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Severely Impaired Polymerization of Recombinant Fibrinogen γ-364 Asp → His, the Substitution Discovered in a Heterozygous Individual

1997; Elsevier BV; Volume: 272; Issue: 47 Linguagem: Inglês

10.1074/jbc.272.47.29596

1083-351X

Nobuo Okumura, Oleg V. Gorkun, Susan T. Lord,

Blood properties and coagulation

During blood coagulation, soluble fibrinogen is converted to fibrin monomers that polymerize to form an insoluble clot. Polymerization has been described as a two-step process: the formation of double-stranded protofibrils and the subsequent lateral aggregation of protofibrils into fibers. Previous studies have shown that γ chain residues Tyr-363 and Asp-364 have a significant role in polymerization, most likely in protofibril formation. To better define the role of these residues, we synthesized three fibrinogens with single substitutions at these two positions: Tyr-363 → Ala, Asp-364 → Ala, and Asp-364 → His. We found that the release of fibrinopeptides A and B was the same for these variants and normal recombinant fibrinogen, showing that all variants had normal fibrin formation. In contrast, we found that polymerization was significantly delayed for both Ala variants and was almost nonexistent for the His variant. Clottability for the Ala variants was only slightly reduced, and fibrin gels were formed. Surprisingly, clottability of the His variant was substantially reduced, and fibrin gels were not formed. Our data suggest that both protofibril formation and lateral aggregation were altered by these substitutions, indicating that the C-terminal domain of the γ chain has a role in both polymerization steps. During blood coagulation, soluble fibrinogen is converted to fibrin monomers that polymerize to form an insoluble clot. Polymerization has been described as a two-step process: the formation of double-stranded protofibrils and the subsequent lateral aggregation of protofibrils into fibers. Previous studies have shown that γ chain residues Tyr-363 and Asp-364 have a significant role in polymerization, most likely in protofibril formation. To better define the role of these residues, we synthesized three fibrinogens with single substitutions at these two positions: Tyr-363 → Ala, Asp-364 → Ala, and Asp-364 → His. We found that the release of fibrinopeptides A and B was the same for these variants and normal recombinant fibrinogen, showing that all variants had normal fibrin formation. In contrast, we found that polymerization was significantly delayed for both Ala variants and was almost nonexistent for the His variant. Clottability for the Ala variants was only slightly reduced, and fibrin gels were formed. Surprisingly, clottability of the His variant was substantially reduced, and fibrin gels were not formed. Our data suggest that both protofibril formation and lateral aggregation were altered by these substitutions, indicating that the C-terminal domain of the γ chain has a role in both polymerization steps. Fibrinogen is a plasma glycoprotein composed of a pair of three polypeptide chains, Aα, Bβ, and γ. The six N termini form a central domain, called E, which can be isolated as a single fragment from a plasmin digest of fibrinogen. The six chains divide into two three-chain sets that emanate in opposite directions from the central E domain as coiled-coil rods that terminate with the C-terminal residues of each chain forming separate domains. The peripheral domains can also be isolated from plasmin digests as the D fragments, which contain residues from all three chains but consist primarily of the C-terminal domains of the Bβ and γ chains.During blood coagulation, fibrinogen is converted to an insoluble fibrin clot by the serine protease thrombin, which cleaves four peptide bonds, releasing two fibrinopeptides A (FpA, Aα 1–16) and two fibrinopeptides B (FpB, Bβ 1–14) and fibrin monomers that polymerize spontaneously. The association of fibrin monomers into a fibrin clot has long been described as a two-step process, where the first step involves half-staggered, end-to-end interactions leading to double-stranded protofibrils and the second step, usually called lateral aggregation, involves the assembly of protofibrils into thick, multi-stranded fibers that branch to form a fibrin network. The final product is a fibrin gel (1Blomback B. Hessel B. Hogg D. Therkildsen L. Nature. 1978; 275: 501-505Crossref PubMed Google Scholar, 2Olexa S.A. Budzynski A.Z. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 1374-1378Crossref PubMed Scopus (151) Google Scholar). The interactions that promote protofibril formation occur between N-terminal α chain residues in the E domain on one fibrin molecule and C-terminal γ chain residues in the D domain on a second fibrin molecule. The interactions that promote lateral aggregation are less well known, although it has been shown that FpB cleavage enhances lateral aggregation (1Blomback B. Hessel B. Hogg D. Therkildsen L. Nature. 1978; 275: 501-505Crossref PubMed Google Scholar, 3Weisel J.W. Veklich Y. Gorkun O. J. Mol. Biol. 1993; 232: 285-297Crossref PubMed Scopus (130) Google Scholar). The enhanced lateral aggregation may, however, be an indirect result of strengthened protofibril interactions that accompany FpB release (4Shainoff J.R. Dardik B.N. Ann. N. Y. Acad. Sci. 1983; 408: 254-268Crossref PubMed Scopus (58) Google Scholar). It has also been shown that the C-terminal domains of the α chain (the αC domains) participate in lateral aggregation and that calcium binding to fibrinogen increases during lateral aggregation (5Okada M. Blomback B. Thromb. Res. 1983; 29: 269-280Abstract Full Text PDF PubMed Scopus (54) Google Scholar, 6Carr M. Gabriel D. McDonagh J. Biochem. J. 1986; 239: 513-516Crossref PubMed Scopus (69) Google Scholar, 7Mihalyi E. Biochemistry. 1988; 27: 976-982Crossref PubMed Scopus (41) Google Scholar, 8Gorkun O.V. Veklich Y.I. Medved L.V. Henschen A.H. Weisel J.W. Biochemistry. 1994; 33: 6986-6997Crossref PubMed Scopus (170) Google Scholar, 9Weisel J.W. Nagaswami C. Biophys. J. 1992; 63: 111-128Abstract Full Text PDF PubMed Scopus (285) Google Scholar, 10Cierniewski C.S. Budzynski A.Z. Biochemistry. 1992; 31: 4248-4253Crossref PubMed Scopus (42) Google Scholar, 11Hasegawa N. Sasaki S. Thromb. Res. 1990; 57: 183-195Abstract Full Text PDF PubMed Scopus (49) Google Scholar).Recent experiments have identified two specific residues in the D domain of the γ chain as participants in protofibril formation. Tyr-363 in the γ chain was identified by photoaffinity labeling with a peptide that mimics the N-terminal α chain residues of the E domain of fibrin (12Yamazumi K. Doolittle R.F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2893-2896Crossref PubMed Scopus (58) Google Scholar). Asp-364 in the γ chain was identified in fibrinogen Matsumoto I (13Okumura N. Furihata K. Terasawa F. Nakagoshi R. Ueno I. Katsuyama T. Thromb. Haemostasis. 1996; 75: 887-891Crossref PubMed Scopus (49) Google Scholar), a dysfibrinogen that was found in a heterozygous individual. Fibrinogen Matsumoto I is a mixture of molecules with normal and variant γ chains that have His at position 364. Polymerization of fibrinogen Matsumoto I is markedly delayed, and this delay can be partially compensated by mixing with normal fibrinogen (13Okumura N. Furihata K. Terasawa F. Nakagoshi R. Ueno I. Katsuyama T. Thromb. Haemostasis. 1996; 75: 887-891Crossref PubMed Scopus (49) Google Scholar). The results suggest that the normal molecules of this fibrinogen support polymerization, whereas the variant molecules do not.In the studies described, here we examined the roles of these two residues by analysis of genetically engineered variants. Using the previously described two-step procedure for synthesis of variant fibrinogens, we synthesized three variants with single amino acid substitutions in the γ chain: Tyr-363 → Ala (Y363A), Asp-364 → Ala (D364A), and Asp-364 → His (D364H). We determined the clottability of these variants and followed the kinetics of thrombin-catalyzed fibrinopeptide release and thrombin-catalyzed polymerization. We compared the variants to one another and to normal recombinant fibrinogen, which has polymerization characteristics comparable to normal plasma fibrinogen (14Gorkun O.V. Veklich Yu.I. Weisel J.W. Lord S.T. Blood. 1997; 89: 4407-4414Crossref PubMed Google Scholar).DISCUSSIONThe data presented here confirmed that the C-terminal γ chain residues 363 and 364 are important participants in polymerization. We found that Ala substitutions at either position 363 or 364 dramatically delayed polymerization. We measured turbidity, which with normal fibrin polymerization gauges the rate of protofibril formation from the lag period, and the rate of fiber formation from the slope of the turbidity change. With both Ala substitutions, the lag periods were lengthened and the slopes were less steep. The 363 Ala substitution was the most normal, and even with this mutant the shortest lag period and the most rapid increase in turbidity were about 3-fold delayed relative to normal fibrinogen under the most favorable conditions. These results suggest that both protofibril formation and fiber formation were affected by the single Ala substitutions. Alternatively, the Ala substitutions may change only one of the polymerization steps and still alter both the lag period and the slope. This possibility has been carefully described by Weisel and Nagaswami (9Weisel J.W. Nagaswami C. Biophys. J. 1992; 63: 111-128Abstract Full Text PDF PubMed Scopus (285) Google Scholar), who showed with simulated data that changing the rate of protofibril formation or the rate of lateral aggregation can change both the lag and the slope of a turbidity curve. For example, if the rate of protofibril formation is slowed, then the lag period is lengthened, and the slope of the turbidity increase is decreased. Similarly, if the rate of fiber formation is slowed, then normal protofibrils form in the normal time frame, but the lateral aggregation of these protofibrils is less efficient, which is seen as both a longer lag period and a reduced slope. Thus, further studies using assays that distinguish these two steps are needed to determine whether one or both steps of polymerization is impaired in these Ala variants. Even though polymerization was delayed with both of the Ala variants, the clottability data and clot appearance, which was similar to the normal clot, indicate that large, insoluble polymers were finally assembled from these molecules.The turbidity curves obtained with the Asp-364 to His substitution were more remarkable. We were able to measure changes in turbidity only at 0.45 mg/ml fibrinogen. At lower fibrinogen concentrations (0.09 mg/ml), where normal polymerization is complete within a few hours, polymers with D364H were not detected even after days of incubation. A delay in D364H fibrin polymerization was predicted from studies with fibrinogen Matsumoto I, which contains normal and variant γ chains, but the striking loss of function found with the homogeneous recombinant variant was unanticipated. The clottability data showed that normal polymers were not formed; no fibrin gel was seen and only half the protein was incorporated into precipitated material. This result was observed even though fibrinopeptide release indicated normal fibrin formation. Analysis of the nonclottable protein suggests that the soluble fibrin molecules were degraded, probably by prolonged exposure to thrombin. Thrombin cleavage of the C-terminal region of α chains has been previously reported (25Yoshida N. Wada H. Morita K. Hirata H. Matsuda M. Yamazumi K. Asakura S. Shirakawa S. Blood. 1991; 77: 1958-1963Crossref PubMed Google Scholar). This extensive proteolysis most likely occurred because the fibrin monomers were not readily incorporated into fibrin polymers, but the proteolysis may subsequently have contributed to the lowered clottability.The normal kinetics for FpB release from these three variants, which show delayed polymerization, was unanticipated. It is generally accepted that the conversion of fibrinogen to fibrin proceeds in an ordered fashion such that FpA is released, desA-fibrin polymers are formed, and FpB is released from these polymers (7Mihalyi E. Biochemistry. 1988; 27: 976-982Crossref PubMed Scopus (41) Google Scholar, 21Higgins D.L. Lewis S.D. Shafer J.A. J. Biol. Chem. 1983; 258: 9276-9282Abstract Full Text PDF PubMed Google Scholar). Consequently, the kinetics of thrombin-catalyzed FpB release depend on polymerization, such that FpB release is delayed when fibrin polymerization is limited. For example, both EDTA and the peptide Gly-Pro-Arg-Pro inhibit polymerization, and both decrease the rate of FpB release from normal fibrinogen (21Higgins D.L. Lewis S.D. Shafer J.A. J. Biol. Chem. 1983; 258: 9276-9282Abstract Full Text PDF PubMed Google Scholar, 26Ruf W. Bender A. Lane D.A. Preissner K.T. Selmayr E. Muller-Berghaus G. Biochim. Biophys. Acta. 1988; 965: 169-175Crossref PubMed Scopus (26) Google Scholar, 27Lewis S.D. Shields P.P. Shafer J.A. J. Biol. Chem. 1985; 260: 10192-10199Abstract Full Text PDF PubMed Google Scholar). Further, two abnormal fibrinogen variants, London I and Ashford, show impaired polymerization of fibrin monomers and exhibit a decreased rate of FpB release. Thus, the normal kinetics for FpB release from the variants studied here (Table I) indicate that an intermolecular structure, either the normal D:E structure found in protofibrils or something that mimics the function of the protofibril structure, was formed at a normal rate. This result is surprising because the residues γ363 and γ364 are thought to support D:E interactions, such that a significant change at these residues would be reflected in the rate of protofibril formation. Our results showed that changes at residues γ363 and γ364 did in fact significantly alter polymerization, so we anticipated a change in D:E interactions and the consequential change in FpB kinetics. The observed normal release of FpB therefore implies that intermolecular structures are formed at a normal rate. Of course, our data are also consistent with the conclusion that these intermolecular structures are not necessary for normal FpB kinetics, but this would be contrary to previous reports.Our results are similar to those seen with several dysfibrinogens with known changes in the C-terminal domain of the γ chain, impaired polymerization associated with normal rate of FpB release (28Ebert R.F. Index of Variant Human Fibrinogens 1994 Edition. CRC Press, Inc., Boca Raton, FL1994Google Scholar). The kinetics of FpB release from these dysfibrinogens must be considered with caution, however, as these variants, like Matsumoto I, were identified in individuals with heterozygous genotypes. Kinetic analysis of a mixture of normal and variant proteins is ambiguous, especially at concentrations of fibrinogen and thrombin that are higher than those used to obtain the data in Table I. Nevertheless, the reports on these dysfibrinogens lend support to the possibility that changes in the C-terminal domain of the γ chain impair polymerization but do not prevent intermolecular interactions needed to support normal FpB release.Recently, high resolution structures have been published for the isolated 30-kDa C-terminal fragment of the human fibrinogen γ chain alone and in a complex with the peptide GPRP (29Yee V.C. Pratt K.P. Cote H.C. Trong I.L. Chung D.W. Davie E.W. Stenkamp R.E. Teller D.C. Structure (Lond.). 1997; 5: 125-138Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 30Pratt K. Cote H. Chung D. Stenkamp R. Davie E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7176-7178Crossref PubMed Scopus (141) Google Scholar). As microcalorimetry data show that the C-terminal domain of the γ chain is folded independently (31Litvinovich S.V. Henschen A.H. Krieglstein K.G. Ingham K.C. Medved L.V. Eur. J. Biochem. 1995; 229: 605-614Crossref PubMed Google Scholar), it is likely that this isolated fragment, which was expressed in the yeast Pichia pastoris, preserves the structure it has in the fibrinogen molecule. These structures, therefore, allows us to speculate on the molecular basis for the functional changes we have observed. A deep pocket, called the polymerization pocket, is defined by four loops, P-1 through P-4. Tyr-363 and Asp-364 lie within loop P-3. As shown in Fig.4, the side chain for Tyr-363 faces into the pocket such that it can participate in D:E interactions. Thus, exchanging the large, aromatic, hydrogen bond-forming side chain of Tyr for the methyl group of Ala is likely to eliminate significant aspects of this intermolecular interaction and thereby abate polymerization. It is reasonable to assume that multiple D-domain residues participate in the D:E interactions, such that Tyr-363 is not the only critical residue. As shown in Fig. 2 and Table II, polymerization with the Y363A variant is abated but not eliminated, and fibrin clots do form. Nevertheless, the extensive delay in polymerization of Y363A demonstrates that this single residue has a consequential role in fibrin clot formation.As shown in Fig. 4, Asp-364 is oriented such that it forms a salt bridge with Arg-375 in loop P4. This electrostatic interaction could anchor the relative orientation of loops P-3 and P-4. As both loops contribute residues that line the polymerization pocket, it is reasonable to expect that changes in the orientation of these loops would alter polymerization. A critical role for Arg-375 is supported by the observation of abnormal fibrin polymerization with the substitution of Gly for Arg-375 in the heterozygous dysfibrinogen Osaka V. Furthermore, the structure obtained in the presence of GPRP showed that Asp-364 forms a salt bridge with the N terminus of the GPRP. Thus, Asp-364 likely participates in two ways: forming an intermolecular salt bridge that is critical to fibrinogen structure and supporting the intermolecular D:E interactions that are critical to fibrin polymerization. Therefore, the substitution of Ala for Asp would be expected to significantly delay polymerization. Our data are also consistent with the recent report by Cote et al. (32Cote H. Pratt K. Chung D. Davie E. Thromb. Haemostasis. 1997; 78: 757Google Scholar) who synthesized and characterized the 30-kDa recombinant fragment with the D364A substitution. They found that the peptide GPRP did not bind to this variant and that this variant fragment was unable to inhibit polymerization of normal fibrin.Interpretation of the results from the substitution of His for Asp-364 is more difficult, because the changes in function were so dramatic. In contrast to the two Ala substitutions, this mutant did not form a fibrin gel under the conditions described here. After long incubations with thrombin, turbidity increases were apparent, but these were not associated with fibrin clot formation. Both the lag period and the rate of turbidity change with this mutant were 10-fold or more longer than that observed with the substitution of Ala at this position. Obviously, the size and potential charge of the histidine side chain differ significantly from alanine, and these characteristics likely contribute to the extreme loss of function, but the rationale for the extensive differences is unclear. The data suggest that the overall structure of the polymerization pocket is changed by this substitution such that no reasonable polymerization site is present. That is, the presence of histidine not only disrupts the salt bridge with Arg-375 but also disrupts the overall conformations of loops P-3 and P-4, with the consequent loss of the polymerization pocket. This conclusion that large structural changes accompany the histidine substitution is supported by our finding that a high Ca2+ concentration was required for binding to the conformation-sensitive IF-1 antibody used for immunopurification of D364H fibrinogen.We interpret our data as indicating that the C-terminal γ chain domain participates in both protofibril formation and fiber formation. Many previous experiments show that protofibrils form when the N terminus of the α chain of one fibrin molecule (exposed after FpA is cleaved from fibrinogen) binds in the polymerization pocket in the C-terminal γ chain domain of a second fibrin molecule. Our data are consistent with this conclusion. In addition, our data indicate that changing the polymerization pocket also changes lateral aggregation. This would be the case if the first interactions, between the polymerization pocket and the N terminus of the α chain, induced a conformational change such that the C-terminal domain of the γ chain takes on a role in the lateral aggregation of protofibrils. This conclusion emanates from a comparison of the data with the two 364 variants. Because the Ala variant was able to form a continuous fibrin gel and the His variant was not, we conclude that sites for both protofibril formation and lateral aggregation are missing in the His variant. As stated above, our results with the Ala variants are also consistent with the conclusion that both protofibril formation and fiber formation are altered by these substitutions. We have initiated experiments that are designed to test whether these single-amino acid substitutions alter more than one step in fibrin clot formation. Fibrinogen is a plasma glycoprotein composed of a pair of three polypeptide chains, Aα, Bβ, and γ. The six N termini form a central domain, called E, which can be isolated as a single fragment from a plasmin digest of fibrinogen. The six chains divide into two three-chain sets that emanate in opposite directions from the central E domain as coiled-coil rods that terminate with the C-terminal residues of each chain forming separate domains. The peripheral domains can also be isolated from plasmin digests as the D fragments, which contain residues from all three chains but consist primarily of the C-terminal domains of the Bβ and γ chains. During blood coagulation, fibrinogen is converted to an insoluble fibrin clot by the serine protease thrombin, which cleaves four peptide bonds, releasing two fibrinopeptides A (FpA, Aα 1–16) and two fibrinopeptides B (FpB, Bβ 1–14) and fibrin monomers that polymerize spontaneously. The association of fibrin monomers into a fibrin clot has long been described as a two-step process, where the first step involves half-staggered, end-to-end interactions leading to double-stranded protofibrils and the second step, usually called lateral aggregation, involves the assembly of protofibrils into thick, multi-stranded fibers that branch to form a fibrin network. The final product is a fibrin gel (1Blomback B. Hessel B. Hogg D. Therkildsen L. Nature. 1978; 275: 501-505Crossref PubMed Google Scholar, 2Olexa S.A. Budzynski A.Z. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 1374-1378Crossref PubMed Scopus (151) Google Scholar). The interactions that promote protofibril formation occur between N-terminal α chain residues in the E domain on one fibrin molecule and C-terminal γ chain residues in the D domain on a second fibrin molecule. The interactions that promote lateral aggregation are less well known, although it has been shown that FpB cleavage enhances lateral aggregation (1Blomback B. Hessel B. Hogg D. Therkildsen L. Nature. 1978; 275: 501-505Crossref PubMed Google Scholar, 3Weisel J.W. Veklich Y. Gorkun O. J. Mol. Biol. 1993; 232: 285-297Crossref PubMed Scopus (130) Google Scholar). The enhanced lateral aggregation may, however, be an indirect result of strengthened protofibril interactions that accompany FpB release (4Shainoff J.R. Dardik B.N. Ann. N. Y. Acad. Sci. 1983; 408: 254-268Crossref PubMed Scopus (58) Google Scholar). It has also been shown that the C-terminal domains of the α chain (the αC domains) participate in lateral aggregation and that calcium binding to fibrinogen increases during lateral aggregation (5Okada M. Blomback B. Thromb. Res. 1983; 29: 269-280Abstract Full Text PDF PubMed Scopus (54) Google Scholar, 6Carr M. Gabriel D. McDonagh J. Biochem. J. 1986; 239: 513-516Crossref PubMed Scopus (69) Google Scholar, 7Mihalyi E. Biochemistry. 1988; 27: 976-982Crossref PubMed Scopus (41) Google Scholar, 8Gorkun O.V. Veklich Y.I. Medved L.V. Henschen A.H. Weisel J.W. Biochemistry. 1994; 33: 6986-6997Crossref PubMed Scopus (170) Google Scholar, 9Weisel J.W. Nagaswami C. Biophys. J. 1992; 63: 111-128Abstract Full Text PDF PubMed Scopus (285) Google Scholar, 10Cierniewski C.S. Budzynski A.Z. Biochemistry. 1992; 31: 4248-4253Crossref PubMed Scopus (42) Google Scholar, 11Hasegawa N. Sasaki S. Thromb. Res. 1990; 57: 183-195Abstract Full Text PDF PubMed Scopus (49) Google Scholar). Recent experiments have identified two specific residues in the D domain of the γ chain as participants in protofibril formation. Tyr-363 in the γ chain was identified by photoaffinity labeling with a peptide that mimics the N-terminal α chain residues of the E domain of fibrin (12Yamazumi K. Doolittle R.F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2893-2896Crossref PubMed Scopus (58) Google Scholar). Asp-364 in the γ chain was identified in fibrinogen Matsumoto I (13Okumura N. Furihata K. Terasawa F. Nakagoshi R. Ueno I. Katsuyama T. Thromb. Haemostasis. 1996; 75: 887-891Crossref PubMed Scopus (49) Google Scholar), a dysfibrinogen that was found in a heterozygous individual. Fibrinogen Matsumoto I is a mixture of molecules with normal and variant γ chains that have His at position 364. Polymerization of fibrinogen Matsumoto I is markedly delayed, and this delay can be partially compensated by mixing with normal fibrinogen (13Okumura N. Furihata K. Terasawa F. Nakagoshi R. Ueno I. Katsuyama T. Thromb. Haemostasis. 1996; 75: 887-891Crossref PubMed Scopus (49) Google Scholar). The results suggest that the normal molecules of this fibrinogen support polymerization, whereas the variant molecules do not. In the studies described, here we examined the roles of these two residues by analysis of genetically engineered variants. Using the previously described two-step procedure for synthesis of variant fibrinogens, we synthesized three variants with single amino acid substitutions in the γ chain: Tyr-363 → Ala (Y363A), Asp-364 → Ala (D364A), and Asp-364 → His (D364H). We determined the clottability of these variants and followed the kinetics of thrombin-catalyzed fibrinopeptide release and thrombin-catalyzed polymerization. We compared the variants to one another and to normal recombinant fibrinogen, which has polymerization characteristics comparable to normal plasma fibrinogen (14Gorkun O.V. Veklich Yu.I. Weisel J.W. Lord S.T. Blood. 1997; 89: 4407-4414Crossref PubMed Google Scholar). DISCUSSIONThe data presented here confirmed that the C-terminal γ chain residues 363 and 364 are important participants in polymerization. We found that Ala substitutions at either position 363 or 364 dramatically delayed polymerization. We measured turbidity, which with normal fibrin polymerization gauges the rate of protofibril formation from the lag period, and the rate of fiber formation from the slope of the turbidity change. With both Ala substitutions, the lag periods were lengthened and the slopes were less steep. The 363 Ala substitution was the most normal, and even with this mutant the shortest lag period and the most rapid increase in turbidity were about 3-fold delayed relative to normal fibrinogen under the most favorable conditions. These results suggest that both protofibril formation and fiber formation were affected by the single Ala substitutions. Alternatively, the Ala substitutions may change only one of the polymerization steps and still alter both the lag period and the slope. This possibility has been carefully described by Weisel and Nagaswami (9Weisel J.W. Nagaswami C. Biophys. J. 1992; 63: 111-128Abstract Full Text PDF PubMed Scopus (285) Google Scholar), who showed with simulated data that changing the rate of protofibril formation or the rate of lateral aggregation can change both the lag and the slope of a turbidity curve. For example, if the rate of protofibril formation is slowed, then the lag period is lengthened, and the slope of the turbidity increase is decreased. Similarly, if the rate of fiber formation is slowed, then normal protofibrils form in the normal time frame, but the lateral aggregation of these protofibrils is less efficient, which is seen as both a longer lag period and a reduced slope. Thus, further studies using assays that distinguish these two steps are needed to determine whether one or both steps of polymerization is impaired in these Ala variants. Even though polymerization was delayed with both of the Ala variants, the clottability data and clot appearance, which was similar to the normal clot, indicate that large, insoluble polymers were finally assembled from these molecules.The turbidity curves obtained with the Asp-364 to His substitution were more remarkable. We were able to measure changes in turbidity only at 0.45 mg/ml fibrinogen. At lower fibrinogen concentrations (0.09 mg/ml), where normal polymerization is complete within a few hours, polymers with D364H were not detected even after days of incubation. A delay in D364H fibrin polymerization was predicted from studies with fibrinogen Matsumoto I, which contains normal and variant γ chains, but the striking loss of function found with the homogeneous recombinant variant was unanticipated. The clottability data showed that normal polymers were not formed; no fibrin gel was seen and only half the protein was incorporated into precipitated material. This result was observed even though fibrinopeptide release indicated normal fibrin formation. Analysis of the nonclottable protein suggests that the soluble fibrin molecules were degraded, probably by prolonged exposure to thrombin. Thrombin cleavage of the C-terminal region of α chains has been previously reported (25Yoshida N. Wada H. Morita K. Hirata H. Matsuda M. Yamazumi K. Asakura S. Shirakawa S. Blood. 1991; 77: 1958-1963Crossref PubMed Google Scholar). This extensive proteolysis most likely occurred because the fibrin monomers were not readily incorporated into fibrin polymers, but the