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 PubMed Scopus Google Scholar, Veklich J.W. PubMed Scopus Google Scholar, J.W. J. Full Text PDF PubMed Scopus Google Scholar, Budzynski A.Z. PubMed Scopus Google Scholar, N. S. Thromb. Res. Full Text PDF PubMed Scopus Google have two residues in the D domain of the γ chain as in protofibril formation. Tyr-363 in the γ chain was by with a peptide that the N-terminal α chain residues of the E domain of fibrin Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus (58) Google Scholar). Asp-364 in the γ chain was in fibrinogen N. Thromb. PubMed Scopus Google a that was found in a Fibrinogen is a of with normal and variant γ chains that have His at Polymerization of fibrinogen is and can be by with normal fibrinogen N. Thromb. PubMed Scopus Google Scholar). The suggest that the normal of fibrinogen polymerization, the variant the studies we the of these two residues by of the described two-step for of variant we synthesized three variants with single substitutions in the γ Tyr-363 → Ala Asp-364 → Ala and Asp-364 → His We the clottability of these variants and the of release and We the variants to one and to normal recombinant fibrinogen, which has polymerization to normal plasma fibrinogen Veklich J.W. PubMed Google data that the C-terminal γ chain residues and are in We found that Ala substitutions at delayed We which with normal fibrin polymerization the of protofibril formation from the and the of formation from the of the both Ala substitutions, the were and the were less The Ala was the most and with the and the most in were delayed to normal fibrinogen the most suggest that both protofibril formation and formation were by the single Ala the Ala substitutions only one of the polymerization and both the and the has been described by and J.W. J. Full Text PDF PubMed Scopus Google with data that the of protofibril formation the of lateral aggregation can both the and the of a the of protofibril formation is the is and the of the is the of formation is normal protofibrils form in the normal but the lateral aggregation of these protofibrils is less which is as both a and a studies that these two are to one both of polymerization is in these Ala polymerization was delayed with both of the Ala the clottability data and clot which was to the normal that insoluble were from these with the Asp-364 to His were We were to in only at fibrinogen. fibrinogen where normal polymerization is a with were not of A in fibrin polymerization was from studies with fibrinogen which normal and variant γ chains, but the of found with the recombinant variant was The clottability data that normal were not fibrin gel was and only the was into result was release normal fibrin formation. of the that the soluble fibrin were by to cleavage of the C-terminal of α chains has been N. M. S. S. 77: PubMed Google Scholar). most likely the fibrin monomers were not into fibrin but the have to the normal for FpB release from these three which delayed polymerization, was It is that the of fibrinogen to fibrin in an that is are and FpB is from these PubMed Scopus Google Scholar, J. Biol. 1983; Full Text PDF PubMed Google Scholar). the of FpB release on polymerization, that FpB release is delayed fibrin polymerization is both and the peptide polymerization, and both the of FpB release from normal fibrinogen J. Biol. 1983; Full Text PDF PubMed Google Scholar, A. PubMed Scopus Google Scholar, J. Biol. Full Text PDF PubMed Google Scholar). two fibrinogen and polymerization of fibrin monomers and a of FpB the normal for FpB release from the variants that an the normal found in protofibrils that the of the protofibril was at a normal result is the residues and are to that a significant at these residues be in the of protofibril formation. Our that at residues and in significantly polymerization, we a in interactions and the in FpB The normal release of FpB that are at a normal data are also with the that these are not for normal FpB but be to are to with with in the C-terminal domain of the γ polymerization with normal of FpB release of Scholar). The of FpB release from these be with however, as these were in with of a of normal and variant is at of fibrinogen and that are to the data in the on these to the that in the C-terminal domain of the γ chain polymerization but not interactions to normal FpB have been for the isolated C-terminal fragment of the fibrinogen γ chain and in a with the peptide Full Text Full Text PDF PubMed Scopus Google Scholar, D. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). data that the C-terminal domain of the γ chain is J. Biochem. PubMed Google it is likely that isolated which was in the the it has in the fibrinogen molecule. to on the for the we have A called the polymerization is by four Tyr-363 and Asp-364 shown in the chain for Tyr-363 into the that it can participate in the chain of for the of Ala is likely to significant of and It is to that residues participate in the that Tyr-363 is not the only shown in and polymerization with the variant is but not and fibrin the in polymerization of that single has a role in fibrin clot shown in Asp-364 is that it a with in the of and both residues that the polymerization it is to that in the of these A role for is by the of fibrin polymerization with the of for in the the in the of that Asp-364 a with the N of the Asp-364 likely in two forming an that is to fibrinogen and the interactions that are to fibrin the of Ala for be to significantly Our data are also with the by D. Thromb. synthesized and the recombinant fragment with the found that the peptide not to variant and that variant fragment was to polymerization of normal of the from the of His for Asp-364 is the in were In to the two Ala substitutions, not form a fibrin gel the described long with thrombin, increases were but these were not with fibrin clot formation. the and the of with were that with the of Ala at the and of the chain significantly from and these likely to the of but the for the is The data suggest that the of the polymerization is by that polymerization is the of not only the with but also the of and with the of the polymerization that accompany the is by that a was for binding to the for of data as indicating that the C-terminal γ chain domain in both protofibril formation and formation. that protofibrils form the N of the α chain of one fibrin molecule is from in the polymerization in the C-terminal γ chain domain of a second fibrin molecule. Our data are with In data that the polymerization also lateral be the the first between the polymerization and the N of the α a that the C-terminal domain of the γ chain on a role in the lateral aggregation of from a of the data with the two the Ala variant was to form a fibrin gel and the His variant was we that for both protofibril formation and lateral aggregation are in the His variant. with the Ala variants are also with the that both protofibril formation and formation are altered by these We have that are to these substitutions 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 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 PubMed Scopus Google Scholar, Veklich J.W. PubMed Scopus Google Scholar, J.W. J. Full Text PDF PubMed Scopus Google Scholar, Budzynski A.Z. PubMed Scopus Google Scholar, N. S. Thromb. Res. Full Text PDF PubMed Scopus Google Scholar). have two residues in the D domain of the γ chain as in protofibril formation. Tyr-363 in the γ chain was by with a peptide that the N-terminal α chain residues of the E domain of fibrin Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus (58) Google Scholar). Asp-364 in the γ chain was in fibrinogen N. Thromb. PubMed Scopus Google a that was found in a Fibrinogen is a of with normal and variant γ chains that have His at Polymerization of fibrinogen is and can be by with normal fibrinogen N. Thromb. PubMed Scopus Google Scholar). The suggest that the normal of fibrinogen polymerization, the variant In the studies we the of these two residues by of the described two-step for of variant we synthesized three variants with single substitutions in the γ Tyr-363 → Ala Asp-364 → Ala and Asp-364 → His We the clottability of these variants and the of release and We the variants to one and to normal recombinant fibrinogen, which has polymerization to normal plasma fibrinogen Veklich J.W. PubMed Google Scholar). data that the C-terminal γ chain residues and are in We found that Ala substitutions at delayed We which with normal fibrin polymerization the of protofibril formation from the and the of formation from the of the both Ala substitutions, the were and the were less The Ala was the most and with the and the most in were delayed to normal fibrinogen the most suggest that both protofibril formation and formation were by the single Ala the Ala substitutions only one of the polymerization and both the and the has been described by and J.W. J. Full Text PDF PubMed Scopus Google with data that the of protofibril formation the of lateral aggregation can both the and the of a the of protofibril formation is the is and the of the is the of formation is normal protofibrils form in the normal but the lateral aggregation of these protofibrils is less which is as both a and a studies that these two are to one both of polymerization is in these Ala polymerization was delayed with both of the Ala the clottability data and clot which was to the normal that insoluble were from these with the Asp-364 to His were We were to in only at fibrinogen. fibrinogen where normal polymerization is a with were not of A in fibrin polymerization was from studies with fibrinogen which normal and variant γ chains, but the of found with the recombinant variant was The clottability data that normal were not fibrin gel was and only the was into result was release normal fibrin formation. of the that the soluble fibrin were by to cleavage of the C-terminal of α chains has been N. M. S. S. 77: PubMed Google Scholar). most likely the fibrin monomers were not into fibrin but the have to the normal for FpB release from these three which delayed polymerization, was It is that the of fibrinogen to fibrin in an that is are and FpB is from these PubMed Scopus Google Scholar, J. Biol. 1983; Full Text PDF PubMed Google Scholar). the of FpB release on polymerization, that FpB release is delayed fibrin polymerization is both and the peptide polymerization, and both the of FpB release from normal fibrinogen J. Biol. 1983; Full Text PDF PubMed Google Scholar, A. PubMed Scopus Google Scholar, J. Biol. Full Text PDF PubMed Google Scholar). two fibrinogen and polymerization of fibrin monomers and a of FpB the normal for FpB release from the variants that an the normal found in protofibrils that the of the protofibril was at a normal result is the residues and are to that a significant at these residues be in the of protofibril formation. Our that at residues and in significantly polymerization, we a in interactions and the in FpB The normal release of FpB that are at a normal data are also with the that these are not for normal FpB but be to are to with with in the C-terminal domain of the γ polymerization with normal of FpB release of Scholar). The of FpB release from these be with however, as these were in with of a of normal and variant is at of fibrinogen and that are to the data in the on these to the that in the C-terminal domain of the γ chain polymerization but not interactions to normal FpB have been for the isolated C-terminal fragment of the fibrinogen γ chain and in a with the peptide Full Text Full Text PDF PubMed Scopus Google Scholar, D. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). data that the C-terminal domain of the γ chain is J. Biochem. PubMed Google it is likely that isolated which was in the the it has in the fibrinogen molecule. to on the for the we have A called the polymerization is by four Tyr-363 and Asp-364 shown in the chain for Tyr-363 into the that it can participate in the chain of for the of Ala is likely to significant of and It is to that residues participate in the that Tyr-363 is not the only shown in and polymerization with the variant is but not and fibrin the in polymerization of that single has a role in fibrin clot shown in Asp-364 is that it a with in the of and both residues that the polymerization it is to that in the of these A role for is by the of fibrin polymerization with the of for in the the in the of that Asp-364 a with the N of the Asp-364 likely in two forming an that is to fibrinogen and the interactions that are to fibrin the of Ala for be to significantly Our data are also with the by D. Thromb. synthesized and the recombinant fragment with the found that the peptide not to variant and that variant fragment was to polymerization of normal of the from the of His for Asp-364 is the in were In to the two Ala substitutions, not form a fibrin gel the described long with thrombin, increases were but these were not with fibrin clot formation. the and the of with were that with the of Ala at the and of the chain significantly from and these likely to the of but the for the is The data suggest that the of the polymerization is by that polymerization is the of not only the with but also the of and with the of the polymerization that accompany the is by that a was for binding to the for of data as indicating that the C-terminal γ chain domain in both protofibril formation and formation. that protofibrils form the N of the α chain of one fibrin molecule is from in the polymerization in the C-terminal γ chain domain of a second fibrin molecule. Our data are with In data that the polymerization also lateral be the the first between the polymerization and the N of the α a that the C-terminal domain of the γ chain on a role in the lateral aggregation of from a of the data with the two the Ala variant was to form a fibrin gel and the His variant was we that for both protofibril formation and lateral aggregation are in the His variant. with the Ala variants are also with the that both protofibril formation and formation are altered by these We have that are to these substitutions one step in fibrin clot formation. The data that the C-terminal γ chain residues and are in We found that Ala substitutions at delayed We which with normal fibrin polymerization the of protofibril formation from the and the of formation from the of the both Ala substitutions, the were and the were less The Ala was the most and with the and the most in were delayed to normal fibrinogen the most suggest that both protofibril formation and formation were by the single Ala the Ala substitutions only one of the polymerization and both the and the has been described by and J.W. J. Full Text PDF PubMed Scopus Google with data that the of protofibril formation the of lateral aggregation can both the and the of a the of protofibril formation is the is and the of the is the of formation is normal protofibrils form in the normal but the lateral aggregation of these protofibrils is less which is as both a and a studies that these two are to one both of polymerization is in these Ala polymerization was delayed with both of the Ala the clottability data and clot which was to the normal that insoluble were from these The with the Asp-364 to His were We were to in only at fibrinogen. fibrinogen where normal polymerization is a with were not of A in fibrin polymerization was from studies with fibrinogen which normal and variant γ chains, but the of found with the recombinant variant was The clottability data that normal were not fibrin gel was and only the was into result was release normal fibrin formation. of the that the soluble fibrin were by to cleavage of the C-terminal of α chains has been N. M. S. S. 77: PubMed Google Scholar). most likely the fibrin monomers were not into fibrin but the have to the The normal for FpB release from these three which delayed polymerization, was It is that the of fibrinogen to fibrin in an that is are and FpB is from these PubMed Scopus Google Scholar, J. Biol. 1983; Full Text PDF PubMed Google Scholar). the of FpB release on polymerization, that FpB release is delayed fibrin polymerization is both and the peptide polymerization, and both the of FpB release from normal fibrinogen J. Biol. 1983; Full Text PDF PubMed Google Scholar, A. PubMed Scopus Google Scholar, J. Biol. Full Text PDF PubMed Google Scholar). two fibrinogen and polymerization of fibrin monomers and a of FpB the normal for FpB release from the variants that an the normal found in protofibrils that the of the protofibril was at a normal result is the residues and are to that a significant at these residues be in the of protofibril formation. Our that at residues and in significantly polymerization, we a in interactions and the in FpB The normal release of FpB that are at a normal data are also with the that these are not for normal FpB but be to Our are to with with in the C-terminal domain of the γ polymerization with normal of FpB release of Scholar). The of FpB release from these be with however, as these were in with of a of normal and variant is at of fibrinogen and that are to the data in the on these to the that in the C-terminal domain of the γ chain polymerization but not interactions to normal FpB have been for the isolated C-terminal fragment of the fibrinogen γ chain and in a with the peptide Full Text Full Text PDF PubMed Scopus Google Scholar, D. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). data that the C-terminal domain of the γ chain is J. Biochem. PubMed Google it is likely that isolated which was in the the it has in the fibrinogen molecule. to on the for the we have A called the polymerization is by four Tyr-363 and Asp-364 shown in the chain for Tyr-363 into the that it can participate in the chain of for the of Ala is likely to significant of and It is to that residues participate in the that Tyr-363 is not the only shown in and polymerization with the variant is but not and fibrin the in polymerization of that single has a role in fibrin clot formation. shown in Asp-364 is that it a with in the of and both residues that the polymerization it is to that in the of these A role for is by the of fibrin polymerization with the of for in the the in the of that Asp-364 a with the N of the Asp-364 likely in two forming an that is to fibrinogen and the interactions that are to fibrin the of Ala for be to significantly Our data are also with the by D. Thromb. synthesized and the recombinant fragment with the found that the peptide not to variant and that variant fragment was to polymerization of normal of the from the of His for Asp-364 is the in were In to the two Ala substitutions, not form a fibrin gel the described long with thrombin, increases were but these were not with fibrin clot formation. the and the of with were that with the of Ala at the and of the chain significantly from and these likely to the of but the for the is The data suggest that the of the polymerization is by that polymerization is the of not only the with but also the of and with the of the polymerization that accompany the is by that a was for binding to the for of fibrinogen. We data as indicating that the C-terminal γ chain domain in both protofibril formation and formation. that protofibrils form the N of the α chain of one fibrin molecule is from in the polymerization in the C-terminal γ chain domain of a second fibrin molecule. Our data are with In data that the polymerization also lateral be the the first between the polymerization and the N of the α a that the C-terminal domain of the γ chain on a role in the lateral aggregation of from a of the data with the two the Ala variant was to form a fibrin gel and the His variant was we that for both protofibril formation and lateral aggregation are in the His variant. with the Ala variants are also with the that both protofibril formation and formation are altered by these We have that are to these substitutions one step in fibrin clot formation. We for for and of for We also for and for in the
No takes yet. Share an insight, caveat, or question.
Okumura et al. (1997) studied this question.
Synapse has enriched 5 related papers. Consider them for comparative context: