THE JOURNAL 0 1986 by The American Society of Biological Chemists,

OF BIOLOGICAL CHEMISTRY , Inc

Vol. 261, No. 1, Issue of January 5, pp. 324-327,1986 Printed in U.S.A.

Human Plasma Prekallikrein IMMUNdAFFINITY PURIFICATION AND ACTIVATION TO a- AND /%KALLIKREIN*

(Received for publication, July 15, 1985)

Danielle Burger, Wolf-Dieter SchleuningS, and Marc Schapirae From the Division de Rhumatologie, H6pital Cantonal Uniuersitaire, I211 Geneve 4, Switzerland and SLaboratoire Central d’Hematologie, Centre Hospitalier Universitaire Vaudois, 101 1 Lausanne, Switzerland

Prekallikrein was purified from human plasma with a final yield of 76% using as the principal step adsorp- tion to immobilized chicken antikallikrein IgY. When purified prekallikrein (3.4 p ~ ) was incubated in the presence of @-Factor XIIa (0.068 pM) for 5 min at 37 “C and pH 7.5, a-kallikrein was obtained. Upon prolonged incubation (0.5-28 h), the M, 52,000 heavy chain of a-kallikrein was progressively cleaved, resulting in the formation of @-kallikrein. The formation of @-kal- likrein was characterized as an autolytic process be- cause it was prevented by specific inhibitors of kalli- krein, including aprotinin and antikallikrein antibody but not by corn trypsin inhibitor, an inhibitor specific for @-Factor XIIa.

Human plasma prekallikrein is a single-chain glycoprotein that exists in two molecular weight variants, M, 88,000 and 85,000 (1-3). Prekallikrein is the zymogen form of plasma kallikrein, a serine protease which plays a central role in the contact phase activation of blood coagulation, fibrinolysis, and inflammation (4,5). Two forms of plasma kallikrein have been described a-kallikrein, obtained by limited proteolysis from purified prekallikrein, consisting of an NH2-terminal heavy chain (Mr 52,000) linked by disulfide bonds to two light chain variants (M, 38,000 and 36,000) (1-3, 6, 7); and p- kallikrein, prepared from acetone-activated plasma, consist- ing of three peptide chains which comprise the normal light chain variants (Mr 38,000 and 36,000) linked by disulfide bonds to two chains of M, 33,000 and 20,000, which are products of a cleavage in the heavy chain (7,8). A cleavage of the kallikrein NH2-terminal heavy chain was also observed when prekallikrein was incubated with P-Factor XIIa for a prolonged period of time (6).

The catalytic properties of a- and P-kallikrein have recently been compared (7). The two forms of kallikrein hydrolyze H - D-Pro-Phe-Arg-p-nitroanilide, activate the zymogen Factor XII, and react with C1-inhibitor at similar rates. In contrast, a-kallikrein has four times greater coagulant activity and cleaves high molecular weight kininogen five times more efficiently than P-kallikrein. Furthermore, a-kallikrein but not p-kallikrein activates blood polymorphonuclear leuko- cytes, as assessed by cell aggregation and elastase release (7).

The mechanism by which (3-kallikrein is formed is poorly understood, although Nagase and Barrett (8) and Hojima et al. (6) have suggested that the cleavage of kallikrein heavy

* This work was supported in part by Grant 3.057.0.84 of from the Swiss National Science 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 “advertisement” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed.

chain may be autolytic. In the present report, we describe a new and specific protocol for the preparation of highly pure prekallikrein and the mechanism of its conversion into a- and P-kallikrein.

EXPERIMENTAL PROCEDURES AND RESULTS’

DISCUSSION

The purification method described in this report yields highly purified human plasma prekallikrein after two steps with 76% recovery (Table I). This represents a significant improvement over previous published methods (1-3, 6, 27). Prekallikrein was entirely in the zymogen form since it ap- peared only as a doublet of M, 86,000 and 83,000 on reducing SDS-PAGE (Fig. 1, right lane), and it did not exhibit amido- lytic activity against the chromogenic substrate S-2302 (Fig. 3, open triangle). Purified prekallikrein had a specific coagu- lant activity of 23 units/mg, a value in agreement with the observations of other investigators (20). Thus, elution of prekallikrein at acidic pH did not damage its coagulant prop- erties. The purification scheme reported here combines speed with high recovery, as do other immunoaffinity techniques.

An intact NHp-terminal heavy chain is required for the appropriate expression of kallikrein coagulant activity, but not for kallikrein amidolytic activity since a-kallikrein, p- kallikrein, and the isolated and alkylated light chain of kalli- krein hydrolyze the chromogenic substrate S-2302 at the same rate (7, 20). Proteolytic cleavage of prekallikrein by P-Factor XIIa was associated with the formation of an active enzyme species possessing the structural and functional properties of a-kallikrein (7), including an intact heavy chain with M, of 52,000 and a coagulant to amidolytic activity ratio of 2.4 (Figs. 2 and 3, time = 5 min). Subsequent analyses of the reaction mixture revealed that a-kallikrein was gradually converted into a species with the characteristics of @-kallikrein (7) . The M, 52,000 heavy chain was progressively cleaved into two components of M , 33,000 and 20,000 and, concomitantly, the coagulant to amidolytic activity ratio decreased to 0.4 (Figs. 2 and 3, time = 30 min to 24 h). Because the kallikreins examined here were obtained by incubating purified proteins under controlled conditions, the results of the present study

Portions of this paper (including “Experimental Procedures,” “Results,” Figs. 1-6, and Table I) are presented in miniprint at the end of this paper. The abbreviations used are: BSA, bovine serum albumin; SDS-PAGE, polyacrylamide gel electrophoresis in the pres- ence of sodium dodecyl sulfate. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Be- thesda, MD 20814. Request Document No. 85M-2308, cite the au- thors, and include a check or money order for $4.40 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

324

Prekallikrein Activation to CY- and P-Kallikrein 325

corroborate our previous findings on the role of kallikrein heavy chain in surface-dependent blood coagulation (7).

The conversion of a-kallikrein into @-kallikrein could result either from autolysis or from proteolysis by @-Factor XIIa or by some unidentified contaminating protease. The latter two possibilities were excluded since the @-Factor XIIa-specific inhibitor corn trypsin inhibitor failed to prevent the formation of @-kallikrein (Fig. 5, lune b), and prekallikrein incubated at 37 "C for 24 h without any addition remained structurally intact (Fig 5, lane d). The formation of @-kallikrein was characterized as autolysis because the M, 52,000 heavy chain remained uncleaved when incubated with kallikrein-specific inhibitors such as aprotinin (Fig. 5 , lune a) and monospecific antikallikrein antibody (Fig. 6, lunes b).

When prekallikrein is activated into kallikrein in normal plasma, the resulting active enzyme species reacts with its substrates, but it is rapidly inactivated by plasma protease inhibitors, predominantly by the a2-glycoprotein C1 inhibitor (21-23). This control system is much less efficient in plasma from individuals with hereditary angioedema (21). These pa- tients, who lack C1 inhibitor (24), suffer from recurrent and long-lasting attacks of mucocutaneous swellings and abdom- inal pain (25). Their interstitial fluid sometimes contains catalytically active kallikrein that is not down-regulated by protease inhibitors (26). Under these circumstances, the au- tolytic cleavage of the M, 52,000 heavy chain described in this communication may serve as an additional lever of control that can reduce or suppress some of the reactions mediated by a-kallikrein.

Acknowledgment-We would like to thank Dr. Ed. Kirby for pro- viding us with purified corn trypsin inhibitor.

REFERENCES

1. Mandle, R. Jr., and Kaplan, A. P. (1977) J. Biol. Chem. 252,

2. Bouma, B. N., Miles, L. A., Beretta, G., and Griffin, J. H. (1980) 6097-6104

Biochemistry 19, 1151-1160

3. Heimark, R. L., and Davie, E. W. (1981) Methods Enzymol. 80,

4. Cochrane, C. G., and Griffin, J. H. (1982) Adu. Zmmunol. 33,

5. Colman, R. W. (1984) J. Clin. Znuest. 73, 1249-1253 6. Hojima, Y., Pierce, J. V., and Pisano, J . J. (1985) J. Biol. Chem.

260,400-406 7. Colman, R. W., Wachtfogel, Y. T., Kucich, U., Weinbaum, G.,

Hahn, S., Pixley, R. A., Scott, C. F., de Agostini, A., Burger, D., and Schapira, M. (1985) Blood 65, 311-318

157-172

241-306

8. Nagase, H., and Barrett, A. J. (1981) Biochem. J. 193, 187-192 9. de Agostini, A., Lijnen, H. R., Pixley, R. A., Colman, R. W., and

Schapira, M. (1984) J. Clin. Inuest. 73, 1542-1549 1U. Kirby, E. P., and McDevitt, P. J. (1983) Blood 61, 652-659 11. Burger, D., Ramus, M.-A., and Schapira, M. (1985) Thromb. Res.,

12. Purdon, A. D., Schapira, M., de Agostini, A., and Colman, R. W.

13. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J.

14. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254 15. Ouchterlony, O., and Nilsson, L. A. (1978) in Handbook of Ex-

perimental Immunology (Weir, D. M., ed) Third Ed., pp. 19.1- 19.44, Blackwell Scientific Publications, Oxford, England

16. Laemmli, U. K. (1970) Nature 227, 680-685 17. Colman, R. W., Mattler, L., and Sherry, S. (1969) J. Clin. Znuest.

18. Hojima, Y., Pierce, J. V., and Pisano, J. J. (1980) Thromb. Res.

19. Fritz, H., and Wunderer, G. (1983) Arzneimittelforsch. 33(I),

20. van der Graaf, F., Tans, G., Bouma, B. N., and Griffin, J. H.

21. Schapira, M., Scott, C. F., and Colman, R. W. (1982) J. Clin.

22. van der Graaf, F., Koedam, J. A., Griffin, J. H., and Bouma, B.

23. Harpel, P. C., Lewin, M. F., and Kaplan, A. P. (1985) J. Biol.

24. Donaldson, V. H., and Evans, R. R. (1963) Am. J. Med. 35.37-

25. Osler, W. (1888) Am. J. Med. Sci. 95. 362-367

in press

(1985) J. Lab. Clin. Med. 105, 694-699

(1951) J. Biol. Chem. 193,265-275

48, 23-32

20, 149-162

479-494

(1982) J. Biol. Chem. 257, 14300-14305

Inuest. 69,462-468

N. (1983) Biochemistry 22, 4860-4866

Chem. 260,4257-4263

44

26. Curd, J. G., Prograis, L. J., Jr., and'cochrane, C. G. (1980) J.

27. Scott, C. F., Liu, C. Y., and Colman, R. W. (1979) Eur. J. Biochem. Exp. Med. 152,742-747

100, 77-83

Rsdi.1 i m n o d i f f u s i o n a s s a y . The p r e k a l l i k r e i n was q u a n t i f i e d e f t e r e a c h s t e p of pre- p a r a r i a by mean. o f r a d i a l h u m d i f f u s i o n a8s.y ( I S ) . l a m u n o p u r i f i e d a n f i k a l l i k r e i n IgY vas used It a f i n d concentration of 40 yg/d i n 1 I ager, 20 mn a o d i m b a r b i t a l pH 8 . 6 m d I I N K 1 . Di f fua iao p roceeded for 48 h . P u r i f i e d p r e k a l l i k r e i n was used as t h e reference.

326 Prekallikrein Activation to a- and &Kallikrein p r e k a l l i k r e i n was a p p a r e n t l y homogeneous, and t h e f i n d y i e l d wa(l 76 X (Table I). P u r i f i e d

condi t ion0 (Fig. 1, l ane b ) . The cor rec t ion o f t he kaOl i l ) " .C t iv~ced pa r t i a l t h rombop las t in p r e k a l l i k r e i n 1,1111 v i s u a l i z e d as a doublet of & 86.WO and 83,WO an SDS-PAGE under reducing

p r e k a l l i k r e i n ( 4 0 &.I) wall 97 X i 3 7. of the t ob ta ined wi th the .me d i l u t i o n s o f normal time of prekllllikrein-deficienL plasma obtained with 10- t o 500-fold d i l v t i o n s o f p u r i f i e d

p o l e d p l a m a c o n t a i n i n g 40.3 * 0 . 6 m / d o f p r e k a l l i k r e i n .

Ac t iva t ion o f p reka l l i k re in to e- and 8-Kal l ikrein

The products o f the incubat ion of pur i f ied p reka l i ik re in wi th 8 -Fac tor XIIa a t pH 7 . 5 and 37% using II 1:SO enzyme LO s u b s t r a t e r s r i o were analyzed at various r imes by SDS-PACE

k r e i n (& 86,000 and 83,000; Fig. 2 , rime I 0) vas comple t e ly conve r t ed i n to n rka l l i k re in . ( F i g . 2 ) and t e s t e d f o r coagulant and amido ly t i c ac t iv i ty (F ig . 3 ) . Wi th in 5 min, p reka l l i -

i . e . i n t o a apeeies with an heavy chain of 3 52.WO and 2 l i g h t c h a i n v a r i a n t s of n 38 WO and 36,000 (Fig. 2 , rime - 5 m i d . Upon p ro longed i ncuba t ion t he i n t ens i fy o f T -ks l ik i e in heavy chain (3 52,0001 decreased, while 2 n e w protein bands of W 33,WO and 20.000 appea- red (F ig . 2 , rime = 30 min t o 28 h ) . P r o t e o l y s i s o f t h e h e a v y e 6 i n wee ala" and semcd t o reach a p l a t e a u a f t e r -20 h. even a f t e r an incubat ion of 48 h, the heavy chain did not d i s a - ppear completely (nor shorn) .

The var ious reac t ion mix ture* were also analyzed for coagulant and amido ly t i c ac t iv i - t y . P r io r t o the addi t ion o f ( -Fac tor X l l n . p r e k a l l i k r e i n vas found LO have a eosgulenr PC-

Ulmg, but no d e t e c t a b l e m i d o l y r i c a c t i v i t y ( F i g . 3 , Open t r i a n g l e ) . Vhen p r e k a l l i k r e i n was t i v i t y o f 0.014 u l m l ( ~ i g . c l o s e d t r i a n g l e ) , i . e . B s p e c i f i c c o a g u l a n t a c t i v i t y o f 23

comple t e ly conve r t ed i n to *ka l l i k re in (F ig . 2 , time 5 m i n ) , t h i s l a t t e r s p e c i e s had eoa- gulant and m i d o l y r i c a c t i v i t i e s o f 0 . 1 1 end 0.046 U/ml, r e s p e c t i v e l y , i . e . s p e c i f i c coagu- l a n t and amidoly t ic Bct iv i t ies o f 183 and 77 Ulng. Upon pro longed incubat ion , ka l l ik re in w i d o l y c i c a c t i v i t y r w a i n e d constant (Fig.3. open c i r c l e s ) . b u t i t s coagulant a c t i v i t y (Fig. 3 , c losed c i rc les) decreased between 5 min and 20-24 h from 0.11 t o 0.018 U h l i . e . by B fac tor o f 6 .

Mechanism of Cleavage of a-Ksllikreio Aewy Chain

t h e f a c t o r ( d responsible for the p ro teo lya ia o f the heavy cha in . P r e l i m i n a r y e x p r i o e n t a S p e c i f i c i n h i b i t o r s o f k a l l i k r e i a and 0-PaCtOI XIIa ( 1 7 , 18) were employed t o d e f i n e

e x m i n e d t h e s p e c i f i c i t y o f corn t r y p s i n i n h i b i t o r and .p ro t in in for inac t iva t ing a- and 8- k a l l i k r e i n as w e l l ae 8-Factor XIIa. 0-Factor XIIa a m i d o l y t i c a c t i v i t y was comple te ly aba l i - Ohed by corn t r y p s i n i n h i b i t o r w i t h i n 15 (I (Fig. 4, open r r i m g l c d , whereas a- and 0-kal l i - k r e i n a c t i v i t y was not af fec ted by Chis inh ib i tor (F ig . 4. open c i r c l e s and open squsre8).

squares) wi thout s f fec t ing 0-Fac tor XIIa r a i d o l y f i c a c t i v i t y (Fig. 4 . cloeed t r iangle . ) . In contract. a p r o t i n i n i n h i b i t e d P and 8 -ka l l i k re in (F ig . 4 . closed c i r c l e s end closed

S i n c e t h e i n h i b i t i o n o f t r y p s i n - l i k e s e r i n e p r o t e a s e s by aprocinin i. revermible (19). t h e r e s i d u a l a c t i v i t y (IO X ) obtained fol lowing preincubat ion of 01- rmd !+kallikrein with epre- r i n i n is l i k e l y t o depend upon the d iaaoc iar ion of the enryae- inhib i tor complex .

heavy chain was exmined by SDS-PAGE .nelyses of the mixture* r e s u l t i n g from a 24 h i n c u b s The e f f e c t o f a p r o t i n i n and =ern f r y p a i n i n h i b i t o r on t h e eleavage of a - k a l l i k r e i n

Cion at 37% o f p r e k d l i k r e i n w i t h 0-Factor XIIa. in the preaence or in the absence o f inh i - b i t o r s . Thhe prellence of 2000 U I m l a p r o t i n i n i n a sys tem conta in ing preka l l ik re in and 8-F~c- tor X X h completely prevented the cleavage of t he I4 52 WO heavy chein (Fig. 5 . l a n e a ) . 1" Contract. corn t r y p s i n i n h i b i t o r (0.1 mglml) a d d e d 3 m i ; a f t e r t h e e d d i t i o n o f 8-Factor XIIa - 5 mi"). d i d not influence t h i s p r o f e o l y r i c cleavage (Fig. 5 . l ane b ) . Thhe p a l f e r n observed t o p r e k a l l i k r e i n . i.e. when p r e k a l l i k r e i n w.8 f u l l y a c t i v a t e d t o a - k a l l i k r e i n ( F i g . 2 , time

using these latter condi t ions was i d e n t i c a l w i t h t h e p a t t e r n found when p r e k a l l i k r e i n "11s incubated with B-FaCtOr XIIII f o r 24 h in the abeence of corn Cryps in inh ib i tor (F ig . 5 , l a n e c ) . Preka l l ik re in i ncvba ted fo r 24 h at 37% in the absence Of @-Factor XI1 and i n h i b i t o r s rwsined unc lewed (F ig . 5 . l a n e d ) . Theme results st rongly l luggest rhar the cleavage of Crka l l ik re in heavy cha in is an a u t o l y t i c p r o c e s s . To fu r the r exp lo re t h i s sugges t ion . p r e k a l l i k r e i n was incubated with 8-Factor XIIa in the p resence o f i lmunopur i f ied

94 - -. w

a n t i k a l l i k r e i n IgY h a v i n g t h e C a p a c i t y o f i n h i b i t i n g k a l l i k r e i n a m i d o l y t i c a c t i v i t y ( d a t a no" presented) . The r e s u l t i n g m i x t u r e w.8 analyzed at v a r i o u s times by SDS-PAGE f o r cleavage of the heavy chain (Fig. 6 ) . In t h e p r e o e n c e o f a n t i k d l i k r e i n IgY. the heavy chain of k a l l i k r e i n (& 5 2 , 0 0 0 ) r w a i n e d unchanged a f t e r 4. 8 , and 24 h of incubat ion and t h e appearance of the % 33,000 and 20,000 cleavage products was not observed (Fig. 6 , lanes b). Control experiments demonstrated that the heavy chain wae cleaved when t h e r e a c t i o n "11s performed in the absence o f IgY ( F i g . 6 , lane. a) or i n t he p re sence o f nom--me I g Y (Fig. 6 , lanes e ) .

86- - 83-

52 -

36- 38-

33-

20-

,O 5 15 30, ,l 2 L 8 12 16 20 24 28 (rnin)

lime (h)

Figure 2. SDS-PACE ana1y.i. o f p r e k a l l i k r e i n a c t i v a t i o n t o LT and & k a l l i k r e i n ab a func- t ion of t ime. The r e a c t i o n was stopped at t h e i n d i c a t e d t i m e a8 described under aper imenfa1 Procedures; & x are i nd ica t ed i n t he l e f t marg in .

J 1.2

0.8

0.4

66- O ? , , , , I

0 a 16 24

0 I 1

tlrne (Ill

45-

30-

20-

Figure 1. SDS-PAGE a n a l y s i s Of prekallikrcin Preparation. *nrik.l l ikrein-Seph.ro.e column

r e r e reduced by boil inn 5 min i n t h e nresence of 0.6 W 8 a c r c a o t o e t h a n o l : h e l u a t e ( l a n e a); p u r i f i e d p r e k a l l i k r e i n e l u t e d from SP-Sephadex ( l a n e b ) ; e o p l e s

Figure 3. Amidolytic and e o a p h n r . e f i v i f i e s o f t h e p r o d u c t s o f p r e k d l i k r e i n a c t i v a t i o n

r imental Procedures; af ter thawing. a l iquoc. are d i l v r e d SO-fold i n 20 mm 1111 a fvncrion Of incubat ion time. The s r a p l e s were obtained ail desc r ibed i n Bpe-

Tria-HC1 pH 7 . 6 . containinp. 150 NaC1 and 2 m d d BSA before mesaurin. their

0 5 10 time (min)

Figure 4 . Inh ib i t i on o f 0 -Fae to r XIIa and k a l l i k r e i n e m i d o l y t i c a c t i v i t i e e a8 a f u n c t i o n o f r e i n c u b s t i o n tine. 43 mlm1 8-Factor xlla ( t r i a n g l e s ) . 5.7 u g l d k a l l i k r e i n c i r c l e a ) or 5.7 w/ml 0 - k a l l i k r e i n (squares) were pre incubafed wi th e i ther 0.38

x of the &dards-are ind ica ted in ' rhe l e f t marg in . . ~ ~ . _

Figure 5.

86- 83-

52-

Prekallikrein Activation to a- and @-Kallikrein 327

38- 36- 33-

20- a b c d

52-

33 -

20 -

a b c a b c, ,a b c , d e U' "

L h 8h 2L h Time

Figure 6. SDS-PACE an.1y.i. of the inhibition of a-kallikrein heavy chain slc.v.ge by anti- kallikrein IsY. 30 ul of prekallikrein (0.8 d m l ) and 30 ul of 10 dl sodium

rere preincubsred for 3 .in at 37%. h e activation started by the addition phosphate pH 7.5. eonrainin8 I PI Hac1 supplemented or not with IO mg11.1 of I g Y

of 10 v1 of I-Faetor XIIa (18 u g l d ) . 20 u1 aliquot. were rewved at the indica- ced t h e . mixed rich 20 111 of SDS-PAGe ample buffer, boiled for I -in LO .top the reaction and kept at -20°C before reduced SDS-PAGe .naly*is. The reastion. r r e carried out in the preeence of buffer (lane. a). mtik*llikrein IgY (lanes b), or nom-iame IgY (lane. c ) . Lane d : mtikallikrein IgY. Lane e : non ilame IgY. & x 10-3 are indicated in the left margin; reducing condition. r r e wed.

Table I : Purifis.tion of Preknllikrein

p1.m. 6090 f 20 4 .13 * 0 . 0 2 I 100

Antikallikrein e1u.t.e 4.87 f 0.21 3.37 f 0.15 1020 82

SP-Saphadex elY.te 3.15 f 0.10 3.15 * 0.05 1475 76