THE JOURNAL OF BIOLOGICAL CHEmlsTRY Vol. 255, No. 9, Iasue of May 10, pp. 3952-3958. 1980 Printed in U. S A .

Immunochemical Studies of Human High Molecular Weight Kininogen and of Its Complexes with Plasma Prekallikrein or Kallikrein*

(Received for publication, October 2, 1979)

Daniele M. Kerbiriou,$ Bonnc? N. Bouma, and John H. Griffin# From the Department of Immunopathology, Research Institute of Scripps Clinic, La Jolla, California 92037 and the Department of Hematology, University Hospital, Utrecht, The Netherlands

Human plasma contains at least two distinct kinino- gens, designated high Mr and low M, kininogens, that differ in their M, and susceptibility to different kal- likreins. By limited proteolysis, plasma kallikrein cleaves high M, kininogen to liberate kinin and gives a molecule containing two disulfide-linked polypeptide chains. Following reduction and alkylation of the two- chain molecule, the heavy chain and the light chain were isolated, and antisera were raised in goats against the alkylated heavy and light chains. The anti-heavy chain antiserum immunoprecipitated both high M, and low M, kininogen, whereas the anti-light chain antise- rum was specific for high M, kininogen. Thus, the heavy chain of kinin-free high M, kininogen and low M, kini- nogen extensively share identical immunologic deter- minants. In contrast, the immunologic determinants of the isolated light chain are unique to high M, kininogen.

Using immunochemical techniques, it was shown that high M, kininogen or the light chain derived from it forms a complex either with prekallikrein or with kal- likrein. Titrations of prekallikrein or kallikrein with increasing amounts of either high M, kininogen or its alkylated light chain indicated that the complexes con- tain equimolar amounts of each molecule. These results show that a single site for prekallikrein or kallikrein binding to high Mr kininogen resides in the light chain region of the molecule.

~ ~~ ~~ ~~ ~

Kininogens are plasma proteins that contain the vasoactive peptides called kinins (1, 2). These peptides are released by limited proteolysis by enzymes known as kallikreins (3, 4). Fractionation of human plasma yields two distinct kininogen species, designated high M, kininogen and low M, kininogen (5-9). These molecules differ in their susceptibility to various kallikreins (5, 7). Both high M, and low M, kininogens are single chain proteins (9-13) and are immunologically related (8, 14). It has been recently shown that human plasmas deficient in high M , kininogen (15, 16) exhibit abnormalities in contact activation reactions, including the surface-depend- ent initiation of the kinin-forming, the intrinsic coagulation, and the fibrinolytic pathways (15-20). The functional role of

21544, NIAID-07007, and NHLBI-16411 from the National Institutes * This work was supported in part by Research Grants NHLBI-

of Health, and by a grant from the Division of Scientific Affairs, North Atlantic Treaty Organization. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Supported by Public Health Service International Research Fel- lowship 5-F05 TWO 2493. Permanent address, Institut de Pathologie Cellulaire, Hopital de Bicbtre, Le Kremlin-Bicbtre, France.

fj Recipient of National Institutes of Health Research Career De- velopment Award NHLBI-00192.

high M, kininogen as a nonenzymatic cofactor in contact activation reactions has been demonstrated (21, 22).

Under some conditions, high M, kininogen co-purifies or co- precipitates with Factor XI (23) or prekallikrein (24, 25). Moreover, purified high M, kininogen that is able to bind to negatively charged surfaces (13) promotes the binding of lZ5I- labeled Factor XI or prekallikrein to negatively charged sur- faces (26). These observations suggest that high M, kininogen plays a role as a surface receptor for Factor XI and prekal- likrein at the sites of their activation by surface-bound acti- vated Factor XI1 (Hageman factor).

Human plasma kallikrein cleaves the M, = 110,OOO high M, kininogen in plasma (13) or in solution (9-13) to give a kinin- free molecule made up of a M, = 65,000 heavy chain and a M, = 44,OOO light chain (13). Following reduction and alkylation, an alkylated light chain can be isolated that quantitatively retains the procoagulant activity of the entire molecule (11, 13).

In this study, immunochemical analyses indicate that the heavy chain of kinin-free high M, kininogen and low M, kininogen extensively share identical immunologic determi- nants. In contrast, the immunological determinants of the light chain are unique to high M, kininogen. Using immuno- electrophoretic techniques, we demonstrate that high M, kin- inogen or its isolated alkylated light chain is able to form complexes with either prekallikrein or kallikrein. Titrations of these complexes indicate equimolar binding. The results thus demonstrate that a single binding site for prekallikrein or kallikrein resides in the light chain moiety of high M, kinino- gen. Preliminary reports of this work have been presented (27, 28).

MATERIALS AND METHODS

All chemicals obtained from commercial sources were the best grade available.

Human high M, kininogen was purified as a single polypeptide chain of M, = 110,000 as described previously (13). After incubation with human plasma kallikrein, the disulfide-linked light and heavy chains of the kinin-free high M, kininogen were reduced, alkylated, and isolated as described previously (13). Partially purified low M, kininogen was obtained by chromatography of fresh human plasma at 20°C on a DEAE-Sephadex column with elution by a combined pH and salt gradient as previously described (29). under conditions in which low M, kininogen which eluted at pH 8.0, 13 to 15 mmho, and was entirely separated from high M, kininogen (elution at pH 7.8 and 20 to 23 mmho).

Human prekallikrein was purified as described below and labeled with using the Bolton-Hunter reagent (30). Using SDS’-polyacryl- amide gel electrophoresis, it was shown that Iz5I-prekallikrein had an apparent molecular weight of 88,OOO in the presence and absence of reducing agents. Kallikrein amidolytic activity was measured as hy-

’ The abbreviations used are: SDS, sodium dodecyl sulfate; p- Factor XII., the active M , = 28,000 form of Factor XI1 (Hageman factor); Bz-, benzoyl-.

3952

Human High M, Kininogen 3953

drolysis of the peptide Bz-Pro-Phe-Arg-p-nitroanilide (Pentapharm or Vega-Fox) (31). Less than 1% of the maximum possible kallikrein amidolytic activity was detected in thoprekallikrein preparation. I2'I-

Kallikrein was prepared by incubating 10 pl of '?-prekallikrein (2 X lo6 cpm), 200 pl of prekallikrein (30 pg), and 20 ,ul of 0.5 M Tris, pH 8.1, with 5 pl of purified 8-Factor XII, (3.5 pg) for 80 min at 37OC. At this time, the kallikrein amidolytic activity had reached a plateau, and addition of another 2.5 pl of P-Factor XII. (1.75 pg) followed by a further 20-min incubation did not increase the activity. Complete cleavage of '251-prekallikrein was verified by SDS-polyacrylamide gel electrophoresis under reducing conditions.

Protein concentration values used for the estimation of the molar ratios in the titration experiments were determined by amino acid analysis of aliquots of the solutions used for the experiments. The carbohydrate content was considered to be similar for the different polypeptide species studied, allowing the calculation of molar ratios with the concentration values given by the amino acid analysis exclusively and using the M , values of 110,000 for high M , kininogen, 44,000 for the light chain of kinin-free high M , kininogen, and 88,OOO for prekallikrein and kallikrein (13, 32).

Preparation of Anti-Heavy Chain and Anti-Light Chain Anti- sera-Either heavy chain or light chain kinin-free high M , kininogen 100 pg in 500 pl or 1 ml of 0.1 M sodium acetate buffer, pH 5.8, were mixed with equal volumes of Freund's complete adjuvant (Difco Laboratories). The emulsified mixtures were injected in multiple subcutaneous sites in two goats. Similar injections were repeated three times at 1-week intervals, 400 ml of blood was collected 7 days after the fmal injection, and the antisera were obtained and analyzed for the presence of antibodies directed against the heavy and light chain. For preparation of the y-globulin fraction of the antisera, 50 ml of the anti-heavy chain and the anti-light chain antisera were heated for 30 min at 56°C. The heated antisera were dialyzed at 4OC against a Tris (0.04 M)/succinate (0.01 M) buffer, pH 8.2, containing 70 t n ~ NaC1, 1 mM EDTA, 1 mM benzamidine, and 0.02% sodium azide (conductivity, 9 mmho). After centrifugation for 30 min at 1300 X g, the dialyzed samples were applied to a DEAE-Sephadex A50 (Phar- macia) column (15 X 2.5 cm) and eluted with the same buffer at a flow rate of 40 ml/h. The y-globulin fractions which did not adhere to the column were collected in 140 ml and were concentrated by precipitation with 50% (v/v) (NH4)2S04. The precipitates were dis- solved in 10 ml of either 0.01 M sodium phosphate or 0.01 M Tris-HC1, pH 7.4, containing 0.4 M NaCl and 0.02% sodium azide, and were dialyzed against the same buffer overnight at 4°C. The protein content of these y-globulin solutions was determined according to Lowry et al. (33) using bovine serum albumin as a standard.

Purification of Prekallikrein-Prekallikrein was purified as de- scribed elsewhere (32, 34) with the following modifications. DEAE- Sephadex chromatography was performed as described elsewhere? The fractions containing prekallikrein that did not adhere to the resin were pooled and subjected to SP-Sephadex chromatography at pH 5.3. SP-Sephadex C-50 (8 g) was allowed to swell overnight in the starting buffer, 0.1 M sodium acetate, 0.08 M NaCl, pH 5.3, 14 mmho conductivity at 22'C. The resin was poured into a siliconized glass column (5 X 30 cm) to give a bed height of 7 cm. The idumn was washed with 2 bed volumes of starting buffer, and the semple, which had been dialyzed for 18 h at 4°C against the starting buffer, was applied. The column was then washed with 1400 ml (10 bed volumes) of starting buffer. Prekallikrein was eluted in a single step using 0.1 M sodium acetate, 0.15 M NaCl, pH 5.3, 18 mmho conductivity at 22°C. The flow rate was 200 ml/h and 5-ml fractions were collected. The next step employed concanavalin A-Sepharose affinity chroma- tography to remove y-globulin from prekallikrein. Prekallikrein ad- hered to this column and was eluted as described elsewhere (32).

This simplified procedure yields prekallikrein preparations in which small amounts of high M, protein, presumably y-globulins, were occasionally observed on nonreduced SDS gels. These proteins were removed by an immunoadsorption technique as follows. An antiserum to the y-globulin fraction that did not adhere to the concanavalin A-Sepharose (as described above) was prepared by four successive weekly injections of 225 pg of protein in Freund's complete adjuvant (Difco) into a rabbit at multiple subcutaneous sites. Blood was collected 7 days after the final injection. The serum was applied at 4°C to a column (1.5 X 5 cm) containing Sepharose to which the Same y-globulin used for the immunization was attached (10 mg/g of

B. N. Bouma, D. M. Kerbiriou, R. A. A. Vlooswijk, and J. H. Griffin, manuscript in preparation.

Sepharose-cyanogen bromide (Pharmacia)). The column was washed with 0.01 M Tris, 0.5 M NaC1, pH 7.4, until the absorbance at 280 nm was zero. The antibodies were then eluted using 3 M NaSCN at a flow rate of 4 ml/h. The fractions containing protein were pooled and dialyzed against 0.01 M Tris, 0.5 M NaCl, pH 7.4. The immunopurified antibodies were coupled to Sepharose-cyanogen bromide (10 mg/g). The insolubilized antibodies were used to remove the contaminant from the prekallikrein preparations. Typically, 0.2 ml of gel was incubated for 1 h at 4°C with constant mixing with 1 mg of prekal- likrein in a volume of 3 ml. The reaction mixture was centrifuged (10 min, 4000 x g) at 4°C and the supernatant containing prekallikrein was collected. The specific activity of the purified prekallikrein was 20 clotting units/mg.

Anti-Prekallikrein y-Globulin-The anti-prekallikrein antibodies used in this study were prepared in goats that were immunized with prekallikrein purified according to the procedure described elsewhere (32). The goat antiserum was absorbed with the human y-globulin fraction that did not adhere to concanavalin A-Sepharose in order to remove a minor contaminating antibody which was detectable when analyzed by double immunodiffusion when large wells were refilled three times with 60 pl of the y-globulin fraction (2 mg/ml). The antiserum was incubated for 1 h at 37'C with l/lOth volume of the human y-globulin fraction (2 mg/ml). After further incubation at 4°C for 18 h, the mixture was centrifuged at 4°C (20 min, 6000 X g). The y-globulin fraction of the absorbed anti-prekallikrein antiserum was then isolated by DEAE-Sephadex chromatography and concanavalin A-Sepharose chromatography as described elsewhere (32).

Laurel1 Rocket Immunoelectrophoresis Procedures-Rocket im- munoelectrophoresis was performed as described by Laurell(35) using 0.9% agarose (Seakem) gels in 25 mM sodium barbital buffer, pH 8.6, containing 0.02% sodium azide. Antibody was mixed with the agarose solution at 54'C. Glass slides (Kodak projector slide cover glass, 8.3 X 10.2 cm) were precoated with 2 ml of 1.5% agarose. The mixture of antibody and agarose (15 m l ) was poured onto a glass slide and allowed to stand after solidification in a moisturized chamber at 4°C for 12 h. Sample aliquots (10 pl) were applied in wells, and electro- phoresis was performed at 7 V/cm for 8 h in a Bio-Rad (model 1415) cooling chamber, at 6"C, using 75 mM barbital buffer, pH 8.6, as electrophoresis buffer.

Two-dimensional immunoelectrophoresis was performed according to Laurel1 (36). Four milliliters of 0.9% agarose solution in 25 mM sodium barbital buffer, pH 9.5, was poured on a surface (2 X 10.2 cm) along the larger length of a glass plate (8.2 X 10.2 cm) for the first dimension electrophoresis. The fmst electrophoresis was performed in the previously described cooled chamber at 6°C using a 75 mM sodium barbital buffer, pH 8.6. The samples, containing 3 pl of Evans blue dye (0.25% in 20% bovine serum albumin) as a marker, were allowed to migrate toward the anode at 7 V/cm for 3 h. Then, 10 ml of a 0.9% agarose solution containing diluted antiserum in 25 mM sodium bar- bital buffer, pH 8.6, at 54OC was poured on the remaining surface for electrophoresis in the second dimension. The second electrophoresis was performed at 7 V/cm for 5 h using the same electrophoresis buffer. After extensive washing with a 0.9% NaCl solution and a final distilled water rinse, the plates were dried and stained with 0.25% Coomassie blue.

Clotting Activity of High M, Kininogen-High M , kininogen clot- ting activity was measured using the two-stage kaolin-activated par- tial thromboplastin time assay ( 2 9 ) employing high M, kininogen- deficient plasma (Fitzgerald trait plasma obtained from George King Biomedical). One unit of high M, kininogen clotting activity is defined as the amount of activity present in 1 ml of a standard pool of normal citrated human plasma.

Amino Acid Analysis-Carefully measured aliquots of protein samples were hydrolyzed in vacuo with 6 N HCI at 105°C for 24 h and then analyzed using a Beckman 121M amino acid analyzer (37). The protein concentration of the samples was determined from the total weight of amino acids in the aliquots, and the carbohydrate content was taken as 15%.

RESULTS

Characterization of Antibodies Against High M, Kinino- gen and Low M, Kininogen-Antibodies were raised in goats against the isolated light chain and the isolated heavy chain moieties of kinin-free human high M, kininogen. The double immunodiffusion patterns of the two antisera tested against high M, kininogen and low M, kininogen are shown in Fig. 1.

3954 Human High M , Kininogen

Both the anti-heavy chain and the anti-light chain antisera gave single precipitating lines with purified high M , kininogen. The anti-heavy chain antiserum also precipitated with par- tially purified low M , kininogen in a single line that showed complete identity with high M , kininogen. In contrast, no precipitation line was observed between low M , kininogen and the anti-light chain antiserum.

The specificities of anti-heavy chain and of anti-light chain antisera were further analyzed by rocket immunoelectropho- resis. As shown in Fig. 2, high or low M , kininogens as well as normal plasma gave single precipitating rockets when electro- phoresed into a gel containing the anti-heavy chain antiserum. The height of the rocket observed with plasma was increased when the level of kininogen was increased by preincubation of plasma with additions of either high or low M , kininogen. In contrast, rocket immunoelectrophoresis using the anti-light chain antiserum showed that high M , kininogen but not low M , kininogen gave a visible rocket. Moreover, addition of high M , kininogen but not low M , kininogen increased the heights of the observed rocket for the plasma sample. The results presented in Figs. 1 and 2 demonstrate that the anti-heavy chain antiserum recognizes high and low M , kininogens, whereas the anti-light chain antiserum appears monospecific for high M , kininogen. Double immunodiffusion of each anti- serum against normal human plasma gave a single visible line of immunoprecipitation, consistent with the data presented in Figs. 1 and 2.

Effect ofAnti-HeaqChain andAnti-Light ChainAntisera on the Procoagulant Activity of High M,. Kininogen-In order to analyze the effect of anti-heavy chain or anti-light chain antibodies on the procoagulant properties of high M , kininogen, the y-globulin fractions of each antiserum were prepared as described under “Materials and Methods.” Ali- quots of normal human plasma (50 p l ) were incubated a t 37°C for 10 min in a total volume of 100 pl in the presence of 30 pg to 1.5 mg of either anti-light or anti-heavy chain y-globulins, and the reaction mixtures were analyzed for their ability to correct the clotting deficiency of high M , kininogen-deficient plasma. As seen in Fig. 3, the procoagulant activity of high M , kininogen in plasma decreased as a function of the amount of anti-light chain y-globulin added. The anti-heavy chain y- globulin a t up to 1.5 mg did not inhibit the procoagulant activity of high M , kininogen, even when the preincubation time was increased to 1 h. However, preincubation of 50 pl of plasma with 3 mg of anti-heavy chain y-globulin for 1 h did inhibit 88% of the procoagulant activity of high M , kininogen. In control experiments, identical or larger amounts of y-glob- ulin fractions prepared from a nonimmunized goat did not

High MW Kininogen

Anti-light Anti-Heavy Chain Chain

I

Low MW Kininogen

FIG. 1. Double immunodiffusion analysis of high and low M , ( M W ) kininogens with ant isera to the isolated light and heavy chains of kinin-free high M , kininogen. Aliquots (20 1-11) of anti- light chain or anti-heavy chain antisera were analyzed by double inmunodiffusion in 1% agarose, pH 7.5, with 20 pl of purified high M , kininogen (5 p g ) or 20 pI of partially purified low M , kininogen (see “Materials and Methods”).

Anti-Heavy Chain B

HMWK LMWK HMWK LMWK Plasma Plafma P d m a

Anti-light Chain

HMWK LMWK HMWK LMWK Plasma + +

Plasma Plasma FIG. 2. Electroimmunodiffusion of high M , kininogen

(HMWK), low M , kininogen ( I N W K ) , and normal human plasma. High M , kininogen (5 p g ) or partially purified low M . kininogen (12 pl) diluted in 25 mM sodium-barbital buffer, pH 8.6, was preincubated in a total volume of 50 pI in plastic tubes in the absence or presence of 25 pI of normal human plasma for 20 min at 37°C. Plasma (25 pl) was incubated in 25 pl of buffer for the control. Each sample (20 pl) was analyzed by rocket immunoelectrophoresis in 0.9Z agarose containing either 3.34 anti-heavy chain antiserum or 3.F$ anti-light chain antiserum.

FIG. 3. Inhibition of high M, kininogen activity by anti-light chain y-globulin (IgC). Samples ( 1 0 0 pl) containing 50 pl of plasma and increasing amounts of anti-light chain y-globulins from 30 p g to 1.5 mg in 0.005 M Tris-HCI buffer, pH 7.4, containing 0.2 M NaCl were incubated at 37°C for 10 min. Aliquots (20 pl) were withdrawn and diluted 20 times to determine the clotting activity of high M , kinino- gen as described under “Materials and Methods.”

Human High M , Kininogen 3955

modify the procoagulant activity of high M , kininogen in plasma under any of the conditions described, indicating that the effects observed were specific for the antibodies present in the immune y-globulin fractions.

Purified high M , kininogen was also preincubated in the presence of the y-globulin fraction of each antiserum and then assayed for residual procoagulant activity. High M , kininogen (50 pI) at 0.5 clotting units/ml was inhibited by 75 or 100% by preincubation for 10 min at 37°C in the presence of 75 or 750 pg, respectively, of anti-light chain y-globulin fractions. The y-globulin fraction of either the anti-heavy chain or of a nonimmune serum, when used in identical amounts, was not able to modify the procoagulant activity of purified high M , kininogen.

Association of the Purified Light Chain of High M , Kini- nogen with Prekallikrein or with Kallikrein-Since the al- kylated light chain isolated from high M , kininogen possesses by itself the procoagulant activity of the entire molecule ( 1 1 , 13), the ability of the isolated alkylated light chain to associate with either prekallikrein or kallikrein was analyzed. The light chain was preincubated for 30 min a t 37°C in the absence or in the presence of equimolar amounts of either "'I-prekal- likrein or '"I-kallikrein. The incubated samples were then analyzed by two-dimensional immunoelectrophoresis using the anti-light chain antiserum in the agarose in the second dimension. As shown in Fig. 4, the immunoprecipitation pat- tern for the isolated light chain was modified by preincubation with either prekallikrein or kallikrein. A precipitating arc appeared that migrated slower than that seen for the light chain alone, indicating the formation of molecular species containing the light chain that migrated slower than the light chain alone.

Autoradiographs of the same stained slides showed the presence of either "'I-prekallikrein or '"'I-kallikrein in these slower migrating species, suggesting that the light chain as- sociated with either molecule during the preincubation. In control experiments, neither prekallikrein alone nor kallikrein alone migrated or was precipitated under these electropho-

Light Chain f\ light Chain Preincubated

With 1251.Prekallikrein 2 Autoradiography

Light Chain Prelncubated With 125l.Prekallikrein D

Light Chain

1251.Kallikrein Preincubated With \

Light Cham Preincubated Autoradiography

With '251.Kall~kre~n - FIG. 4. Crossed immunoelectrophoresis of the isolated light

chain of kinin-free high M . kininogen preincubated with prekallikrein or with "'1-kallikrein using anti-light chain an- tibodies. Alkylated light chain (1.4 pg) was incubated for 30 min at 37°C in 50 pI of 25 mM sodium barbital buffer, pH 8.6, in the absence or presence of 3.2 pg of either lrsI-prekallikrein (200,000 cpm) or "'1- kallikrein (170,200 cpm). The incubated samples were analyzed by two-dimensional immunoelectrophoresis as described under "Mate- rials and Methods." The anti-light chain antiserum was diluted to 5 1 in 0.9% agarose for electrophoresis in the second dimension.

retic conditions. The autoradiographs indicated the presence of slowly migrating, heterogeneous radiolabeled material in the first dimension. This could reflect the progressive and slow dissociation of molecular complexes during electropho- resis in the first dimension.

Titrations of High M,. Kininogen and its Isolated Light Chain with Prekallikrein or Kallikrein-Prekallikrein and kallikrein did not migrate in agarose upon electrophoresis at pH 8.6. However, as seen above and elsewhere,' association of prekallikrein or kallikrein with high M , kallikrein or with its isolated light chain gave negatively charged complexes that are able to migrate at this pH. Taking advantage of these electrophoretic properties, titrations of the binding of prekal- likrein or kallikrein with high M , kininogen or i t s light chain were performed by using Laurel1 rocket immunoelectropho- resis (35) employing the y-globulin fraction of an anti-prekal- likrein antiserum. Prekallikrein or kallikrein (0.5 pg) was incubated for 20 min at 37°C in plastic tubes in the absence or presence of 0.14 to 4.6 pg of high M , kininogen or 0.05 to 1.8 pg of alkylated light chain in 20 pl of 25 m M sodium barbital buffer, pH 8.6. Aliquots (10 pl) were analyzed by one-dimen- sional rocket immunoelectrophoresis in agarose containing anti-prekallikrein. As shown in Fig. 5, prekallikrein alone did not migrate under these conditions, but it migrated after preincubation with either high M , kininogen or its light chain. The heights of the rockets increased as a function of increasing high M , kininogen or light chain concentration until a maxi- mum height was reached. Results similar to those in Fig. 5 were observed when prekallikrein was replaced by kallikrein. The observed rocket height as a function of the molar ratio of high M, kininogen or of its light chain to prekallikrein is seen in Fig. 6a. The titration data are fit using two straight lines as shown in Fig. 6a, with the intersection of the two lines at

High Mr Kininogen Molar Ratio

Prekallikreln Versus 0 0.24 0.48 0.72 0.96 1.14 3.9 5.8 7.7

Molar Ratio Llghl Chain

p,k.~~~~ 0 0.24 0.49 0.73 0.98 1.96 3.9 5.9 7.8

FIG. 5. Titration of prekallikrein with high M , kininogen or its isolated light chain using Laurel1 rocket electrophoresis (35) with anti-prekallikrein antibodies. Aliquots (10 pl) contain- ing 0.25 p g of prekallikrein or kallikrein were incubated with increas- ing amounts of high M , kininogen or i t s light chain, as described in the text, and were then analyzed by rocket immunoelectrophoresis. The electrophoresis was performed as described under "Materials and Methods" in 0.9% agarose containing 2.5% anti-prekallikrein y- globulin. As a control, 0.96 p g of prekallikrein or of kallikrein were preincubated with either 2.4 p g of alkylated heavy chain or 2.2 pg of alkylated light chain. Prekallikrein or kallikrein preincubated with heavy chain did not migrate upon immunoelectrophoresis, whereas 1.5-cm rockets were observed for the samples preincubated with the light chains.

3956 Human High M, Kininogen

* O { Y l High Mr Kininogen

I t A

E E

g o 0 1 2 3 4 5 6 7 8 I .=

Molar Ratio of ProteinlPrekallikrein .d a?

l0!+-"-l

Light Chain

o i i i i i i b i i Molar Ratio of ProteinlKallikrein

FIG. 6. Titration of prekallikrein and kallikrein with high M, kininogen or its light chain measured by Laurell rocket heights (35). The rocket heights corresponding to the migration of prekallikrein and kallikrein in the presence of increasing amounts of high M, kininogen or its isolated light chain were measured after immunoelectrophoresis into agarose containing anti-prekallikrein y- globulin as decribed in the legend to Fig. 5. The molar ratios were calculated based on protein concentrations measured by amino acid analysis and using the molecular weights of 88,000 for prekallikrein and kallikrein (32), 110,OOO for high M, kininogen, and 44,000 for the isolated light chain (13).

molar ratios of high M , kininogen or of light chain versus prekallikrein corresponding to 1.2 -+ 0.2 and 0.9 k 0.2, respec- tively. In the case of the kallikrein titrations, the molar ratios of high M , kininogen or of its light chain to kallikrein were determined to be 1.1 k 0.2 and 0.9 f 0.2, respectively, at the points of intersection of the two straight lines.

DISCUSSION

Human plasma contains at least two distinct kininogens, high M, kininogen and low M, kininogen, that are single polypeptide chains of M, = 110,OOO (9-13) and of M, = 50,000 to 78,000 (7,9), respectively. Human plasma kallikrein cleaves high M, kininogen in plasma (13) or in solution (9-13) to give a kinin-free molecule made up of a M, = 65,000 heavy chain and a M, = 44,000 light chain (13). Following reduction and alkylation of the kinin-free molecule, the alkylated light chain can be separated from the alkylated heavy chain. As reported here, the antiserum raised against the isolated alkylated heavy chain purified from kinin-free high M, kininogen precipitates both high M, kininogen and low M, kininogen. In contrast, the anti-light chain antiserum exclusively precipitates high M, kininogen. Thus, it is possible to prepare an unadsorbed antiserum that is monospecific for high M, kininogen. This antiserum is a very specific tool for analysis of high M, kininogen and can be used to show the existence of specific complexes between high M, kininogen and other molecules in plasma2 and in purified systems. In double immunodiffusion analysis, the anti-heavy chain antiserum gives precipitation lines with both high M, kininogen and low M , kininogen in a reaction indicating complete immunological identity between these two molecules.

Antibodies raised against low M, kininogen have previously

shown that low M, kininogen cross-reacts with apparent com- plete identity with high M, kininogen using such antibodies (6). Other immunological studies attempted to elucidate the structural similarities and differences between the two kinds of kininogens in human plasma (8,14). These studies indicated that the two kininogens share a large number of determinants. Kat0 et al. (38) using antiserum against the isolated heavy chain of bovine high M, kininogen showed apparent complete immunological identity between the isolated heavy chain of bovine high M, kininogen and the isolated heavy chain of bovine low M, kininogen. These results and the results pre- sented in this paper for the human molecule indicate that the heavy chain of high M, kininogen and low M, kininogen extensively share identical immunological determinants. Moreover, we demonstrate here that the immunological de- terminants of the light chain of human high M, kininogen are unique to this molecule and are not apparent in low M , kininogen.

Some authors have immunized animals with high M , kini- nogen and obtained anti-high M , kininogen antisera that also precipitated low M, kininogen (11, 12, 39-42). Monospecific anti-high M, kininogen antibodies were prepared from these antisera either by immunoaffinity chromatography (42) or by adsorption with either purified low M, kininogen (41) or with Fitzgerald trait plasma that contains low M, kininogen but lacks high M, kininogen (11). The concentration of high M, kininogen in normal plasma determined by Laurell rocket immunoelectrophoresis (35) using our anti-light chain antise- rum was 70 pg/nd2 Other studies using adsorbed anti-high M, kininogen antisera report the value to be 90 p g / d by hemag- glutination inhibition reactions (41) or by radioimmunoassays (42).

Preincubation of high M, kininogen with the y-globulin fraction of the anti-light chain antiserum inhibits the proco- agulant properties of the molecule. Much higher protein con- centrations of the anti-heavy chain y-globulin fractions are needed to inhibit the high M, kininogen procoagulant activity. Analyses of the procoagulant activity of the alkylated heavy and light chains isolated from human (11, 13) and bovine (43, 44) kinin-free high M, kininogen indicated that the alkylated light chain quantitatively retains the procoagulant activity of the native molecule, whereas the isolated heavy chain is inactive. The purification of both kinds of antibodies would be necessary to establish a quantitative comparison of their potencies to inhibit high M , kininogen procoagulant activity. However, the fact that the anti-light chain antibodies strongly inhibit the procoagulant activity shows that the formation of complexes between anti-light chain antibodies and high M, kininogen is sufficient to abolish the procoagulant activity of the entire molecule.

The anti-light chain antiserum was used to analyze by crossed immunoelectrophoresis the association between the isolated alkylated light chain of high M, kallikrein and pre- kallikrein or kallikrein. Preincubation of the alkylated light chain with '"1-prekallikrein or with '"1-kallikrein resulted in the formation of lz5I-1abeled complexes that exhibit electro- phoretic mobilities at pH 8.6 that are intermediate between those of prekallikrein or kallikrein alone and the light chain alone. This qualitative result indicates that the light chain moiety of high M, kininogen is, by itself, able to bind to either prekallikrein or kallikrein.

Interactions among these molecules were quantitatively studied by Laurell rocket immunoelectrophoresis (35) in aga- rose gel containing anti-prekallikrein antibodies. Preincuba- tion of prekallikrein or kallikrein with increasing concentra- tions of high M, kininogen or light chain increases the rocket heights in a manner that shows that the formation of com-

t 1 High MW Kininogen

Active

Human High M , Kininogen

l ight Chain Active

1 2 l o w MW Kininogen

Inactive

Heavy Chain Inactive

3957

FIG. 7. Structural models for the polypeptide structure of high M, (MW) and low M, kininogens. The models represent both molecules as sin- gle polypeptide chains and are based on the studies of the primary structures of bovine high M , and low M , kininogens (50.51) and on the structural, physiolog- ical, and immunochemical analysis of hu- man high M, and low M , kininogens. Cleavage by human plasma kallikrein liberates kinin from high M , kininogen and subsequently an NHz-terminal heavy chain can be separated from a COOH-terminal light chain after reduc- tion and alkylation. The alkylated heavy chain of high M , kininogen and low Mr kininogen are not procoagulant and ex-

The alkylated light chain of high M , tensively share identical determinants.

kininogen is unique to this molecule and possesses the procoagulant activity.

plexes is proportional to the amount of protein ligand added. Maximum rocket heights for prekallikrein or kallikrein are reached when equimolar amounts of high M, kininogen or of its alkylated light chain are present. A maximum number of one binding site for high M, kininogen or its light chain on prekallikrein or kallikrein is calculated from such experiments. In normal human plasma, there is a slight molar excess of high M , kininogen over prekallikrein.' It therefore seems reasonable that a one-to-one complex of prekallikrein and high M, kininogen is the predominant physiological form of these molecules.

Previous observations, using other techniques, of high M , kininogen complexes containing kallikrein or prekallikrein have been reported. In attempts to purify human kallikrein and prekallikrein, Wendel et al. (45) and Nagasawa and Na- kayasu (46) showed that these molecules co-eluted during gel filtration of plasma proteins with other molecules as acidic complexes of M , = 180,000 (45) or M, = 300,000 (46). Kallikrein or prekallikrein was further isolated from these complexes as a y-globulin of M , = 98,000. Mandle et al. (24) later demon- strated that prekallikrein and high M, kininogen co-eluted at an apparent M , of 285,000 in gel filtration experiments. Don- aldson et al. (25) removed both prekallikrein and high M , kininogen from plasma by using either anti-kallikrein or anti- high M , kininogen antibodies. While this work was in progress, Colman and Scott (47) have also reported that prekallikrein and high M, kininogen form a complex under conditions of immunoelectrophoresis. Kat0 et al. (48) showed that bovine prekallikrein or kallikrein associates with bovine high M , kininogen. Thompson and Kaplan (49) recently studied the binding of prekallikrein to high M , kininogen or its light chain when the latter molecules were adsorbed to the surface of a plastic test tube. The dissociation constant was approximately lo-@ M for the surface-bound complex. The total concentration of prekallikrein or kallikrein in solution in our titration exper- iments (Figs. 5 and 6) was 3 X M. The immunochemical measurements of complex formation indicate the equimolar stoichiometry of molecules in the complexes but do not allow an accurate estimate of the asociation equilibrium constants. Further studies using other techniques will be necessary to determine whether the binding constant for prekallikrein dif- fers from that of kallikrein or whether the isolated alkylated light chain is significantly different from the native high M , kininogen molecule.

Schematic polypeptide structural models seen in Fig. 7 describe the respective properties of high and low M , kinino-

gens and of the heavy and light chain regions of high M , kininogen. These models are based on the distinctive immu- nochemical properties of the molecules described in this pa- per, on structural studies of the bovine (38,50,51) and human (8, 9, 12-14) kininogens, and on the procoagulant properties of the isolated alkylated light chains of bovine (43, 44) and human (11-13) high M, kininogen. Human high M, kininogen is a single polypeptide chain susceptible to cleavage by human plasma kallikrein to liberate bradykinin and give a two-chain disulfide-linked molecule. Reduction and alkylation of the kinin-free molecule allows separation of an NHz-terminal heavy chain that is inactive in coagulation assays and that is structurally similar to the inactive low M, kininogen from a COOH-terminal light chain.

High M, kininogen enhances the activation of prekallikrein by surface-bound activated Factor XI1 (21,22) and, in plasma,

I-prekallikrein binds to negatively charged surfaces in the presence but not in the absence of high M, kininogen (26). These observations taken together suggest that prekallikrein, bound to high M, kininogen in plasma, can be bridged by this molecule to a negatively charged surface where activation by surface-bound activated Factor XI1 will occur. The alkylated light chain of high M, kininogen is, as discussed above, as procoagulant as the native high M, kininogen and also pos- sesses the histidine-rich region (52) that is likely to be respon- sible for the binding of the native molecule to negatively charged surfaces. It is then not surprising that the light chain region of high M , kininogen possesses the binding site for prekallikrein or kallikrein as shown here.

125

Acknowledgments-The skillful technical assistance of Riek A. A. Vlooswijk is gratefully acknowledged. Discussions with Dr. Theodore Zimmerman were most helpful. The support and encouragement of Dr. Charles G. Cochrane is gratefully acknowledged.

REFERENCES

1. Werle, E., and Berek, U. (1948) Angew. Chem. 60A, 53 2. Rocha e Silva, M., Beraldo, W. T., and Rosenfeld, G. (1949) Am.

3. Pierce, J. V., and Webster, M. E. (1961) Biochem. Biophys. Res.

4. Werle, E., Trautschold, I., and Leysath, G. (1961) Hoppe-Seyler's

5. Jacobsen, S. (1966) Nature (Lond.) 210,98-99 6. Pierce, J. V., and Webster, M. E. (1966) in Hypotensive Peptides

(Erdos, E. G., Back, N., Sicuteri, F., and Wilde, A. F., e&) pp. 130-138, Springer-Verlag, New York

7. Habal, F. M., Movat, H. Z., and Burrowes, C. E. (1974) Biochem.

J. PhysioZ. 156,261-273

Commun. 5,353-357

Z. Physiol. Chem. 326,174-176

3958 Human High M , Kininogen

Pharmacol. 23,2291-2303 8. Habal, F. M., Underdown, B. J., and Movat, H. Z. (1975) Biochem.

Pharmacol. 24, 1241-1243 9. Nagasawa, S., and Nakayasu, T. (1974) in Chemistry and Biology

of the Kallikrein- Kinin System in Health and Disease (Pisano, J. J., and Austen, K. F., eds) Department of Health, Education, and Welfare Publication No. (NIH) 76-791, pp. 139-143, Na- tional Institutes of Health, Bethesda, Md.

10. Saito, H. (1977) J. Clin. Inuest. 60, 584-594 11. Thompson. R. E., Mandle, R., Jr., and Kaplan, A. P. (1978) J.

12. Nakayasu, T., and Nagasawa, S. (1979) J. Biochem. (Tokyo) 85,

13. Kerbiriou, D. M., and Griffin, J. H. (1979) J. Biol. Chem. 254,

14. Pierce, J . V. (1968) Fed. Proc. 27, 52-57 15. Wuepper, K. D., Miller, D. R., and Lacombe, M. J. (1975) J. Clin.

Invest. 56, 1663-1672 16. Colman, R. W., Bagdasarian, A,, Talamo, R. C., Scott, C. F.,

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

17. Saito, H., Ratnoff, 0. D., Waldmann, R., and Abraham, J . P. (1975) J. Clin. Inwest. 55, 1082-1089

18. Lacombe, M. J., Varet, B., and Levy, J. P. (1975) Blood 46, 761- 768

19. Schiffman, S., Lee, P., and Waldmann, R. (1975) Thromb. Res. 6, 451-454

20. Donaldson, V. H., Check, H. L., Miller, M. A., Movat, H. Z., and Habal, F. (1976) J . Lab. Clin. Med. 87, 327-337

21. Griffin, J . H., and Cochrane, C . G. (1976) Proc. Natl. Acad. Sci. U. S. A. 73,2554-2558

22. Meier, H. L., Pierce, J. V., Colman, R. W., and Kaplan, A. P. (1977) J. Clin. Invest. 60, 18-31

23. Thompson, R. E., Mandle, R., Jr., and Kaplan, A. P. (1977) J. Clin. Invest. 60, 1376-1380

24. Mandle, R. J., Colman, R. W., and Kaplan, A. P. (1976) Proc. Natl. Acad. Sci. U. S. A. 73,4179-4183

25. Donaldson, V. H., Kleniewski, J., Saito, H., and Sayed, J . K. (1977) J. Clin. Invest. 60, 571-583

26. Wiggins, R. C., Bouma, B. N., Cochrane, C. G., and Griffin, J . H. (1977) Proc. Natl. Acad. Sci. U. S. A . 74,4636-4640

27. Kerbiriou, D. M., and Griffin, J. H. (1979) Fed. Proc. 38, 847 28. Bouma, B. N., and Vlooswijk, R. A. A. (1979) Thromb. Haemo-

29. Griffin, J. H., and Cochrane, C. G. (1976) Methods Enzymol. 45,

Exp. Med. 147,488-499

249-258

12020-12027

stasis 432, 263

56-65

30. Bolton, A. E., and Hunter, W. M. (1973) Biochem. J. 133, 529- 539

31. Amundsen, E., Svendsen, L., Vennerod, A. M.. and Laake, K. (1974) in Chemistry and Biology of the Kallikrein-Kinin Sys- tem in Health and Disease (Pirano, J. J., and Austen, K. F., eds) Department of Health, Education, and Welfare Publica- tion No. (NIH) 76-791, pp. 215-220, National Institutes of Health, Bethesda, Md.

32. Bouma, B. N., Miles, L. A., Beretta, G., and Griffin, J. H. (1980) Biochemistry, in press

33. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275

34. Bourna, B. N., and Griffin, J. H. (1977) Thromb. Haemostasis 38, 136

35. Laurell, C.-B. (1966) Anal. Biochem. 15, 45-52 36. Laurell, C.-B. (1965) Anal. Biochem. 10, 358-369 37. Moore, S., and Stein, W. H. (1963) Methods Enzymol. 6,819-831 38. Kato, H., Han, Y. N., Iwanaga, S., Suzuki, T., and Komiya, M.

(1976) J. Biochem. (Tokyo) 80, 1299-1311 39. Habal, F. M., and Movat, H. Z. (1972) Res. Commun. Chem.

Pathol. Pharmacol. 4,477-486 40. Hamberg, U., Elg, P., Hissinen, E., and Stelwagen, P. (1975) Int.

J. Pept. Protein Res. 7, 261-280 41. Kleiniewski, J., and Donaldson, V. H. (1977) Proc. SOC. Exp. Biol.

Med. 156, 113-117 42. Proud, D., Pierce, J. V., Peyton, M. P., and Pisano, J . J . (1979)

Fed. Proc. 38, 686 43. Wuepper, K. D., Miller, D. R., Han, Y. N., Kato, H., and Iwanaga,

S. (1978) Fed. Proc. 37, 1587 44. Scicli, A. G., Waldmann, R., Guimaraes, A,, Scicli, E., Carretero,

0. A., Kato, H., Han, Y. N., and Iwanaga, S. (1979) J. Exp. Med. 149,847-855

45. Wendel, V., Vogt, W., and Seidel, G. (1972) Hoppe-Seyler’s 2. Physiol. Chem. 353, 1591-1606

46. Nagasawa, S., and Nakayasu, T. (1973) J . Biochem. (Tokyo) 74,

47. Colman, R. W., and Scott, C. G. (1979) Clin. Res. 27, 460A 48. Kato, H., Sugo, T., Ikari, N., Hashimoto, N., Iwanaga, S., and

49. Thompson, R. E., and Kaplan, A. P. (1978) Circulation 58,11-211 50. Han, Y. N., Kato, H., Iwanaga, S., and Suzuki, T. (1976) J.

51. Han, Y. N., Kato, H., Iwanaga, S., and Komiya, M. (1978) J.

52. Kerbiriou, D. M., and Griffin, J . H. (1979) Thromb. Res. 42,252

401-403

Fujii, S. (1979) Thromb. Haemostasis 42, 262

Biochem. (Tokyo) 79, 1201-1222

Biochem. (Tokyo) 83,223-235