• Nie Znaleziono Wyników

Carp fibrinogen and its terminal plasmin degradation products

N/A
N/A
Protected

Academic year: 2021

Share "Carp fibrinogen and its terminal plasmin degradation products"

Copied!
10
0
0

Pełen tekst

(1)

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 BIOPHYS1CA 11, 1996

Tadeusz Krajewski, Paweł Nowak, Jacek Golański

CARP FIBRINOGEN AND ITS TERMINAL PLASMIN DEGRADATION PRODUCTS

The isolation and characterization of carp (Cyprinus carpio) plasma fibrinogen and its plasmin degradation products are described. Alike other vertebrate species, carp fibrinogen is a dimeric protein and consists of three different pairs of disulfide-bonded polypeptide chains. Aa, B/f, and y. In contrast to mammalian fibrinogen, the B0 chain of carp fibrinogen has apparently a higher molecular weight than Aa chain. The relatively large size of the carp B/J chain results from the unusually large size of the N H 2-terminal B fibrinopeptide released by thrombin cleavage of fibrinogen. As in mammalian species, plasmin digestion of carp fibrinogen produced as main terminal end- products two classes of fragments D and E. It was found that fragment D (D,) inhibited fibrin monomer polymerization not only in homo- but in heterologous system as well.

1. INTRODUCTION

H um an fibrinogen is a soluble glycoprotein (M r 340 000) built up of three pairs o f non-identical polypeptide chains (Aa, B/i, y) which form tw o-half molecule units held together by three disulfide bonds at the N H 2-terminal portions o f polypeptides [2, 3, 17, 18]. In native molecule there exist different functional dom ains (two D and one E) im portant for protofibril form ation [4, 17, 18]. Following throm bin-m ediated rem oval of two pairs o f fibrinopeptides, A and B, from the A a and Bfi chains of the fibrinogen, respectively, activated fibrin m onomers appeared which spon­ taneously polymerize. The general mechanism of fibrin form ation has been suggested to be based on the interaction o f preexisted binding sites located in the CO OH -term inal region (domain D ) o f the fibrinogen molecule with polymerizing sites situated in the N H 2-terminal part (dom ain E) o f the neighboring m olecule [16, 25] and polym er assembly comm ences with form ation o f double-strended fibrils. The fibrils subsequently associate laterally, form ing thick fibers of fibrin [7, 29-32, 37, 39].

(2)

Plasmin digestion of human fibrinogen gives as m ain terminal end-products two classes o f carbohydrate-containing fragments D and E representing the C O O H -term inal and N H 2-term inal ends (central nodule) o f m olecule, respectively [5, 15, 28, 35], Depending on the degree of degradation o f the y-chain, fragm ent D may exist as D, (94 000), D 2 (88 000) or D 3 (83 000). Digestion o f hum an fibrinogen in the presence of calcium ions results in fragm ent D ,. W hen proteolysis is carried out in the presence o f ED TA (EG TA ) D 3 is the m ain product of hydrolyzate [38], It has been shown th at fragm ent D , but not D 3 strongly inhibited fibrin m onom ers polyme­ rization [26, 27, 39]. We found in a com parative investigation that fragm ent D, of duck fibrinogen also exerts inhibitory effect on the above process [23].

The aim o f this study was to characterize submolecular com position and some physico-chemical properties of carp fibrinogen as well as its plasm in degradation products (D,, D 3) and com pare the effect o f D, and D 3 fragm ents on polymerization o f carp fibrin m onom ers (homologous system) and hum an fibrin m onom ers (heterologous system).

2. MATERIAL AND METHODS

Blood collection. Nine parts of fish blood obtained by direct cardiac puncture after the heart was surgically exposed, were taken into one p art o f 10% sodium citrate containing 0.1 M e-aminocaproic acid. 50 u K l/m l o f trasylol and 15 /ig/ml phenylm ethanesulphonyl fluoride.

Fibrinogen preparation. Fibrinogens (fish and hum an) were isolated from fresh blood plasm a according to the m ethod of D o o l i t t l e et al. [12]. Fibrinogen preparations were dissolved in 0.14 M N aC l buffered with 0.01 M sodium phosphate. pH 7.4 and passed through a Sepharose-lysine column to remove plasminogen. Both isolated fibrinogens were 93-95% coagulable with bovine throm bin. Fg preparations were characterized by polyacrylam ide gel electrophoresis according to the m ethod o f W e b e r and O s b o r n [41].

Protein concentration was determined by the m ethod of I t z h a k e and G i l l [21].

Isolation o f fragm ents D ¡(D Ca+2) and D3(I)edta). H um an or carp fib­ rinogens (15 m g/m l) in 1 m M Tris/H C l buffer, pH 7.4, containing 0.15 M N aC l and 5 mM CaCl2 were digested with hum an plasmin at 0.05 cesein units/m g o f fibrinogen for 18 h at 37°C. Fragm ents D, were isolated from the digest by chrom atography on Lys-Sepharose by the m ethod o f R u p p et al. [36]. D 3 rem nants obtained from fragm ents D , by further plasm in hydrolysis in the presence o f 5 m M ED TA were purified on Sephadex G-100. M olecular weights of fish and hum an fragm ents were estimated by SD S-PAG E.

(3)

Inhibition o f fibrin monomers polymerization. Throm bin fibrin m onom ers were prepared from carp and hum an fibrinogen according to the m ethod o f B e l i t s e r et al. [1]. Polymerization o f fibrin m onom ers was m easured spectrophotom etrically at 350 nm [6]. In typical experiment, 0.1 ml aliquit o f the fragm ents D, or D 3 in 0.15 M Tris/H C l buffer was mixed with 0.8 ml of the same buffer in a 10 mm long quartz cell and the base line was recorded at 350 nm. A t zero time the recorder was started, 0.1 ml o f fibrin m onom ers (1.5 mg/ml in 0.02 M acetic acid) was added, mixed well and the increase of absorbance was recorded for 15 min. To m easure the rate o f polymerization o f fibrin m onom ers alone, 0.1 ml of fibrin m onom ers was added to 0.9 ml o f the buffer. The maximum rate of polymerization was calculated from the slope o f the steepest part o f the curve and expressed as percent o f that for fibrin m onom ers alone.

3. RESULTS

SDS-polyacrylamide gel electrophoresis of unreduced and reduced samples o f carp fibrinogen are presented in Fig. 1. The results indicate that this protein is built up of 3 pairs o f non-identical subunits (M r 48 000, 50 500, 55 500) bound by S-S bridges, corresponding to 3 pours o f m am m alian fibrinogen chains. M olecular weight o f native carp fibrinogen was calculated to be about 310 000.

Identification o f carp fibrinogen subm its. In order to relate the three observed polypeptides to the three subunits o f carp fibrinogen, we investigated which polypeptide bands are sensitive to digestion with specific proteolytic enzymes know n to selectively digest particular subunits of fibrinogen. Throm bin splits off the A and B fib- rinopeptides from the A a and B/J subunits of fibrinogen, giving rise to the a and p chains of fibrin. The y subunit is not digested by this enzyme. The snake venom protease- batroxobin selectively digests A a subunit o f fibrinogen but not o f the B/J and y.

In our electrophoresis gel system undigested both hum an and carp fibrinogens were resolved each o f them into three bands corresponding to Aa, B/f and y chains (Fig. 2, line F). The same figure presents polyacrylamide gel electrophoresis

1

%

15“»

✓ 55 500 5 S 3 -5 0 5 0 0 '4 8 000

Fig. 1. SDS polyacrylamide gels after electrophoresis of unre­ duced (7% gel) and reduced (15% gel) carp fibrinogen sta­ ined with Coomasie brillant

(4)

digestion with batroxobin. As we can see proteolysis of hum an fibrinogen with the enzyme, results in cleavage o f largest polypeptide only and in the appearance of new band of slightly lower m olecular weight. In case o f carp fibrinogen the m iddle band was attacked by batroxobin and new faster migrating band overlopping y chain appeared (Fig. 2, lane FB).

F F B F T - -•hC ~i-g —* - 6~X A<C -BP • «C

B

Fig. 2. Selective enzymatic digestion or carp (A) and human (B) fibrinogens. Coomassie blue-stained proteins after SDS-polyacrylamide gel electrophoresis of: undigested fibrinogen (lane F), fibrinogen digested with batroxobin (I®116 fibrinogen digested with thrombin

(lane FT)

Digestion of hum an and carp fibrinogens by throm bin results in cleavage o f the largest polypeptides as well as the middle one in both cases (Fig. 2, lane FT), giving rise to faster m igrating bands. As it was shown above (Fig. 2) batroxobin fails to digest the largest polypeptide of carp fibrinogen. Since this chain is digested by throm bin, we can deduct that it m ust be the B/J. M olecular weights of polypeptide subunits of carp fibrinogen and fibrin estimated by SDS-polyacrylamide gel electrophoresis are given in Tab. 1.

T a b l e 1 Molecular weights of polypeptide subunits of carp fibrinogen and fibrin

Subunits

Aa a B/J P y FpA FpB

(5)

A part from that visible on the top o f the gels additional slow m igrating bands (lanes FB and FT ) designed as y-y and p-a correspond to very well known gamm a dimer and polymers a chains, respectively. It is the result o f crosslinking o f m onom ers by fibrin stabilizing factor [34],

i x

- Y

D

E

10,10 232 140 94 67 43 30 r3 Fg 5 15 30 60 120

Incub ation lim e (mini

• ■ • f t S IS 68 120 Incubation lim e [ m i n i m.w 10 -<232 *140 H 04 -j 67 43 30 -3

B

Fig. 3. SDS-polyacrylamide gels after electrophoresis of unreduced samples of plasmin digest of carp (A) and human (B) fibrinogen (Fg). The same type and size of plasmic degradation products in both cases are seen (X,Y, D, E). However, carp fibrinogen appears to be degraded by plasmin

at faster rate

Plasmin degradation products o f carp fibrinogen. Fig. 3 shows typical SDS polyacrylamide gel electrophoresis pattern of both unreduced hum an and carp fibrinogen digest with plasmin with respect to time. Carp fibrinogen shows significant increase in mobility already at 15 min. and becomes a m ixture of intact fibrinogen and X , Y , D, E fragm ents. A t this time hum an fibrinogen indicates a very small degree o f conversion to degradation

(6)

(appearance of D and E fragm ents on the gel). This analysis indicates that fibrinogen isolated from carp plasma is inherently less ressistant to plasmin th an hum an. However, it is w orth to notice th at both fragm ents (called D ,) occupy alm ost the same position on the gel and have mol. weight of 94 kD a. W hen fragm ent D , was repeatedly hydrolyzed by plasmin in the presence o f E D T A a m ore degraded form D 3 o f m olecular weight of 80 k D a was isolated from the hydrolyzate digest (not shown).

Effect o f fragm ents D l and D } on fibrin monomer polymerization. After throm bin removal o f amino-term inal FpA and FpB from fibrinogen new binding sites appear in central nodul (E) and m onom ers polymerize to form fibrin clot. The m ain effort o f this part of our work was to evaluate the influence o f D, and D 3 fragm ents on this process. It was found that D, fragm ent introduced into m onom er incubation m ixture inhibited fibrin m onom er polymerization in both hom o- i heterologous system (Fig. 4 and 5). A 50% decrease of m aximal reaction rate was observed at m olar ratio o f these fragm ents to carp fibrin m onom er of I : l and 1 : 2.5 for carp and hum an D ,, respectively (Fig. 4). Similar results were obtained when hum an fibrin m onom er was used. In this case hom ology fragm ent D, (human) decreased maximum rate of polymerization to 50% at approximately 1 : 1 m olar ratio o f D, to fibrin m onom er (FM ). The inhibitory effect of carp fragm ent D , on this process was lower. 50% inhibition was estimated to occur at an approxim ately 1 : 3.5 m olar ratio o f FM to carp D, (Fig. 5). The same experiments carried out with fragments D 3 indicated th at these degraded form s o f fragm ents D, were completely devoid of antipolim erizing activity (Fig. 4 and 5).

4. DISCUSSION

In all m am m alian species which have been studied up to now fibrinogen has dimeric structure with a total m olecular weight o f 340 000 and the relative sizes o f polypeptide chains from largest to smallest are A a, Bp and y. Fibrinopeptides removed from fibrinogen of m am m alas are relatively constant in size with nearly all o f them comprising 13-21 amino acids and thus having m olecular weight in the range o f 1000-2000 [9, 10].

C arp fibrinogen possesses high degree o f sim ilarity to m am m alian fibrinogen in respect to m olecular weight (310 000), submolecular structure (3 pairs nonidentical polypeptide chains) and amino acid com position (not shown). In contrast to m am m alian coagulable protein BP chain o f carp fibrinogen has a higher apparently m olecular weight than the A a chain. The y-chain has the lowest m olecular weight in the carp protein, as in th at

(7)

_ .Fragment D . MOLAR RATIO ( ^ - -■ — )

' C a rp F M '

Fig. 4. The effect of fragments D , and D 3 of carp and human fibrinogen on the rate of carp fibrin monomer polymerization. Various ratios of the fragments to fibrin monomers (abscissa) were tested. The formation of fibrin polymers was monitored in a spec- trophotometr at 350 nm. For each run the maximum reaction rate was calculated from the slope of the steepest part of the curve and expressed as percent o f that for fibrin monomer alone (100% on

„ /Fragment D x MOLAR RATIO (-7—=--- — ) v Human FM '

Fig. 5. The effect of fragments D , and D 3 of human and carp fibrinogen on the rate of human fibrin monomer polymerization

(8)

o f other species. Therefore the sequence o f carp fibrinogen subunits on the gel (Fig. 1) should be following: B/J, A a and y. Digestion of carp fibrinogen by throm bin (Fig. 2) dem onstrated th at relatively large size o f the Bfi chain results from unusually large size of the fibrinopeptide B split o f from carp fibrinogen (Tab. 1). The high m olecular weight of fibrinopeptide B has also been found by W a n g h et al. [19, 40] during selective enzymatic digestion of Xenopus fibrinogen as well as by D o o l i t t l e and C o t t r e l l in case o f lamprey fibrinogen [11].

C arp fibrinogen alike goose and duck fibrinogens [23] appeared to be m ore sensitive to plasmin degradation in com parison with than m am m alian fibrinogens [24], W hether it is due to the some properties of the structure o f the carp fibrinogen itself o r to a fibrinolytic system, which is m ore active in carp plasm a [22], is not known yet.

Inhibition o f carp fibrinogen m onom ers polymerization and assembly o f fibrin clot by D, fragm ent o f carp fibrinogen, but not D 3 is in agre­ ement with earlier observations related to hum an protein [13]. 0 1 e x a and B u d z y n s k i [32] isolated from y chain o f fragm ent D ,, peptide built up of 38 amino acid residue peptide encompassing positions 374 to 411, which compited for the binding site in E dom ain of neighbouring fibrin m onom er and inhibited protofibril form ation. H o r o w i t z et al. [20] found that 23-residue peptide originating from the carboxy-term inal region o f y-chain inhibits fibrin m onom er polymerization and contains polymerization site within y-chain segment 374-396. This site does not overlap with segments of the y-chain that responsible for platelet ag­ gregation ( y ^ , , ) . The above described polymerization site is absent in all the D 3 rem nants (shortened y-chain) isolated from different kinds of fibrinogens and therefore these fragments D 3 are not active in the process.

Similar observations dealing with inhibitory effect o f duck D , fragm ent but n o t D 3 both on duck fibrin m onom er polymerization (homologous) and pig fibrin m onom er polymerization (heterologous system) have also been successfully carried out by us [23], Our latest finding with D, rem nant o f carp fibrinogen (Fig. 5) indicated th at this fragm ent reacts not only with carp fibrin m onom er but also exhibits inhibitory effect on hum an fibrin m onom er (heterologous system), however, this was observed to smaller extend.

Evidence presented here together with our earlier finding concerning the hom ology o f polym erization dom ain in vertebrate fibrinogens strongly support the hypothesis that, in spite of some distinguishing characteristics for each o f the fibrinogen types, structures providing polym erization sites rem ain unchanged during the evolution o f vertebrate fibrinogen and are uncom m only conservative [8, 33]. As it was proved by C i e r n i e w s k i et

(9)

al. [7] the polymerization site is formed simply both a linear sequence of am ino acid residues in a segment o f the y-chain and native tertiary structure. It m eans therefore th a t two elements are essential for the expression o f polym erization sites in the structural D dom ain.

5. REFERENCES

[1] B e i i t s e r V. A., T o l s t y k n T. V., T s a r j u k V. M., P o z d n j a k o v a T. M. (1975), Thromb. Res., 7, 797-806.

[2] B lo rn b a c k B., B l o m b a c k M. (1972), Ann N. Y. Acad. Sei., 202, 77-97 [31 B l o m b a c k B„ H e s s e l B., H o g g D. (1976), Thromb, Res., 8, 639-658.

[4] B l o m b a c k B„ H e s s e l B., H o g g D., T h e r k i l d s e n L. (1978), „Nature” , 275, 501-505.

[5] B u d z y ń s k i A. Z., M a r d e r V. J. (1977), Thromb. Haemostas., 38, 793-800. [6] B u d z y ń s k i A. Z., O l e x a S. A., P a n d y a B. V. (1983), Ann N. Y. Acad. Sei., 408,

301-314.

PI C i e r n i e w s k i C. S., B u d z y ń s k i A. Z. (1992), „Biochemistry” , 31, 4248-4253. 181 C i e r n i e w s k i C. S., K r a j e w s k i T., J a n i a k A. (1980), Thromb. Res., 19, 599-607. [9] D o o l i t t l e R. F. (1976), Federation. Proc., 35, 2145-2149.

[10] D o o l i t t l e R. F. (1983), Ann N. Y. Acad. Sei., 408, 13-26.

[11] D o o l i t t l e R. F., C o t t r e l l B. A. (1974), Biochem Biophys. Res. Commun., 60, 1090-1096.

[12] 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. (1967), Arch. Biochem Biophys., 118, 456-467.

[13] D r a y - A t t a l i L., L a r r i e u M. J. (1977), Thromb. Res., 10, 575-586.

[14] F u r l a n M., R u p p C., B e c k E. A. (1983), Biochem Biophys. Acta, 742, 25-32. [15] G a f f n e y P. J„ B r a s h e r M. (1973), Biochem Biophys. Acta, 295, 308-313.

[16] H a n t g a n R., M c D o n a g h J., H e r m a n s J. (1983), Ann N. Y. Acad. Sd., 408, 344-367. [17] H e n s c h e n A., L o t t s p e i c h F., K e h l M., S o u t h a n C. (1983), Ann N. Y. Acad.

Sei., 408, 28-43.

[18] H o e p r i c h P. D„ D o o l i t t l e R. F. (1983), „Biochemistry” , 22, 2049-2055.

[19] H o l l a n d L. J., W a n g h L. J., S p o l s k i R. J., W e i s e l J. W. (1984), J. Biol. Chem., 259, 3757-3762.

[20] H o r o w i t z B. H., V a r a d i A., S c h e r a g a H. A. (1984), Proc. Natl. Acad. Sei. USA, 81, 5980-5984.

[21] I t z h a k e R. F., G i l l D. M. (1964), Anal. Biochem, 9, 401-410.

[22] K r a j e w s k i T., C i e r n i e w s k i C. S., W r o n a U. (1981), 1RCS. J. Med. Sei., 9, 806-807. [23] K r a j e w s k i T., N o w a k P., C i e r n i e w s k i C. (1980), Biochem Biophys. Acta, 622,

94-104.

[24] K r a j e w s k i T., N o w a k P., C i e r n i e w s k i C. S. (1985), Acta Biochim. Pol., 32, 145-154.

[25] K u d r y k B., C o l l e n D., W o o d s K. R., B l o m b a c k B. (1974), J. Biol. Chem., 429, 3322-3325.

[26] L a r r i e u M. J., D r a y L., A r d a i l l o u N. (1972), Br. J. Haematol., 22, 719-733. [27] L a r r i e u M. J., D r a y L., A r d a i l l o u N. (1975), Thromb. Diathes. Haemorrh., 34,

(10)

[29] M e d v e d L. V., L i t v i n o v i c h S. V., U g a r o v a T. P., L u k i n o v a N. I., K a l i k - h e v i c h V. N„ A r d e m a s o v a Z. A. (1993), FEBS. Lett., 320, 239-242.

[30] M o s e s s o n M. W. (1990), J. Lab. Clin. Med., 116, 8-17.

[31] O l e x a S. A., B u d z y ń s k i A. Z. (1980), Proc. Natl. Acad. Sci., USA, 77, 1374-1378. [32] O l e x a S. A., B u d z y ń s k i A. Z. (1981), J. Biol. Chem., 256, 3544-3549.

[33] P a n Y , D o o l i t t l e R. F. (1992), Proc. Natl. Acad. Sci. USA, 89, 2066-2070. [34] P i s a n o J. J., F i n l a y s o n J. S., P e y t o n M. P. (1968), „Science”, 160, 892-893. [35] P i z z o S. V., S c h w a r t z M. L., H i l l R. L., M c K e e P. (1972), J. Biol. Chem., 247 636-645. [36] R u p p C., S i e v i R., F u r l a n M. (1982), Thromb. Res., 27, 117-121. [37] U g a r o v a T. P., B u d z y ń s k i A. Z. (1992). J. Biol. Chem., 267, 13687-13693. [38] V a n R u i j v e n - V e r m e e s J. A. M., N i e u w e n h u i z e n W., H a v e r k a t e F.,

T i m a n G. (1979), Hoppe-Seyler’s Z. Physiol. Vhem., 360, 633-637. [39] V a r a d i A., S c h e r a g a H. A. (1986), „Biochemistry” , 25, 519-528.

[40] W a n g h L. J., H o l l a n d L. J., S p o l s k i R. J., A p r i s o n B. S., W e i s e l J. W. (1983), J. Biol. Chem., 258, 4599-4605.

[41] W e b e r K., O s b o r n M. (1969), J. Biol. Chem., 244, 4406-4410.

Came in editorial office Department of Biochemistry

„Folia biochimica et biophysica” University o f Łódź, Poland

30.07.1993

Tadeusz Krajewski, Paweł Nowak, Jacek Golański FIBRYNOGEN KARPIA I JEGO KOŃCOWE PRODUKTY

PLAZM INOW EJ DEGRADACJI

W pracy opisano sposób izolowania fibrynogenu karpia (Cyprinus carpio) oraz produktów jego plazminowej degradacji, a także dokonano charakterystyki tych białek. Stwierdzono, że podobnie jak u innych gatunków kręgowców, fibrynogen karpia składa się z trzech par nieidentycznych łańcuchów polipeptydowych, Aa, B/l i y. W przeciwieństwie jednak do fibrynogenu ssaków, łańcuch B/i fibrynogenu karpia posiada wyższą masę cząsteczkową niż łańcuch Aa. Stosunkowo duży rozmiar łańcucha B/i fibrynogenu karpia wynika z wysokiej masy cząsteczkowej N H 2-końcowego fibrynopeplydu B, odłączanego z fibrynogenu przez trombinę. Tak jak u ssaków, trawienie fibrynogenu karpia plazminą prowadzi do otrzymania dwóch głównych końcowych produktów degradacji: fragmentów D i E. Wykazano, że fragment D (D,) hamuje polimeryzację monomerów fibryny nie tylko w układzie homo-, ale także w heterologicznym.

Cytaty

Powiązane dokumenty

Kolejne dwa rozdziały („D er D eutsche O stm arken-V erein” oraz „Die polnische A ntw ort”), stanowiące zasadniczą część omawianej pracy, przedstawiają strukturę

nietypowych lub trud- nych, oznaczonych przez autorów podręczników specjalnymi piktogramami (np. Mamy więc do czynienia z tzw. orientowaniem uczniów na typ zadania. zjawisko

przynajmniej co drugie dziecko (56,3%) stwierdziło, że atrakcyjność tych dzieł bywa różna, a 5,6% innych uważa, że szkolne lektury w ogóle nie są intere- sujące. Jeżeli

1 (nie rozstrzyga się o ich pra­ wach i obowiązkach ani nie są osobami, które mogą być rozstrzygnięciem organu administracji w tej sprawie bezpośrednio dotknięci)

Effects of second-phase nano-particles, MgO-dopant and particle size of alumina powder on the microstructure, and in turn on the resultant density, hardness, fracture toughness

Eyring plots (top panels) and temperature dependence of the KIEs (bottom panels) on the reactions of PETNR (A), TOYE (B), and XenA (C) with natural coenzymes and synthetic

zm arł w Szczecinie

[r]