Photooxidation of fibrinogen in the presence of methylene blue and its effect on polymerization

161

Biochimica et Biophysica Acta, 532 (1978) 1 6 1 - - 1 7 0 © E l s e v i e r / N o r t h - H o l l a n d Biomedical Press

BBA 37811

PHOTOOXIDATION OF F I B R I N O G E N IN THE PRESENCE OF METHYLENE BLUE AND ITS E F F E C T ON POLYMERIZATION

Y U J I I N A D A *, B I R G I T H E S S E L a n d B I R G E R B L O M B A C K

Department of Blood Coagulation Research, Karolinska Institute, Stockholm, Sweden and The New York Blood Center, New York, N.Y. (U.S.A.) (Received April 2 5 t h , 1977)

Summary Human fibrinogen was illuminated in the presence of methylene blue. The resulting photooxidized fibrinogen was devoid of polymerization activity and thrombin-induced coagulability. The initial rate of the thrombin catalysed release of fibrinopeptides from photooxidized fibrinogen was normal. It was shown that illumination of photooxidized fibrinogen and photooxidized fragment N-DSK caused the modification of histidine residues. Tryptophan residues were also modified. When fibrinogen was photooxidized immediately after the addition of thrombin, the capacity to polymerize was lost. The inhibition of polymerization was less marked when oxidation was initiated at the time when polymerization began or thereafter. Photooxidized fibrinogen acts as an inhibitor of the polymerization of fibrin monomers. Photooxidized fibrinogen has affinity for thrombin-activated fibrinogenSepharose and thrombin-activated fragment N-DSK-Sepharose. When the former conjugate is illuminated in the presence of methylene blue its affinity for fibrinogen is decreased. It is concluded that the fragment N-DSK domain of fibrinogen is affected by photooxidation.

Introduction Fibrinogen, after activation with thrombin, forms an ordered fibrin network which is subsequently cross-linked by factor XIIIa [1--4]. Kudryk et al. [5,6]

* On leave from Laboratory of Biological Chemistry, Tokyo Institute of Technology, Ookayama, Meguroku, Tokyo, Japan Abbreviahons- N-DSK, (A~1-51, B~I-118, 9"1-78)2 ref. 4; N-DSKa, (c~17-51, ~15-118, 71-78) 2 ref. 4.

162

and York and Blomb/ick [7] prepared conjugates of Sepharose and fibrin monomers as well as conjugates of Sepharose and the NH2-terminal part {fragment N-DSK) of fibrinogen and studied the affinity of these conjugates to different fragments of fibrinogen in order to identify the binding domains. One of the domains is contained within fragment N-DSK which is located in a central portion of the fibrinogen molecule. The other domain is located in plasmic fragment D. There are two identical fragment D-domains situated at opposite extremities in the molecule [4]. The fragment N-DSK domain requires activation with thrombin in order to become active (fragment N-DSKa), whereas the fragment D domains do not require activation. Thus, fibrinogen contains two complementary binding domains which may be of importance for the initial alignment of activated fibrinogen molecules to form fibrin [2,4]. By modifying tyrosine and/or lysine residues in fibrinogen some insight has been obtained into their functional importance for polymerization [8--11]. Modification of histidine residues in proteins has been achieved by several investigators using photooxldation in presence of methylene blue [12,13] and rose bengal [ 14]. In one study on the photooxidation of fibrmogen, it was observed that irradiation in the presence of h e m a t o p o r p h y n n prolonged the thrombin clotting time [ 15]. The authors suggested that the change in activity was accompanied by destruction of histidine residues. The importance of histidine residues for the polymerization of fibrinogen was previously suggested by Ferry [16] on the basis of a study of polymerization activity at different pH. In order to obtain more information on the importance of histidine residues in the polymerization reaction, we have performed a study on the photooxldation of human fibrinogen in the presence of methylene blue. In our study, we have made an a t t e m p t to correlate the changes in function as regards polymerization with the release of fibrinopeptides and the number of amino acid residues being modified. We have also investigated the effect of photooxidation on the affinity characteristics displayed by thrombin-treated fibrinogen- and fragment N-DSK-Sepharose. Materials and Methods

Biomaterials and chemicals Human fibrinogen, isolated by the method of Blomb/ick and Blomb~ick [17], was obtained from IMCO Corp., Stockholm, Sweden. The molar extinction coefficient e = 5.1 • 10 s M -~ • cm -1 at 278 nm was calculated from E I ~ m = 15.0 [18] and the molecular weight of fibrinogen (340000). Bovine thrombin with the activity of 207 NIH units/mg of protein was prepared essentially as described earlier [ 19]. Methylene blue, 3,7-bis(dimethylamino)-phenzathionium chloride, was used as a photosensitizer for the oxidation of fibrinogen. Fragment N-DSK was obtained from fibrinogen treated with cyanogen bromide [20]. Fibrin monomer-Sepharose conjugates and fragment N-DSKa-Sepharose conjugates were prepared as previously described; fibrinogen or fragment N-DSK was coupled to CNBr-activated Sepharose 4B and each insolubilized conjugate was subsequently activated with thrombin [5,6,21]. Photooxidation o f fibrinogen, fragment N-DSK and conjugates Photooxidation experiments were performed as follows: mixtures of 2--15 •

163 10 -6 M fibrinogen and 2--10 • 10 -s M methylene blue in 0.08--0.15 M Tris .. HC1 (pH 6.9) containing 0.05--0.10 M NaCl and 0.015--0.04 M CaC12 were illuminated with 60 W or 150 W incandescent lamps from a distance of 15--60 cm. During illumination, the sample solutions were kept in ice-water. The sample was shielded from radiation heat by putting a glass plate or a Petri dish containing cold water between the light-source and the sample. (a) In some experiments 1 ml samples were added to cuvettes and 20 pl of methylene blue (0.6 mg/ml; see Figs. 1, 2, 3) were added. Polymerization activity was determined spectrophotometrically at various times during illumination. (b) In other experiments 25 ml of the fibrinogen solution (in a 100 ml beaker, diameter 5 cm) was mixed with 0.5 ml methylene blue (0.6 mg/ml) and illuminated as above, for up to 2 h (see Fig. 4). The samples were thereafter dialyzed against distilled water or 0.025 M Tris. HC1 (pH 7.3) containing 0.125 M NaCI. Photooxidized thrombin-activated fibrinogen-Sepharose was prepared by illuminating the conjugate in the presence of methylene blue with a 150 W lamp at a distance of 30 cm for 1 h. During illumination the sedimented conjugate was suspended in 0.05 M Tris. HC1 buffer, pH 7.6, containing 0.1 M NaCI, 0.005 M EDTA, 0.025 M e-aminocaproic acid and Trasylol (5000 KIU */1) {Buffer I) [21].

Analysis of polymerization activity of photooxidized fibrinogen To an aliquot of fibrinogen solution was added 0.1 volume of thrombin solution (5 NIH units/ml). Polymerization was assessed spectrophotometrically by measuring the absorbance of the sample solution at 450 nm vs. time, using a Beckman DK-2A-Ratio Recording Spectrophotometer. In these experiments optical density changes of 0--0.5 A were obtained during 10 min. The maxim u m rate of absorbance change (A A/t) was taken as a measure of polymerization activity of the sample.

Determination of fibrinopeptides released from fibrinogen and photooxidized fibrinogen The a m o u n t of fibrinopeptides released from fibrinogen and photooxidized fibrinogen was determined by NH2-terminal amino acid analysis essentially as described previously [22]. To 1.4 ml of fibrinogen (5.0 mg/ml) in 0.025 M Tris • HC1 (pH 7.2) containing 0.125 M NaCl was added 10 ~l of a thrombin solution (4 NIH units/ml). Coupling was performed by adding 200 pl of sample solution to a mixture of 200 pl urea (10 M), 0.15 M Tris • HC1 (pH 9.5), 400 pl distilled pyridine and 1 #1 of phenyliso[3SS]thiocyanate (1.46 molfl, 27.9 Ci/ mol). Incubation was carried o u t for 1 h at 40°C. After extraction of the mixture with benzene and precipitation of the coupled protein with acetone, the protein was hydrolyzed in 1 M HC1. The phenylthiohydantoins were extracted with ethylacetate and were subjected to thin-layer chromatography. Phenylthiohydantoins on the chromatogram were extracted with 95% ethanol and the radioactivity counted in a Beckman Liquid Scintillator, LS-200 B.

* KaUikrein inhibitor unit.

164

Amino acid analysis Amino acid composition of fibrinogen and fragment N-DSK before and after photooxidation was determined using the Technicon Autoanalyzer Model TSM-1 after hydrolysis with 5.7 N HC1 at l l 0 ° C for 22 h. Tryptophan was determined spectrophotometrically [23,24].

Affinity characteristics of fibrinogen and photooxidized fibrinogen to various Sepharose conjugates The fibrin monomer-Sepharose and fragment N-DSKa-Sepharose conjugates were suspended in Buffer I (see above), and poured into a column (0.4--0.6 cm 2 × 4--9 cm). 0.5--7 ml of fibrinogen solutions were added to each column and eluted with Buffer ! at a flow-rate of 0.13 ml/min. Elution of adsorbed protein was performed with Buffer I containing 8 M urea (Buffer II). Fractions (2 ml) were collected and the absorbance at 280 nm of each fraction was recorded with the UA-5 Absorbance Monitor, Isco, Lincoln, Nebraska. Results

Inhibitory effect of light on the clotting activity Fig. 1 shows a plot of the polymerization activity of fibrinogen against the incubation time in light (curves A and C) and in the dark (curve B). Judging by

E

15

O4

o

5 ~

10

o

c___ = (..)

-.~

02

rn FF 0 ~n rn <[

5

20

30

Irradtahon Time In M,n

4 .

.

.

.

,,,,, , .

0

o

10

3

40

4

0

TIME (mm)

Fig. 1. I n h i b i t i o n o f p o l y m e r i z a t i o n b y p h o t o o x l d a t l o n o f f i b n n o g c n . C u r v e A. P o l y m e r i z a t i o n a c t l w t y of f i b r m o g e n a f t e r a d d i t i o n o f t h r o m b m . S a m p l e i l l u m i n a t e d m p r e s e n c e of m e t h y l e n e b l u e w i t h a 60 W i n c a n d e s c e n t l a m p f r o m a d i s t a n c e o f 30 c m a t 1 5 ° C f o r v a r i o u s p e r i o d s . C u r v e B: p o l y m e r i z a t i o n a c t i v i t y of f l b r i n o g e n w i t h t h r o m b m in p r e s e n c e o f m e t h y l e n e b l u e m t h e d a r k . C u r v e C p o l y m e r i z a t i o n a c t i v i t y of f l b r i n o g e n i l l u m i n a t e d m t h e p r e s e n c e o f m e t h y l e n e blue a f t e r s t a n d i n g f o r 30 rain in t h e d a r k . 1 m l s a m p l e s o l u t i o n s w e r e u s e d . C o n c e n t r a t x o n s : 2.1 10 -6 M f l b r l n o g e n , 1.8 1 0 - 5 M m e t h y l e n e blue a n d 0 . 5 N I H u n i t t h r o m b m p e r m l . F o r f u r t h e r d e t a i l s see M e t h o d s a n d M a t e r i a l s . Fig. 2. I n h i b i t i o n o f p o l y m e r i z a t i o n b y p h o t o o x l d a t i o n f o r t i m e i n t e r v a l s f o l l o w i n g t h e a d d i t m n of t h r o m b m . A b s o r b a n c e c h a n g e s w e r e m e a s u r e d a t 4 5 0 n m . C u r v e 1" f i b r i n o g e n - t h r o m b i n s y s t e m in t h e d a r k in t h e p r e s e n c e o f m e t h y l e n e b l u e . C u r v e 2: t h e s a m e as c u r v e 1, e x c e p t t h a t t h r o m b i n h a d b e e n p r e v i o u s l y i l l u m i n a t e d f o r 2 m i n m t h e P r e s e n c e of m e t h y l e n e b l u e . C u r v e 11" f l b r i n o g e n a n d m e t h y l e n e b l u e w e r e i l l u m i n a t e d f o r 2 m i n , w h e r e a f t e r t h r o m b l n w a s a d d e d . C u r v e s 3 - - 1 0 . t h r o m b m w a s a d d e d to f i b r i n o g e n in t h e p r e s e n c e of m e t h y l e n e b l u e a n d t h e s a m p l e s w e r e e x p o s e d to I L l u m i n a t i o n at 60 W, at a d i s t a n c e of 15 c m f o r 9 0 s b e g i n n i n g at v a r i o u s t i m e s f o l l o w i n g t h e a d d i t i o n o f t h r o m b m . T h e v e r t i c a l lines i n d i c a t e t h e t i m e s f o r i m t i a t l o n o f i l l u m i n a t i o n . C o n c e n t r a t i o n s : 1.8 • 10 -6 M f l b r i n o g e n , 3 • 10 -5 M m e t h y l e n e b l u e , a n d 1 N I H u m t t h r o m b i n / m l . F o r f u r t h e r d e t a i l s see M e t h o d s a n d M a t e r i a l s .

165 the lack of increase in absorption after addition of thrombin, the illumination of fibrinogen in the presence of methylene blue for approximately 30 min caused a complete loss of ability to polymerize. On the other hand, the polymerization activity was fully retained after incubation in the dark. When the sample which had been kept in dark was illuminated, the polymerization activity was decreased (curve C). Fig. 2 represents experiments in which the fibrinogen solution was photooxidized for different periods following the addition of thrombin. Curve 1 shows the polymerization activity after the addition of thrombin in the dark. The polymerization activity of fibrinogen with thrombin in the absence of methylene blue shows an identical profile (not shown in Fig. 2). Curve 11 shows that methylene blue alone has negligible effect on the absorption pattern of fibrinogen. In order to test whether thrombin was affected by photooxidation, thrombin in the presence of methylene blue was Illuminated for 2 rain and its polymerizing activity on fibrinogen was subsequently measured. The result is shown in curve 2. No appreciable inactivation of thrombin occurred. The particular experiment shown in curve 2 suggests that photooxidation results in fact in a slight increase in the activity of thrombin. However, in other experiments no such effect could be demonstrated. In the experiments shown in curves 3--10 the samples were exposed to illumination for 90 s beginning at various times after the addition of thrombin. Following the light-exposure (shown by dashed lines in Fig. 2) the absorbance was again recorded in the dark. These experiments show that polymerization activity is inhibited by light-exposure after the addition of thrombin. However, the inhibition of the polymerization process is most pronounced in the cases where illumination was started prior to the appearance of polymerization. This can be seen by comparing curves 3--6 and curves 7--10. Samples of fibrinogen solution ( 0 . 4 - - 1 . 8 . 1 0 -s M) were illuminated in the presence of methylene blue for 20 and 60 s, respectively, whereupon the polymerization activity was determined. Fig. 3 shows the double reciprocal plot of the rate of the absorbance increase at 450 nm against fibrinogen concentration, where curve A was obtained in the dark and curves B and C after exposure to light for 30 and 60 s, respectively. This experiment shows that the photooxidized fibrinogen inhibits the polymerization of fibrinogen.

Thrombin-catalysed release of fibrinopeptides from the photooxidized fibrinogen The following experiment was performed in order to test whether fibrinopeptides were released from photooxidized fibrinogen by the action of thrombin. Fibrinogen, photooxidized for a b o u t 2 h, did neither clot nor show any absorbance increase after the addition of thrombin. Release of fibrinopeptides by thrombin was determined by NH2-terminal analysis. Increase in NH2-terminal glycine during digestion reflects the release of fibrinopeptides A and/or B [19,22]. Fig. 4 shows the release of fibrinopeptides from photooxidized fibrinogen (curve A) and normal fibrinogen (curve B). The initial increase in the ratio (Gly/Tyr %) for the photooxidized fibrinogen showed the same increase as was found for normal fibrinogen. The results indicate that the

166 70--

A/

60-50

16

C

12

~4o

,~,3o

>

B

~8

~.

1c

I '1

I 2

]/$

I 3

I 4 X 105 I"1

oI

i 10

i

i

20

30

T~ me m M m

Fig. 3. D o u b l e r e c i p r o c a l p l o t o f p o l y m e r i z a t i o n a c t w t t y vs. f t b r i n o g e n c o n c e n t r a t i o n . C u r v e s A: in t h e d a r k , B a n d C: l U u m i n a t i o n f o r 30 s a n d 6 0 s, r e s p e c t i v e l y . A f i b r i n o g e n s o l u t i o n (1.76 • 10 - s M) m 0 . 1 5 M T r i s • H C I ( p H 6 . 9 ) c o n t a i n i n g 0.1 M N a C I a n d 0 . 0 4 M CaCl 2 w a s d i l u t e d 2, 30 4 a n d 5 t i m e s , r e s p e c t w e l y , w i t h the b u f f e r a n d 0 . 0 2 m l o f m e t h y l e n e b l u e ( 1 . 8 8 • 10 -3 M) w a s a d d e d t o 1 ml a h q u o t s o f t h e f l b r i n o g e n s o l u h o n s . I l l u m i n a t i o n w a s c a r ~ e d o u t w i t h a 1 5 0 W i n c a n d e s c e n t l a m p f r o m a d i s t a n c e of 15 c m . T e m p e r a t u r e 20°C. F o r f u r t h e r d e t a i l s see M e t h o d s a n d M a t e r i a l s . Fig. 4. R e l e a s e o f f i b r m o p e p t l d e s f r o m p h o t o o x t d m e d a n d i n t a c t f i b r l n o g e n . C u r v e A: 1 . 4 - 1 0 - s M p h o t o o x i d i z e d f i b n n o g e n . P h o t o o x i d a t l o n o f f l b n n o g e n ( 2 5 m l ) w a s p e r f o r m e d w~th a 1 5 0 W i n c a n d e s c e n t l a m p at a d i s t a n c e o f 15 c m f o r 2 h. C u r v e B. 1.4 • 10 - s M f i b r i n o g e n . A c l o t w a s f o r m e d 12 m m a f t e r t h e a d d i t i o n o f t h r o m b m . F o r f u r t h e r d e t a i l s see M e t h o d s a n d M a t e r i a l s .

inhibitory effect of photooxidation on the polymerization activity is not manifested in the proteolytic process catalysed by thrombin but rather in the polymerization phase following the release of fibrinopeptides.

Amino acid compositions of photooxidized fibrinogen and fragment N-DSK Table I shows the amino acid composition of fibrinogen and fragment N-DSK which were photooxidized for 2 h, as well as those of intact fibrinogen and intact fragment N-DSK. In photooxidized fibrinogen the number of histidine residues was approximately 40% of that in native fibrinogen. The presence of histidine residues in photooxidized fragment N-DSK could not be demonstrated. The number of tryptophan residues as determined spectrophotometrically appeared to be lower in fibrinogen photooxidized for 2 h than in intact fibrinogen. Most of the other amino acids in the acid hydrolysate appeared not to be significantly changed by photooxidation. The values for cystine in acid hydrolysates are somewhat unreliable b u t the absence of cysteic acid would indicate insignificant modification of this residue. The somewhat lower value for methionine after photooxidation may indicate that some modification of this residue had occurred.

Affinity of fibrinogen and photooxidized fibrinogen to photooxidized and nonphotooxidized fibrin monomer-Sepharose and to fragment N-DSKa-Sepharose In one set of experiments the affinity of normal fibrinogen and photo-

167 TABLE I AMINO ACID COMPOSITIONS OF F I B R I N O G E N AND F R A G M E N T N-DSK BEFORE AND A F T E R PHOTOOXIDATION P h o t o o x l d a t i o n o f f l b r m o g e n ( 2 5 m l ) was p e r f o r m e d b y t l l u m m a t l o n w i t h 150 W i n c a n d e s c e n t l a m p f r o m a d m t a n c e o f 15 c m for 2 h. F r a g m e n t N - D S K was t U u m m a t e d wxth a 150 W l a m p f r o m 30 c m d i s t a n c e for 1 h. E a c h s a m p l e was h y d r o l y z e d w i t h 5.7 N HC1 at l l 0 ° C for 22 h.

AsP Thr Set Glu Pro Gly Ala Cys/2 Val Met Ile Leu Tyr Phe His Lys Arg T r p *** Yield.%

Flbrmogen (vmol/mg protein)

F r a g m e n t N-DSK ( p m o l / m g protein) *

Non-photooxtdlzed

Non-photooxxdlzed

0.933 0.383 0.438 0.877 0.271 0.662 0.299 0.028 0.326 0.168 0.266 0.445 0.253 0.229 0.137 0.637 0.461 0.241 76.8

Photooxldlzed 0.889 0.361 0.448 0.840 0.261 0.704 0.311 0.069 0.343 0.149 0.302 0.500 0.254 0.232 0.037 0.631 0 455 0.113 75 8

0 731 0.206 0.342 0.843 0.206 0.452 0 446 0.166 0.337 t r a c e ** 0 192 0.479 0.166 0.140 0.063 0.413 0.415 -62 2

Photooxxdzzed 0.661 0.194 0.303 0.701 0.184 0.393 0 373 0.130 0.306 trace * 0.157 0.413 0.142 0.114 trace 0.338 0.354 -52.6

*

* C a l c u l a t e d o n p r o t e m free o f c a r b o h y d r a t e s a n d o t h e r p r o s t h e t i c g r o u p s . ** D e t e r m i n e d as h o m o s e r m e . *** D e t e r m i n e d s p e c t r o p h o t o m e t r m a i l y [ 2 4 ] .

i i~Buffer I

~ I

0

~

J,l(.--BufferII

AA

10 Number 20 Tube

30

Fig. 5. A f f i n i t y of f i b r i n o g e n f o r p h o t o o x i d i z e d fibrin m o n o m e r - S e p h a r o s e c o n j u g a t e a n d fibrin m o n o m e r - S e p h a r o s e c o n j u g a t e . C u r v e A: e l u t i o n p a t t e r n o f f i b r i n o g e n ( 1 0 rag) p a s s e d t h r o u g h a c o l u m n ( 0 . 3 6 c m 2 × 7 c m ) o f fibrin m o n o m e r - S e p h a r o s e . C u r v e B: e l u t t o n p a t t e r n o f f l b r i n o g e n ( 1 0 rag) p a s s e d t h r o u g h a c o l u m n ( 0 . 3 6 e m 2 X 7 c m ) o f p h o t o o x i d i z e d fibrin m o n o m e r - S e p h a r o s e . T h e c o l u m n s w e r e e q u i l i b r a t e d w i t h B u f f e r I ( 0 . 0 5 M T n s • HCI, p H 7.6, c o n t a i n i n g 0.1 M NaCl, 0 . 0 0 5 M E D T A , 5 0 0 0 U/1 T r a s y l o l a n d 0 . 0 2 5 M e - a m i n o c a p r o m a m d ) a n d a b s o r b e d p r o t e i n s e l u t e d w i t h B u f f e r II ( B u f f e r I c o n t a i n i n g 8 M u r e a ) . F o r p h o t o o x l d a t l o n of t h e c o n j u g a t e s see Materials a n d M e t h o d s , " P h o t o o x l d a t l o n o f f l b r i n o g e n , f r a g m e n t N-DSK and conjugates, b".

168

oxidized fibrinogen (prepared as in "Photooxidation of fibrinogen, fragment N-DSK and conjugates, b " ) was tested on fibrin monomer-Sepharose columns. Five mg portions of the different fibrinogens in 3 to 7 ml of Buffer I were applied to the column (0.6 cm 2 × 4 cm). Adsorbed protein was eluted using Buffer II (containing urea). The results showed that normal fibrinogen and photooxidized fibrmogen were adsorbed to the same extent. In another set of experiments, the affinities of fibrinogen and photooxidized fibrinogen were tested on fragment N-DSKa-Sepharose under similar conditions. As before both types of fibrinogen were adsorbed to the same extent. In a third set of experiments, fibrin monomer-Sepharose was illuminated in the presence of methylene blue and the affinity of the photooxidized conjugate for fibrinogen was subsequently tested. The results are shown in Fig. 5. Curves A and B represent the elution profiles obtained with the conjugate before and after photooxidation, respectively. Comparison of the profiles reveals that photooxidation has drastically reduced the bindi.~g capacity of the conjugate for fibrinogen. Discussion

It is well-known that photochemical oxidation in the presence of a sensitizing dye causes the modification of amino acid residues in proteins (cf. ref. 25). Histidine, tyrosine, tryptophan, methionine and cystine have been shown to react during oxidation. Using proflavine as photosensitizer oxidation of histidine residues is usually the fastest reaction at neutral pH whereas oxidation of tyrosine residues is most pronounced at higher pH values [26]. Tryptophan and methionine residues are preferentially oxidized below pH 4 [26]. Zieve and Solomon [ 15] found that irradiation of fibrinogen in the presence of hematoporphyrin resulted in prolongation of the clotting time. They postulated that this effect was due to destruction of histidine residues although this was not shown in their experiments. The present study shows that photooxidation of fibrinogen in the presence of methylene blue is accompanied by a rapid loss of thrombin-induced coagulability of fibrinogen. This effect cannot be ascribed to an influence on the thrombin catalyzed step since the enzyme was shown to be unchanged during photooxidation and the proteolytic cleavage occurred at a normal initial rate in photooxidized fibrinogen. The fact that the initial rate was not changed suggests that the release of fibrinopeptide A was normal [1]. It is therefore likely that photooxidation has produced a structural change in the fibrinogen molecule which hampers the polymerization process that follows the release of fibrinopeptide A. This proposed structural change is likely to involve histidine and/or tryptophan residues. In fact, extensive photooxidation of fibrinogen was accompanied by modification of a b o u t 70% of the histidine residues. As judged from spectrophotometrical analysis, a b o u t 50% of the tryptophan residues were changed. The inhibitory effect of photooxidation on polymerization seems to depend to some extent on the time at which the photooxidation is initiated during the polymerization. The results shown in Fig. 2 indicate that the polymerization is almost completely abolished when illumination is performed immediately after

169

the addition of thrombin. One possible interpretation of these results is that illumination at an early stage in the thrombin-catalyzed reactions prevents the unfolding of a polymerization site(s) which is hidden in the intact molecule. The site itself when unfolded is relatively unaffected by photooxidation. An alternative explanation is in keeping with previous suggestions [2,4--7] that two discrete sets of sites are involved in the polymerization of fibrinogen. One set of sites becomes operative u p o n the release of fibrinopeptide A whereas the second set of sites is activated on the release of fibrinopeptide B, which occurs later. It is possible that the first site is more susceptible to photooxidation than the second site. Thus, the inhibition of polymerization will be diminished in cases where the oxidation is initiated late in the polymerization procedure. Work is now in progress to further elucidate this problem by measuring the release of fibrinopeptides A and B and relating this to the polymerization reaction. The present results also give some hints regarding the functional dommns in fibrinogen which are affected by photooxidation. Previous work in our laboratory has demonstrated that there exist two distinct functional domains in the fibrinogen molecule: one contained in the fragment N-DSK-portion of the molecule and the other in the Fragment D-region [2,4--7]. This conclusion was based primarily on affinity studies as it was found that fibrin monomer-Sepharose conjugates and fragment N-DSKa-Sepharose conjugates had affinity to fibrinogen and plasmic Fragment D [ 5--7]. In the present study we found that these conjugates displayed affinity towards n o t only normal fibrinogen but also to photooxidized fibrinogen. We interprete the results to mean that the Fragment D-domain is functional in photooxidized fibrinogen. In contrast, however, when fibrin monomer-Sepharose conjugate was illuminated in the presence of methylene blue, this conjugate lost its affinity for fibrinogen. This experiment was interpreted to mean that the fragment N-DSK-domain in fibrinogen is rendered more or less non-functional by photooxidation. The experiments shown in Fig. 3 may then be interpreted to mean that the photooxidized fibrinogen acts as an inhibitor by combining with the fragment N-DSK-domams of activated fibrinogen molecules and thereby blocking the growth of the polymer. The amino acid analysis of photooxidized fibrinogen suggests that histidine is most easily oxidized b u t tryptophan residues seem also to be oxidized to an appreciable extent. Experiments now in progress (Hessel, B., et al., in preparation) suggest that modification of histidine residues rather than tryptophan residues is correlated with the decrease in polymerization activity. The accuracy of the amino acid analysis does not permit at the present time any definite conclusion concerning the modification of methionine and cystine residues during photooxidation. The fact that all histidines are readily oxidized in fragment N-DSK may indicate that the loss of polymerization activity is due to the modification of these residues in the fragment N-DSK-domain of fibrinogen. It should be mentioned that a role for histidine residues in the polymerization of fibrin monomers has been suggested by Mihalyi [27] and Sturtevant et al. [28]. These authors suggested that in the polymerization of thrombin-activated fibrinogen, the imidazole groups of histidine residues in the activated fibrinogen molecule form hydrogen-bonds with phenolic groups of tyrosine residues in the molecule.

170 Work is now in progress to identify the functional domains in fibrinogen which are affected by photooxidation and also to identify the location of the modified residues in the primary structure.

Acknowledgements The authors wish to thank Dr. Bengt G~rdlunu, Mrs Elvy Andersson, Sonja SSderman, Lisbeth Therkildsen, Helga Messel, Miss Lena WikstrSm and Mr. Peter Wolf for their help in executing this work. This work was supported by grants from the National Institutes of Health (HL07379-11) and the Swedish Medical Research Council (B76-13x-02475-10C).

References 1 Blomback, B. (1967) m Blood Clotting E n z y m o l o g y (Seegers, W.H., ed.), pp. 143--215, Academic Press Inc., New York 2 Blomb~ck, B. and Blomb~ck, M. (1972) Ann. New York Acad. Sci. 202, 77--97 3 DooHttle, R.F. (1973) in Advances in Protein Chemistry, pp. 1--109, Academic Press, New York 4 Blombbck, B., Hogg, D.H., G~rdlund, B., Hessel, B. and K udryk, B. (1976) Thrombosis Research Suppl. II, 8, 329--346 5 Kudryk, B.J., CoHen, D., Woods, K.R. and Blomback, B. (1974) J. Biol. Chem. 249, 3322--3325 6 Kudryk, B., Reuterby, J. and Blomblick, B. (1973) Thrombosis Res. 2, 297--304 7 York, L.L. and Blombitck, B. (1976) Thrombosis Res. 8 , 6 0 7 - - 6 1 8 8 Laka, K. and Mlhalyl, E. (1949) Nature 163, 66 9 Caspary, E.A. (1956) Biochem. J. 62, 507--512 10 Phillips, H.M. and York, J.L. (1973) Biochemistry 12, 36 37--3642 11 Mihalyi, E. and Albert, A. (1971) Biochemistry 10, 237--242 12 Ray Jr., W.J. and Koshland, Jr., D.E., (1962) J. Biol. Chem. 237, 2493--2505 13 Martinez-Carrion, M., Turano, C., Riva, F. and FaseHa, P. (1967) J. Biol. Chem. 242, 1426--1430 14 Westhead, E.W. (1965) Biochemistry 4, 2139--2144 15 Zieve, P.D. and Solomon, H.M. (1966) Am. J. Physiol. 210, 1391--1395 16 Ferry, J.D. (1952) Proe. Natl. Acad. Sci. U.S. 38, 566--569 17 Blomb//ck, B. and Blombbck, M. (1957) Ark. Kern. 10, 415--443 18 Mihalyi, E. (1968) Bmchemistry 7 , 2 0 8 - - 2 2 3 19 Blombdck, B. and Yamashina, I. (1958) Ark. Kern. 12, 299--319 20 Blomb/ick, B., GrSndahl, N.J., Hessel, B., Iwanaga, S. and WaH~n, P. (1973) J. Bml. Chem. 248, 58 06--5820 21 Heene, D.L. and Matthias, F.R. (1973) Thrombosis Res. 2, 137--154 22 Hogg, D.H. and Blomb/tck, B. (1974) Thrombosts Res. 5 , 6 8 5 - - 6 9 3 23 Edelhoch, H. (1967) Biochemistry 6, 1 9 4 8 - - 1 9 5 4 24 Bencze, W.L. and Schmid, K. (1957) Analyt. Chem. 29, 1193--1196 25 Means, G.E. and Feeney, R.E. (1971) m Chemical Modification of Protein, pp. 165--174, HoldenDay-Inc, San Francisco 26 Sluyterman, L.A. (1962) Blochim. B1ophys. Acta 60, 557--561 27 MLhalyi, E. (1954) J. Biol. Chem. 2 0 9 , 7 3 3 - - 7 4 1 28 Sturtevant, J.M., Laskowskl, Jr., M., Donnelly, T.H. and Scheraga, H.A. (1955) J. Am. Chem. Soc 77, 6168--6172