12P HUMAN B L O O D P L A T E L E T A G G R E G A T I O N B Y E S T E R S OF 12'DEOXYPHORBOL
W i l l i a m s o n , *J. Westwick and F . J . E v a n s , Department of Pharmacognosy , The S c h o o l of Pharmacy, 29-39 Brunswick S q u a r e , London, WCIN IAX, *Department of P h a r m a c o l o g y , The R o y a l C o l l e g e o f S u r g e o n s , L i n c o l n ' s I n n F i e l d s , London. E.M.
OH
'CH,OH 20
E s t e r s of p h o r b o l ( F i g u r e 1 1 , a r e p o t e n t tumour p r o m o t e r s and i n f l a m m a t o r y a g e n t s ( E v a n s and S o p e r 1 9 7 8 ) . Platelet a g g r e g a t i o n i s a s i m p l e model which c a n be u s e d t o s t u d y t h e r e l e a s e of i n f l a m m a t o r y m e d i a t o r s . Since 1 2 - 0 - t e t r a decanoylp h o r b o l - 1 3 - a c e t a t e ( T P A ) h a s b e e n shown t o i n d u c e p l a t e l e t a g g r e g a t i o n (Zucker e t a 1 1974) w e have i n v e s t i g a t e d t h e p l a t e l e t a g g r e g a t i n g p r o p e r t i e s of a s e r i e s of c l o s e l y r e l a t e d compounds. Blood f r o m male d o n o r s was c o l l e c t e d i n t o sodium c i t r a t e and p l a t e l e t - r i c h p l a s m a ( P R P ) p r o d u c e d by centrifugation. Ten e s t e r s , d i s s o l v e d i n a c e t o n e t o g i v e a f i n a l a c e t o n e c o n c e n t r a t i o n of less t h a n 0.5%, w e r e added t o c i t r a t e d PRP and p l a t e l e t a g g r e g a t i o n m o n i t o r e d a s d e s c r i b e d by Westwick The compounds w e r e examined i n a c o n c e n t r a t i o n and W e b b ( 1 9 7 8 ) . r a n g e of 0 . 0 5 t o 1 0 p m o l e s . 0.5% a c e t o n e s o l u t i o n d i d n o t produce p l a t e l e t aggregation. TPA i n d u c e d 50% maximum aggregation a t a c-centration of 0 . 1 3 i m o l a r , w h i l e p h o r b o l , t h e p a r e n t a l c o h o l , was i n a c t i v e a t 6 u m o l a r . F o u r C-13 m o n o e s t e r s o f 1 2 - d e o x y p h o r b o l p r o d u c e d 50% a g g r e g a t i o n i n c o n c e n t r a t i o n s of 1 . 1 9 t o 6 u m o l a r , b u t a f u r t h e r f o u r d i e s t e r s i n which t h e C-20 p r i m a r y hydroxy g r o u p was a c e t y l a t e d had no e f f e c t s a t a s i m i l a r c o n c e n t r a t i o n range. Two c l o s e l y r e l a t e d compounds, r e s i n i f e r a t o x i n and t i n y a t o x i n , which have a r o m a t i c e s t e r g r o u p s a t C-20, a l s o f a i l e d t o p r o d u c e a g g r e g a t i o n of p l a t e l e t s . From c o m p a r i s o n of t h e a g g r e g a t i n g e f f e c t s o f TPA and 1 2 - d e o x y p h o r b o l e s t e r s i t c a n be s e e n t h a t t h e removal of a n a c y l g r o u p a t C-12 of t h e n u c l e u s r e d u c e s t h e p o t e n c y of t h e compound i n t h e p l a t e l e t aggregation test. F u r t h e r m o r e a f r e e p r i m a r y hydroxy g r o u p a t C-20 and a n e s t e r f u n c t i o n a t C-13 a r e n e c e s s a r y f o r i n d u c t i o n of human p l a t e l e t a g g r e g a t i o n .
193-233 E v a n s , F . J . and S o p e r , C . J . ( 1 9 7 8 ) L l o y d i a 41: 1 2 : 937-978 Westwick, J. and Webb, H. ( 1 9 7 8 ) Thomb. R e s . Z u c k e r , M . B . , T r o l l , W. and B e l l m a n , S. ( 1 9 7 4 ) C e l l . B i o l . 60: 325-336
W i l l i a m s o n , *J. Westwick and F . J . E v a n s , Department of Pharmacognosy , The S c h o o l of Pharmacy, 29-39 Brunswick S q u a r e , London, WCIN IAX, *Department of P h a r m a c o l o g y , The R o y a l C o l l e g e o f S u r g e o n s , L i n c o l n ' s I n n F i e l d s , London. E.M.
OH
'CH,OH 20
E s t e r s of p h o r b o l ( F i g u r e 1 1 , a r e p o t e n t tumour p r o m o t e r s and i n f l a m m a t o r y a g e n t s ( E v a n s and S o p e r 1 9 7 8 ) . Platelet a g g r e g a t i o n i s a s i m p l e model which c a n be u s e d t o s t u d y t h e r e l e a s e of i n f l a m m a t o r y m e d i a t o r s . Since 1 2 - 0 - t e t r a decanoylp h o r b o l - 1 3 - a c e t a t e ( T P A ) h a s b e e n shown t o i n d u c e p l a t e l e t a g g r e g a t i o n (Zucker e t a 1 1974) w e have i n v e s t i g a t e d t h e p l a t e l e t a g g r e g a t i n g p r o p e r t i e s of a s e r i e s of c l o s e l y r e l a t e d compounds. Blood f r o m male d o n o r s was c o l l e c t e d i n t o sodium c i t r a t e and p l a t e l e t - r i c h p l a s m a ( P R P ) p r o d u c e d by centrifugation. Ten e s t e r s , d i s s o l v e d i n a c e t o n e t o g i v e a f i n a l a c e t o n e c o n c e n t r a t i o n of less t h a n 0.5%, w e r e added t o c i t r a t e d PRP and p l a t e l e t a g g r e g a t i o n m o n i t o r e d a s d e s c r i b e d by Westwick The compounds w e r e examined i n a c o n c e n t r a t i o n and W e b b ( 1 9 7 8 ) . r a n g e of 0 . 0 5 t o 1 0 p m o l e s . 0.5% a c e t o n e s o l u t i o n d i d n o t produce p l a t e l e t aggregation. TPA i n d u c e d 50% maximum aggregation a t a c-centration of 0 . 1 3 i m o l a r , w h i l e p h o r b o l , t h e p a r e n t a l c o h o l , was i n a c t i v e a t 6 u m o l a r . F o u r C-13 m o n o e s t e r s o f 1 2 - d e o x y p h o r b o l p r o d u c e d 50% a g g r e g a t i o n i n c o n c e n t r a t i o n s of 1 . 1 9 t o 6 u m o l a r , b u t a f u r t h e r f o u r d i e s t e r s i n which t h e C-20 p r i m a r y hydroxy g r o u p was a c e t y l a t e d had no e f f e c t s a t a s i m i l a r c o n c e n t r a t i o n range. Two c l o s e l y r e l a t e d compounds, r e s i n i f e r a t o x i n and t i n y a t o x i n , which have a r o m a t i c e s t e r g r o u p s a t C-20, a l s o f a i l e d t o p r o d u c e a g g r e g a t i o n of p l a t e l e t s . From c o m p a r i s o n of t h e a g g r e g a t i n g e f f e c t s o f TPA and 1 2 - d e o x y p h o r b o l e s t e r s i t c a n be s e e n t h a t t h e removal of a n a c y l g r o u p a t C-12 of t h e n u c l e u s r e d u c e s t h e p o t e n c y of t h e compound i n t h e p l a t e l e t aggregation test. F u r t h e r m o r e a f r e e p r i m a r y hydroxy g r o u p a t C-20 and a n e s t e r f u n c t i o n a t C-13 a r e n e c e s s a r y f o r i n d u c t i o n of human p l a t e l e t a g g r e g a t i o n .
193-233 E v a n s , F . J . and S o p e r , C . J . ( 1 9 7 8 ) L l o y d i a 41: 1 2 : 937-978 Westwick, J. and Webb, H. ( 1 9 7 8 ) Thomb. R e s . Z u c k e r , M . B . , T r o l l , W. and B e l l m a n , S. ( 1 9 7 4 ) C e l l . B i o l . 60: 325-336
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