Fink, Edwin

Motta, G.

Sampaio, C. A. M.

Sampaio, Misako U.

English

Open Access LMU

AAS 38/11 Recent Progress on Kìnins <0 1992 Birkhâuscr Verlag Basel 265 HUMAN PLASMA KALLIKRBIN PROCESSING: PROTEOLYSIS AS AN ALTERNATIVE CONTROL Motta G, Fink* E, Sampaio MU and Sampalo CAM Depto. Bioquimica, Escola P a u l i s t a de Medicina, S. Paulo, SP, B r a z i l , and *Dept. C l i n i c a l Chemistry and C l i n i c a l Biochemistry, U n i v e r s i t y of Munich, Munich, Germany SUMMARY Human plasma k a l l i k r e i n (HuPK) was detected i n normal non-a c t i v a t e d and d i l u t i o n - a c t i v a t e d plasma by immunoblotting, using p o l y c l o n a l antibodies. In non-activated plasma, the predominatly detected p r o t e i n corresponds to p r o k a l l i k r e i n (Mr 80,000-90,000). A c t i v a t e d plasma, besides k a l l i k r e i n , contains l a r g e r proteins (Mr > 130,000) that p o s s i b l y represent complexes between k a l l i k r e i n and proteinase i n h i b i t o r s . Plasma a l s o contains species (Mr 43,000) which corresponds to k a l l i k r e i n heavy chain. In a c t i v a t e d plasma, monoclonal antibodies against k a l l i k r e i n heavy chain detected, besides these same bands described above, two a d d i t i o n a l bands (Mr 30,000 and 20,000) pos s i b l y correspondent to fragments of k a l l i k r e i n heavy chain. 266 G. Motta et aJ. INTRODUCTION Human plasma k a l l i k r e i n i s a serine proteinase present i n plasma as p r o k a l l i k r e i n , a glycoprotein zymogen synthesized by l i v e r as a s i n g l e polypeptide chain with 619 aminoacid residues. The a c t i v a t i o n of p r o k a l l i k r e i n to k a l l i k r e i n requires the cleavage of the peptide bond A r g 3 7 1 - I l e 3 T 2 i n the s i n g l e chain, forming a molecule with a heavy chain (371 residues) and a l i g h t chain (248 residues) held together by d i s s u l f i d e bridges (1). P r o k a l l i k r e i n c i r c u l a t e s i n plasma complexed with high molecular weight kininogen as a bimolecular complex 1:1 (2), being the heavy chain of p r o k a l l i k r e i n bound to the l i g h t - c h a i n of high molecular weight kininogen (3). P r o k a l l i k r e i n i s activa t e d i n blood by a c t i v a t e d f a c t o r XII (4). K a l l i k r e i n i t s e l f a ctivates f a c t o r XII at the e a r l y phase of i n t r i n s i c blood coagulation (5), and hydrolyzee high molecular weight kininogen l i b e r a t i n g the hypotensive peptide bradykinin (6). Plasma k a l l i k r e i n also p a r t i c i p a t e s i n some reactions of f i b r i n o l y s i s (7) and complement systems* (8), and promotes the aggregation of p l a t e l e t s (9) and leucocytes (10). Plasma k a l l i k r e i n can be found i n two d i f f e r e n t molecular weight species (Mr 88,000 and 85,000) (11). The NH2-terminal heavy chain (43,000) i s important f o r the s p e c i f i c a c t i v i t y of k a l l i k r e i n and i s s i m i l a r to the heavy chain of f a c t o r XI (12); the COOH-terminal l i g h t chain (Mr 36,000-33,000) lodges the Human Plasma Kallikrein Processing 267 c a t a l y t i c s i t e of k a l l i k r e i n , and i s s i m i l a r to t r y p s i n (1). K a l l i k r e i n i s i n a o t i v a t e d e i t h e r by plasma i n h i b i t o r s such as C l - i n h i b i t o r , alpha 2-macroglobulin, antithrombin I I I (13) and p r o t e i n C i n h i b i t o r (14), or by the l i v e r , where i t i s recognized by i t s alpha-galactose residues (15). In t h i s report, we studied the p r o t e o l y s i s of human plasma k a l l i k r e i n i n a c t i v a t e d human plasma, as an a l t e r n a t i v e c o n t r o l of plasma k a l l i k r e i n metabolization. MATERIAL AND METHODS Human plasma k a l l i k r e i n (HuPK) was i s o l a t e d from Cohn's f r a c t i o n IV (16) or f r e s h plasma (17). The materials were submitted to SBTI-Sepharose chromatography (0.02 M t r i s - H C l , 0.3 M NaCl, pH 8.0 buffer) and the adsorbed k a l l i k r e i n was eluted by benzamidine 1.0 M. The pr o t e i n concentration was ca l c u l a t e d according to the mesurement of the e x t i n c t i o n c o e f f i c i e n t ( E 2 S 0 , 1 X = 1 0 . 6 ) , determined by Nagase and B a r r e t t (18). The heavy and l i g h t chains of plasma k a l l i k r e i n were i s o l a t e d by reduction w i t h d i t h i o t h r e i t o l , a l k y l a t i o n w i t h iodoacetamide (19) and separated on SBTI-Sepharose. P o l y c l o n a l antibodies against HuPK were r a i s e d i n ra b b i t s . The antiserum was p u r i f i e d on HuPK-Sepharose (0.05 M t r i s - H C l , 0.15 M NaCl, pH 8.0 b u f f e r ) , and the bound a n t i b o d i e s were eluted with 0.2 M glycine-HCl, pH 2.0 bu f f e r , and the pH of 268 G. Motta et al. the c o l l e c t e d samples was kept at 8.0 by a d d i t i o n of 1.0 M t r i s . Antibody against r a b b i t IgG (second antibody) was r a i s e d i n goats, and anti-serum was p u r i f i e d on anti-HuPK-Sepharose, by the same procedure. Monoclonal antibodies (PR 9) against heavy chain of HuPK, k i n d l y provided by Prof. Dr. Werner M u l l e r - K s t e r l ( U n i v e r s i t y of Mainz-Qermany), were p r e c i p i t a t e d from a s c i t i c f l u i d s with saturated ammonium s u l f a t e s o l u t i o n , pH 7.0 (11:9, v/v). The s o l u t i o n was kept at 4°C during 60 minutes and centrifuged at 3,500 X g at 4°C f o r 30 minutes. The p e l l e t was ressuspended i n 50% ammonium s u l f a t e s o l u t i o n and recentrifuged, under the same conditions. This p e l l e t was ressuspended i n 0.01 M t r i s - H C l , pH 7.5 buffer and d i a l y z e d against the same b u f f e r , overnight at room temperature. The antibodies were cromatographed on a column (100 ml) of DHAE-Sepharose CL 6B (0.01 M t r i s - H C l , pH 7.5 b u f f e r ) , and eluted w i t h a sodium c h l o r i d e gradient from 0 to 0.25 M. The PK9 antibodies were eluted w i t h 0.14 M sodium c h l o r i d e . Antibodies were l a b e l l e d with iodine-125 using as oxidant reagents e i t h e r Iodo-beads (Pierce) or Chloramine T (20). The s p e c i f i c a c t i v i t y of the p o l y c l o n a l or monoclonal antibodies was 10-20 uCi/ug, and i n each experiment 2 x 10° cpm (100 rig of protein) were used. Human normal blood was c o l l e c t e d d i r e c t e l y into p l a s t i c tubes mixed with sodium c i t r a t e s o l u t i o n 3.8%, 9:1 (v/v). The Human Plasma Kallikrein Processing 269 sample was centrifugea at 1,500 x g, during 15 minutes at 4°C, and the separated plasma was aliquoted and kept frozen at -20°C. For a c t i v a t i o n , plasma samples (2 u l ) were thawed, d i l u t e d i n water i n a f i n a l volume of 200 u l , and incubated at 37°C during 15 minutes. The a c t i v a t i o n r e a c t i o n was stopped by f r e e z i n g , and the samples were submitted to electrophoresis. Control samples of non activated plasma (2 u l ) were thawed and d i l u t e d d i r e c t l y i n electrophoresis b u f f e r . Sodium dodecyl s u l f a t e Polyacrylamide g e l electrophoresis (SDS-PAGE) was performed i n slab gels from 5% to 15%, according to the method of Laemmli (21), with 3% stacking gels. Apparent molecular weights were determined by running standard proteins of known molecular weights (range 14,000 to 200,000) on the g e l . Immunoblotting was performed according to the modification described by Burnette et a l . (22) or according to Kyhse-Andersen (23). The immunodetection of the antigen was achieved by a double antibody r e a c t i o n : the n i t r o c e l l u l o s e membranes were incubated with anti-HuPK antibody (20 ug/ml) and the complexes were developed with 1 2 ö I - s e c o n d antibody (2 x 10 s cpm per assay). A l t e r n a t i v e l y , the immunodetection of the antigens was achieved by reaction with the monoclonal antibody against heavy chain 1 2 Ö I - P K 9 (2 x 10° cpm per assay). 270 G. Motta et al. RESULTS Human plasma k a l l i k r e i n was p u r i f i e d by a described procedure (16, 17) and the two known forms, alpha and beta, were prepared; both préparâtion8 resulted i n s i n g l e bands on non reduced gel elec t r o p h o r e s i s (Figure l a ) . a b 1 2 3 4 5 6 K a l l i k r e i n Heavy chain Light chain Fragments of heavy chain FIGURE 1: SDS-polyacrylamide electrophoresis of k a l l i k r e i n (HuPK) forms. a. Non reduced g e l 1. alpha-HuPK (15 ug) 2. alpha-HuPK and beta-HuPK (20 ug) 3. beta-HuPK (10 ug) b. Reduced gel 4. alpha-HuPK (15 ug) 5. alpha-HuPK and beta-HuPK (20 ug) 6. beta-HuPK (10 ug) Human Plasma Kallikrein Processing 271 A f t e r reduction with DTT, alpha-HuPK presente a heavy chain (Mr 45,000) and a l i g h t chain (Mr 36,000 and 33,000); beta-HuPK has i t s heavy chain hydrolyzed i n t o two bands (Mr 30,000 and 20,000), and a l i g h t chain (Mr 36,000 and 33,000). On an immunoblotting using anti-HuPK and 12°I-eecond antibodies, i n non-activated plasma ( f i g u r e 2a) p r o k a l l i k r e i n gives a strong band (Mr 80,000) and a l s o weak (Mr > 150,000) bands. The 100-fold d i l u t e d plasma ( f i g u r e 2b) presents the same p r o k a l l i k r e i n - k a l l i k r e i n band (Mr 80,000), and the high molecular bands (Mr > 130,000), that represent k a l l i k r e i n -i n h i b i t o r complexes. The activa t e d plasma also e x h i b i t s a band (Mr 43,000) that corresponds to the p u r i f i e d heavy-chain of the enzyme used as marker i n the experiment ( f i g u r e 2d); t h i s band does not coincide with IgG fragments which may contaminate k a l l k r e i n preparations ( f i g u r e 2c). The same experiment indicated i n f i g u r e 2 was performed with a monoclonal antibody against heavy chain (PK9). D i l u t e d human plasma (1:100) was incubated with p u r i f i e d HuPK or i t s i s o l a t e d heavy chain and the antigens were immunoprecipitated with 12ÖI-PK9 (fi g u r e 3). 272 G. Motta et al. a b c d P r o k a l l i k r e i n or k a l l i k r e i n Heavy chain FIGURE 2: A c t i v a t i o n of human normal plasma by d i l u t i o n (1:100). a. human plasma (2 u l ) b. human plasma (2 u l ) d i l u t e d 1:100 c. human IgG (6.5 ug) d. i s o l a t e d heavy chain of HuPK (1.0 ug) Immunoprecipitation: anti-HuPK Development: 12°I-second antibodies In a c t i v a t e d plasma the monoclonal antibodies (PK9) i d e n t i f i e d k a l l i k r e i n (Mr 80,000) and l a r g e r proteins (Mr > 130,000); i n t h i s case, the higher molecular weight complexes correspond to k a l l i k r e i n - i n h i b i t o r e complexes, and not to eventual complexes between free l i g h t chain of HuPK and i n h i b i t o r s , because the monoclonal PK9 used to develop the b l o t t i n g does not react with l i g h t chain (24). The a d d i t i o n of i s o l a t e d k a l l i k r e i n heavy chain to act i v a t e d plasma causes a strong enhancement of the weak pr o t e i n band (Mr 43,000), showing that the band corresponds to heavy chain. Human Plasma Kallikrein Processing 273 1 2 3 4 5 K a l l i k r e i n Heavy chain FIGURE 3: D i l u t e d human plasma (1:100) incubated with p u r i f i e d k a l l i k r e i n and k a l l i k r e i n heavy chain. 1. human normal plasma (2 u l ) 2. human normal plasma (2 u l ) and HuPK (0.5 ug) 3. human normal plasma (2 u l ) and i s o l a t e d heavy chain (0.3 ug) 4. HuPK (0.5 ug) 5. i s o l a t e d heavy chain (0.3 ug) Development: "°I-PK9 Plasma (2 u l ) was d i l u t e d (1:100) and incubated with increasing q u a n t i t i e s of p u r i f i e d HuPK, and the antigens were immunoprecipitated with 12°I-PK9 (f i g u r e 4). The increasing q u a n t i t i e s of p u r i f i e d k a l l i k r e i n added to activated plasma causes a strong enhancement of the k a l l i k r e i n - i n h i b i t o r s complexes bands (Mr > 130,000), as w e l l as the heavy chain band (Mr 43,000), and also the appearance of smaller proteins bands (Mr 30,000 and 20,000). These r e s u l t s are the f i r s t i n d i c a t i o n of the hyd r o l y s i s of k a l l i k r e i n heavy chain, i n plasma. 274 G. Motta et al. 1 2 3 4 5 6 Heavy chain K a l l i k r e i n Heavy chain fragmente FIGURE 4: Diluted human plasma (1:100) incubated with d i f f e r e n t q u a n t i t i e s of HuPK. 1. human plasma (2 u l ) 2. human plasma (2 u l ) and HuPK (0.5 ug) 3. human plasma (2 u l ) and HuPK (1.0 ug) 4. human plasma (2 u l ) and HuPK (2.5 ug) 5. HuPK (2.5 ug) 6. HC (0.2 ug) Development: 1 2 Ö I - P K 9 DISCUSSION Beta-HuPK derives from a clevage i n the heavy chain of the alpha-HuPK, and one p o s s i b i l i t y f o r t h i s m o d i f i c a t i o n i s to occur to keep pure k a l l i k r e i n s o l u t i o n s at room temperature. The beta form has the same a c t i v i t y on synthetic substrates, but Human Plasma Kallikrein Processing 275 a l p h a - k a l l i k r e i n cleaves high molecular weight kininogen (HMWKgn) r e l e a s i n g b r a y k i n i n , f a s t e r than the beta form (25). Contact of plasma with negatively charged surfaces, such as glass or k a o l i n , causes ths a c t i v a t i o n of the i n t r i n s i c pathway of blood c l o t t i n g , by bringing together k a l l i k r e i n , kininogen and faotor XII on the surface. However, d i l u t i o n induces a c t i v a t i o n by decreasing the i n h i b i t o r concentration i n plasma and thus promoting a c t i v a t i o n of the key c l o t t i n g enzymes. In our experiments the a c t i v a t i o n was confirmed by the formation of complexes, equivalent to those formed by incubation of k a l l i k r e i n and p u r i f i e d i n h i b i t o r s (13). Only i n the activa t e d plasma the protein band corresponding to the heavy chain (Mr 43,000) was detected by both p o l y c l o n a l and monoclonal antibodies (26). The addition of large q u a n t i t i e s of a c t i v e k a l l i k r e i n i n a c t i v a t e d plasma brings evidences that the transformation of alpha to b e t a - k a l l i k r e i n proceeds i n the plasma, as i t was observed f o r p u r i f i e d samples of k a l l i k r e i n . The l a r g e r protein bands (Mr > 130,000) probably correspond to complexes of k a l l i k r e i n and plasma proteinase i n h i b i t o r s . The polypeptide chains with Mr < 43,000 may i n d i c a t e that k a l l i k r e i n or i t s heavy chain i s being processed i n act i v a t e d plasma, r e s u l t i n g i n shorter fragments. No corresponding free l i g h t chain i s found i n ac t i v a t e d plasma containing f r e e heavy chain. One possible explanation f o r t h i s f i n d i n g i s an extensive hydrolysis of the l i g h t chain, and no immunnodetectable fragments would be a v a i l a b l e f o r re a c t i o n 276 G. Motta et al. w i t h p o l y c l o n a l antibodies. Or, a l t e r n a t i v e l y , the formation of l i g h t c h a i n - i n h i b i t o r s complexes might occur but d i f f i c u l t to resolve from the HuPK-inibitor complexes. Thus, the c o n t r o l of k a l l i k r e i n a c t i v i t y , e s p e c i a l l y i n the blood c l o t t i n g cascade, might also be exerted by a p r o t e o l y t i c degradation of i t s molecule. The r e s u l t i n g free heavy chain might a l t e r the surface binding a b i l i t y of high molecular weight kininogen, and the whole a c t i v i t y of the contact phase would be lowered. 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CoIman RW and Kaplan AP

Human plasma kallikrein processing: Proteolysis as an alternative control

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Human plasma kallikrein processing: Proteolysis as an alternative control

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