Proc. Nati. Acad. Sci. USAVol. 73, No. 11, pp. 4179-4183, November 1976Immunology

Identification of prekallikrein and high-molecular-weight kininogenas a complex in human plasma

(Hageman factor/bradykinin generation/contact activation)

ROBERT J. MANDLE*, ROBERT W. COLMANt, AND ALLEN P. KAPLAN*** Allergic Diseases Section, Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda,Maryland 20014; and t The Coagulation Unit of the Hematology-Oncology Section, Department of Medicine, University of Pennsylvania, Philadelphia, Pa.19104

Communicated by K. Frank Austen, August 19, 1976

ABSTRACT Prekallikrein and high-molecular-weight ki-ninogen were found associated in normal human plasma at amolecular weight of 285,000, as assessed by gel filtration onSephadex G-200. The molecular weight of prekallikrein inplasma that is deficient in high-molecular-weight kininogen was115,000. This prekallikrein could be isolated at a molecularweight of 285,000 after plasma deficient in high-molecular-weight kininogen was combined with plasma that is congeni-tally deficient in prekallikrein. Addition of purified 125I-labeledprekallikrein and high-molecular-weight kininogen to the re-spective deficient plasma yielded a shift in the molecular weightof prekallikrein, and complex formation could be demonstratedby incubating prekallikrein with high-molecular weight kini-nogen. This study demonstrates that prekallikrein and high-molecular-weight kininogen are physically associated in plasmaas a noncovalently linked complex and may therefore be ad-sorbed together during surface activation of Hageman factor.The complex is disrupted when these proteins are isolated byion exchange chromatography.

Activation of Hageman factor upon a negatively charged sur-face initiates the intrinsic coagulation pathway, the fibrinolyticpathway, and the generation of the vasoactive peptide bra-dykinin. Recent investigations from several laboratories haveshown that the proteins of the kinin-forming pathway, namely,prekallikrein (1, 2) and high-molecular-weight (HMW) kini-nogen (3-5), are both required for optimal activation andfunction of Hageman factor. Since prekallikrein and HMWkininogen are intimately associated functionally, it appearedpossible that they might be physically associated in plasma. Inthis paper we demonstrate that prekallikrein and HMW kini-nogen circulate in plasma as a noncovalently linked complex.Formation of this complex was observed: (a) when prekalli-krein-deficient plasma was combined with plasma deficientin HMW kininogen, (b) after prekallikrein-deficient plasmaand plasma deficient in HMW kininogen were reconstitutedwith prekallikrein and HMW kininogen, respectively, and (c)when purified prekallikrein was incubated with HMW kini-nogen.

MATERIALS AND METHODSApoferritin and catalase (Calbiochem, San Diego, Calif.), bluedextran, ovalbumin, ribonuclease A, and chymotrypsinogen(Pharmacia Fine Chemicals, Inc., Piscataway, N.J.), chloramineT (Matheson, Coleman and Bell Co., Norwood, Ohio), sodi-um[125I] iodide (New England Nuclear Co., Boston, Mass.), andhuman transferrin (Sigma Chemical Co., St. Louis, Mo.) wereobtained as indicated. Plasma deficient in Hageman factor andcontaining 0.38% sodium citrate was obtained from Sera TecBiologicals, New Brunswick, N.J. Prekallikrein-deficient plasma

(Fletcher trait) was a gift from Dr. C. Abildgaard (Universityof California, Davis, Calif.). Kininogen-deficient plasma wasobtained from Ms. Williams and was collected as described(3).

Preparation of Plasma Proteins. Fresh plasma used for theisolation of prekallikrein and HMW kininogen was collectedin 0.38% sodium citrate. Hexadimethrine bromide (3.6 mg) in0.1 ml of 0.15 M saline was added for each 10 ml of blooddrawn. The tubes were then centrifuged at 900 X g for 20 minat 40 and the plasma was separated with plastic pipettes. Plasticcolumns and test tubes were used throughout the chromato-graphic procedures to minimize contact activation of Hagemanfactor and nonspecific adsorption to glass surfaces. Sampleswere concentrated by ultrafiltration (Amicon Corp., Lexington,Mass.) through a UM-10 membrane.Hageman Factor Fragments. Prealbumin fragments of

Hageman factor were purified by chromatography of plasmaon QAE-Sephadex twice, Sephadex G-100, and SP-Sephadex,and elution from alkaline disc gels after electrophoresis as re-ported (6).

Prekallikrein. Two liters of fresh plasma were dialyzedagainst 0.003 M phosphate buffer (pH 8.3) and passed over a20 X 100 cm column of QAE-Sephadex equilibrated with thesame buffer. The effluent, containing a mixture of proteins ofgamma globulin mobility, was then fractionated by sequentialchromatography on SP-Sephadex and Sephadex G-150, as de-scribed (7), followed by passage over an immunoadsorbentprepared with antisera to human IgG and ,B2 glycoprotein I.

For preparation of the immunoadsorbent, 100 ml of sheepantiserum to human IgG and 132 glycoprotein I was made 45%in ammonium sulfate and stirred at 24° for 1 hr. The mixturewas centrifuged at 1200 X g for 90 min at 40, and the precipi-tate was washed twice in 45% ammonium sulfate. The washedprecipitate was dissolved in distilled water, dialyzed againstthree changes of 10 liters of 0.003 M phosphate buffer-0. 15 MNaCl (pH 7.5) for 24 hr at 40, and coupled to 250 ml of Seph-arose 4B by the cyanogen bromide method (8). The Sepharosewas poured into a 5 X 20 cm plastic column, washed with onecolumn volume of 1.0% ethanolamine, and then equilibratedin 0.003 M phosphate buffer-0.35 M NaCl (pH 8.0). Sampleswere dialyzed against this buffer prior to application to theimmunoadsorbant.HMW Kininogen. HMW kininogen was isolated by chro-

matography of normal human plasma on QAE-Sephadex A-50,ammonium sulfate precipitation, fractionation on SP-Sephadex,and Sephadex G-200 gel filtration according to the method ofHabal et al. (9). The preparation was quantitated functionallyby its ability to selectively correct the coagulation defect inWilliams trait plasma as described (3). It was free of any de-tectable Hageman factor or prekallikrein.The protein content of samples was approximated by ab-

4179

Abbreviation: HMW kininogen, high-molecular-weight kininogen.f To whom reprint requests should be addressed.

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sorbance at 280 nm with Al%m assumed to equal 10, or wasdetermined by the Lowry method (10); the color reaction wasread at an optical density of 700 nm in a Beckman spectro-photometer. Gel filtration on Sephadex G-150 (7), alkaline discgel electrophoresis (11), and sodium dodecyl sulfate gel elec-trophoresis (12) were performed as described. Prekallikrein wasradioiodinated by the Chloramine T method (13) using sodium[125I]iodide. The iodinated protein was immediately fraction-ated on a 2 X 100 cm column of Sephadex G-50 equilibratedin 0.003 M phosphate buffer-0.15 M NaCl (pH 8.0). The initialpeak of radioactivity was completely separated from the peakof free iodine and contained over 99% trichloroacetic acid-precipitable counts. The 125I-labeled prekallikrein was mixedwith a 100-fold excess of nonradiolabeled prekallikrein in orderto assess binding to HMW kininogen. Radioactivity was de-termined in a sodium iodide well scintillation counter (NuclearChicago-Searle, Des Plaines, Ill., model 1185) with automaticsubtraction of background and an efficiency of 82%.

Coagulation Assays. The partial thromboplastin time wasmeasured by the method of Proctor and Rapaport (14). Hage-man factor, prekallikrein, and HMW kininogen were deter-mined by a modification of the procedure for determiningpartial thromboplastin time, with congenitally deficient plasma(3).Assays of Kinin-Forming Proteins. The proteolytic activity

of kallikrein was routinely measured by its ability to releasebradykinin from heat-inactivated plasma (11). Twenty-fivemicroliters of kallikrein source were incubated with 0.2 ml ofsubstrate for 2 min of 370 and the bradykinin generated wasquantitated by bioassay as described (11).

Prekallikrein was determined by incubation of 25 ,l ofproenzyme source with 25 ,ul of Hageman factor fragments (25jig/ml) for 5 min at 370 and the kallikrein generated was de-termined.

Unactivated Hageman factor was assayed for its kinin-gen-erating capacity by adding 10 ,l of sample to 200 ,g of plasmadeficient in Hageman factor containing 0.9 mg/ml of EDTA.Fifty microliters of a suspension of kaolin in 0.15 M NaCl (10mg/ml) were added, the mixture was incubated for 2 min at370C and applied to the bioassay. Incubation of the kaolinsuspension with the plasma deficient in Hageman factor gen-erated no detectable bradykinin. Activated Hageman factorwas assayed in the same manner, with 0.15 M NaCl in place ofthe kaolin suspension.

Fibrinolytic Assays. Plasminogen, plasmin, and plasminogenproactivator were assayed as described (15).

Sephadex G-200 Gel Filtration of Normal and DeficientPlasmas. A 2.6 X 95 cm Pharmacia K26/100 column of Se-phadex G-200 was equilibrated with 0.01 M Tris-HOl buffer(pH 7.0) made 0.15 M in NaCl and 10-1M in EDTA. The col-umn was run at either 40 or 370, the elution rate with upwardflow was 10 ml/hr, 4-ml samples were applied, and 3.1-mlfractions were collected. Molecular weight was determined bygel filtration according to the method of Andrews (16). Thestandards used and their molecular weights by gel filtrationwere apoferritin (460,000), catalase (195,000), bovine serumalbumin (65,000), and chymotrypsinogen (25,000).

RESULTS

Four milliliters of normal plasma were fractionated on a 2.6 X95 cm column of Sephadex G-200 at 40 and the fractions wereassayed for prekallikrein, HMW kininogen, and unactivatedHageman factor by their ability to correct the partial throm-boplastin time of congenitally deficient plasma. As shown in

E

8'IC

0.70.60.50.40.30.20.1

0.70.60.50.40.30.20.1

50 60 70 80 90 100 110 120

TUBE NO.

Ew

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0co

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'S0.

FIG. 1. Sephadex G-200 gel filtration of normal human plasma(upper panel) and plasma deficient in Hageman factor (lower panel).The column fractions were assayed for prekallikrein (A), HMW ki-ninogen (o), and unactivated Hageman factor (0): Protein contentwas estimated by the Lowry method and the color reaction was readat 700 nm (0).

the upper panel of Fig. 1, the prekallikrein andHMW kinino-gen eluted in the same fractions, between the first and secondprotein peaks at an approximate molecular weight of 285,000,while unactivated Hageman factor was eluted between thesecond and third protein peaks at a molecular weight of110,000-120,000. When plasma deficient in Hageman factorwas fractionated in an identical fashion (lower panel, Fig. 1),prekallikrein and HMW kininogen were again found togetherat a molecular weight of 285,000. The elution position of pre-kallikrein and Hageman factor in the normal plasma chro-matogram was then confirmed, as assessed by their kinin-generating ability. The location of each protein as assessed bykinin generation was identical to its elution profile as assessedby the coagulation assay, and there was no detectable activekallikrein or activated Hageman factor in these fractions.When plasma deficient in HMW kininogen was fractionated

on Sephadex G-200, prekallikrein was found at a molecularweight of 115,000 and overlapped the Hageman factor peak(Fig. 2, upper panel). When prekallikrein-deficient plasma wasfractionated on Sephadex G-200 (Fig. 2, center panel), unac-tivated Hageman factor was found at a molecular weight of115,000, while the HMW kininogen eluted at a molecularweight of 200,000. When equal volumes of HMW kininogen-deficient plasma and prekallikrein-deficient plasma were mixedand 4 ml was fractionated on Sephadex G-200, the prekallikreinwas then found with HMW kininogen at a molecular weightof approximately 285,000 (Fig. 2, lower panel). It appearedpossible that the association of prekallikrein with HMW kini-nogen was a cold (4°)-dependent phenomenon; thereforenormal plasma was centrifuged at 370, immediately appliedto a jacketed Sephadex column, and chromatographed at 370.Prekallikrein was again found in association with HMW kini-nogen. Chromatography of normal plasma at 240 or use ofphosphate rather than Tris buffers did not affect the result.

In Fig. 3 is shown an alkaline disc gel electrophoresis andsodium dodecyl sulfate gel electrophoresis of the preparationof purified human prekallikrein at 1 mg/ml. A single band isobserved in the alkaline disc gel, while a major band with asmall minor band below is observed in the sodium dodecyl

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0.70.60.50.40.30.20.1

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4 .'5 E6 -7w

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FIG. 2. Sephadex G-200 gel filtration of plasma deficient inHMW kininogen (upper panel) prekallikrein-deficient plasma(middle panel), and a mixture of prekallikrein-deficient plasma andplasma deficient in HMW kininogen (lower panel). The columnfractions were assayed for prekallikrein (A), HMW kininogen (o),and unactivated Hageman factor (0).

sulfate gel. The minor band may represent a contaminatingprotein or may be prekallikrein that has been digested duringpurification. Functional assessment of this prekallikrein dem-onstrated that it contained no detectable kallikrein, Hagemanfactor, kininogen, factor XI, or plasminogen. It did, however,contain plasminogen proactivator activity. The molecularweight of the highly purified human prekallikrein as assessedby sodium dodecyl sulfate gel electrophoresis was 104,000, a

value which was similar to the molecular weight of prekallikreinin the plasma deficient in HMW kininogen. These resultssuggested that plasma prekallikrein is normally complexed withHMW kininogen and that this complex is independent of thepresence of unactivated Hageman factor.We next attempted to reconstitute the prekallikrein-deficient

plasma and plasma deficient in HMW kininogen with the re-

spective proteins isolated from normal plasma to determinewhether binding would be observed. Fifty microliters

! w~~~~~~w

FIG. 3. Alkaline disc gel electrophoresis (top) and sodium dodecylsulfate gel electrophoresis (bottom) of 30 jg of purified human pre-

kallikrein.

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50 60 70 80 90 100 110 120TUBE NUMBER

FIG. 4. Sephadex G-200 gel filtration of 12-I-labeled prekallikreinadded to plasma deficient in HMW kininogen (upper panel), Se-phadex G-200 gel filtration of 125I-labeled prekallikrein plus nonra-

diolabeled prekallikrein added to prekallikrein-deficient plasma(center panel), and Sephadex G-200 gel filtration of 125I-labeledprekallikrein and HMW kininogen added to plasma deficient inHMW kininogen. The prekallikrein coagulant activity is shown bythe shaded area.

of 125I-labeled prekallikrein were mixed with 1 ml of plasmadeficient in HMW kininogen and fractionated on SephadexG-200. 25I-labeled prekallikrein eluted as a major peak of ra-

dioactivity at a molecular weight of 115,000, which corre-

sponded to the peak of prekallikrein procoagulant activity (Fig.4, upper panel). The 125I-labeled prekallikrein was next mixedwith an excess of nonradiolabeled prekallikrein. The mixturewas added to 2 ml of prekallikrein-deficient plasma and incu-bated at 24° for 10 min, and the plasma was then fractionatedon Sephadex G-200. Two peaks of radioactivity were obtained;the first peak was eluted at a molecular weight of 285,000, andthe second peak at a molecular weight of 115,000 (Fig. 4, centerpanel). When the column was assayed for prekallikrein by a

coagulation assay, a peak of prekallikrein activity was foundat 285,000 and activity could be detected through the 100,000molecular weight region. One milliliter of plasma deficient inHMW kininogen was next reconstituted with HMW kininogensuch that plasma levels were achieved. Fifty microliters of125I-labeled prekallikrein were added to the plasma; the mixturewas incubated at 240 for 10 min and fractionated on SephadexG-200 (Fig. 4, lower panel). Again, peaks of '25I-labeled pre-

kallikrein were observed at molecular weight 285,000 and115,000, while the prekallikrein in the plasma deficient inHMW kininogen that was previously found at molecular weight115,000 (Fig. 4, upper panel) was now identified at a molecularweight of 285,000.

Finally the mixture of 125I-labeled prekallikrein and non-

radiolabeled prekallikrein was incubated with HMW kininogenfor 10 min at 240 and then fractionated on Sephadex G-200. As

HMW KININOGENDEFICIENT PLASMA

PREKAWKREINDEFICIENT PLASMA

PREKALLIKREIN DEFICIENT PLASMA

HMW KININOGEN DEFICIENT PLASMA

An,~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

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EC000(N4

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,::::::::....t50 60 70 80 90 100 110

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TUBE NUMBER6o

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FIG. 5. Sephadex G-200 gel filtration of '25I-labeled prekallikrein plus nonradiolabeled prekallikrein after incubation with a 10-fold excessofHMW kininogen. The partial thromboplastin time (min) of the prekallikrein added is shown by the shaded area.

shown in Fig. 5, '25I-labeled prekallikrein was found complexedwith the HMW kininogen and a second peak of radioactivitywas also found at molecular weight 115,000. When the columnfractions were assayed for prekallikrein functionally, virtuallyall of the activity was found at a molecular weight of 285,000.HMW kininogen was found functionally from tube 70 to 90;however, a trough separating complexed and uncomplexedkininogen was not clearly distinguished in the presence of excess

kininogen.

DISCUSSION

Colman et al. (3) and Wuepper et al. (4) have recently reportedpatients who possessed unique abnormalities of the Hage-man-factor-dependent pathways. In each instance, the patientwas found to be deficient in kininogen and the factor thatcorrected each of the functional abnormalities was identifiedas a high-molecular-weight form of human kininogen. Twoother patients with the identical functional abnormalities havebeen reported (5, 17), and they have also been found to lackHMW kininogen (5). Patients with Fletcher trait are deficientin prekallikrein (1) and possess a diminished rate of surface-'dependent coagulation and fibrinolysis which has been shownto reflect a diminished rate of formation of activated Hagemanfactor (2). Although kallikrein can be shown to directly cleaveHageman factor in the fluid phase (18, 19), kallikrein does notactivate Hageman factor in human plasma unless a suitablesurface is present (20).

Recent reports by Liu et al. (21), Meier et al. (22), Websteret al. (23), Kaplan et al. (24), and Griffen and Cochrane (25)have shown that HMW kininogen augments the function ofactivated Hageman factor and HMW kininogen is a cofactorrequired for kallikrein to activate surface-bound Hagemanfactor. Thus, it appeared that prekallikrein and HMW kini-nogen act together to yield a normal rate of Hageman factoractivation and HMW kininogen is further necessary for optimalfunction of the activated Hageman factor. We have now shownthat prekallikrein and HMW kininogen normally circulate inplasma as a complex and they may therefore be adsorbed bynegatively charged surfaces together. Unactivated Hagemanfactor was not found associated with this complex. Liu et al. (21)have presented evidence that the effect of HMW kininogenupon.the function of activated Hageman factor is to enhance

the activity of the active site, and a stoichiometric relationshipbetween activated Hageman factor and HMW kininogen hasbeen observed (22, 24, 25). In addition, it is also possible thatHMW kininogen enhances the interaction of Hageman factorwith prekallikrein by sterically positioning the prekallikreinto facilitate its cleavage.

Nagasawa and Nakayasu (26) have previously reported thathuman plasma prekallikrein was found at a molecular weightof approximately 300,000 and first suggested that it must cir-culate complexed to some other protein. We find a complex ofmolecular weight 285,000 containing prekallikrein and HMWkininogen. However, the molecular weight of the complex(285,000) was less than that predicted for the sum of the reac-tants (315,000). It is possible that the apparent molecular weightof the complex or the reactants by gel filtration is different fromits true molecular weight. Habal et al. (27) have reported amolecular weight of 210,000 for their purified HMW kinino-gen, which is similar to the value we find for HMW kininogenin prekallikrein-deficient plasma or partially purified HMWkininogen (200,000). However, they report a molecular weightof only 110,000 for purified HMW kininogen in guanidineSepharose 4B. Thus, HMW kininogen may contain two non-covalently bound subunits of equal size or its molecular weight,as determined by Sephadex G-200 gel filtration, is considerablygreater than its true value.We were able to demonstrate binding of prekallikrein to

HMW kininogen by mixing prekallikrein-deficient plasma andplasma deficient in HMW kininogen or by reconstituting thesedeficient plasmas with purified components. We did not obtaincomplete binding of '25I-labeled prekallikrein in the presenceof an excess of HMW kininogen although most of the nonra-diolabeled prekallikrein added was complexed, as assessed bya functional assay. This may reflect the presence of denaturedprekallikrein in the radiolabeled material and/or the presenceof a nonbinding contaminant having the same molecularweight. The complex of prekallikrein and HMW kininogen isdissociated when plasma is fractionated by ion exchangechromatography, suggesting that the binding is in part attrib-utable to a charge interaction between the alkaline prekallikrein[isoelectric point of 8.75 (7)] with the very acidic HMW kini-nogen [isoelectric point 4.3 (9)]. Divalent cations do not appearto be required for binding since 10-3M EDTA did not alter thechromatographic patterns of the plasma.

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Prekallikrein and HMW kininogen are intimately associatedfunctionally since they react in sequence to liberate the va-soactive peptide bradykinin and react together upon surfacesto enhance the activation and function of Hageman factor.Patients that are deficient in prekallikrein have normal levelsof HMW kininogen. However twobf the patients with HMWkininogen deficiency have diminished levels of circulatingprekallikrein (3, 17). This may reflect a shortened half-life ofprekallikrein when not associated with HMW kininogen or thegenes coding for the synthesis of these proteins may be linked.We have not ruled out the possibility that other proteins mayalso participate in the interaction between prekallikrein andHMW kininogen, although molecular weight considerationssuggest that this is unlikely. Plasma proteins other than pre-kallikrein may also complex with HMW kininogen, as suggestedby the observation that in one of three gel filtrations of pre-kallikrein-deficient plasma, a second HMW kininogen peakat a molecular weight of approximately 400,000 was observed;this will require further investigation.

1. Wuepper, K. D. (1973) J. Exp. Med. 138, 1345-1355.2. Weiss, A. S., Gallin, J. I. & Kaplan, A. P. (1974) J. Clin. Invest.

53,622-633.3. Colman, R. W., Bagdasarian, A., Talamo, R. C., Scott, C. F.,

Seavey, M., Guimaraes, J. A., Pierce, J. V. & Kaplan, A. P. (1975)J. Clin. Invest. 56,1650-1662.

4. Wuepper, K. D., Miller, K. R. & LaCombe, M. J. (1975) J. Clin.Invest. 56, 1663-1672.

5. Donaldson, V. H., Glueck, H. I., Miller, M. A., Movat, H. Z. &Habal, F. (1976) J. Lab. Clin. Med. 87,327-337.

6. Kaplan, A. P., Spragg, J. S. & Austen, K. F. (1971) in Second In-ternational Symposium, Biochemistry of the Acute AllergicReactions, eds. Austen, K. F. & Becker, E. L. (Blackwell ScientificPublications, Ltd., Oxford, England), pp. 279-278.

7. Kaplan, A. P., Kay, A. B. & Austen, K. F. (1972) J. Exp. Med. 135,81-97.

8. Axen, R., Dorath, J. & Ernback, S. (1967) Nature 214, 1302-1330.

9. Habal, F. M., Movat, H. Z. & Burrowes, C. E. (1974) Biochem.Pharmacol. 23, 2291-2303.

10. Lowry, 0. H., Rosebrough, N. J., Farr, A. C. & Randall, R. J.(1951) J. Biol. Chem. 193,265-275.

11. Kaplan, A. P. & Austen, K. F. (1970) J. Immunol. 105, 802-811.

12. Weber, K. & Osborn, M. (1969) J. Biol. Chem. 244, 4406-4412.

13. Greenwood, F. C., Hunter, W. M. & Glover, J. S. (1963) Biochem.J. 89, 114-123.

14. Proctor, R. R. & Rapaport, S. I. (1961) Am. J. CGn. Pathol. 35,212-219.

15. Kaplan, A. P. & Austen, K. F. (1972) J. Exp. Med. 136, 1378-1393.

16. Andrews, P. (1965) Biochem. J. 96,595-606.17. Saito, H., Ratnoff, 0. D., Waldmann, R. & Abraham, J. P. (1975)

J. Clin. Invest. 55,1082-1084.18. Cochrane, C. G., Revak, S. D. & Wuepper, K. D. (1973) J. Exp.

Med. 138, 1564-1583.19. Bagdasarian, A., Lahiri, B. & Colman, R. W. (1973) J. Biol. Chem.

248,7742-7747.20. Kaplan;A. P., Meier, H. L., Yecies, Y. D. & Heck, L. W. (1976)

in Fogarty International Center Proceedings, The KallikreinSystem in Health and Disease, eds. Pisano, J. J. & Austen, K. F.(U.S. Government Printing Office, Washington, D.C.), No. 27,in press.

21. Liu, C. Y., Bagdasarian, A., Meier, H. L., Scott, C. F., Pierce, J.V., Kaplan, A. P. & Colman, R. W. (1976) Fed. Proc. 35, 692(abstr.).

22. Meier, H. L., Webster, M. E., Liu, C. Y., Colman, R. W. & Ka-plan, A. P. (1976) Fed. Proc. 35, 692 (abstr.).

23. Webster, M. E., Guimaraes, J. A., Kaplan, A. P., Colman, R. W.& Pierce, J. V. (1976) Adv. Exp. Med. Biol. 70, 285-299.

24. Kaplan, A. P., Meier, H. L. & Mandle, R., Jr. (1976) Seminars inThrombosis and Haemostasis 3,1-26.

25. Griffin, J. H. & Cochrane, C. G. (1976) Proc. Natl. Aced. Sci. USA73,2554-2558.

26. Nagasawa, S. & Nakayasu, T. (1973) J. Biochem. 74, 401-403.27. Habal, F. M., Underdown, B. J. & Movat, H. Z. (1975) Biochem.

Pharmacol. 24, 1241-1243.

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