lmmunochemistry, 1977, Vol. 14. pp. 405-414. Pergamon Press. Printed in Great Britain

H U M A N FIBRINOGEN--IMMUNOLOGICAL PROPERTIES OF THE SUBUNITS BENGT GARDLUND Department of Blood Coagulation Research, Karolinska Institutet, S-104 01 Stockholm, Sweden (First received 18 October 1976; in revised form 5 January 1977) Abstract--The immunochemical properties of the purified chains of human fibrinogen have been investigated. Antisera against the chains have been raised in rabbits and immunodiffusion and crossedimmunoelectrophoresis studies were performed on the chains. No immunological cross-reaction between the chains could be detected. The chains of fibrinogen were found to be electrophoretically heterogeneous and complete or partial immunological identity was observed between the presumably intact chains and chain derivatives which appeared as contaminants in the chain preparations. Electrophoretic heterogeneity of the non-purified chains of reduced and carboxymethylated fibrinogen was also demonstrated.

INTRODUCTION The three chains of human fibrinogen Ac~, B/~ and 7 have mol. wts of 64,000, 57,000 and 48,000, respectively (McDonagb et al., 1972). It has been suggested that these chains are derived from one common ancestral gene. This is supported by the fact that even with the limited amino acid sequence known, a striking homology between the three chains has been observed (Doolittle, 1970; Doolittle, 1973; Hessel, 1975). Consequently, one would expect the chains to have antigenic sites in common and would thus show immunological cross-reaction. Studies on the chains of bovine fibrinogen using anti-fibrinogen serum or antisera to the purified chains in crossed-absorption studies and passive hernagglutination tests have suggested immunological cross-reaction between the Ac~- and Bfl-chains (Gollwitzer et al., 1972, 1975). On the other hand, Lefvert et al. (1974), employing a radioimmunoassay for the A~-chain did not observe any cross-reaction between the A~- and B/~-chains of human fibrinogen, The aim of this work has been to establish if crossreaction occurs between chains of human fibrinogen and in that case to identify the cross-reacting parts of the chains. The intention with this study was also to elucidate the suitability of the crossed-immunoelectrophoresis method for studying the chains of fibrinogen.

gomery, 1970; McDonagh et al., 1972). Polyacrylamide gel electrophoresis in SDS was performed alternatively with gels and buffer containing 8 M urea after incubating the samples with 8 M urea at 60°C for 1 hr or 6 hr. Amino acid analysis Amino acid analysis was performed after acid hydrolysis on a Technicon Auto Analyzer (BlomNick et al., 1972). Thrombin digestion For thrombin digestion before NH2-terminal analysis, 2rag of the chains was dissolved in 60#1 of 0.2M NH4HCO3, 6M urea, pH 8.5, and slowly diluted with 120 ,ul of 0.2 M NH4HCO ~, pI-/8.5. Thrombin, 1800 NIH units/mg (Hogg & Blomb/ick, 1974) was added to a final concentration of 180NIH units/ml. The samples were digested at 37°C for 4 hr and were then freeze-dried. NH 2-terminal attal),sis For quantitative NH2-terminal analysis with phenyl(35S)isothiocyanate (Collen et al., 1975), approx 2rag of the sample was dissolved in a Tris-urea-pyridine coupling buffer and an aliquote was withdrawn prior to coupling lbr determination of the protein concentration by amino acid analysis. After washing the coupled protein with benzene it was precipitated with acetone. Cleavage and cyclization was achieved using 1 M HCI. The phenylthiohydantoin (PTH) amino acids were extracted with ethylacetate and were chromatographed on thin-layer plates. The radioactive spots were eluted and quantitated by liquid scintillation counting (Collen et al., 1975).

Immunological techniques Antisera against the respective chains were produced by immunizing rabbits by foot pad injections of 0,2 ml protein Fibrinogen and fibrinogen chains suspension in each foot pad [1-3 mg/ml in equal volumes Human fibrinogen, fraction 1-4 (Blomb~ick & Blomb/ick, of Freund's complete adjuvant (Difco, Detroit, MI, U.S.A.) 1956) was reduced and carboxymethylated as described and 0.15 M NaC1]. At two-week intervals, the injections before (Murano et al., 1971). Highly purified chains of were repeated twice, now with Freund's incomplete adjufibrinogen were isolated by chromatography on carboxy- vant. Five weeks later, the rabbits were boosted by intramethylcellulose (Murano et al., 1971). muscular injections with the same immunogen and one week later, blood was collected by marginal ear vein inciPolyacrylamide gel electrophoresis sions. Five weeks after a bleeding, the boosting and bleedPolyacrylamide gel electrophoresis at acidic pH and ing could be repeated. The antisera were coded with a polyacrylamidc gel electrophoresis in sodium dodecyl sul- number referring to the rabbit and a letter for each confalc (SDS) with or without dithiothreitol (DTT) in 7% gels. secutive bleeding. In earlier experiments, Freund's complete was performed as described earlier (Brummel & Mont- adjuvant was used throughout the immunization. How405 MATERIAL AND METHODS

406

BENGT GARDLUND

ever, this procedure generally led to necrotic, slow-healing ~ounds which often became infected and it was therefore not used in this study. Absorption of antisera was performed a~ 37 C for 60 rain with varying amounts of antigen (20 500 ~g/'ml serumk Absorption of antigen was carried out in the same way (2 4 ml antiserum/rag antigen). After absorption the precipitates were removed by centrifugalion. ('rossed-immunoelectrophoresis was performed essentially according to Weeke (1973a, 1973h) on 10 x 10cm glass plates with a 1 mm layer of agarose (Miles-Sera~,ac, Berks, U.K) in 0.04 M barbital buffer, pH 8.6. In a corner of the gel, 2cm from the right edge and 1.5 cm from the lower edge, 5 td of the sample was applied in a 3 mm well. A water-cooled electrophoresis apparatus (Desaga, Heidelberg, West Germany) was used with 0,04 M barbital hafl~:r, pH 8.6. as electrode buffer. The elcctrophoresis was run with the right edge as cathode for 1 hr with 12 V/cm after which the upper 4/5 of the gel was removed and replaced with the same agarose solution containing antiserum. in the second dimension, electrophoresis was run for about 20 hr with 2 V/era with the lower edge as cathode. The plates were then covered with wet filter papers and dry tissue paper and excess moisture was removed by u light uniform pressure for 15 rain. The thin agarosc fihn was washed with 0.15 M NaC1 and then with wa~er for 15 rain. The plates were air dried and stained for 5 rain in ethanol-acetic acid water (9 : 2: 9) containing Coomassic Brilliant Blue (2.5 g/l). The plates were destained in ethanol acetic acid water (5:2:9). The photographs of the plates show the lower edge at full length. [mmunodiffusion was performed essentially as described (Ouchterlony. 1948) in 2.5 mm thick 0.6'~;~ agar (Difco, Detroit, MI. U.S.A.) in 0.11 M NaCI, 0.04 M Tris. pH 7.2, Of the sample (0.5 mg/ml), 25pl and of tire antiscrum. 5(~ 100jd was added to each 4ram well. After 24 48 hr at 20 C, the plates were either photographed under darkfield illumination or washed, dried and stained in essentially the same way as that described for the crossedimmun oelectrophoresis plates. For immunological tests, the fibrinogen chains were dissolved in 0.04 M barbital buffer, pH 8.6, containing 6 M urea and were diluted during stirring by dropwise addition of the same buffer without urea to give a final urea concentration of 2 M. With this gentle dilution procedure, the chains remained in solution in 2 M urea. The protein concentration in the final solution was determined by amino acid analxsis.

There were trace a m o u n t s of material having tool. wts of a b o u t twice the respective chains (cf. Fig. 1). These bands were not diminished by reduction with D T T or by pre-incubating the samples in 8 M urea lor I h r at 6 0 C and running the SDS gel electrophoresis in 8 M urea. However, pre-incubation of the samples in 8 M area for 6 h r at 6 0 C rendered the SDS gels virtually free from higher mol. wt material. It can thus be concluded that these bands represent firmly associated non-covalently b o u n d aggregates of the respective chains,

N tf ~-levminal cmalysis The results of the quantitative NHz-terminal analyses of the chains are shown in Table 1. The yields of P T H - a m i n o acids, a b o u t 50% for all three chains, arc consistent with the yields normally obtained by using this method, As expected, the A:~-chain preparation was demonstrated to have alanine and aspartic acid as the main NH2-terminal a m i n o acids (Blomhiick et al,, 19661. The NH2-terminal glycine which is usually also found in small a m o u n t s m fibrinogcn may be derived from a ~-chain lacking fibrinopcptide A (Kierulf & Abildgaard, 1971). After thrombin digestion of the Ae-chain, glycine and valinc appeared as the main NH2-terminal amino acids as a result of cleavage of A r g - G l y a n d A r g Val bonds, respectively (Blombiick et aL, 1967). Since n o N H , - t c r m i n a l tyrosine could be demonstrated in the

Aa

Chemicols MI chemicals used were of analytical grade.

RESULTS AND DISCUSSION

PoIyacrylamide gel electrophoresis Judging from the SDS gel electrophoresis patterns. the purified fibrinogen chains were essentially free from c o n t a m i n a t i o n from the other chains under optimal conditions for b a n d resolution (Fig. l). In the 7-chain preparation, a trace a m o u n t of material with lower tool. wt t h a n the ),-chain was observed, supposedly representing a degraded y-chain or a cont a m i n a n t derived from the A~- or Bfl-chains. O n overloading the SDS gels, faint additional b a n d s appeared, a m o n g them, bands in the Bfl-chain preparation corresponding to the A~- and y-chain b a n d s and in the Ae- and y-chain preparations bands corresponding to the Bfl-chain band. Essentially the same patterns were observed on polyacrylamide gel electrophoresis at acidic pH.

Fig. I. Polyacrylamide gel electrophoresis in SDS of purified fibrinogen chains. Electrophoresis in 7% gel matrix in 0.1'~, SDS of 15 pg of protein. Cathode and origin are uppermost.

Immunology of Fibrinogen Chains Act-chain preparation, it can be concluded that it is essentially free from y-chain contaminants. Presence of contaminating B/j-chain cannot be excluded. The intact B/j-chain with NH2-terminal pyrrolidone carboxylic acid (Blomb~ick et al., 1966) gave only trace amounts of PTH-amino acids. However, after thrombin treatment glycine appeared as the main NH2-terminal (Table 1). The NH2-terminal tyrosine is most likely due to contaminating y-chain. The trace amounts of NH2-terminal alanine and valine may represent chains partially degraded from the NH2-terminal end. The y-chain gave tyrosine as the main NH2-terminal together with trace amount of glycine (Table 1). The glycine could be derived from degraded Actand/or Bj~-chains devoid of fibrinopeptides. After thrombin treatment the amount of NH2-terminal glycine increased. This is most likely a consequence of the liberation of fibrinopeptide B from contaminating B/J-chain.

hnmunodiffusion experiments

407

showed a single precipitin line with the y-chain preparation but also with the Bfl-chain preparation showing a pattern of complete identity. Against a mixture of B/J- and y-chains, this antiserum gave a single precipitin fine showing a double spur with the B/j-chain precipitin line (Fig. 2d). No reaction was observed with the Act-chain. Absorption of the anti-Act serum was performed with varying amounts of the B/j-chain preparation. When the anti-Act serum was absorbed with a sufficient amount of the B/j-chain preparation to prevent a reaction with the B/j-chain preparation, the antiserum did not react with the Act-chain preparation either. Likewise, absorption of the anti-B/j serum with the y-chain preparation abolished the reaction of the antiserum with the B/j-chain preparation as well as with the y-chain preparation. Using anti-B/j serum as absorbant, the y-chain preparation was depleted of material reactive with anti-Bfl serum without affecting the reaction with the anti-y serum. These immunodiffusion experiments do not give evidence for cross-reaction between the chains. Conclusions concerning the purity of the chains and the antisera as judged by NHz-terminal analysis and immunodiffusion studies agree and are summarized as follows: the different antisera to the chains seem to be monospecific. The Act-chain preparation probably contains a small amount of B/j-chain, the B/j-chain preparation is contaminated with small amounts of Act- and y-chains, and the y-chain preparation is contaminated with B/j-chain material.

A total of 25 rabbits were immunized with the three fibrinogen chains, giving serum drawn at 67 occasions. The antisera were tested on immunodiffusion against the immunogen and the other chains. No significant difference was observed between the antisera drawn at different occasions from the same rabbit and for the anti-Act and anti-B/j sera, the antisera from different rabbits did not display major differences. The anti-Act serum usually gave rise to a weak, diffuse precipitin line against the immunogen on immunodiffusion (Fig. 2a bottom). Anti-Act serum did Crossed-immunoelectrophoresis of the isolated chains not react with the y-chain preparation but gave a In order to further study the contaminants and weak precipitin line with the B/j-chain preparation, possible cross-reaction of the chains, crossedapparently identifying with the Act-chain preparation immunoelectrophoresis was performed with the (Fig. 2a bottom). In most cases, the rabbits im- chains against the different antisera. Anti-Act serum munized with the B/j-chain produced serum with a gave a sharp symmetrical arch with the Act-chain (Fig. good titre of antibodies, giving a single, distinct preci- 3a) but also with the B/j-chain preparation. No reacpitin line with the B/j-chain (Fig. 2a top). The anti-B/j tion was found with the y-chaim When the Act- and serum also reacted with the y-chain preparation and Bt3-chains are run together against the anti-Act serum, faintly with the A~-chain preparation giving lines of the arches seem to fuse (Fig. 3b). The anti-B/j serum complete identity with the B/j-chain preparation gave a distinct precipitin arch with the B/j-chain (Fig. (Figs. 2a top and 2b). The immunodiffusion pattern 3c) but also a reaction with the Act-chain preparation. shown in Fig. 2c demonstrates the non-identity of the When the A~- and B/j-chains were run together precipitates obtained with the anti-Act and anti-B/j a g a i n s t this antiserum, the arches showed a pattern sera. The rabbits immunized with the 7-chain pro- of complete identity (Fig. 3d). When anti-Act and duced poor antiserum with little or no precipitating anti-B/j sera were used together against the mixture antibodies. Only 2 rabbits out of 9 produced anti- of the two chains, two continuous precipitin lines serum with a good titre of antibodies. The antiserum were observed (Fig. 3e). Thus, it can be concluded Table 1. NH2-terminal amino acids of the chains of fibrinogen before and after thrombin digestion

Amino acid Ala Asp Gly Val Tyr

Act-chain 0.34 0.09 0.08

Molar yield of NHz-terminal amino acids Thrombin Thrombin digested digested As-chain Bfl-chain BE-chain 7-Chain 0.04

0.03

0.04

0.23 0.07

0.03 0.03 0.06

0.46 0.05 0.07

Thrombin digested 7-chain

0.03

0.07

0.44

0.43

The figures represent molar yield of PTH-amino acids after elution from thin-layer chromatograms without correction for loss during analysis. The caleulations of yields were based on mol. wts of 64,000 for the Act-chain, 57,000 for the Bfl-chain and 48,000 for the v-chain.

408

BENGT GARDLUND that the Bfl-chain preparation is contaminated by a Ac~-chain derivative with an electrophoretic mobility similar to that of the Bfl-chain, and that the A~-chain preparation includes Bfl-chains with electrophoretic mobility similar to the A:~-chain. No immunological cross-reaction could be detected between the A~.- and Bfl-chains. The 7-chain run against the anti-Bfi serum showed a double arch. In Fig. 4a the profile is shown of the Bfl- and 7-chains run together against the anti-Bfl serum. It would appear that the ;,-chain preparation contains two populations of Bfi-chain-like material. One is probably immunologically intact Bfl-chain and the other, with partial immunological identity with the Bfl-chain, may be a degraded Bfi-chain which has an incomplete set of epitopes. The experiments with the anti-7 serum supported this interpretation. Using the anti- 7 serum, no cross-reaction between the Briand 7-chains was detected. This antiserum gave a single precipitin arch against the ?,-chain {Fig. 4b) and a continous asymmetrical arch against a mixture of the Bfi- and ;'-chains (Fig. 4c). The two precipitin protiles in Figs. 4a and 4c both appeared when anti-B# and anti-?' sera were used together against the mixture of the two chains on the same plate. No reaction was found with anti-;' serum against the A>chain.

aBB BI3

Fig. 2(aL

Crossed-immunoelectrophore,~is o/reduced and carhoxvmethylated ¢ibrino,qen In the crosscd-immunoclectrophoresis experiments. thc chains of fibrinogen show charge heterogeneity. To elucidate whether this heterogeneity is artefactually produced during isolation of the chains. reduced and carboxymethylated fibrinogen (CM Fbg) was tested in the crossed-immunoelectrophoresis system. The A~,-chain of CM Fbg gave a broader arch

Fig. 2(b).

7

Bfl Fig. 2(d).

aBi3'

;oAa Fig. 2{cl.

Fig. 2. lmmunodiflusion of purilied tibrinogen chains against antisera to the chains {a d). A0~-chain, 25#1, 0.5mg/ml (A~): Bfi-chain 25H1, 0.5mg/ml (Bfi): T-chain, 251d, 0.5mg/ml (7): Bfl-chain, 0.25mg/ml and ),-chain. 0.25 mg/ml, 25 itl (Bfl/71; anti-Ax serum 158H), 3 x 25 HI (aAc0: anti-Bfl serum (59J). 2 x 25/4 (aB/4): anti-7 serum il04A), 4 x 25td (aTI. The precipitates in Fig. 2d were stained with ('oomassie Brilliant Blue.

Immunology of Fibrinogen Chains

409

O

Fig. 3(a).

Fig. 3(b).

Fig.~ 3(c).

t M M 14/f~

B

BENGT GARDLUND

41

Human fibrinogen--immunological properties of the subunits.

lmmunochemistry, 1977, Vol. 14. pp. 405-414. Pergamon Press. Printed in Great Britain H U M A N FIBRINOGEN--IMMUNOLOGICAL PROPERTIES OF THE SUBUNITS...
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