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W1994382696 abstract "During cleavage of fibrinogen by thrombin, fibrinopeptide A (FpA) release precedes fibrinopeptide B (FpB) release. To examine the basis for this ordered release, we synthesized A′β fibrinogen, replacing FpB with a fibrinopeptide A-like peptide, FpA′ (G14V). Analyses of fibrinopeptide release from A′β fibrinogen showed that FpA release and FpA′ release were similar; the release of either peptide followed simple first-order kinetics. Specificity constants for FpA and FpA′ were similar, demonstrating that these peptides are equally competitive substrates for thrombin. In the presence of Gly-Pro-Arg-Pro, an inhibitor of fibrin polymerization, the rate of FpB release from normal fibrinogen was reduced 3-fold, consistent with previous data; in contrast, the rate of FpA′ release from A′β fibrinogen was unaffected. Thus, with A′β fibrinogen, fibrinopeptide release from the β chain is similar to fibrinopeptide release from the α chain. We conclude that the ordered release of fibrinopeptides is dictated by the specificity of thrombin for its substrates. We analyzed polymerization, following changes in turbidity, and found that polymerization of A′β fibrinogen was similar to that of normal fibrinogen. We analyzed clot structure by scanning electron microscopy and found that clots from A′β fibrinogen were similar to clots from normal fibrinogen. We conclude that premature release of the fibrinopeptide from the N terminus of the β chain does not affect polymerization of fibrinogen. During cleavage of fibrinogen by thrombin, fibrinopeptide A (FpA) release precedes fibrinopeptide B (FpB) release. To examine the basis for this ordered release, we synthesized A′β fibrinogen, replacing FpB with a fibrinopeptide A-like peptide, FpA′ (G14V). Analyses of fibrinopeptide release from A′β fibrinogen showed that FpA release and FpA′ release were similar; the release of either peptide followed simple first-order kinetics. Specificity constants for FpA and FpA′ were similar, demonstrating that these peptides are equally competitive substrates for thrombin. In the presence of Gly-Pro-Arg-Pro, an inhibitor of fibrin polymerization, the rate of FpB release from normal fibrinogen was reduced 3-fold, consistent with previous data; in contrast, the rate of FpA′ release from A′β fibrinogen was unaffected. Thus, with A′β fibrinogen, fibrinopeptide release from the β chain is similar to fibrinopeptide release from the α chain. We conclude that the ordered release of fibrinopeptides is dictated by the specificity of thrombin for its substrates. We analyzed polymerization, following changes in turbidity, and found that polymerization of A′β fibrinogen was similar to that of normal fibrinogen. We analyzed clot structure by scanning electron microscopy and found that clots from A′β fibrinogen were similar to clots from normal fibrinogen. We conclude that premature release of the fibrinopeptide from the N terminus of the β chain does not affect polymerization of fibrinogen. fibrinopeptide A fibrinopeptide B fibrinopeptide A′ (FpA with a G14V mutation) FpA′ substituted on the N terminus of the α chain FpA′ substituted on the N terminus of the β chain base pair(s) high performance liquid chromatography Gly-Pro-Arg-Pro acetate salt peptide Fibrinogen is a 340-kDa plasma protein that is involved in the final phase of the coagulation cascade. Fibrinogen consists of two pairs of three polypeptide chains (Aα, Bβ, and γ) that fold to produce a trinodular protein with two distal (D) nodules connected to a central nodule (E) by coiled-coil regions. The central nodule of the molecule consists of the N termini of all six polypeptide chains, and the D nodules consist predominantly of the C termini of the β and γ chains, each folded into a globular domain. To initiate polymerization, the serine protease thrombin cleaves four specific Arg–Gly bonds at the N termini of both the Aα and Bβ chains, releasing fibrinopeptides A (FpA)1 and B (FpB), respectively. The release of FpA, a 16-residue peptide, exposes the “A” site, which noncovalently interacts with the “a” site in the γ chain of the D nodule of another molecule. This A:a interaction results in the linear arrangement of half-staggered, double-stranded protofibrils (1Ferry J.D. Morrison P.R. J. Am. Chem. Soc. 1947; 69: 388-400Crossref PubMed Scopus (222) Google Scholar). The release of FpB, a 14-residue peptide, exposes the “B” site (2Blomback B. Hessel B. Hogg D. Therkildsen L. Nature. 1978; 275: 501-505Crossref PubMed Google Scholar, 3Hurlet-Jensen A. Cummins H.Z. Nossel H.L. Liu C.Y. Thromb. Res. 1982; 27: 419-427Abstract Full Text PDF PubMed Scopus (41) Google Scholar, 4Ruf 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), which presumably interacts with a “b” site in the β chain of the D nodule of another molecule (5Everse S.J. Spraggon G. Veerapandian L. Doolittle R.F. Biochemistry. 1999; 38: 2941-2946Crossref PubMed Scopus (76) Google Scholar). This B:b interaction is thought to be responsible for lateral aggregation of protofibrils to form fibers (2Blomback B. Hessel B. Hogg D. Therkildsen L. Nature. 1978; 275: 501-505Crossref PubMed Google Scholar) and to be analogous to the A:a interaction; however, the mechanism of this interaction is not yet well understood. The final product of this polymerization is a complex, branching network of fibers.The interactions of thrombin with fibrinogen have been extensively studied (Refs. 6Hogg D.H. Blomback B. Thromb. Res. 1978; 12: 953-964Abstract Full Text PDF PubMed Scopus (39) Google Scholar and 7Kaminski M. McDonagh J. J. Biol. Chem. 1983; 258: 10530-10535Abstract Full Text PDF PubMed Google Scholar; for a review, see Ref. 8Binnie C.G. Lord S.T. Blood. 1993; 81: 3186-3192Crossref PubMed Google Scholar). Thrombin contains three domains that interact with fibrinogen: the active site, an apolar specificity pocket, and a fibrinogen-binding exosite. The exosite, also called the fibrinogen recognition site, confers the specificity with which thrombin binds to fibrinogen. Upon binding, thrombin cleaves FpA and initiates polymerization. FpA release from fibrinogen follows first-order kinetics, described by the kinetic constantk 1. In contrast, FpB is released from fibrinogen at a slow initial rate (3Hurlet-Jensen A. Cummins H.Z. Nossel H.L. Liu C.Y. Thromb. Res. 1982; 27: 419-427Abstract Full Text PDF PubMed Scopus (41) Google Scholar, 9Martinelli R.A. Scheraga H.A. Biochemistry. 1980; 19: 2343-2350Crossref PubMed Scopus (81) Google Scholar, 10Hanna L.S. Scheraga H.A. Francis C.W. Marder V.J. Biochemistry. 1984; 23: 4681-4687Crossref PubMed Scopus (52) Google Scholar), which is then increased upon polymerization (2Blomback B. Hessel B. Hogg D. Therkildsen L. Nature. 1978; 275: 501-505Crossref PubMed Google Scholar, 9Martinelli R.A. Scheraga H.A. Biochemistry. 1980; 19: 2343-2350Crossref PubMed Scopus (81) Google Scholar, 10Hanna L.S. Scheraga H.A. Francis C.W. Marder V.J. Biochemistry. 1984; 23: 4681-4687Crossref PubMed Scopus (52) Google Scholar, 11Eckhardt T. Nossel H.L. Hurlet-Jensen A. LaGamma K.S. Owen J. Auerbach M. J. Clin. Invest. 1981; 67: 809-816Crossref PubMed Scopus (24) Google Scholar) and the depletion of FpA as a substrate for thrombin. This efficient release of FpB, subsequent to FpA release, follows first-order kinetics and is described by the kinetic constantk 2, assuming that the release of fibrinopeptides A and B from thrombin occurs through two successive first-order processes.Crystallographic data have depicted the contacts between FpA and thrombin (12Stubbs M.T. Oschkinat H. Myr I. Huber R. Angliker H. Stone S.R. Bode W. Eur. J. Biochem. 1992; 206: 187-195Crossref PubMed Scopus (196) Google Scholar); but, to date, similar data are not available for thrombin and FpB. The sequence of FpB is different from that of FpA (13Henschen A. Lottspeich F. Kehl M. Southan C. Ann. N. Y. Acad. Sci. 1983; 408: 28-43Crossref PubMed Scopus (206) Google Scholar) such that FpB should require different contacts with thrombin (12Stubbs M.T. Oschkinat H. Myr I. Huber R. Angliker H. Stone S.R. Bode W. Eur. J. Biochem. 1992; 206: 187-195Crossref PubMed Scopus (196) Google Scholar). Because we do not have structural information, our understanding of FpB release is indirect and based on kinetic studies that examined the timing of FpB release. These studies have shown that the majority of FpB is released from fibrin after FpA has been removed (3Hurlet-Jensen A. Cummins H.Z. Nossel H.L. Liu C.Y. Thromb. Res. 1982; 27: 419-427Abstract Full Text PDF PubMed Scopus (41) Google Scholar, 14Higgins D.L. Lewis S.D. Shafer J.A. J. Biol. Chem. 1983; 258: 9276-9282Abstract Full Text PDF PubMed Google Scholar) and that the rate of FpB release is enhanced upon polymerization of des-A polymers (2Blomback B. Hessel B. Hogg D. Therkildsen L. Nature. 1978; 275: 501-505Crossref PubMed Google Scholar, 3Hurlet-Jensen A. Cummins H.Z. Nossel H.L. Liu C.Y. Thromb. Res. 1982; 27: 419-427Abstract Full Text PDF PubMed Scopus (41) Google Scholar, 4Ruf 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, 15Lewis S.D. Shields P.P. Shafer J.A. J. Biol. Chem. 1985; 260: 10192-10199Abstract Full Text PDF PubMed Google Scholar). To date, however, the mechanism responsible for the delay in FpB release remains undetermined.To extend our understanding of the mechanism of thrombin on fibrinopeptide B and its resulting effect on polymerization, we have designed a variant recombinant fibrinogen (A′β) that contains a fibrinopeptide A-like substrate on the N termini of the β chains. This fibrinogen was designed to probe the mechanism of fibrinopeptide release and more specifically to determine whether the delayed release of FpB is a consequence of its affinity for thrombin or of its location on the N terminus of the β chain. Our studies have revealed that it is the specificity of thrombin for FpB that is responsible for the order of fibrinopeptide release during fibrin polymerization.DISCUSSIONWe have synthesized a recombinant fibrinogen (A′β) to determine what effect the substitution of FpA′ on the N terminus of the β chain has on the kinetics of fibrinopeptide release. The release of FpA and FpA′ from A′β fibrinogen occurred from the beginning of the reaction and followed first-order kinetics, indicating that both FpA and FpA′ are equally competitive substrates for thrombin and have cleavage sites that are accessible to thrombin at the beginning of the reaction. Thus, the assumption used to characterize FpB release, that FpB release depends on prior FpA release, does not accurately describe the release of FpA′ from A′β fibrinogen. In addition, the similar rate of release of either FpA′ or FpB from the β chain indicates that despite the fibrinopeptide placed on the N terminus of the β chain, the rate of cleavage of the Arg–Gly bond by thrombin remains constant. Taken together, we conclude that it is thrombin, in its interaction with FpB, that is responsible for the delay in efficient FpB release during fibrin polymerization, i.e. the specificity of thrombin for FpB, and not its location on the N terminus of the β chain, accounts for the kinetics of fibrinopeptide release from fibrinogen.Additional experiments support the conclusion that FpA′ release from A′β fibrinogen is dictated by thrombin specificity and not fibrin polymerization. We found that FpA′ release is qualitatively different from FpB release from normal fibrinogen, indicating that the release of FpA′ from the β chain is not dependent on prior polymerization of des-A monomers. This conclusion is supported by our studies in the presence or absence of GPRP, an inhibitor of fibrin polymerization. Our results showed that FpB release was impaired in the presence of GPRP, whereas FpA′ release was not affected. Together, these results indicate that FpA′ release from A′β fibrinogen is independent of fibrin polymerization. Thus, we have created a fibrinogen in which the delay in the rate enhancement of fibrinopeptide release from the β chain has been eliminated.We present the following models of the interaction of thrombin with fibrinogen, which accommodate our findings with A′β and normal fibrinogens. Initially, thrombin is oriented such that all three fibrinogen interaction sites (the apolar specificity pocket, fibrinogen-binding exosite, and active site) are aligned to accommodate FpA as the preferred substrate. Upon binding to A′β fibrinogen, thrombin is best suited for FpA or FpA′ cleavage, regardless of the placement of the peptide on the α or β chain; thus, all four fibrinopeptides are cleaved simultaneously and with similar affinity. In normal fibrinogen, the thrombin active site is specific for FpA, cleaving it efficiently to initiate polymerization, whereas thrombin cleavage of FpB is much less efficient. During polymerization, however, efficient cleavage of FpB becomes favorable, i.e. FpB release becomes more efficient upon the appearance of des-A polymers (2Blomback B. Hessel B. Hogg D. Therkildsen L. Nature. 1978; 275: 501-505Crossref PubMed Google Scholar, 3Hurlet-Jensen A. Cummins H.Z. Nossel H.L. Liu C.Y. Thromb. Res. 1982; 27: 419-427Abstract Full Text PDF PubMed Scopus (41) Google Scholar, 4Ruf 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, 15Lewis S.D. Shields P.P. Shafer J.A. J. Biol. Chem. 1985; 260: 10192-10199Abstract Full Text PDF PubMed Google Scholar). We propose two possible mechanisms, as depicted in Fig.8, for this shift to efficient cleavage of FpB. Either 1) upon the release of FpA, thrombin changes its conformation such that FpB can be accommodated and cleaved efficiently, or 2) the local conformation of FpB is altered over the course of polymerization, which correctly orients the Arg14–Gly15 bond for efficient cleavage within the active site of thrombin.Although our results do not give us reason to favor one possibility over the other, previous studies lend credence to both. Studies on thrombin binding to exosite-binding fragments of hirudin, heparin cofactor II, and the thrombin receptor have shown that exosite interaction can allosterically modify the active site of thrombin (28Naski M.C. Fenton J.W., II Maraganore J.M. Olson S.T. Shafer J.A. J. Biol. Chem. 1990; 265: 13484-13489Abstract Full Text PDF PubMed Google Scholar, 29Dennis S. Wallace A. Hofsteenje J. Stone S.R. Eur. J. Biochem. 1990; 188: 61-66Crossref PubMed Scopus (59) Google Scholar, 30Hortin G.L. Trimpe B.L. J. Biol. Chem. 1991; 266: 6866-6871Abstract Full Text PDF PubMed Google Scholar, 31Liu L.W. Vu T.K. Esmon C.T. Coughlin S.R. J. Biol. Chem. 1991; 266: 16977-16980Abstract Full Text PDF PubMed Google Scholar, 32De Cristofaro R. Rocca B. Bizzi B. Landolfi R. Biochem. J. 1993; 289: 475-480Crossref PubMed Scopus (32) Google Scholar). Because thrombin binds to fibrinogen in the exosite, it is possible that conformational changes that occur in fibrinogen during polymerization can indirectly affect the thrombin active site. Alternatively, conformational changes in the fibrinogen molecule have been proposed (2Blomback B. Hessel B. Hogg D. Therkildsen L. Nature. 1978; 275: 501-505Crossref PubMed Google Scholar, 3Hurlet-Jensen A. Cummins H.Z. Nossel H.L. Liu C.Y. Thromb. Res. 1982; 27: 419-427Abstract Full Text PDF PubMed Scopus (41) Google Scholar, 14Higgins D.L. Lewis S.D. Shafer J.A. J. Biol. Chem. 1983; 258: 9276-9282Abstract Full Text PDF PubMed Google Scholar, 23Ng A.S. Lewis S.D. Shafer J.A. Methods Enzymol. 1993; 222: 341-358Crossref PubMed Scopus (39) Google Scholar, 33Hantgan R.R. Hermans J. J. Biol. Chem. 1979; 254: 11272-11281Abstract Full Text PDF PubMed Google Scholar, 34Weisel J.W. Veklich Y. Gorkun O. J. Mol. Biol. 1993; 232: 285-297Crossref PubMed Scopus (130) Google Scholar, 35Veklich Y.I. Gorkun O.V. Medved L.V. Niewenhuizen W. Weisel J.W. J. Biol. Chem. 1993; 268: 13577-13585Abstract Full Text PDF PubMed Google Scholar) and measured (36Henschen-Edman A.H. Cell. Mol. Life Sci. 1997; 53: 29-33Crossref PubMed Scopus (19) Google Scholar) during fibrin polymerization, and these changes could reposition the E domain such that the scissile bond in FpB is properly oriented for efficient cleavage by thrombin. Whether it is conformational changes in fibrinopeptide B, thrombin, or both, these studies suggest that either model is possible and reemphasize that we can only conclude from our studies that it is the specificity of thrombin for the fibrinopeptides that dictates the rate and timing of their release.Production of A′β fibrinogen also allowed us to evaluate the effect of early exposure of the B site on fibrin polymerization. If early exposure of the B site did affect polymerization, we would expect a polymerization curve similar to that for fibrin monomer polymerization, when the A and B sites are exposed from the start of the reaction. Our results did not follow this pattern, thus suggesting that exposure of the B site alone does not directly influence polymerization. Rather, the participation of the B site in polymerization likely depends on certain polymerization events. Previous studies (33Hantgan R.R. Hermans J. J. Biol. Chem. 1979; 254: 11272-11281Abstract Full Text PDF PubMed Google Scholar, 37Weisel J.W. Nagaswami C. Biophys. J. 1992; 63: 111-128Abstract Full Text PDF PubMed Scopus (285) Google Scholar) suggest that this event is the polymerization of des-A monomers to form protofibrils of a critical length.In summary, by making this fibrinogen with essentially four fibrinopeptides A, we have synthesized a model substrate that eliminated the delay in fibrinopeptide release from the β chain. Thus, the normal delay in fibrinopeptide B release likely arises from a specific interaction between thrombin and FpB. We therefore conclude that the kinetics of fibrinopeptide release are dictated by the affinity of thrombin for its substrates. In addition, our work suggests that early exposure of the B site does not affect the polymerization process. Fibrinogen is a 340-kDa plasma protein that is involved in the final phase of the coagulation cascade. Fibrinogen consists of two pairs of three polypeptide chains (Aα, Bβ, and γ) that fold to produce a trinodular protein with two distal (D) nodules connected to a central nodule (E) by coiled-coil regions. The central nodule of the molecule consists of the N termini of all six polypeptide chains, and the D nodules consist predominantly of the C termini of the β and γ chains, each folded into a globular domain. To initiate polymerization, the serine protease thrombin cleaves four specific Arg–Gly bonds at the N termini of both the Aα and Bβ chains, releasing fibrinopeptides A (FpA)1 and B (FpB), respectively. The release of FpA, a 16-residue peptide, exposes the “A” site, which noncovalently interacts with the “a” site in the γ chain of the D nodule of another molecule. This A:a interaction results in the linear arrangement of half-staggered, double-stranded protofibrils (1Ferry J.D. Morrison P.R. J. Am. Chem. Soc. 1947; 69: 388-400Crossref PubMed Scopus (222) Google Scholar). The release of FpB, a 14-residue peptide, exposes the “B” site (2Blomback B. Hessel B. Hogg D. Therkildsen L. Nature. 1978; 275: 501-505Crossref PubMed Google Scholar, 3Hurlet-Jensen A. Cummins H.Z. Nossel H.L. Liu C.Y. Thromb. Res. 1982; 27: 419-427Abstract Full Text PDF PubMed Scopus (41) Google Scholar, 4Ruf 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), which presumably interacts with a “b” site in the β chain of the D nodule of another molecule (5Everse S.J. Spraggon G. Veerapandian L. Doolittle R.F. Biochemistry. 1999; 38: 2941-2946Crossref PubMed Scopus (76) Google Scholar). This B:b interaction is thought to be responsible for lateral aggregation of protofibrils to form fibers (2Blomback B. Hessel B. Hogg D. Therkildsen L. Nature. 1978; 275: 501-505Crossref PubMed Google Scholar) and to be analogous to the A:a interaction; however, the mechanism of this interaction is not yet well understood. The final product of this polymerization is a complex, branching network of fibers. The interactions of thrombin with fibrinogen have been extensively studied (Refs. 6Hogg D.H. Blomback B. Thromb. Res. 1978; 12: 953-964Abstract Full Text PDF PubMed Scopus (39) Google Scholar and 7Kaminski M. McDonagh J. J. Biol. Chem. 1983; 258: 10530-10535Abstract Full Text PDF PubMed Google Scholar; for a review, see Ref. 8Binnie C.G. Lord S.T. Blood. 1993; 81: 3186-3192Crossref PubMed Google Scholar). Thrombin contains three domains that interact with fibrinogen: the active site, an apolar specificity pocket, and a fibrinogen-binding exosite. The exosite, also called the fibrinogen recognition site, confers the specificity with which thrombin binds to fibrinogen. Upon binding, thrombin cleaves FpA and initiates polymerization. FpA release from fibrinogen follows first-order kinetics, described by the kinetic constantk 1. In contrast, FpB is released from fibrinogen at a slow initial rate (3Hurlet-Jensen A. Cummins H.Z. Nossel H.L. Liu C.Y. Thromb. Res. 1982; 27: 419-427Abstract Full Text PDF PubMed Scopus (41) Google Scholar, 9Martinelli R.A. Scheraga H.A. Biochemistry. 1980; 19: 2343-2350Crossref PubMed Scopus (81) Google Scholar, 10Hanna L.S. Scheraga H.A. Francis C.W. Marder V.J. Biochemistry. 1984; 23: 4681-4687Crossref PubMed Scopus (52) Google Scholar), which is then increased upon polymerization (2Blomback B. Hessel B. Hogg D. Therkildsen L. Nature. 1978; 275: 501-505Crossref PubMed Google Scholar, 9Martinelli R.A. Scheraga H.A. Biochemistry. 1980; 19: 2343-2350Crossref PubMed Scopus (81) Google Scholar, 10Hanna L.S. Scheraga H.A. Francis C.W. Marder V.J. Biochemistry. 1984; 23: 4681-4687Crossref PubMed Scopus (52) Google Scholar, 11Eckhardt T. Nossel H.L. Hurlet-Jensen A. LaGamma K.S. Owen J. Auerbach M. J. Clin. Invest. 1981; 67: 809-816Crossref PubMed Scopus (24) Google Scholar) and the depletion of FpA as a substrate for thrombin. This efficient release of FpB, subsequent to FpA release, follows first-order kinetics and is described by the kinetic constantk 2, assuming that the release of fibrinopeptides A and B from thrombin occurs through two successive first-order processes. Crystallographic data have depicted the contacts between FpA and thrombin (12Stubbs M.T. Oschkinat H. Myr I. Huber R. Angliker H. Stone S.R. Bode W. Eur. J. Biochem. 1992; 206: 187-195Crossref PubMed Scopus (196) Google Scholar); but, to date, similar data are not available for thrombin and FpB. The sequence of FpB is different from that of FpA (13Henschen A. Lottspeich F. Kehl M. Southan C. Ann. N. Y. Acad. Sci. 1983; 408: 28-43Crossref PubMed Scopus (206) Google Scholar) such that FpB should require different contacts with thrombin (12Stubbs M.T. Oschkinat H. Myr I. Huber R. Angliker H. Stone S.R. Bode W. Eur. J. Biochem. 1992; 206: 187-195Crossref PubMed Scopus (196) Google Scholar). Because we do not have structural information, our understanding of FpB release is indirect and based on kinetic studies that examined the timing of FpB release. These studies have shown that the majority of FpB is released from fibrin after FpA has been removed (3Hurlet-Jensen A. Cummins H.Z. Nossel H.L. Liu C.Y. Thromb. Res. 1982; 27: 419-427Abstract Full Text PDF PubMed Scopus (41) Google Scholar, 14Higgins D.L. Lewis S.D. Shafer J.A. J. Biol. Chem. 1983; 258: 9276-9282Abstract Full Text PDF PubMed Google Scholar) and that the rate of FpB release is enhanced upon polymerization of des-A polymers (2Blomback B. Hessel B. Hogg D. Therkildsen L. Nature. 1978; 275: 501-505Crossref PubMed Google Scholar, 3Hurlet-Jensen A. Cummins H.Z. Nossel H.L. Liu C.Y. Thromb. Res. 1982; 27: 419-427Abstract Full Text PDF PubMed Scopus (41) Google Scholar, 4Ruf 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, 15Lewis S.D. Shields P.P. Shafer J.A. J. Biol. Chem. 1985; 260: 10192-10199Abstract Full Text PDF PubMed Google Scholar). To date, however, the mechanism responsible for the delay in FpB release remains undetermined. To extend our understanding of the mechanism of thrombin on fibrinopeptide B and its resulting effect on polymerization, we have designed a variant recombinant fibrinogen (A′β) that contains a fibrinopeptide A-like substrate on the N termini of the β chains. This fibrinogen was designed to probe the mechanism of fibrinopeptide release and more specifically to determine whether the delayed release of FpB is a consequence of its affinity for thrombin or of its location on the N terminus of the β chain. Our studies have revealed that it is the specificity of thrombin for FpB that is responsible for the order of fibrinopeptide release during fibrin polymerization. DISCUSSIONWe have synthesized a recombinant fibrinogen (A′β) to determine what effect the substitution of FpA′ on the N terminus of the β chain has on the kinetics of fibrinopeptide release. The release of FpA and FpA′ from A′β fibrinogen occurred from the beginning of the reaction and followed first-order kinetics, indicating that both FpA and FpA′ are equally competitive substrates for thrombin and have cleavage sites that are accessible to thrombin at the beginning of the reaction. Thus, the assumption used to characterize FpB release, that FpB release depends on prior FpA release, does not accurately describe the release of FpA′ from A′β fibrinogen. In addition, the similar rate of release of either FpA′ or FpB from the β chain indicates that despite the fibrinopeptide placed on the N terminus of the β chain, the rate of cleavage of the Arg–Gly bond by thrombin remains constant. Taken together, we conclude that it is thrombin, in its interaction with FpB, that is responsible for the delay in efficient FpB release during fibrin polymerization, i.e. the specificity of thrombin for FpB, and not its location on the N terminus of the β chain, accounts for the kinetics of fibrinopeptide release from fibrinogen.Additional experiments support the conclusion that FpA′ release from A′β fibrinogen is dictated by thrombin specificity and not fibrin polymerization. We found that FpA′ release is qualitatively different from FpB release from normal fibrinogen, indicating that the release of FpA′ from the β chain is not dependent on prior polymerization of des-A monomers. This conclusion is supported by our studies in the presence or absence of GPRP, an inhibitor of fibrin polymerization. Our results showed that FpB release was impaired in the presence of GPRP, whereas FpA′ release was not affected. Together, these results indicate that FpA′ release from A′β fibrinogen is independent of fibrin polymerization. Thus, we have created a fibrinogen in which the delay in the rate enhancement of fibrinopeptide release from the β chain has been eliminated.We present the following models of the interaction of thrombin with fibrinogen, which accommodate our findings with A′β and normal fibrinogens. Initially, thrombin is oriented such that all three fibrinogen interaction sites (the apolar specificity pocket, fibrinogen-binding exosite, and active site) are aligned to accommodate FpA as the preferred substrate. Upon binding to A′β fibrinogen, thrombin is best suited for FpA or FpA′ cleavage, regardless of the placement of the peptide on the α or β chain; thus, all four fibrinopeptides are cleaved simultaneously and with similar affinity. In normal fibrinogen, the thrombin active site is specific for FpA, cleaving it efficiently to initiate polymerization, whereas thrombin cleavage of FpB is much less efficient. During polymerization, however, efficient cleavage of FpB becomes favorable, i.e. FpB release becomes more efficient upon the appearance of des-A polymers (2Blomback B. Hessel B. Hogg D. Therkildsen L. Nature. 1978; 275: 501-505Crossref PubMed Google Scholar, 3Hurlet-Jensen A. Cummins H.Z. Nossel H.L. Liu C.Y. Thromb. Res. 1982; 27: 419-427Abstract Full Text PDF PubMed Scopus (41) Google Scholar, 4Ruf 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, 15Lewis S.D. Shields P.P. Shafer J.A. J. Biol. Chem. 1985; 260: 10192-10199Abstract Full Text PDF PubMed Google Scholar). We propose two possible mechanisms, as depicted in Fig.8, for this shift to efficient cleavage of FpB. Either 1) upon the release of FpA, thrombin changes its conformation such that FpB can be accommodated and cleaved efficiently, or 2) the local conformation of FpB is altered over the course of polymerization, which correctly orients the Arg14–Gly15 bond for efficient cleavage within the active site of thrombin.Although our results do not give us reason to favor one possibility over the other, previous studies lend credence to both. Studies on thrombin binding to exosite-binding fragments of hirudin, heparin cofactor II, and the thrombin receptor have shown that exosite interaction can allosterically modify the active site of thrombin (28Naski M.C. Fenton J.W., II Maraganore J.M. Olson S.T. Shafer J.A. J. Biol. Chem. 1990; 265: 13484-13489Abstract Full Text PDF PubMed Google Scholar, 29Dennis S. Wallace A. Hofsteenje J. Stone S.R. Eur. J. Biochem. 1990; 188: 61-66Crossref PubMed Scopus (59) Google Scholar, 30Hortin G.L. Trimpe B.L. J. Biol. Chem. 1991; 266: 6866-6871Abstract Full Text PDF PubMed Google Scholar, 31Liu L.W. Vu T.K. Esmon C.T. Coughlin S.R. J. Biol. Chem. 1991; 266: 16977-16980Abstract Full Text PDF PubMed Google Scholar, 32De Cristofaro R. Rocca B. Bizzi B. Landolfi R. Biochem. J. 1993; 289: 475-480Crossref PubMed Scopus (32) Google Scholar). Because thrombin binds to fibrinogen in the exosite, it is possible that conformational changes that occur in fibrinogen during polymerization can indirectly affect the thrombin active site. Alternatively, conformational changes in the fibrinogen molecule have been proposed (2Blomback B. Hessel B. Hogg D. Therkildsen L. Nature. 1978; 275: 501-505Crossref PubMed Google Scholar, 3Hurlet-Jensen A. Cummins H.Z. Nossel H.L. Liu C.Y. Thromb. Res. 1982; 27: 419-427Abstract Full Text PDF PubMed Scopus (41) Google Scholar, 14Higgins D.L. Lewis S.D. Shafer J.A. J. Biol. Chem. 1983; 258: 9276-9282Abstract Full Text PDF PubMed Google Scholar, 23Ng A.S. Lewis S.D. Shafer J.A. Methods Enzymol. 1993; 222: 341-358Crossref PubMed Scopus (39) Google Scholar, 33Hantgan R.R. Hermans J. J. Biol. Chem. 1979; 254: 11272-11281Abstract Full Text PDF PubMed Google Scholar, 34Weisel J.W. Veklich Y. Gorkun O. J. Mol. Biol. 1993; 232: 285-297Crossref PubMed Scopus (130) Google Scholar, 35Veklich Y.I. Gorkun O.V. Medved L.V. Niewenhuizen W. Weisel J.W. J. Biol. Chem. 1993; 268: 13577-13585Abstract Full Text PDF PubMed Google Scholar) and measured (36Henschen-Edman A.H. Cell. Mol. Life Sci. 1997; 53: 29-33Crossref PubMed Scopus (19) Google Scholar) during fibrin polymerization, and these changes could reposition the E domain such that the scissile bond in FpB is properly oriented for efficient cleavage by thrombin. Whether it is conformational changes in fibrinopeptide B, thrombin, or both, these studies suggest that either model is possible and reemphasize that we can only conclude from our studies that it is the specificity of thrombin for the fibrinopeptides that dictates the rate and timing of their release.Production of A′β fibrinogen also allowed us to evaluate the effect of early exposure of the B site on fibrin polymerization. If early exposure of the B site did affect polymerization, we would expect a polymerization curve similar to that for fibrin monomer polymerization, when the A and B sites are exposed from the start of the reaction. Our results did not follow this pattern, thus suggesting that exposure of the B site alone does not directly influence polymerization. Rather, the participation of the B site in polymerization likely depends on certain polymerization events. Previous studies (33Hantgan R.R. Hermans J. J. Biol. Chem. 1979; 254: 11272-11281Abstract Full Text PDF PubMed Google Scholar, 37Weisel J.W. Nagaswami C. Biophys. J. 1992; 63: 111-128Abstract Full Text PDF PubMed Scopus (285) Google Scholar) suggest that this event is the polymerization of des-A monomers to form protofibrils of a critical length.In summary, by making this fibrinogen with essentially four fibrinopeptides A, we have synthesized a model substrate that eliminated the delay in fibrinopeptide release from the β chain. Thus, the normal delay in fibrinopeptide B release likely arises from a specific interaction between thrombin and FpB. We therefore conclude that the kinetics of fibrinopeptide release are dictated by the affinity of thrombin for its substrates. In addition, our work suggests that early exposure of the B site does not affect the polymerization process. We have synthesized a recombinant fibrinogen (A′β) to determine what effect the substitution of FpA′ on the N terminus of the β chain has on the kinetics of fibrinopeptide release. The release of FpA and FpA′ from A′β fibrinogen occurred from the beginning of the reaction and followed first-order kinetics, indicating that both FpA and FpA′ are equally competitive substrates for thrombin and have cleavage sites that are accessible to thrombin at the beginning of the reaction. Thus, the assumption used to characterize FpB release, that FpB release depends on prior FpA release, does not accurately describe the release of FpA′ from A′β fibrinogen. In addition, the similar rate of release of either FpA′ or FpB from the β chain indicates that despite the fibrinopeptide placed on the N terminus of the β chain, the rate of cleavage of the Arg–Gly bond by thrombin remains constant. Taken together, we conclude that it is thrombin, in its interaction with FpB, that is responsible for the delay in efficient FpB release during fibrin polymerization, i.e. the specificity of thrombin for FpB, and not its location on the N terminus of the β chain, accounts for the kinetics of fibrinopeptide release from fibrinogen. Additional experiments support the conclusion that FpA′ release from A′β fibrinogen is dictated by thrombin specificity and not fibrin polymerization. We found that FpA′ release is qualitatively different from FpB release from normal fibrinogen, indicating that the release of FpA′ from the β chain is not dependent on prior polymerization of des-A monomers. This conclusion is supported by our studies in the presence or absence of GPRP, an inhibitor of fibrin polymerization. Our results showed that FpB release was impaired in the presence of GPRP, whereas FpA′ release was not affected. Together, these results indicate that FpA′ release from A′β fibrinogen is independent of fibrin polymerization. Thus, we have created a fibrinogen in which the delay in the rate enhancement of fibrinopeptide release from the β chain has been eliminated. We present the following models of the interaction of thrombin with fibrinogen, which accommodate our findings with A′β and normal fibrinogens. Initially, thrombin is oriented such that all three fibrinogen interaction sites (the apolar specificity pocket, fibrinogen-binding exosite, and active site) are aligned to accommodate FpA as the preferred substrate. Upon binding to A′β fibrinogen, thrombin is best suited for FpA or FpA′ cleavage, regardless of the placement of the peptide on the α or β chain; thus, all four fibrinopeptides are cleaved simultaneously and with similar affinity. In normal fibrinogen, the thrombin active site is specific for FpA, cleaving it efficiently to initiate polymerization, whereas thrombin cleavage of FpB is much less efficient. During polymerization, however, efficient cleavage of FpB becomes favorable, i.e. FpB release becomes more efficient upon the appearance of des-A polymers (2Blomback B. Hessel B. Hogg D. Therkildsen L. Nature. 1978; 275: 501-505Crossref PubMed Google Scholar, 3Hurlet-Jensen A. Cummins H.Z. Nossel H.L. Liu C.Y. Thromb. Res. 1982; 27: 419-427Abstract Full Text PDF PubMed Scopus (41) Google Scholar, 4Ruf 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, 15Lewis S.D. Shields P.P. Shafer J.A. J. Biol. Chem. 1985; 260: 10192-10199Abstract Full Text PDF PubMed Google Scholar). We propose two possible mechanisms, as depicted in Fig.8, for this shift to efficient cleavage of FpB. Either 1) upon the release of FpA, thrombin changes its conformation such that FpB can be accommodated and cleaved efficiently, or 2) the local conformation of FpB is altered over the course of polymerization, which correctly orients the Arg14–Gly15 bond for efficient cleavage within the active site of thrombin. Although our results do not give us reason to favor one possibility over the other, previous studies lend credence to both. Studies on thrombin binding to exosite-binding fragments of hirudin, heparin cofactor II, and the thrombin receptor have shown that exosite interaction can allosterically modify the active site of thrombin (28Naski M.C. Fenton J.W., II Maraganore J.M. Olson S.T. Shafer J.A. J. Biol. Chem. 1990; 265: 13484-13489Abstract Full Text PDF PubMed Google Scholar, 29Dennis S. Wallace A. Hofsteenje J. Stone S.R. Eur. J. Biochem. 1990; 188: 61-66Crossref PubMed Scopus (59) Google Scholar, 30Hortin G.L. Trimpe B.L. J. Biol. Chem. 1991; 266: 6866-6871Abstract Full Text PDF PubMed Google Scholar, 31Liu L.W. Vu T.K. Esmon C.T. Coughlin S.R. J. Biol. Chem. 1991; 266: 16977-16980Abstract Full Text PDF PubMed Google Scholar, 32De Cristofaro R. Rocca B. Bizzi B. Landolfi R. Biochem. J. 1993; 289: 475-480Crossref PubMed Scopus (32) Google Scholar). Because thrombin binds to fibrinogen in the exosite, it is possible that conformational changes that occur in fibrinogen during polymerization can indirectly affect the thrombin active site. Alternatively, conformational changes in the fibrinogen molecule have been proposed (2Blomback B. Hessel B. Hogg D. Therkildsen L. Nature. 1978; 275: 501-505Crossref PubMed Google Scholar, 3Hurlet-Jensen A. Cummins H.Z. Nossel H.L. Liu C.Y. Thromb. Res. 1982; 27: 419-427Abstract Full Text PDF PubMed Scopus (41) Google Scholar, 14Higgins D.L. Lewis S.D. Shafer J.A. J. Biol. Chem. 1983; 258: 9276-9282Abstract Full Text PDF PubMed Google Scholar, 23Ng A.S. Lewis S.D. Shafer J.A. Methods Enzymol. 1993; 222: 341-358Crossref PubMed Scopus (39) Google Scholar, 33Hantgan R.R. Hermans J. J. Biol. Chem. 1979; 254: 11272-11281Abstract Full Text PDF PubMed Google Scholar, 34Weisel J.W. Veklich Y. Gorkun O. J. Mol. Biol. 1993; 232: 285-297Crossref PubMed Scopus (130) Google Scholar, 35Veklich Y.I. Gorkun O.V. Medved L.V. Niewenhuizen W. Weisel J.W. J. Biol. Chem. 1993; 268: 13577-13585Abstract Full Text PDF PubMed Google Scholar) and measured (36Henschen-Edman A.H. Cell. Mol. Life Sci. 1997; 53: 29-33Crossref PubMed Scopus (19) Google Scholar) during fibrin polymerization, and these changes could reposition the E domain such that the scissile bond in FpB is properly oriented for efficient cleavage by thrombin. Whether it is conformational changes in fibrinopeptide B, thrombin, or both, these studies suggest that either model is possible and reemphasize that we can only conclude from our studies that it is the specificity of thrombin for the fibrinopeptides that dictates the rate and timing of their release. Production of A′β fibrinogen also allowed us to evaluate the effect of early exposure of the B site on fibrin polymerization. If early exposure of the B site did affect polymerization, we would expect a polymerization curve similar to that for fibrin monomer polymerization, when the A and B sites are exposed from the start of the reaction. Our results did not follow this pattern, thus suggesting that exposure of the B site alone does not directly influence polymerization. Rather, the participation of the B site in polymerization likely depends on certain polymerization events. Previous studies (33Hantgan R.R. Hermans J. J. Biol. Chem. 1979; 254: 11272-11281Abstract Full Text PDF PubMed Google Scholar, 37Weisel J.W. Nagaswami C. Biophys. J. 1992; 63: 111-128Abstract Full Text PDF PubMed Scopus (285) Google Scholar) suggest that this event is the polymerization of des-A monomers to form protofibrils of a critical length. In summary, by making this fibrinogen with essentially four fibrinopeptides A, we have synthesized a model substrate that eliminated the delay in fibrinopeptide release from the β chain. Thus, the normal delay in fibrinopeptide B release likely arises from a specific interaction between thrombin and FpB. We therefore conclude that the kinetics of fibrinopeptide release are dictated by the affinity of thrombin for its substrates. In addition, our work suggests that early exposure of the B site does not affect the polymerization process. We gratefully acknowledge Li Fang Ping and Kasim McLain for excellent technical assistance in protein purification and production. We also gratefully acknowledge John Weisel, Chandrasekaran Nagaswami, and Yuri Veklich for teaching us the electron microscopic techniques and Victoria Madden and Bob Bagnell for technical assistance in this endeavor. We thank Frank Church for the generous donation of human α-thrombin." @default.
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W1994382696 title "Recombinant Fibrinogen Studies Reveal That Thrombin Specificity Dictates Order of Fibrinopeptide Release" @default.
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