Some molecular and functional changes in high molecular weight kininogen induced by plasmin and trypsin

THROMBOSIS RESEARCH 25; 387-399, 1982 0049-3848/82/050387-13$03.00/O Printed in the USA. Copyright (c) 1982 Pergamon Press Ltd. All rights reserved.

SOME MOLECULAR AND FUNCTIONAL CHANGES IN HIGH MOLECULAR WEIGHT KININOGEN INDUCED BY PLASMIN AND TRYPSIN"

Jozef Kleniewski, M.D., Ph.D.** Virginia H. Donaldson, M.D. and Constance J. Wagner, B.S. *Departments of Pediatrics and Medicine, University of Cincinnati College of Medicine, and the Children's Hospital Research Foundation, Cincinnati, Ohio 45229 **Present Address: Department of Cell Pathophysiology, Post Graduate Medical School, Warsaw, Marymoncka 99, Poland

(Received 22.9.1981; in revised form 10.1.1982. Accepted by Editor O.D. Ratnoff) ABSTRACT When purified human HMW-kininogen was digested by plasmin, its specific antigenic properties were initially enhanced and then gradually destroy. ed, but its clot-promoting activity (Fitzgerald factor activity) was only slightly decreased. When endogenous serum plasminogen was activated by streptokinase, similar alterations in specific HMW-kininogen antigens and Fitzgerald factor activity occurred. In contrast, trypsin induced increased antigenic properties initially, but readily destroyed the Fitzgerald factor activity and less readily destroyed the specific HMW-kininogen antigenic properties in purified HMW-kininogen and in nor. ma1 human serum. When normal serum was treated with streptokinase, the antigenic properties shared by HMW and LMW-kininogens were in Sephadex G-200 fractions of lower molecular weight than in the case of untreated serum, but the elution volumes of specific HMW-kininogen antigens and Fitzgerald factor activity were not significantly altered. When prekallikrein-deficient serum was subjected to the same G-200 gel filtration process, there was a broad overlap in the elution volumes of antigens shared by both HMW and LMW-kininogens with specific HMW-kininogen antigenic and coagulant properties, which remained after streptokinase treatment of the serum. Despite the disparate rates of destruction of the antigenic and clot-promoting portion of HMW-kininogen by proteases these properties did not separate from one another during ion exchange chromatography.

INTRODUCTION The high molecular weight kininogen (HMW-kininogen) (1) in human plasma Key Words:

HMW-kininogen, plasmin, trypsin, kininogens, plasma kininogens 387

388

PROTEOLYSIS OF HMW-KISINOGEN

Vo1.25, ?lo.j

plays a key role in Hageman factor-mediated activation of coagulation, fibrinthe conversion of prekallikrein to kallikrein and subsequent kinin olysis, Two different antigenic determinants have been located generation (2,3,4,5). on the HMW-kininogen molecule (7), one of which is common to both high and low molecular weight kininogen (LM’W-kininogen) and is located on the heavy-chain portion of the molecule not active in contact-initiated clotting. The other antigen which is specific for HMW-kininogen (7) is located on the light chain portion of the molecule which is responsible for facilitating Hageman factordependent plasma reactions (6,8). Recent studies (9) demonstrated that upon exposure of HMW-kininogen to plasmin, the concentration of specific HMW-kininogen The present studies were undertaken to examine further antigens was increased. the effect of plasmin on the HMW-kininogen molecule to attempt to define the functional and structural alterations in the molecule induced by plasmin. Despite the cleavage of purified HMW-kininogen or of the HMW-kininogen in normal HMW-kininogen antigenic human serum by plasmin, and ultimate loss of specific Tryptic determinants, the coagulant functions of HMW-kininogen persisted. digestion of HMW-kininogen, on the other hand, destroyed both the coagulant and the antigenic properties of the kininogen following an increased expression of the concentration of the’antigenie determinants. MATERIALAND METHODS Fresh human plasma for isolation of high molecular weight kininogen (HMWkininogen) was obtained from the blood drawn into l/SO volume of 0.5 M sodium Plasma from a person with citrate, pH 5.0, containing 50 mMbenzamidine. which is markedly deficient in Fitzgerald hereditazy deficiency of kininogens, factor clotting activity (S), was obtained from blood drawn into l/SO volume (G.E. Dri-film, SC-87) glassof 0.5 M citrate buffer, pH 5. Silicone-coated oil (200 cetipoids viscosity, General ware and syringes were used, and silicone Citrated plasma deficient in Electric Co.) was used to coat plastic vessels. Fletcher factor (a prekallikrein found in normal plasma) was obtained from George King Bio-Medical, Inc., Kansas City, Kansas. Platelet poor plasma was separated by centrifugation of blood at 200 xg for 20 min. and recentrifugation Disodium ethylene-diaminetetraacetate of the plasma at 20,000 xg for 10 min. (EDTA) was added to a concentration of 1 mMand hexadimethrine bromide (Polybrene) HMW-kininogen was isolated by batch adsorption and in an amount of 50 pg/ml. elution from QAE-Sephadex A-SO followed by chromatography on the same resin, according to the procedure described by Saito (10). The fractions rich in HMWkininogen clotting activity were pooled, precipitated at 60% saturation with were chromatographed sequentially on QAEammonium sulfate and, after dialysis, by Sephadex A-SO and CM-Sephadex C-SO, by modifying the procedures described Kerbiriou and Griffin (8) for HMW-kininogen purification on DEAE-Sephadex A-50 This kininogen gave a single band in SDS and SP-Sephadex C-50 respectively. activity of polyacrylamide gel electrophoresis (Figure 4), and had a specific 5 U/mg protein; 1 unit was that amount of HMW-kininogen clotting activity in 1 ml of normal human plasma in a specific assay for this activity (Fitzgerald factor) (5). Humanplasminogen was isolated by affinity chromatography (11). Plasmin was produced by incubation of 0.5 Christensen units of streptokinase with each 1 pg of plasminogen in 0.05 M Tris buffer pH 8.0 containing 0.1 M NaCl at 37OC activity of the plasminogen was 23.5 C.T.A. U/mg for 10 min. The specific using a substrate of alpha casein. protein, The presence of the antigen common to both plasma kininogens was detected by double immunodiffusion in 1% agarose gel pH 8.6, using goat antiserum (7).

The

PROTEOLYSIS

X0.5

Vo1.25,

antigen

specific

inhibition buffer

assay used

for (7,9),

for sample to prevent

HZW-kininogen using pooled and antiserum plasminogen

of heparin, indicator cells. Ten different by l/10 and ranging from l/20 quantitation measurement

mide

,

SDS polyacrylamide as described by The

(Factor the

of 0.5:; (0.45 of differences disc Weber,

clotting activity XII), P,T.h. (Factor

modified

partial

was normal

quantified by human plasma

dilution activation

dilutions to l/200

mcg) of of 0.5X

of each were tested

the amount of in concentration.

test (7).

was

time

(13)

and

The

M EACX and aggregation

35 U/ml of

preparation differing This allowed accurate

HMW-K in

HMW-kininogen (Fitzgerald XI), and Fletcher factor

thromboplastin

hemagglutination as a reference.

10 -3 contained and nonspecific

gel electrophoresis et al. (12). of

389

OF %W-SINi?iOGEN

normal

performed

plasma,

in

7.5;:

and

acryls-

factor), Hageman factor were quantified using

plasma

from

individuals

with

severe hereditary deficiency of the factor in question as a substrate. To measure Fletcher factor activity, the period of incubation before recalcificaThe capacity of test fractions to tion was reduced from 8 min. to 1 min. shorten the clotting time of a deficient substrate plasma provided an estimate of the amount of that clotting activity in the test fraction. Protein with a Gilford serum albumin

concentration was estimated by using 240 spectrophotometer, solution as a standard.

absorbance dilutions

at 280 nm, of crystalline

as

measured bovine

In an attempt to separate the coagulant from the antigenic portion of E,MW-K, purified HXW-kininogen (4 mg) was digested with freshly activated SK-plasmin for 25 min. at 37OC, and the mixture depleted of plasmin by adding lysine-Sepharose whicll was sedimented after 10 min. by centrifugation. After reduction with GTT, alkylation with iodoacetamide and dialysis, the digestion products were chromatographed upon a column of SP-Sephadex C-50, using a linear gradient of 0.1 to 0.6 M sodium chloride in a ph 5.8, 0.05 M sodium acetate buffer for elution. Effluent fractions were tested for Fitzgerald factor clotting activity and examined in SDS gels. Sepharose 4B, Sephadex C-200, QAE-Sephadex A-50, CM-Sephadex C-50, SPSephadex C-50 were obtained from Pharmacia Fine Chemicals Inc., Piscataway N.J.; acid washed kaolin and agarose from Fisher Scientific Co., Cincinnati, Ohio; tannic acid from Matheson Coleman and Bell, Norwood, Ohio; bovine serum albumin and lysine from Calbiochem, San Diego, Calif.; polyacrylamide from BioRad Labs. ; Richmond, Calif. ; crystalline salt-free trypsin from Worthington Biochemical Corp., Freehold, N.J. ; Tris HCL, soya bean trypsin inhibitor (SBTI) and iodoacetamide from Sigma Chemical Co., St. Louis, Xo.; Centrolexphospholipid from Central Soya, Chicago, Ill.; sodium dodecyl sulphate (SDS) and dithiothreitol; (DTT) from Eastman Kodak Co., Rochester, N.H. ; Polybrene from the Aldrich Chemical Co., Inc., Milwaukee, Wise.; benzamidine from MCB Manufacturing Chemists Inc., Darmstadt, Germany; and streptokinase from Lederle Laboratories, Pearl River, N.Y. Purified human a-thrombin was the gift of Dr. John Fenton, New York State Dept. of Health Laboratories, Albany, N.Y. The a-thrombin has a specific activity of 2.1 NIH U/up (14). All chemical reagents were of analytical grade.

RESULTS When

purified

HMW-kininogen

was

digested

with

streptokinase-activated

plasminogen (21 ug/ml mixture) the 120,000 molecular veig'ntband gradually disappeared and was no longer detectable in SDS gal electrophoresis after 60 min. The left gel, Figure 4, contains purified HMV-kininogen of 120,000 molecular weight. A fragment of 108,000 molecular weight appeared within 10 min. (Figure 1, seconl band, left gel), and other fragments of 92,000, 85,000 and 61,000 molecular weight were apparent after 30 min. digestion (Figure 1, second gel). After 60 min., only the 61,000 fragment was visible (Figure 1, third and fourth gels). Despite this disruption in the molecule, the coagulant properties of HMW-kininogen were easily detected even after these mixtures were incubated for 2 hrs. (not shown). The specific antigenic properties of HMW kininogen were increased after digestion with plasmin for 5 min., but after 10 min. they were progressively destroyed (Figure 2). The antigenic properties of HMW-kininogen in unfractionated serum incubated with streptokinase also increased after 5 min. and then deterioriated, despite retention of clot-promoting activity (Figure 3). These differences in antigen concentrations are significant for 10 dilutions of serum or HMW-R ranging from 0.5 to 5% of starting concentrations were tested, allowing detection of 0.5% changes in concentration. Similar changes in antigen concentrations were found in normal plasma after incubation with streptokinase, despite almost no loss in coagulant activity.

FIG. 1 SDS polyacrylamide gel electrophoresis of HMW-kininogen digested by plasmin: 350 ug of human HMW-kininogen dissolved in 0.7 ml of 0.05 M Tris pH 8.0 and O.lM NaCl was mixed with 0.06 ml of this buffer containing 16 ug of streptokinase-activated human plasmin (23.5 CTA U/mg) in Tris-sodium chloride. Plasminogen, 21 ug/ml, was activated with 16 units of streptokinase at 26OC for 20 min. At 10, 30, 60 and 120 min. (gels 1,2,3 and 4 respectively) of incubation at 37OC, samples of 4 different volumes were taken to contain 40,60,92 and 115 ug of HMW-kininogen

*01.23,

77

-

PROTEOLYSIS

x0.5

OF >B'WKISI:;OGEX

391

respecitvely, to obtain sufficient concentrations of digestion products to be visilalized in gels. Each of these and a sample of 40 '_g of undigested HYtikininogsn was boiled for 2 min. in 17; SDS and 1% 3 msrcaptoethanol and the sizes Human plasminogen (MJJ of component proteins compared during electrophoresis. 90,000), bovine serum albumin (MW 68,000) and human alpha thrombin (X4 37,000) in highly purified states were used as indicators of the electrophoretic behavior of proteins of these sizes in SDS gels (right gel, V5).

3-t 05io Time

I

i

30

60

of lncubahon

(mm)

FIG. 2 Fitzgerald factor clotting and specific antigenic activity of human HMWkininogen digested by plasmin: A sample of a mixture of 0.05 ml of HMW-kininogen (500 ng/ml) and plasmin (0.5 U/ml) or HMW-kininogen and 0.15 M NaCl (as described for Fig. 1) were taken at various times of incubation at 37OC, stirred after mixing with 0.1 ml of settled lysine-Sepharose for 5 min. at room temperaturre and the mixtures added to 2.0 ml of barbital-saline buffer containing 10-2 M EACA. The resin was separated by centrifugation and supernatants examined for clotpromoting activity attributable to HMW-kininogen (Fitzgerald factor) and for antigens in a hemagglutination-inhibition assay (7). FIG. 3 The influence of plasminogen activation on specific human HMU-kininogen antigen and Fitzgerald factor activity in NHS: 2.5 ml of normal human serum (NHS) was added to 0.15 ml of 0.15 M NaCl or the same volume of this solution containing 150 U of streptokinase. During incubation at 37OC, 0.5 ml samples taken at the intervals shown were stirred for 10 min. with 0.5 ml of settled lysineSepharose. The serum was separated by centrifugation, and HMW-kininogen antigen and Fitzgerald factor remaining were quantified (7).

When purified EE<-kininogen bias exposed to trppsin (10 ~.g per ml of mixture). the rate of loss of these two properties was reversed. There was rapid destruction of the H?iW-kininogen clotting properties and a transient rise followed by, a loss of antigenic properties (Figures 4,5). The HHW-Xininogen moiecule was cleaved by trypsin, producing an early fragment of 40,000 molecular weight within 5 min. (Figure 4) which lacked coagulant activity and was apparently resistent to further tryptic digestion.

Rome

of

lncubotm

(mtn)

FIG. 4 SDS polyacrylamide gel electrophoresis of human H?lW-kininogen digested by trypsin: 19 vol. of HMW-kininogen (0.5 mg/ml) dissolved in 0.1 M NaCl was mixed with 1 vol. of Tris buffer containing 200 ug/ml of trypsin or alone. Samples of 0.1 ml taken at different times of incubation at 37OC were added with 0.025 ml of SBTI (80 ug/ml), boiled for 2 min. in 1% SDS and 1% 8 mercaptoethanol and examined following electrophoresis in SDS-polyacrylamide disc gels. The gels from left to right contained samples taken after 0, 5, 10, 30 and 60 min. incubation. The gel to the right contained plasminogen, bovine serum albumin and thrombin; cathode on top. FIG. 5 Digestion of HMW-kininogen by trypsin; effect upon Fitzgerald factor clotting and HMW-kininogen antigenic activity: The samples of HMW-kininogen treated by trypsin and control samples incubated with buffer were prepared as in the experiment described in Figure 4.

~01.25, No.5

PROTEOLYSIS OF HMW-KININOGEN

393

These observations implied that the functional properties and specific antigen(s) of HH&kininogen are not in an identical part of the light chain Therefore, attempts were made to isolate separate fragportion of the molecule. ments containing functional and antigenic properties._ Plasmin-digestedpurified HMW-K was reduced and alkylated and chromatographedon SP-Sephadex C-50 (see methods) and effluent fractions were dialyzed and tested for Fitzgerald factor clotting activity and examined in SDS gels. Fitzgerald factor clotting activity was found in fractions containing 0.2 to 0.3 M sodium chloride and persisted in fractions containing 2 cleavage fragments of molecular weights of 48,000 and 58,000, which were the smallest plasmin-generatedfragments associated with coagulant activity. The functional properties of RMW-kininogenwere not separated from its specific antigenic properties by this procedure. Fitzgerald factor clotting activity was found in all fractions which had clearly detectable HMWkininogen antigens. The gel filtration behavior of RMW-kininogen and LMW-kininogenantigens in unfractionatedplasma and serum were characterized using a column of SephadexG-200. Antigens shared by HMW and LMW-kininogenswere found in fractions in two different regions of the effluent volume containing substances of 115,000 to 150,000 molecular weight (Figure 6). Fitzgerald factor clotting activity and specific HMW-kininogenantigen(s) were found only in the heavier fractions containing substances of 125,000 to 140,000 molecular weight (160-180ml, Figure 6, upper and middle panels). There was no difference in the filtration behavior of these properties in normal serum as compared to normal plasma (Figure 6). When the plasminogen in the normal serum was first activated by streptokinaseand the serum then filtered upon the same G-200 column, the effluent fractions which contained Fitzgerald factor clotting activity and specific HMW-kininogenantigens no longer contained the shared kininogen antigens located on the heavy chain (Figure 6, lower panel); the shared antigens were found only in fractions containing substances of molecular weights of 50,000 to 70,000 (230-260 ml), and may represent only LMW-kininogenmolecules in the serum. Since it was possible that the HMW-kininogen of higher molecular weight was complexed with prekallikrein (17-19), prekallikrein-deficient serum was filtered over the same G-200 column in an identical experiment. The properties of kininogens (Fitzgerald factor activity, HMW-kininogen antigen, and shared kininogen antigens) eluted in overlapping fractions of molecular weights of 120,000 to 280,000 (110-190 ml effluent, Figure 7a), as if some of the RMW-kininogen was complexed with proteins other than prekallikrein, possibly with PTA (20). When prekallikrein deficient serum which had been incubated with streptokinase was filtered over the Sephadex G-200 column, Fitzgerald factor activity was again eluted over a large volume (Figure 7b), in contrast to behavior in streptokinase treated normal serum (Figure 6), and much of the HMW-kininogen clotting activity was in lower molecular weight fractions than in the case of normal serum handled identically. Shared kininogen antigens were found in many of the same fractions as those containing specific HMW-kininogen antigens in both streptokinase and untreated prekallikrein deficient serum.

394

PROTEOLYSIS OF HMW-KINIEOGEN

GEL FILTRATION

BEHAVIOR

SERUM : G-200;EFFECT

OF HMW-KININOGEN

Vo1.25, So.5

IN PLASMA 45,000

158.000 MW

100

AND

OF STREPTOKINASE

180

MO

Volume

220

MW

260

(ml)

?? PrOleanCoM KO280)

1

A

Fttzqerald Fock~ Activity ( Y. of thot m Normal PL

0

HMW-Kinieaqen Antiqen ( % of Uvaf in Normal PL)

5

Kininoqsn Anttqenr OetacWls

5x Cone Fractions

FIG. 6

Sephadex 6200 filtration of normal human plasma (NHP), normal human serum 2 ml of NHP, 2 ml of NHS or 2 ml of NHS pre(MIS), treated with streptokinase: incubated with 60 U/ml of streptokinase were added to 1 ml of settled lysineSepharose and the mixture stirred for 10 min. at room temperature. The supernatants obtained were filtered through a 2.7 x 72 cm column of Sephadex G-200 equilibrated with 0.05 M Tris pH 8.0 in 0.1 M NaCl. Flow rate = 15 ml/h., Fractions = 5 ml each. Protein concentration, Fitzgerald factor and HMW-kininogen antigens were measured in unconcentrated samples. For the detection of the antigen common to both species of plasma kininogens (in double agarose diffusion), the samples were precipitated with addition of ammonium sulfate to 60% of saturation, the precipitate dissolved and dialyzed against 0.05 Tris pH 8.0 in 0.1 M NaCl and the volume of dialyzed precipitate obtained was made to l/5 volume of the original fraction used. Substances of molecular weight of 158,000 (adolase) eluted from this column in a peak at 158 ml, of 45,000 (ovalbumin) at 269 ml and of 25,000 (chymotrypsin) at 318 ml.

SEPHADEX

G-200

FILTRATION

Protwn

0 A

Gmc.

OF PREKALLIKREIN

F

HMW-

Q 8

m

Kinlnoqen

DEFICIENT’ SERUM

(00280)

Fltzgemld Factor Adivity(%afmut

0

Kininaqen

Antiqen

Antigens

in nannal plaemo)

Dekbbh

DetecMSe:

158,000

0.6

‘0 .:\ * s a

395

PROTEOLYSIS OF HMW-KININOGEN

vo1.25, X0.5

5x Cone

Fractwts

MW

3.0

0.4

20

‘0 0.2 Q

I.0

8 100

140

180

220

Volume

SEPHADEX G-200 FILTRATION OF PREKALLIKREIN TREATED WITH STREPTOKINASE 0

Protein

*

Fi:zqarold

Cone

0

HMW-

Eza

Kininoqm

260

(ml)

DEFICIENT

SERUM

(00 2801

Factor oCtiwty(%

Klnlnoqen

Antiqen

ofmat

in normal plosmo)

Detectable

Antiqens Oetecfoble: 5s Cow

Fractions

I60 Volume

220 (ml

260

1

FIG. 7a Sephadex G-200 filtration of prekallikrein-deficient serum which was not treated with streptokinase. The filtration process was carried out just azn the experiment shown in Figure 6 using the same G-200 column and 2 ml of serum. The molecular weight of substances eluting at 158 ml was 158,000, at 269 ml was 45,000 and at 318 ml was 25,000. FIG. 7b Sephadex G-200 filtration of prekallikrein deficient serum treated with SK: The experiment was performed just as that with NHS treated with SK described for Figure 6, and employed the same G-200 column used in the experiment shown in Figure 7a.

396

“ROTEOLYSIS OF HMW-KININOGEX

Vo1.25,

No.5

DISCUSSION Since plasmin can release kinin directly from HMW-kininogen (22,24), it is a plasma kininogenase. When HMW-kininogen was digested by plasmin for 60 min. several fragments were released, but there was little loss of its clot-promoting activity (Figures 1,2). When plasma kallikrein cleaved human HMW-kininogen into two chains, its clot-promoting activity remained intact (6,8), but the fragments released when HMW-kininogen was digested by plasmin were of lower molecular weight (61,000, 58,000 and 48,000) than those which Thompson, et al. (6) reported to be released by plasma kallikrein (108,000, 92,000, 85,000). In the present studies the chromatography fractions containing the smallest plasmin-generated HMW-kininogen fragments that had Fitzgerald factor clotting activity were those containing 48,000 and 58,000 molecular weight materials. Fractions containing tryptic digestion fragments of 40,000 molecular weight lacked HMW-kininogen clotting activity. Since the concentration of HMW-kininogen antigens varied significantly, during plasmin digestion, but there was little change in clot-promoting activity (Figures 2,3), the antigenic sites must be distinct from clot-promoting portions of the molecule. These two properties were also distinguished from one another during tryptic digestion of HMW-kininogen, for the rate of inactivation of the antigenic determinants differed from that of the functional properties of this molecule. In contrast to the effect of plasmin, trypsin rapidly destroyed the clotting activity, but the HMW-kininogen antigenic determinants were destroyed more slowly When tryptic digests of HMW-kininogen were examined in SDS polyacry(Figure 5). a fragment having a molecular weight of 40,000 was lamide gel electrophoresis, released within 5 min. which was apparently resistant to further tryptic digestion, and lacked clot-promoting activity. The loss of clotting activity, then! occurred when HMW-kininogen fragments were cleaved to 40,000 molecular weight. Saito (10) has also reported increased anodal electrophoretic mobility of HMWkininogen exposed briefly to trypsin. The antigenic properties shared by HMWand LMW-kininogen reside in the heavy while those specific for HMW-kininogen as well as chain portion of the molecule, its functional (procoagulant) property reside in the light chain region (6,8). There was no difference in the gel filtration behavior of all three of these When endogproperties in normal serum as compared to normal plasma (Figure 6). enous plasminogen in normal serum was activated with streptokinase, the filtration behavior of the coagulant and specific HMW-kininogen antigen(s) did not change appreciably, but the shared antigens were all in fractions of lower molecular contained LMW-kininogen as well (Figure 6, lower panel). weight which probably may have cleaved the shared antigens of the heavy Thus, the plasmin generated chain region from the rest of the molecule either by direct attack, or by an indirect path in which Hageman factor was cleaved releasing fragments which activate prekallikrein and the resulting kallikrein then digested HMW-kininogen (15,161. Attempts to chromatographically separate the coagulant activity from the specific in purified reduced and alkylated plasmin digests of HMW-kininogen antigen(s), This antibody may well recognize HMW-kininogen (not illustrated) were unsuccessful. multiple antigenic determinants on the light chain of HMW-K, making it unlikely that the antigenic and functional parts of the chain could be separated. The effect of prekallikrein-HMW-kininogen complexes (18,191, on gel filtration behavior of HMW-kininogen was examined by comparing its behavior in prekallikreinFitzgerald factor clotting activity deficient serum with that in normal serum. in prekallikrein-deficient serum were found and specific HMW-kininogen antigens in overlapping Sephadex G-200 fractions which also contained shared HMWand LMW-

Vo1.25,

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PROTEOLYSIS OF HMW-KININOGEZ

397

kininogen antigens (Figure 7a); shared kininogen antigens were actually in higher molecular weight fractions than in the G-200 fractions of normal serum, as if kininogens may form complexes with proteins not identified in this study in The HMW-kininogen was probably at least partly plasma deficient in prekallikrein. complexed with P.T.A. In streptokinase-treated prekallikrein-deficient serum HMW-kininogen coagulant and antigenic properties were largely in 2 peaks which also contained shared kininogen antigens (Figure 7b) and substances of about Some of the shared antigens were in lighter 92,000 and 140,000 molecular weight. G-200 fractions (60,000 molecular weight). Fractions containing LMW-kininogen were identified as those having shared antigens but lacking HMW-kininogen clotting We cannot explain the difference in the elution activity in this experiment. pattern of proteins (h-280) between normal and prekallikrein-deficient serums.

The difference in gel filtration behavior of kininogen antigens in streptokinase-treated normal and prekallikrein-deficient serums may be due to the indirect generation of kallikrein in the normal serum by plasmin through the cleavage of Hageman factor into fragments which activate prekallikrein (15,16). This pathway could lead to further digestion of HMW-kininogen. Some of the shared kininogen antigens in streptokinase-treated prekallikrein-deficient serum were concentrated in G-200 fractions containing materials of lower molecular weight than in untreated prekallikrein-deficient serum; plasmin might have directly cleaved the heavy chain region of the HMW-kininogen in this serum. It is not clear why low molecular weight fragments found in SDS gels having HMW-kininogen clotting activity cleaved from HMW-kininogen by plasmin (Figure 1) in plasma or serum were found in the same effluent volumes of Sephadex G-200 streptokinase-treated serum as of the untreated serum (Figure.6). Cleavage fragments of HMW-kininogen that have functional and antigenic properties may have formed complexes with other proteins (21). Alternatively, they may aggregate with one another when released, leading to their elution with heavier proteins during gel filtration. Such aggregates might dissociate during SDS gel electrophoreis, so that they behave as lighter proteins when examined in this manner. Support for this possibility was derived in an experiment, not illustrated, in which both antigenic and procoagulant properties of undigested purified HNWkininogen were eluted from the Sephadex G-200 column beginning at a volume of 190 ml, containing materials of 120,000 molecular weight, but the same properties in HMW-kininogen digested by plasmin to a point at which the 120,000 molecular weight band was not longer detectable in the SDS gel, were eluted in volumes of 126 to 209 ml (90,000 to 190,000 molecular weight). Since some cleavage fragments were in fractions containing materials of higher molecular weight than the undigested HMW-kininogen, the cleavage fragments probably aggregated.

These observations confirm earlier studies showing that plasmin does digest kininogen molecules directly (24) and also emphasize the relative resistance of the clot-promoting function of this molecule to proteolysis by plasmin. These experiments do not determine if cleavage sites are located within regions of disulfide linkages; all electrophoretic studies were done in SDS and S-mercaptoethanol. The relative resistance or: the protion of the HMW-kininogen molecule which participates in Hageman factor-dependent reactions to plasmin, which can evolve in plasma, may be physiologically important because these reactions may occur in vivo in disease states complicated by circulating proteolytic activity.

398

PROTEOLYSIS OF HNW-KIXNOGEI';

Vo1.25,

So.5

REFEREXES 1.

JACOBSEN, S. Separation of two different substrates for plasma kininforming enzymes. Nature (London) 210338-43, 1966.

2.

SAITO, H., RATNOFF, O.D., WALDMANN, R., ABRAHAM, J.P. Fitzgerald trait: Deficiency of a hitherto unrecogrized agent, Fitzgerald factor, participating in surface-mediated reactions of clotting, fibrinolysis, generation of kinins, and the property of diluted plasma enhancing vascular permeability (PF/DIL). J. Cl&.-Invest. -55:1082-1089, 1975. _

3.

WLJEPPER,K.D., MILLER, D.R., LACOMBE, M.J. Fleaujeac trait deficiency of human plasma kininogen. J. Clin. Invest. -56:1663-1672, 1975.

4.

COLMAN, B.R., BAGDASARIAN, A., TALAMO, R.C., SCOTT, C.F., SEAVAY, M., GUIMARAES, J.P., PIERCE, J.R., KAPLAN, A.P. Williams trait: Human kininogen deficiency with diminished levels of plasminogen proactivator and prekallikrein associated with abnormalities of the Hageman factordepencent pathways. J. Clin. Invest. -56:1650-1662, 1975.

5.

DONALDSON, V.H., GLUECK, H.J., MILLER, M.A., MOVAT, H.Z., HABAL, F. Kininogen deficiency in Fitzgerald trait: Role of high molecular weight kininogen in clotting and fibrinolysis. J. Lab. Clin. Med. -87:327-337, 1976.

6.

THOMPSON, R.E., MANULE, JR., R., KAPLAN, A.A. Characterization of human high molecular weight kininogen procoagulant activity associated with the light chain of kinin-free high molecular weight kininogen. J. Exp. Med. 147:488-499, 1978.

7.

KLENIEWSKI, J., DONALDSON, V.H. Quantitation of human high molecularweight kininogen (HMW-Kgn) by specific hemagglutination inhibition reaction. Proc. Sot. Exp. Biol. Med. 156: 113-117, 1977.

8.

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