Proc. Natl. Acad. Sc. USAVol. 77, No. 5, pp. 2372-2376, May 1980Chemistry

Location of peptide fragments in the fibrinogen molecule byimmunoelectron microscopy

(antigen-antibody complex/disulfide knot/fragment H/fibrinogen structure)

J. N. TELFORD*, J. A. NAGYt, P. A. HATCHERt, AND H. A. SCHERAGAt**Section of Biochemistry, Molecular and Cell Biology, and tBaker Laboratory of Chemistry, Cornell University, Ithaca, New York 14853

Contributed by H. A. Scheraga, February 4, 1980

ABSTRACT Antibodies to the disulfide knot fragment ofbovine fibrinogen have been used to locate the site of thisfragment within the intact fibrinogen molecule. The antibodieswere isolated from rabbit antifibrinogen antisera by affinitychromatography. Electron micrographs of reaction mixtures ofbovine fibrinogen and antibodies against the disulfide knotfragment showed pairs of fibrinogen molecules crosslinked byantibody molecules as well as higher order antibody-fibrinogencomplexes. From an electron microscopic investigation of thecrosslinked material, we conclude that the disulfide knot lieswithin the central nodule of the trinodular fibrinogen molecule.Antibodies to fragment H were used in the same manner to lo-cate this fragment within the outer nodules of the human fi-brinogen molecule.In connection with our studies of the interaction of thrombinwith fibrinogen (1, 2) and of the polymerization of fibrinmonomer (3, 4), it is important to locate the sites of the fibrin-ogen molecule that are involved in various functions such asthrombin-binding, polymerization, fibrin stabilization, andplasmin degradation. From a series of physicochemical (5, 6)and electron microscopic (4, 7-10) observations, the lineartrinodular structure of fibrinogen appears established. In ad-dition, electron microscope observations of dimers, trimers,tetramers, etc. of fibrin monomer (4) have verified the con-clusions from earlier physicochemical studies (11) that theseintermediate polymers arise by a lateral association with partialoverlapping, giving two parallel end-to-end chains withstaggered junctions. In this paper, we use the technique ofimmunoelectron microscopy (12-16) to locate two peptidefragments within the trinodular structure of fibrinogen. Thesetwo fragments are the "disulfide knot" (N-DSK) (17) andfragment H (18). The former is the disulfide-linked NH2-ter-minal portions of the Aa, Bf3, and -y chains (Mr - 58,000)produced by CNBr cleavage of fibrinogen (17), and fragmentH (Mr 20,000) is a plasmin-digestion product of fibrinogen(18). By comparison of the NH2-terminal sequence of fragmentH given by Harfenist and Canfield (18) with the amino acidsequence reported by Doolittle et al. (19), it appears thatfragment H corresponds to residues 240-424 of the Aachain.

MATERIALS AND METHODSBovine fibrinogen (96% clottable) was prepared from Milesfraction I (lot 90, 75% clottable) or Sigma fraction I (lot117C-0140, 82% clottable) as described (20) and was dialyzedagainst 10mM Na2HPO4/3mM KH2PO4/120mM NaCl, pH7.5 (phosphate-buffered saline), 5 mM H3B04/1.25 mMNa2B4O7.10H20/140 mM NaCl, pH 8.5 (borate/saline buffer),or 50 mM ammonium formate (pH 8.5) for immunization,

quantitative precipitation, or electron microscopy, respectively.The reduced and alkylated material exhibited three bands(corresponding to the Aa, BB, and y chains) on NaDodSO4/7.5% polyacrylamide gel electrophoresis (21). All other chem-icals were of reagent grade or better.Human fibrinogen was purchased from AB Kabi (Stockholm,

lot 1981-07-01-57031, grade L, 95% clottable). It was dissolvedin 50 mM ammonium formate (pH 8.5) and dialyzed overnightat 4°C against this salt solution.

Fibrinogen concentrations were determined from the ab-sorbance at 280 nm with an extinction coefficient of 1.6 mlmg'- cm-1 (22). The concentrations of DSK and rabbit IgGwere determined in the same manner, with extinction coeffi-cients of 1.18 (23) and 1.67 ml mg-' cm-1 (24), respectively.Clottability was measured by the method of Laki and Gladner(25).The disulfide knot (DSK), which represents approximately

16% of the entire molecule and contains the sections of the Acaand B#. chains from which fibrinopeptides A and B are hy-drolyzed by the action of thrombin, was first prepared fromhuman fibrinogen by Blomback et al. (17). Their procedure,as modified by Hageman and Scheraga (20), was used (with oneadditional modification) to obtain the DSK of bovine fibrino-gen. The Sephadex G-100 column, used previously, was re-placed by a Sephacryl S-200 Superfine (Pharmacia) column (5.5X 100 cm) equilibrated with 0.1 M ammonium acetate buffer(pH 3.5). The column was eluted at a flow rate of 60 ml/hr, and15-min fractions were collected.Fragment H, a polypeptide that arises early in the digestion

of human fibrinogen by plasmin, was isolated and character-ized by Harfenist and Canfield (18). Edman degradationshowed that it consists mainly of a single polypeptide chainwhose NH2-terminal sequence is Met-Glu-Leu-Glu-Arg-Pro-Gly-Gly-Asn-Glu-, with a minor contaminating subfragmentbeginning at residue 254 (18). This fragment H pool was usedby Canfield and coworkers (private communication) to elicitantibodies against this portion of the fibrinogen molecule.An affinity chromatography column (1.1 X 21 cm) con-

taining bovine fibrinogen was prepared by binding 60 mg ofthis protein to 6 g of CNBr-activated Sepharose 4B (Pharmacia),and another column (1.3 X 9 cm) containing the bovine DSKwas prepared by binding 25 mg of this fragment to 2.5 g ofCNBr-activated Sepharose 4B (Pharmacia). The procedure wasthat recommended by the manufacturer (26).

Antisera against bovine fibrinogen were produced by im-munization of four young New Zealand white female rabbits.For the primary injection, 0.30 mg of fibrinogen per ml in 0.9%

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2372

Abbreviations: DSK (or N-DSK), disulfide knot; phosphate-bufferedsaline, 10 mM Na2HPO4/3 mM KH2PO4/120mM NaCI, pH 7.5; bo-rate/saline buffer, 5 mM H3BO4/1.25 mM Na2B407.10H20/140mMNaCl, pH 8.5.t To whom requests for reprints should be addressed.

Proc. Natl. Acad. Sci. USA 77 (1980) 2373

saline was first filtered through a 0.22-,um Millex filter, thenmixed 1:1 (vol/vol) with Freund's complete adjuvant (GIBCO)and emulsified in a Branson Microtip Sonicator; 3.2-3.6 ml perrabbit was injected in 0.1-ml doses intradermally in rows alongthe shaven backs of the rabbits, with an additional 0. 1-ml in-tramuscular dose in each hind leg. For the first booster injection,0.20 mg of fibrinogen in phosphate-buffered saline wasemulsified in Freund's incomplete adjuvant (GIBCO), (1:1,vol/vol) with a Vortex mixer and administered in 1.0-ml in-tramuscular doses in each hind leg after 4 weeks. Ten to four-teen days after this booster injection, blood was collected byincision of the marginal ear vein. The booster injection andbleeding schedule were then repeated every 3 weeks for about6 months. Antisera were isolated by centrifugation of the bloodat 40C. IgG fractions of the antisera were obtained by ammo-nium sulfate precipitation (27); they were dissolved in and di-alyzed against borate/saline buffer and stored in small volumesat -20'C.The IgG fraction of the antisera (20-50 ml) was passed

through the fibrinogen affinity column at room temperatureat a flow rate of 20 ml/hr, and 2-ml fractions were collected.The column was washed with 50 ml of borate/saline buffer, 15ml of 0.1 M sodium acetate/0.5 M NaCI, pH 5.5, and 10 ml of0.5 M NaCI, pH 5.5. Antibodies bound to the column wereeluted in a sharp peak with 0.1 M disodium citrate/0.5 M NaCl,pH 2.2, and were collected and immediately neutralized intubes containing 125 ,l of 4.0 M Tris (28). The column waswashed with 0.1 M sodium acetate/0.5 M NaCl, pH 1.8, andthen equilibrated in borate/saline buffer between each use. Theprotein content of the effluent was determined by the Folin-Lowry reaction (29), with bovine serum albumin as a reference.The presence of antibodies was also checked by immunodif-fusion against bovine fibrinogen. The large antifibrinogenfractions were pooled and dialyzed against borate/saline buffer,concentrated in a collodion bag (UH 100/25, Schleicher &Schuell), and passed through a Millipore filter to remove a smallamount of precipitate. Controls were also run on the fibrino-gen-affinity column with nonimmunized rabbit antiserum.

Anti-DSK was prepared by passing 42 ml of antifibrinogen(0.8 mg/ml) through the DSK-affinity column by the sameprocedure as above. The anti-DSK was dialyzed against 50 mMammonium formate (pH 8.5) for electron microscopy.

Double immunodiffusion according to Ouchterlony (30) wasperformed on 0.9% agarose immunodiffusion discs (Miles).Protein samples were in borate/saline buffer, phosphate-buf-fered saline, or ammonium formate. Whole antifibrinogenantiserum was also analyzed by the quantitative precipitationmethod described previously (27).

Antiserum against fragment H was prepared by Canfield andcoworkers, and kindly provided to us as a frozen solution. Theantiserum had been prepared by immunizing rabbits with afragment H-ovalbumin conjugate. The frozen solution wasthawed, and the IgG fraction was obtained by ammoniumsulfate precipitation as described (27). The IgG fraction wasdissolved in ammonium formate and dialyzed against thisbuffer.

Antigen-antibody complexes were prepared for electronmicroscopy in the following manner. Complexes of bovine fi-brinogen with anti-DSK were formed by adding 400 Al of bo-vine fibrinogen (1.7 mg/ml) to 120 ul of anti-DSK antibodies(0.25 mg/ml), both in ammonium formate, and incubating at37°C for 30 min (this is a 10:1 molar ratio of fibrinogen toanti-DSK antibody).Human fibrinogen (0.5 ml, 0.4 mg/ml) was incubated with

0.5 ml of the IgG fraction of the rabbit serum containing anti-bodies against fragment H (0.7 mg/ml) for 30 minat 370C. The

reaction mixture was freed of excess globulin by passagethrough a Sephadex G-200 (Pharmacia) column (1.1 X 30 cm)equilibrated with ammonium formate. The fibrinogen-anti-body complexes, which appeared with the void volume of thecolumn, were then prepared for electron microscopy. In otherexperiments, human fibrinogen (0.5 ml, 4.0 mg/ml) was in-cubated with rabbit IgG (0.5 ml, 7 mg/ml). The visible pre-cipitate that formed was centrifuged and the supernatantcontaining the excess nonspecific globulin was removed. Theprecipitate was resuspended in a solution of fibrinogen (0.5 ml,4.0 mg/ml) in ammonium formate, reprecipitated and resus-pended twice more, and diluted 1:10 in ammonium formate.The sample was then prepared for electron microscopy.

All samples were diluted with glycerol (1:1, vol/vol) (10) andthen shadowed with platinum/palladium by the followingprocedure. Approximately 0.25 ml of sample was placed in astandard nebulizer and sprayed onto l-cm2 pieces of freshlycleaved mica that had been coated with carbon by the indirectevaporation method of Whiting and Ottensmeyer (31). Thecarbon-coated mica supporting the sample was immediatelytransferred to a rotary shadowing device custom fitted to anMRC Series V-4 evaporator (Materials Research, Orangeburg,NY) and evacuated to 3 x 10-5 torr (1 torr = 1.33 X 102 pas-cals). The samples were rotary shadowed at an angle of 100. Thecarbon film was floated off the mica onto the surface of distilledwater, picked up from below onto no. 400 mesh copper grids,and blotted dry with filter paper.The samples were examined in an AEI EM6B electron mi-

croscope operated at an accelerating voltage of 80 kV, at amagnification of 40,000-60,000. The instrument magnificationwas calibrated with a Waffle-type carbon grating having 54,684lines per inch. The negatives from the electron microscopeplates were photographically enlarged to a final magnificationof 142,500. Measurements of the sizes of the fibrinogen mole-cules were made with a lOX precision loupe having a graduatedscale of 10 divisions per cm.

RESULTS AND DISCUSSIONAffinity Chromatography. The elution profile obtained in

the affinity chromatography purification of antibodies againstbovine fibrinogen is presented in Fig. 1. This profile was ob-tained by passing the heterogeneous IgG fraction of the rabbitantisera through the fibrinogen affinity column and elutingwith the solvents indicated in Materials and Methods.Nonimmunized control rabbit IgG is shown for comparison.

30 40Fraction Number

FIG. 1. Affinity chromatography of IgG (from rabbits immunizedwith bovine fibrinogen) on the bovine fibrinogen/Sepharose column.0, Antifibrinogen IgG; 0, control serum; - - -, pH/salt gradient.

Chemistry: Telford etA

Proc. Natl. Acad. Sci. USA 77 (1980)

Nonspecific IgG appeared in the first large peak. The materialfrom tubes 39-51 (containing anti-fibrinogen antibodies) waspooled, dialyzed against borate/saline buffer, passed througha Millipore filter, and applied to the DSK affinity column. Theisolated anti-DSK antibodies represented ;30% of the antifi-brinogen antibodies that were added to the DSK affinitycolumn.

Immunodiffusion. Fig. 2 illustrates a double-immunodif-fusion experiment with several different proteins. Antifibri-nogen serum gave rise to a strong precipitin line against bovinefibrinogen and a weak precipitin line against DSK. Antifibri-nogen antibodies (from tubes 39-51 of Fig. 1), dialyzed againstammonium formate, showed a strong diffuse precipitin lineagainst bovine fibrinogen. Anti-DSK antibodies gave a sharpprecipitin line against bovine fibrinogen and a weak line againstDSK. Control serum showed no precipitin line with either bo-vine fibrinogen or with DSK. Purified antifibrinogen did notappear to give a precipitin line against DSK. With a higherconcentration of DSK (12 mg/ml) and a 10-fold increase in theconcentration of anti-fibrinogen in other experiments, however,precipitin lines were visible against antifibrinogen.

Quantitative Precipitation Analysis. These analyses, withwhole anti-bovine fibrinogen antiserum, were carried out, asin ref. 27, to determine the number of antibodies bound perfibrinogen molecule. By using an average Mr of 150,000 forantibody molecules and one of 340,000 for fibrinogen, a valueof 35 for the minimum molar ratio of antibody to fibrinogenwas obtained. This represents a minimum value for the numberof antibodies bound per fibrinogen molecule because someantigenic determinants could overlap or lie close to each other(i.e., by competition or steric hindrance between antibodymolecules of different specificities, the binding of one antibodycould prevent the binding of another). Thus, there are _035antigenic sites per fibrinogen molecule.

Electron Microscopy of Bovine Fibrinogen. Fig. Sa showsa representative field of rotary-shadowed fibrinogen molecules

FIG. 2. Photograph of disk used in immunodiffusion experiment.Center well, DSK (25 AI, 10 mg/ml in phosphate-buffered saline); 1and 4, bovine fibrinogen (25 AI, 1.7 mg/ml in borate/saline buffer);2, purified anti-bovine fibrinogen (25 AI, 0.5 mg/ml in borate/salinebuffer); 3, anti-bovine fibrinogen antiserum (25 Al); 5, normal rabbitserum (control; 25 gl); 6, anti-DSK (25 Al, 0.6 mg/ml in ammoniumformate). Although no precipitin line is visible between 2 and thecenter well, it was visible in other immunodiffusion experiments witha 10-fold increase in purified antifibrinogen concentration.

P A it .

. _4j, I /

El't 'V-,M 6

Af

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FIG. 3. Electron micrographs (bar = 1000 A). (a) Bovine fibrin-ogen prepared as in the text and rotary shadowed. Concentration offibrinogen was 50 ,g/ml; (b) Complex of bovine fibrinogen with pu-rified anti-DSK antibodies. Arrows indicate fibrinogen-antibodycomplexes. (c) Fibrinogen-antibody complexes, with an interpretivedrawing given below each frame.

distributed randomly over the whole area. The individualmolecules appear as trinodular rods. The structures of themolecules, dried in the presence of glycerol (10), seemed to bebetter preserved compared with those dried with no glycerolpresent.The overall length of the shadowed fibrinogen is 495 ± 24

A; the two outer nodules are 142 + 12 A in diameter and thecenter nodule is 109 + 16 A in diameter. These dimensions ofrotary-shadowed fibrinogen molecules were corrected for theshell of platinum/palladium that must surround the molecule.By estimating the shell in these preparations to be 20 A thick(10), the molecules in our preparation are 455 A long (±24 A);the two outer nodules are 102 A in diameter and the centernodule is 69 A in diameter. The corrected measurements agreewith the overall dimensions of fibrinogen reported previously(4, 7-10, 32, 33).

10r."374 Chemistry: Telford et al.

Proc. Natl. Acad. Sci. USA 77 (1980) 2375

0; * -.f

..,.. . %*-"Oao

....

;S;.r:0P.

.fW.W., .4- S e

FIG. 4. Electron micrographs (bar = 1000 A). (a) Human fi-

brinogen prepared as in the text and rotary shadowed. Concentrationof fibrinogen was 40 ,ug/ml. (b) Complexes of human fibrinogen withantibodies to fragment H. Arrows indicate fibrinogen-antibodycomplexes. (c) Fibrinogen-antibody complexes showing individualfibrinogen molecules with attached antibody, and antibody-linkedfibrinogen complexes, with an interpretive drawing given below eachframe.

Antibody-Bovine Fibrinogen Complexes. Fig. 3b is an

electron micrograph of bovine fibrinogen that had been incu-bated with anti-DSK antibodies. Fibrinogen is no longer dis-tributed randomly, but appears arranged predominantly indimeric and oligomeric groups which we have interpreted as

antibody-crosslinked complexes. The gallery of enlargementsin Fig. Sc shows pairs of fibrinogen molecules crosslinked withone or more anti-DSK antibody molecules and higher-ordercomplexes of fibrinogen and antibody. The anti-DSK appearsto croslink the fibrinogen molecules at only one specific region.This binding site rs in the central nodule of the trinodularstructure of the fibrinogen molecule.

Because the width of an Fab arm of an IgG molecule isroughly 40 A (34), we cannot expect to resolve the antibody

molecule in a preparation of antibody-crosslinked fibrinogenthat has been rotary shadowed because of the insufficient res-olution of this technique.Antibody-Human Fibrinogen Complexes. An electron

micrograph of human fibrinogen, again depicting a trinodularstructure with a corrected overall length of 457 i 30 A, is shownin Fig. 4a. Fig. 4b shows reaction mixtures of fibrinogen in-cubated with antibodies against fragment H, depicting severalforms of fibrinogen-antibody complexes. Pairs of fibrinogenmolecules, as well as higher-order complexes, interpreted asbeing crosslinked by anti-fragment H antibody molecules, maybe observed in the gallery of Fig. 4c. In contrast to Fig. 3b, Fig.4c shows that antibody against fragment-H crosslinks the fi-brinogen molecule at the outer nodules.

Several facts indicate that fragment H must be near thesurface of the fibrinogen molecule. (i) In physiological regu-lation of fibrinolysis, plasmin can attack fibrinogen and releasefragment H quite readily (18, 35); (ii) this peptide is highlyhydrophilic (18); and (iii) fragment H contains the crosslinkingacceptor sites necessary for the in vivo stabilization of fibrin byFactor XIII (18, 36, 37). These acceptor sites include residues328 and 366 of the Aa chain (19, 37). The immunoelectronmicroscopic evidence presented here is in agreement with thelocation of fragment H on the surface of the intact fibrinogenmolecule.

Conclusion. On the basis of the observations reported here,it appears that the bovine DSK (containing the NH2-terminalportions of the Aa, Bj3, and y chains, including the Arg-Glybonds hydrolyzed by thrombin) is located in the central noduleof fibrinogen, and that human fragment H (consisting of resi-dues 240-424 of the Aa chain) is located in the outer nodule.

Note Added in Proof. The thickness of the shell of platinum/pal-ladium that surrounds the shadowed fibrinogen molecules has beencalibrated with ferritin molecules of known diameter that had beenrotary shadowed by the same procedure as used for the fibrinogenpreparations. The thickness of the shell of platinum/palladium wasfound to be 25 ± 3 A. Therefore, the corrected dimensions of the fi-brinogen molecule, reflecting the contribution of two thicknesses ofa 25-A-thick platinum/palladium shell.surrounding each molecule,are: length, 445 A; diameter of outer nodules, 92 A; diameter of centernodule, 59 A.

We are indebted to W. E. Fowler for a prepublication copy of ref.10, which provided the suggestion to use glycerol in preparations offibrinogen prior to rotary shadowing. We also thank Dr. R. E. Canfieldfor the gift of anti-fragment H antiserum and Mr. T. W. Thannhauserfor performing nitrogen analyses. This work was supported by researchgrants from the National Heart, Lung and Blood Institute of the Na-tional Institutes of Health (HL-01662) and from the National ScienceFoundation (PCM75-08691).,J.A.N. was a National Institutes of HealthPredoctoral Trainee, 1977-1980.

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