Structural difference between polymerized and non-polymerized fragment X, obtained by plasmin digest of fibrinogen

Structural difference between polymerized and non-polymerized fragment X, obtained by plasmin digest of fibrinogen Hiroko Sate* and Joel K. Swadesh Department of Polymer Chemistry, Kyoto University, Yoshida-honmachi, Kyoto 606-01, Japan and Department of Biochemistry, Temple University School of Medicine, Philadelphia, PA 19104, USA (Received 25 February 1993; revised 13 July 1993) Fragment X (LMrFX) was obtained as low molecular weight preparations from a late stage 2 plasmin digest of humanjbrinogen. The thrombin-treated LMrFXpreparations, which resulted in impairedpolymerization, were further subfractionated into polymerized and non-polymerized components. The fractions were examined by SDS- PAGE and immunochemical methods. In polymerized fractions, more peptide bands were observed on SDS-PAGE in the reduced state than in non-polymerized fractions. Both fractions contained a similar number of internal cleavages in the Au, BP and y chains, which are linked by disulJide bonds. Thus, the partial dejciencies in polymerization sites of the carboxy terminal region of the y chain and the amino terminal portions of the B#I chain, as well as internal cleavage, were considered to participate in the impairment of the thrombin-induced polymerization of LMrFX. Keywords: Fibrinogen; fragment X; plasmin digest; antibodies; internal cleavage; polymerization sites

Introduction Fibrinogen-derived fragment X as well as fragments Y, D, and E are fibrinogen degradation products (FDP). Fragment X is regarded as a blood coagulable protein, while fragments Y, D, and E are known to inhibit coagulation. However, compared with fibrinogen, fragment X has been reported to form mechanically weaker clots’ with markedly less ADP-induced platelet aggregation2. We have purified the low molecular weight fragment X (LMrFX) preparations obtained from a late stage 2 plasmin digest of human fibrinogen3p4. The preparations caused gradual depolymerization after the thrombininduced polymerization4. Such incomplete polymerization should result from the impairment of polymerization sites on LMrFX. Plasmin hydrolyses peptide bonds on the carboxyl terminal side of Lys and Arg residues in the hydrophilic regions of the fibrinogen molecule. Despite the abundance of both residues in fibrinogen and a less specific enzyme of plasmin, fragments X, Y, D and E have been reported to have a relatively conserved molecular weight (M,). Fragment X species, ranging from M, 285000 to 240000”5, were obtained from stages 1 and 2 by the plasmin digest of fibrinogen. LMrFX has been found to lose the central to carboxyl terminal region of the Aa chain’ and the amino terminal region of the B/? chain6*‘, and to be hydrolysed partially on so-called coiled-coil structures, connecting the E and D domains*,9. To elucidate the molecular structure of LMrFX, we used immunochemical methods to predict the assignment of * To

whom correspondence

should be addressed

0141-8130/93/060323-05 0 1993 Butterworth-Heinemann

Limited

at Kyoto University.

major peptides of LMrFX, referring to previous papers on cleavage sites of fibrinogen by plasmin. Because of the numerous peptides in the LMrFX preparations and the relatively high number (32%)” of B/? and y chains, it was difficult to separate and identify them by high performance liquid chromatography and amino acid sequencing methods.

Experimental The plasmin digestion of human fibrinogen (Kabi, Sweden) was carried out by the addition of streptokinase and 27 mM CaCl, for 60min at 37 “C, as described previously4, in a late stage 2 plasmin digest. LMrFX preparations were purified through columns packed with Sepharose CL6B (Pharmacia) and then Ultrogel AcA 34 (LKB Instruments), and pooled on 0.15 M Tris-HCl at pH 7.4 with 0.02% NaN, and 25 kIU ml-’ aprotinin (Trasylol; Bayern, Germany). Murine monoclonal antibody 9C311 was used as the primary antibody for the enzyme-linked immunosorbent assay (ELISA)“. LMrFX was coated onto Immulon 2 plates (Dynatech Lab. Inc., VA, USA). The horseradish peroxidase-conjugated goat anti-mouse IgG (Cappel, West Chester, PA, USA) was used as the secondary antibody. Colour development was allowed to proceed for about 10 min, and wells were read at 405 nm with a Bio-Rad Model 2550 EIA plate reader connected to a Macintosh Plus computer. Disulfide bonds of fragment X were reduced with 1.4% 2-mercaptoethanol (Eastman, Rochester, NY, USA) and incubated at 100 “C for 5 min. SDS (O.l%)-PAGE was

Int. J. Biol. Macromol., 1993, Vol. 15, December

323

Structure of fibrinogen-LMrFX: H. Sato and J. Swadesh performed according to the Laemmli protocol ~3. Reducing 10-15% gradient SDS-PAGE gels were stained with Coomassie brilliant blue (CBB; Merck, Darmstadt, Germany) and scanned on an LKB 2202 Ultroscan laser densitometer operating at 632.8 nm. For the molecular weight determination by SDS-PAGE and staining by CBB, the SDS-6 Dalton Mark VI Mr standard kit (Sigma, MO, USA), Trasylol, and Act, Bfl, and 7 chains of fibrinogen were used. Pre-stained electrophoretic standards were used for Western blotting kits of mid-range and low-range M, standards (Diversified Biotech, Newtown Center, MA, USA). Immunoblotting14'l 5 was performed on samples that had been disulfide-reduced and electrophoretically transferred to nitrocellulose at 30 mA at 4°C. The murine monoclonal antibody 9C3, polyclonal rabbit antisera to the Act chain, the peptide Bfll-ll8, and the ? chain of human fibrinogen ~6-a9 were used as the primary antibodies for Western blotting. Polyclonal rabbit antisera against peptides 71-78 and 795 264, and the cyanogen bromide degradation fragments of the ? chain were a generous gift of Dr Edward F. Plow (Research Institute of Scripps Clinic, La Jolla, CA, USA). The primary antibodies were typically used at a dilution of 1:1000. Horseradish peroxidase-conjugated goat antirabbit (Bio-Rad Lab., CA, USA) or goat anti-mouse IgG (Cappel) were diluted 1:500 as the secondary antibodies. Peptide samples were visualized by the reaction of horseradish peroxidase with hydrogen peroxide and 4-chloro-l-naphthol (Sigma). Polymerization of LMrFX was induced by adding bovine thrombin (Parke Davis, N J, USA). Polymerized and non-polymerized species of LMrFX were obtained as precipitate and supernatant subfractions, abbreviated as ppt-x and sup-x, respectively, by centrifugation (6500 g for 5 min) at the point of maximum turbidity on the turbidity-time curves. Protein concentrations of LMrFX preparations and fibrinogen were estimated by using the absorption coefficient of 1.5 mg protein for a 1 cm light path at 280 nm.

Results Assay for the central region of the Act chain in L M r F X Plasmin digests the Act chain of fibrinogen extensively to form fragment X until the carboxy terminus Act206 corresponds to the Act chain of fragment D 2°. The epitope of the monoclonal antibody 9C3 is located in the peptide region Act240-26811 (Kd=352pM) 17. The amount of undigested central region of the Act chain in LMrFX was estimated by ELISA; the reactivity of enzyme bound to antigen was proportional to fibrinogen concentrations below 0.2/~g ml- 1. By comparison of the initial slopes of the reactivity for LMrFX and fibrinogen, the amount of epitope on LMrFX against the monoclonal antibody 9C3 was estimated to be 4.6% of that of fibrinogen. Origin of major peptides in L M r F X Subfractions of LMrFX, ppt-x and sup-x, were investigated by reducing SDS-PAGE (Figure 1 and Table 1), where the M r of seven major bands 1, 2, 3, 4~, 5, 6 and 7a corresponded to 48000, 39700, 33000, 20900, 14000, 9700 and 7500, respectively. The occurrence of more than four bands suggests internal cleavage of LMrFX, i.e. hydrolysis in trinodule-connecting regions.

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~pt-x

~3

I .,I

4a

3

2

I sup-x

I

I

I

I

Migration Distance

I

I

I

I

(upside)

Figure 1 Densitometric scan of polypeptide chains: supernatant (sup-x) and precipitates (ppt-x) of thrombin-treated LMrFX.

The vertical axis is absorbance, and the horizontal axis is migration (cm) to the left, the low molecular weight. Numbers on peaks correspond to those in Table 1

By thrombin treatment of LMrFX, two bands were shifted to bands 4 a and 7a at slightly lower positions (data not shown); that is, those peptides derived from the Act chain are considered to have a lower molecular weight owing to the release of fibrinopeptide A (Actl-16). Immunoblotting techniques appear useful to distinguish the origin of the seven major disulfide-reduced bands. Moreover, the seven major bands can be assigned on the basis of Mr values obtained by three different methods: first, the M r of each peptide on CBB-stained bands; second, the M, evaluated on the immunoblotting data; and third, the calculated M, of a peptide derived from the reported sites digested by plasmin 6'a'2°-24. The amino acid sequence of the peptides was based on the nucleotide sequences coded for the Act chain 25, the BE chain 26, and the ? chain 27 for human fibrinogen. A wide band (51 kDa to 46 kDa peptides) corresponding to the 48 000 band (Table 1) was obtained on SDS-PAGE. Since the Mr of the intact ?1-411 + C H O , where CHO represents carbohydrate, is 48 300, the 51 kDa peptides should be derived only from the BE chain. As seen in Figure 2A, B, and C, the 46 kDa peptides probably contain somewhat digested BE chains and the intact 7 chain, because of the reactivity not only with anti-Bill118 antibodies but also with anti-? antibodies, anti?95-264 antibodies, and anti-?l-78 antibodies (data not shown). Thus, the 46 kDa peptides are concluded to be derived from BE and ? chains. In Figure 2A, the 38 kDa and 33 kDa peptides are generated by plasmin digestion, contain some portion of the sequence of the peptide Bfll-ll8, and are not found in the sequences of the Bfl chain. Cross-reactivity of the antibodies with the ? chain was suggested by the presence of two bands (the upper clear band and the lower faint band) in the lane of fibrinogen, reacting with the anti-Bfll-ll8 antibodies. As seen in Figure 2B, anti-? chain antibodies visualize two bands in the fibrinogen standard. Because of genomic homology between the BE and ?

Structure of fibrinogen-LMrFX: H. Sato and J. Swadesh Table 1 Putative assignment of major bands for SDS-PAGE of the thrombin-treated LMrFX in the reduced state SDS-PAGE No. (reduced)

No. of major bands

Mr from SDS-PAGE

Proposed peptide chain and Mr

1

48000

B # 4 3 - , 54-461+CHO (52000-50900)

71-411 + CHO (48300)

2

39700

Bfl134-461 +CHO (39500)

763-411 (39500)

3

33000

4a

20900

Act17-206 (22900)

5

14000

Act105-206 (12700)

Act

Bfl

ppt-x sup-x

1

3

"~---~,~

/

786-356 (30700)

~ ~

4a

6 7a

9700

~ 6 ~ 7

Bf143-, 54-120 or -122 (8700-7300)

7500

a

71-53, -58 or -62+CHO (7900-8900)

Act17-78 (7500)

Brackets indicate the calculated M,

Antisera: Antigen:

B~H m '

F px

T

sx

!

F

px

Aa

Tgs-z64

sx

~

i

F

px

sx

|

r

F

!

px

SX

~1"

Mr

Mr

51KD---, 46 --

38

.....

33

.....

0

.......

-

~

-

B?K D 53

....

23

.... 14

5

.... 7.5

.... (A)

(B)

(C)

(D)

Figure 2 Western blot analysis of each band of Act, Bfl and ? chains using antibodies against (A) Bfll-ll8 peptide, (B) Y chain, (C) 795-264 peptide, and (D) Act chain. Numbers indicate the M, estimated from pre-stained Mr markers. F, px and sx indicate fibrinogen, ppt-x and sup-x after centrifugation of the thrombin-treated LMrFX

chains, as shown above, six different antisera against the 7 chain and its fragments were surveyed to find suitable antisera without cross-reactivity and with sensitivity to antigen. As Fioure 2C shows, the anti-y95-264 antibodies

did not cross-react with the fibrinogen Bfl chains. They reacted with the 46kDa, 33kDa, and 2 0 k D a bands obtained with ppt-x, indicating that they are peptides derived from the ~ chain. The 33 kDa and 20 kDa peptides

Int. J. Biol. Macromol., 1993, Vol. 15, December

325

Structure of fibrinogen-LMrFX: H. Sato and J. Swadesh may be the 786-356 and 7109-302 peptides, respectively. These cleavages are unexpected, because the calcium ion is believed to inhibit cleavage at these sites by plasmin digestion 2s. The cleavages may have occurred during extensive chromatography, because the chromatographic buffers contained no calcium ions. The 38 kDa peptide (Figure 2A and B), a major band migrating at 39 700 on CBB-stained SDS-PAGE (Table 1), may correspond to both residues of Bfl134-461 + C H O and 763-411. However, the peptide should not react against anti-Bill-118 antibodies. The peptide of 763-411 may cross-react with anti-Bill-118 antibodies (calculated Mr= 39.5 kDa). The remnant peptides smaller than 10 kDa, linked with disulfide bonds, appear in both ppt-x and sup-x, CBB-stained and reacted with antisera against the peptide Bill-118 and the 7 chain. In immunoblotting, the monoclonal antibody 9C3 and anti-Act chain antibodies showed the same pattern at the positions of the 67 kDa and 53 kDa peptides for both sup-x and ppt-x (data not shown). The low molecular weight peptide bands did not clearly react with the monoclonal aatibody 9C3 and anti-Act chain antibodies. However, another major band at the 14 kDa position may have been derived from residues A~105-206, whose remnant peptide, linked with disulfide bonds, corresponds to A~17-78. Another major band remaining at the 9.7 kDa position may have been derived from peptides

such as 71-53 + C H O , Bfl43-120, and Bfl54-120, which should exist as the remnant peptides of 763-411 or 786-356, and Bf1134-461+CHO. Hence, the putative assignment of peptide bands of LMrFX is summarized in Table 1.

Discussion

Internal cleavage of peptides between disulfide bonds Internal cleavage of fragment X appears in the late stage 2 plasmin digest 6,s, and may be estimated from the CBB-stained peptides on SDS-PAGE 29, and the results are shown in Table 1. The extent of internal cleavage (CVintAa) in the Aa chain of sup-x and ppt-x was calculated from the area (A) of peaks 4, 5 and 7 of Figure 1 using equation (1):

(1)

CVintAa = (A5 -+- A7)/(A4 + A5 + A7)

The average extent of the internal cleavage (C Vinta fl + 7)' expressed by equation (2), in the Bfl and 7 chains was calculated as an average over both chains. Because fragments derived from Bfl and 7 chains are not resolved on SDS-PAGE CVintBfl+7 ---

(A2 + A3 + A6)/(A1 + A2 + A3 + A6)

(2)

Table 2 Internal cleavage of subfractions in thrombin-treated LMrFX, compared with fragment Y Chains

ppt-x

sup-x

FragmentY

A~ Bfl/7

43.5±7.4% 33.3±4.9%

41.8±6.2% 36.4±6.6%

76.2±0.1% 54.2±1.7%

As shown in Table 2, the extent of internal cleavage in ppt-x and sup-x was the same within experimental error. Therefore, cleavages internal to the peptide chains of LMrFX are unlikely to greatly influence the course of polymerization by the thrombin treatment.

R42

K53

Pln

~

o E56

q

Pln

R57 R44

K47

K54

K58 Figure 3 Molecular model, drawn using P E P M O D 33"34, for the peptide region Bfl40-59 (GYRARPAK AAATQKKVER KA) which is predicted to be the a-helical region; (O) carbon and (O) nitrogen atoms. A, Ala; E, Glu; G, Gly; K, Lys; P, Pro; Q, Gin; R, Arg; T, Thr; V, Val; Y, Tyr. Arrow marks indicate cleavage sites in the early stage by plasmin (Pln)

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Int. J. Biol. Macromol., 1993, Vol. 15, December

Structure o f f i b r i n o g e n - L M r F X : H. Sato and J. Swadesh Partial digestion in the carboxy terminal portion o f the chain

Plasmin digestion at the linkage ),356-357 and the consequent loss of the c a r b o x y terminal portion ~,357~11 should impair the polymerization site a a°'31 and should significantly alter the course of depolymerization, after the thrombin-induced polymerization of L M r F X . The extent of digestion in this i m p o r t a n t segment of the chain in ppt-x is estimated to be 19% from equation (3): '~def-C

=

A. Pixley for the densitometric measurements, Drs Czeslaw S. Cierniewski, Jerome L. Gabriel, S. Shaukat Husein, and John H. Weisel for helpful discussions, and Mr Joseph O'Brien for the computer analysis. Professor Sadaaki Iwanaga, Kyushu University, is cordially thanked for progressive opinions and discussions. This study was supported by grant HL36221 from the National Heart, Lung, and Blood Institutes, National Institutes of Health, Bethesda, Maryland, USA.

(A3/33)/{A1/(48 x 2) + A2/(39.7 x 2) + A3/33)}

(3) Taking into account the internal cleavage, the theoretical molecular weight of L M r F X was calculated to be 242 000. In addition, fragment Y used in this study was obtained from the same late stage 2 plasmin digest used to isolate L M r F X , and b a n d assignments were performed similarly. Moreover, it is reasonable that the extent of internal cleavage is greater than 50% in the Aat and the unresolved Bfl and 7 chains of fragment Y (Table 2). Digestion in the amino terminal region o f the Bfl chain

The a m i n o terminal region of the Bfl chain is rich in Arg and Lys residues. The segment Bfl41-59 is empirically predicted to form an s-helix despite the existence of Pro-45, a breaker residue for an or-helical structure, on the basis of the probability of each residue in protein c o n f o r m a t i o n 32. A molecular model for the segment Bfl40-59 is illustrated (Figure 3), assuming the formation of s-helix. The side groups of Arg 42 and Lys 53 face the same side. F r o m i m m u n o c h e m i c a l studies 19, segment Bfll-53 was presumed to be a surface-exposed area, which seems to cause its eminent susceptibility to plasmin. Thus, the 51 k D a b a n d and the upper portion of the thick 46 k D a b a n d are considered to correspond to peptides B f 1 4 3 - 4 6 1 + C H O and B f 1 5 4 - 4 6 1 + C H O , respectively (Table 1). H a r d l y any 46 k D a peptides were derived from the Bfl chain in the ppt-x (Figure 2A), which indicates less digestion in the a m i n o terminal region of the Bfl chain in the ppt-x than in the sup-x. On the other hand, peptides in the ppt-x derived from the ~ chain were m o r e degraded than those in the sup-x, as seen in Figure 2B. In conclusion, the less-digested a m i n o terminal portion of the Bfl chain seems to play an i m p o r t a n t role in the thrombin-induced polymerization of L M r F X . The partial deficiencies at polymerization sites such as the carboxy terminal region of the y chain and the amino terminal portions of the Bfl chain, as well as internal cleavage in the rigid rod-like structure of fibrinogen, were considered to participate in the depolymerization of L M r F X .

Acknowledgements The authors are grateful to Dr Andrei Budzynski for guidance, useful discussions, and financial support. We thank Dr Robin

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