THE JOURNAL OF Bro~ocrcn~. CHEMISTRY Vol. 254, No. 23, Issue of December 10, pp. 12020-12027, 1979 Printed m U.S.A.

Human High Molecular Weight Kininogen STUDIES OF STRUCTURE-FUNCTION RELATIONSHIPS AND OF PROTEOLYSIS OF THE MOLECULE OCCURRING DURING CONTACT ACTIVATION OF PLASMA*

(Received for publication, May 18, 1979)

Danitile M. KerbiriouS and John H. Griffin8

From the Department of Immunopathology, Scripps Clinic and Research Foundation, La Jolla, California 92037

Human high M, kininogen was purified from normal plasma in 35% yield. The purified high M, kininogen appeared homogeneous on polyacrylamide gels in the presence of sodium dodecyl sulfate and mercaptoetha- no1 and gave a single protein band with an apparent M, = 110,000. Using sedimentation equilibrium techniques, the observed M, was 108,000 & 2,000.

Human plasma kallikrein cleaves high iW, kininogen to liberate kinin and give a kinin-free, two-chain, di- sulfide-linked molecule containing a heavy chain of apparent M, = 65,000 and a light chain of apparent iW, = 44,000. The light chain is hi&dine-rich and exhibits a high affinity for negatively charged materials. The isolated alkylated light chain quantitatively retains the procoagulant activity of the single-chain parent mole- cule.

‘261-Human high M, kininogen undergoes cleavage in plasma during contact activation initiated by addition of kaolin. This cleavage, which liberates kinin and gives a two-chain, disulfide-linked molecule, is dependent upon the presence of prekallikrein and Factor XII (Hageman factor) in plasma. Addition of purified plasma kallikrein to normal plasma or to plasmas de- ficient in prekallikrein or Factor XII in the presence or absence of kaolin results in cleavage of high i%Z, ki- ninogen and kinin formation.

Human plasma contains substances that release the vaso- dilator, bradykinin (1). This substance, a nonapeptide that is able to diminish blood pressure (1, 2), induce smooth muscle contraction (1, 2), and provoke pain (3), is released after cleavage of precursor protein molecules, kininogens, by pro- teolytic enzymes (4, 5). Human plasma contains at least two distinct forms of kininogen that differ in molecular weight (6- 10) and in susceptibility to various kallikreins (6, 8).

Exposure of plasma to negatively charged surfaces initiates intrinsic coagulation (ll), the kinin-forming pathway (12), fibrinolysis (I3), and the generation of vasoactive peptides (14). It has been recently shown that human plasmas deficient in high M, kininogen (15, 16) exhibit abnormalities in contact activation reactions, including the kinin-forming, intrinsic co-

* This work was supported in part by Research Grants NHLBI- 21544, NIAID-07007, and NHLBI-16411 from the National Institutes of Health. This is Publication 1787 from the Department of Immu- nopathology Scripps Clinic and Research Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “adver- tisement” in accordance 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.

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

agulation, and fibrinolytic pathways (15-20). High M, ki- ninogen isolated from bovine plasma is able to correct the contact activation abnormalities of such plasmas (21-22). The functional role of high M, kininogen as a nonenztiymatic cofac- tor in contact activation of intrinsic coagulation and fibri- nolysis has been demonstrated (23, 24).

Bovine high M, kininogen is a single-chain molecule, which has been partially sequenced by Iwanaga and his colleagues (25,26), who also analyzed the relationships between structure and function of the molecule (21, 22, 27, 28). In this study we describe a purification procedure for human high M, kininogen that yields a highly purified single-chain molecule. We dem- onstrate that the two-chain, kallikrein-cleaved, kinin-free high M, kininogen as well as the light chain isolated from this cleaved molecule quantitatively retain the procoagulant activ- ity of the native single-chain molecule. We also demonstrate that human high M, kininogen undergoes cleavage in plasma during contact activation and that this cleavage, which liber- ates kinin and gives a two-chain, disulfide-linked molecule, is dependent upon the presence of prekallikrein and Factor XII (Hageman factor). A preliminary report of this work has been presented (29).

MATERIALS AND METHODS

Purification of High M, Kininogen-High M, kininogen was iso- lated from titrated fresh human plasma. The plasma (2.2 liters) was obtained as previously described (30) and dialyzed at 20°C against the starting buffer (40 mM Tris, 10 mM succinic &id, pH 8.2, contain- inp 1 mM EDTA, 1 mM benzamidine. 50 ucln/ml of Polvbrene (Aldrich Chemicals), 0.02% NaN3, and 13 rnM’Nabfi for chromatography on a column (10 x 40 cm) containing 140 g of DEAE-Sephadex A-50 (Pharmacia). The protein fraction that did not adhere to the resin contained prekallikrein and Factor XI and flowed through the column in the starting buffer. The column was then washed with a buffer containing the same components, but made 121 mM in Tris, 44 mM in succinic acid, pH 7.7, and 103 mu NaCl (15 mmho conductivity). This buffer effected the elution of plasminogen and Factor XII. After extensive washing with this buffer, the high M, kininogen was eluted at 480 ml/h with a buffer containing the same components as above but made 193 mM Tris, 75 mM succinic acid, pH 7.4 and 182 mM NaCl (24 mmho). Fractions containing high M, kininogen were pooled and brought to 50% saturation of (NH&S04 by addition of solid (NH&SO4 at 4°C. After stirring for 90 min, the precipitate was collected by centrifugation for 1 h at 2000 X g. The precipitate was dissolved in 130 ml of buffer containing 50 mM sodium acetate, 75 mM

NaCl, 0.5 mM EDTA, 0.5 mu benzamidine, and 0.01% NaNa at pH 5.3. After extensive dialysis in this buffer, a slight precipitate was eliminated by centrifugation for 20 min at 1200 x g, and the sample was then applied to an SP-Sephadex C-50 column (2.5 x 13 cm). After the sample entered the gel, the column was washed at 30 ml/h with 450 ml of a buffer containing 0.1 M sodium acetate, 150 mM NaCl, 1 mM EDTA, 1 mM benzamidine, and 0.02% NaN3, pH 5.3. A linear gradient elution was then performed with 150 ml of the latter pH 5.3 buffer in the stirred proximal chamber and 150 ml of an identical buffer but containing 500 mM NaCl in the distal chamber.

Clotting Activity of High M, Kininogen-High M, kininogen clot- ting activity was measured by two-stage kaolin-activated partial

12020

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thromboplastin times (31) using 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 titrated human plasma.

Protein concentrations were determined by the Lowry method (32) using bovine serum albumin (Sigma) as reference protein.

Polyacrylamide Gel Electrophoresis-Electrophoresis was carried out in the presence of SDS’ on 7.5% or 10% polyacrylamide gels (6 mm x 8 cm) according to Weber et al. (33). The gels were stained for proteins with Coomassie blue G-250.

Ultracentrifugal Analysis-Sedimentation equilibrium experi- ments were performed through the courtesy of Dr. Sally Hoch (De- partment of Cellular Biology), using a Beckman/Spinco model E analytical ultracentrifuge equipped with a scanner, ultraviolet optics, and a multiplexer. The samples were centrifuged using 12-mm filled Epon double-sector cells equipped with sapphire windows coated with silicone (Aquasil, Pierce Chemical Co.). High M, kininogen (0.4 mg/ ml) in 0.1 M sodium acetate, 0.15 M NaCl, 1 mM EDTA, and 0.02% azide, pH 5.3, was centrifuged at 12’C for 26 h at 10,006 rpm. The sample was shown to be at equilibrium since scans taken at 22 h and 26 h showed no more detectable variation than that expected from experimental error. At the end of the run, the speed was increased to 50,000 rpm to deplete the meniscus; after deceleration to the experi- mental velocity to 10,000 rpm, measurements were made for base-line corrections. The slope of a plot of the natural logarithm of the optical density at 280 nm as a function of the square of radial distance was linear, and the M, was determined from the slope of such plots. The M, value was calculated assuming c = 0.718 g/liter.

Human Plasma Kallikrein-Prekallikrein was purified from fresh human plasma as described elsewhere.’ Kallikrein was obtained by activation of the precursor molecule.* The kallikrein activity was usually quantitated as amidolytic activity by the hydrolysis of the synthetic substrate Bz-Pro-Phe-Arg-paranitroanilide (Vega Fox).*

Radiolabeling of High M, Kininogen-High M, kininogen was labeled with either ‘25I or I31 I using the chloramine-T method (34). Radioactivity was measured with a Searle automatic y-spectrometer (model 1195). The high M, kininogen retained its procoagulant activ- ity after the radiolabeling procedure and contained from 1 to 10 pCi/

I%. Preparation and Purification of the Heavy and Light Chains of

High M, Kininogen-Twelve milligrams of high M, kininogen in 12 ml were dialyzed into 0.1 M Tris-HCl buffer, pH 8.1 containing 0.14 M NaCl. The dialyzed sample was incubated in 10.2 ml for 5 h at room temperature with human plasma kallikrein at 8 pg/ml. Analysis of the reaction mixture on reduced SDS-polyacrylamide gels showed a complete cleavage of high M, kininogen under these conditions. The cleaved sample was dialyzed into 0.2 M Tris-HCl, pH 8.7, containing 4 M urea and 0.5 mM EDTA. The material was then reduced by incubation for 1 h in the presence of 50 mM dithioerythritol (Pierce Chemical Co.) under nitrogen and then alkylated for 45 min in the dark by addition of 120 mM iodoacetamide. After elimination of the reagents by dialysis against 0.1 M sodium acetate, pH 5.8, 80 mM NaCl, 1 mM EDTA, 1 mM benzamidine, 0.02% NaNa, and 4 M urea, the polypeptide chains were renatured by dialysis into the same buffer without urea. The sample was then applied to a SP-Sephadex C-50 column (1.5 x 7 cm) equilibrated in the same buffer. Five bed volumes of buffer were used to wash the column. The bound material was then eluted at a flow rate of 10 ml per h with a linear gradient using 30 ml of the starting buffer in the proximal chamber and an identical buffer containing 1 M NaCl in the distal chamber. The eluted fractions were analyzed for protein content and procoagulant activity.

Amino Acid Analysis-Twenty-microgram samples were hydro- lyzed in uacuo with 6 M HCl at 105°C for 24 h and were analyzed on a Beckman 121M amino acid analyzer (35). Half-cystine was deter- mined as cysteic acid following performic acid oxidation (36). Tryp- tophan was analyzed in separate alkaline hydrolysates (37).

Kinin Bioassays-Kinin activity was measured as uterus-stimulat- ing activity on isolated rat uterus. The uterus was obtained from virgin rats that were injected intraperitoneally with 100 pg of dieth- ylstilbestrol diluted at 1 mg/ml in mineral oil at 18 h prior to being sacrificed. The isolated organ was suspended in a bath of 20-ml capacity, in De Jalon’s solution containing 0.15 M NaCl, 5.4 mM KCl, 0.54 mu CaC12, 5.55 mM glucose, 6 mM NaHC03, pH 8.07 at 30°C.

’ The abbreviation used is: SDS, sodium dodecyl sulfate. ’ B. N. Bouma, L. A. Miles, G. Beretta, and J. H. Griffin, manuscript

in preparation.

The activity was measured by the latent period between administra- tion of the fluid being tested and the onset of contraction. The sensitivity of the response was calibrated with a standard solution of bradykinin (Sandoz Pharmaceuticals) by adding 0.5 to 5 ng to the bath.

Kinin Liberation and Clotting Activity of High M, Kininogen Incubated With Kallikrein-High M, kininogen (final concentration 230 pg/ml) was incubated at 37°C with plasma kallikrein (final concentration 0.65 pg/ml) in 1.6 ml of 10 mM Tris-HCl buffer, pH 8.1, and 50-~1 aliquots were withdrawn as a function of time and mixed with a 25-pl solution containing 76 pg/ml of soybean trypsin inhibitor (Sigma) and 2.4 mM orthophenanthroline. Under these conditions the soybean trypsin inhibitor blocked kallikrein activity immediately. One to 5 ~1 of each sample containing 0.13 to 0.66 pg of high M, kininogen were then tested for kinin content in the rat uterus bioassay, and 10 ~1 of each sample were diluted 1 to 60 into Tris-buffered saline containing 1 mg/ml of bovine serum albumin for analysis of high M, kininogen procoagulant activity.

Kinin Liberation and Cleavage of High M, Kininogen in Plasma During Contact Actiuation-Seventy microliters of lZ51-high M, ki- ninogen (6 PCi, 0.2 clotting unit/ml) in Tris-buffered saline were mixed with the same volume of either normal human plasma or prekallikrein-deficient or Factor XII-deficient plasmas in plastic tubes. To initiate contact activation, this 140~~1 sample was mixed in a siliconized glass tube with 87 ~1 containing 16 mg/ml of kaolin, 5 times diluted rabbit brain cephalin (Sigma), and 20 IMI CaC12 in Tris- buffered saline. Ten-microliter aliquots were withdrawn as a function of time and rapidly added to siliconized glass tubes in a boiling water bath containing 100 ,sl of 1% SDS, 8 m urea, and 1% P-mercaptoeth- anol. The samples were boiled for 8 min and electrophoresed on 10% SDS-polyacrylamide gels. The gels were then sliced and the radioac- tivity of the slices determined. Data were recorded on punchtape and analyzed on a Hewlett-Packard 9810A calculator and plotter. In order to determine the kinetics of kinin liberation in plasma during contact activation, identical experiments without the radiolabeled high M, kininogen were performed in a total volume of 2 ml. As a function of time, 100~~1 aliquots were withdrawn and rapidly added to 200 ~1 of boiling 0.9% NaCl (12). This heat treatment was sufficient to quench totally the liberation of kinin. After the samples were held in a boiling water bath for at least 5 min, each sample was brought to room temperature and assayed in the kinin bioassay.

RESULTS

Preparation of Human High Mr Kininogen-The purifi- cation steps and the yield for a typical preparation of human high M, kininogen are shown in Table I. The purification steps are described under “Materials and Methods.” The DEAE-Sephadex chromatography step allowed the separa- tion of high M, kininogen from Factor XI and prekallikrein. Factor XII and plasminogen were subsequently eluted with the 15mmho buffer. High M, kininogen was eluted from the DEAE-Sephadex at a conductivity of 24 mmho. The bulk of the remaining contaminating proteins was then eliminated during the sulfopropyl-Sephadex chromatography. At pH 5.3, high M, kininogen bound to the sulfopropyl-Sephadex column, while the other proteins did not, and was subsequently eluted with a NaCl gradient at 23-mmho conductivity. This purifi- cation procedure gave high M, kininogen in 35% yield with a lOOO-fold purification. Based on the observed specific activity of 16 clotting units/mg, the concentration of high M, kininogen in plasma is estimated to be 61 pg/ml. Over a 2-year period,

TABLE I

Purification of human high M, kininogen

,T^Llme Total " "11 Clotting Speciti c

protein activity clottin) RtXOV- :

activitl f -Y

ml g units units/mg of % protein

Plasma 2200 138.6 2200 0.0159 DEAE-Sephadex 1340 3.6 2077 0.574 94 Ammonium sulfate 160 1.34 1460 1.09 66

precipitation SP-Sevhadex 48 0.048 770 16.0 35

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12022 Human High M, Kininogen

determinations of the specific clotting activity of four different preparations of high iW, kininogen using different samples of deficient plasma have given values ranging from 13 to 19 clotting units per mg.

Molecular Weight of High M, Kininogen-In SDS-poly- acrylamide gels in the absence of reducing agents, high M, kininogen migrated as a single protein band, as shown in Fig. 1, corresponding to an apparent M, of 110,000. The presence of reducing agents did not modify the apparent M,, showing that the molecule was isolated as a single polypeptide chain.

The M, of native high M, kininogen determined by sedi- mentation equilibrium methods was 108,000 + 2,000.

Cleavage of Human High M, Kininogen by Human Plasma Kallikrein-The proteolytic cleavage of high M, kininogen in solution by purified kallikrein was studied. A mixture of unlabeled and 1311-labeled high M, kininogen at 2.5 clotting units/ml was incubated in the presence of 0.19 pg/rnl of kallikrein, at pH 8.1. Aliquots were withdrawn at a function of time and rapidly added to an excess of boiling hot SDS to quench the reaction. The samples were then analyzed by electrophoresis on SDS-polyacrylamide gels in the presence and absence of mercaptoethanol. As seen in Fig. 2 on unre- duced gels, the protein corresponding to the 110,000 M, species disappeared with increasing time, while two other species of slightly faster mobility were progressively formed. The anal- ysis of the samples on reduced SDS gels showed that the cleaved molecules were made up of disulfide-linked poly- peptide chains. After 165min incubation (Fig. 2), the M, 110,000 form completely disappeared, showing that all the high M, kininogen single-chain molecules were cleaved, and two molecular species with apparent M, values of 65,000 and 44,000 were seen on reduced SDS gels.

In order to quantitate the kinetics of cleavage of 1311-high M, kininogen by kallikrein, the stained gels were sliced and

Unred Red

Reference Proteins

FIG. 1. Human high M. kiuiuogen on 7.5% SDS-polyacryl- amide gels. The gels were run in the presence or absence of 1% /3- mercaptoethanol and contained 11 pg of protein

Unreduced SDS Gels

110,000-

I /

1 x

-Ref.

Reduced SDS-Gels

110,000-

-Ref. 65,000-

44.000-

0 7.5 30 90 165

Time (min]

FIG. 2. Cleavage of high M, kininogen by purified human plasma kallikrein. In a plastic tube, a mixture of unlabeled and 13’1- labeled high M, kininogen (5.5 pCi/rnl, 2.5 clotting units/ml) was incubated in presence of 0.19 pg/ml of human plasma kallikrein at 37°C in 0.01 M Tris-HCl buffer, pH 8.1, in a total volume of 860 ~1. Forty-microliter samples were withdrawn as a function of time and added to 50 d of boiling 1% SDS, 8 M urea with or without 1% B- mercaptoethanol. The s&ples were boiled for 8 min and electropho- resed on 7.5% SDS-polyacrylamide gels. Each sample contained 4 pg of bovine serum albumin (Mr 68,000) as an internal reference.

the radioactivity profiles on reduced gels were analyzed as a function of time. The results indicated that under the condi- tions used 50% of the single-chain form of high M, kininogen was cleaved in 17 min and that >90% of the radioactive molecules at 110,000 M, was cleaved in 90 min. In these experiments the cleavage of 1311-high M, kininogen as judged in the radioactivity profiles showed a good correlation to cleavage as judged by gel staining.

In order to analyze kinin liberation in relation to proteolysis of high M, kininogen by kallikrein, the presence of kinin in the incubated samples was examined. A parallel experiment was performed in which the kallikrein activity was stopped as a function of time by adding 50-~1 aliquots of the reaction mixture to 25 ~1 of soybean trypsin inhibitor and orthophen- anthroline (see under “Materials and Methods”). The samples were then tested for procoagulant activity and for the presence of kinin. Kallikrein progressively liberates kinin from high M, kininogen, and the amount of kinin maximally reached 1% of the weight of incubated high M, kininogen. The clotting activity of the kinin-free high M, kininogen molecule remained

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Human High M, Kininogen 12023

invariably the same, showing that kinin release does not result in any loss of procoagulant activity.

Isolation of a Kinin-containing Cleaved High M, Kinino- gen-In approximately half of our preparations of high n/i kininogen a disulfide-linked, two-chain form of high M, kinin- ogen copurified with the single-chain molecule. This species migrated on unreduced SDS gels as a single protein band exhibiting a migration slightly faster than the single-chain molecule. This two-chain form of high M, kininogen contained kinin. The intermediate form of the high M, kininogen ob- served during kallikrein cleavage (Fig. 2) exhibited an identical mobility on SDS-polyacrylamide gels in the absence of reduc- ing agent.

Purification of the Heavy and Light Chains of Kinin-free High M, Kininogen-High M, kininogen (12 mg) was cleaved by human plasma kallikrein under conditions described under “Materials and Methods” to provide material for isolation of the polypeptide chains of the kinin-free molecule. As deter- mined by a rat uterus bioassay, kinin was entirely liberated from high M, kininogen, while the clotting activity of this molecule remained unchanged. The kinin-free high M, kinin- ogen migrated as a single band on an SDS-polyacrylamide gel in the absence of reducing agent. Analysis of this same sample on a reduced SDS-polyacrylamide gel verified (Fig. 3) that the cleaved high M, kininogen was a disulfide-linked, two- chain molecule made up of a light chain and a heavy chain with apparent M, values of 44,000 and 65,000. For the isolation of the heavy and light chains, kinin-free high M, kininogen was reduced and alkylated as described under “Materials and Methods.” The alkylated light chain and heavy chain were then separated by sulfopropyl-Sephadex chromatography. At a 13-mmho conductivity at pH 5.8, the heavy chain did not bind to the resin, while the light chain was retained. A NaCl

Kinin. Native Free Heavy light HMrK HMrK Chain Chain

gradient was then applied to elute the light chain. The elution peak appeared at a conductivity of 40 mmho. As seen in Fig. 3, the isolated alkylated heavy and light chains migrated as single bands on SDS-polyacrylamide gels and exhibited the respective apparent M, values of 65,000 and 44,000.

Activities of the Isolated Heavy and Light Chains of High M, Kininogen-The clotting activities of the isolated alkyl- ated heavy and light chains were determined and compared to the activities of native and kinin-free high M, kininogen. The results (Table II) show that the isolated light chain exhibited a clotting of 31 units per mg or 1.4 units per nmol, a specific clotting activity identical with that of the native molecule. Thus, the light chain accounts for the entire activity of the native and of the kinin-free molecules. The heavy chain did not show any detectable activity. These quantitative re- sults indicate that the procoagulant activity of high M, kinin- ogen resides exclusively in the light chain region of the mole- cule without any apparent participation of the heavy chain region in this activity.

Amino Acid Composition of Human High M, Kininogen and of Its Heavy and Light Chains-The amino acid com- positions of high M, kininogen and of the isolated heavy and light chains were determined and are shown in Table III. In general, the amino acid compositions of the human molecules are very close to those of bovine high M, kininogen and of the heavy and light chains isolated after cleavage of this molecule by human urinary kallikrein (26). In particular, the 11.2% histidine content of the human light chain is very high, and it reflects most of the histidine content of the native molecule. Interestingly, this proportion of histidine is the same as that reported for the bovine fragment that contains an unusual histidine-rich region named Fragment 1.2 (26).

TABLE II

Procoagulant activity of high M, kininogen and its fragments Aliquots of the different molecular species were analyzed in clotting

assays as described under “Materials and Methods.” The molecular weights were estimated to be 110,000,44,099, and 65,000 for the native or kinin-free high M, kininogen, the light chain, and the heavy chain, resoectivelv.

110,000-

-65,000

-44,000

FIG. 3. Reduced 10% SDS-polyacrylamide gels of native high M, kininogen (H&K), kinin-free high M, kininogen, and iso- lated alkylated heavy chain and light chain. Fifteen micrograms of native high M, kininogen or kinin-free high M, kininogen and 10 pg of isolated heavy or light chain were analyzed in presence of 1% /.?-mercantoethanol.

Form of high M, ki- Native high Kinin-free

ninogen M, kininogen hi$,o$ef- yw; Heavy

chain

Clotting units per 13 12 31 Cl

ClZing units per 1.4 1.3 1.4 (0.06 nmol

TABLE III Amino acid composition of human high M, kininogen

Amino acid Residues per 100 residues

Native Heavv chain Lieht chain

Lysine Histidine Arginine Aspartic acid Tbreonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan Half-cystine

7.8 4.5 3.5

10.9 7.9 7.2

12.5 6.8 7.7 5.0 4.8 0.95 4.7 6.0 2.5 3.6

1.0 2.7

7.4 a.7 1.3 11.2 2.8 1.4

12.3 12.5 8.2 6.7 6.4 7.6

13.7 10.2 5.3 8.4 5.1 12.7 6.0 2.5 6.0 1.9 0.73 1.0 5.6 4.2 5.9 6.0 3.7 0.73 4.1 2.8 0.80 0.86 4.73 0.57

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12024 Human High 34, Kininogen

Cleavage of High M, Kininogen in Plasma-The func- tional role of high M, kininogen as a cofactor in the activation of surface-bound Factor XII by kallikrein as well as in the activation of Factor XI or prekallikrein by surface-bound activated Factor XII has been previously demonstrated (23, 24). It has also been shown that prekallikrein is very rapidly activated to kallikrein during the contact activation process2 (23). Since purified high M, kininogen is susceptible to cleav- age by purified human plasma kallikrein, the question arose of whether or not the newly generated kallikrein cleaves the single-chain high M, kininogen during contact activation. To analyze this question, ‘251-high M, kininogen was added to normal plasma or to plasmas deficient to prekallikrein or Factor XII. As described under “Materials and Methods,” contact activation of plasma was induced by kaolin, and aliquots withdrawn as a function of time were analyzed on reduced SDS-polyacrylamide gels and in kinin bioassays. As seen in Fig. 4, the radioactivity profiles of the gels indicate that the single-chain high M, kininogen was rapidly cleaved. Maximum cleavage was reached in less than 100 s after kaolin was added. The kinetics of cleavage shown in Fig. 5 indicate that following an initial lag period half of the high M, kinin- ogen molecules were cleaved at 36 s. Kinin appearance very closely paralleled the cleavage of high M, kininogen. The maximum amount of kinin liberated was 0.19 pg/rnl (Fig. 5). Assuming kinin represents 1% of the mass of high M, kinino- gen and correcting for the dilution of plasma in these experi- ments, it is calculated that the kinin released reflects cleavage of 62 pg/ml of high M, kininogen in the original plasma. In

4T 100 Set L!d!lL-

50 Set ILL-

40 Set L L J 0 10 20 30 40 50

SOS Gel Slice Number

FIG. 4. SDS-polyacrylamide gel profiles of “‘I-high M, ki- uinogen in normal plasma during contact activation. Contact activation of normal human plasma containing ““I-high M, kininogen was induced by kaolin as described under “Materials and Methods.” Ten-microliter aliquots were withdrawn as a function of time and added to 100 $ of boiling 1% SDS, 8 M urea, and 1% P-mercaptoeth- anal. The gels were sliced and analyzed for their radioactivity profiles. In controls, it was verified that the denaturing solution eluted >95% of the radiolabeled material from kaolin and that the presence of kaolin in the samples loaded on the gels did not influence the profiles observed.

Time (Secl

FIG. 5. Kinetics of kinin formation and cleavage of ‘%high M, kininogen during contact activation. The radioactivity of the protein band corresponding to the uncleaved 110,000 M, molecule on the gel profiles shown in Fig. 4 was quantitated to determine the percentage of cleavage of ?-high M, kininogen in normal plasma (W). Identical experiments were performed in studies of lz51- high M, kininogen added either to prekallikreiu-deficient plasma (A- - -A) or Factor XII-deficient plasma (U - -0). The kinin ap- pearance as a function of time in normal plasma during contact activation was defined using kinin bioassays as described under “Ma- terials and Methods” (0- - -0).

prekallikrein-deficient plasma or Factor XII-deficient plasma, no cleavage of high M, kininogen and no kinin release were observed. Addition of purified plasma kallikrein to plasmas deficient in either prekallikrein or Factor XII resulted in cleavage of the ‘251-high M, kininogen and a release of kinin. This indicates that kallikrein was sufficient for high M, kinin- ogen cleavage and kinin release and that Factor XII is not necessary if prekallikrein has been converted to kallikrein.

Structure of Kinin-free High M, Kininogen Cleaved in Plasma Compared To That Cleaved by Purified Kallikrein in Solution-A comparative analysis of the structure of 1251- high M, kininogen cleaved in plasma to that of the kinin-free molecule generated by purified kallikrein in solution was performed to determine whether or not the single chain pro- tein was cleaved identically under each set of conditions. To generate the molecular species arising from the cleavage of high M, kininogen in plasma, 15 ~1 of normal human plasma containing 1251-high M, kininogen was incubated for 2 min at 37°C in the presence of 5 mg/ml of kaolin in 60 ~1 of Tris- buffered saline. The kaolin in this reaction mixture bound 55% of the lz51-high M, kininogen. This kaolin was washed extensively by T&-buffered saline containing 1 mg/ml of bovine serum albumin. Then the proteins were eluted from the surface and electrophoresed on reduced 10% SDS-poly- acrylamide gels. A sample of ‘251-high M, kininogen cleaved in a purified system by plasma kallikrein was similarly analyzed on reduced 10% SDS-polyacrylamide gel. As seen in Fig. 6, high M, kininogen obtained from plasma incubated with ka- olin was a cleaved molecule made up of two polypeptide chains that exhibited the same apparent M, values as the heavy and light chains formed when high M, kininogen was cleaved in solution by plasma kallikrein. In gel profiles not shown, the ‘251-high M, kininogen found in the plasma super- natant at the end of the incubation was also entirely cleaved. Moreover, as seen in Fig. 6, the light chain was poorly labeled using the chloramine-T method. This is probably due to the fact that the tyrosine content of the light chain is low (0.7%) compared to that of the heavy chain (3.7%).

The Mobility of Kinin-free High M, Kininogen on Reduced SDS-Polyacrylamide Gels is Influenced by Plasma Pro-

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Human High M, Kininogen 12025

110,000 65,000 44,000 15 I 1 1

T F 1 h - KAOLIN-BOUND

HMrK FROM PLASMA

- - CLEAVAGE BY PURlFlE0 KALLIKREIN

SDS Gel Slice Number

FIG. 6. Comparison of ‘ZSI-(kinin-free) high M, kininogen (HM,.K) cleaved on kaolin in plasma (--) to that cleaved by purified kallikrein in solution (- - -) on reduced 10% SDS- polyacrylamide gels. Fifteen microliters of normal human plasma containing lZ51-hiah M, kininoaen (0.4 &Ji, 0.2 ug) were incubated as described-in the &xt in presence of kaolin: To &e washed kaolin was added 40 ~1 of 5% SDS, 4 M urea, 50 mM EDTA, and 2.5% /3- mercaptoethanol. After boiling for 10 min, the sample was analyzed on SDS-polyacrylamide gel (-). lZ51-High M, kininogen (0.2 PCi, 3 clotting units/ml) was incubated for 30 min at 37°C in the presence of 25 pg/ml of plasma kallikrein in a total volume of 55 ~1 in 0.01 M

Tris-HCl buffer, pH 8. The incubated sample was boiled for 8 min in presence of 50 ~1 of 1% SDS, 8 M urea, 1% /3-mercaptoethanol, and 0.1 M EDTA and analyzed on SDS-polyacrylamide gels (- - -).

teins-The reduced SDS-polyacrylamide gels profiles of kinin-free high M, kininogen seen in Fig. 4 did not show distinct peaks for the heavy and light chains in the samples that underwent cleavage in the plasma. This observation suggested that the presence of plasma proteins in the loaded sample might influence the migration of the cleavage prod- ucts. In order to explore this possibility, high M, kininogen was cleaved by plasma kallikrein either in the absence or in the presence of other plasma proteins and was analyzed on reduced SDS-polyacrylamide gels. In the presence of 16 pg/ ml of plasma kallikrein, ‘251-high M, kininogen (15 @i/ml, 0.28 clotting unit/ml) was incubated for 35 min at 37”C, either in Tris-buffered saline, pH 7, or in prekallikrein-deficient plasma diluted 1 to 3 with Tris-buffered saline in a total volume of 25 ,ul. The incubated samples were then diluted 4- fold and boiled in a reducing SDS solution. At the same time, 1311-high M, kininogen (20 &i/ml, 0.28 clotting unit/ml) was incubated for 35 min at 37°C in the presence of 16 pg/ml of plasma kallikrein in Tris-buffered saline in a total volume of 100 ~1. This sample was then diluted 2-fold and boiled in a reducing SDS solution. Two 50-/d aliquots were withdrawn from this 1311-labeled reaction mixture and mixed with 50 ~1 of the lz51-high M, kininogen that had been cleaved either in Tris-buffered saline or in the plasma. The two mixed samples were then analyzed on SDS-polyacrylamide gels. Fig. 7A shows the gel profiles of 1311-high M, kininogen and ‘251-high M, kininogen incubated with kallikrein and electrophoresed in the absence of plasma proteins. Both 1311-high M, kininogen and ‘251-high M,-kininogen were cleaved and made up of light and heavy chains of apparent M, 44,000 and 65,000. However, as seen on Fig. 7B, the profile of ‘251-high M, kininogen incubated with kallikrein and electrophoresed in the presence of plasma proteins is similar to the profiles seen on Fig. 4, showing the cleavage of high M, kininogen in normal plasma during contact activation. The profile seen in Fig. 7A of 1311- high M, kininogen cleaved in a purified system is changed in Fig. 7B indicating that simply the presence of 240 pg of plasma proteins during the electrophoresis modified the apparent mobility of both the heavy and light chains of high M, kinin- ogen.

- A 65,000 + 44,000

20- + CLEAVE0 ffl ABSENCE OF

- 110,000 PLASMA PROTEINS

$

- - CLEAVE0 IN

-CLEAVED IN

10 20 30 40 50 SDS Gel Slice Number

FIG. 7. Influence of the presence of plasma proteins on the mobility of the heavy and light chains of kinin-free high M. kininogen on reduced SDS gels. The ‘*‘I-labeled samples and the 1311-labeled samples incubated under the conditions described in the text were mixed with SDS under reducing conditions and analyzed on 7.5% SDS-polyacrylamide gels. A, mixture of 50 ~1 of ?-high M, kininogen (- - -) and of 50 ~1 of 13’1-high M, kininogen (----) both cleaved by purified kallikrein in solution. B, mixture of 50 ~1 of lZ51- high M, kininogen cleaved by kallikrein in prekallikrein-deficient plasma (- - -) and of 50 ~1 of 13’1-high M, kininogen cleaved by purified kallikrein in solution (---).

DISCUSSION

Human high M, kininogen purified as described here is a single polypeptide chain of M, 110,000. Previously, highly purified high M, kininogen composed of two disulfide-linked chains of 70,000 and 50,000 M, was isolated (8) although Nagasawa and Nakayasu reported the isolation of a single- chain molecule (10). Initially, we were able to isolate a two- chain form (23). Other authors reported obtaining a mixture of two distinct species of high M, kininogen (38, 39). The two species were kinin-containing molecules in either a one-chain or a two-chain form (38, 39). Thompson et al. reported the isolation of high M, kininogen mainly as a single polypeptide chain (40). Taken together, these results suggest that human high M, kininogen is originally a single chain 110,000 M, molecule that is proteolytically converted either in uivo or during the isolation procedure into a two-chain species.

The major kininogenase of plasma circulates as a zymogen, prekallikrein, that is activated to kallikrein by limited prote- olysis2 by activated Factor XII (41). Incubation of high M, kininogen with purified human plasma kallikrein was per- formed and showed, in agreement with other reports, that kinin is progressively liberated, and that a two-chain, disul- fide-linked high M, kininogen is formed (10, 38-40). In the kallikrein cleavage studies, a form of high M, kininogen ap- peared on mu-educed SDS-polyacrylamide gels that had a mobility that was intermediate between the native single- chain molecule and the final two-chain product. This inter- mediate form of the molecule may be identical with the two- chain form of high M, kininogen that contains kinin. Following cleavage by plasma kallikrein, high M, kininogen is made up of a heavy chain of apparent M, 65,000 and a light chain of apparent M, 44,000 that are disulfide linked. Following reduc-

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12026 Human High M,. Kininogen

tion and alkylation, the heavy and light chains can be isolated using chromatography on a sulfopropyl-Sephadex column at pH 5.8. Analysis of the procoagulant activity of the two-chain, kinin-free molecule and of the alkylated isolated heavy and light chains indicates that the alkylated light chain quantita- tively retains the procoagulant activity of the native molecule and of the two-chain disultide-linked molecule. The alkylated heavy chain does not show any detectable procoagulant activ- ity. This quantitative estimation of the respective activities of high M, kininogen and its fragments is consistent with the previous qualitative observations that human kinin-free high M, kininogen as well as alkylated light chain shorten the clotting time of high M, kininogen-deficient plasma (40).

Suzuki and his colleagues purified bovine high n/l, kininogen as a single-chain molecule (42, 43) and demonstrated that human urinary kallikrein cleaves the molecule resulting in kinin liberation and giving a two-chain molecule containing an NHp-terminal heavy chain and a COOH-terminal light chain (26, 44). Waldmann et al. (27) and Wuepper et al. (28) showed that this kinin-free high M, kininogen and the carbox- ymethylated light chain are as procoagulant as the native bovine molecule when measured in human high M, kininogen- deficient plasma. However, when bovine high M, kininogen is cleaved by bovine plasma kallikrein, the procoagulant activity is entirely lost (21, 22). This loss of activity is correlated with the release, along with kinin, of a histidine-rich glycopeptide fragment (25, 26,43,44). Sequence studies indicated that this histidine-rich fragment is situated in the NHz-terminal end of the light chain region of bovine high M, kininogen (25).

This report presents the amino acid composition of human high M, kininogen and of its fragments formed by cleavage with human plasma kallikrein. In general, the amino acid compositions of the human polypeptide chains are very similar to those of the corresponding bovine polypeptide chains (26). The histidine content of the isolated human light chain is 11.2% and reflects most of the histidine content of the native molecule. This proportion of histidine is the same as that observed in the bovine polypeptide chain obtained by cleavage of that molecule with human urinary kallikrein (26). Histidine- rich polypeptide fragments have been isolated from proteo- lytic digests of human high M, kininogen (45). It appears, therefore, that the active human light chain contains the unusual histidine-rich region. It is particularly noteworthy that human plasma kallikrein does not readily liberate the histidine-rich region from human high M, kininogen as shown here and elsewhere (39), whereas bovine plasma kallikrein readily releases the histidine-rich fragment from bovine high M, kininogen with a consequent loss of procoagulant activity (uide supra) .

kinetic studies of surface-dependent reactions, it was shown that high M, kininogen functions as a stoichiometric cofactor in the activation of Factor XI as well as of prekallikrein by surface-bound activated Factor XII (23,24). Moreover, it was observed that high M, kininogen also facilitated the limited proteolysis of surface-bound Factor XII by kallikrein (23,46). It was, therefore, suggested that Factor XII and high M, kininogen associate with each other on a negatively charged surface and that this association facilitates the action of acti- vated Factor XII on its substrates. It was shown that high M, kininogen associates in solution with prekallikrein (47) or with Factor XI (48) and that high M, kininogen is indeed necessary for the binding of prekallikrein or Factor XI to negatively charged surfaces in the milieu of plasma (49). As demonstrated here, kaolin added to plasma containing lz51-high M, kininogen binds the labeled molecule. Moreover, a region of high M, kininogen, the light chain, was isolated that is rich in histidine and that binds fiiy to negatively charged resins. Thus, it appears that high M, kininogen binds to negatively charged surfaces through this histidine-rich region and acts as a sur- face-bound receptor for Factor XI and prekallikrein where these molecules are activated by surface-bound activated Fac- tor XII (49, 50). Factor XI, molecules remain bound at the surface sites of activation, in contrast to kallikrein molecules that readily dissociate into the surrounding space (49, 51). Using immunological techniques we have recently shown that the isolated light chain is by itself able to bind prekallikrein.3 This emphasizes the essential and sufficient role of the light chain moiety of high M, kininogen for the contact activation cofactor activities of the parent molecule.

Since high M, kininogen is rapidly cleaved in plasma during contact activation, the question arises of whether or not the molecule has to be cleaved to be procoagulant. Kato et al. (52) recently reported that the rate of kallikrein generation in a purified system containing bovine Factor XII, bovine plasma prekallikrein, and kaolin is more effectively accelerated in the presence of a nicked bovine high M, kininogen than in the presence of the native single chain form of the molecule. Thus, these authors suggested that limited proteolysis of high M, kininogen during the early phase of contact activation diges- tion may transform this molecule into an “active” high M, kininogen. Further studies will be required to establish whether conformational changes in human high M, kininogen due to limited proteolysis are essential or relevant for the activity of this molecule as a cofactor in contact activation.

When contact activation is induced by addition of kaolin to plasma, rapid cleavage of lz51-human high M, kininogen and liberation of kinin occur (Fig. 5). The time course of kinin appearance closely parallels the cleavage of ?-high M, kinin- ogen. This cleavage in plasma gives a disulfide-linked molecule similar to the molecule cleaved by kallikrein in purified reac- tion mixtures as judged by reduced SDS-polyacrylamide gel electrophoresis. In prekallikrein-deficient or Factor XII-defi- cient plasma, no cleavage of high M, kininogen was observed upon kaolin addition. However, addition of purified plasma kallikrein to plasmas deficient in either prekallikrein or Factor XII resulted in cleavage of high M, kininogen and in the release of kinin. Thus, it appears that the plasma kallikrein generated during contact activation is responsible for the rapid cleavage of high M, kininogen in plasma during contact activation and for the release of kinin.

Acknowledgments-The support and encouragement of Dr. Charles G. Cochrane is gratefully appreciated. The assistance of Dr. Sallie Hoch in the determination of the M, of high M, kininogen using the ultracentrifuge is gratefully acknowledged.

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D M Kerbiriou and J H Griffinactivation of plasma.

relationships and of proteolysis of the molecule occurring during contact Human high molecular weight kininogen. Studies of structure-function

1979, 254:12020-12027.J. Biol. Chem. 

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