Secretion of biologically active recombinant fibrinogen by yeast.

Fibrinogen (340 kDa) is a plasma protein that plays an important role in the final stages of blood clotting. Human fibrinogen is a dimer with each half-molecule composed of three different polypeptides (Aα, 67 kDa; Bβ, 57 kDa; , 47 kDa). To understand the mechanism of fibrinogen chain assembly and secretion and to obtain a system capable of producing substantial amounts of fibrinogen for structure-function studies, we developed a recombinant system capable of secreting fibrinogen. An expression vector (pYES2) was constructed with individual fibrinogen chain cDNAs under the control of a Gal-1 promoter fused with mating factor Fα1 prepro secretion signal (SS) cascade. In addition, other constructs were prepared with combinations of cDNAs encoding two chains or all three chains in tandem. Each chain was under the control of the Gal-1 promoter. These constructs were used to transform Saccharomyces cerevisiae (INVSC1; Matα his3-Δ1 leu2 trp1-289 ura3-52) in selective media. Single colonies from transformed yeast cells were grown in synthetic media with 4% raffinose to a density of 1 × 108 cells/ml and induced with 2% galactose for 16 h. Yeast cells expressing all three chains contained fibrinogen precursors and nascent fibrinogen and secreted about 30 μg/ml of fibrinogen into the culture medium. The Bβ and chains, but not Aα, were glycosylated. Glycosylation of Bβ and chains was inhibited by treatment of transformed yeast cells with tunicamycin. Intracellular Bβ and chains, but not the Aα chains in secreted fibrinogen, were cleaved by endoglycosidase H. Carbohydrate analysis indicated that secreted recombinant fibrinogen contained N-linked asialo-galactosylated biantennary oligosaccharide. Recombinant fibrinogen yielded the characteristic plasmin digestion products, fragments D and E, that were immunologically indistinct from the same fragments obtained from plasma fibrinogen. The recombinant fibrinogen was shown to be biologically active in that it could form a thrombin-induced clot, which, in the presence of factor XIIIa, could undergo chain dimerization and Aα chain polymer formation.

Human fibrinogen is a large plasma glycoprotein with diverse physiological functions. Its primary roles are in the final stages of blood coagulation, when it forms a fibrin clot and participates in platelet aggregation. Fibrinogen is a dimeric molecule with each half-molecule composed of three different polypeptides. The A␣ chain has 610, the B␤ 461, and the ␥ 411 amino acid residues. The B␤ and ␥ chains are N-glycosylated. The six chains are connected by 29 disulfide bonds. The primary structure of fibrinogen is known, and structural studies indicate that fibrinogen is elongated and trinodal. The central E domain contains the NH 2 termini of the six polypeptide chains, and the two terminal "D" nodes are formed by carboxylterminal globular domains of the B␤ and ␥ chains and a small (12-kDa) segment of the A␣ chain. The COOH-terminal regions of the A␣ chain extend beyond the globular domains of the B␤ and ␥ chains and may fold back and contribute to the structure of the central node. Between the central and terminal domains the three chains are coiled together in an ␣-helical, rope-like manner. This ␣-helical area, which occupies about 111 amino acids in each chain, is termed the "coiled-coil" region and is flanked at either end by a set of interchain disulfide bonds called the "disulfide rings." Unfortunately, to date, high resolution structural analysis has not been achieved since fibrinogen crystals have diffracted poorly. Nevertheless, a combination of biochemical and electron microscopy studies have reached a consensus on the general structure as described above. The structure and physiology of fibrinogen has been reviewed (1)(2)(3)(4)(5)(6)(7).
Biologically active recombinant fibrinogen has been expressed in several mammalian cell systems (8 -11), and the individual component chains of fibrinogen have been expressed in Escherichia coli (12)(13)(14). Although recombinant fibrinogen mutants have been used for structure/function studies (15)(16)(17)(18) the procedures are hampered by the fact that prokaryotic systems do not assemble the fibrinogen chains and that transfected mammalian cells only secrete small amounts of biologically active fibrinogen. To obtain substantial quantities of biologically active fibrinogen for structure/function studies and to develop a system in which a genetic approach to understanding fibrinogen chain assembly could be undertaken, we have expressed fibrinogen in yeast.
Construction of Expression Vectors-Expression vectors containing fibrinogen cDNAs for single chains, for combinations of two chains, and for all three chains were inserted into multiple cloning sites at the 3Ј-end of the Gal-1 promoter fused to the MF␣1 1 prepro secretion signal (SS) 1 cascade in pYES2 plasmid (see Fig. 1). To prepare pYES2A␣, pYES2B␤, and pYES2␥, full-length cDNAs were released by appropriate restriction enzymes from previously described constructs (8) and ligated to pYES2 plasmid at the 3Ј-end of the Gal-1-SS promoter. Other constructs, pYES2A␣B␤, pYES2A␣␥, pYES2B␤␥, and pYES2A␣B␤␥, were made by ligating fibrinogen chain cDNAs in tandem, each under the control of the Gal-1-SS promoter. Elution of DNA fragments from agarose gel, dephosphorylation of plasmids with calf intestinal phosphatase, fill-in reaction with Klenow fragment, and ligation were performed by standard procedures (22) and as described previously (8,17).
Transformation-Transformation of S. cerevisiae (INVSC1) with pYES2 vectors containing fibrinogen cDNAs was performed by the alkali-cation method, and the cells were plated on SC-ura plates (23). Single colonies from each plate were grown in SC-ura medium containing 4% raffinose at 30°C with vigorous shaking overnight and kept as a stock culture. Transformed yeast cells, with the above described constructs , were named INVSC1A␣, INVSC1B␤, INVSC1␥,  INVSC1A␣B␤, INVSC1A␣␥, INVSC1B␤␥, and INVSC1A␣B␤␥.
Expression and Treatment with Tunicamycin-Stock culture was grown in 5 ml of SC-ura medium overnight at a density of 1 ϫ 10 8 cells/ ml. The cells were harvested by centrifugation for 5 min at 500 ϫ g, resuspended in SC-ura medium containing 2% galactose, and grown for an additional 16 h for induction of fibrinogen chain synthesis. The cells were again harvested, resuspended in SC-ura-Met medium containing 50 Ci/ml of L-[ 35 S]methionine, and incubated for 1 h at 30°C. In some cases, the cells were preincubated with medium containing 10 g/ml of tunicamycin for 1 h and then incubated with medium containing 10 g/ml tunicamycin and L-[ 35 S]methionine for an additional hour. When determining intracellular fibrinogen, the cells were harvested, washed with phosphate-buffered saline, lysed with 0.5 ml of IP buffer (50 mM Tris-HCl, pH 7.4, 1% Triton X-100, 0.2% SDS, 150 mM NaCl, 5 mM EDTA, 10 units/ml Trasylol, 1 mM phenylmethylsulfonyl fluoride, 0.1 mM tosylphenylalanyl chloromethyl ketone, 1 g/ml soybean-trypsin inhibitor) and 200 mg of acid-washed glass beads (0.5 mm diameter)/10 8 cells by vortexing twice for 45 s (24). The cell lysate was diluted to 1 ml with water and centrifuged at 15,000 ϫ g for 15 min at 4°C. Fibrinogen was isolated by immunoprecipitation from the soluble fraction with a rabbit polyclonal antibody to human fibrinogen (Dako Corporation, Carpenteria, CA) as described elsewhere (8).
Endoglycosidase H Treatment-INVSC1A␣B␤␥ cells were metabolically labeled with L-[ 35 S]methionine for 1 h as described above. Radioactive fibrinogens were isolated from the incubation medium and from cell lysates by immunoprecipitation using polyclonal antibody to fibrinogen. The immune complex was treated with 2 mIU/ml of endoglycosidase H at 37°C overnight, reimmunoprecipitated, and separated on SDS-PAGE as described previously (21,25).
Secretion of Fibrinogen-Yeast cells transformed with pYES2A␣B␤␥ and grown from single colonies were inoculated in 50 ml of SC-ura medium containing 4% raffinose and grown overnight at 30°C. The cells were then induced with 2% galactose and incubated for an additional 16 h. The culture medium was centrifuged at room temperature for 5 min at 500 ϫ g. The pH of the medium was adjusted to 7.0 with 1 M Tris-HCl buffer, pH 8.0, and a mixture of protease inhibitors (10 units/ml Trasylol, 1 mM phenylmethylsulfonyl fluoride, 0.1 mM tosylphenylalanyl chloromethyl ketone, 1 g/ml soybean-trypsin inhibitor, 1 g/ml pepstatin) was added.
Isolation and Purification of Fibrinogen from the Culture Medium-Fibrinogen was isolated from the culture medium by absorption on a protamine sulfate-Sepharose 6B column (10 ml) calibrated with buffer A (50 mM Tris-HCl, pH 7.4, 5 mM ⑀-amino caproic acid, 5 mM EDTA) (11). The column was washed with buffer A containing 0.8 M NaCl, and bound fibrinogen was eluted with 0.1 M sodium acetate, pH 4.5. The pH of eluted fibrinogen was adjusted to 7.0 with 1 M Tris-HCl, pH 8.0.
Quantitation of Secreted Fibrinogen-The amount of fibrinogen secreted in the medium was measured by competition enzyme-linked immunosorbent assay using two different fibrinogen chain-specific monoclonal antibodies, 1-8C6 (anti-B␤ 1-21) and Fd4 -7B3 (anti-fibrinogen fragment D). The specificities of the antibodies have been described (26).
Carbohydrate Structure of Recombinant Fibrinogen-The N-linked oligosaccharide analyses of purified recombinant fibrinogen was per-FIG. 1. Expression vectors containing fibrinogen chain cDNAs. The fulllength cDNAs for individual fibrinogen chains were inserted into multiple cloning sites at the 3Ј-end of Gal-1-SS promoter (pYES2A␣, pYES2B␤, and pYES2␥). In the other constructs, combinations of two chains (pYES2A␣B␤, pYES2A␣␥, and pYES2B␤␥) and all three chains (pYES2A␣B␤␥) were inserted in tandem. Each arrow indicates the cleavage site of the secretion signal formed by Glyko, Inc. (Novato, California) using a FACE ® (Fluorophore Assisted Carbohydrate Electrophoresis) N-linked oligosachharide sequencing kit. Recombinant fibrinogen, purified from the culture medium, was lyophilized and resuspended in 170 l of water to a concentration of 2.35 mg/ml. The protein was denatured with 0.5% SDS, reduced with 1% ␤-mercaptoethanol, and digested with peptide-N-glycosidase F (PNGase F) at 37°C for 48 h. The released oligosaccharides were isolated by ethanol precipitation, labeled with fluorescent tag, and separated by electrophoresis in a 40% polyacrylamide gel. The oligosaccharide was characterized by sequential digestion with specific glycosidases and comparison of their electrophoretic mobility against standard N-linked oligosaccharides, as described in the manufacturer's protocol. Sialic acid was measured by the thiobarbituric acid assay (27) and by Glyko's monosaccharide composition kit.
The above digest was mixed with an equal volume of buffer A (40 mM Tris-HCl, 110 mM NaCl, 0.1% NaN 3 , pH 7.5) and applied to a 2-ml column containing approximately 40 mg of agarose-conjugated (Pierce AminoLink 228) anti-fragment D monoclonal antibody (Fd4 -7B3). Flow was stopped for 2 h to allow maximum binding. Nonadsorbed protein was removed by extensive washing with buffer A. Adsorbed protein was eluted with 4 ml of 3 M NaSCN in buffer A.
The above nonadsorbed fraction was applied to a 2-ml column containing approximately 40 mg of agarose-conjugated anti-fibrinogen fragment E monoclonal antibody (2N3H10). The sample was recycled several times over this column to allow maximum binding. Nonadsorbed protein was removed by extensive washing with buffer A. Adsorbed protein was eluted with 4 ml of 3 M NaSCN in buffer A.
Factor XIIIa Cross-linking-Secreted recombinant fibrinogen was treated with thrombin (6.8 NIH units/ml) with or without factor XIIIa (1.0 units/ml) to determine its ability to form a thrombin-induced clot and to cross-link. The fibrin complexes were separated by SDS-PAGE and detected by staining with Coomassie Blue and by Western immunoblots using several chain-specific antibodies: 1C2-2 (anti-fibrinogen A␣/fibrin ␣) (28), Ea3 (anti-fibrinogen B␤/fibrin ␤) (26), T2G1 (antifibrin ␤) (29), and 4 -2 (anti-fibrinogen ␥/fibrin ␥-dimer) (29). In human plasma fibrinogen the B␤ and ␥ chains, but not A␣, are N-glycosylated. To determine if correct N-glycosylation occurred the transformed cells expressing single chains were incubated in the absence or presence of tunicamycin, which prohibits N-glycosylation. Tunicamycin had no effect on A␣ chain, but B␤ and ␥ had faster electrophoretic mobilities, indicating that they lacked N-linked carbohydrates ( Fig. 2A).

Expression of Individual Fibrinogen Chains and N-Glycosy
INVSCIA␣B␤␥ expressed all three fibrinogen chains, and analysis of intracellular fibrinogen complexes on nonreduced SDS-PAGE demonstrated the presence of several fibrinogen precursors. Free A␣, B␤, and ␥, two chain complexes (B␤-␥ and A␣-␥), a three-chain half-molecule, and dimeric fibrinogen accumulated intracellularly (Fig. 2B). The intracellular complexes were characterized by their estimated molecular weights, based on SDS-PAGE, and the radioactive bands were excised and re-electrophoresed in reduced conditions to determine the chain compositions (data not shown).
On treatment with tunicamycin the A␣ chain expressed by INVSCIA␣B␤␥ had a mobility similar to A␣ chains from untreated cells, but both B␤ and ␥ chains had faster electrophoretic mobilities. Tunicamycin also affected the electrophoretic mobilities of the higher molecular weight complexes. In tunicamycin-treated cells the two-chain and three-chain  fibrinogen complexes were not as distinct as the corresponding complexes from untreated cells (Fig. 2B).
Endoglycosidase H Treatment of Intracellular and Secreted Fibrinogen-Nascent glycoproteins present in the endoplasmic reticulum contain mannose-rich carbohydrate side chains, which are later trimmed and further processed in the trans Golgi compartment before secretion occurs. The mannose-rich oligosaccharides, but not the fully processed side chains, are cleaved from glycoproteins by endoglycosidase H. To determine the carbohydrate nature of intracellular and secreted fibrinogen, INVSCIA␣, INVSCIB␤, and INVSCI␥ cells were metabolically labeled with L-[ 35 S]methionine, and intracellular and secreted fibrinogen were treated with endoglycosidase H. Analysis on reduced SDS-PAGE showed that intracellular B␤ and ␥ chains, but not A␣, were cleaved by endoglycosidase H. By contrast the secreted fibrinogen chains were not affected by endoglycosidase H, which is to be expected if the N-linked carbohydrates were fully processed (Fig. 3).
Carbohydrate Structure and Composition-Digestion with peptide-N-glycosidase F for 48 h at 37°C released two oligosaccharides (Fig. 4, panel A, lane 3, asterisks) in the region of the gel where N-linked sugars migrate. A third band that migrated similarly to band 3 of the partial wheat starch digest (Fig. 4, panel A, lane 1) could be maltotetraose (Fig. 4,  Based on the relative migration of standard oligosaccharides, as compared with the bands obtained from partial digestion of wheat starch (data not shown), the main N-linked oligosaccharide (marked by an asterisk) obtained by peptide-Nglycosidase F digestion is consistent with it being an asialogalactosylated biantennary oligosaccharide. Subsequent experiments confirmed the structure. Digestion with neuraminidase III, (panel B, lane 2) had no effect on the major band, indicating lack of sialic acid, and treatment with endoglycosi- dase H (panel B, lane 3) also did not affect the mobility of the N-linked oligosaccharide, indicating, as was shown in a previous experiment (Fig. 3), that secreted recombinant fibrinogen glycoprotein is not of the high mannose type. Partial digestion with ␤- galactosidase (panel B, lane 4) showed the starting material and the appearance of two lower bands. This suggests that at least two galactose monomers were cleaved.
Further confirmation of the oligosaccharide structure was obtained by complete digestion with ␤-galactosidase (panel C, lane 2), which demonstrates removal of approximately two galactose units. Treatment of the starting material with a combination of ␤-galactosidase and a hexosaminidase (hexosaminidase III) (panel C, lane 3) indicated complete removal of galactose and GlcNAc from the nonreducing end of the starting oligosaccharide. Taken together these results are consistent with the recombinant fibrinogen being a glycoprotein that is not of the high mannose type but contains an asialo-galactosylated biantennary oligosaccharide.
The absence of sialic acid was confirmed by assaying the recombinant yeast fibrinogen by the thiobarbituric acid method (27). Plasma fibrinogen, used as a control, yielded about 1.25 mol of sialic acid/mol of fibrinogen, but sialic acid was not detected in yeast fibrinogen. Also a monosaccharide composition assay, performed by Glyko Inc., failed to detect sialic acid (data not shown). It was not possible, however (because of a high background of glucose, which was also present in the blank sample and is possibly due to contamination from the dialysis membranes), to accurately determine the molar concentration of the sugars (data not shown). Secretion of Fibrinogen-After induction with galactose for 16 h the media of INVSCIA␣B␤␥ cells was collected and neutralized, and the amount of fibrinogen was determined. Quantitation was performed using two different monoclonal antibodies with specificities to different domains of fibrinogen. One of the monoclonal antibodies (1-8C6) reacted with B␤ chain at amino acid residues 1-21, and the other (Fd4 -7Bc) recognizes a plasmin digest fragment of fibrinogen (fragment D). Using both antibodies, human plasma fibrinogen and recombinant fibrinogen gave identical curves (Fig. 5). Transformed cells (10 8 cells/ml) secreted 25-30 g/ml after 16 h of induction with galactose.
In some cases the secreted fibrinogen was isolated from the incubation medium by affinity chromatography using prota-mine sulfate conjugated to Sepharose. Fibrinogen was the principal protein product present in the incubation medium although there was a large amount of low molecular weight material, which did not bind to the protamine-sulfate column and which absorbed at 280 nm (Fig. 6A). Analysis of the component fibrinogen chains by SDS-PAGE indicated that they had similar electrophoretic mobilities as human plasma fibrinogen (Fig. 6B) and that only B␤ and ␥, and not A␣, reacted with periodic acid-Schiff stain.
Clotting Properties of Recombinant Fibrinogen-Secreted recombinant fibrinogen, purified from the incubation medium by affinity chromatography on a protamine-Sepharose sulfate column, was treated with thrombin or with thrombin and factor XIIIa. The recombinant fibrinogen formed a visible fibrin clot, which was solubilized in an SDS-containing buffer with dithiothreitol. The solubilized proteins from the fibrin clot were separated by SDS-PAGE and analyzed by Western immunoblots using two different monoclonal antibodies. One of the monoclonal antibodies reacted with the ␥ chain of fibrin(ogen) and ␥ dimer from fibrin, and the other reacted with the ␤ fibrin chain. The results are shown in Fig. 7. As controls, reduced plasma fibrinogen and untreated recombinant fibrinogen are shown in lanes 2 and 3. The A␣ chain of plasma and recombinant fibrinogen, as is often the case, was partially degraded (panel A). On treatment with thrombin, in the absence of factor XIIIa, a similar pattern to the control samples was noted. Removal of fibrinopeptides A and B was not expected to show a marked difference in electrophoretic mobility of the ␣ and ␤ chains as compared with A␣ and B␤. Western blots with antibody to the ␤ chain (panel B, lane 4) and to ␥ chain (panel C, lane 4) also showed that these chains are present in the fibrin clots. On treatment with factor XIIIa, the ␥ chain was not detected by Coomassie Blue staining (panel A, lane 5) and was markedly reduced as determined by Western blot with antibody to ␥ chain. There was a concomitant appearance of ␥ dimer (panel C, lane 5). As a control it was noted that factor XIIIa had no effect on the ␤ chain (panel B, lane 5). Taken together these results demonstrate that recombinant fibrinogen forms a thrombin-induced clot and undergoes factor XIIIacatalyzed cross-linking.
Structure of Recombinant Fibrinogen Determined by Plasmin Digestion-Plasmin digestion of plasma fibrinogen yields well defined fragments that are dependent on the structural integrity of fibrinogen and the correct assembly of its component chains. To determine whether recombinant fibrinogen, on treatment with plasmin, yields fragments D and E, which are derived from the terminal and central domains of dimeric fibrinogen, purified recombinant fibrinogen was digested with plasmin. The digest was subsequently fractionated by affinity chromatography using antibodies specific to fragments D and E. Adsorbed proteins were further characterized by SDS-PAGE and Western immunoblots. Recombinant fibrinogen, like plasma fibrinogen, yielded fragments D and E (Fig. 8), indicating that they have similar structures. DISCUSSION Human fibrinogen has been expressed in a number of different recombinant systems (8 -11). Although these procedures usually only produce small amounts of secreted fibrinogen they can be scaled up, using cells in suspension and roller bottles, to yield sufficient quantities to study structure/function relationships. The yeast system offers an advantage in that it is more easily adaptable to express and secrete milligram quantities of fibrinogen. The fibrinogen expressed in yeast is biologically active in that it forms a thrombin-induced clot and undergoes factor XIIIa cross-linking. Carbohydrate processing, composition, and sequence was determined by several different methods. Periodic acid-Schiff staining of the separated chains, treatment of transformed yeast cells with tunicamycin, and endoglycosidase H digestion of intracellular and secreted fibrinogen showed that only B␤ and ␥ chains are glycosylated. In addition, tunicamycin and endoglycosidase H treatment suggest that initial N-linked glycosylation of recombinant fibrinogen occurs in a manner similar to that in hepatocytes. Tunicamycin treatment only affected the processing of B␤ and ␥ chains, and digestion with endoglycosidase H indicated that mannose-rich fibrinogen precursors are present in the ER and are processed before secretion occurs. Carbohydrate analysis demonstrated that recombinant fibrinogen, unlike plasma fibrinogen, does not contain terminal sialic acid but otherwise may be similar in composition and sequence to plasma fibrinogen (30). These results are in keeping with the synthesis of N-linked glycans by yeast. The early stages of N-glycosylation in yeast and animal systems are similar, but further oligosaccharide processing, which occurs in the Golgi, differs. In yeast, mannose-rich oligosaccharides are usually formed, although galactose and N-acetylglucosamine residues may be added (31).
Our studies indicate that recombinant yeast fibrinogen is not of the high mannose variety and is similar but not identical to that of plasma fibrinogen, since it lacks terminal sialic acid.
Biological activity of recombinant fibrinogen was shown by its ability to form a thrombin-induced clot and to undergo factor XIIIa-catalyzed cross-linking of fibrin chains. In addition the response of recombinant fibrinogen to thrombin and factor Xllla shows that recombinant fibrinogen has a structure similar to that of plasma fibrinogen. Cleavage of fibrinopeptides A and B by thrombin, polymerization, and correct alignment of ␣ and ␥ chains for participation in factor XIIIa-catalyzed crosslinking, requires proper chain assembly and folding. Further evidence that recombinant fibrinogen has a structure similar to plasma fibrinogen was obtained by determining that fragments D and E are produced when recombinant fibrinogen is digested with plasmin. Fragments D and E are characteristic products when fibrin(ogen) is treated with plasmin and can only be obtained if the fibrinogen chains are organized in the correct configuration.
Fibrinogen chains are assembled in a series of stepwise reactions in which single chains are linked into two-chain complexes, followed by the addition of a third chain to form halfmolecules, which are subsequently joined to produce dimeric fibrinogen (19 -21, 32-34). In the yeast recombinant system, the same intermediates that accumulate in HepG2 cells are noted. Free A␣, B␤, and ␥ chains, two-chain complexes (A␣⅐␥ and B␤⅐␥), and half-molecules as well as dimeric fibrinogen accumulated in transformed yeast cells. This suggests that the sequence of chain assembly in yeast is similar to that in mammalian cells and that this recombinant system will be useful for studying the mechanisms of fibrinogen chain assembly and folding. Folding and assembly probably involve several chaperones present in the endoplasmic reticulum, and the yeast system allows the use of a genetic approach to studying this process. Yeast mutants, defective in secretory factors or chaperones, can be prepared and used to analyze chain assembly, folding, and secretion (35)(36)(37)(38)(39)(40).
Knowledge gained from congenital dysfibrinogens, from analyzing evolutionary conserved domains, and from biochemical and structural determinations has led to assignment of specific domains as important in the functional properties of fibrinogen. However, many of these assignments were reached by inference and have not been unambiguously elucidated. It is obvious that the recombinant systems provide the opportunity to mutate specific domains and study functional modifications. The yeast system should prove useful in this regard since it produces relatively large amounts of secreted fibrinogen that is biologically active. Purified fibrinogen from yeast culture medium was digested with plasmin. The digested material was divided into two parts. The first part was adsorbed with antibody to fragment D, and the second part was adsorbed with antibody to fragment E coupled to Agarose columns. The bound materials from these two affinity columns were eluted and run on SDS-PAGE followed by Coomassie Blue staining and Western blot analyses. Panel A, protein stain; panel B, immunoblot reacted with anti-fragment D; panel C, immunoblot reacted with anti-fragment E. Lane 1, plasmin digest of yeast fibrinogen; lane 2, material absorbed by anti-fragment D; lane 3, material absorbed by anti-fragment E.