Effect of gamma irradiation on blood plasma fibrinogen

A C T A U N I V E R S Ï T A T I S L O D Z I E N S I S FOLIA BIOCHIMICA ET BIOPHYSICA 2 , 1983

T a deus z K r a j e w s k i , C z esła w S . C i er n i ew sk i

EFFECT OF GAMMA IRRADIATION ON BLOOD PLASMA FIBRINOGEN

The e f f e c t o f gamma i r r a d i a t i o n on c l o t t i n g o f p la sm a f i b r i n o g e n h a s been i n v e s t i g a t e d . C r i t i c a l a l t e r a t i o n s o f m e c ha n ica l p r o p e r t i e s o f th e f i b r i n c l o t formed b oth from b l oo d p la sm a and i s o l a t e d f i b r i n o g e n ( 60C o -s o u r c e ), were i d e n t i f i e d by th r o m b e l a st o g r a p h y . The d o s e - r e s p o n s e (0 -3 KGy) a n a l y s i s o f th e p o l y p e p t i d e c o m p o si tio n o f p u r i f i e d f i b r i n o g e n showed t h e g r a d u a l d i s a p p e a r a n c e o f th e A ot c h a i n . T h is p o l y p e p t i d e was th e most s e n s i t i v e one t o i o n i z i n g i r r a d i a t i o n and was p r o b a b ly d i r e c t l y i n v o l v e d in th e a g g r e g a t i o n o f f i b r i n o g e n m o l e c u le s . T h is was n o t th e c a s e when whole p la sm a was s u b j e c t e d t o i r r a d i a t i o n .

I n t r o d u c t i o n

I r r a d i a t e d f i b r i n o g e n e x h i b i t s the enhanced ten d enc y t o form i n s o l u b l e a g g r e g a t e s and t h i s phenomenon i s p r o b a b l y a s s o c i a t e d w ith the l o s s o f i t s c l o t t a b i l i t y [ 1 - 5 ] .

The s t u d i e s r e p o r t e d h er e have been d i r e c t e d tow ard s an é v a l u -t a -t i o n o f -th e n a -t u r e o f p o s -t - i r r a d i a -t i o n a l -t e r a -t i o n s o f f i b r i n o g e n s t r u c t u r e and p r o p e r t i e s . We a tt em p t ed t o c o r r e l a t e a l t e r a t i o n s o f f i b r i n o g e n s u b u n i t s o f f i b r i n o g e n and f i b r i n w ith th e l o s s o f c l o t t i n g p r o p e r t i e s o f b loo d p la sm a and p u r i f i e d f i b r i n o g e n a f t e r i r r a d i a t i o n . M a t e r i a l s and methods

Human c i t r a t e d p la sma was employed. F r e s h l y c o l l e c t e d by v e n i -p u n c tu r e , 9 -p a r t s o f b l oo d were drawn i n t o t u b e s c o n t a i n i n g 1 -p a r t o f 3 . 8

%

sodium c i t r a t e .

F i b r in o g e n was i s o l a t e d from f r e s h human p la sm a by th e c o l d e t h a n o l p r e c i p i t a t i o n t e c h n iq u e [ 6 ] and was f u r t h e r p u r i f i e d by p r e c i p i t a t i o n w i th ammonium s u l f a t e a t 2696 o f s a t u r a t i o n . F i b r i n o -gen p r e p a r a t i o n s d i s s o l v e d i n 0 .1 4 M NaCl c o n t a i n i n g 0 .0 1 M p h osp h a te b u f f e r , pH 7 .1 ( P B S ) , were 97% c o a g u l a b l e by thrombin and d i d n ot show any c o n t a m in a t io n w ith o t h e r p l as m a p r o t e i n s a s ju d g e d by SDS p o l y 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 [ 7 ] .

T rea tmen t o f b l oo d p la sm a and f i b r i n o g e n . A l i q u o t s o f th e same human p lasm a f i b r i n o g e n s o l u t i o n s (5 mg/ml, 10 mg/ml, 15 mg/ml) in PBS were s u b j e c t e d t o i r r a d i a t i o n . The i r r a d i a t i o n was c a r r i e d ou t i n a Gamma C e l l a t d i f f e r e n t d o s e s ( 0 . 2 5 , 0 . 5 0 , 0 . 7 5 , 1 . 0 , 2 . 0 and 3 . 0 KGy) a t a d ose r a t e o f 0 .9 0 KGy p e r hour w h ile kee p in g th e te m p e ra tu re a t 4 °C . The tu b e s were k e p t und er th e 6 0Co s o u r c e w ith a i r a s g a s p h a s e .

T h r om b ela s tog r ap h y . The H e l l i g e t h r o m b e la s to g r a p h was u s e d i n t h e s e e x p e r im e n ts . T h r e e - t e n th s o f c i t r a t e d p la s m a was p i p e t t e d i n t o th e c e l l s and a f t e r 5 min. ( f o r te m p e ra t u re e q u i l i b r a t i o n ) 0 . 0 6 ml o f CaCl2 s o l u t i o n a t c o n c e n t r a t i o n 1.29% was a dd ed . The r e a g e n t s were mixed by r a p i d l y moving th e p i s t o n up and down s i x tim e s b e f o r e f i n a l l y l ow er in g i t i n t o c l o t t i n g m ix tu r e s. The throm- b e l a s t o g r a p h i c r e c o r d i n g s were a llo w e d t o p ro c ee d f o r 90 min. A lthoug h th e r e s u l t s were q u i t e r e p r o d u c i b l e , one o f th e th re e c e l l s a lw a ys c o n t a i n e d a c o n t r o l c l o t t i n g m ix tu re ( u n i r r a d i a t e d sam ple o f th e same b lo o d p l a s m a ) i n eac h e x p e rim e n t. T h r om b ela st o- g r a p h ic t r a c i n g s were r e c o r d e d and th e f o ll o w in g p a r a m e t e rs were d ete r m i n ed : r e a c t i o n time ( r ) , c l o t f o r m a t i on tim e ( k ) , maximal a m p l itu d e (MA) and e l a s t i c i t y o f f i b r i n g e l (ME) [ 8 ] .

F i b r i n o g e n c o n c e n t r a t i o n s o f p la sm a sa m p le s b e f o r e and a f t e r i r r a d i a t i o n were d e ter m i n ed . Plasma was c l o t t e d by thrombin. F i b r i n c l o t s were then e x h a u s t i v e l y washed ou t w ith PBS and d i s s o l v e d in 0 .1 M NaOH. The c o n te n t o f c l o t t e d p r o t e i n in b oth c a s e s was e s t i -mated by a m i c r o b i u r e t method [ 9 ] .

Thrombin c l o t t i n g time o f i r r a d i a t e d f i b r i n o g e n was d eterm in ed s p e c t r o p h o t o m e t r i c a l l y a t 600 nm [ 1 0 ] .

P r e p a r a ti o n o f f i b r i n from i r r a d i a t e d p la s m a . N o n c r o s s l in k e d f i b r i n was formed by thrombin a c t i o n on b l o o d p la sm a i n th e p r e -s en c e o f e x c e -s -s EDTA. One hundred j i l o f p l a-s m a wa-s mixed w ith 100 p i o f 0 . 5 M EDTA, pH 7 .1 and th en 10 p i o f thromb in (4 0 NIH -u/ml) was ad d ed . A f t e r th e c l o t was formed i t was washed w it h PBS and

d i s s o l v e d i n 0 .1 M p h os ph a te b u f f e r , pH 7 .1 c o n t a i n i n g 8 M u r e a and 196 SDS [ 1 1 ] . P o l y a c ry la m i d e g e l e l e c t r o p h o r e s i s was p erform ed in

5%

and

7%

g e l s in th e p r e s e n c e o f 0.1% SDS [ 7 ] . Sam ple s o f f i -b r in o g e n and f i -b r i n were red uc ed w ith 2

%

2 - m e rc a p to e th a n o l f o r 3 h ou rs a t 3 7 °C . G e l s were s t a i n e d w ith C oom assie B r l l l a n t B l u e .

R e s u l t s

I r r a d i a t i o n o f p u r i f i e d f i b r i n o g e n . The i s o l a t e d f i b r i n o g e n d i s s o l v e d a t d i f f e r e n t c o n c e n t r a t i o n s i n PBS was e xp ose d t o gamma i r r a d i a t i o n . The in d uc ed a l t e r a t i o n s o f i t s s u b u n it s t r u c t u r e were c o n t r o l l e s by th e a n a l y s i s i n SDS p o l y a c r y la m i d e g e l s . E l e c t r o -p h o r e t i c -p a t t e r n s o f i r r a d i a t e d f i b r i n o g e n a r e -p r e s e n t e d i n Fig . 1. When un red uced i r r a d i a t e d f i b r i n o g e n was r u n , h i g h m o l ec u la r w eig h t a g g r e g a t e s a p p ea re d a t th e top o f th e g e l s . These were n ot d i s p e r s e d even i n th e p r e s e n c e o f 8 M u r e a and

1%

SDS. The q u a n t i -t y o f a g g r e g a -t e d p r o -t e i n s i n c r e a s e d w i-th -th e d o se up -t o 3 KGy. However, no o b v io u s c ha ng es i n th e f i b r i n o g e n s o l u t i o n c o u ld be o b se r v e d . A f t e r r e d u c t i o n o f d i s u l f i d e bonds in f i b r i n o g e n w ith 2 - m er c a p t oe th a n ol th e r e was a g r a d u a l l o s s o f th e Aot c h a i n in th e t o t a l c o m p o s i ti o n . Fo llo w in g e xp o su r e o f f i b r i n o g e n t o .3 KGy th e r e was a lm o s t a c om p lete l a c k o f th e Aa, c h a i n i n th e m ol ec u le and o n ly th e B|3 and

j

c h a in s were d e t e c t a b l e i n th e g e l s . S in c e SDS g e l e l e c t r o p h o r e s i s o f th e same b ut unred uced sa m p le s o f f i b r i n o g e n , h a s n ot r e v e a l e d th e p r e s e n c e o f p l a s m i c f r a g m e n ts , t h i s may s u g -g e s t t h a t a d i s a p p e a r e n c e o f th e Aa c h a in i s n o t c a u s ed by p r o -t e o l y -t i c d e g r a d a -t i o n . T h e r e f o r e we presumed -t h a -t Aoi. p o l y p e p t i d e may be d i r e c t l y i n v o l v e d i n th e f o r m a t io n o f f i b r i n o g e n a g g r e g a t e s which a re w e ll v i s i b l e on g e l s ( F i g . 1 ) even a f t e r r e d u c t i o n w ith 2 -m e r c a p to e t h a n o l. A l t e r a t i o n s i n s u b u n i t s t r u c t u r e o f th e m olec ul e were a s s o c i a t e d w ith th e ul o s s o f p o ul y m e r i z i n g a c t i v i t y o f f i -b ri n o g e n . F ig u r e 2 shows a p l o t o f th e p o l y m e r i z a t i o n a c t i v i t y a g a i n s t th e tim e o f i n c u b a ti o n o f f i b r i n o g e n w ith throm b in . F i b r i -nogen was i r r a d i a t e d in th e ra n g e 0 . 7 5 - 2 . 0 KGy. The d o se above 3 . 0 KGy c a u s e d a c om pl ete l o s s o f th e a b i l i t y o f f i b r i n o g e n t o p o l y -m e ri z e .

I r r a d i a t i o n o f f i b r i n o g e n in human p l as m a. The e f f e c t o f gamma i r r a d i a t i o n on c l o t t i n g o f f i b r i n o g e n in human p la sm a by r e c a l c i

-F i g . 1 . D e n si to m e tr i e s c a n s o f SDS p o l y a c r y l a m i d e g e l s o f c o n t r o l f i b r i n o g e n ( a ) and f i b r i n o g e n e xp o s ed t o 1 KGy ( b ) , 2 KGy ( c ) and 3 KGy ( d ) . G el s were s t a i n e d w ith C ooma ssie B r i l l a n t Blue and

a f t e r d e s t a i n i n g t h e y were scan ned a t 560 nm

Wykresy d en sy tom etry cz n e żelów p o liak ryl oam id ow y c h po r o z d z i a l e k o n tr ol n e g o f ib r y n o g e n u ( a ) o ra z f ib ry n og e n u nap rom ienion ego dawką 1 KGy ( b ) , 2 KGy ( c j o ra z 3 KGy ( d ) . Że le barwiono b łę k it e m

Cooma-s Cooma-s i e a po od b a rw ien iu denCooma-sytometrowano p rz y d ł u g o ś c i f a l i 5Ó0 nm JJeHCHTOMeTpHMHbie rpauJWKH nojiHaKpujiaMHUHUx rejiefl nocjie pa3^e;ieHHa KOHTpojibHoro jHÓpHHoreHa ( a ) u i&HÖpHHoreHa o6jiyMeHHoro ąo3oft 1 -K3y ( b ) , 2 K3y ( c )

u

.3 K3y ( d ) . r e j i n

Kparaeime

Jia3ypbio C oom a ssie

a nocjie oÖecuTBereHHH neHCHTOMeTpHpoBaim ripn ÄJiHHe bojihh 560 nm

f i c a t i o n , a s r ec o rd e d by th r o m b e l a s t o g r a p h ic m e a s u r e m e n ts ,i s shown in F i g . 3 and T ab . 1.

I t can be s een t h a t gamma i r r a d i a t i o n r e d u c e s s i g n i f i c a n t l y th e th r o m b e la st o g r a p h y maximum a m p l it u d e . T h is s u g g e s t s t h a t th e r e i s a s i g n i f i c a n t r e d u c t io n o f c l o t s t r e n g t h when p lasm a i s exposer)

1.0 KGy

20 KGy

F i g . 2 . The p o l y m e r i z a t i o n o f i r r a d i a t e d f i b r i n o g e n in d u c ed by thrombin a c t i o n

P o l i m e r y z a c j a na prom ienion ego f i b r i n o g e n u indukowana d z i a ła n i e m trombiny

nojiHMepn3aiiHH ofijiyMeHHoro imOpHHoreHa HanyunpoBaHa AettcTBaeM TpoMÓHHa

time (min)

F i g . 3. ^ t y p i c a l th r o m b e l a sto g ra p n y r e c o r d i n g o f c o n t r o l pla sm a (P^) and a f t e r i t s ex p o su re t o 3 KGy

Z a p i sy t r o m b o e l a s t o g r a f i c z n p w y kr z e p ia n ia ko n t ro l n eg o fib ry n og en u ( pK) o ra z na promienionego dawką 3 KGy ^P300^

TpoM6o3jacTorpari)H'łHbie BacBSpTUBaHaH xoHTpojibHoro $n6pnHoreHa (P )

u oO^yneHHoro ^030ii 3 K3y (Pj q q)

t o a d o se o f 0 .2 5 KGy o r more. However, a f t e r i r r a d i a t i o n a t 3 KGy t h e p la s m a sa m p le s were s t i l l c l o t t a b l e and g a ve a normal t h r o m b e l a s t o g r a p h ic p a t t e r n ( e x c e p t th e a m p l i t u d e ) . I t i s n ot e -worthy t h a t t h i s was a ccomp anied by th e p r o l o n g a t i o n o f p r o th -rombin ti m e o f i r r a d i a t e d p l as m a when compared t o c o n t r o l p la sm a ( T a b . 1 ) .

T a b l e 1

The e f f e c t o f gamma i r r a d i a t i o n on t h r o m b e la sto g ra p h y p a r a m e t e r s , p rothrom b in tim e and f i b r i n o g e n c o n c e n t r a t i o n o f human p la sm a

( b a s e d on 8 -1 2 d e t e r m i n a t i o n s i n each e x p e ri m e n ta l g ro u p ) Wpływ p rom ieniow ania gamma na p a ra m e try t r o m b o e l a s t o g r a f i c z n e ,

c z a s protrombinowy i s t ę ż e n i e f ib ry n o g e n u w p l a ź m ie l u d z k i e j (w o p a r c i u o 8 -1 2 ozna cz eń w k a ż d e j g r u p i e d o ś w ia d c z a ln e j) B flH H H H e r a u M a - o O jr y u e H H H H a i p o M f i o a j i a c T o r p a t i i H u e c K H e n a p a M e i p u , n p o - T p 0 M Ó H H 0 B 0 e B p eM H u K O i je H T p a i ;n io < J )H 6 p H H o re H a b o n a s M e q e j i o B e M e c K o t t K p o B H ( H a 0 C H 0 B e 8 - 1 2 H S M e p eH H H b ic a a c ą o t t o n H T H o i i r p y n n e ) Dose

Thromb elastog ram

Prothrom-b i n ti m e F i b r i n o -gen mg/100 ml r k MA ME C o n tr ol 12 2 6 8 .5 2 1 7 .5 1 6 .5 257 0 . 2 5 KGy 12 2 6 4 .0 1 1 7 . 7 2 0 . 9 255 0 . 7 5 KGy 12 2 5 8 .0 1 1 4 .0 2 1 .5 260 1 .0 KGy 12 2 5 3 . 0 1 1 2 .7 2 3 . 0 260 2 . 0 KGy 12 3 4 7 . 0 8 8 . 6 2 4 .0 257 3 . 0 KGy 12 3 4 4 . 0 7 8 . 5 2 8 . 0 260 C r i t i c a l a l t e r a t i o n s o f m e c ha n ic a l p r o p e r t i e s o f t h e f i b r i n c l o t o f i r r a d i a t e d p l as m a a r e d o m en st ra ted i n F i g . 4 . The expo-s u r e t o d o expo-s e expo-s up t o 1 KGy b ro u g h t a b o u t a expo-s t r i k i n g l o expo-s expo-s o f th e

e l a s t i c i t y o f t h e i r r a d i a t e d f i b r i n . T reatme nt o f p la sm a w ith h ig h e r d o s e s d id n o t i n f l u e n c e a p p r e c i a b l y th e m ec h a n ic a l

s t r e n g t h o f t h e r e s u l t a n t g e l s . The e l a s t i c i t i e s rema ine d p r a c t i -c a l l y un-changed ( F i g . 4 ) .

S i n c e t h e m ec h a n ic a l s t r e n g t h o f t h e f i b r i n g e l f o l l o w i n g i r r a d i a t i o n a t 1 KGy r ea c h e d on ly a b ou t 50% o f th e maximum o b s e r -ved f o r c o n t r o l p la s m a , e x p e rim e nt s were a t te m p te d t o e x p l a in t h i s phenomenon. F i r s t o f a l l , t h e r e was no e vid en c e f o r v a r i a

-F i g . The e f f e c t o f gamma i r r a d i a t i o n on t h e e l a s t i c i t y o f f i -b r i n g e l s formed from p la sm a exp osed t o d o s e s o f 0 .2 5 - 3 KGy. The e l a s t i c i t y was c a l c u l a t e d a s s ta n d a r d p a r a m e te r ME from thrombe- l a s t o g r a p h i c r e c o r d i n g s (mean v a l u e s were c a l c u l a t e d from 10

de-t e r m i n a de-t i o n s )

Wpływ p rom ienio wa n ia gamma na e l a s t y c z n o ś ć ż e l u w łóknika utworz o-nego z o s o c z a krwi nap ro mienioneg o dawkami 0 . 2 5 - 3 KGy. E l a s t y c z -ność ż e l u w y l ic z a n o ja k o p a ra m e te r ME z z a p isów t r o m b o e l a s t o g r a -

f i c z n y c h ( s ą t o w a r t o ś c i ś r e d n i e 10 oz n ac z eń )

BJiHíiHHe o6jiy<eHHfl raMMa Ha ajiac T h u h oc tł re jia (JwOpHHa B03HHKiner0 H3 njiasMu KpoBH o6^y^eHHoii n,03aMn 0 , á 5 - 3 xCGy. SaacTHUHocTb

reza

b u c-

uüTano Ka.K napaweTp jCE H3 TpoMfíoojiacTorpaiín'iHLix 3 alinee# ( s t o cpej^— HHe BeJIHMHHU 10 oOOSHa^eHHÍt)

t i o n s in f i b r i n o g e n c o n c e n t r a t io n o f i r r a d i a t e d b lo o d p lasm a (Ta b . 1 ) . T h i s seems t o i n d i c a t e t h a t c h an g es i n t h e q u a n t i t y o f c o a g u l a b l e p r o t e i n do n o t p r o v i d e a b a s i s f o r e x p l a i n i n g th e l a r g e d i f f e r e n c e s o b se r v e d in f i b r i n g e l r i g i d i t y . To st ud y t h i s f u r t h e r , c r o s s - l i n k e d and n o n c r o s s - l i n k e d f i b r i n were p r ep a r e d from c o n t r o l and i r r a d i a t e d p l a s m a . T hese were n e x t a n a l y s e d by SDS p o l y a c r y la m i d e g e l e l e c t r o p h o r e s i s t o c h a r a c t e r i z e t h e d os e - —dependence o f th e c ha ng es in su b u n i t s t r u c t u r e o f th e m o le c u le . The r e s u l t s o f t h e s e a n a l y s i s a r e p r e s e n t e d i n F i g 5 . The g e l s f o r n o n c r o s s - l i n k e d f i b r i n o b t a i n e d from i r r a d i a t e d and- from c o n t r o l

p la sm a show th e p r e s e n c e o f un m od i fied a , (3 and y c h a i n s . The s c a n s f o r i r r a d i a t e d f i b r i n s i n F i g . 5 d e m o n stra te c o n s i d e r a b l e

/f -

dimer f o rm a ti o n which r e s u l t s from th e a c t i o n o f F a c t o r X l l l a , g e n e r a t e d d e s p i t e t h e p r e s e n c e o f e x c e s s EDTA. T h i s a l s o shows t h a t t h e r e i s no d i f f e r e n c e in th e m o b i l i t y and c o n c e n t r a

-F i g . 5 . D e n s i to m e tr i e s c a n s o f SDS p o ly a c ry la m i d e e l e c t r o p h o r e s i s g e l s o f r ed uc e d , n o n c r o s s - l i n k e d f i b r i n i s o l a t e d from c o n t r o l human p la s m a ( a ) and from p lasm a i r r a d i a t e d a t 1 KGy ( b ) , 2 KGy

( c ) and 3 KGy ( d )

Wykresy d ensyto m etryc z ne żelów po lia kr yl a m id o w y c h po r o z d z i a l e zredukowanego włókn ika n i e s t a b i l i z o w a n e g o otrzymanego z l u d z k i e -go o s o c z a k o n tr o ln eg o ( a ) or az z prób ek o s o c z a napromienionyc h

dawkami 1 KGy ( b ) , 2 KGy ( c ) o ra z 3 KGy ( d )

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cofcpaneHHoro HecTaöHJiH30BaHHoro $n6pnHa nojiy^eHHoro «3

'iejioBe'ie-CKOii KOHTpOJIbHOÖ IUia3MM ( a ) H H3 npOÖM njia3MN 06ay»ieHH0ił A03a,\lH

t i o n o f c o n s t i t u e n t c h a i n s o f f i b r i n o f i r r a d i a t e d p la s m a a s e s -ti m a t e d on t h e b a s i s o f d e n s i t o m e t r i c s c a n n i n g . S in c e t h e s e g e l s showed a s m a ll d eg r ee o f y - y d im e r i z a t i o n i t may be s u g g e s t e d r F i g . 6 . D e n s i to m e t r i c s c a n s o f SDS p o ly a c r y l a m i d e g e l s o f reduc ed human f i b r i n o g e n ( a ) , re d uc e d , c r o s s - l i n k e d f i b r i n p r e p a r e d from

c o n t r o l p la sm a ( b ) and from i r r a d i a t e d p la sm a a t 3 KGy ( c) Wykresy d en sytom etryc z ne ¿ słó w p olia kr yl a m id ow y c h po r o z d z i a l e zredukowanego f ib r yn og e n u lu d z k ie g o ( a ), zredukowanego włóknika s ta b i l iz o w a n e g o otrzymanego z o s o c z a k o n tr o l n e g o ( b) o ra z z o

so-c z a nap rom ienion ego dawką 3 KGy ( so-c )

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coicpaąeHHoro ‘«•JioBeMecKoro $HÖpnHoreHa (a), coKpanjeHHoro c i a ö n - jiHsoBaHHoro $łi6pnHa noayueHHoro na kohtpojibhoK ruia3Mu ’vb) h H3 njia-

t h a t gamma i r r a d i a t i o n even a t a s h ig h a d o se a s 3 KGy d oe s n ot damage F a c t o r X H I a n o r i m p a i r i t s f u n c t i o n .

F i g u r e 6 shows th e e l e c t r o p h o r e t i c p a t t e r n s on SDS g e l s o f c l o t s formed from p la sm a f o l l o w in g gamma i r r a d i a t i o n . The c l o t s were made by th e s im u l ta n eo u s a d d i t i o n o f c a lc iu m i o n s t o th e p la sm a s am p le s e xp ose d t o d o s e s o f 3 KGy. The c o n t r o l p a t t e r n shown in th e same f i g u r e i n d i c a t e s th e f o rm a ti on o f c r o s s - l i n k e d j -

y

d im ers and a - m u l t i m e r s . Of th e thre.e c o n s t i t u e n t c h a i n s , only (3 p o l y p e p ti d e rema ined i n t a c t in a l l c a s e s . The a b i l i t y to form o t-p ol y m er s i s unchanged even when th e d os e 3 KGy was ap-p l i e d . D i s c u s s i o n In p r e s e n t s t u d i e s th e f o r m a t i o n o f h ig h m o l e c u la r w eig ht a g g r e g a t e s i n th e i r r a d i a t e d sa m p l es o f f i b r i n o g e n was e vid e n c ed by SDS p o l y 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 . . In a d d i t i o n i t was o b se r v ed t h a t f o rm a ti o n o f a g g r e g a t e s i s i n i t i a t e d a t , t h e same d os e a s t h e d i s a p p e a r e n c e o f th e Aa, c h a in i n red uc ed sa m p les o f i r r a d i a t e d f i b r i n o g e n . Thus i t may be su p p os e d t h a t t h e i r r a d i a -t i o n - i n d u c e d a g g r e g a -t e s r e p r e s e n -t co m pl exes o f f i b r i n o g e n mole-c u l e s l i n k e d by A a p o l y p e p ti d e c h a i n s . T h i s was n o t t h e c a s e when whole p la sm a was ex p o se d t o gamma i r r a d i a t i o n . P o l y p e p t id e c o m p o si tio n o f f i b r i n i s o l a t e d from i r r a d i a t e d p la sm a d i d n ot show any d i f f e r e n c e s when compared t o t h a t o f u n i r r a d i a t e d p l a -sma.

The i n i t i a l e f f e c t o f gamma i r r a d i a t i o n on c l o t t i n g o f p l a s -ma f i b r i n o g e n i s d e t e c t a b l e by t h r o m b e l a st o g r a p h y . S in c e sa m p le s o f th e same p la sm a were a lw a y s s u b j e c t e d t o i r r a d i a t i o n , a l a r g e number o f f a c t o r s which can i n f l u e n c e t h e t h r o m b e l a s to g r a p h i c t r a c i n g s were, i n th e same c o n c e n t r a t i o n . Hence th e i n t e r p r e t a -t i o n o f c h an g e s i n -th r o m b e la s -to g r a p h y s h o u ld n o-t be amb ig uous. Gamma i r r a d i a t i o n o f b lo o d p la sm a c a u s e d th e m o d i f i c a t i o n o f t h r o m b e l a s t o g r a p h ic p a r a m e t e r s s i m i l a r t o t h a t o b se rv e d by T y - 1 e r [1 2 ] f o r d e c r e a s e d a c t i v i t y o f F a c t o r X l l l a . However, c om plete jf' - d i m e r i z a t i o n and a l m o s t c om pl ete ot - p o l y m e r i z a t i o n c ou ld be o b s e rv e d on 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 when

sam-p i e s o f c r o s s - l i n k e d f i b r i n o b t a i n e d from sam-p la sm a ex sam-p ose d a t 3 KGy were run ( F i g . 6 ) . T h e r e f or e t h e s e m o d i f i c a t i o n s o f th e s o -l i d i t y o f th e c -l o t a f t e r i r r a d i a t i o n ca nn ot be e x p l a i n e d by an i n a c t i v a t i o n o f F a c t o r X l l l a .

In ag reement with o t h e r s [ 5 ] ou r e xp er im en ts d em on str ate d t h a t due t o th e p r e s e n c e o f a l a r g e number o f d i s u l f i d e b r i d g e s th e f i b r i n o g e n m o lec u le i s very s e n s i t i v e t o i r r a d i a t i o n . However damaging e f f e c t on s t r u c t u r e and b i o l o g i c a l a c t i v i t y o f f i b r i n o gen was much s m a l l e r when whole p la sm a i n s t e a d o f i s o l a t e d p r o -t e i n was i r r a d i a -t e d . I -t may s u g g e s t t h a t som e.p la sm a components can somehow n o n s p e c i f l c a l l y p r o t e c t f i b r i n o g e n t o gamma i r r a d i a -t i o n e f f e c -t s . N e v e r t h e l e s s i n b oth c a s e s th e g e n e r a l c h a n ge s in m e c ha n ica l p r o p e r t i e s o f f i b r i n c l o t s were th e same. The f i b r i n g e l s p rod u ce d from i r r a d i a t e d s u b s t r a t e s were more t r a n s p a r e n t and s i g n i f i c a n t l y l e s s r i g i d . Acknowledgements We e x p r e s s ou r g r a t i t u d e t o Ms M. J a n u s z k i e w ic z - C y p e r l i n g f o r h e r e x c e l l e n t t e c h n i c a l a s s i s t e n c e d u ri n g p erf o rm in g t h e s e s t u -d i e s an-d t o Dr R. W ie rz b ic k i f o r e x p e r t a s s i s t e n c e a t thromb- e l a s t o g r a p h i c e x p e r im e n t s. T h i s s tu d y was s u p p o r te d by P r o j e c t R . I I I . 1 3 . 3 . 1 . REFERENCES [ 1 ] S c h e r a g a H. A ., N i m s L. P . , Arch. Biochem. B i op h y s . 36 , 3 36 -3 44 (1 9 5 2 ) . [ 2 ] S o w i ń s k i R . , O k a r e n k o L . , K o e n i g V. L . , Rad. R es. 1 1 , 9 0-10 0 ( 1 9 5 8 ) . [ 3 ] S o w i ń s k i R . , O k a r e n k o Ł . , K o e n i g V. L . , Rad. R es. 9 , 2 29 -2 3 9 ( 1 9 5 8 ) . [ 4 ] K o e n i g V. L . , S o w i ń s k i R. , O k a r e n -k o L . ,* F r e 1 i n g I . , W e i G. C . , Rad. R es. 1 3, 4 32 -4 4 4 (1 9 6 0 ) .

[ 5 ] K a m a t H. N. , N a d k a r n i G. B . , Rad. R e s. 4 9, 3 8 1-3 90 (1 9 7 2 ) . [ 6 ] D o o l i t t l e R. F., S c h u b e r t D., S c h w a r t z S . A ., A rch. Blochem. B iop h y s. 1 1 8 . 4 5 6-4 67 ( 1 9 6 7 ) . [ 7 ] M c D o n a g h J . , M e s s e l H. , M c D o n a g h R. P . , M u r a n o G. , B l o m b a c k B . , Blochem. B io p h y s . A c ta 2 5 7 , 13 5 -1 42 (1 9 7 2 ). [ 8 ] E g e b l a d K. , A s t r u p T . , B r i t . J . H aematol. 13 , 1 0 -16 (1 9 6 7 ) . [9] I t z h a k i R. F. , G i l l D. M. A nal. Blochem.

9_,

4 0 1-4 10 ( 1 9 6 9 ) . [ 1 0 ] C i e r n i e w s k i C. , K r a j e w s k i T . , Thromb. R es. 1 3 , 6 1 5-62 3 ( 1 9 7 8 ) . [ 1 1 ] C i e r n i e w s k i C. , K r a j e w s k i T . , K l i m -c z a k J . , Thromb. R e s. 7 , 2 97 -3 0 3 ( 1 9 7 5 ). [ 1 2 ] T y 1 e r H. N. Thromb. D i a th . Haemorrh. 2 2 , 3 98 -3 99 (1 9 6 9 ) . Department o f B i o c h e m i s tr y I n s t i t u t e o f B io c h e m i st r y and B i o p h y s i c s U n i v e r s i t y o f Łódź T adeu sz K r a j e w s k i , C zesław C i e rn i e w s k i WPŁYW PROMIENIOWANIA GAMMA NA FIBRYNOGEN OSOCZOWY

W p ra c y bad ano wpływ p rom ie niow an ia gamma na w y kr z e p ia n ie f i - brynogenu osocz ow eg o. Za pomocą t r o m b o e l a s t o g r a f i i wykryto duże zmiany we w ł a ś c i w o ś c i a c h mechanicznych s kr z e p u w łók n ika , o t r z y -manego z o s o c z a krwi l u b z wyizolowanego f ib r y n o g e n u , poddanych p r z e d wykrzepieniem d z i a ł a n i u prom ien i jo n i z u j ą c y c h ( 60C o ). Ana-l i z a sk ła d u p oAna-l ip ep ty d o w eg o c z ą s t e c z k i f ib ry n o g e n u , n ap ro m ien io-n e j różio-nymi dawkami (0 - 3 KGy), w y ka za ła stopniowy u by tek z a w a rto-ś c i ła ń c u c h a A a l f a . Ta p o d j e d n o s tk a o k a z a ł a s i ę być n a j b a r d z i e j w ra ż l iw ą na d z i a ł a n i e p rom ie niow ania j o n i z u j ą c e g o i prawdopodob-n i e b e z p o ś r e d prawdopodob-n i o b i e r z e u d z i a ł w a g r e g a c j i c z ą s t e c z e k f i b r y n o -genu.

T a f l e y m K p a e B C K H , 4 e c J i a B I J e p n e B C K H

BJIHHHHE TAMMA ORJiyMEHHH HA ITJIA3MATÎWECKMÎÎ $HEPHH0rEH

B HacTOHmeit p afioT e HccjieflOBaHO b ^h hu h s raMMa oOjiy^eHHa Ha

c E e p T U B a H H e r u ia 3 M a T H < i e c K o r o ( J w O p H H o r e H a . r i 0 K a 3 a H 0 , î t o H a H Ô o ji e e 'l y B C T B H T e j i b H o ü n o ^ t e f lH H H U e i t M O Jie K y jiu $ n 6 p H H o r e H a H a n e f t c T B u e h o h h- 3 H p y » n e r o H S J iy ^ e H H a H B J i a e T C H u e n b Aot. IT p H H H M a er O H a H e n o c p e n c - t B e H H o e j n i a c T H ë b 0 0 p a 3 0 B a H H H B u c o K O M O ji e K y jw p H H x a r p e r a T O B b o O j i y - u e H H u x n p e n a p a T a x $ > H 6 p H H o r e 'H a . I l o c j i e o f i j i y q e H H a , $ K 6 p n H o r e H o O p a - 3 y e T C r y C T O K , K O T O p O # O M eH b O T JIH M a eT C H M eX aH H H eC K H M H C B O ÎÎC T B a M H o t o r y c T K a B b 3 H H K i n e r o h s K O H T p o j ib H o r o $ H < 5 p H H o r e H a .