A C T A U N I V E R S I T A T I S L O D Z I E N S I S FOLIA BIOCHIMICA ET BIOPHYSICA 2 , 1983
B a rb a r a Wachowicz, T adeusz K r a je w s ki THE PROTEINS SECRETED BY WASHED PIG PLATELETS *
The aim o f t h i s work was t o s tu d y th e amounts and com-p o s i t i o n o f com-p r o t e i n s r e l e a s e d from washed com-p i g p l a t e l e t s a f t e r t r e a t m e n t o f p l a t e l e t s w ith d i f f e r e n t a g g r e g a t i n g a g e n t s . R e l e a s e d p r o t e i n m a t e r i a l was s e p a r a t e d on Sep ha- r o s e 4B, S e p h a r os e 6B, Seph adex G-200 and by means o f SDS- p o ly a c r y l a m i d e g e l e l e c t r o p h o r e s i s . Among th e r e l e a s e d p ro -t e i n s alb um in , f i b r i n o g e n and s p e c i f i c p l a -t e l e -t p r o -t e i n -
(1 th r om b o g lo b u l in c o n s t i t u t e th e main components. As a r e -s u l t o f r e l e a -s e r e a c t i o n c a u -s ed by thrombin th e p r e s e n c e o f h i g h m o l e c u l a r p r o t e i n - thromb ospondin was o b se r v e d .
I n t r o d u c ti o n
B lo od p l a t e l e t s re sp o n d t o s t i m u l i in v a r i o u s w ays. When ex-p o se d t o thromb in, c o l l a g e n o r o t h e r s u b s t a n c e s they s e c r e t e s e -l e c t i v e -l y th e c o n t e n ts o f t h e i r g r a n u -l e s . I t i s w e l l e s t a b l i s h e d t h a t s e c r e t e d compounds c o n t a in a l s o d i f f e r e n t p r o t e i n s which p a r t i c i p a t e i n h a e m o s t a s i s , in f la m m a ti on and c e l l growth [ 6 , 7 ] . C l a s s i f i c a t i o n o f r e l e a s e d p l a t e l e t p r o t e i n s i s b a s e d on c h a r a c -t e r i s -t i c s o f -th e p r o -t e i n s , b i o s y n t h e t i c o r i g i n , l o c a l i z a t i o n and f u n c t io n [ 6 ]. S e c r e t e d p r o t e i n s have been c l a s s i f i e d i n t o f o u r g r o u p s : - p r o t e i n s i d e n t i c a l o r s i m i l a r t o p l a sm a p r o t e i n s , - l y s o so m a l enzymes, * T h i s work was s u p p o r te d by P r o j e c t R . I I I . 1 3.
- c a t i o n i c p r o t e i n s ,
- p l a t e l e t s p e c i f i c p r o t e i n s . .
The p u r p o s e o f t h i s p a p e r i s t o d e ter m in e amounts and cha-r a c t e cha-r i z e th e p cha-r o t e i n s cha-r e l e a s e d fcha-rom washed p i g p l a t e l e t s upon th e d i f f e r e n t s t i m u l i . '
M a t e r i a l and methods
Blood was ta ke n from p i g s i n t o 1% EDTA, 0 . 1 4 M NaCl, pH 7 . 4 (1 volume o f a n t i c o a g u l a n t t o 9 volumes o f b l o o d ) . B lo od was cen-t r i f u g e d a cen-t 120 x g f o r 20 min. P l a t e l e t r i c h p la sm a was removed and c e n t r i f u g e d a t 1200 x g f o r 20 min. The p l a t e l e t p oo r p la sm a was d i s c a r d e d and th e p l a t e l e t s were r e su s p e n d ed i n t h e f i r s t w ashing s o l u t i o n t h a t c o n s i s t e d o f 0.1 1 M NaCl, 4 . 3 mM K-^HPO^, 4 . 3 mM Na-jHPO^,. 2 4 .4 mM NaHgPO^, 5 .5 mM g l u c o s e , pH 6 . 5 [1 ]. Any re d c e l l s a t th e bottom o f th e tu b e were n o t r e su s p e n d ed with th e p l a t e l e t s and t h i s r e s u l t e d i n some l o s s o f p l a t e l e t s . The p l a t e l e t s u s p e n s i o n was th en c e n t r i f u g e d a t 1200 x g f o r 20 min and th £ p l a t e l e t s r e su sp e n d e d i n t h e sec ond washing s o l u t i o n c o n s i s t i n g o f 0 .1 4 M NaCl, 15 mM T r i s -H C l , 5 .5 mM g l u c o s e , pH 7 . 5 [ 1 ] .
The p r o c e d u r e o f washing w ith second b u f f e r was r e p e a t e d t w i c e . The p l a t e l e t s u s p e n s io n was then c e n t r i f u g e d a t 1200 x g f o r 20 min. The f i n a l s u s p e n s io n medium was 0 . 1 0 9 M NaCl, 4 . 3 mM K2HP04 , 16 mM Na2HP04 , 8 . 3 mM NaH2 P04 , 5 . 5 mM g l u c o s e , pH 7 . 4 . The p l a t e l e t s u sp e n s io n was d i v i d e d i n t o f o u r eq u a l p a r t s and p l a t e l e t a g g r e g a t i o n was in d uc ed by t h e a d d i t i o n o f d i f f e r e n t i n d u c e r s :
- b o vin e thrombin (Biomed Serum and V ac c i ne M a n u f a c tu r e r s in L u b l i n ) a t f i n a l c o n c e n t r a t i o n s : 1, 3, 5 , 7 . 5 o r 10 u n i t s p e r mg o f p l a t e l e t p r o t e i n , w it h o u t o r w i th c a l c i u m i o n s (C a C l2 ) a t f i n a l c o n c e n t r a t i o n 10 mM,
- c o l l a g e n from c h ic ken tend on p r e p a r e d a c c o r d in g t o S t i -l -l e r e t a l . [ 8 ] and u s e d a t c o n c e n t r a t i o n 5 o r 10 p g p e r mg o f p l a t e l e t p r o t e i n ,
- ADP (Sig m a London Ch emical Co. L t d ) a t th e c o n c e n t r a t i o n 1 0 " 3 M o r 10“ ^ M,
- one p a r t o f p l a t e l e t s u s p e n s io n was i n c u b a te d w ith b u f f e r ( c o n t r o l ) .
A f t e r th e i n c u b a t i o n tim e ( 0 . 5 - 5 min, 3 7 °C ) th e a g g r e g a t e d p l a t e l e t s were c e n t r i f u g e d a t 1200 x g f o r 20 min a t 4 °C . The s u p e r n a t a n t c o n t a in in g th e s o l u b l e p l a t e l e t r e l e a s e p r o d u c t s was c a r e f u l y s e p a r a t e d .
In th e c o n t r o l s u p e r n a ta n t and s u p e r n a t a n t a f t e r a g g r e g a t i o n a s w e l l a s i n s u s p e n s io n s o f p l a t e l e t s , p r o t e i n was d eterm in ed by m i c r o b i u r e t method [ 2 ] u s in g b ov in e serum albumin a s a s ta n -d a r-d .
In some e xp e r im e n ts l o s s o f l a c t i c d eh y d rog ena se ( E . C . 1 . 1 . 1 . 2 ) from p l a t e l e t s was m easured by th e o p t i c a l t e s t w ith py ru-v a t e [ 3 ].
Ch romatograph ic a r a l y s i s : p l a t e l e t "su sp ensions (7 0 0 - 1 5 0 0 mg o f p l a t e l e t p r o t e i n ) were in c u b a te d f o r 2 min w ith a g g r e g a t i n g a g e n t s : thromb in (10 u . p e r mg o f p l a t e l e t p r o t e i n w ith 10 mM CaCl~) o r c o l l a g e n (10 pg p e r mg o f p l a t e l e t p r o t e i n ) o r ADP (1 0 M). A f t e r c e n t r i f u g i n g t h e s u p e r n a ta n t was s t o r e d ov e rn ig h t a t 4°C arnd formed p r e c i p i t a t e s were removed by c e rn t r i f u -g a t i o n . T h i s p r e c i p i t a t e d m a t e r i a l p a r t i c u l a r l y ab und ant a f t e r thrombin a c t i o n , was f i b r i n . The c l e a r c o n c e n t r a t e d s o l u t i o n c o n t a i n in g a b o u t 40 mg o f p r o t e i n was a p p l i e d on S ep h a r os e 4B column. The column ( 1 .8 x 60 cm) was e q u i l i b r a t e d and e l u a t e d w ith 3 . 8 mM b o r a t e , 25 mM T r i s , 1 mM EDTA, 0 . 1 5 M NaCl, pH 8 .8 a t 30 m l/ h . F r a c t i o n s o f 3 ml were c o l l e c t e d and a b s o rb a n c e a t 280 nm was e s t im a t e d .
The t h r o m b i n - r e l e a s e d p r o t e i n s d e r i v e d from 2 - 3 s e p a r a t e d p r e p a r a t i o n s were a n a l y s e d f o r p r e s e n c e o f (3 - th ro m b og lo b u li n u s in g rec h rom a tog rap h y o f f r a c t i o n 3 o b t a i n e d from S e p h a r o s e 4B [ 9 ] . Rechromatography was c a r r i e d ou t on Seph ad ex G-200 u s i n g th e same e l u t i o n b u f f e r a s by S ep h a r os e 4B [ 5 ].
Gel' e l e c t r o p h o r e s i s o f p r o t e i n s :
- l i b e r a t e d from p l a t e l e t s d u ri n g w ashing p r o c ed u r e , - r e l e a s e d from p l a t e l e t s a f t e r thromb in a c t i o n , - t o t a l p r o t e i n s o f p la s m a .
S o l u t i o n a f t e r sec ond wash in g o f p l a t e l e t s was c o n c e n t r a t e d , d i a l y s e d a g a i n s t 10 mM T r i s -H C l , pH 7 . 4 and a n a l y s e d by means o f S D S -p o ly a c r y la m id e g e l e l e c t r o p h o r e s i s . The p r o t e i n s r e l e a s e d
by th romb in and t o t a l p r o t e i n s o f p l a t e l e t p oo r p la sm a (2 0 0 pg o f p r o t e i n ) were a n a l y s e d i n th e same way.
S D S- p o ly a c ry l a m id e g e l e l e c t r o p h o r e s i s was c a r r i e d ou t in 0 .0 1 M p h o sp h a te b u f f e r e s s e n t i a l l y d e s c r i b e d by W e b e r and O s b o r n [ 1 0 ] . M o l e c u l a r w ei gh t d e te r m i n a ti o n was made by u s i n g s t a n d a r d p r o t e i n s : re d u c ed and n on -re d u c ed f i b r i n o g e n , serum a lb um in , myoglobin and cytochrom c .
R e s u l t s
Washed p i g p l a t e l e t s upon th e a d d i t i o n o f t h e h ig h concen-t r a concen-t i o n o f a g g r e g a concen-t i n g a g e n concen-t s r e l e a s e a g r e a t number o f p r o t e i n s from t h e i r g r a n u l e s . I t i s a s p e c i f i c p r o c e s s s i n c e no l i b e r a -t i o n o f l a c -t i c d e hy d rog en a se a c -t i v i -t y was s i m u l ta n o u s l y o b s e r -ved (T a b . 1 ) .
T a b l e 1
The e f f e c t s o f thromb in , c o l l a g e n and ADP on l o s s o f l a c t a t e d eh yd ro gen a se (LDH) from washed p i g p l a t e l e t s
Wpływ trom bin y , k ol ag en u i ADP na spa d ek deh ed rog ena zy mleczanow ej (LDH)
z przemywanych wieprzowych p ł y t e k
Der E f f e k t von Thrombin, K o ll a g e n und ADP a u f den V e r l u s t von LDH a u s gewaschenen S c h w e i n e b l u tp l ä t tc h e n C o n t ro l Thrombin C o ll a g e n ADP 1 . 9 1 . 8 2 .1 1 . 9 6 . 6 6 . 2 6 . 9 7 .1 1 1 . 4 1 2 .1 1 1 .9 1 1 .3 N o t e : P ig p l a t e l e t s u s p e n s io n (15 2 0 mg o f p l a t e l e t p r o -t e i n i n ml) was d i v i d e d i n t o 4 p a r t s . Each p a r t was in c u b a te d w ith thromb in (1 0 u n i t s p e r mg), c o l l a g e n ( 1 0 -jag p e r mg) o r ADP ( 1 0 -3 M) f o r 2 min a t 37°C and th en c e n t r i f u g e d . The a c t i v i t y o f l a c t a t e d eh yd rog en a se was d eterm in ed i n t o t a l s u p e r n a t a n t a f -t e r p l a -t e l e -t s c e n -t r i f u g a -t i o n and e x p r e s s e d i n Wróblewski u n i -t s . C o n t r o l l i n c u b a ti o n was p erfo rm ed i n th e same c o n d i t i o n s a s th e a b se n c e o f a g g r e g a t i n g a g e n t s .
The e f f e c t s o f t e s t e d a g g r e g a t i n g a g e n t s on amounts o f r e l e a -s ed p r o t e i n -s from wa-shed p i g p l a t e l e t -s a r e p r e -s e n t e d i n T ab . 2 .
T a b l e 2
The p r o t e i n r e l e a s e d from washed p i g p l a t e l e t s a f t e r a c t i o n o f a g g r e g a t i n g a g e n t s . V a l u e s e x p r e s s e d a s th e p e r c e n t a g e o f t o t a l
p l a t e l e t p r o t e i n
B i a ł k a z przemywanych wieprzowych p ł y t e k po r e a k c j i a g r e g a c j i . W a rt oś ci podano w p r o c e n ta c h b i a ł k a c a ł k o w i te g o p ł y t e k Die P r o t e i n e f r e i g e s e t z t e n a u s S c h w e i n e b l u t p la tt c h e n nach d e r
Wirkung von A g g r e g a t i o n s f a k t o r e n . Die Werte s i n d i n
%
von G e s a m tp l a tt c h e n p r o t e i n e g eg eb en%
p r o t e i n r e l e a s e d from p i g p l a t e l e t s by Thrombin u n i t s NIH/mg o f p l a t e l e t p r o t e i n Ca 1 u . 3 u . 5 u . 10 u. X 5 . 9 7 . 8 9 . 7 1 2 . 9 1 1 .6 1 6 .0 1 4 . 9 2 3 . 0 SD 0 . 5 0 . 4 0 . 2 0 . 6 0 . 7 0 . 4 0 . 6 1 . 4%
p r o t e i n r e l e a s e d from p i g p l a t e l e t s by C o n t ro l ADP c o l l a g e n (w ith ou t a g g r e g a t i n g ) 1Q- 4 M 1 - 3 M 5pg/mg
o f p l a t e l e t a g e n t s p r o t e i n _______ X 2 . 5 5 . 8 9 .3 1 0 .0 SD 0 . 3 0 . 6 0 . 5 1 .2 N o t e : Washed p i g p l a t e l e t s (3 0 - 5 0 mg o f p l a t e l e t p r o t e i n ) were i n c u b a te d w ith thromb in a t d i f f e r e n t c o n c e n t r a t i o n (w it h and w ith o u t 10 mM C a C l? )» w ith ADP o r c o l l a g e n i n a medium c o n s i s t i n g o f 0 .1 0 9 M NaCl, 4 . 3 mM KH2P O i ,l 6 mM Na,HP04, 8 . 3 mM NaH2P0 4, 5 .5 mM g l u c o s e , pH 7 . 4 . C o n tr ol i n c u b a t i o n w i th ou t any a g g r e g a -t i n g a g e n -t was c a r r i e d ou -t i n -th e same ways. P r o t e in c o n te n t was m easured by m i c r o b iu r e t method [ 2 ] i n s u p e r n a t a n t a f t e r p l a t e l e t c e n t r i f u g a t i o n .As we can s e e thrombin a t th e h i g h e s t c o n c e n t r a t i o n (1 0 u . NIH p e r mg o f p l a t e l e t p r o t e i n ) c a u s e s th e r e l e a s e o f 1 4 . 9 + 0 . 6 and 2 3 .0 +1.4% o f t o t a l p l a t e l e t p r o t e i n s i n th e a b se n c e and i n th e p r e s e n c e o f c a lc iu m i o n s , r e s p e c t i v e l y . C o ll a g e n and ADP in u sed
c o n c e n t r a t i o n s r e l e a s e d c o n s i d e r a b l y l e s s p r o t e i n s . I t i s a l s o i n d i c a t e d t h a t p l a t e l e t s bo th d u ri n g th e washing p r o c e d u r e and s u sp en d in g in b u f f e r l o s e some p r o t e i n s .
The amounts o f s e c r e t e d p r o t e i n s depend on n a t u r e , d o se o f a g g r e g a t i n g f a c t o r a s w e ll a s on te m p e r a tu r e and p r e s e n c e o f
F i g . 1 . D i f f e r e n t amounts o f p r o t e i n r e l e a s e d from washed p i g p l a t e l e t s a f t e r t r e a t m e n t w ith d i f f e r e n t a g g r e g a t i o n a g e n t s . P la -t e l e -t s u s p e n s i o n ( 2 . 5 mg o f p l a -t e l e -t p r o -t e i n i n ml) was in c u b a -t e d w i-th -thromb in ( 7 . 5 u . NIH/mg o f p l a -t e l e -t p r o -t e i n ) a -t 37°C - o - a t 37°C w ith 1 mM EDTA - • - and a t 4 C - A - . On th e r i g h t s i d e th e amount o f r e l e a s e d p r o t e i n s a t 3 7 ° C a f t e r tr e a t m e n t with thromb in ( 7 . 5 u . NIH/mg o f p l a t e l e t p r o t e i n ) - o - , c o l l a g e n - □ - a t
c o n c e n t r a t i o n 25 p g /m l and ADP - A - ( 1 0 “£ M) i s p r e s e n t e d
I l o ś c i b i a ł k a uwolnionego z prz emytych p ł y t e k w iep rz a w wyniku d z i a ł a n i a różnych czynników a g r e g u ją c y c h . Z a w ies in ę p ł y t e k ( 2 , 5 mg b i a ł k a p ły tkow eg o/m l) inkubowano z tr om b in ą ( 7 . 5 u . NIH/mg b i a ł k a p ł y t e k ) w 37°C - o - , w 37°C z 1 mM EDTA - • - i w 4°C - A - . Z p ra w ej s t r o n y i l o ś ć b i a ł k a uwolnionego w 3 7 ° C w yn iku d z i a ł a n i a trombiny ( 7 , 5 u . NIH/mg b i a ł k a p ły tko w eg o ) - o - , k ola g en u - n - w
s t ę ż e n i u 25 fig/ml i ADP - A - ( 1 0 “ Ł M)
Die Menge d e r f r e i g e s e t z t e n P r o t e i n e a u s gewaschenen Schweine-b l u t p l ä t t c h e n nach d e r Wirkung von v e r s c h i e d e n e n A g g r e g a t i o n s f a - k to r e n . B l u t p l ä t t c h e n s u s p e n s i o n e n ( 2 . 5 mg von P l ä t t c h e n p r o t e i n e in ml) wurden m it Thrombin ( 7 . 5 u/mg von P r o t e i n e - 3 7 ° C ) - o - , mit Thrombin zusammen m it 1 mM EDTA - • - und
U°C
-A - i n k u b i e r t . Es w ird d i e Menge d e r f r e i g e s e t z t e n P r o t e i n e ( r e c h t s ) nac h Wirkung ( 3 7 ° C ) d e r Thrombin - o - , - o - K o ll a g en ( 2 5 Ug p ro ml und ADPEDTA ( F i g . 1 ) . A l l th e v a l u e s p r e s e n te d a r e c o r r e c t e d f o r spon-ta n e o u s l i b e r a t i o n o f p r o t e i n and add ed p r o t e i n . The means o f 5-8 e x p e ri m e n ts w ith SD ha ve been scivpn.
4 5 0 0 0 0 3 0 0 0 0 0 2 5 0 0 0 a b c F i g . 2 . S D S -p ol y a c ry la m id e g e l e l e c c r o p h o r e s i s (5% g e l ) o f p ro -t e i n s r e l e a s e d from p l a -t e l e -t s a f -t e r -thromb in a c -t i o n ( a ) , p la sm a p r o t e i n s ( 5 p i o f p l a s m a ) ( b ) and p r o t e i n s l i b e r a t e d from p l a -t e l e -t s -t o -th e b u f f e r d ur in g wash ing p r o c e d u re ( c ) E l e k t r o f o r e z a w ż e lu poliakryloamidowy m (5%) w o b e c n o śc i SDS b i a -ł e k uwolnionych z p -ł y t e k w wyniku d z i a -ł a n i a tromb iny ( a ) , b i a łe k plazmy (5 p i p laz m y) ( b ) i b i a ł e k uwolnionych z p ł y t e k p od c z a s
przemywania ( c )
S D S - P o l y a k r y l a m i d e g e l e l e k t r o p h o r e s e von: a - f r e i g e s e t z t e n Ge-s a m t p r o t e i n e , b - P l a s m a p r o t e i n e , c P r o t e i n e a u s d e r F l ü s s i g
-k e i t nach den z w ei te n Washen d e r P l ä t t c h e n
A f t e r washing th e p l a t e l e t s and removing them, t h e su p e rn a -t a n -t c o n -t a i n s n o -t on ly p la s m a p r o -t e i n s b u -t -th e p r o -t e i n s d e r iv e d from p l a t e l e t s to o ( F i g . 2 ) . Among th e p l a t e l e t p r o t e i n s l i b e r a t e d t o th e washing medium t h e r e i s a low m o l e c u l a r p r o t e i n f r a c -t i o n (a b o u t 25 000 d a l t o n s ) . T h is p r o t e i n i s a b s e n t i n th e p l a -sma b u t p r e s e n t i n th e m a t e r i a l r e l e a s e d by thrombin ( F i g . 2 ) .
During o u r e x p e rim e nt s we have n o t i c e d t h a t p r o t e i n m a t e r i a l
■
fra c tio n num ber F i g . 3 . S e p h a r o s e 4B p r o f i l e o f th e r e l e a s e d p r o t e i n s (.d eprived o f f i b r i n o g e n ) from t h e s u p e r n a ta n t o f th r o m b i n - a g g r e g a te d
wash-ed p i g p l a t e l e t s (10 u . NIH p e r mg o f p l a t e l e t p r o t e i n ) . The con-c e n t r a t e d s u p e r n a t a n t ( 6 ml) c o n t a i n i n g 40 mg o f p r o t e i n was ap-p l i e d t o a S e ap-p h a r os e column (6 0 x 2 . 5 cm) and g e l f i l t r a t i o n was p erf orm ed a t a f low r a t e o f 25 ml/h u s i n g 25 mM T r i s - H C l , 1 mM EDTA, 3 . 8 mM b o r a t e , 0 .1 5 M NaCl, pH 8 . 8 , 3 ml f r a c t i o n s were
c o l l e c t e d
P r o f i l r o z d z i a ł u na S e p h a r o z ie 4B b i a ł e k (pozbawionych f i b r y n o - genu) uwolnionych p o d c z a s a g r e g a c j i przemytych p ł y t e k w ie p rz a spowodowanej tro m b iną (1 0 u . NIH/mg b i a ł k a p ł y tk o w e g o ). Zatężony s u p e r n a ta n t (6 ml) z a w i e r a j ą c y 40 mg b i a ł e k n a n o sz o n o'n a kolum-nę (6 0 x 2 , 5 cm) w yp ełnioną S ep h a r oz ą i prowadzono s ą c z e n i e na ż e l u s t o s u j ą c sz y b k oś ć e l u c j i 25 m l/h i 25 mM T ri s - H C l , 1 mM EDTA, 3 , 8 mM b o ra n , 0 , 1 5 M NaCl, pH 8 , 8 ja k o e l u e n t. Z b ier an o
f r a k c j e o o b j ę t o ś c i 3 ml
Ein t y p i s c h e s E l u t i o n p r o f i l (S e p h a r o s e 4 B ) d e r f r e i g e s e t z t e n Pro-t e i n e (ohne F i b r in o g e n ) a u s dem S u p e r n a Pro-ta n Pro-t d e r T hrombinagg re-g i e r t e n B l u t p l ä t t c h e n . Das k o n z e r t r i e r t e S u p e rn a ta n t' (6 ml) m it 40 mg von P r o t e i n e wurde a u f S e p h a r os e 4B a u f g e g e b e n und m it G e sc h w i n d ig k e i t von 25 m l/h f r a k t i o n i e r t . 3 ml F r a k t i o n e n wurden
A 280 nm
fra c tio n n um be r F i g . 4 . S e p h a r o s e 4B p r o f i l e s o f th e p r o t e i n s r e l e a s e d from wash-ed p i g p l a t e l e t s by c o l l a g e n - A - ( 25 pg p e r ml) and by ADP - o - (1 0 -4 m) . C o n c e n t ra t ed s u p e r n a t a n t was ch rom atograp hed a s d e s
-c r i b e d i n F i g . 3
P r o f i l e r o z d z i a ł u c h r om a to g r a f ic z n e g o na S e p h a r o z i e 4B b i a ł e k u- wolnionych z prz emytych p ł y t e k w wyniku d z i a ł a n i a ko lag e nu - A - (2 5 jj g / m l ) i ADP - o - ( 1 0 " * M). Warunki r o z d z i a ł u z a t ęż o n e g o su -
p e m a t a n t u j a k w o p i s i e r y s . 3
Ein t y p i s c h e s E l u t i o n p r o f i l (S e p h a r o s e 4B) d e r f r e i g e s e t z t e n Pro-t e i n e nach d e r I n ku b a Pro-ti o n d e r gewaschenen S c h w e in e b l u tp l ä t t c h e n m it K o l l a g e n -A - 25 p g /m l und m it ADP (10“ * M) - o - . Das konz e n t r i e r t e S u p e rn a ta n t wurde wie f r ü h e r ( F i g . 3 ) c h rom a tog ra
A 2 80 nm
fra ction numbe r
F i g . 5 . R echrom atography o f th e low m o le c u la r p r o te in f r a c t i o n r e l e a s e d by throm bin (p ea k 3 , F i g . 3 ) on Seph ad ex G-200 (1 00 x x 2 .5 cm ). G el f i l t r a t i o n was p erfo rm ed a t a flo w r a t e 10 m l/h w ith th e same b u f f e r a s d e s c r i b e d un d er F i g . 3 . 3 ml f r a c t i o n s
were c o l l e c t e d
R e c h r o m a t o g r a f ia - f r a k c ji b i a łe k n isk o c z ą ste c z k o w y c h uw olnionych w wyniku d z i a ł a n i a trom b in y ( r y s . 3 , s z c z y t 3 ) na S e p h a d e k sie G- -2 0 0 (1 00 x 2 , 5 cm ). S ą c z e n ie na ż e lu prowadzono p rz y sz y b k o śc i e l u c j i 10 m l/h s t o s u j ą c ten sam b u f o r ( r y s . 3 ) . Z b ie ran o f r a k c j e
o o b j ę t o ś c i 3 ml
D ie A uftren nun g a u f Seph ad ex G-200 (100 x 2 . 5 cm) d e r niederm ol e k u ol a r e n P r o t e in e nach T h r o m b in in d u z ie rte r F r e i s e t z u n g . G e ol f i ol -t r a -t i o n wurde m i-t G e sc h w in d ig k e i-t von 10 m l/h , P u f f e r wie f r ü h e r
( F i g . 3 ) verw en d et. 3 ml F ra k tio n e n wurden g esam m elt
r e l e a s e d by throm b in c o n ta in s f i b r in o g e n which c o n s t i t u t e s ab ou t one te n th o f t o t a l p r o t e i n s e c r e te d by p l a t e l e t s . The f ib r in o g e n h a s been c l o t t e d o v e rn ig h t a t 4 °C .
trac tion number
F i g . 6 . S e p h a ro se 6B p r o f i l e o f th e p r o t e i n s r e l e a s e d by throm bin (10 u . NIH p e r mg o f p l a t e l e t p r o t e i n ) . G el f i l t r a t i o n was p e r
-form ed in th e same way a s on S e p h a ro se 4B (s e e F i g . 3)
P r o f i l r o z d z ia łu na S e p h a r o z ie 6B b i a ł e k uw olnionych w wyniku d z i a ł a n i a trom biny (10 u . NIH/mg b i a ł k a p ły tk o w e g o ). Warunki s ą
-c z e n ia na ż e lu t a k i e same, ja k w p rzy pad ku Seph a rozy 4B ( r y s . 3 ) Ein t y p is c h e s E l u t i o n p r o f i l a u f S e p h a ro se 6B P r o t e in e nach Throm-b i n i n d u z i e r t e r F re is e tz u n g (1 0 u . p ro m g). G e l f i l t r a t i o n wurde
w ie f ü r h e r m i t t e l s S ep h a ro se 4B d u rc h g e fü h rt
C h rom atoh rap hic a n a l y s i s r e v e a le d t h a t S ep h a ro se 4B e lu ti o n p r o f i l e s o f p r o t e i n r e l e a s e d from p i g p l a t e l e t s by d i f f e r e n t
s t im u l i seem t o be r a th e r s i m i l a r . Whenever r e l e a s e r e a c t i o n was c a u se d by th rom b in , c o ll a g e n o r ADP, th e th r e e d i s t i n c t p ea k s were o b ta in e d ( F i g . 3 and F i g . 4 ) . 450 000 300 000 Al bumina 2 5 0 0 0 14 doo F i g . 7 . S D S-p o ly a c ry la m id e g e l e l e c t r o p h o r e s i s g e l ) o f p l a t e -l e t p r o t e i n s r e -le a s e d a f t e r th rom bin a c t i o n : a - p r o t e i n s o f f r a c t i o n 4 ( F i g . 6 ) , b - t o t a l p r o t e i n s r e l e a s e d from p l a t e l e t s a f t e r th rom bin a c t io n
E le k t r o f o r e z a w ż e lu pollakry loam idow ym (5%) z a w iera ją cy m SDS b i a łe k p łytkow ych uw olnionych w wyniku d z i a ł a n i a tro m b in y : a b i a ł -ka f r a k c j i 4 ( r y s . 6 ) , b - c a łk o w ite b i a ł k o uw olnione z p ły te k
w wyniku d z i a ł a n i a trom b in y
S D S - p o ly a k rv la m id e g e le le k tro p h o re s e von P r o t e in e d e r Sch w ein e- b l u t p la t t c h e n : a - F r a k t io n 4 ( F i g . 6 ) , b - f r e i g e s e t z t e G esam
t-p r o t e in e
H owever, th e p r o t e i n s r e l e a s e d by ADP c o n ta in e d e xtrem e ly sm a ll amounts o f h ig h m o le c u la r p r o t e i n (p ea k 1 , F i g . 4 ) . Low m o le c u la r f r a c t i o n (p ea k 3 ) a p p ea red t o be th e l a r g e s t one o f them a l l , p a r t i c u l a r l y in c a s e o f throm b in a c t i o n ( F i g . 3 ) . T h is f r a c
-* \
X
t i o n a lw a y s c o n ta in e d (3-th ro m b o g lob u lin a s a component. T h is c o u ld be d em o n strated by means o f rech rom atog rap h y on Sep h ad ex G- -2 0 0 a c c o r d in g to M o o r e e t a l . [ 5 ] ( F i g . 5 , peak C ).
The h e t e r o g e n ity o f p r o t e in m a t e r i a l s e c r e t e d from p l a t e l e t s by throm bin was shown in F i g . 6 and F i g . 7 . As a r e s u l t o f g e l f i l t r a t i o n on S ep h a ro se 6B, 5 d i s t i n c t p e a k s were o b ta in e d ( F i g . 6 ) . The h ig h m o le c u la r w eig h t f r a c t i o n (p e ak 1) was a b o u t 450 000 d a lt o n s w h ile f r a c t i o n 2 seemed to have m o le c u la r w eigh t ap p ro-x im a te ly 300 000 d a l t o n s . The co m p o sitio n o f low m o le c u la r f r a c -ti o n (p e a k 4 , F i g . 6 ) was ve ry c h a n g e a b le . U su a lly were ob se rve d 3 main b a n d s: 25 000 d a l t o n s , 14 000 d a lt o n s and a band c o r r e s -pon d ing to alb um in .
D isc u s s io n
P l a t e l e t r e l e a s e r e a c t io n was i n v e s t i g a t e d by m easu rin g p ro -t e i n r e l e a s e d from c e l l s i n -t o -th e e x -t r a c e l l u l a r p h a se . The p l a -t e l e -t s were washed and resu sp en d ed in an a r -t i f i c i a l medium to remove p la sm a p r o t e i n s . However, some p a r t o f p l a t e l e t p r o t e i n s was removed in t o th e w ashin g s o l u ti o n d u r in g t h e s e p r o c e d u re s .
The tre a tm e n t o f p i g p l a t e l e t s w ith a g g r e g a t in g a g e n t s r e -s u l t -s in th e r e l e a s e o f g r e a t amount o f p r o t e i n s from c e l l g r a -n u l e s . The amount o f s e c r e t e d p r o t e i n s depend s on ty p e and con-c e n t r a ti o n o f a g g r e g a t i n g a g e n ts . Thrombin t r e a t e d p l a t e l e t s s e -c r e te d th e g r e a t e s t amount o f p r o t e in p a r t i -c u l a r l y in th e p r e -sen c e o f c a lc iu m i o n s .
The ch rom a tog ra p h ic p r o f i l e s o f th e s e p a r a t io n o f r e le a s e d compounds on S ep h ar ose 4B a r e g e n e r a ll y s i m i l a r , however th e r e a r e some d i f f e r e n c e s . The A D P -treated p l a t e l e t s r e l e a s e only ve ry sm a ll amounts o f h ig h m o le c u la r p r o t e i n f r a c t i o n . T h is f r a c t i o n i s e xtrem e ly d i s t i n c t in th ro m b in -r e le a s e d m a t e r i a l and c o rre sp o n d to "th rom bin s e n s i t i v e p r o t e i n " c a l l e d throm b ospon- d in - a g ly c o p r o t e i n d e sc r ib e d r e c e n t ly by L a w l e r e t a l .
[ 4 ] . The low m o le c u la r f r a c t i o n o b ta in e d by s e p a r a t io n on Se p -h a r o se -h as a r a t -h e r v a r i a b l e c o m p o sitio n . However i t can be s e p a r a t e d on Sep h adex G-200 in t o th r e e d i s t i n c t p e a k s . Peak I i s p o s s i b l y Ig G, p eak I I i s serum alb um in and p eak I I I seam s to be (3-th ro m b o g lo b u lin [ 5 , 9 ] .
We su p p o se t h a t th e m ajo r component o f th ro m b in t re a t e d p ro -t e i n s i s -throm bospon din w i-th i -t s d e g r a d a -tio n p r o d u c -ts o f m olecu-l a r w eig h t a b o u t 300 000 (d im e r ), 150 0 00 d a lt o n s (monom er), a l -bumin and (3 -th ro m b o g lob u l i n .
The c o m p o sitio n o f p r o t e i n s s e c r e t e d from washed p ig p l a t e -l e t s r e se m b -le s th e c o m p o sitio n o f m a t e r i a -l r e l e a s e d from human p l a t e l e t s . REFERENCES [1 ] B e a n z i g e r N. L . , B r o d i e G. N . , M a j e-r u s P. w. , P ro c. N a t. S c i . USA 6 8 , 231-240 (1 9 7 1 ). [2 ] I t z h a k e F. R. , G i l l D. M ., A nal. Biochem . 9 , 401-401 (1 9 6 4 ).
[3 ] K w i a t k o w s k a J . , O ksyd ored u k taz y , e d . S z c z e -k l i -k E , [ i n . : ] Enzym olog ia k l i n i c z n a , Warszawa 204
(1 9 7 4 ) .
[4 ] L a w 1 e r J . W., S l a y t e r H. S . , C o 1 i g a n J . E . , vF. B i o l . Chem. 86 09 -8 6 16 (1 9 7 8 ).
[5 ] M o o r e S . , P e p p e r D. S . , G a s h J . D. , Biochim . B iop hys . A cta . 3 79 , 3 6 0 -3 69 (1 9 7 5 ).
[6 ] N i e w i a r o w s k i S . , Thrombos, H a e m o sta sis 38, 9 2 4-93 8 (1 9 7 7 ). [7 ] N o s s (1 9 7 8 ). e 1 H. L. , Thrombos. H a em o stasis 4 0 . 16 8-17 4 [8 ] S t i l 1 e r R. A . , B e l a m a r i c h F. A . , S h e -p r o D. , Thrombos D ia t h e s. H aemorrh. 3 2 , 6 85 -6 9 4 (1 9 7 4 ). [ 9 ] W a c h o w i c z B. , K r a j e w s k i T ., Thrombos. H a e m o stas is 4 2 , 1-7 (1 9 7 9 ) . [1 0 ] W e b e r K ., 0 s b o r n M ., [ i n : ] P r o t e i n s , ed . N e u r a t h H ., New Y ork, 19 7-2 33 (197 5.).
D ep artm ent o f B io c h e m istry I n s t i t u t e o f B io c h e m istry and B io p h y s ic s U n iv e r s it y o f Łódź
B a rb a ra Wachowicz, T ad eusz K ra je w sk i
BIAŁKA UWOLNIONE Z PRZEMYTYCH KRWINEK PŁYTKOWYCH WIEPRZA
Badano i l o ś ć o ra z s k ła d b i a łe k uw alnian ych z przem ytych p ły -te k wieprzowych po in k u b a c ji z różnymi czynnikam i a g r e g u ją c y m i. Uw alniane z w iąz k i b iałk ow e r o z d z ie la n o na S e p h a ro z ie 4B, S ep - h a r o z ie 6B, S e p h a d ex ie G-200 o ra z w ż e lu p oliakrylam id ow ym . Wśród uw aln ian ych b i a ł e k p r z e w a ż a ją : f ib r y n o g e n , alb um in a o raz s p e c y fi c z n e b i a łk o p ły tkow e (3-th ro m b o g lo b u lin a .
W wyniku r e a k c ji u w a ln ia n ia stym ulow an ej p r z e z trom b in ę , stw ie rd z o n o ob ec n ość w ysokoc ząstecz kow eg o b i a łk a - tro m b osp on d i- ny .
B a rb a ra Wachowicz, T ad eu sz K ra je w sk i
FREIGESETZTE PROTEINE AUS GEWASCHENEN SCHWEINEBLUTPLÄTTCHEN
Es wurden d ie Menge und Zusammensetzung d e r f r e i g e s e t z t e n P r o t e in e a u s gew aschenen S c h w e in e b lu tp lä tt c h e n u n te rs u c h t und d er E i n f l u s s von v e rs c h ie d e n e n A g g r e g a tio n s fa k t o r e n b e o b a c h te t. Das f r e i g e s e t z t e P r o t e in m a t e r ia l wurde m i t t e l s G e l f i l t r a t i o n a u f S ep h a ro se 4B, S e p h a ro se 6B, a u f Sep h adex G-200 f r a k t i o n i e r t und m i t t e l s S D S - p o ly a k r y la m id e g e le le k tr o p h o re s e a n a l y s i e r t .
Man z e i g t e , d a s s d i e a u s S c h w e in e b lu tp lä ttc h e n f r e i g e s e t z t e P ro te in e a l s Grundkomponenten F ib r in o g e n , Albumin und e in sp e -z i f i s c h e P l ä t tc h e n p r o te i n - (3 -T h rom b og lob u lin e n t h a l te n . Nach d er T h ro m b in -in d u z ie rten F r e is e t z u n g r e a k t i o n i s t auch d a s hochmole-k u la r e s P r o te in - Thrombospondin b e o b a c h te t.
