Turnover of Humanand Monkey Plasma Kininogens in

Rhesus Monkeys

TADATAKAYAMADA, DAVID A. WING, JACK V. PIERCE, and GEORGEW. PETTIT,Bacteriology, Pathology, and Animal Assessment Divisions, U. S. Army MedicalResearch Institute of Infectious Diseases, Fort Detrick, Frederick, Maryland21701, and The Section on Physiological Chemistry, Laboratory of Chemistry,National Heart, Lung, and Blood Institute, National Institutes ofHealth, Bethesda, Maryland 20014

A B S T RA C T The normal metabolic turnover ofplasma kininogens was studied by measuring the disap-pearance of intravenously administered radiolabeledhuman and monkey plasma kininogens from thecirculation of healthy adult rhesus monkeys. Curvesobtained by plotting log radioactivity against timecould be expressed as double exponential equations,with the first term representing diffusion, and thesecond, catabolism. No significant difference betweenthe turnovers of human and monkey kininogens wasobserved. The difference between the t112 of highmolecular weight kininogen (25.95±1.60 h) (mean+SEM) and that of low molecular weight kininogen(18.94±1.93 h) was only marginally significant (P< 0.05). In contrast, a highly significant (P < 0.001)difference in their mean catabolic rates (1.12±0.08 d-lfor high molecular weight kininogen vs. 2.07±0.09 d-lfor low molecular weight kininogen) was observed.These differences between the two kininogens wereattributed to differences in their distribution betweenthe intra- and extravascular pools. Studies of kininogenturnover will be useful in elucidating the in vivo func-

This work was presented in part at the VIth InternationalCongress on Thrombosis and Haemostasis, Philadelphia,Pa., 1 July 1977.

In conducting the research described in this report,the investigators adhered to the "Guide for the Care and Useof Laboratory Animals," as promulgated by the Committeeon the Revision of the Guide for Laboratory Animal Facilitiesand Care of the Institute of Laboratory Animal Resources,National Research Council. The facilities are fully accreditedby the American Association for Accreditation of LaboratoryAnimal Care. The views of the authors do not purport toreflect the positions of the Department of the Army or theDepartment of Defense. Reprint requests should be ad-dressed to Dr. Yamada, Gastroenterology Division (691/111 C), Wadsworth VA Hospital, Los Angeles, Calif. 90073.

Received for publication 26 August 1977 and in revisedform 17 August 1978.

tions of the various kininogens in health as well asduring clinical illness.

INTRODUCTION

The kinins are a group of polypeptides known pri-marily for their potent vasodilatory actions in addi-tion to their ability to cause increased capillarypermeability (1, 2). They are generated in plasmathrough enzymatic cleavage of a precursor protein,kininogen, by plasma kallikrein (3). Plasma kallikreinis formed from its precursor, prekallikrein, and thisconversion is dependent upon the presence of acti-vated Hageman factor (blood coagulation Factor XII)(4). Because of their profound physiologic actions,the kinins have been implicated in the pathophysiol-ogy of a wide variety of clinical disorders which in-clude carcinoid syndrome (5), hereditary angioedema(6), dumping syndrome (7), Gram-negative septicemia(8), and disseminated intravascular coagulation (9).Documentation of the involvement of the kinin sys-tem in these various disorders, however, has beenlimited to the observation of elevated plasma kininand kallikrein activities, and the depletion of theirprecursor proteins, kininogen and prekallikrein. Suchobservations only imply kinin system activation. Nodata regarding the specific kinetics of kinin systemproteins in these disorders are available.

Recent advances in the isolation and purification ofplasma kininogens (10) have enabled us to examinethe normal turnover of these proteins. By intravenousinjection into rhesus monkeys of radiolabeled, purifiedhuman and monkey kininogens, and subsequent meas-urement of their disappearance, we have endeavoredto characterize the kinetics of these proteins in plasma.We hope to use this information to study kininogenturnover in various disease states to clarify the roleof the kinin system, if any, in their pathogenesis.

The Journal of Clinical Investigation Volume 63 January 1979 -45-52 45

METHODSIsolation of kininogens. Kininogens were purified with

the affinity chromatographic methods of Pierce and Gui-maraes (10) as outlined in Fig. 1. 2 liters of either humanor monkey blood were drawn into plastic receptacles whichcontained 1 part 3.8% sodium citrate, 0.1% hexadimethrinebromide (Aldrich Chemical Co., Inc., Milwaukee, Wis.),and 0.01 Mbenzamidine chloride (Calbiochem, San Diego,Calif.), to 9 parts whole blood. The samples were centri-fuged at 25°C for 10 min at 2,000 g, and the plasma supernate(520 ml) was chromatographed on a 2.6- x 40.0-cm columnof L-lysine coupled to Sepharose 4B (Pharmacia Fine Chemi-cals, Div. of Pharmacia Inc., Piscataway, N. J.) which wasprepared as described by Deutsch and Mertz (11). Theplasminogen-free plasma (580 ml) thus obtained was stirredinto 4 liters of a solution that contained 50 g dry weightof DEAE-cellulose (DE-23, Whatman Chemicals, Divisionof W& R Balston, Maidstone, Kent, England), 0.1% hexa-dimethrine, and 1 mMbenzamidine; titrated to pH 6.0by the addition of 5 N HCI. The resulting slurry was stirredvigorously for 2 h, allowed to settle for 30 min, decantedonto a coarse-fritted Buchner funnel, and washed with 5liters of 0.01% hexadimethrine-1 mMbenzamide solution.The washed adsorbent was suspended with 67 ml of 3.0 MTris-HCl buffer, pH 6.0, and enough 0.01% hexadimethrine-1 mMbenzamidine to give a final volume of 250 ml. Afterstirring for 1 h, the suspension was filtered in a coarse-fritted Buchner funnel and washed with 500 ml of 0.4 MTris-HCl, 0.01% hexadimethrine, and 1 mMbenzamidinebuffer, pH 6.0. The eluate, called prep A, was adjustedto pH 7.2, and applied at 25°C to an affinity column (3.1

Plasma

I (L-lysine Sepharose chromatography)

Plasminogen-free plasma

I (DEAE-cellulose batch adsorption)

Prep A

I (Affinity chromatography with immobilized antikininogenantibody)

Prep B

(DEAE-cellulose chromatographv: elut ionwith phosphate buffer gradient)

Bl B2 B3 B4

(Bio-Gel A-O.5m gelfiltration)

Bla BlE B2a B26 B3ct B36 B4a B4B B4Y

FIGURE 1 Isolation procedure for plasma kininogens.

x 13.9 cm) which was prepared by coupling purified sheepanti-human low molecular weight kininogen to cyanogenbromide-activated Bio-Gel A-50m (Bio-Rad Laboratories,Richmond, Calif.) by the method of March et al. (12). Thecolumn was washed with a buffer which contained 0.4 MTris-HCl, 0.01% hexadimethrine, and 1 mMbenzamidine,pH 6.0, and eluted with 8 M guanidine HCI. The purifiedkininogen thus obtained, called prep B, was dialyzed against0.05 Mphosphate buffer, pH 6.0, for 24 h at 25°C and chro-matographed on a DEAE-cellulose column (0.9 x 30.0 cm)at 4°C. Four main A280 peaks coincident with peaks of kinino-gen activity, called Bl, B2, B3, and B4, were eluted with alinear phosphate buffer gradient, 0.05-0.30 M, pH 6.0. Essen-tially no difference was observed between human and monkeykininogens in the number or position of the protein peaks.Approximately 48% of the kininogen activity of the initialplasma was recovered in the total activities of B1-B4. Gelfiltration of each of the peaks with Bio-Gel A-0.5m revealedseparate kininogen subfractions, called a, 3, and y whichcorresponded to proteins of molecular weight 80,000, 160,000,and 225,000, respectively. Whereas B 1, B2, and B3 con-tained nearly equal amounts of the a and /3 forms, B4consisted mainly of the y protein (59%) with some a (1%)and /3 (40%) proteins. Hence, for the purposes of our study,we will refer to B4 as high molecular weight (HMW)lkininogen, and a pooled sample consisting of BL, B2, andB3 as low molecular weight (LMW) kininogen, unless thea, /3, or y speciation is otherwise noted.

Sheep antisera to human LMWkininogens I and II wereprepared by the method of Pierce and Webster (13). Theantisera were purified by immunoprecipitation followed bychromatography on hydroxyapatite in 8 Murea.2 A reaction ofidentity was obtained by Ouchterlony double diffusion withhuman and monkey HMWand LMWkininogens againstthe purified monospecific antibody.

Kinin-generating activity of the various kininogen frac-tions, both before and after radioiodination, was measuredby bioassay on guinea pig ileum by the direct method ofPrado et al. (14) (modified by Webster and Prado [15] andPierce and Guimaraes [10]). A 3-cm segment of ileum wassuspended in 5 ml of Tyrode's solution at 32°C. An amountof kininogen sample estimated to release 30 ng of lysyl-bradykinin (kallidin) was added to the bath followed by 10,lp of partially purified human urinary kallikrein (10 tosylL-arginine methyl ester U/ml) prepared by the method ofPierce and Guimaraes (10). The magnitude of isotonic con-traction of ileum, loaded with 1 g of tension, caused bythe kallidin released from kininogen, was measured with adisplacement transducer (Narco Bio-Systems, Inc., Houston,Tex.). Results were quantified with a standard curve of ilealcontractions elicited by known quantities of a standard solu-tion of kallidin (Schwarz/Mann Div, Becton, Dickinson &Co., Orangeburg, N. Y.).

The ability of our HMWkininogen preparation to cor-rect the abnormality in activated partial thromboplastin timeobserved in HMWkininogen-deficient plasma was examinedwith a modification of the semiquantitative functional assayinitially described by Lacombe et al. (16). A test tube thatcontained 0.10 ml of HMWkininogen-deficient plasma(George King Bio-Medical, Inc., Salem, N. H.), 0.05 ml ofsample, and 0.10 ml of kaolin 10 mg/ml Thrombofax

'Abbreviations used in this paper: ev/iv, extravascular/intravascular pool ratio(s); Fc, mean catabolic rate(s); Fe, i,return flow; Fi, e, capillary transfer rate; HMW,high molec-ular weight; LMW, low molecular weight.

2 Pierce, J. V. Manuscript in preparation.

46 T. Yamada, D. A. Wing, J. V. Pierce, and G. W. Pettit

300 -

200 -

0.0

x

I

0.2 0.4

KININOGEN (/1g / TEST)

FIGURE 2 Correction of prolonged activated Iboplastin time in HMWkininogen-deficient paddition of HMWkininogen (x - - - x) ankininogen (0 0).

(Ortho Diagnostics, Inc., Raritan, N. J.) was inmin at 37°C. 0.05 ml of 0.04 M CaCl2 was theclotting time was measured in a fibrometer (Bi(& Falcon Products, Becton, Dickinson & Co.,Md.). Results are depicted in Fig. 2.

Iodination of kininogens. Proteins for kineti(radiolabeled with 1251 with the microdiffusioGruber and Wright (17). A 50-ml Ehrlenme)modified to contain an inner chamber by Ex 2.3-cm plastic vial to its bottom. 1 ml of ation at an approximate concentration of 1 mg/nfrom the extirittion coefficients of the variou:was placed in the outer chamber of the flask,;0.002 M KI and 1 mCi of Na[P25I] (50 mCi/ml, rNuclear, Boston, Mass.) were placed intichamber. The flask was sealed with a rubhvaccine stopper and 0.2 ml of a 1:20 dilutioacid-dichromate solution (0.27 M Na2Cr2O7 inwas added to the inner chamber with a syrin4-inch 20-gauge needle. The flask was rotat25°C so that no mixing between the two ccurred. After 1 h, the radiolabeled protein was resyringe through a 4-inch needle, and separalreacted iodine by gel filtration on a 1.5- x 5.5-Sephadex G-25 (Pharmacia Fine Chemicals, DivInc., Piscataway, N. J.). Efficiency of labelitechnique was generally low (4.6-9.7%), but thretained practically all of their kinin-genera(96.7-99.2%) after iodination. The functionaHMWkininogen in correcting the coagulationof HMWkininogen-deficient plasma was esaltered (Fig. 2).

Animal studies. Adult male rhesus monkimulatta), which weighed 3.5-6.0 kg, werebefore injection of radiolabeled kininogen, ewas administered 1 ml of a saturated solutionfor the duration of the study, all fluid intaketo a solution that contained 0.02% Nal. Duririnjection of kininogen, and for each subsesampling, the monkeys were anesthetized wiHCl (10 mg/kg, i.m.). A preparation of iodinate

in an amount not exceeding 5% of the total blood contentof the kininogen species injected, as estimated from therelative amounts of each specie purified from plasma, wasadministered to each animal via the saphenous vein. 2min after injection, a 1.8-ml blood sample was withdrawnfrom a femoral vein into a siliconized glass, 2-ml Vacu-tainer tube (Becton, Dickinson & Co., Rutherford, N. J.)which contained 0.2 ml of 3.8% sodium citrate, 0.1% hexadi-methrine bromide, and 0.01 M benzamidine, and this wasconsidered the 0 time sample. Subsequent samples werewithdrawn at 1-4, 6, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, and48 h after injection of radiolabeled kininogen.

From each 2-ml sample, 0.4 ml of whole blood was re-moved and stored at -70°C. The remaining blood wascentrifuged at 25°C for 10 min at 2,000 g, and 0.4 ml ofplasma supernate was removed and stored at -70°C. Asecond 0.4-ml aliquot of plasma was mixed with 0.4 ml of

-1-,r--r-,-1 0.2 M Tris-acetate, 0.01 M EDTA buffer, pH 6.0, and 0.10.6 0.8 1.0 ml of antibody against human LMWkininogen. The mix-

ture was incubated at 4°C for 16 h, then centrifuged at2,000 g for 10 min at 4°C. The resulting supernatant solu-

partial throm- tion, and three subsequent washes of the precipitate withlasma by the 1 ml of 0.2 M Tris, 0.1 M EDTA buffer, pH 6.4, wered [125I]HMW poured over a 0.22-,m Millipore filter (Millipore Corp.,

Bedford, Mass.). The filter and the remaining precipitatewere placed in a single tube and stored at -70°C. At theend of each 48-h study, all the stored preparations were

cubated for 8 counted in a gammacounter (model 1185, Searle Diagnostics,n added, and Inc., subsid. of G. D. Searle & Co., Des Plaines, Ill.).o Quest, BBL The injection schedule of radiolabeled kininogens was asCockeysville, follows: 6 monkeys received human prep B (a mixture of HMW

and LMWkininogens), 6 received human HMWkininogen, 4cstudies were received human LMWkininogen, 4 received human B3a,3n method of 4 received human B4y, 4 received monkey HMWkinino-yer flask was gen, and 4 received monkey LMWkininogen.gluing a 1.3- Data analysis. Turnover of kininogen was assessed withprotein solu- the plasma slope method described in the multiple pipe-

nl, calculated line model for interstitial albumin distribution by Reevekininogens, and Bailey (18). The radioactivity of each sample with-

and 0.2 ml of drawn from the monkeys, expressed as a percentage of theNew England radioactivity of the initial (time 0) sample for each monkey,o the inner was plotted on a logarithmic scale against time after injec-er skirt-type tion of radiolabeled kininogen. In each case, the disap-n of a stock pearance curve of injected kininogen from plasma could be36 N H2SO4) expressed as a double exponential equation, Y = C,e-atge through a + C2e-bt, where Y represents percent of initial counts perLed gently at minute, t represents time, Cl and C2 represent the constants~hambers oc- of the two exponentials, and a and b are the rate constants.moved with a The equation that best fits the data for each test wasted from un- calculated by linear regression analysis using the Simula-cm column of tion Analysis and Modeling (SAAM) program of Bermanof Pharmacia and Weiss (19) in a Univac 1108 computer (Sperry Univac,ng with this Blue Bell, Pa.) at the National Bureau of Standards.ie kininogens Correlation coefficients for the multiple regressions wereating activity calculated with the method described by Dixon andl activity of Massey (20).

abnormality In the double exponential model for fibrinogen turn-sentially un- over, the first, rapid exponential is hypothesized to repre-

sent initial ditiusion of labeled protein from the intra-eys (Macaca to the extravascular pool. The second, slower exponential

used. 24 h is thought to represent disappearance of fibrinogen from,ach monkey the intravascular pool as a result of catabolism. Assuming aof KI orally; similar hypothesis for kininogen, we used the followingwas limited

ng the initialquent blood 3The A280 peak B3 actually consisted of two subpeaks,ith ketamine- called B3.1 and B3.2. The B3a used for our studies wasd kininogen, B3.2a.

Turnover of Plasma Kininogens 47

O - 7

equations as derived by Regoeczi (21) to calculate meancatabolic rate(s) (Fc), which represents the fraction ofintravascular-injected labeled protein catabolized in 24h;capillary transfer rate (Fi, e), which represents the frac-tion of intravascular protein transferred to the extravascularpool in 24 h; return flow (Fe, i), which represents thefraction of extravascular protein returned to the intra-vascular pool in 24 h; and extravascular/intravascular poolratio(s) (ev/iv), which represents the ratio between extra-and intravascular pool size:

Fc = x 24 d-1Cl/a +C2/b

Fi, e = (Cla + C2b) x 24 - Fc d-l,

Fe, i = (a + b) x 24 - (Fc + Fi, e) d-1,

Fi eev/iv= 'Fe, i

C1 and C2, as before, represent the constants for the twoexponentials and a and b represent the rate constants.Biologic t112 was calculated with the standard equation:t1/2= ln 2/b. The rate constant of the second exponential(b) which expresses the rate of catabolism was used toobtain t112. All statistical comparisons of data were madewith Student's t test.

RESULTS

The disappearance curves of radiolabeled humankininogens from plasma of rhesus monkeys could beexpressed as double exponential equations (Fig. 3).Like fibrinogen (22, 23), kininogen had a highmetabolic rate, and first-order diffusion and cataboliccharacteristics. HMWand LMWkininogens hadsimilar configurations for their turnover curves, butconsiderable differences in their kinetics existed. Asexpected, prep B, a mixture of HMWand LMWkininogens, appeared to share characteristics of bothforms of kininogens. A comparison of kinetic param-eters between HMWand LMWkininogens is depictedin Table I. Plasma tl12 of HMWkininogens was25.95±+1.60 h (mean± SEM), while that of LMWkinino-gen was 18.94±1.93 h; a difference that was onlymarginally significant (P < 0.05). On the other hand, ahighly significant difference (P < 0.001) existed be-tween their Fc. Because plasma t1/2 is a measure ofcatabolism of only the kininogen in the intravascularpool, and Fc is dependent upon both intra- and extra-vascular pools of kininogen, it follows that the ob-served differences between HMWand LMWkinino-gens result from differences either in intravascular-extravascular flux or in pool size. A significant differ-ence in ev/iv existed, with HMWkininogen being par-titioned primarily into the intravascular pool, and LMWkininogen being more equally distributed betweenthe two pools. Although Fi, e of HMWkininogen wasslower than that of LMWkininogen, the differencewas not significant. No significant difference was foundin Fe, i for the two kininogens.

20

a.E

10--j

z

e 5-05lt -0.0291tprep B U- y 48.3e.05111 51.20

Ti 24.24 h

-0.524t -0.0272tHMW KININOGEN 0--- y=43.20 * 56.20

Ti=25.95 h

LMW KININOGEN A- -- y-611e0 65+385e5O375t

Tia18.94 h

0 10 20 30 40 50

TIME (HOURS)

FIGuRE 3 Turnover curves for human HMW,LMW, andmixed (prep B) kininogens. Radiolabeled proteins wereadministered intravenously to rhesus monkeys, and theirdisappearance from the plasma on a log scale was plottedagainst time. The points represent mean+SE of 6, 4, and 6studies for kininogens of high, low, and mixed molecularweight, respectively. The equations that best fit the datapoints and the plasma t1,2 for each of the kininogens aredepicted at the bottom.

The adequacy of the model for describing the sys-tem with double exponential equations was testedby two methods. First, the sum of the constants forthe two exponentials (C1 and C2) of each of the disap-pearance curves was measured. Theoretically, thisvalue should approach 100% if there are no additionalexponentials required for proper fit of the disap-pearance curves to the calculated equations. Theresults shown in Table II indicate that two exponentialsare all that are required to provide a good fit for theturnover curves. The second method used to evaluateadequacy of fit was simple calculation of correlation

48 T. Yamada, D. A. Wing, J. V. Pierce, and G. W. Pettit

TABLE IComparison of Kinetic Parameters for Disappearance of Radiolabeled HMWvs.

LMWKininogens from Plasma*

Kininogen t112 Fc Fi, e Fe, i ev/iv

HMW 25.95+1.60 1.12+0.08 4.73+1.02 7.20±1.02 0.63+0.05LMW 18.94+1.93 2.07+0.09 7.40+2.64 7.11±2.49 1.05+0.07

P <0.05 <0.001 NS NS <0.01

* Values given are mean+SE.

coefficients for multiple regressions. Correlation(r) values for the double exponentials were in excessof 0.9900 for all of the studies (Table III). Equallynotable was the absence of any improvement in fitof the curves to triple exponential equations.

Measurement of turnover by following the disap-pearance of plasma radioactivity was compared withthe disappearance of whole blood radioactivity andantibody-precipitated kininogen antigen radioactivity.Less than 10% of total radioactivity remained inthe supernate after immunoprecipitation of kininogen.Turnover curves obtained by measuring whole bloodand immunoprecipitated kininogen radioactivity werenot significantly different from plasma studies, norwere they significantly different from each other.This was true for all forms of kininogen tested.

Because so-called HMWkininogen and LMWkininogen are each mixtures of kininogens of variousmolecular weights, their turnover curves were com-pared with those of the pure 80,000 and 225,000molecular weight proteins, B3a and B4y. As shown inFig. 4, the turnover curves for B3a and B4y were notsignificantly different from their counterparts of lesserpurity, which indicates that the heterogeneous com-position of our HMWand LMWkininogen prepara-tions did not alter the basic kinetic characteristicsof their respective major components.

TABLE IISum of the Constants (C1 + C2) of the Two Exponentials

Describing the Disappearance of LabeledKininogen from Plasma

Prep B HMWkininogen LMWkininogenStudy Cl + C, C, + C, Cl + C,

1 99.2 97.5 99.62 98.5 97.6 99.83 100.0 99.9 100.04 99.9 100.0 99.95 100.0 100.06 100.0 99.8

Mean±SD 99.6+0.6 99.1±1.2 99.6+0.5

The data discussed have referred exclusively toturnover of human kininogens. Because of the phylo-genetic similarity between monkeys and humans,and because of the identity in the measured bio-chemical and antigenic characteristics of their kinino-gens, we assumed that human kininogens wouldhave similar turnover kinetics compared with mon-key kininogens when studied in rhesus monkeys.The accuracy of this assumption was tested and ourresults are depicted in Fig. 5. The disappearancecurves for radiolabeled monkey HMWand LMWkininogens fell within the 95% confidence limits of thecurves for their corresponding human kininogens.While there was no difference in their plasma t1/2,Fc of monkey LMWkininogen, 1.10+0.14 d-l wassignificantly lower (P < 0.001) than that of humanLMWkininogen, 2.07+0.09 d-1. There were no signifi-cant differences in Fi, e or in Fe, i to explain thelower catabolic rate, but the ev/iv for monkey LMWkininogen, 0.64+0.03, was significantly smaller (P< 0.01) than that for human LMWkininogen, 1.052+0.068.

DISCUSSION

Wehave characterized the normal turnover of kinino-gens by measuring their disappearance from the plasma

TABLE IIICorrelation Coefficients (r) Obtained by Fitting

Disappearance of Labeled Kininogen to2 vs. 3 Exponentials

Prep B HMWkininogen LMWkininogen

Study 2 exp* 3 exp 2 exp 3 exp 2 exp 3 exp

r r r r r r

1 0.9961 0.9963 0.9506 0.9915 0.9963 0.99672 0.9950 0.9951 0.9949 0.9953 0.9953 0.99623 0.9999 0.9999 0.9997 0.9998 O.9998 0.99984 0.9988 0.9998 0.9995 0.9996 0.9997 0.99985 0.9996 0.9999 0.9989 0.99896 0.9996 0.9998 0.9985 0.9985

* Exponentials.

Turnover of Plasma Kininogens 49

0 10 20 30 40

TIME (HOURS)

FIGuRE 4 Comparison of disappearance curves for radio-labeled B4y vs. HMWkininogen (A), and B3a vs. LMWkininogen (B). The 95% confidence limits for the turnovercurves of HMWand LMWkininogens are represented bythe shaded areas, and the circles (0) represent mean±SEof 4 studies each for B4y and B3a.

of rhesus monkeys after bolus injection. The relativelyrapid metabolic rate observed for kininogens indi-cated that kinetic analysis by the plasma slope methodwould be appropriate for the study of their turnover.Because terminal hydrolysis products of radioiodinatedproteins are excreted at finite rates, methods of pro-tein turnover analysis which involve measurement ofurine radioactivity (24) can be inaccurate in the caseof rapidly metabolized proteins (23). An inevitablelag period between catabolism and excretion exists forany protein; this lag period is short in the case ofslowly metabolized proteins, but can be significantin rapidly metabolized proteins, and can result in a

100 MONKEY HMWKININOGEN MONKEY LMW KININOGEN

80

60-

40-

_20

Z,

220 30 40 50

FIGURE 5 Comparison of turnover curves for monkey HMW(A) and LMW(B) kininogens vs. their corresponding hu-man proteins. The 95% confidence limits for the turnovercurves of human HMWand LMWkininogens are repre-sented by the shaded areas, and the circles (0) representmean+SE of 4 studies each for monkey HMWand LMWkininogens.

disequilibrium between catabolism and excretion.Such circumstances could create the erroneous appear-ance of a site of extravascular protein sequestrationbefore catabolism. In addition to theoretical reasonsfor avoiding urine radioactivity measurements, thetechnical problem of obtaining accurate urine samplesfrom monkeys without surgical intervention wasvirtually insurmountable.

Our results indicated that turnover of plasmakininogens conformed to double exponential equa-tions. In measuring turnover by the plasma slopemethod, we assumed a simple system consisting oftwo pools: the intravascular pool into which radio-labeled kininogen was injected, and the single extra-vascular pool into which the injected kininogendiffused, or was catabolized. As we did not attemptcomplicated multicompartmental modeling analysisduring our present study, the pools did not representspecific anatomic compartments, but were poolsonly in a kinetic sense. According to our assump-tion, kininogen could exit from the intravascularpool by only two processes: diffusion or catabolism.If our double exponential hypothesis were correct,the sum of the constants for the fraction of injectedprotein that diffuses (Cl) and the fraction that iscatabolized (C2) should approximate 100%. As our dataindicated, the average values for C1 + C2 were closeto the predicted value, which suggests that therewere no overlooked components to the equation de-scribing kininogen turnover in plasma. Weconfirmedthis observation by testing the fit of our data totriple exponentials, and observed no notable improve-ment in correlation coefficient. These results are incontradistinction to those observed in studies offibrinogen turnover which, although said to conformto double exponential equations, indicate the pres-ence of a small (2-3%) third component in theirturnover curves (23).

Analysis of our kininogen turnover curves wassimplified, and accuracy was improved considerablywith the SAAMcomputer program. Without the pro-gram, the constants for the double exponential equa-tions would have had to be estimated by graphicmeans. By extrapolation of the terminal portion ofthe turnover curves to time 0, the y-intercept (C2) andslope (-b) of the second exponential could be ob-tained, and by subtraction of the second exponentialfrom the total curve, the constants (Cl and -a) forthe first exponential could be calculated. With theSAAMprogram, we were able to perform iterationsof the data to obtain constants for the curve thatprovided the best fit of our data to more than twoexponentials without difficulty.

To follow the disappearance of radiolabeled kinino-gen from the intravascular pool, we compared measure-ments of whole blood, plasma, and antibody-precipi-

50 T. Yamada, D. A. Wing, J. V. Pierce, and G. W. Pettit

n

-i

0 10 20 30 40 50

A LB

0 10 20 30 40 50 O I1TIME (HOUR)

tated kininogen radioactivities. There were no differ-ences in turnover curves obtained with any of thethree methods of analysis, although the last methodwas clearly the most specific. We were not able tomeasure the radioactivity of functionally activekininogen as opposed to kininogen antigen. In studiesof fibrinogen turnover, this problem is not en-countered because plasma samples can be clottedand the radioactivity of the fibrin contained withinthe clots counted. However, fibrinogen turnover curvesobtained by measuring plasma radioactivity werenearly identical to those obtained by measuringradioactivity of clottable protein (25). While we cannotexclude the possibility that kininogen may continueto circulate in the intravascular pool as measurableantigen even after partial catabolism, it is unlikely,as a common pathway for diffusion and catabolism issuggested by the observation that the catabolicpattern for kininogen was superimposed by a similarkinetic for its diffusion.

The differences we observed between the kineticsof HMWand LMWkininogens cannot be explainedsimply on the basis of molecular size, becausealbumin, with a molecular weight less than that ofLMWkininogen, has a tl12 nearly four times as long(20). The differences in Fc of the two kininogenswere attributable primarily to differences in theirev/iv. It is tempting to speculate about the functionsof the two kininogens in relation to their pool distri-bution, but we have little data with which to supportsuch conjectures. However, the importance of HMWkininogen in the coagulation process has been welldocumented (26-29), although the specific in vivofunctions of the various kininogens are otherwiseunknown. In addition, Pierce and Guimaraes (10) aswell as Habal et al. (30) have corroborated earlierobservations by Jacobsen and Kriz (31) that HMWkininogen is the preferred substrate for plasma kalli-krein. These two observations are consistent with thepredominant intravascular distribution of HMWkininogen, but do not shed further light on the rela-tionship between distribution and function of thekininogens.

Turnover of rhesus monkey kininogens was similarto that of human kininogens when measured in mon-keys. This was to be expected in view of the simi-larities between the kininogens of the two species.However, Regoeczi has shown that in rabbits, humanfibrinogen disappeared at 1.45 times the rate ofhomologous fibrinogen before immunologic clearancemechanisms were activated (32). Similar studies inmonkeys that were injected with human fibrinogenshowed its clearance rate to be 1.3 times faster thanthat of the homologous fibrinogen, despite the absenceof any detectable immune response (23). The differ-ence in clearance rates was attributable to differences

in diffusion rates as well as in ev/iv. Wedid not observeany significant differences in plasma tl/2 between mon-key and human kininogens, but the former had aslower catabolic rate than its human counterpart,and this difference was apparently related to theformer's significantly greater distribution into theintravascular pool. The differences in ev/iv betweenthe heterologous and homologous LMWkininogensmay reflect differences in their functional charac-teristics in monkeys.

In their description of a patient with Flaujeacfactor deficiency (16), a disease later shown byWuepper et al. to be caused by deficiency in HMWkininogen (27), Lacombe et al. reported that the func-tional abnormality in coagulation observed in theirpatient could be corrected in vivo by the infusion offresh frozen plasma. The factor(s) in fresh frozenplasma which corrected the functional deficiency hada t1/2 of 6.5 days. Although this observation is ap-parently at variance with our t1/2 for HMWkininogenof 25.95 h, several important differences betweenLacombe's study and ours must be considered: (a)their study reports in vivo functional t1/2, whereasour t1/2 was derived from plasma disappearancecurves; (b) the exact substance of which t1/2 was in-ferred in Lacombe's study is unclear, because theyused fresh frozen plasma for infusion rather thanpurified HMWkininogen; (c) their t1/2 curve wasdrawn from three points in a single patient after oneinfusion of fresh frozen plasma; and (d) a speciesdifference in metabolic turnover of HMWkininogencannot be excluded.

Because of their unique and potent physiologicactions, the kinins have been implicated in the patho-physiology of a number of severe clinical illnesses.Evidence of kinin system activation in these illnesses,however, has been inferential for the most part, be-cause turnover of the proteins involved has notbeen evaluated. We have studied the syndrome ofdisseminated intravascular coagulation in Salmonellatyphimurium-infected rhesus monkeys and, as inother illnesses with suspected kinin system partici-pation, our evidence of kinin activation has beenlimited to the indirect observations of elevatedkallikrein and diminished prekallikrein and kininogen(33). Weare presently attempting to clarify the associa-tion between kinins and clinical illness by directstudy of kininogen turnover in our infected monkeys,and by comparing with the normal studies presentlydiscussed. In this fashion, we hope to be able todocument the involvement of the kinin system in dis-seminated intravascular coagulation and to evaluatevarious potential therapeutic measures in relation totheir effect on the kinin system. The technique formeasuring kininogen turnover should also be ap-plicable to the study of other illnesses in which the

Turnover of Plasma Kininogens 51

kinin system is thought to play an important patho-physiologic role.

ACKNOWLEDGMENTSThe authors wish to express their appreciation to Dr. J.David Gangemi, Mr. Edgar W. Larson, and Dr. William R.Beisel for their helpful criticism; Mrs. Phebe W. Summersfor her editorial help; and Messrs. Glen A. Higbee andDwayne D. Oland for their help with the computeranalysis of our data. Wegratefully acknowledge the techni-cal assistance of Dr. Moacir de Sa Pereira; Messrs. CharlesK. Ambrose and Steven A. Tobery; and Ms. CathleenSchmidt.

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52 T. Yamada, D. A. Wing, J. V. Pierce, and G. W. Pettit