fibrin formation (crosslinked fibrin/thrombin/fibrinogen/fibrinogen -fibrin degradation products/clot formation)

Transcription

1 Proc. Natl. Acad. Sci. USA Vol. 77, No. 3, pp , March 1980 Biochemistry Evidence for four different polymerization sites involved in human fibrin formation (crosslinked fibrin/thrombin/fibrinogen/fibrinogen -fibrin degradation products/clot formation) STEPHANIE A. OLEXA AND ANDREI Z. BUDZYNSKI Specialized Center of Research in Thrombosis and Department of Biochemistry, Temple University Health Sciences Center, Philadelphia, Pennsylvania Communicated by Sidney Weinhouse, December 13, 1979 ABSTRACT The mechanism of association and the organization of human fibrin were studied by using affinity chromatography. Insolubilized fibrinogen, fibrin monomer, and crosslinked fibrin were used to localize the binding sites on fibrinogen and fibrin derivatives. Four different polymerization sites have been distinguished. A binding site ("a"), available without thrombin action, is present on the fibrinogen fragment D domain. The complementary ("A") is inoperative in fibrinogen and requires thrombin for activation; it is located on the fibrinogen NH2-terminal domain. A third polymerization site ("b") appears to be formed by the alignment of the fragment D domains on two fibrin monomer molecules upon polymerization; this site functions without thrombin mediation and the alignment is stabilized by the Factor XIIIa-catalyzed crosslink bonds. The "b" site is complementary to another thrombinactivated site ("B") on the fibrinogen NHllterminal domain. The two thrombin activable sites, "A" and "B", are distinguishable, although they are located in the same fibrinogen domain. Activation of fibrinogen by thrombin causes the release of fibrinopeptides A and B, followed by the formation of an ordered fibrin polymer, probably due to the association of complementary binding sites (1). Recent investigations have focused on defining areas of the fibrin monomer molecule that participate in polymerization by identifying binding properties of degradation products of fibrinogen or fibrin. Binding sites have been localized on fragment D* and in the NH2-terminal domain of fibrin (2-5). It was suggested that the COOH terminus of the fragment D y-chain remnant contains a binding region (2). The localization of the NH2-terminal binding site is being actively pursued and questions about whether the site is monoor bivalent and about the role of the fibrinopeptides are unresolved. Accumulating data support a hypothesis that the removal of fibrinopeptides A and B activates separate binding sites on the NH2-terminal region of the fibrinogen molecule (6-9). These sites are not equivalent because the site revealed by the loss of fibrinopeptide A is sufficient to induce polymerization of fibrin but the site activated by the loss of fibrinopeptide B will not initiate clotting under physiological conditions. We have shown that the binding sites on the NH2-terminal and fragment D domains of the fibrinogen molecule can be examined in a soluble system (10). Binding sites localized on fragments E1 and E2t were complementary to those on fragment DD*, but did not bind to fragment D from fibrinogen or from noncrosslinked fibrin. This indicated that fragments E1 and E2 contained sites specifically complementary to the aligned domains of the two fragment D moieties in fragment DD. Fragment DD also complexed with fibrinogen, fragments The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C solely to indicate this fact. X and Y, and NH2-terminal disulfide knot (NDSK) after these species were treated with thrombin (10). The purpose of the present work was to investigate the interactions of fibrinogen, fibrin, and their derivatives with short fibrin oligomers. The steric alignment of the fibrin monomer molecules appears to be an important event in the generation of a new binding site. The aligned fibrin monomers were covalently stabilized by Factor XIIIa-induced crosslinking. The prepared insolubilized crosslinked fibrin oligomers were utilized in conjunction with insolubilized fibrinogen and fibrin monomer to examine the availability of binding sites on fibrinogen and fibrin derivatives, and were used to localize and characterize fibrin polymerization sites. MATERIALS AND METHODS Reagents and Biological Preparations. Fibrinogen, grade L, was from A. B. Kabi, Stockholm, Sweden; CNBr-Sepharose 4B and Sepharose CL-6B were from Pharmacia. Human thrombin, H-i, was kindly provided by David L. Aronson, Bureau of Biologics, Food and Drug Administration. Trasylol (Aprotinin) was from Mobay Chemical (New York). Hirudin was from Calbiochem, and DE-52 cellulose was from Whatman. Pevikon C-879 was from Mercer (New York). Preparation of Insolubilized Fibrinogen. Coupling of fibrinogen to cyanogen bromide-activated Sepharose 4B (Seph-Fbg) and its conversion to insolubilized fibrin monomer (Seph-FM) was carried out according to the method of Heene and Matthias (11). Binding efficiency was approximately 80% and approximately 200 mg of protein was bound per ml of packed resin. Crosslinking of Insolubilized Fibrin Monomers. Fibrinogen (200 mg) was dialyzed against 0.05 M Tris-HCl, ph 7.6/0.1 M NaCl/25 mm CaC12/25 units of trasylol per ml, mixed with 1 ml of packed Seph-FM resin containing approximately 200 mg of bound fibrin monomer, and stirred gently at room temperature for 3 hr. The resin was washed with the same buffer until all unbound fibrinogen was eluted and then it was mixed with 1374 Abbreviations: NDSK, NH2-terminal disulfide knot; Seph-Fbg, Seph-FM, and Seph-XF denote fibrinogen, fibrin monomer, and crosslinked fibrin oligomers, respectively, bound covalently to Sepharose. * Fragments Di (stage 2) and D3 (stage 3) are sequential degradation products from the COOH-terminal region of fibrinogen or noncrosslinked fibrin of MrS 103,500 and 86,500, respectively. Fragment DD is a plasmic degradation product of crosslinked fibrin, consisting of two fragment D1 moieties joined in the y chains by Factor XIIIa-induced crosslink bonds (Mr 190,000). t Fragments El, E2, and E,3 from crosslinked fibrin are of Mr 60,000, 55,000, and 50,000, respectively. Fragments El and E2 can bind to fragment DD forming a (DD)E complex. Fragment E species from a fibrinogen digest resemble fragment E3 in Mr. charge, and the lack of ability to bind to fragment DD.

2 Biochemistry: Olexa and Budzynski 1,ul of 2-mercaptoethanol, 100 units of thrombin, CaCl2 (10 mm final concentration), and 0.5 mg of human Factor XIII (550 units/mg of protein) purified according to the method of Loewy and colleagues (12). The mixture was incubated for 3 hr at room temperature with gentle stirring. The resin (Seph- XF) was packed into a column and rinsed alternately with 25 ml of 0.05 M Tris-HCl, ph 7.6/0.1 M NaCl/25 units of trasylol per ml (buffer 1) and 25 ml of 0.05 M Tris-HCl, ph 4.1/1.0 M NaCl/6 M urea/25 units of trasylol per ml (buffer 2), repeating the cycle three times. Approximately 0.1 ml of each of the three resins (Seph-Fbg, Seph-FM, and Seph-XF) was reduced in 1.0 ml of solution containing 9 M urea, 3% sodium dodecyl sulfate, and 3% 2-mercaptoethanol and electrophoresed on sodium dodecyl sulfate/ 7% polyacrylamide gels under reducing conditions. Binding Experiments. Species to be tested were dialyzed in buffer 1, after which the protein concentration was adjusted to 25 mg/ml. The protein (5 mg; 0.2 ml) was applied to an affinity column (0.6 X 3 cm) containing 0.5 ml of packed resin that had been equilibrated in buffer 1. Samples (1 ml) were collected (flow rate, 20 ml/hr) until the A2.w3 was less than Bound protein was eluted with buffer 2. Fractions eluted with buffers 1 and 2 were separately pooled, concentrated to 1.0 ml, and dialyzed against the buffer, and the protein concentration was determined. Recovery of protein was %. The conditions for affinity chromatography were kept constant for all three resins. Binding of proteins to resins in batch was done by incubating 0.5 ml of packed resin with 100 mg of the protein in 10 ml of buffer 1 at room temperature for 3 hr with gentle agitation. The resin was then poured onto a column and rinsed with buffers 1 and 2 as outlined. Purification of Degradation Products from Fibrinogen and Fibrin. Plasmic digests of fibrinogen and noncrosslinked fibrin were labeled as stage 1, stage 2, or stage 3 according to the content of specific degradation products (13) as determined by polyacrylamide gel electrophoresis. Fragment X from stage 1 and stage 2 digests of fibrinogen and fragment Y from the latter digest were purified according to the method of Marder and colleagues (13). Fragments D(stage 2), E(stage 2), D(stage 3), and E(stage 3) were obtained from stage 2 and stage 3 digests of fibrinogen or noncrosslinked fibrin by preparative electrophoresis on a Pevikon block (14). NDSK was isolated from cyanogen bromide-degraded fibrinogen by a modification of the method of Blomback and colleagues (10, 15). Fragment A (16) was purified by column gel filtration on Sepharose CL-6B (17). Fragment DD, the (DD)E complex, and a polymer remnants were isolated by gel filtration on Sepharose CL-6B (18, 19). Fragments El, E2, and E3 were isolated by a combination of column gel filtration on Sepharose CL-6B and ion exchange chromatography on DE-52 cellulose (10). Thrombin treatment of fragments was carried out (5), after which the thrombin was inhibited by the addition of 100 antithrombin units of hirudin per unit of thrombin prior to binding studies. Binding of thrombin-treated fragments to Seph-Fbg was done in the presence of 25 antithrombin units/ml of hirudin. Polyacrylamide gel electrophoresis was done on 7% polyacrylamide gels (0.5 X 9 cm) containing 0.1% sodium dodecyl sulfate (20) and on Tris glycine (9%) gels (21). Staining was done with Coomassie brilliant blue and gels were scanned with a densitometer (Densicord 552, Photovolt, New York). Quantitative protein determination was carried out by direct ultraviolet spectrophotometry at 280 nm (absorption coefficients at 1 mg/ml; fibrinogen, 1.5; fragment D and DD, 2.0; fragment E, 1.2) or by spectrophotometry at 630 nm of trichloroacetic acid-precipitated, amido black-stained protein Proc. Natl. Acad. Sci. USA 77 (1980) 1375 (22). Molar concentrations were calculated from the protein concentration and the molecular weights: fibrinogen, 340,000; fragment X, 250,000; fragment Y, 150,000; fragments D, 103,500 (stage 2) and 86,500 (stage 3); fragment A, 21,000; NDSK, 58,000; fragment DD, 190,000; (DD)E, 250,000; and fragments E1, E2, and E3, 60,000, 55,000 and 50,000, respectively. RESULTS Insolubilized Crosslinked Fibrin (Seph-XF). Seph-FM incubated in batch with 100 mg of fibrinogen adsorbed 17 mg of fibrinogen per 100 mg of fibrin monomer attached to the Sepharose. The binding of fibrinogen to the insolubilized fibrin monomer presumably occurs by a mechanism similar to that responsible for the formation of fibrinogen-fibrin monomer complexes in solution. This result indicated that 83% of the insolubilized fibrin monomer molecules was probably functionally unreactive. After removal of nonadsorbed fibrinogen and incubation with factor XIII, thrombin and CaC12, some of the y chains formed dimers. Fig. 1 shows that, after reduction of Seph-Fbg and Seph-FM, free : and By polypeptide chains were present whereas a majority of the a chain was bound to the resin and did not enter the gel. Seph-XF contained in addition to these chains y-chain dimer. Because the Ayy crosslink bond occurs intermolecularly (23) and only after the proper alignment of fibrin monomer molecules (24), the Seph-XF preparation must contain species arranged as in a crosslinked fibrin clot. A number of derivatives can be formed after the crosslinking reaction of Seph-FM loaded with fibrinogen. In the original Seph-FM pool, 17% of the attached material, rather than 100%, was functionally active; however, after incubation with fibrinogen, probably no free active fibrin monomer was available. An insolubilized fibrin dimer was probably formed but could be further crosslinked to similar species. Therefore, the Seph-XF preparation probably contained a majority of unreactive insolubilized fibrin monomer and some species of either fibrin dimers, trimers, or tetramers. To differentiate among the latter possibilities, the ratio of the Fyy to y chains was cal- -yy a, atop U, C co.0 CD CU Seph-Fbg Seph-FM Seph-XF Gel length FIG. 1. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of reduced Seph-Fbg, Seph-FM, and Seph-XF. The presence of -yy chains in Seph-XF indicates that this material contains structures resembling crosslinked fibrin.

3 1376 Biochemistry: Olexa and Budzynski culated from the formula of each and multiplied by the fraction of y chains involved in a complex between Seph-FM and fibrinogen. The product gave the actual experimental ratio of,yy per total y chains. For example, if fibrin dimers would represent the predominant crosslinked species, the experimental ratio of yy chains per total By chains should be 0.145; for trimers the ratio would be and for tetramers, Two batches of Seph-XF were reduced and tested on sodium dodecyl sulfate/7% polyacrylamide gels under reducing conditions. The ratio of dimer y chains to total y chains was determined by scanning of gels and was found to be i (SD) This indicates that the Seph-XF preparation contains reactive species having an average of 4.6 fibrin monomer molecules per crosslinked oligomer. Affinity Chromatography on Seph-Fbg, Seph-FM, and Seph-XF. Fibrinogen and fibrin derivatives were tested for binding on the three affinity resins. The amount of protein bound was quantitated and expressed in nanomoles. Each derivative was tested in duplicate on two preparations of each resin. Table 1 contains the mean ± SD of the results. In order to standardize the experimental conditions, sample volume, column size, and flow rate were kept constant. There was no loss of binding capacity of the resins after repeated usage. Fibrinogen did not bind to Seph-Fbg or Seph-XF either on columns or in batch, but was bound to Seph-FM (Table 1, line 1). Fragments X (stage 2) and Y and NDSK bound to Seph-Fbg but only after the fragments were thrombin treated (lines 3-6, 16, and 17). Therefore, thrombin activated or uncovered binding sites on the NH2-terminal region of these molecules that are complementary to sites on intact fibrinogen. Thrombin treatment of fragment X (stage 2) increased the binding of this fragment to Seph-FM by about 9%; however, binding to Seph-XF was increased by 36% (lines 3 and 4). Likewise, there was a 20% increase in binding of fragment Y to Seph-FM and an 85% increase in binding to Seph-XF after fragment Y was treated with thrombin (lines 5 and 6). After NDSK was incubated with thrombin the binding to Seph-FM and to Seph-XF Proc. Natl. Acad. Sci. USA 77 (1980) was increased 14-fold (lines 16 and 17). These data indicate that fragments X and Y and NDSK contain two types of sites, one that is available without thrombin action and one that is revealed by this enzyme. Because the total bound protein and the increase in binding after thrombin treatment was significantly greater for NDSK than for fragments X or Y, the thrombindependent sites probably account for a larger proportion of the affinity of NDSK to the insolubilized proteins than of the other fragments. Similarly, after thrombin action the increase in binding of fragments X and Y to Seph-XF was significantly greater than the increase in binding to Seph-FM. This suggested that thrombin-activated binding sites must play a relatively more important role in the binding to crosslinked fibrin than in the binding to single fibrin monomer molecules. Fragment D from fibrinogen or noncrosslinked fibrin and fragment DD from crosslinked fibrin did not bind to Seph-Fbg but did bind to Seph-FM (Table 1, lines 7-10 and 19). These species apparently bound to the thrombin-activated sites on the NH2-terminal region of fibrin monomer. Fragment D1 (stage 2) bound to a greater extent with Seph-FM than with Seph-XF. Fragment D1 (stage 2) of Mr 103,500 had significantly higher binding than that of fragment D3 (stage 3). The latter preparation was of Mr 86,500, suggesting that plasmic degradation of fragment D removed or modified the binding sites (2). There was no evidence for a thrombin-activated site on fragment D. Fragments E isolated from either stage 2 or stage 3 digest of fibrinogen did not bind to any of the insolubilized proteins; a lack of binding was also found after treatment with thrombin (Table 1, lines 11-14). In contrast to this result, fragments E1 and E2t purified from a plasmic digest of crosslinked fibrin bound to Seph-XF but did not bind to Seph-Fbg or Seph-FM (Table 1, lines 20-21). The amount of fragments E1 and E2 bound to Seph-XF was on the molar basis the highest of all derivatives and amounted to 20 and 16 nmol, respectively. Only 11.9 nmol of thrombin-treated NDSK was bound under the same conditions. Fragment E3 from a crosslinked fibrin digest Table 1. Binding of fibrinogen and fibrin derivatives to Seph-Fbg, Seph-FM, and Seph-XF Protein bound to Seph-Fbg Seph-FM Seph-XF Derivative Source Treatment mg nmol mg nmol mg nmol 1. Fbg Fragment X (st. 1) Fbg ± Fragment X (st. 2) Fbg Fragment X (st. 2) Fbg T Fragment Y Fbg Fragment Y Fbg T 0.75 ± Fragment D1 (st. 2) Fbg ± Fragment D1 (st. 2) Fbg T Fragment D3 (st. 3) Fbg Fragment D3 (st. 3) Fbg T Fragment E (st. 2) Fbg FragmentE (st. 2) Fbg T FragmentE (st. 3) Fbg FragmentE (st. 3) Fbg T Fragment A Fbg NDSK Fbg NDSK Fbg T (DD)E XF Fragment DD XF ± FragmentEl XF Fragment E2 XF Fragment E3 XF a-chain polymer remnants XF Fbg, fibrinogen; XF, crosslinked fibrin; T, thrombin; st., stage.

4 Biochemistry: Olexa and Budzynski did not have binding capacity similar to that of fragments E from a fibrinogen digest. This would imply that the plasmic cleavage of fragment E2 to E3 removes or alters polymerization binding sites. The (DD)E complex, which contains fragment El or E2 as the E moiety, did not bind with any resin, indicating that the binding sites on both E and DD moieties appeared to be mutually saturated in the complex. Fragment A and remnants of a chain polymers did not bind with fibrinogen or with fibrin derivatives. DISCUSSION Two processes may be involved in fibrin network formation: a linear end-to-end association of fibrin monomer molecules and a lateral side-to-side aggregation of strands (25). It has been speculated (26) that the two processes could be regulated by the differential release rates of fibrinopeptides A and B by thrombin. Recent reports favor this concept (6-9) and postulate that two sets of binding sites may be engaged to direct the linear and lateral association of the monomers, respectively. The data presented in Table 1 with Seph-Fbg and Seph-FM further support this concept and demonstrate the presence of polymerization sites that are complementary to each other. One binding site is associated with the fragment D moiety present in fragments derived from the COOH terminus of fibrinogen (27, 28). The binding site, called "a" in Fig. 2, is unaffected by thrombin and is exposed in fibrinogen, as shown by the binding of fibrinogen to Seph-FM as well as in several studies using different systems (2-5, 10). The complementary binding site marked "A" in Fig. 2 is not available on fibrinogen and its derivatives containing the NH2-terminal region of the parent molecule, but it is exposed or activated by thrombin. The conclusion that thrombin activates a polymerization site in the NH2-terminal region of fibrinogen corroborates observations in other investigations (2-10, 33). Thrombin-treated NDSK bound to Seph-Fbg, Seph-FM, and Seph-XF, confirming the presence of a binding site on the NH2-terminal region. Before thrombin treatment there was some binding of NDSK to the resins, suggesting the presence of another binding site either specific or exposed after denaturation with formic acid used for the preparation of NDSK (15). Our earlier observation on the binding properties of fragments E1 and E2 isolated from a plasmic digest of human crosslinked fibrin (10) demonstrated their affinity for fragment DD but not for fragments D. This binding difference suggested that crosslinked fibrin may carry additional affinitive regions. In order to test this hypothesis a short oligomer, consisting of approximately four Factor XIIIa-crosslinked fibrin monomer units attached covalently to Sepharose (Fig. 1), has been synthesized. This material provided a matrix resembling a strand of crosslinked fibrin and as such offered an experimental system enabling binding studies of fibrinogen and fibrin derivatives to an early antecedent of a fibrin clot. Blomback and colleagues (9) proposed that, in addition to the primary polymerization site revealed by the release of fibrinopeptide A, the release of fibrinopeptide B activates a second polymerization domain that interacts with another site in a neighboring molecule. These authors inferred that linear polymerization takes place through two sets of complementary binding sites. Experimental basis for this is a delay of fibrinogen Detroit (34) polymerization until removal of approximately 10% of fibrinopeptide B. The data in the present work indicate that the second set of polymerization sites "B" and "b", the former perhaps revealed by the loss of fibrinopeptide B, are not responsible for end-to-end alignment of fibrin monomer molecules but contribute to the lateral aggregation of fibrin strands (Fig. 2). Fi bri nogen Proc. Natl. Acad. Sci. USA 77 (1980) 1377 Thrombin Linear fibrin polymer It FE Fibrin monomer Lateral fibrin polymer FIG. 2. Model for human fibrin polymerization. Fibrinogen is a bivalent molecule depicted as a flexible banana model (29, 30) with an available polymerization site on the fragment D domain of the molecule "a". Upon cleavage of fibrinopeptides A and B by thrombin, fibrin monomer is formed and a three-nodule form (31, 32) is accentuated in this figure. By this reaction two sets of binding sites ("A" and "B") on the NH2-terminal domain of the molecule become available. Polymerization sites depicted by broken letters are on the opposite side of the molecule from sites referred to by solid letters. The "A" sites are complementary to the "a" sites on the fragment D domain. The binding of these sites induces linear polymerization of the molecules. The linear polymerization of fibrin monomers results in the formation of a new bivalent polymerization site "bb." The crosslink bonds between two y chains of the neighboring fibrin monomer molecules either may participate in the binding site or may stabilize the sites on aligned fibrin monomer molecules. The "b" site is complementary to the thrombin-activated site on the NH2-terminal domain of fibrin monomer "B." The two "B" sites on the second (upper) layer of fibrin bind to the "bb" sites on the first (lower, shaded) layer. Meanwhile, the alignment of the fibrin monomer molecules on the second (upper) layer results in the formation of "bb" binding sites that will enable the addition of a third layer of fibrin. Therefore, the binding of "A" to "a" sites promotes linear polymerization of the fibrin monomer molecules as well as fibrin strand branching, whereas the binding of "BB" to "bb" sites allows lateral aggregation. The interaction of these four sites represents a major mechanism for fibrin polymerization; however, other interactions such as those responsible for the ordered precipitation of fibrinogen by protamine sulfate may also play a role. Affinity chromatography on Seph-XF (Table 1) gave the essential information about the additional fibrin polymerization sites. The binding of fragments E1 and E2 to Seph-XF but not to Seph-Fbg or Seph-FM suggested that the fragments E contain binding sites ("B" in Fig. 2) complementary to a site on polymerized fibrin that is not available on fibrinogen or fibrin monomer. This site ("b" in Fig. 2) is also operative in fragment DD because fragments E1 and E2 bound in solution with fragment DD but not with fragment D (10). The binding site "b" may be at or near the Factor XIIIa-induced crosslink bonds or these covalent bonds may align and stabilize the sites on the two fragment D moieties. Because fragments E1 or E2 bound only to the aligned fragment D domains, but not to single fragment D molecules, this suggests that there is an order of association of the polymerization sites. The binding between sites "A" and "a" would occur as soon as thrombin exposes site "A" (Fig. 2). However, the association between sites "B" and "b" cannot occur even after the cleavage of both fibrinopeptides. The critical step is the alignment of the fragment D domains in the fibrin fiber, which generates "b" binding sites.

5 1378 Biochemistry: Olexa and Budzynski The data presented in this work indicate that there are four different binding sites involved in fibrin polymerization and that there is an order of association of these sites (Fig. 2). First, a thrombin-activated site "A" is located on the NH2-terminal region of fibrinogen, exemplified by the binding of thrombin-treated fragments X and Y and NDSK to Seph-Fbg (Table 1). Second, there is a site "a" on the COOH terminus of fibrinogen localized on the fragment D domain that is not affected by thrombin and is complementary to the "A" site, shown by the binding of fibrinogen fragments X, Y, and D to Seph-FM but not to Seph-Fbg (Table 1). Third, the site "b" specific to polymerized fibrin is formed by the aligned fragment D moieties, exemplified by the binding of Fragments E1 and E2 to Seph-XF but not to Seph-FM (Table 1). Fourth, there is a thrombin-activated site "B" on the NH2-terminal region localized on fragments E1 and E2 and NDSK that is complementary to the "b" site newly formed in polymerized fibrin. This latter site is also shown by the greater increase in binding of fragments X and Y to Seph-XF than to Seph-FM after thrombin treatment (Table 1). The sites "A" and "B" can be distinguished, although both are located on the NH2-terminal region of fibrinogen and both are thrombin activated. Thrombin-treated NDSK contains both "A" and "B" sites, because this molecule binds to Seph-FM and Seph-XF as well as forms a complex with fragment DD in solution (10). However, fragments E1 and E2 contain the "B" site whereas the "A" site is not available, perhaps due to proteolytic cleavages by plasmin, because these fragments cannot bind to Seph-Fbg or Seph-FM or form a complex in solution with fragment D. The sites "a" and "b" can be distinguished because "a" is present on the COOH-terminal region of fibrinogen in fragment D and "b" is present on the aligned fragment D regions of fibrin (fragment DD). These results support a model of fibrin polymerization (outlined in Fig. 2) that describes not only the linear elongation of fibrin strands but also the lateral aggregation of strands of fibrin. Because there are two different polymerization sites activated by thrombin, one probably involved in strand elongation and the other in lateral aggregation, it is possible that the former site is exposed by the removal of fibrinopeptide A and the latter, by removal of fibrinopeptide B as suggested by Blombick and colleagues (9). The slow rate of fibrinopeptide B removal by thrombin in the liquid phase would serve to ensure the orderly association of polymerization sites with predominant formation of linear polymers of fibrin under physiological conditions. 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