phospholipid vesicles induced by cadmium: characterization of the reconstituted protein-membrane complex. Steven D. CARSON and William H. KONIGSBERG.
197
Bioscience Reports I, 197-205 (1981) l~inted in Great Britain
C o a g u l a t i o n f a c t o r III ( t i s s u e f a c t o r ) i n t e r a c t i o n w i t h phospholipid v e s i c l e s induced by c a d m i u m : c h a r a c t e r i z a t i o n of the r e c o n s t i t u t e d p r o t e i n - m e m b r a n e c o m p l e x Steven D. CARSON and William H. KONIGSBERG Department of Molecular Biophysics and Biochemistry, Yale University, 333 Cedar Street, New Haven, CT 06510, U.S.A. (Received 30 January 1981)
Coagulation factor III ( t i s s u e f a c t o r ) is a m e m b r a n e g l y c o p r o t e i n which serves as a cofactor in the proteolytic activation of f a c t o r X and f a c t o r IX by f a c t o r VIIa. Mixing of human placental factor III apoprotein with vesicles of bovine brain phospholipids does not p r o d u c e significant reconstitution of factor III activity, but, when the m i x t u r e of a p o p r o t e i n and v e s i c l e s is made 5 mM with CdCI2, the apoprotein is incorporated into the vesicles. Ultracentrifugation on sucrose density g r a d i e n t s d e m o n s t r a t e d that the active factor III-lipid complex formed by reconstitution with v e s i c l e s had a d e n s i t y i n d i s t i n g u i s h a b l e f r o m t h a t of t h e c o m p l e x formed by detergent dialysis. Vesicles i s o l a t e d a f t e r centrifugation were shown to range in diameter from 20 nm to over 100 nm using the electron microscope. Gel f i l t r a t i o n showed that factor-III activity was associated with all size-classes of vesicles. The presence of factor III activity in the smaltest vesicles argues for a specific cadmium-mediated reconstitution of the apoprotein w i t h phospholipid vesicles. Coagulation factor III (tissue f a c t o r ) , present in the membrane or reconstituted into phospholipid vesicles, a c c e l e r a t e s t h e p r o t e o l y t i c activation of both factor X and factor IX by factor VIIa (1,2,3). The assembly of factor III into phospholipid vesicles during d e o x y c h o l a t e d i a l y s i s is promoted by CdCI 2 (4), and although the apoprotein does not interact significantly with preformed phospholipid vesicles, it can be i n d u c e d to do so by the addition of CdCl 2 (5). The control of factor-III activity is not well understood but is known to be a f f e c t e d by the particular phospholipids bound to the apoprotein (1,6-9) and by the amount of lipids p r e s e n t ( 1 0 ) . This r e p o r t d e m o n s t r a t e s t h e c a d m i u m - i n d u c e d i n c o r p o r a t i o n of human p l a c e n t a l factor III into preformed phospholipid vesicles and describes t h e r e s u l t a n t p r o t e i n membrane complexes.
Experimental Procedures F a c t o r III was p r e p a r e d f r o m heptane-butanol-extracted acetone powders of human placentas obtained at parturition (l 1). The factor III was e x t r a c t e d with Triton X-t00, followed by ammonium sulfate p r e c i p i t a t i o n , and c h r o m a t o g r a p h y on P h e n y l - S e p h a r o s e , Con A - S e p h a r o s e , and D E A E - c e l l u l o s e ( 5 ) . F a c t o r - I I I a c t i v i t y was determined with the two-stage clotting assay described by Pitlick and 9
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Nemerson ( l l ) . The final factor-III preparation had a specific activity of 1.7 x 105 units/mg, which is a 2 0 0 0 - f o l d p u r i f i c a t i o n from t h e a c e t o n e powder (assuming 100% extraction with the Triton X-100). We estimate that a 100 0 0 0 - f o l d p u r i f i c a t i o n will be r e q u i r e d to achieve homogeneity. F a c t o r I I I - m e m b r a n e r e c o n s t i t u t i o n by deoxycholate dialysis was accomplished as d e s c r i b e d e l s e w h e r e ( 4 , 5 ) . For i n c o r p o r a t i o n of f a c t o r III into p r e f o r m e d vesicles~ mixed phospholipids from bovine brain ( l l ) dissolved in 0.25% sodium deoxycholate at 2.5 mM w e r e p l a c e d in a c o n i c a l m i c r o c e n t r i f u g e t u b e , c o v e r e d with dialysis membrane, and floated on 0.05 M Tris, 0.1 M NaCI, 0.001% NaN 3, pH 7.6, overnight at 4~ Phospholipid concentration was determined by phosphate analysis as described (11,12). The r e c o n s t i t u t i o n m i x t u r e (final volume 0.5 ml) contained 50 pl of 0.5 M Tris, I M NaCl, 0.01% "// I
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NAN3, pH 7.6; 10 pl of the factor-III preparation (0.38 pg of protein); and test amounts of the dialyzed phospholipids. A f t e r lO rain a t 2tt~ CdCI2 was added from a 50 mM stock to give a final concentJ~ation of 5 mM c a d m i u m (or t e s t c o n c e n t r a t i o n s as i n d i c a t e d ) . Coagulation assays were conducted after an additional 10 rain at 24~ Results Early experiments (1) had shown that factor III bound phospholipids in the presence, but not in the absence, of deoxycholate. Experiments d e s c r i b e d here have confirmed that mixing factor-III apoprotein with preformed vesicles does not restore factor-III a c t i v i t y . A d d i t i o n of CdCI2 to the mixture, however, promotes the interaction of factor III and phospholipid vesicles, which restores r activity. Optimum c o n c e n t r a t i o n s for r e s t o r a t i o n of activity were determined for lipid vesicles (Fig. 1) and for CdCI~ (Fig. 2). Freshly prepared stocks of lipids and CdCI 2 i n c r e a s e d the r e p r o d u c i b i l i t y of their respective optima. The o p t i m u m c o n c e n t r a t i o n s of lipid and C d C I 2 w e r e i n d e p e n d e n t of each other (Figs. 1 and 2), and the CdC12 optimum
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Fig. 3. Sucrose-gradient fractionation of factor-Ill complexes. Ultracentrifugation was conducted as previously described (4) except that factor-Ill sample volumes were 300 pl and the centrifugation was conducted at 40 000 rev/min at 20~ for 22 h. The sucrose gradient contained 0.05 M Tris, 0.i M NaCI, 0.001% NAN3, pH 7.6. The factor-Ill samples compared were relipidated before centrifugation by deoxycholate dialysis with CdCI 2 (lower panel) and by CdCl2-mediated assembly into preformed v e s i c l e s (upper panel). The gradient fractions were assayed directly for factor activity. w a s i n d e p e n d e n t of t h e a m o u n t of f a c t o r - I I I a p o p r o t e i n . When the a p o p r o t e i n c o n c e n t r a t i o n was i n c r e a s e d 2.5-fold, t h e o p t i m u m C d C I 2 c o n c e n t r a t i o n r e m a i n e d near 5 mM. The lipid o p t i m u m , h o w e v e r , was i n c r e a s e d to 0.5 m M , d e m o n s t r a t i n g that the factor-III activity recovered by a s s e m b l y i n t o p r e l o r m e d v e s i c l e s is d e p e n d e n t on t h e r a t i o of phospholipid to p r o t e i n (211 to 1 [ w / w ] at the o p t i m u m in t h e s e e x p e r i m e n t s ) , as had been found for r e c o n s t i t u t i o n by d e t e r g e n t
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Fig. 4. Electron micrograph of factor-Ill complexes. The vesicles contained in the p e a k f a c t o r - I l l fraction (number 17) after ultracentrifugation of the apoprotein assembled into preformed vesicles (Fig. 3, upper panel) are shown. The vesicles were allowed to settle on carbon-coated grids, were stained with 1% phosphotungstic acid/O.05 M Tris, and were examined with a Philips EM300 at 80 kV. The bar corresponds to i00 nm.
dialysis ( 5 , 8 , 1 2 ) . In s i m i l a r e x p e r i m e n t s using CaCI 2 and MgCI2, CaCI~ (at 20 raM) produced a 3-fold increase in f a c t o r - I I I a c t i v i t y , considerably less than the increase seen with CdCI2. The reconstituted factor III prepared using preformed vesicles was c o m p a r e d with factor III reconstituted by deoxycholate dialysis (with added CdCI~) by ultracentrifugation on sucrose density gradients. Both m e t h o d s for r e c o n s t i t u t i n g f a c t o r - I I I a p o p r o t e i n with phospholipid vesicles produced active complexes which banded between 13 and 16% s u c r o s e (Fig. 3). F r a c t i o n 17 of the s a m p l e r e c o n s t i t u t e d from preformed vesicles contained v e s i c l e s with d i a m e t e r s ranging from about 20 nm to more than 100 nm as revealed by electron microscopy (Fig. #).
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R e c o n s t i t u t e d f a c t o r III was g e l - f i l t e r e d on B i o - G e l A l 5 m to determine which size-classes of vesicles were associated with factor-III activity. For comparison, factor-III apoprotein alone and mixed with vesicles without CdCI 2 were also g e l - f i l t e r e d . Direct coagulation a s s a y s of the column fractions d e t e c t e d significant factor-IIl act i vi t y only in fractions containing vesicles reconst i t ut ed in the p r e s e n c e of c a d m i u m ( F i g . 5) . T he Iactor-III act i vi t y was present in fractions eluted at the excluded volume and in all subsequent fractions through t h o s e in which b a c t e r i o p h a g e Gt~ (di am et er 28 to 30 nm [ 1 5 ] ) had eluted during c a l i b r a t i o n o* t h e c o l u m n . The r e l a t i v e I a c t o r - I I I a c t i v i t y which e l u t e d a t t h e e x c l u d e d v o l u m e and in subsequent fractions varied quantitatively among experiments, but the demonstrat i o n of f a c t o r - I l I a c t i v i t y a s s o c i a t e d w i t h all vesicle sizes was a reproducible finding. No Iactor-III activity was d e t e c t e d in fractions wh ich e l u t e d a f t e r the vesicles until further relipidation was carried out to reveal the presence of factor-III apoprotein which had not been r eco n s titu te d in the initial reaction. The apoprotein eluted just prior tO albumin, even in the presence of phospholipid vesicles (Fig. 5A, B).
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Discussion
The c a d m i u m - p r o m o t e d a s s e m b l y of f a c t o r - I I I a p o p r o t e i n into preformed phospholipid vesicles is an unexpected observation, since the a p o p r o t e i n does not i n t e r a c t a p p r e c i a b l y with phospholipid vesicles (1,5) and is not significantly induced to do so by CaCI 2 or MgCI 2. The vesicle-reconstituted factor III-lipid complex is very similar to the active complex f o r m e d by d e o x y c h o l a t e d i a l y s i s . Both modes of reconstitution have a CdCI 2 optimum near 5 mM, show optimum lipid concentrations at similar l i p i d - t o - p r o t e i n r a t i o s ( % 5 ) , and p r o d u c e f a c t o r - I I I vesicles with indistinguishable densities on sucrose densitygradient ultracentrifugation. The persistent association of f a c t o r I l l reconstituted with vesicles using CdCI 2 during prolonged centrifugation and on gel f i l t r a t i o n in the absence of CdCI 2 demonstrated that the i n d u c e d a s s o c i a t i o n is not r e a d i l y reversible. Electron microscopy revealed that the sizes of the vesicles in a single sucrose d e n s i t y fraction with f a c t o r - I l l a c t i v i t y spanned the range of diameters from about 20 nm to well over 100 nm. The 20-nm vesicles are r e m i n iscent of those observed by Huang after sonication and gel f i l t r a t i o n o:f phosphatidylcholine vesicles on Sepharose 4B (16).
Fig. 5. Sizing of reconstituted vesicles by gel filtration. A column (1.7 x 20 cm) of Bio-Gel Al5m, equilibrated in 0.05 M Tris, 0 . 1 M NaCI, 0.001% NAN3, pH 7.6, was allowed to flow by gravity at approximately 15 ml/hr. Fractions were collected at 4-min intervals. Factor-Ill samples were prepared for gel filtration using 25 ~i of the apoprotein with the appropriate increase (2.5-fold) of phospholipids to p r o d u c e optimum reconstitution (see 'Results'). Phospholipid in the fractions was estimated according to Bartlett (13). Fractions were assayed directly for factor-Ill activity, and were then individually reconstituted and a s s a y e d again to detect any apoprotein which had not been reconstituted in the initial reaction. Relipidation of the individual fractions was done by combining I00 ~i of each fraction with 25 pl of mixed lipids in 0.25% sodium deoxycholate, I0 mM Tris~ pH 7.5. After i0 min, 12 ~I of 50 mM CdCI~ was added. The samples were assayed for coagulant activity after 10 min. During c a l i b r a t i o n of the AlSm column, the elution of bacteriophage G4 was monitored by assaying fractions for plaque-forming ability on E. co2i C (14). The preparations presented were (A) factor-Ill apoprotein alone~ (B) factor-Ill apoprotein mixed with phospholipid vesicles, and (C) f a c t o r III m i x e d with phospholipid vesicles and made to 5 mM CdCI 2 for reconstitution. Reference standards were bovine serum albumin (Alb), horse-spleen ferritin (Ft), and bacteriophage G4 (#G4).
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CARSON & KONIGSBERG
T h e d e m o n s t r a t i o n t h a t calcium~ which e f f e c t i v e l y p r o m o t e s vesicle-vesicle fusion ( [ 7 - 1 9 ) , does not p r o m o t e r e c o n s t i t u t i o n of factor-III activity nearly as well as does cadmium, suggested that the assembly of the apoprotein into the lipid bilayers involves a specific i n t e r a c t i o n r a t h e r t h a n r a n d o m i n c o r p o r a t i o n during vesicle-vesicle fusion. Confirmation of this i n t e r p r e t a t i o n , h o w e v e r , r e q u i r e d t h e demonstration of factor-III activity associated with the 20-nm vesicles. Gel filtration on Bio-Gel AlSm revealed that factor-III a c t i v i t y was indeed associated with the smallest vesicles observed as well as with the larger lipid structures. Although factor-III a c t i v i t y may b e c o m e a s s o c i a t e d with t h e l a r g e r vesicles by incorporation into the 20-nm vesicles followed by v e s i c l e - v e s i c l e fusi on, t h e c a d m i u m - m e d i a t e d apoprotein-vesicle assembly mechanism may o p e r a t e not only with the 20-nm vesicles but with the more extensive m e m b r a n e s o b s e r v e d as well. As previously discussed (5), the singular ability of cadmium to promote f a c t o r III-vesicle assembly may be t h e r e s u l t of c a d m i u m c h l o r i d e c o m p l e x e s rather than the divalent cation. Indeed, several preliminary experiments have indicated that the ratio of cadmium to c h l o r i d e is m o r e i m p o r t a n t f or r e c o n s t i t u t i o n of f a c t o r III-vesicle complexes than is the absolute concentration of cadmium. T h e s e e x p e r i m e n t s have led to a rapi d ( 2 0 - m i n ) m e t h o d f o r reconstituting factor-III activity from the apoprotein and phospholipid vesicles, and demonstrated that cadmium can induce interaction of the apoprotein with vesicles. The centrifugation and gel filtration results can be used for preparation of f act or III-vesicle complexes of defined size and lipid-to-protein ratio. This is especially important for studies of factor-III activity, since f a c t o r VIIa (the enzym e for which f a c t o r III serves as a c o f a c t o r ) and f a c t o r X (the subst rat e) both bind t o phospholipid surfaces (20), and this may help explain data obtained in studies of factor-X activation kinetics (Dr. Yale N em erson, p e r s o n a l communication). Excess phospholipids inhibit the activation of f a c t o r X, possibly by sequestering f a c t o r X on v e s i c l e s which c o n t a i n no f a c t o r III ( 1 0 ) . Factor-III apoprotein not incorporated into vesicles may also a f f e c t the activation of f a c t o r X by r e t a i n i n g f u n c t i o n a l e n z y m e or s u b s t r a t e binding sites. Additionally, fact or-X activation may require binding of factors X and VII to vesicles containing f a c t o r III. The r e a c t i o n could t h e r e f o r e be influenced by geometric constraints imposed upon the l i p i d - c o f a c t o r - e n z y m e - s u b s t r a t e complex by the size or c ur vat ur e of the lipid surface on which these components in ter act. The influence of vesicle size and st ruct ure can be illustrated by e m p h a s i z i n g t h a t t h e e x t e r n a l phospholipid surface available for these reactions is approximately 30% great er in a system c o n t a i n i n g only 20-nm v e s i c l e s t ha n in a system containing larger vesicles in which the bilayer approaches a planar g e o m e t r y (21). The spacing of lipid head groups, packing of acyl chains, and the a s y m m e t r y of lipid distribution across the bilayer are also influenced by vesicle size (21). These considerations demonstrate the importance of physically defined f a c t o r III-containing vesicles with which to investigate further aspects of factor-III activity and its regulation.
Acknowledgements I wish to thank Dr. G. Nigel Godson for the stocks of phage G~ and E. c o l i C, and Dr. Eleanor Spicer for her assistance in setting up
COAGULATION FACTOR III INTERACTION WITH VESICLES
205
t h e p l a q u e as s ay. I also t h a n k Mr. Doug Keene for the el ect ron micrographs. Mrs. S h a r o n C a r s o n p r o v i d e d e x c e l l e n t t e c h n i c a l assistance. This work was supported in part by grant HL 22957; S.D.C. was supported by National Research S e r v i c e A w a r d 1 F32 HL 0603t4-01 from the National Heart, Lung, and Blood Institute. References I. 2. 3.
Pitlick FA & Nemerson Y (1970) Biochemistry 9, 5105-5113. Nemerson Y (1966) Biochemistry 5, 601-608. Osterud B & Rapaport SI (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 5260-5264. 4. Carson SD & Konigsberg WH (1980) Science 208, 307-309. 5. Carson SD & Konigsberg WH (1980) Thrombos. Haemostas. 44~ 12-15. 6. Nemerson Y (1968) J. Clin. Invest. 47, 72-80. 7. Hvatum M & Prydz H (1969) Thrombos. Diathes. Haemorrh. 21, 217-222. 13. Nemerson Y (1969) J. Clin. Invest. 48, 322-331. 9. Wijngaards G, van Deenen LLM, & Hemker HC (1977) Biochim. Biophys. Acta 488, 161-171. i0. Nemerson Y, Zur M, Bach R, & Gentry R (1980) in Regulation of Coagulation (Mann KB and Taylor FB, eds), pp 193-202, Elsevier, New York. 11. Pitlick FA & Nemerson Y (1978) Meth. Enzymol. 45~ 37-48. 12. Chen PS Jr, Toribara TY, & Warner H (1956) Anal. Chem. 28, 1756-1758. 13. Bartlett GR (1959) J. Biol. Chem. 234, 466-469. 14. Godson GN~ Fiddles JC, Barrel BG, & Sanger F (1978) in The Single-Stranded DNA Phages (Denhardt DT, Dressler D, & Ray DS, eds)~ pp 51-86~ Cold Spring Harbor. 15. Godson GN (1978) in The Single-Stranded DNA Phages (Denhardt DT, Dressler D~ and Ray DS, eds), pp 103-112, Cold Spring Harbor. 16. Huang C-H (1969) Biochemistry 8, 344-352. 17. Liao M-J & Prestegard JH (1979) Biochim. Biophys. Acta 550, 157-173. 18. Holz RW & Stratford CA (1979) J. Membrane Biol. 46, 331-358. 19. Papahadjopoulos D~ Vail WJ, Pangborn WA, & Poste G (1976) Biochim. Biophys. Acta 448, 265-283. 20. Nelsestuen GL, Kisiel W, & DiScipio RG (1978) Biochemistry 17, 2134-2138. 21. Tanford C (1980) The Hydrophobic Effect, 2nd ed, pp 114-117, John Wiley and Sons~ New York.
