Inhibition by CTINH of Hageman Factor

Fragment Activation of

Coagulation, Fibrinolysis, and Kinin Generation

ALAND. SGmREIBE, ALLEN P. KAPLAN, and K. FRANKAUSTEN

From the Departments of Medicine, Harvard Medical School and Robert B.Brigham Hospital, Boston) Massachusetts 02120

A B S T R A C T Highly purified inhibitor of the firstcomponent of complement (C1INH) was shown to in-hibit the capacity of active Hageman factor fragments toinitiate kinin generation, fibrinolysis, and coagulation.The inhibition of prealbumin Hageman factor fragmentsobserved was dependent upon the time of interaction ofthe fragments with C1INH and not to an effect uponkallikrein or plasmin generated. The inhibition of thecoagulant activity of the intermediate sized Hagemanfactor fragm.ent by C1INH was not due to an effect onPTA or other clotting factors. The inhibition by C1INHof both the prealbumin and intermediate sized Hagemanfactor fragments occurred in a dose response fashion.The C1INH did not appear to be consumed when theactivity of the Hageman factor fragments was blocked,although the fragments themselves could no longer berecovered functionally or as a protein on alkaline discgel electrophoretic analysis. These results suggest thatthe C1INH may have an enzymatic effect on the frag-ments or that an additional site on C1INH is involvedin Cl inactivation.

INTRODUCTIONThe activation of Hageman factor results in the initia-tion of coagulation (1), kinin generation (2, 3), andfibrinolysis (4, 5) by activation of pre-plasma thrombo-

Dr. Kaplan was formerly, National Institutes of HealthSpecial Research Fellow (2 F03 AI 42810) ; his presentaddress is the Section Head, Allergic Diseases, Laboratoryof Clinical Investigation, National Institute of Allergy andInfectious Diseases, National Institutes of Health, Bethesda,Md.

Received for publication 13 November 1972 and in revisedform 24 January 1973.

plastin antecedent (pre-PTA)1" (1), prekallikrein (6),and plasminogen proactivator (7, 8). These three plasmaproenzyme substrates are activated both by intact acti-vated Hageman factor and by fragments present inserum or derived from activated intact Hageman factorexperimentally (6, 9). The inhibitor of the first com-ponent of complement (C1INH) is recognized to in-hibit kallikrein (10-12), plasmin (11), PTA (13), andthe coagulant activity of activated Hageman factor (13).An additional critical site by which C1INH regulatesthese effector systems of tissue injury is shown to beits capacity to inhibit the action of the Hageman factorfragments upon each of these three plasma proenzymes.

METHODSAntisera to al antitrypsin and a2 macroglobulin (a2M)(Behring Diagnostics Inc., Woodbury, N. Y.); hexadi-methrine bromide (polybrene) (Aldrich Chemical Co., Inc.,Milwaukee, Wis.); enzodiffusion fibrin plates and strepto-kinase (Hyland Div., Travenol Laboratories, Inc., CostaMesa, Calif.) were obtained as indicated. Hageman factor-deficient plasma and PTA-deficient plasma were supplied bySera-Tec Biologicals, New Brunswick, N. J. Concentrationof various chromatographic fractions was performed byultra filtration using UM-10 membranes (Amicon Corp.,Lexington, Mass.) in either 500 ml, 50 ml, or 10 ml ca-pacity Amicons as appropriate. All phosphate buffer used

1 In order to avoid ambiguity and confusion as to thestate of activation of the molecule we have referred to theprecursor of PTA as pre-PTA throughout the text. Otherworkers may consider PTA as the precursor form andrefer to the activated molecule as activated PTA.

'Abbreviations used in this paper: a2M, a2 macroglobu-lin; C1INH, inhibitor of the activated first component ofcomplement; EACI, erythrocyte-bound C1; PTA, plasmathromboplastin antecedent; PTT, partial thromboplastintime; QAE, Quartenary aminoethyl; SE Sephadex, Sulpho-ethyl Sephadex; SP Sephadex, Sulphopropyl Sephadex.

1402 The Journal of Clinical Investigation Volume 52 June 1973.1402-1409

was 0.0035 Mphosphate buffer pH 7.8-8.0 containing 110 mgNaH2PO4 H20 and 388 mg Na2HPO4 per liter. All phos-phate-buffered saline was 0.0035 M phosphate buffer con-taining 0.15 MNaCl.

Plasma was prepared for the isolation of plasma proen-zymes and C1INH in ethylenediaminetetraacetic acid (ED-TA) and polybrene as described (6). The conversion ofpre-PTA (1), prekallikrein (6), and plasminogen pro-activator (7, 8) by the Hageman factor fragments wasmeasured in terms of subsequent coagulation, bradykininformation, and fibrinolysis. The coagulant activity of theHageman factor fragment was determined by preparingtwo-fold falling dilutions of the preparation of Hagemanfactor fragment and relating its concentration to the partialthromboplastin time (PTT) achieved with Hageman factor-deficient plasma (Fig. 1).

Hageman factor prealbumin fragments were prepared asfollows: plasma dialyzed against 0.0035 M phosphate bufferpH 8.0 containing 0.06 M NaCl was applied to Quartenaryaminoethyl (QAE) Sephadex (5 X 100 cm) equilibrated with0.0035 M phosphate buffer pH 8.0. The column was batcheluted with the dialysis buffer, concentrated to the startingvolume, activated by stirring in a glass beaker for 24 h at4VC, dialyzed against 0.0035 M phosphate buffer pH 8.0,and rechromatographed on QAE Sephadex with a linearsalt gradient of 2,500 ml of dialysis buffer and 2,500 mlof 0.0035 M phosphate buffer pH 8.0 containing 0.3 MNaCl. The column was run at 50 ml/h and 12 ml fractionswere collected. The Hageman factor fragments eluted be-tween 0.23 and 0.28 M NaCl, were concentrated to 4 ml,and were fractionated at 30 ml/h on Sephadex G-100 (5 X100 cm) equilibrated with 0.0035 M phosphate buffer pH8.0 containing 0.15 M NaCl. The Hageman factor pre-albumin fragments eluted at a mol wt of 32,500, were con-centrated to approximately 20-25 ,ug/ml, aliquoted, andstored at -70'C for routine use. Disc gel electrophoresis(14) revealed prealbumin bands with only trace contamina-tion with albumin.

An intermediate sized Hageman factor fragment was iso-lated from human plasma by applying plasma dialyzedagainst 0.0035 Mphosphate buffer pH 8.0 containing 0.04 MNaCl to QAE Sephadex (5 X 100 cm) equilibrated with0.0035 M phosphate buffer pH 8.0. The column was batcheluted with the dialysis buffer, concentrated to the startingvolume, activated by stirring in a glass beaker for 24 h at4VC, dialyzed against 0.0035 M phosphate buffer pH 8.0,and rechromatographed on QAE Sephadex with a linearsalt gradient of 2,500 ml of dialysis buffer and 2,500 mlof 0.0035 M phosphate buffer pH 8.0 containing 0.3 MNaCl. 12 ml fractions were collected at 50 ml/h. Theintermediate sized Hageman factor fragment eluted be-tween 0.08 M and 0.10 M NaCl, was concentrated to 4 ml,and was fractionated at 30 ml/h on Sephadex G-100 (5 X100 cm) equilibrated with 0.0035 M phosphate buffer pH 8.0containing 0.15 M NaCl. The intermediate sized Hagemanfactor fragment eluted at a mol wt of 80,000 and wasdivided and stored at - 70'C for further use. Assessmentof the preparation by disc gel electrophoresis revealed twobands in the fl-globulin region, one corresponding to theHageman factor fragment and the other to a transferrincontaminant.

Plasminogen was prepared by affinity chromatography of100 ml of plasma utilizing lysine-sepharose columns ande-aminocaproic acid elution (8, 15). After dialysis against0.0035 M phosphate buffer pH 8.0 containing 0.15 M NaCl,the preparation was fractionated by Sephadex G-100 gel

50

25

KC.K.

l0

5

2.5

100 50 25 12.5 63 1.6

%HAGEMANFACTORFRAGMENr

FIGURE 1 Correction of the partial thromboplastin time ofHageman factor-deficient plasma by the intermediate sizedHageman factor fragment.

filtration at 30 ml/h utilizing a 5 X 100 cm column equili-brated with the dialysis buffer. Fractions containing plas-minogen as assessed by streptokinase activation followed byapplication to fibrin plates were pooled, concentrated, andstored at - 70'C. Disc gel electrophoresis revealed a singlebroad band identified as plasminogen by functional analysisof an unstained sliced replicated disc gel. There were no

contaminating proteins detected. Plasmin was prepared byactivating 500 ul of plasminogen (100 ,ug/ml) with 50 ;Ld(1,500 U) of streptokinase for 30 min at 30'C and assayedon human fibrin plates (8).

The plasminogen proactivator was isolated by applying 80ml of dialyzed human plasma to QAE Sephadex (5 X 100cm) equilibrated in 0.0035 M phosphate buffer pH 8.0. Thecolumn was batch eluted with the dialysis buffer, concen-trated to 10 ml, dialyzed against 0.0035 M phosphate bufferpH 6.0, and applied to Sulphoethyl (SE) Sephadex (3.5 X30 cm) equilibrated with the dialysis buffer. The plasmino-gen proactivator was eluted with a linear salt gradient of2,000 ml of equilibrating buffer and 2,000 ml of equilibratingbuffer containing 0.35 M NaCl. The column was run at 50ml/h and 10 ml fractions were collected. The plasminogenproactivator eluted between 0.12 and 0.14 M NaCl and was

pooled, concentrated to 25 ml, and fractionated on Sepha-dex G-150 (2.5 X 150 cm) equilibrated in 0.0035 M phos-phate buffer pH 8.0 containing 0.15 M NaCl. Fractionationwas performed by upward flow at 10 ml/h and 2.5 ml frac-tions were collected. The plasminogen proactivator elutedat a mol wt of approximately 100,000 and was pooled,concentrated to 10 ml and stored at - 70°C. The prepara-tion contained trace prekallikrein and IgG contaminationwhen assessed by bioassay and Ouchterlony or alkaline discgel electrophoretic analysis, respectively. The plasminogenproactivator did not contain plasminogen or PTA.

Purification of C1INH. 80 ml of plasma dialyzed against0.0035 Mphosphate buffer pH 7.8 were applied to a 5 X 100cm column of QAE Sephadex equilibrated with the samebuffer. The column was washed with 600 ml of equilibrating

Inhibition of Hageman Factor Fragments by C1INH 1403

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FIGURE 2 Isolation of

140 180 220 260 300 340 380 420

pi

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TUBE NUMBERC1INH by chromatography of human plasma on

0.250 -

0.210 _

0.170 I

0.130 i23

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buffer and eluted with a linear salt gradient ofequilibrating buffer and 2,500 ml of 0.0035 Mbuffer pH 7.8 containing 0.3 M NaCl. The crun at 50 ml/h and 10 ml fractions were colC1INH was assayed functionally in microtiterC1INH peak eluted at 0.15 M NaCl (Fig. 2)containing C1INH were pooled, concentrated tolyzed for 5 h against 0.05 M sodium acetate bufand applied to a 3.5 X 30 cm column of SulphopSephadex equilibrated with the same buffer. Thewashed with 200 ml of equilibrating buffer, and

0.3

a 0.2 _

oi

10 20 30

FIGuRE 3 Isolation of C1INHSephadex G-200 gel filtration.

was batch eluted by washing the column with approximately200 ml of 0.05 M sodium acetate buffer pH 5.0 containing0.09 M NaCl. The column was run at 50 mI/h and frac-tions were collected in glass tubes containing 5 ml of 2 MTris Cl buffer pH 8.0 to total 13 ml per tube, in order to

raise the final pH above 7.2. The C1INH pool was concen-

trated to 4 ml and applied to a 5 X 100 cm column of Sepha-dex G-200 equilibrated in 0.0035 M phosphate buffer pH7.8 containing 0.15 M NaCl. Fractionation was performedby upward flow at 10 ml/h and 10 ml fractions were col-lected. C1INH eluted along the ascending limb of the

14

12

Pa,/- 4

\\bi4 !b

40 50 60 70 80 90 100 110 120

TUBENUMBERobtained by QAE and SP Sephadex chromatography by

1404 A. D. Schreiber, A. P. Kaplan, and K. F. Austen

QAE Sephadex.

second protein peak as shown in Fig. 3. The fractions con-taining C1INH were concentrated to 2.0 ml and furtherfractionated by Sephadex G-150 (2.5 X 150 cm) gel filtrationequilibrated in 0.0035 M phosphate buffer pH 7.8 containing0.15 M NaCl. Fractionation was performed by upward flowat 10 ml/h and 2.5 ml fractions were collected. No opticaldensity at 280 nm was observed; fractions containingCIJNH eluted at 55% of bed volume and were pooled(total volume 27 ml) and stored at -70'C for furtherstudies. This preparation had no detectable al antitrypsinor a2M as assessed by Ouchterlony analysis or electroim-munodiffusion utilizing monospecific antisera to each. Thishighly purified C1INH preparation (6,000 U/ml) gave adouble band on alkaline disc gel electrophoresis in aregion from which unstained gels yielded active C1INHupon elution (Fig. 4).

The C1INH was assessed functionally by its ability toinhibit the hemolytic activity of erythrocyte bound C1(EAC1) by either microtiter plate or tube titrations (16)as indicated and was quantitated by immunodiffusion (17,18). The titer of 1,613 U/flg of C1INH obtained is com-parable to the activity in normal serum.

RESULTS

Inhibition of the action of Hageman factor frag-ments upon prekallikrein, plasminogen proacti-vator, and pre-PTAPrekallikrein. 10 Al of Hageman factor fragments

were incubated with increasing volumes of highly puri-fied C1INH (1,000 U/ml) at 37°C for 30 min. Eachmixture was brought to a final volume of 160 Al andincubated for 2 min at 37°C with fresh human plasmaas a source of prekallikrein and kininogen, and thebradykinin generated was determined. As illustrated inFig. 5, as the amount of C1INH was increased, thebradykinin generating activity of the Hageman factorfragments was progressively decreased. In order to dis-tinguish inhibition by C1INH of the Hageman factor

c _k

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1 150

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SL/CE NUMBER

FIGURE 4 Disc gel electrophoresis of C1INH obtained byQAE Sephadex (Fig. 2), SP Sephadex, Sephadex G-200(Fig. 3), and Sephadex G-150 chromatography.

fragments from direct inhibition of kallikrein, the Hage-man factor fragments were incubated with C1INH(1,000 U/ml) for varying time intervals before intro-duction into fresh plasma. No inhibition of bradykiningeneration was observed when the Hageman factor frag-ments and C1INH were preincubated for up to 2 min,the time interval subsequently used to assess the brady-kinin generating capacity of the Hageman factor frag-ments in fresh plasma. After 2 min of preincubation ofthe fragments with C1INH, a progressive decrease inthe capacity of the fragments to generate bradykininfrom fresh plasma was observed (Fig. 6). These kineticstudies reveal that the effect of C1INH is on the Hage-man factor fragments and not upon kallikrein generated.

250 IAI of Hageman factor fragments were incubatedwith either 250 Al of C1INH (10,000 U) or with bufferalone for 90 min at 37°C. In addition, 250 #l of CIINHwere incubated with buffer under the same conditions.Replicate 225 #l samples from each mixture were thenassessed by disc gel electrophoresis; one disc gel was

400 600

Cf1INH (Units/rm/800

FIGURE 5 Inhibition by C1INH of the ability of the prealbumin Hageman factor fragmentsto generate bradykinin from fresh plasma.

Inhibition of Hageman Factor Fragments by CIINH

--I5(

2!

1405

500

375

250

125

0 4 8 12 16 20 24 28 32

INCUBATION (min)

FIGURE 6 Kinetics of inhibition by C1INH (closed circles)of the capacity of the prealbumin Hageman factor fragmentsto generate bradykinin from fresh plasma. Open circles referto prealbumin Hageman factor fragments not treated withC1INH.

stained with Coomassie blue stain (Colab Lab, Inc.,Chicago Heights, Ill.) and the replicate gel slicedinto 1 mmsections, crushed, eluted in 200 Al 0.15 Msodium chloride, and assayed for prekallikrein acti-vating activity. Prekallikrein activating activity was

found in slices 7-9 of the disc gel of Hageman factor

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D/SC GEL SECT/ON(O.0 mms/ices)

fragments incubated in buffer alone. No prekallikreinactivating activity was detected in the disc gel eluatesfrom Hageman factor fragments preincubated withCTINH (Fig. 7). In addition, the stained gel of theHageman factor fragments incubated in buffer containedprealbumin bands at the position corresponding to slices7-9, whereas no prealbumin bands were present in thedisc gel of Hageman factor fragments preincubatedwith C1INH.

Plasminogen proactivator. 10 ,A of Hageman factorfragments were incubated with 200 A4 of C1INH (1,000U/ml) in phosphate-buffered saline (pH 7.8) or withbuffer alone for 30 min at 370C. 5 p1 were removedfrom each incubation mixture and added to 50 A4 ofplasminogen proactivator; the mixture was placed at40C for 15 h to permit conversion of plasminogen pro-activator to plasminogen activator. 20 Al of this mixturewere then incubated with 20 p1 of highly purified plas-minogen (200 Ag/ml) for 1 h at 370C. The Hagemanfactor fragments incubated in buffer alone generated10.5 og/ml of plasmin, while the Hageman factor frag-ments preincubated with C1INH yielded only 2.5 pzg/mlof plasmin. This result is attributed to inhibition of theability of the Hageman factor fragments to generateplasminogen activator from the plasminogen proactiva-tor. At the concentration of C1INH utilized, no inhibi-tion of plasmin (50 og/ml) obtained by interaction ofplasminogen with either plasminogen activator or strep-tokinase was demonstrable.

Pre-PTA. For these experiments a higher molecularweight Hageman factor fragment (mol wt = 80,000)possessing greater pre-PTA activating activity than the

;kl,Z)i:.,jcr't"llI-

FIGURE 7 Elution of prekallikrein activating activity after CiIVH (Units/mi) x10-5disc gel electrophoresis of prealbumin Hageman factor frag- FIGURE 8 Inhibition by C1INH of the ability of the inter-

ments treated with C1INH (closed circles) or buffer (open mediate size Hageman factor fragment to correct the clot-circles). ting deficiency of Hageman factor-deficient plasma.

1406 A. D. Schreiber, A. P. Kaplan, and K. F. Austen

_

I 1-I1 I

2 60-

20

0 5 15 30 60

MINUTES

FIGURE 9 Kinetics of inhibition by C1INH of the capacityof the intermediate size Hageman factor fragment to correctthe clotting deficiency of Hageman factor-deficient plasma.

prealbumin fragments (mol wt = 32,500) was utilized.25 1ul of twofold falling dilutions of C1INH (50,000 U/ml) were incubated for 80 min at 370C with 25 1 ofHageman factor fragment and the correction of thePTT of Hageman factor-deficient plasma determined.As shown in Fig. 8, as the concentration of the C1INHincreased, there was progressive inhibition of the co-agulant activity of the Hageman factor fragment.

A time-course of inhibition of the coagulant activityof the Hageman factor fragment by C1INH was studiedas follows. 125 ul of Hageman factor fragment wereincubated with either 125 A1 of C1INH at a concentra-tion of 50,000 U/ml in phosphate-buffered saline pH7.8 or in buffer alone at 370C. 50 14 samples of eachmixture were then removed at designated intervals, andthe ability to correct the PTT of Hageman factor-de-ficient plasma determined. Addition of kaolin did notlead to further activation or a decrease in PTT, as thematerial was completely active. No inhibition of the Hage-man factor fragments occurred at 0 time, while inhibitionwas marked by 5 min and tended to reach a plateauafter 15 min of interaction of C1INH with Hagemanfactor fragment (Fig. 9). This experiment indicatesthat the effect of C1INH is on the Hageman factor frag-ment and not upon the PTA generated or later clottingfactors.

ClINH consumptionIn order to ascertain whether the inhibition of the

Hageman factor fragments by C1INH results in con-sumption of CiINH, residual C1INH was measuredby its ability to inhibit Cl. 75 Al of an 80,000 mol wt

Hageman factor fragment preparation undiluted and di-luted 1: 2 with phosphate-buffered saline pH 7.8 wereincubated with either 75 Al of C1INH (5,000 U/ml)or buffer alone at 37'C for 30 min. These mixtures werethen examined for their prekallikrein and pre-PTA ac-tivating ability using fresh plasma and Hageman factor-deficient plasma, respectively, as described above. TheC1INH inhibited 100% of the kinin generating activityand 50% of the coagulant activity in the undiluted prep-aration. In addition, 75 Al of C1INH were incubatedwith 75 Al of a partially purified preparation of kalli-krein, and 10 Al of the mixture were examined fortheir ability to generate bradykinin from 200 Al of heat-inactivated plasma, using a 2 min incubation; the kalli-krein was 90% inhibited. As shown in Fig. 10, incuba-tion of C1INH with either the undiluted Hagemanfactor fragment or Hageman factor fragment diluted1:2 did not change the C1INH titer even though thekinin generating and coagulant ability of the frag-ments was markedly reduced. The partially purifiedkallikrein preparation produced a decrease in the C1INHtiter from 4,700 to 600 U/ml.

The effect of increasing the ratio of Hageman factorfragments to C1INH was examined by incubating 10,20, or 30 1l of the 80,000 mol wt Hageman factor frag-ment preparation with 10 Al of the C1INH preparation(5,000 U/ml) for 30 min at 370C in a total volume of40 Al for each reaction mixture. 10 1l of C1INH werealso incubated with the partially purified kallikreinpreparation under the same conditions. As in the previ-ous experiment, inhibition of kallikrein (100%) wasassociated with C1INH consumption. 100% inhibition

2

0.5-

02

2 4 8 16 32

CTNmw RELAIV/ ClA'ENWTR7A7W

FIGURE 10 The effect of Hageman factor fragment (openand closed boxes) and kallikrein (open triangles) on therecovery of CiINH as measured by an effective moleculetitration. C1INH incubated in buffer alone is indicated bythe open circles.

Inhibition of Hageman Factor Fragments by C1INH 1407

PTA or the other clotting components generated (Fig.9). Thus, the CTINH inhibited the action of intermedi-ate and small Hageman factor fragments upon three ofits naturally occurring substrates, prekallikrein, plas-minogen proactivator, and pre-PTA, at concentrationsof C1INH comparable to those present in normal plasmaand under experimental conditions in which its effecton other enzymes in the reaction sequences was not afactor.

Recent studies have also revealed that partially puri-fied C1INH suppresses the esterase activity of 37,000mol wt Hageman factor fragments on benzoyl arginineethyl ester (19). Forbes, Pensky, and Ratnoff havedemonstrated that incubation of C1INH with C1 ester-

| I | Is | ase reverses the ability of C1INH to inhibit either acti-2 4 8 16 32 64 vated Hageman factor or activated PTA in clotting

assays and suggest a common site on C1INH for Hage-man factor, PTA, and CT1 esterase (13). However, de-

l The effect of an increased ratio of Hageman struction by Cl esterase as has been suggested by Loos,agment to C1INH (closed squares and open and Wolf, and Opferkuch (20) might also account for thiscles) and kallikrein (closed triangles) on the re- observation.

C1INH as measured by an effective molecule The interaction of C1INH with kallikrein was pre-C1INH incubated in buffer alone is indicated by viously observed to deplete both functions from the re-;quares. action mixture (12) when kallikrein was measured as

an esterase and C1INH assessed by an effective mole-iageman factor fragments in terms of kinin cule titration. In contrast, in the present experiments

inhibition of the 80,000 mol wt Hageman factor frag-) ment by C1INH was not associated with consumptionDISCUSSION of the CTINH (Figs. 10 and 11). The functional in-

tegrity of the inhibitor after interaction with the 80,000purified C1INH (Fig. 4) was shown to inhibit mol wt Hageman factor fragment was established by itscity of active Hageman factor fragments to action in interfering with the hemolytic titration of Cf.cinin generation, fibrinolysis, and coagulation. Even though the C1INH did not appear to be consumedMH had been previously recognized to inhibit when the prealbumin fragments were blocked, the frag-

(10-12), plasmin (11), and PTA (13), it ments themselves could no longer be recovered eitherntial to distinguish an action at these points functionally or as a protein on disc gel electrophoretic

effect of the CiINH on the Hageman factor analysis of the reaction mixture (Fig. 7). These resultss. TheC*INH inhibited the kinin generating suggest the CiINH may have an enzymatic effect onof the Hageman factor fragments in whole the fragments, but might also be explained by an addi-

a dose response fashion (Fig. 5). The experi- -.2 ,1 . ,0 ., ... ,1 tional site on C1INH involved in Cl inactivation.

mental conditions were such that the inhibition ob-served was dependent upon the time of interaction ofthe fragments with C1INH (Fig. 6), and not to an

effect on the kallikrein generated. Similarly, a concen-

tration of CfINH having no effect upon plasmin or theplasminogen activator was capable of protecting highlypurified plasminogen proactivator from activation by theprealbumin Hageman factor fragments. Utilizing an inter-mediate sized Hageman factor fragment, it was possible toshow that the C1INH yielded a dose-dependent inhibitionof the capacity to correct the coagulation defect of Hage-man factor-deficient plasma (Fig. 8). The inhibitionobserved was dependent upon the time of interactionof the fragment with C1INH and not to an effect upon

ACKNOWLEDGMENTS

Wewould like to thank Dr. Peter H. Schur for quantitativeimmunodiffusion of C1INH.

This paper was supported by grant AI-07722 from theNational Institutes of Health, and a grant from The JohnA. Hartford Foundation, Inc. Dr. Schreiber was supportedby Training Grant AI-00366 from the National Institutes ofHealth.

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1408 A. D. Schreiber, A. P. Kaplan, and K. F. Austen

2

ZI0.5

0.2

0.1

FIGURE 11factor fraclosed circovery oftitration.the open s

of the Egeneratio(Fig. 11.

Highly pthe capa(initiate kAs CIINkallikreinwas esseifrom thefragmentactivity (

plasma in

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Inhibition of Hageman Factor Fragments by C1INH 1409