SEARCH:   Advanced

Blood, 1 June 2004, Vol. 103, No. 11, pp. 4157-4163. Prepublished online as a Blood First Edition Paper on February 5, 2004; DOI 10.1182/blood-2003-12-4296. This Article Abstract Full Text (PDF) All Versions of this Article: 2003-12-4296v1 103/11/4157    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Okumura, N. Articles by Lord, S. T. Search for Related Content PubMed PubMed Citation Articles by Okumura, N. Articles by Lord, S. T. Related Collections Hemostasis, Thrombosis, and Vascular Biology Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY Substitution of the -chain Asn308 disturbs the D:D interface affecting fibrin polymerization, fibrinopeptide B release, and FXIIIa-catalyzed cross-linking Nobuo Okumura, Oleg V. Gorkun, Fumiko Terasawa, and Susan T. Lord From the Laboratory of Clinical Chemistry, Department of Biomedical Laboratory Sciences, School of Health Sciences, Shinshu University, Matsumoto, Japan; and the Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill.    Abstract Top Abstract Introduction Materials and methods Results Discussion References   Crystallographic structures indicate that -chain residue Asn308 participates in D:D interactions and indeed substitutions of Asn308 with lysine or isoleucine have been identified in dysfibrinogens with impaired polymerization. To probe the role of Asn308 in polymerization, we synthesized 3 variant fibrinogens: Asn308 changed to lysine (N308K), isoleucine (N308I), and alanine (N308A). We measured thrombin-catalyzed polymerization by turbidity, fibrinopeptide release by high-performance liquid chromatography, and factor XIIIa–catalyzed cross-linking by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. In the absence of added calcium, polymerization was clearly impaired with all 3 variants. In contrast, at 0.1 mM calcium, only polymerization of N308K remained markedly abnormal. The release of thrombin-catalyzed fibrinopeptide B (FpB) was delayed in the absence of calcium, whereas at 1 mM calcium FpB release was delayed only with N308K. Factor XIIIa–catalyzed - dimer formation was delayed with fibrinogen (in absence of thrombin), whereas with fibrin (in presence of thrombin) - dimer formation of only N308K was delayed. These data corroborate the recognized link between FpB release and polymerization. They show fibrin cross-link formation likely depends on the structure of protofibrils. Together, our results show substitution of Asn308 with a hydrophobic residue altered neither polymer formation nor polymer structure at physiologic calcium concentrations, whereas substitution with lysine altered both.    Introduction Top Abstract Introduction Materials and methods Results Discussion References   Fibrinogen is a 340-kDa glycoprotein that circulates in the blood at 5.88 to 11.76 µM (2-4 g/L). It is composed of a pair of 3 polypeptide chains, A,B, and , connected by 29 intrachain and interchain disulfide bonds.1 The 6 chains are arranged into 3 globular nodules. The central E nodule contains the N-termini of all chains and the distal D nodules contain the C-termini of the , , and a short segment of the chains. Coiled coils composed of all 3 chains link the E and D nodules.2 The C-terminal residues of A chains form globular nodules that are associated with one another, as well as with the E nodule.3 Recent crystallographic studies have provided high-resolution structures of the C-terminal region of the chain, which contains functional sites for fibrin polymerization, calcium binding, platelet aggregation, and factor XIII (FXIII) cross-linking.4-6 During coagulation, thrombin cleaves fibrinogen, releasing fibrinopeptides A (FpA) and B (FpB) from the N-termini of the A and B chains, respectively, and converting fibrinogen to fibrin monomers (for reviews, see Blomback1 and Doolittle2). Fibrin monomers polymerize spontaneously through a 2-step process, with a second step being calcium-dependent.7,8 The first step begins with the release of FpA. This exposes the "A" site, which interacts with the "a" site in the D nodule of another molecule, leading to the formation of half-staggered, double-stranded protofibrils.9,10 Within protofibrils, individual fibrin molecules also interact through the D nodule sites called the D:D interface.11-13 Subsequently, during the second step, the release of FpB exposes the "B" site, which likely interacts with the "b" site in the D nodule of another molecule to promote lateral aggregation of the protofibrils.5,6,8,10 During fibrin assembly FXIIIa catalyzes the formation of intermolecular isopeptide bonds, thereby cross-linking the individual fibrin molecules within the fibrin matrix.14 The final product is a complex, stable, insoluble, branching network of fibrin fibers and bundles of fibers. High-resolution crystal structures have identified the D nodule residues that likely compose the "a" and "b" sites, as well as several calcium-binding sites.6,15,16 These and subsequent crystal structures also identified the residues that likely form the D:D interface.16,17 This interface is unusual in 2 ways: it is not symmetric as might be expected for interactions between identical nodules and it is not supported by a significant network of hydrogen bonds or salt links. Nevertheless, specific residues within the chain appear to be critical to this interface. The analysis of dysfibrinogens has provided useful information for understanding the molecular details that promote thrombincatalyzed polymerization.18-21 Analysis of 2 dysfibrinogens with substitutions at -chain residue 308, Asn308 to lysine and Asn308 to isoleucine, indicated that this residue has an important role in polymerization.22-25 As is the case with most dysfibrinogens, these substitutions occurred in individuals who are heterozygous for the mutation. This complicates functional analysis because fibrinogen in these individuals is a complex mixture of normal and abnormal molecules.26,27 We have circumvented this complication by synthesizing recombinant variant fibrinogens and have examined the changes in function associated with changes in primary structure.28 Here we describe analysis of 3 variant fibrinogens with substitutions at Asn308 in the chain: N308K and N308I, analogous to dysfibrinogens, and N308A. Our data indicate that this residue has an important role in the D:D interactions that are critical for normal polymerization.    Materials and methods Top Abstract Introduction Materials and methods Results Discussion References   Materials All chemicals were reagent grade from Sigma (St Louis, MO), unless otherwise noted. Human -thrombin and FXIII were purchased from Enzyme Research Laboratories (South Bend, IN). Preparation of variant fibrinogens The construction of mutant expression vectors and the synthesis and purification of the recombinant fibrinogens have been described.29-31 Briefly, recombinant fibrinogens were expressed in Chinese hamster ovary (CHO) cells cultured in serum-free medium. Fibrinogen was purified from harvested culture medium by immunoaffinity chromatography using a calcium-dependent monoclonal antibody (IF-1; Iatron Laboratories, Tokyo, Japan). Fibrinogen was eluted with 5 mM EDTA (ethylenediaminetetraacetic acid), and fractions were pooled and dialyzed against 20 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), pH 7.4; 0.12 M NaCl (HBS), first with 1 mM CaCl2, and subsequently against HBS without added calcium. Dialyzed samples were aliquoted and stored at -80° C. The fibrinogen concentration was determined from the A280, assuming a 1 mg/mL solution has an absorbance of 1.51.32 As described previously, normal recombinant fibrinogen purified by this procedure is 96% clottable.30 Thrombin-catalyzed fibrin polymerization Polymerization was followed by turbidity at 350 nm using a UV-110-02 spectrophotometer (Shimadzu, Tokyo, Japan). Reactions were performed in a final volume of 100 µL as described elsewhere.30 Briefly, fibrinogen (90 µL at 0.3 mg/mL) in HBS, with or without added CaCl2, was mixed with human -thrombin (10 µL at 0.5 U/mL) and changes in turbidity were monitored at ambient temperature. Two parameters, lag period and the maximum slope, were obtained from the turbidity curves, as described elsewhere.33 The reactions were performed in triplicate. Thrombin-catalyzed fibrinopeptide release Thrombin-catalyzed fibrinopeptide release was monitored by reversedphase high-performance liquid chromatography (HPLC), essentially as described.34,35 Briefly, fibrinogen (0.1 mg/mL) in HBS, with or without 1 mM CaCl2, was incubated with human -thrombin (0.005 U/mL) at ambient temperature and the reactions stopped by incubation at 100° C. Fibrinopeptides were separated by HPLC and their concentrations determined from the peak areas measured with the ISCO Chemresearch 2.4 software. The data were fit to first-order kinetics (DeltaGraph; Deltapoint, Monterey, CA), assuming the release of FpB followed the release of FpA. Specificity constants, kcat/Km, were determined as described.34,35 Reactions were performed in triplicate. FXIIIa-catalyzed cross-linking of fibrin or fibrinogen FXIII (50 U/mL) was activated with human -thrombin (1 U/mL) for 60 minutes at 37° C in HBS with 5 mM CaCl2. To examine cross-linking of fibrin, fibrinogen (final concentration, 0.47 mg/mL) was incubated at 37° C with FXIIIa (final concentration, 3.3 U/mL) and human -thrombin (final concentration, 0.07 U/mL) in HBS and 0.67 mM calcium. To examine cross-linking of fibrinogen, hirudin (10 U/mL) was added to thrombin- -activated FXIIIa (final concentration, 3.3 U/mL) prior to incubation with fibrinogen (final concentration, 0.47 mg/mL) in HBS and 0.67 mM calcium. The reactions were stopped at various times by addition of an equal volume of sodium dodecyl sulfate (SDS) sample buffer with 2-mercaptoethanol and incubation (5 minutes) at 100° C. Samples equivalent to 4.7 µg fibrinogen were separated on 8% SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie brilliant blue R-250.31    Results Top Abstract Introduction Materials and methods Results Discussion References   Synthesis and characterization of recombinant fibrinogens We synthesized 3 variant fibrinogens with single amino acid substitutions in the C-terminal region of the chain: N308K, N308I, and N308A, where Asn308 is changed to lysine, isoleucine, and alanine, respectively. The variant fibrinogens were synthesized in cultured CHO cells and purified from the culture medium, as described.27,35 SDS-PAGE run under reducing conditions (Figure 1) showed the usual pattern of 3 bands corresponding to the A, B and chains, except, as previously reported,24,31 the migration of the -chain band in N308K and N308I was faster than normal. The unusual -chain migration was most obvious in fibrinogen Matsumoto II (N308K) purified from plasma, which showed 2 distinct bands corresponding to the normal and variant chains in this heterozygous individual (compare lane 5 to lane 1 or 6 in Figure 1). Similar shifts in band mobility have been reported for several dysfibrinogens with substitutions or small deletions in the chain.36-41 View larger version (29K): [in this window] [in a new window]   Figure 1.. Characterization of recombinant fibrinogens. Coomassie-stained 10% SDS-PAGE run under reducing conditions; normal recombinant fibrinogen (lane 1), N308K fibrinogen (lane 2), N308I fibrinogen (lane 3), N308A fibrinogen (lane 4), plasma fibrinogen from Matsumoto II patient (lane 5), and plasma fibrinogen from normal individual (lane 6). Positions of the molecular markers are indicated on the left (M).   Thrombin-catalyzed fibrin polymerization Thrombin-catalyzed fibrin polymerization was monitored as the change in turbidity at 350 nm, as described in "Materials and methods." Representative curves are shown in Figure 2. We measured 2 parameters from these curves: the lag time, which reflects the formation of protofibrils, and the maximum slope (Vmax), which reflects the rate at which protofibrils laterally aggregate with each other to form a fibrin fiber. When the experiments were performed in 20 mM HEPES, pH 7.4, 0.12 M NaCl and no added calcium (Figure 2A), polymerization was abnormal for all 3 variants. Polymerization of N308I fibrinogen was the most impaired, with the longest lag time and the slowest rise in turbidity. Compared to normal fibrinogen, the lag times of N308A and N308K fibrinogens are longer by 2- and 3.5-fold, respectively. The slopes of each of the polymerization curves were approximately the same as normal. Thus, under these conditions, the substitution of the bulky, hydrophobic isoleucine side chain resulted in the greatest delay in polymerization. As seen in Figure 2B, the results were markedly different in the presence of 1 mM CaCl2. The increase in calcium concentration led to a substantial improvement such that the polymerizations of N308A and N308I fibrinogens became similar to the polymerization of normal fibrinogen. The turbidity curves of N308A and N308I were similar to one another and to the curve of normal fibrinogen. In contrast, the curve with N308K fibrinogen remained markedly different from normal because the increase in calcium concentration only modestly improved the turbidity profile. In this respect, the addition of calcium improved polymerization of N308K fibrinogen to the same extent as the addition of calcium improved polymerization of normal fibrinogen. View larger version (17K): [in this window] [in a new window]   Figure 2.. Thrombin-catalyzed fibrin polymerization. Polymerization of fibrinogen (0.27 mg/mL) was initiated with thrombin (0.05 U/mL) and the change in turbidity at 350 nm was followed with time. Representative polymerization curves of normal (), N308K (), N308I (), and N308A () fibrinogens in the absence of added CaCl2 (A) and in the presence of 1 mM CaCl2 (B) are shown.   We further characterized the influence of calcium by performing polymerization experiments at several calcium concentrations. The average values for lag time and Vmax from 3 experiments are plotted in Figure 3 as a function of calcium concentration. The concentration of calcium had a marked influence on the polymerization of both hydrophobic substitutions and was greatest for the isoleucine substitution. For example, with N308I fibrinogen, the lag period was 6-fold longer and the Vmax 5-fold slower in the absence of added calcium relative to 1 mM CaCl2. With N308A fibrinogen the lag period was 1.9-fold longer and the Vmax 2.3-fold slower in the absence of added calcium relative to 1 mM CaCl2. The comparable values for normal fibrinogen were a 1.5-fold longer lag period and a 1.3-fold slower Vmax. As a result, at 1 mM CaCl2 polymerization of N308I fibrinogen and N308A fibrinogen was essentially normal. Overall with the variants N308I and N308A the most substantive influence of calcium occurred at less than 0.1 mM CaCl2. In contrast, with N308K fibrinogen, the changes with calcium concentration were analogous to those seen with normal fibrinogen; the lag period was 1.1-fold longer and the Vmax 1.6-fold slower in the absence of added calcium relative to 1 mM CaCl2. As a result, the difference between normal fibrinogen and N308K was relatively constant at all calcium concentrations, such that the respective curves in Figure 3 parallel one another. These experiments also showed that raising the calcium concentration to 5 mM had a minimal impact on polymerization of all 4 fibrinogens (Figure 3). View larger version (19K): [in this window] [in a new window]   Figure 3.. Influence of CaCl2 concentration on polymerization parameters. Thrombin-catalyzed fibrin polymerization was performed as indicated for Figure 2. For each calcium concentration, polymerization was performed 3 times. Average values of lag time (A) and Vmax (B) for normal (), N308K (), N308I (), and N308A () fibrinogens were determined as described in "Materials and methods."   Calcium dependence of fibrinopeptide release Thrombin-catalyzed fibrinopeptide release was monitored by HPLC and the data plotted as percent of peptide released as a function of time, as described in "Materials and methods." Because polymerization showed an unusual dependence on calcium concentration, we examined fibrinopeptide release with no added calcium and 1 mM CaCl2. Average specificity constants, kcat/Km, from 3 experiments are given in Table 1. As expected, the kinetics of FpA release was the same for all 4 fibrinogens and the rate of FpA release was not influenced by calcium. View this table: [in this window] [in a new window]   Table 1.. Specificity constants of thrombin-catalyzed fibrinopeptide release in the presence and the absence of added CaCl2   In contrast, the kinetics of FpB release from the variant fibrinogens was different from normal fibrinogen, and a strong calcium dependence of FpB release was apparent with N308A and N308I fibrinogens. In the absence of added calcium, FpB release from all variants was slower than normal with N308A being closest to normal and N308K being the most different. At 1 mM calcium, FpB release from N308I was indistinguishable from normal, whereas FpB release from N308A was slightly faster than normal. In contrast, FpB release from N308K at 1 mM calcium was not different from FpB release in the absence of added calcium, such that FpB release remained slower than normal. Thus, for all the variants the kinetics of FpB release correlated directly with polymerization. Under conditions where polymerization was impaired, FpB release was also slower, and when polymerization was normal, then FpB release was also normal. This correlation has been reported previously and was interpreted as indicating that FpB is released from small fibrin polymers.9,42-44 FXIIIa-catalyzed cross-linking of fibrin or fibrinogen Cross-linking of fibrin and chains was performed in the presence of FXIIIa and thrombin, and the reaction products analyzed by SDS-PAGE as described in "Materials and methods." Because FXIIIa is a calcium-dependent enzyme,45,46 we measured cross-linking only in the presence of calcium. The data are presented in Figure 4. With normal fibrin (Figure 4; N308), the expected - dimer and -polymer bands were readily apparent. As previously reported, the - dimer appeared first, weakly evident at the earliest time point, 2 minutes, and the 2 -polymer bands appeared later, evident at 5 minutes.47 With longer incubation, the intensity of - dimer and -polymer bands increased, whereas the intensity of - and -chain bands decreased. With N308I and N308A fibrins, the - dimer and 2 -polymer bands were evident after 2 and 20 minutes, respectively. As with normal fibrinogen, the intensities of these bands increased with time, whereas the intensities of the - and -chain bands decreased. In contrast, with N308K fibrin cross-link formation was delayed, the - dimer and -polymer bands being evident only after 20 and 60 minutes, respectively (Figure 4; N308K). The FXIIIa-catalyzed cross-linking of fibrin correlated with polymerization similarly to the correlation of FpB release with polymerization. When polymerization was impaired (N308K fibrin) the cross-linking was delayed. When polymerization was normal (N308A and N308I fibrins) the cross-linking was normal. View larger version (83K): [in this window] [in a new window]   Figure 4.. FXIIIa-catalyzed cross-linking of fibrin. Cross-linking of fibrin by FXIIIa was examined with 8% SDS-PAGE, under reducing conditions as described in "Materials and methods." Fibrinogen (0.47 mg/mL) was mixed with FXIIIa (3.3 U/mL) and thrombin (0.07 U/mL), and the reaction was incubated for the specified time at 37° C in 20 mM HEPES, pH 7.4; 0.12 M NaCl, and 0.67 mM CaCl2 buffer. The reduced fibrin chains (, , , cross-linked - dimer, and cross-linked -chain polymers) are indicated on the right side of the gels.   To circumvent the role of polymerization and examine the influence of the substitution per se, we examined FXIIIa-catalyzed cross-linking of fibrinogen.48 FXIII was first activated with thrombin, hirudin was added to inhibit the thrombin, the enzyme mixture was added to the fibrinogen solutions, and reaction products analyzed by SDS-PAGE; the data are presented in Figure 5. With all 4 fibrinogens, - dimer and polymers were visible in aliquots removed at the earliest time point, 10 minutes. With normal fibrinogen (Figure 5; N308), the intensity of the - dimer band had increased noticeably through the observed period. In contrast, with all 3 N308 variant fibrinogens the intensity of the - dimer band remained essentially constant throughout the incubation. These results indicated that all the substitutions influenced crosslinking of soluble fibrinogen. This differs from the cross-linking of polymerized fibrin. We conclude that polymerization, rather than the substitution per se, influenced the fibrin cross-linking data shown in Figure 4. Specifically, the data show that - dimer formation was normal for the N308A fibrin and N308I fibrin variants because polymerization of these variants was normal at this calcium concentration. In contrast, - dimer formation with N308K fibrin was delayed because polymerization of this fibrin was delayed. View larger version (82K): [in this window] [in a new window]   Figure 5.. FXIIIa-catalyzed cross-linking of fibrinogen. Cross-linking of fibrinogen by FXIIIa was examined with 8% SDS-PAGE, under reducing conditions as indicated for Figure 4, except that hirudin was added to FXIIIa prior to addition to fibrinogen and no thrombin was added to fibrinogen. The reduced fibrinogen chains (A, B, , cross-linked - dimer, and cross-linked -chain polymers) are indicated on the right side of the gels.      Discussion Top Abstract Introduction Materials and methods Results Discussion References   Our results support the conclusion that the C-terminal -chain residue Asn308 is important for normal D:D interactions and thus for normal polymerization. We examined 3 variants at this position, and in the absence of added calcium, each showed impaired polymerization. In contrast, at 0.1 mM calcium, polymerization of 2 of the variants, N308A and N308I, was only slightly impaired, whereas polymerization of N308K differed markedly from normal. These data suggest that the lysine substitution directly disrupts a critical aspect of polymerization, whereas the isoleucine or alanine substitutions do so only indirectly, in conjunction with calcium binding. Thus, the hydrophobic variants may disrupt calcium binding and thereby a critical aspect of polymerization, or they may disrupt a critical aspect of polymerization that is evident only in the absence of calcium. This remarkable calcium dependence was also apparent in the kinetics of thrombin-catalyzed FpB release, such that impaired FpB release always correlated with impaired polymerization. These results lend strong support to the previous conclusion that FpB is cleaved from small fibrin polymers, not fibrin monomers.9,42-44 Similarly, FXIIIa-catalyzed - dimer formation in fibrin was impaired only whenever polymerization was impaired. Because FXIIIa is a calcium-dependent enzyme, we did not examine the influence of calcium on - dimer formation. Nevertheless, because FXIIIa-catalyzed cross-linking of fibrinogen (Figure 5) was equally abnormal for all variants, we conclude that the data showed impaired fibrin cross-link formation (N308K; Figure 4) correlated with impaired polymerization (N308K; Figure 2B). Furthermore, these data suggest that the D:D conformation in the fibrin polymer is a more effective substrate for FXIIIa-catalyzed - dimer formation than the D:D conformation in fibrinogen, presumably because the D:D/E complex is a more effective substrate. Because the mutations at Asn308 in chain have been identified in several heterozygous dysfibrinogens, we anticipated the impaired polymerization of the recombinant variant proteins. We were, however, surprised that polymerization of these homogeneous recombinant variants was relatively normal. In our previous studies with recombinant fibrinogens analogous to the heterozygous dysfibrinogens Vlissingen/Frankfurt IV and Matsumoto I, polymerization of the recombinant proteins was substantially more impaired than the polymerization of fibrinogen isolated from these heterozygous patients.26,39,49 Indeed, neither recombinant protein polymerized at all. Data from the recombinant proteins and their dysfibrinogen counterparts are summarized in Table 2. The nearnormal polymerization of the N308I variant at physiologic calcium concentration might have been anticipated from the preliminary report on fibrinogen Baltimore III, where it was noted that addition of more than 7 mM calcium to patient plasma normalized the clotting time.50 However, subsequent analysis of fibrinogen purified from the heterozygous Baltimore III patient showed no disparity between calcium binding to the normal and abnormal chains.22 Analysis of the patient's fibrinogen in the absence of added calcium also showed that the kinetics of FpB release did not correlate with fibrin monomer polymerization, because the former was normal whereas the latter was delayed (Table 2).50 In contrast, with homozygous recombinant N308I fibrinogen, the Baltimore III substitution, both polymerization and FpB release were impaired in the absence of added calcium, and both were essentially normal as calcium concentration was increased to 0.1 mM. This difference likely reflects the experimental conditions because FpB release from the patient's fibrinogen was measured at higher fibrinogen and thrombin concentrations where fibrinopeptide release is rapid such that the decreased rate of FpB release might not be apparent (Table 2). View this table: [in this window] [in a new window]   Table 2.. Summary of variant fibrinogens and their functions   As reported previously, at 1 mM CaCl2 polymerization of the recombinant N308K was delayed relative to polymerization of fibrinogen isolated from the heterozygous Matsumoto II patient (Table 2).27 Further, the parallel calcium dependence seen when comparing normal fibrinogen and the N308K variant could have been anticipated from previous calcium-binding studies that showed normal calcium binding to fibrinogen isolated from the heterozygous Kyoto I patient (Table 2).24 In these studies, Yoshida et al24 measured calcium binding to D1 fragments isolated from the patient's fibrinogen (N308K substitution) and found essentially identical binding to normal and variant fragments, with Kd = 3.2 µM for the variant and 2.8 µM for the normal fragment. Thus, polymerization of the homogenous recombinant lysine variant did mimic polymerization of fibrinogen isolated from the analogous heterozygous patients, although the former was more impaired than the latter.24,27 As reported here, the delayed polymerization of N308K was associated with the delayed FpB release and FXIIIa-catalyzed fibrin cross-linking, which cannot be corrected with calcium. The role of residue Asn308 in polymerization has been considered in 2 reviews that correlated dysfibrinogens with crystallographic structures of fibrinogen fragments.19,21 The side chain of Asn308 forms hydrogen bonds with the backbone atoms of Tyr278 and of Gly309. These hydrogen bonds would be absent in any of the variants we examined. Introduction of a positively charged lysine at 308 would further destabilize the complex interactions, as the positively charged side chain of Arg275 also forms hydrogen bonds with the backbone of Gly309. Positive charge repulsion is also likely with Lys321. In the FXIIIa cross-linked D-dimer structure, a cluster of residues including Asn308, Gly309, Gly268, Arg275, and Met310 is found at or near the D:D interface between adjacent molecules.11 These residues are likely to be buried during formation of protofibrils, such that the altered polymerizations seen here reflect impaired protofibril formation. Therefore, it would be expected that substitutions at 308 would disrupt the initial alignment of fibrin monomers into protofibrils, and thereby impair FpB release. Further, the disruption of the D:D interface would be expected to influence FXIIIa-catalyzed - dimer formation in fibrin. The analyses of N308K fibrinogen were consistent with these expectations. Furthermore, our analyses with N308A and N308I fibrinogens suggest a hydrophobic side chain can be accommodated within the normal D:D interface, implying the positive charge on lysine mediated the impaired polymerization of N308K fibrinogen. In conclusion, our results show substitution of Asn308 in chain of fibrinogen with a hydrophobic residue isoleucine and alanine altered neither polymer formation nor polymer structure at physiologic calcium concentrations, whereas substitution with lysine, had altered both.    Acknowledgements   The authors acknowledge S. Kani for helping in the preparation of the figures.    Footnotes   Submitted December 19, 2003; accepted January 26, 2004. Prepublished online as Blood First Edition Paper, February 5, 2004; DOI 10.1182/blood-2003-12-4296. Supported by a grant from the Charitable Trust Clinical Pathology Research Foundation of Japan (N.O.) and National Institutes of Health grant HL-31048 (S.T.L.). The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734. Reprints: Nobuo Okumura, Laboratory of Clinical Chemistry, Department of Biomedical Laboratory Sciences, School of Health Sciences, Shinshu University, 3-1-1 Asahi, Matsumoto 390-8621, Japan; e-mail: nobuoku{at}gipac.shinshu-u.ac.jp.    References Top Abstract Introduction Materials and methods Results Discussion References   Blomback B. Fibrinogen and fibrin-proteins with complex roles in hemostasis and thrombosis. Thromb Res. 1996;83: 1-75.[CrossRef][Medline] [Order article via Infotrieve] Doolittle RF. Fibrinogen and fibrin. Ann Rev Biochem. 1984;53: 195-229.[Medline] [Order article via Infotrieve] Veklich YI, Gorkun OV, Medved LV, Nieuwenhuizen W, Weisel JW. Carboxyl-terminal portions of the alpha chains of fibrinogen and fibrin. Localization by electron microscopy and the effects of isolated C fragments on polymerization. J Biol Chem. 1993;268: 13577-13585.[Abstract/Free Full Text] Pratt KP, Cote HCF, Chung DW, Stenkamp RE, Davie EW. The primary fibrin polymerization pocket: three-dimensional structure of a 30 kDa C-terminal chain fragment complexed with the peptide Gly-Pro-Arg-Pro. Proc Natl Acad Sci U S A. 1997;94: 7176-7181.[Abstract/Free Full Text] Everse SJ, Spraggon G, Veerapandian L, Riley M, Doolittle RF. Crystal structure of fragment double-D from human fibrin with two different bound ligands. Biochemistry. 1998;37: 8637-8642.[CrossRef][Medline] [Order article via Infotrieve] Kostelansky MS, Betts L, Gorkun OV, Lord ST. 2.8 Å crystal structures of recombinant fibrinogen fragment D with and without two peptide ligands: GHRP binding to the "b" site disrupts its nearby calcium-binding site. Biochemistry. 2002;41: 12124-12132.[CrossRef][Medline] [Order article via Infotrieve] Boyer MH, Shainoff JR, Ratnoff OD. Acceleration of fibrin polymerization by calcium ions. Blood. 1972;39: 382-387.[Abstract/Free Full Text] Blomback B, Hessel B, Hogg D, Therkildsen L. A two-step fibrinogen-fibrin transition in blood coagulation. Nature. 1978;275: 501-505.[CrossRef][Medline] [Order article via Infotrieve] Hantgan RR, Hermans J. Assembly of fibrin. A light scattering study. J Biol Chem. 1979;254: 11272-11281.[Abstract/Free Full Text] Olexa SA, Budzynski AZ. Evidence for four different polymerization sites involved in human fibrin formation. Proc Natl Acad Sci U S A. 1980;77: 1374-1378.[Abstract/Free Full Text] Spraggon G, Everse S, Doolittle RF. Crystal structures of fragment D from human fibrinogen and its crosslinked counterpart from fibrin. Nature. 1997;389: 455-462.[CrossRef][Medline] [Order article via Infotrieve] Matsuda M, Baba M, Morimoto K, Nakamikawa C. `Fibrinogen Tokyo II' an abnormal fibrinogen with an impaired polymerization site on the aligned DD domain of fibrin molecules. J Clin Invest. 1983;72: 1034-1041.[Medline] [Order article via Infotrieve] Mosesson M, Siebenlist K, DiOrio J, Matsuda M, Hainfeld JF, Wall JS. The role of fibrinogen D-domain intermolecular association sites in the polymerization of fibrin and fibrinogen Tokyo II (275 Arg Cys). J Clin Invest. 1995;96: 1053-1058.[Medline] [Order article via Infotrieve] Folk JE, Finlayson JS. The -(-glutamyl) lysine crosslink and the catalytic role of transglutaminases. Adv Prot Chem. 1977;31: 1-133.[Medline] [Order article via Infotrieve] Cote HCF, Pratt KP, Davie EW, Chung DW. The polymerization pocket "a" within the carboxyterminal region of the chain of human fibrinogen is adjacent to but independent from the calcium binding site. J Biol Chem. 1997;272: 23792-23798.[Abstract/Free Full Text] Everse SJ, Spraggon G, Veerapandian L, Doolittle RF. Conformational changes in fragments D and double-D from human fibrin(ogen) upon binding the peptide ligand Gly-His-Arg-Proamide. Biochemistry. 1999;38: 2941-2946.[CrossRef][Medline] [Order article via Infotrieve] Doolittle RF. X-ray crystallographic studies of fibrinogen and fibrin. J Thromb Haemost. 2003;1: 1559-1565.[CrossRef][Medline] [Order article via Infotrieve] Henschen A, Kehl M, Southan C. Genetically abnormal fibrinogens-strategies for structure elucidation, including fibrinopeptide analysis. Curr Probl Clin Biochem. 1984;14: 273-320.[Medline] [Order article via Infotrieve] Cote HCF, Lord ST, Pratt KP. -Chain dysfibrinogenemias: molecular structure-function relationships of naturally occurring mutations in the chain of human fibrinogen. Blood. 1998;92: 2195-2212.[Free Full Text] Matsuda M, Sugo T, Yoshida N, et al. Structure and function of fibrinogen: insights from dysfibrinogens. Thromb Haemost. 1999;82: 283-290.[Medline] [Order article via Infotrieve] Everse SJ, Spraggon G, Doolittle, RF. A three-dimensional consideration of variant human fibrinogens. Thromb Haemost. 1998;80: 1-9.[Medline] [Order article via Infotrieve] Bantia S, Bell WR, Dang CV. Polymerization defect of fibrinogen Baltimore III due to a Asn308 Ile mutation. Blood. 1990;75: 1659-1663.[Abstract/Free Full Text] Okumura N, Furihata K, Terasawa F, Ishikawa S, Ueno I, Katsuyama T. Fibrinogen Matsumoto II: 308 Asn Lys (AAT AAG) mutation associated with bleeding tendency. Br J Haematol. 1996;94: 526-528.[CrossRef][Medline] [Order article via Infotrieve] Yoshida N, Terukina S, Okuma M, Moroi M, Aoki N, Matsuda M. Characterization of an apparently lower molecular weight -chain variant in fibrinogen Kyoto I. The replacement of -asparagine 308 by lysine which causes accelerated cleavage of fragment D1 by plasmin and the generation of a new plasmin cleavage site. J Biol Chem. 1988; 263: 13848-13856.[Abstract/Free Full Text] Grailhe P, Boyer-Neumann C, Haverkate F, Grimbergen J, Larrieu MJ, Angles-Cano E. The mutation in fibrinogen Bicetre II (Asn308 Lys) does not affect the binding of t-PA and plasminogen to fibrin. Blood Coag Fibrinolysis. 1993;4: 679-687.[Medline] [Order article via Infotrieve] Hogan KA, Lord ST, Okumura N, et al. A functional assay suggests that heterodimers exist in two C-terminal -chain dysfibrinogens: Matsumoto I and Vlissingen/Frankfurt IV. Thromb Haemost. 2000;83: 592-597.[Medline] [Order article via Infotrieve] Okumura N, Terasawa F, Fujita K, Fujihara N, Tozuka M, Koh C-S. Evidence that heterodimers exist in the fibrinogen Matsumoto II (308N K) proband and participate in fibrin fiber formation. Thromb Res. 2002;107: 157-162.[CrossRef][Medline] [Order article via Infotrieve] Lord ST, Gorkun OV. Insight from studies with recombinant fibrinogens. Ann N Y Acad Sci. 2001;936: 101-116.[Medline] [Order article via Infotrieve] Okumura N, Furihata K, Terasawa F. Nakagoshi R, Ueno I, Katsuyama T. Fibrinogen Matsumoto I: A 364 Asp His (GAT CAT) substitution associated with defective fibrin polymerization. Thromb Haemost. 1996;75: 887-891.[Medline] [Order article via Infotrieve] Gorkun OV, Veklich YI, Weisel JW, Lord ST. The conversion of fibrinogen to fibrin: recombinant fibrinogen typifies plasma fibrinogen. Blood. 1997;89: 4407-4414.[Abstract/Free Full Text] Okumura N, Terasawa F, Fujita K, Tozuka M, Ota H, Katsuyama T. Difference in electrophoretic mobility and plasmic digestion profile between four recombinant fibrinogens, 308K, 308I, 308A, and wild type (308N). Electrophoresis. 2000;21: 2309-2315.[Medline] [Order article via Infotrieve] Mihalyi E. Physicochemical studies of bovine fibrinogen, IV: ultraviolet absorption and its relation to the structure of the molecule. Biochemistry. 1968;7: 208-223.[CrossRef][Medline] [Order article via Infotrieve] Furlan M, Rupp C, Beck EA. Inhibition of fibrin polymerization by fragment D is affected by calcium, Gly-Pro-Arg and Gly-His-Arg. Biochim Biophys Acta. 1983;742: 25-32.[CrossRef][Medline] [Order article via Infotrieve] Ng AS, Lewis SD, Shafer JA. Quantifying thrombin-catalyzed release of fibrinopeptides from fibrinogen using high-performance liquid chromatography. Methods Enzymol. 1993;222: 341-358.[Medline] [Order article via Infotrieve] Lord ST, Strickland E, Jayjock E. Strategy for recombinant multichain protein synthesis: fibrinogen B-chain variants as thrombin substrates. Biochemistry. 1996;35: 2342-2348.[CrossRef][Medline] [Order article via Infotrieve] Terukina S, Yamazumi K, Okamoto K, Yamashita H, Ito Y, Matsuda M. Fibrinogen Kyoto III: a congenital dysfibrinogen with a aspartic acid-330 to tyrosine substitution manifesting impaired fibrin monomer polymerization. Blood. 1989;74: 2681-2687.[Abstract/Free Full Text] Miyata T, Furukawa K, Iwanaga S, Takamatsu J, Saito H. Fibrinogen Nagoya, a replacement of glutamine-329 by arginine in the -chain that impairs the polymerization of fibrin monomer. J Biochem (Tokyo). 1989;105: 10-14.[Abstract/Free Full Text] Yoshida N, Hirata H, Imaoka S, Matsuda M, Yamazumi K, Asakura S. Effect of calcium on the mobility of -chain from fibrinogen Osaka V on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Thromb Res. 1994;73: 79-82.[CrossRef][Medline] [Order article via Infotrieve] Hogan KA, Gorkun OV, Lounes KC, et al. Recombinant fibrinogen Vlissingen/Frankfurt IV. The deletion of residues 319 and 320 from the chain of fibrinogen alters calcium binding, fibrin polymerization, cross-linking, and platelet aggregation. J Biol Chem. 2000;275: 17778-17785.[Abstract/Free Full Text] Koopman J, Haverkate F, Briet E, Lord ST. A congenitally abnormal fibrinogen (Vlissingen) with a 6-base deletion in the -chain gene, causing defective calcium binding and impaired fibrin polymerization. J Biol Chem. 1991;266: 13456-13461.[Abstract/Free Full Text] Mullin JL, Brennan SO, Ganly PS, George PM. Fibrinogen Hillsborough: a novel Gly309Asp dysfibrinogen with impaired clotting. Blood. 2002; 99: 3597-3601.[Abstract/Free Full Text] Nossel HL, Hurlet-Jensen A, Liu CY, Koehn JA, Canfield RE. Fibrinopeptide release from fibrinogen. Ann N Y Acad Sci. 1983;408: 269-278.[CrossRef][Medline] [Order article via Infotrieve] Lewis SD, Shields PP, Shafer JA. Characterization of the kinetic pathway for liberation of fibrin-opeptides during assembly of fibrin. J Biol Chem. 1985;260: 10192-10199.[Abstract/Free Full Text] Ruf W, Bender A, Lane DA, Preissner KT, Selmayr E, Muller-Berghaus G. Thrombin-induced fibrinopeptide B release from normal and variant fibrinogens: influence of inhibitors of fibrin polymerization. Biochim Biophy Acta. 1988;965: 169-175.[Medline] [Order article via Infotrieve] Muszbek L, Adany R, Mikkola H. Novel aspects of blood coagulation factor XIII, I: structure, distribution, activation, and function. Crit Rev Clin Lab Sci. 1996;33: 357-421.[Medline] [Order article via Infotrieve] Ariëns RA, Lai T-S, Weisel JW, Greenberg CS, Grant PJ. Role of factor XIII in fibrin clot formation and effects of genetic polymorphisms. Blood. 2002;100: 743-754.[Abstract/Free Full Text] McKee PA, Mattock P, Hill R. Subunit structure of human fibrinogen, soluble fibrin, and cross-linked insoluble fibrin. Proc Natl Acad Sci U S A. 1970; 66: 738-744.[Abstract/Free Full Text] Kanaide H, Shainoff JR. Cross-linking of fibrinogen and fibrin by fibrin-stabilizing factor (factor XIIIa). J Lab Clin Med. 1975;85: 574-597.[Medline] [Order article via Infotrieve] Okumura N, Gorkun OV, Lord ST. Severely impaired polymerization of recombinant fibrinogen -364Asp His, the substitution discovered in a heterozygous individual. J Biol Chem. 1997;47: 29596-29601. Ebert RF, Bell WR. Fibrinogen Baltimore III: congenital dysfibrinogenemia with a shortened -subunit. Thromb Res. 1988;51: 251-258.[CrossRef][Medline] [Order article via Infotrieve] Marchi R, Loyau S, Angles-Cano E, Weisel JW. Structure and properties of clots form fibrinogen Bicetre II (308Asn Lys). Increased permeability due to a larger pores, thicker fibers and decreased rigidity. Ann N Y Acad Sci. 2001;936: 125-128.[Medline] [Order article via Infotrieve] CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This article has been cited by other articles: T. A. Dugan, V. W.-C. Yang, D. J. McQuillan, and M. Hook Decorin Modulates Fibrin Assembly and Structure J. Biol. Chem., December 15, 2006; 281(50): 38208 - 38216. [Abstract] [Full Text] [PDF] S. Kani, F. Terasawa, K. Yamauchi, M. Tozuka, and N. Okumura Analysis of fibrinogen variants at {gamma}387Ile shows that the side chain of {gamma}387 and the tertiary structure of the {gamma}C-terminal tail are important not only for assembly and secretion of fibrinogen but also for lateral aggregation of protofibrils and XIIIa-catalyzed {gamma}-{gamma} dimer formation Blood, September 15, 2006; 108(6): 1887 - 1894. [Abstract] [Full Text] [PDF] This Article Abstract Full Text (PDF) All Versions of this Article: 2003-12-4296v1 103/11/4157    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Okumura, N. Articles by Lord, S. T. Search for Related Content PubMed PubMed Citation Articles by Okumura, N. Articles by Lord, S. T. Related Collections Hemostasis, Thrombosis, and Vascular Biology Social Bookmarking                   What's this?

	Copyright © 2004 by American Society of Hematology         Online ISSN: 1528-0020