Sepharose was usually added after chromatography on UltrogelAcA 54, and immune adsorption on a column of Sepharose-antihumanIgG was used when necessary. This kallikrein was homogeneous inSDS electrophoresis gels (Fig. 1), and had specific activity as high as235-446 U of coagulant activity (I U being the amount in 1.0 ml ofpooled normal human plasma) per mg protein.

Human Hageman factor. Hageman factor was extracted fromnormal plasma using minor modifications of previously publishedprocedures (10-12). All test tubes and vessels were either coated withsilicone (GE-Dri film, SC-87, Pierce Chemical Co., Rockford, IL) ormade of polypropylene to prevent surface activation of Hagemanfactor. Blood was drawn into polypropylene bottles containing 1/50 offinal volume of a solution of 0.5 M citrate buffer, pH 5.0, 0.1 Mbenzamidine hydrochloride, and 100 mg hexadimethrine bromide(Polybrene, Aldrich Chemical Co.) per 10 ml of solution. Plasma wasrendered platelet deficient by recentrifugation of cell-free plasma at12,000 g in a Sorvall RC-2B centrifuge for 20 min at 2°C. TheHageman factor was isolated by sequential chromatography uponcolumns of QAE-Sephadex A-50, DEAE cellulose, SP-Sephadex C-50,and AcA 54 Ultrogel. All equilibrating and eluting buffers used inQAE-Sephadex A-50 and DEAE Sephadex chromatography contained2 mM benzamidine HC1, 50 mg Polybrene/liter, 0.1 mM disodiumEDTA, 0.1 M sodium chloride, and 0.02% sodium azide. Columns

MM''U'

Figure 1. SDS polyacrylamide gel (7.5%) electrophoresis of Hagemanfactor (left) isolated from normal plasma by the method described,having a specific coagulant activity of 95 U/mg protein. Kallikrein(center), isolated as described, had a specific coagulant activity of 440U/mg protein. The plasminogen preparation (right) had a sp act of23 CTA U/mg. The samples applied to gels contained: kallikrein, 320gg, Hageman factor, 100 ,g, plasminogen, 40 Mg. These gels were runseparately; dye markers are anodal (down).

and glassware were coated with silicone. Fractions containing 0.25 Uof Hageman factor activity per milliliter or more were pooled, examinedin SDS polyacrylamide gels and the specific activity of the Hagemanfactor determined. The specific activity was usually >100 U/mg ofprotein. An SDS gel of the product is shown in Fig. 1.

Plasminogen. Plasminogen was prepared by affinity chromatographyaccording to the method of Deutsch and Mertz (13). This materialhad a sp act of 17-21 CTA U/mg protein.

The plasminogen used in experiments testing the interactions withdysfunctional Cl-INH as analyzed on SDS-polyacrylamide gel electro-phoresis (PAGE) containing a gradient of acrylamide concentrations(Fig. 4) was the generous gift of Dr. Eileen Remold-O'Donnell of theCenter for Blood Research, Boston, MA.

Cl. Already activated ClI, was prepared according to the methodof Bing et al. (14, 15). It gave a homogeneous single band in SDS-PAGE and had a sp act of 1,060 U/mg protein (16).

Enzymatic assaysAssays for enzymatic activity using the synthetic chromogenic substrateswere performed in a 1-cm cuvette in a Gilford model 240 spectropho-tometer (Gilford Instruments, Inc., Oberlin, OH) with a jacketedcuvette holder maintaining the temperature of the assay at 37°C.Synthetic chromogenic substrates were dissolved in distilled water, asrecommended by the manufacturer, unless otherwise noted. The totalvolume of the assay mixture was 600 Al.

Hageman factor. Hageman factor was activated by incubation ofthe purified protein in barbital-saline buffer, pH 7.4, containing 1%bovine serum albumin, with an equal volume of 10' M ellagic acidin a silicone-coated test tube at 37°C. Preliminary tests showedincubation for 2 h before assay was required for optimal activation,and these conditions were used throughout.

HFf were generated spontaneously during storage at -70°C andthawing of solutions of purified Hageman factor in pH 5.2 acetatebuffer. This process was visualized by SDS-PAGE, the original singlecathodal band, of a molecular weight of -80,000, disappearing as twomore anodal bands were generated. At this point the coagulant activityof Hageman factor had also largely decayed.

Dysfunctional Cl-Inhibitor Activities 125

Hageman factor and HFf were assayed in 5 mM Tris buffer, pH7.8, using either S-2302 or Na-BZ-Pro-Phe-Arg-PNA HCI as a substrate.

Kallikrein. Kallikrein was assayed using the substrates and conditionsdescribed above for Hageman factor.

Plasmin. Plasmin was prepared by incubating, at room temperatureand pH 7.4 for 10 min, three parts of plasminogen with one part ofSK (1,000 U/ml). Its activity was measured with the substrate S-2251in 0.05 M Tris, pH 7.4, containing 0.012 M sodium chloride.

For experiments on the interaction of dysfunctional Cl-INH withplasmin in SDS gels containing an acrylamide gradient, purifiedplasminogen was activated with purified SK as described by Remold-O'Donnell and Lewandrowski (17). The SK was dissolved in H20 andplasminogen activated at a 1:200 (microgram/microgram) for 15 minat 370C in 15 mM Tris-HCl buffer, pH 8.1 containing 37 mM lysineand 25% glycerol. It was fully activated as judged by analysis underreducing conditions on SDS-PAGE.

Before initiation of the assay, plasmin, Hageman factor, HFf orkallikrein was incubated in buffer at 370C for 5 min. Substrate wasthen added and the solution rapidly mixed by inversion. The releaseof chromogenic PNA was read at 410 nm at 1-min intervals. Wheninhibitor proteins were being assessed they were added to enzymesbefore the 5-min preincubation period. Because the activity of the SK-plasmin deteriorated significantly at 4°C or at 37°C during andbetween assays, it was necessary to investigate the inhibition ofplasminusing a carefully controlled procedure. Each dysfunctional C1-INHprotein was therefore assessed individually, by simultaneous assay in aset of cuvettes, using a freshly activated SK-plasmin mixture, and anuninhibited (buffer) plasmin standard in each individual assay.

Cls activity was initially measured using the substrate CBZ-L-Tyr-O-PNA. This was dissolved in acetone at 1 mM and incubated, at37°C for 2 min, in the assay cuvette with 0.005 M Tris, pH 8.05,containing 0.09 M NaCl. The reaction was started by the addition ofCli. Cli inhibition was measured in assays reported here (Table IIand Fig. 2) according to the method of Levy and Lepow (16) usingALTEE as substrate.

The rate of hydrolysis of the chromogenic substrates by eachenzyme, expressed as nanomoles of PNA released per minute permicrogram enzyme, was calculated from the data obtained during thefirst 5 min of assay. Table I summarizes the rates of hydrolysis of thesynthesis substrates used. The rate of hydrolysis by mixtures containinginhibitor proteins was also calculated, and the inhibitory activityexpressed as a decrease in nanomoles of PNA hydrolyzed per minuteper microgram inhibitor protein. The percent inhibition of eachdysfunctional protein with respect to inhibition by the normal inhibitorwas then calculated. In amidolytic assays, two or more concentrationsof each Cl-INH were tested (Table II) and 40 ,g or more of each Cl-INH were used in assays using ALTEE as a substrate for Cli (16).

Table I. Specific Amidolytic or Esterolytic ActivitiesofPurified Enzymes Tested with Synthetic Substrates

Enzyme Substrate Rate of hydrolysis

nmol PNA released/min per sg enzyme

Kallikrein Pro-Phe-Arg-PNA 4.509+0.76(S-2302)

HFa (Factor Xlla)(ellagic acidactivated) Pro-Phe-Arg-PNA 0.57±0.069

HFf Pro-Phe-Arg-PNA 0.296±0SK-activated Val-Leu-Lys-PNA 0.447±0.025

plasminogen (S-225 1)Cli CBZ-L-tyrosine-O-PNA 0.087±0.023CIs ALTEE* 1.06±U/,gg+0.26

* Esterolytic assay used (16).

Coagulation assays to quantify Hageman factor or kallikrein duringfractionation were carried out by methods described (10) in which thecorrection of delayed clotting of a plasma of a person with a severehereditary deficiency ofthe coagulation factor in question was measured.For these assays, column fractions were diluted 1:5 or 1:20 in eitherdistilled water, or, when measuring Hageman factor, in barbital-salinebuffer, pH 7.4, containing 1% bovine serum albumin. Fractionscontaining Hageman factor were added to a mixture of kaolin andCentrolex-O phospholipid in the presence of albumin or other protein.

Assays for Cli hydrolytic activity using the standard assay of Levyand Lepow (16) were performed in 0.067 M phosphate, pH 7.4, andthe final acid released titrated in a microformol titration using aRadiometer automatic titrator and pH meter, model PHM 82, with aTTT 80 titrator and an ABU 80 autoburette (Radiometer, Copenhagen,Denmark).

SDS-polyacrylamide gradient gels were made by an adaptation ofthe method ofLaemmli (18). The gradient was 7.5 to 12.5% acrylamideprepared from stocks of 29.4% acrylamide and 0.6% bis-acrylamide.Polymerization was initiated with ammonium persulfate and NNN,N'-tetramethyl-ethylene diamine (Eastman Kodak Co., Rochester, NY) ata final concentration of 0.036 and 0.017%, respectively. After poly-merization of the gradient gel (usually within 50 min) of acrylamideand bis-acrylamide, a stacking gel of 3.5% acrylamide prepared fromthe same stock solution with identical concentrations of ammoniumpersulfate and NNN',N'-tetramethyl-ethylene diamine (Eastman KodakCo.) was added. The gels were electrophoresed vertically at roomtemperature at 3-3.5 V/cm for 18 h or until the tracking dye waswithin 1-2 cm from the bottom of the gel. In this system Cli (M, =87,000) and plasmin (M, = 77,000) migrated at about the sameposition corresponding to an -65,000-70,000 Mr. This system, however,was determined optimal for resolution of free inhibitor from complexand/or free enzyme and degradation products derived by apparentaction of the proteases on the inhibitor. In standard 7.5 and 10% SDS-PAGE gels both proteases had molecular weights corresponding topreviously reported values (13, 14), and exhibited no evidence ofdegradation under any of those conditions (data not shown). Theenzymes and inhibitors were incubated together at 37°C for 15 min,SDS added and incubation continued for 25 min before electrophoresis.

ResultsFunctional studies. As shown in Table II, purified dysfunctionalC7-INH proteins exhibited a wide range of inhibitory activityagainst the enzymes tested. The different dysfunctional C7-INH proteins are indicated by the first two letters of eachfamily name. With the exception of C7-INH Ri, all gave asignificant degree of inhibition of Cli (19-90%) (Table II)using an esterolytic assay (ALTEE substrate). In an assaymeasuring Cli activity by esterolysis of CBZ-L-tyrosine-O-PNA, the depression of the hydrolytic rate of the C-is by theCI-INH proteins was in the range of l0-' to 10-' nmol ofsubstrate hydrolyzed per minute per microgram inhibitor, butthe inhibitor proteins depressed ALTEE hydrolysis by Clifrom 6.7 to 100.5 nmol/min per Ag inhibitor. Although itappears that ALTEE is a more satisfactory substrate fordetermining the inhibition of Cli, it is possible that certain ofthe dysfunctional proteins block the hydrolysis of ALTEEmore effectively than of CBZ-L-tyrosine-O-PNA. The levels ofinhibition of preparations of C7 by infractionated serumsamples from the patients studied are shown in column 2,Table II.

Inhibition of the amidolytic properties of purified humanplasma kallikrein by dysfunctional CI-INH proteins also variedwidely compared to the activity of the normal C7-INH (TableII). Inhibition values ranged from 1% (Ri) to 64% (Za) of thatprovided by normal Ci-INH. There was a wide variation in

126 Donaldson, Harrison, Rosen, Bing, Kindness, Canar, Wagner, and Awad

Table 1I. Inhibitory Activities ofDysfunctional CJ-INH Proteins Against Plasma Enzymes: Percent Normal CJ-INH Activity

Enzyme inhibition by purified C6-INH proteinsDysfunctional Serum inhibitionC0-INH of Ci (16) CHt Kallikrein HFa HFf SK-Plasmin

Da 0 75 50 70 194 20.2(72-86) (45-55) (57-97) (118-276) (20-47)

Ta 0-15 90 10 93 24 1.3(65-91) (0-21) (78-108) (24, 24) (0-5)

We 0 19 33 97 39 1.8(2-35) (30-35) (97, 97) (35-43) (0-5)

Ri 0-30 4 1 0 0 1.2(1-10) (0-2.6) (0) (0) (0-5)

At 0 50 2.5 39 46 0.35(30-70) (0-5) (30-49) (40-52) (0-1.4)

Bo 0 61 16 64 64 0.7(32-100) (14-18) (57-70) (53-79) (0-3)

Mo <20 52 6 119 70 2.2(22-81) (0-11) (119-120) (67-73) (0-6)

Za 0-20 19 64 687 (NT) 0(0-32) (41-97) (483-893) (0)

Normal 100 100 100 100 100 100

Each dysfunctional CI-INH protein is designated by the first two letters of each family name. Ranges in multiple assays in parentheses. NT,not tested. The inhibition of each enzyme by dysfunctional CI-INH proteins is given as a percentage of inhibition by normal CI-INH, whichwas derived from the depression of the rate of hydrolysis (nanomoles per minute per microgram of inhibitor) of synthetic substrate. Each dys-functional protein was tested from two to six times against each enzyme. The amount of enzyme used in assays was: Cl , 28 Mg; kallikrein, 0.13Mug; HFa, 2.3 Mig; HFf, 2.1 Mg; plasmin, 4.9 ,ug. ClI was tested with ALTEE as substrate; kallikrein, HFa and HFf with S-2302, and SK-plasminwith S-2251. The amounts of each CI-INH protein tested were as follows: ClH assay: Normal CI-INH, 20, 40 Mg; CI-INH Da, Ta, We, Ri, At,Bo, Mo, 40 Mg; CI-INH Za, 40, 80 ,gg. Kallikrein assay: Normal CI-INH, 6 Mg; All dysfunctional CI-INH proteins, 6, 12 gg. HFa assay: Nor-mal CI-INH, 12 Mig; CI-INH Da, We, Mo, 6 Mg; CI-INH Ta, At, Bo, Za, 6, 12 Mg; CI-INH Ri-18 jug. HFf assay: Normal, 6 Mg; CI-INH Da,8.4, 16.8 Mug; All other Cl-INH's, 6, 12 usg. Plasmin assay: Normal CI-INH, 6, 7, 12, 14 and 36 Mg; CI-INH Da, 17, 68 Mg; CI-INH Ta, We, Ri,10, 20 Mg; At, Bo, Mo, Za, 6, 12 fug. The mean value of percent inhibition provided by each inhibitor protein is given with the range of valuesobtained in different assays in parentheses beneath the mean value. Column 2 indicates the amount of inhibition of Cl provided by sera fromwhich the CT-INH proteins were isolated. This information has been published previously (1, 3, 31-33).

the ratios of anti-CIi to anti-kallikrein activity among dys-functional inhibitors. For example, Ci-INH Ta exhibited ahigh degree of inhibition of ClIs esterolytic activity (90%normal), but little antikallikrein activity (10% normal). Asimilar ratio was found with Ci-INH At, which inhibited Cliesterolytic activity -50% as well as normal CI-INH, but had<3% of the antikallikrein activity of the normal CI-INHprotein. In contrast, CI-INH Za had only 19% of the normalamount of activity against Cls, but considerably more anti-kallikrein activity (64%).

When 40 ug of CI-INH Ta was added to Ta plasma, noinhibition of ClIs could be measured, but the protein had aspecific activity against ClH of 12 U/mg of inhibitor beforebeing added to the plasma from which it was derived. When40 ,g of a stored preparation of normal CI-INH was addedto Ta plasma, the specific inhibition of ClIs was 52 U/mg ofthis protein, as it was in mixtures containing buffer in placeof Ta plasma. Therefore, the anti-ClIs activity of the CI-INHTa was markedly reduced by the Ta plasma.

When the amidolytic activity of ellagic acid HFa, FactorXIIa was measured, six of eight dysfunctional CI-INH proteinshad >60% of the inhibitory activity found in the normal Cl-

INH. Only one (Ri) had essentially no inhibitory activity. Theinhibition of HFf by dysfunctional proteins did not parallelthe inhibition of HFa, and most had reduced activity againstHFf. CI-INH Da, however, had enhanced activity against HFf.CI-INH Mo had essentially normal activity against bothactivated forms of Hageman factor, but its action against Cliand kallikrein was only 52 and 6%, respectively, of that of thenormal inhibitor. Cl-INH Za had exceptionally high anti-HFaactivity, but little anti-Cls activity.

All of the dysfunctional CY-INH proteins were deficient intheir capacity to block the amidolytic activity of SK-plasmin(Table II), but Ci-INH Da had more activity than any of theothers. In examining the inhibition of the amidolytic activityof SK-plasmin, each CI-INH protein was examined individuallywith its own standard SK-plasmin mixture run simultaneously.This was necessary because the activity of the SK-plasmindeteriorated significantly at 40C or at 370C during and betweenassays.

The levels of inhibition by each dysfunctional Ci-INHprotein against each enzyme tested are summarized diagram-matically in Fig. 2. These data are expressed as a depressionin hydrolytic rate of each enzyme in nanomoles, or units in

Dysfunctional Cf-Inhibitor Activities 127

A

- 180

_

a)- 140 CY

+tl_ c

._

- 100 X

80 E

-60

- 40

200

0

c1§

- 1.0_

C)+C

-04_Z.c

z

-0.6

Tc- E

CL

0.2

0.1t

Kallikrein

Enzyme

->0.2-0.200

-0.190

-0.14

Norm CINH

SK PL

Fragment

Fragment

0.120CM

0.10

0.08 E

f-C

.048

0.02 =

I

0.01

N -9

I a

Norm _N CI INH

ClINH TA

SK- PL

HFa(Xlla) HFf

Figure 2. A diagrammatic representation of the inhibition of Cli(let), kallikrein, ellagic acid-activated HFa, and HFf by normal Cl-INH and dysfunctional CI-INH proteins. The transverse line in eachset indicates the mean value of inhibition by purified normal Cl-INH and the shaded boxes indicate ±2 SD. N, the number of assaysdone with purified normal C1-INH in each set of experiments.Individual points (*) represent the mean value of inhibition of enzy-matic activity by each dysfunctional protein in two to six experi-ments. The ratios of amounts micrograms of inhibitor protein toenzyme used were: Cli assay: 0.8-2.8:1; kallikrein: 46-92:1; HFf,2.9-8:1; HFa: 8-16:1; SK-plasmin: 1.2-13.8:1. (see legend, Table II).The substrate used for ClH was ALTEE; for kallikrein and HFa,S-2302 was used.

the case of ClI, per minute per microgram of inhibitor protein,rather than as a percentage of the activity of the normalprotein. In each set of Hageman factor assays, the standarddeviation of repeated assays using normal CI-INH was 0,probably because the same preparation of Hageman factor atthe same thawing was used in these experiments.

SDS gel analyses ofCHs and plasmin with CJ-INH proteins.Since plasmin can cleave normal CI-INH (8), several of thedysfunctional Cl-INH proteins were examined in SDS disc gelelectrophoresis to see if they were more extensively cleaved byplasmin than the normal inhibitor, or if they were cleaveddifferently than normal CI-INH. Mixtures used in experimentsmeasuring antiplasmin activity were boiled in SDS and appliedto the gels. Normal Cl-INH inhibited plasmin and was partiallycleaved by the amount of plasmin used (Fig. 3, panel A). Acomplex that has an approximate molecular weight of 145,000formed between normal Ci-INH and plasmin, and cleavagefragments having apparent molecular weights of 81,000 and69,000 were released (Fig. 3, panel A, gels 1 and 2). Cl-INHRi, which had essentially no antiplasmin activity, did not form

11-160

I" -_ C INHEREm- i

so

3 ;-I

Ci INHN_

We

2Z 3

U- 43

- l +-V

-30

--) 0.1

- 144

Figure 3. Samples of dysfunctional and normal CI-INH proteinswere incubated with 12 Ag of SK-plasmin and pH 7.4, 0.15 M Tris-HCI buffer at 37'C for 5 min. Then 50 Al of 0.01 M S-2251 was

added and the chromogen released was measured during a continu-ous titration at 370C for 5 min. Each sample was then transferred to100 Ml of 10% SDS and the mixtures placed in a boiling water bathfor 3 min. These were stored at -30'C until all samples had beenincubated and boiled. The amounts of inhibitor proteins in theincubation mixtures were: normal, 36 jig, Ta, 50 Mg, Ri, 50 Mg, andWe, 100 Mg. The contents of each mixture were then applied to SDSgels and electrophoresis carried out according to the procedure ofLaemmli under nonreduced conditions (18). The samples shown inpanel A were run simultaneously in disc gels, and those in panel Brun simultaneously in a different set of disc gels. Panel A: 1, SK-plasmin; 2, SK-plasmin and normal CI-INH, treated as above; 3, Cl-INH Ri; 4, mixture of C1-INH Ri and SK-plasmin; 5, molecularweight (Mr) markers Xl03. Panel B: 1, normal Cl-INH; 2, Ci-INHTa; 3, mixture of CI-INH Ta and SK-plasmin; 4, CI-INH We; 5,C1-INH We and SK-plasmin; 6, SK-plasmin used with C1-INH We.Numbers to the right indicate M, X 10' derived from mobility of amixture of proteins of known Mr (Pharmacia Fine Chemicals) run

concurrently (see panel A).

visible complexes with the plasmin and there was no apparentcleavage of the inhibitor (Fig. 3, panel A, gels 3 and 4). Thepreparation of CI-INH Ta contained two readily visible con-

taminants, the anodal of which was albumin (Fig. 3, panel B,gels 2 and 3). Ci-INH We was also contaminated with albumin(Fig. 3, panel B, gel 4). CI-INH Ta did not form complexeswith plasmin that were detectable in these SDS gels, and was

128 Donaldson, Harrison, Rosen, Bing, Kindness, Canar, Wagner, and Awad

- 400

- 380

- 340

- 300

- 280

- 220

(3

>1L_

o70._C

UM-.C.

U)

not apparently cleaved by the enzyme (Fig. 3, panel B, gel 3).CI-INH We, on the other hand, was cleaved by the plasminand small amounts of complexes having approximate molecularweights of -150,000 were visible in these mixtures, eventhough the inhibitor did not block the action of plasmin (Fig.3, panel B, gels 4 and 5). Cleavage fragments were releasedfrom Ci-INH We of apparent molecular weights of 102,000,89,000, and 61,000 after incubation with plasmin (Fig. 3,panel B, gel 5). Thus, fragments cleaved from C1-INH Weappeared to have different molecular weights than those releasedfrom normal Ci-INH by plasmin.

These interactions were then examined in thin-layer SDSgels in which the concentration of polyacrylamide was graded(Fig. 4). In these experiments, mixtures of Cli or plasmin andinhibitors were not boiled in SDS before being applied to thegels, and varied ratios of plasmin to inhibitor proteins weretested.

The interaction of normal CI-INH with Cls and plasminis shown in Fig. 4, panel A. At molar ratios of 23:1, 3:1, and1:1, Cli formed a complex with Ci-INH, with no evidence ofproteolytic degradation of the inhibitor. In contrast, plasminin molar ratios of inhibitor to enzyme of 3:1 or 1:1 formed

both a complex with CI-INH and also caused limited proteolysisof the inhibitor to a lower molecular form, in agreement withthe observations of Harpel and Cooper (8). CI-INH Ta formeda high molecular weight complex with ClIs when mixed atratios of about 3:1 (Fig. 4 B, lane 3) and was cleaved by theCls (lane 3). A mixture containing CI-INH Ta and plasminat molar ratios of 23:1, inhibitor to enzyme, showed noevidence of complex formation, but by changing the inhibitorto enzyme ratio to 3:1, CI-INH Ta could be shown to form ahigh molecular weight complex and was simultaneously con-verted to a lower molecular weight form, which we call I'according to the terminology of Weiss and Engel (19) (Fig. 4B, lanes 5 and 6). When CI-INH Ta and plasmin were mixedin approximately equimolar amounts (0.74:1), there appearedto be an increase in the formation of I', but the predominantspecies was a higher molecular weight complex (Fig. 4 B, lane6). C1-INH We formed a complex with Cli and with plasminwhile being converted to a lower molecular weight form byeach enzyme. Although CI-INH We as isolated was somewhatmore heterogeneous in these gels than the other dysfunctionalinhibitors, it was still possible to demonstrate that this particularinhibitor was proteolytically degraded by both enzymes, in

INTERACTION OF CiINH AND DYSFUNCTIONAL C1INH WITH CIs AND PLASMIN

A CIMWx10-3

I 2 3 4 t

92 5- ~ft'w I66.2- - IF

P1450-

B-C1 INH-Ta1 2 3 4 5 6

_ m -C

C-C IINH-We1 2 34 5 6 7

- _0cI-we -I,

PI

-c- I

IKCIsPl

31.0-

21 5-14.4-

Li L._ .

Cls Pi

D C INH-Ri

MWx10-3 1 2 4- 6

92 566.2-

-C

IUCls Pi

E Cl INH-Zo1 2 3 4 5 6

-c

a-c_ PI*-ClS P

AP

45.0-

31 0-

21.5-144-

L

C1s PILI J

Cls Pi

11 .

Cl Pi

C1 INH-Do1 2 3 4 5 6

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_-

Cls Pi

Figure 4. Interactions of Cli andSK-activated plasmin (SK-PI) withCl-INH proteins: Thin-layer gelscontaining polyacrylamide in gradedconcentrations from 7.5 to 12% (topto bottom) and 1% SDS. The cath-ode is at the top of each set. Thesamples in the lanes are as follows:Panel A: lane 1, 7.6 Atg normal Ci-INH; lane 2, 7.6 ug normal Cl-INH+ 2.3 gg ClI; lane 3, 7.6 gg normalCl-INH + 0.24 Ag SK-Pl; lane 4,7.6 .g normal CI-INH + 1.9 ugSK-Pl; lane 5, 7.6 gg normal Cl-INH + 7.5 jsg SK-Pl. Panel B: lane3 contains 15.1 gg CI-INH Ta +

- I 4.6 ug ClI. Other mixtures are as in- I' panels D-F. Panel C (CI-INH We):- C Is As panel A except that lane 4 con-

Pi tains 6.2 Ag ClIs; lanes 5, 6, and 7contain SK-PL mixtures as in otherpanels. Panels D-F: lane 1, normalCI-INH, 15.1 Mug; lane 2, dysfunc-tional Cl-INH, 15.1 Mg; lane 3, dys-functional CI-INH, 7.6 yg + 2.3 ,gC1s; lane 4, dysfunctional Cl-INH,7.6 Mg + 0.24 Ag SK-P1; lane 5,dysfunctional CI-INH, 7.6 Mg + 1.9jAg SK-Pl; lane 6, dysfunctional Cl-INH, 7.6 Ag + 7.5 Ag SK-P1.

Dysfiinctional Cl-Inhibitor Activities 129

contrast to the behavior of normal or other dysfunctionalinhibitors except Cl-INH Ta. With Cl-s at a 3:1 molar ratioof inhibitor to enzyme, a higher molecular weight complexformed and the CI-INH We was converted to a lower molecularweight form. (Fig. 4 C, lane 3). When Cli and CI-INH Wewere present in equimolar amounts, no intact inhibitor re-mained, either in a complex or a proteolyzed form (Fig. 4 C,lane 4). Similarly with plasmin at a molar ratio of 23:1,inhibitor to enzyme, there appeared to be conversion to alower molecular weight form (Fig. 4 C, lane 5). At a molarratio of 3:1 inhibitor to enzyme, the proteolysis of CI-INHWe by plasmin was very evident, as multiple bands were foundprobably ranging in molecular weight from 100,000 to -60,000(Fig. 4 C, lane 6). At 0.74:1 inhibitor to enzyme, all of theinhibitor was in the lower molecular weight form or waspresent as a higher molecular weight complex (Fig. 4 C, lane7). As previously noted (see Methods), it was difficult to obtainan accurate estimate of the molecular weight of the fragmentsbecause in the gradient gels the proteins used did not migrateaccording to their true molecular weight. CI-INH Ri formeda trace of a higher molecular weight complex when incubatedwith ClI at a molar ratio of 3:1 inhibitor to enzyme (Fig. 4D, lane 3). CI-INH Ri appeared to be much more susceptibleto limited proteolysis by plasmin, being completely convertedto the I' form when incubated with plasmin at inhibitor toenzyme ratios of 3:1 and 0.74:1 (Fig. 4 D, lanes 5 and 6).There appeared to be a small amount of a higher molecularweight complex at higher enzyme concentrations (poorlyvisible). C1-INH Za at threefold molar excess over ClI formeda small amount of complex with the Cls (Fig. 4 E, lane 3).Addition of small amounts of plasmin led to proteolyticconversion of I to I' (Fig. 4 E, lane 4). At 0.74:1 and 3:1 molarratios of inhibitor to enzyme, no intact C1-INH Za remained,being completely converted to I' with a small amount of highermolecular weight complex remaining (Fig. 4 E, lanes 5 and6). Ci-INH Da, is partially complexed with albumin (3) asreadily seen in the lane containing purified inhibitor (Fig. 4 F,lane 2). ClH formed little if any complex with Cl-INH Da(Fig. 4 F, lane 3), but this dysfunctional inhibitor was rapidlydegraded at all concentrations of plasmin (Fig. 4 F, lanes 4 to6). Apparently the complex of albumin with CI-INH Da issusceptible to plasmin proteolysis (Fig. 4 F, lanes 5 and 6). Atapproximately equimolar amounts ofC1-INH Da and plasminthere was a suggestion of formation of a high molecular weightcomplex. (Fig. 4 F, lanes 5 and 6).

Table III summarizes the observations on formation ofcomplexes between C l-s and plasmin and the inhibitor proteinsand designates those inhibitors cleaved by each enzyme, underconditions of these experiments.

Discussion

Each dysfunctional Ci-INH protein exhibited a unique spec-trum of activity against Cls, active forms of Hageman factor(HFa and HFf), kallikrein, and plasmin. Several purifieddysfunctional inhibitors inhibited Cls to a greater degree thanthey inhibited kallikrein (Ta, Da, At, Mo), but one (Za)demonstrated the opposite ratio of activities against theseenzymes, and one (We) inhibited both enzymes to a similardegree. All but CI-INH Ri had significant, or normal inhibitoryactivity against activated forms of Hageman factor, but theamount of activity against HFa and HFf could not be correlated

Table III. Summary ofComplex Formation andCleavage of Inhibitor Proteins with CLi and Plasmin

Enzyme

CIH Plasmin

Complex ComplexInhibitor formation Cleavage formation Cleavage

Normal + 0 + +Ta + + + +We + + + +Ri i 0 0 +Da + 0 + +Za + 0 + +

+, Complex or cleavage visualized in graded SDS gels described inMethods. 0, No visible complex between inhibitor and enzyme orcleavage of inhibitor. ±, Trace of complex or cleavage visualized.

with that directed against kallikrein or ClIs. These data indicatethat there are different structural requirements for the inhibitionof each enzyme by CI-INH. Normal and dysfunctional Cl-INH proteins are single chain molecules, with the exceptionof CI-INH Da, part of which exists in plasma complexedthrough disulfide linkages with albumin (3, 7). Cl-INH Tawas unique among dysfunctional proteins in that earlier workindicated that it was of a higher molecular weight than normalor other dysfunctional Cl-INH proteins examined to date(7, 20).

CI-INH Ri was distinct because of its markedly limitedinhibitory function against all of the enzymes tested. This maybe due to a mutation that makes the molecule incapable ofbinding firmly to the proteases tested. Experiments in progresswill characterize the interactions, if any, of this protein withthe other enzymes. Cl-INH Da also had unique functionalfeatures, for it was the only preparation of dysfunctionalinhibitor that had significant activity against plasmin. CI-INHRi apparently failed to form a covalent complex with plasmin,which was visible in SDS gels (Fig. 3, panel A; Fig. 4 D), as ifthis inhibitor had defective binding sites required for theinhibition of plasmin. Some visible complex was formed withCli under different conditions of electrophoresis (Fig. 4 D).CI-INH Ta also failed to form a visible complex with plasminin the SDS disc gels (Fig. 3, panel B) but complexes werevisible under different experimental conditions (Fig. 4 B). Ci-INH We was cleaved by plasmin, even though it did notinhibit the enzyme (Figs. 3 B and 4 C). This cleavage actuallyoccurred at concentration ratios of plasmin to inhibitor atwhich no additional anodal fragments were generated fromthe normal CI-INH. Moreover all of the Ci-INH We wasdegraded into low molecular weight components with increasingplasmin concentrations (Fig. 4 C). Thus, C1-INH We isapparently excessively susceptible to plasmin digestion. Cl-INH We and Ta were both subject to cleavage by ClS incontrast to all other inhibitors examined (Fig. 4 C, lane 4).

Harpel et al. (8) found that Cl-INH could inhibit plasminby a different mechanism than that leading to inhibition ofCls as seen in Fig. 4 A. In their studies, single chain CI-INHmolecules formed equimolar inhibited complexes with plasmin,as ocurred with ClIs. In addition, plasmin could produce alower molecular weight CI-INH molecule (I', Fig. 4 A) that

130 Donaldson, Harrison, Rosen, Bing, Kindness, Canar, Wagner, and Awad

could also form an inhibited complex with the plasmin.Cleavage of other antiproteases is known to occur during theformation of inhibited complexes with certain enzymes(21-23).

Cls was not thought to cleave normal CI-INH beforeforming an inhibited stoichiometric complex and neither Harpelor Sim et al. (8, 24) detected cleavage of CI-INH by Cli.Weiss and Engel (19) recently reported that normal Ci-INHwas cleaved by Cls and this was enhanced by heparin. We,however, failed to demonstrate cleavage of normal CI-INH byCl-s in the presence or absence of heparin. Cleavage of C7-INH by Cls is, therefore probably not directly related to theformation of an inhibited complex (25). Only CI-INH Weand Ta were cleaved by Cls in the present experiments (Fig.4, B and C).

When conditions of electrophoresis were varied and gradedconcentrations of polyacrylamide were used in SDS gel studies(Fig. 4), further information regarding the variability of theinteractions of the dysfunctional proteins tested and plasminand Cls were obtained, some of which was not evident in theSDS gels shown in Fig. 3 that were derived from kineticexperiments. In these studies, high molecular weight complexeswere found in mixtures of CI-INH Ta, We, Za, and Da whenthese proteins were incubated with Cls or with increasingamounts of plasmin. A high molecular weight complex wasonly faintly visible, visible between Cls and CI-INH Za (Fig.4). Moreover, an I' fagment of CI-INH was generated in eachof these mixtures except CI-INH We, as was seen with thenormal Cl-INH as reported by Harpel and Cooper (8). Thesegels were done with mixtures of enzymes and inhibitor wereincubated in SDS and applied to graded polyacrylamide con-centrations in gels without boiling, which may account forsome of the additional detail not seen in Fig. 3. It is clear thatin several instances when sufficient plasmin was used, complexescan be formed with all dysfunctional CI-INH proteins testedin these gels, except Cl-INH Ri, as seen in Fig. 3 (panel A).These studies confirm the heterogeneity of these proteins andsupport the likelihood that CI-INH We is unduly susceptibleto cleavage by plasmin, for this protein was broken into fourfragments in mixtures containing 1.9 jug of plasmin in contrastto the effect of plasmin on the other dysfunctional and normalproteins. CI-INH Ri did not form visible complexes with threedifferent concentrations of plasmin, but the I, or normalinhibitor band, was readily converted to r despite lack ofdemonstrated inhibitory effect. Similar cleavage occurred inthe case of CI-INH Za and Da. It is noteworthy that the highmolecular weight complex of CI-INH Da with albumin didnot protect it from plasmin cleavage (Fig. 4, panel F, lanes 5and 6).

Cl-INH Za was distinct in that it had an extraordinarilyhigh activity against HFa, while its activities against Cli,plasmin and kallikrein were reduced. This enhanced activityagainst HFa is reminiscent of the altered function found in"antithrombin Pittsburgh" (26, 27). Antithrombin Pittsburghis an al proteinase inhibitor variant that acquired potentantithrombin activity because ofa single amino acid substitutionof an arginine for a methionine (27). This substitution wasapparently sufficient to generate the potent antithrombin activityand induce a fatal hemorrhagic diathesis in the propositus(27). It is unlikely that the variable properties of dysfunctionalCl-INH proteins reported here could be explained on thebasis of a single amino acid substitution.

Earlier reports suggested dysfunctional CI-INH proteinsmight inhibit kallikrein (28), and clearly demonstrated heter-ogeneous electrophoretic and functional properties of severaldysfunctional CI-INH proteins from patients with HANE (3).Some dysfunctional proteins interacted with Cls when the ClIwas added to unfractionated plasma while others failed to bindto CIs (3). Heterogeneity of structure of dysfunctional proteinshas also been demonstrated in studies of isolated preparationsof Ci-INH (7, 8).

The information obtained from studies of purified reagentscan not be applied directly to events which occur in unfrac-tionated plasma. In our earlier description of the interactionsof CI-INH in plasmas from patient We and three others, notdescribed here, there was no apparent interaction between Cl-INH and kallikrein, Hageman factor, or high molecular weightkininogen (29). On the other hand, CI-INH proteins in plasmafrom patients We, Da and Ri interacted with streptokinase-activated plasmin (29), in contrast to the present observations.The behavior of purified CT-INH proteins from these individ-uals differed from their behavior in plasma, in part due todifferences in the conditions of assay. Furthermore, all of thedysfunctional proteins were isolated from plasmas ofindividualswith excessive consumption of the natural substrates of C!(C4 and C2), because of defective regulation of C1. Therefore,the ability of purified CI-INH Da and Ta to inhibit Cli isprobably due to differences between their interactions with thephysiologic enzymes which can form in plasma (macromolec-ular Ci or Cli-Cls complexes) and its purified Cli subcom-ponent.

CI-INH Ta may also be susceptible to digestion by one ormore serum proteases to which normal CI-INH is resistant,for the anti-Cls activity of Ci-INH Ta was lost when it wasadded to Ta serum, whereas that of the normal CI-INH wasnot. The in vivo behavior of three ofthe dysfunctional proteinsdescribed herein has been reported (5). CI-INH Ta and Atbehaved similarly in being cleared more slowly than thenormal inhibitor from the vascular space of normal individuals,patients with HANE (5), and other patients with excessive Clactivation (30, 31). This suggested that catabolism was inde-pendent of inhibitory functions of these proteins. CI-INH Wewas cleared more rapidly than CI-INH Ta and At, as if Cl-INH We may be excessively susceptible to proteolytic digestionby enzymes such as plasmin, which may play a role in itscatabolism.

These studies confirm earlier reports of genetically deter-mined heterogeneity of dysfunctional CI-INH proteins foundin HANE plasmas, and additionally demonstrate a complexityof interactions with plasma proteases which was not previouslyappreciated. Work in progress will characterize the interactionsof dysfunctional Ci-INH proteins and other plasma proteasesand assess the relevance of these interactions to the pathogenesisof hereditary angioneurotic edema.

AcknowledgmentsWe thank Dr. Marco Cicardi and Dr. Peter Lachmann for plasmasfrom patients in their respective countries, Italy and England. Weappreciate Augustine Merriweather's technical expertise in preparingHageman factor and kallikrein. Anne F. Marr and Judy P. Wier haveenabled us to complete this manuscript by their word processingexpertise.

This work was supported by research grants from the U. S. PublicHealth Service (HL-15690, AI-21163, RR-02172, and AM-17351),

Dysfunctional Cf-Inhibitor Activities 131

The March of Dimes, a Wellcome Research Travel Grant (Dr. Rosen),and by the Children's Hospital Research Foundation, Cincinnati, OH.

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132 Donaldson, Harrison, Rosen, Bing, Kindness, Canar, Wagner, and Awad