Variant Fibrinogens Research Articles

Heterozygous variant fibrinogen γA289V (Kanazawa III) was confirmed as hypodysfibrinogenemia by plasma and recombinant fibrinogens.

Congenital fibrinogen disorders are classified as afibrinogenemia, hypofibrinogenemia, dysfibrinogenemia, and hypodysfibrinogenemia. However, difficulties are associated with discriminating between dysfibrinogenemia, hypofibrinogenemia, and hypodysfibrinogenemia using routine analyses. We previously reported a heterozygous variant fibrinogen (γA289V; Kanazawa III) as hypodysfibrinogenemia; however, the same variant had previously been described as hypofibrinogenemia. To clarify the production of γA289V fibrinogen, we expressed recombinant γA289V (r-γA289V) fibrinogen and compared it with wild-type (WT) and adjacent recombinant variant fibrinogens. Target mutations were introduced into a fibrinogen γ-chain expression vector by site-directed mutagenesis, and the vector was then transfected into Chinese hamster ovary cells to produce recombinant fibrinogen. Fibrinogen was purified from the plasma of the proposita, and culture media and fibrinogen functions were analyzed using fibrin polymerization, plasmin protection, and FXIIIa-catalyzed fibrinogen cross-linking. The fibrinogen concentration ratio of the culture media to cell lysates was markedly lower for r-γA289V fibrinogen than for WT. Because the secretion of recombinant γF290L (r-γF290L) fibrinogen was similar to WT, we compared r-γF290L fibrinogen functions with WT. The fibrin polymerization of Kanazawa III plasma (K-III) fibrinogen was significantly weaker than normal plasma fibrinogen. Moreover, K-III fibrinogen showed a markedly reduced "D:D" interaction. However, all functions of r-γF290L fibrinogen were similar to WT. An in silico analysis confirmed the above results. The present results demonstrated that γA289 is crucial for the γ-module structure, and the γA289V substitution markedly reduced fibrinogen secretion. Moreover, K-III fibrinogen showed markedly reduced fibrin polymerization and "D:D" interactions. γA289V fibrinogen was confirmed as hypodysfibrinogenemia.

Screening Method for Hepatic Fibrinogen Storage Disease Associated with Congenital Hypofibrinogenemia

Background: Hepatic endoplasmic reticulum (ER) storage disease (HERSD), which causes the accumulation of variant proteins that induce liver cirrhosis, was originally reported with a homozygous deficiency in A1AT (α1-antitrypsine, G342K, Z-mutation). The molecular mechanisms responsible for the A1AT Z mutation causing hepatocyte injuries have been examined extensively, and have been associated with increased ER retention as a conformational disease, the activation of ER-specific stress responses, up-regulated mitochondrial autophagy, and mitochondrial injury and apoptosis. Fibrinogen storage disease (FSD) or HERSD associated with hypofibrinogenemia was initially reported in patients with the γG284R variant (Brennan SO: Am J Pathol 2000; 157: 189-96). Another three types of heterozygous γ-chain variant fibrinogens in the C terminal region (γT314P, deletion of γG346-Q350, and γR375W) have also been identified, with several cases of γR375W being analyzed and designated as the Aguadilla mutation. We previously established γR375W variant fibrinogen-producing Chinese hamster ovary (CHO) cells, and subsequently compared the synthesis of variant fibrinogens and morphological characteristics of these cells with those of wild-type fibrinogen-producing cells. The γR375W variant secreted less fibrinogen and producing cells indicated two types of aberrant cytoplasmic inclusion bodies, namely large-granular bodies and fibrous forms by immunofluorescence. We also demonstrated that these inclusion bodies were derived from ER using confocal laser scanning microscopy (CLSM) and transmission electron microscopy (Kobayashi T: Thromb Res 2014; 133: 101-7).Objectives: The aim of the present study was to determine whether the generation of cell lines and subsequent immunostaining were applicable as a screening method for FSD. We establish cell lines reported as FSD other than γR375W and also reported as hypofibrinogenemia, but not FSD.Methods: The expression vectors coding variant γ-chains were altered by oligonucleotide-directed mutagenesis and stably transfected into CHO cells expressing normal human fibrinogen Aα- and Bβ-chains (AαBβ-CHO cells). The synthesis of fibrinogen (media and cell lysates) was measured by ELISA for each cloned cell line and morphological characteristics were observed under immunofluorescence microscopy. The expression vector coding γR375W was single transfected into CHO cells or co-transfected with a normal Aα- or Bβ-chain expression vector.Results: The medium/cell lysate fibrinogen ratios of variant cells reported as FSD were markedly lower (γG284R: 0.05 ± 0.01, γT314P: 0.08 ± 0.04, and deletion of γG346-Q350: 0.08 ± 0.05) than those of normal cells (0.77 ± 0.22). In γT314P and deletion of γG346-Q350-fibrinogen-producing cells, immunofluorescence staining showed aberrant cytoplasmic inclusion bodies including the large-granular and fibrous forms observed in γR375W-producing cells. However, immunofluorescence staining for γG284R-fibrinogen-producing cells did not show aberrant cytoplasmic inclusion bodies including the large-granular and fibrous forms observed in γR375W-produing cells.The medium/cell lysate fibrinogen ratios of variant cells reported as hypofibrinogenemia and non-FSD were also markedly lower (γS313N: 0.07 ± 0.01, γC326S: 0.01 ± 0.00, γC326Y: 0.00 ± 0.00, γC326A: 0.02 ± 0.00, γM336I: 0.07 ± 0.01, γA341D: 0.06 ± 0.01, and γN345D: 0.05 ± 0.00) than those of normal cells. Immunofluorescence staining showed normal cytoplasmic patterns for γC326Y-, γM336I-, and γA341D-CHO cells, but large-granular forms (less than 10%) for γS313N-, γC326S-, γC326A-, and γN345D-CHO cells.Although CHO cells transfected with the γR375W vector into AαBβ-CHO cells showed many aberrant inclusion bodies including the fibrous form, CHO cells transfected with not only the singular γR375W vector, but also the combined γR375W and normal Aα- or Bβ-chain expression vector did not have any aberrant cytoplasmic inclusion bodies.Conclusion: These results demonstrated that the establishment of variant fibrinogen-producing CHO cells and observation of inclusion bodies with the fibrous form by immunostaining may be applicable as a screening method for FSD. Furthermore, aberrant cytoplasmic inclusion bodies were formed by the assembly of the variant γ-chain into normal Aα- and Bβ-chains. DisclosuresNo relevant conflicts of interest to declare.

Differences in the function and secretion of congenital aberrant fibrinogenemia between heterozygous γD320G (Okayama II) and γΔN319-ΔD320 (Otsu I)

BackgroundWe encountered two patients with hypodysfibrinogenemia and designated them as Okayama II and Otsu I. Although the affected residue(s) in Okayama II and Otsu I overlapped, functionally determined fibrinogen levels and the ratio of functionally to immunologically determined plasma fibrinogen levels were markedly different. MethodsDNA sequence and functional analyses were performed for purified plasma fibrinogen. A recombinant protein was synthesized in Chinese hamster ovary (CHO) cells to determine the secretion of variant fibrinogens. ResultsA heterozygous A>G in FGG, resulting in γ320Asp>Gly for Okayama II, and a heterozygous deletion of AATGAT in FGG, resulting in the deletion of γAsn319 and γAsp320 (γΔN319-ΔD320) for Otsu I, were obtained. SDS-PAGE and Coomassie staining revealed that the variant γ-chain was not clear in Okayama II, but was clearly present in Otsu I. The lag period for the fibrin polymerization of Okayama II was slightly slower than that of the normal control, whereas Otsu I fibrinogen indicated no polymerization within 30min. Both variant γ-chains were synthesized in CHO cells and assembled into fibrinogen; however, the fibrinogen concentration ratio of the medium/cell lysate of γ320Gly was six-fold lower than that of γΔN319-ΔD320. ConclusionsWe concluded that the plasma fibrinogen of Okayama II, constituted by a lower ratio of the variant γ-chain, led to the almost normal functioning of fibrin polymerization. However, the plasma fibrinogen of Otsu I, with a higher ratio of the variant γ-chain, led to marked reductions in fibrin polymerization.

Fibrinopeptide A release is necessary for effective B:b interactions in polymerisation of variant fibrinogens with impaired A:a interactions

Fibrin polymerisation is mediated by interactions between knobs 'A' and 'B' exposed by thrombin cleavage, and holes 'a' and 'b'. We demonstrated markedly delayed thrombin-catalysed fibrin polymerisation, through B:b interactions alone, of recombinant γD364H -fibrinogen with impaired hole 'a'. To determine whether recombinant variant fibrinogens with no release of fibrinopeptide A (FpA) polymerise similarly to γD364H -fibrinogen, we examined two variant fibrinogens with substitutions altering knob 'A', Aα17A- and Aα17C-fibrinogen. We examined thrombin- or batroxobin-catalysed fibrinopeptide release by HPLC, fibrin clot formation by turbidity and fibrin clot structure by scanning electron microscopy (SEM) and compared the results of the variants with those for γ D364H-fibrinogen. Thrombin-catalysed FpA release of Aα17A-fibrinogen was substantially delayed and none observed for Aα17C-fibrinogen; fibrinopeptide B (FpB) release was delayed for all variants. All variant fibrinogens showed substantially impaired thrombin-catalysed polymerisation; for Aα17A-fibrinogen it was delayed less, and for Aα17C more than for γD364H -fibrinogen. No variants polymerised with batroxobin, which exposed only knob 'A'. The inhibition of variant fibrinogens' polymerisation was dose-dependent on the concentration of either GPRP or GHRP, and both peptides that block holes 'b'. SEM showed that the variant clots from Aα17A- and γD364H-fibrinogen had uniform, ordered fibres, thicker than normal, whereas Aα17C -fibrinogen formed less organised clots with shorter, thinner, and tapered ends. These results demonstrate that FpA release per se is necessary for effective B:b interactions during polymerisation of variant fibrinogens with impaired A:a interactions.

Fibrinogen residue γAla341 is necessary for calcium binding and ‘A-a’ interactions

The fibrinogen γ-module has several important sites relating to fibrinogen function, which include the high affinity calcium binding site, hole 'a' that binds with knob 'A', and the D:D interface. Residue γAla341, which is located in the vicinity of these sites, is altered in three variant fibrinogens: fibrinogen Seoul (γAla341Asp), Tolaga Bay (γAla341Val), and Lyon III (γAla341Thr). In order to investigate the impaired polymerisation of fibrinogens γAla341Asp and γAla341Val to understand the role of γAla341 in fibrin polymerisation and fibrinogen synthesis, we have expressed γAla341Asp and γAla341Val in Chinese hamster ovary (CHO) cells, purified these fibrinogens from the culture media and performed biochemical tests to elucidate their function. Expression in CHO cells was similar for these variants. For both variants the kinetics of thrombin-catalysed FpA release was not different from normal fibrinogen, while FpB release was slower than that of normal. Thrombin-catalysed polymerisation of both variants was dependent on the calcium concentration. At physiologic calcium (1 mM) the variants showed impaired polymerisation with a longer lag period and a slower Vmax than normal fibrinogen. Scanning electron micrographs showed the clots were less organised than normal, having thicker and more twisted fibers, and larger pores. Analysis by SDS-PAGE showed that factor XIIIa-catalysed γ and α chain cross-linking was delayed, and plasmin-catalysed lysis was not reduced by the presence of 5 mM calcium or 5 mM GPRP (Gly-Pro-Arg-Pro). Our data indicate that fibrinogen residue γAla341 is important for the proper conformation of the γ-module, maintaining calcium-binding site and 'A-a' interactions.

Heterozygous Bβ-chain C-terminal 12 amino acid elongation variant, BβX462W (Kyoto VI), showed dysfibrinogenemia

A heterozygous patient with dysfibrinogenemia with slight bleeding and no thrombotic complications was diagnosed with fibrinogen Kyoto VI (K-VI). To elucidate the genetic mutation(s) and characterize the variant protein, we performed the following experiments and compared with identical and similar variants that have already been reported. The proposita's PCR-amplified DNA was analyzed by sequencing and her purified plasma fibrinogen underwent SDS-PAGE followed by immunoblotting, fibrin polymerization, and scanning electron microscopic observation of fibrin clot and fibers. Sequence analyses showed that K-VI fibrinogen substituted W (TGG) for terminal codon (TAG), resulting in 12 amino acid elongation 462-473 (WSPIRRFLLFCM) in the Bβ-chain. Protein analyses indicated that the presence of some albumin-binding variant fibrinogens and a dimeric molecule of variant fibrinogens reduced fibrin polymerization, with a thinner fiber and aberrant fibrin network. These results are almost the same as for the identical variant of Magdeburg, however, different from the similar variant of Osaka VI [12 amino acid elongation 462-473 (KSPIRRFLLFCM) in the Bβ-chain] in the presence of variant forms and clot structure. We speculate the side-chain difference at 462 residues, W in K-VI, K in Osaka VI, and/or the difference in the presence of disulfide bridged forms of variant fibrinogens, led to the notable difference in the fibrin bundle network. Although a strong evolutional and structural association between Bβ-chain and γ-chain molecules is established, the corresponding recombinant 15 residue elongation variants of the fibrinogen γ-chain showed reduced assembly and secretion.

Molecular Mechanisms Affecting Fibrin Structure and Stability

Fibrin structure and stability have been linked to many thrombotic diseases, including venous thromboembolism. Analysis of the molecular mechanisms that affect fibrin structure and stability became possible when the crystal structure of fibrinogen was solved. Biochemical studies of natural and recombinant variant fibrinogens have examined the interactions that mediate the conversion of soluble fibrinogen to the insoluble fibrin network. These studies identified intermolecular interactions that control fibrin structure, although some critical events remain ambiguous. Studies show that fibrin structure modulates the enzymatic lysis of the fibrin network, so the molecular mechanisms that control structure also control stability. Studies show that the mechanical stability of the fibrin clot depends on the properties of the fibrin monomer, leading investigators to explore the molecular basis of the monomer's mechanical properties. The work summarized here provides insights that might allow the development of pharmaceuticals and treatments to modulate fibrin structure and stability in vivo and thereby prevent or limit thrombotic disease.

Fibrinogen residue γAla341 Is Necessary for Calcium Binding and ‘A-a’ interaction.

AbstractAbstract 1154Fibrinogen, a 340-kDa glycoprotein has essential roles in blood coagulation and platelet aggregation. Fibrinogen is a complex molecule consisting of 3 pairs of Aα, Bβ, and γ chains intertwined to form a tri-nodular molecule with 2 terminal D regions and a central E region. The fibrinogen γ-nodule, a part of D region, has several important sites relating to fibrinogen function, which are the high affinity calcium binding site, hole ‘a’ that binds with knob ‘A’, and the D:D interface. Residue γAla341, which is located in the vicinity of those sites and conserved between all available species, is altered in two variant fibrinogens: fibrinogen Seoul (γAla341Asp) (Song et al., Clin Appl Thromb Hemost 2006) and fibrinogen Tolaga Bay (γAla341Val) (Davis et al., Thromb Haemost 2007). Fibrinogen Seoul showed hypodysfibrinogenemia, and fibrinogen Tolaga Bay hypofibrinogenemia. We have expressed these two variant fibrinogens in CHO cells, purified them from the culture media and performed biochemical tests to elucidate their function. Thrombin-catalyzed kinetics of FpA release was not different (p-value, 0.3, n=3) from normal fibrinogen. Average specificity constant, kcat/Km for FpA was 7.4±1.8, 5.7±1.8, and 8.1±1.2 (mean±SD, 106M-1s-1) for normal, γAla341Val, and γAla341Asp, respectively. However, FpB release from both variants was slower than that of normal (p-value, 0.005 in One Way ANOVA; p-value, 0.006 and 0.002 for normal vs γAla341Val, and normal vs γAla341Asp, respectively in multiple comparison). Average specificity constant for FpB was 3.6±1.1, 1.7±0.4, and 1.4±0.3 (mean±SD, 106M-1s-1) for normal, γAla341Val, and γAla341Asp, respectively. We measured fibrin polymerization by turbidity with the final thrombin and fibrinogen concentration being 0.2mg/mL and 0.1U/mL, respectively. At 10mM calcium we saw no turbidity rising with either variant. Both variants showed impaired polymerization with a longer lag period and a slower Vmax than normal fibrinogen at physiologic 1mM calcium. Lag period, which reflects protofibril formation, for normal, γAla341Val, and γAla341Asp was 110±30, 2,500±150, and 1,200±40, respectively (mean±SD, sec). Vmax, which reflects lateral aggregation, was 81±26, 15±3, and 3±1 (mean±SD, 10-5s-1) for normal, γAla341Val, and γAla341Asp. With the FXIIIa cross-linking, measured by SDS-PAGE, we found that γ and α chain cross-linking was delayed in both variants. We tested the calcium binding and the functionality of ‘a’ polymerization site with the plasmin protection assay. Both variants were not protected from plasminolysis in the presence of 1mM calcium or 0.55mM GPRP, indicative of impaired binding of calcium and knob ‘A’. Given these results, both fibrinogen Seoul and Tolaga Bay likely have a conformational change in their calcium and GPRP binding sites resulting in the impaired fibrin polymerization. In conclusion, we think fibrinogen residue γAla341 is important for calcium binding, ‘A-a’ interactions and the conformation of the γ-nodule. Disclosures:No relevant conflicts of interest to declare.

From Fibrinogen to Fibrin: Dynamic Light Scattering to Probe the Mechanism of Protofibril Assembly Into Fibers.

AbstractAbstract 1156The conversion from fibrinogen to fibrin, and fibrin clot structure and function, are important problems in cardiovascular research. The mechanism of fibrin clot formation remains undefined after more than 50 years of study. We have developed a new method to monitor polymerization and examine the products formed prior to gelation. We monitored polymerization by dynamic light scattering (DLS), a sensitive method to measure formation of small complexes. We “stopped” thrombin-catalyzed polymerization at different phases by crosslinking the polymers with formaldehyde and analyzed the products by DLS, agarose gel electophoresis (AGE) and transmission electron microscopy (TEM). DLS of the formaldehyde fixed products showed the products were stable and were analogous to the polymerization reactions with about a 2 minute delay in the average radius. As shown in the table, AGE showed a decrease in the numbers of small molecules and the appearance of larger molecules with polymerization. Preliminary TEM results showed a similar progression with AGE and clearly displayed the polymerization process from fibrin monomers, oligomers, protofibrils, then fibers. About 6% protofibrils were observed at 2 minutes and continuously increased with polymerization. Small amounts of fibers appeared from 4 minutes, and increased both in sizes and numbers with polymerization. At 10 minutes, about 8% fibers were observed and the length of large fibers reached around 3 μm. TEM also showed that after 7 minutes, the polymerization products were relatively heterogeneous with a high dispersity of sizes. With these experiments we followed the polymerization process step by step and determined the changes between steps by comparing adjacent-time products. Additionally, our laboratory specializes in synthesizing recombinant variant fibrinogens that have proven useful in studying functional attributes of fibrinogen. By DLS, we examined polymerization of variants in calcium binding sites: the single substitution gE132A (in the b2 calcium site) and the double substitutions gD318A,D320A (in the high affinity calcium site) and gD298A,D301A (in the g2 calcium site). For gD298A,D301A the DLS profile was similar to normal fibrinogen with a slower formation of protofibrils, as expected for the slightly prolonged lag time. For gE132A DLS showed a more rapid rise and higher plateau than normal fibrinogen. The initial polymerization is the same as normal fibrinogen shown in DLS, indicating the rate of protofibril formation for gE132A is the same as normal. After protofibril formation, the rapid rise in DLS for gE132A preceded the rapid rise for normal fibrin, indicating that assembly of the variant protofibrils into fibers was faster than normal. For gD318A,D230A fibrinogen we saw a slight change in DLS, such that the average radius increased 2-fold in 8.5 hours. This change in radius was not different after 13 hours, indicating a stable complex was formed. Further experiments are needed to characterize this complex. Considered together, these initial studies show that DLS can provide additional insight into the influence of structure on polymerization. The comparison of the normal fibrinogen to variant fibrinogens will help us to better understand the role of functional sites in polymerization and identify the residues of fibrinogen that influence protofibril formation and lateral aggregation.Table 1AGE result of polymerization products crosslinked by formaldehyede at different timepoints.ConstituentPercent composition10 min0 min2 min4 min7 minmonomer97.4%84.4%83.7%80.3%56.8%dimer2.6%14.3%12.5%11.7%22.1%trimer1.3%3.8%3.9%13.0%tetramer2.5%3.6%pentamer1.6%3.5%hexamer1.0% Disclosures:No relevant conflicts of interest to declare.

Recombinant variant fibrinogens substituted at residues γ326Cys and γ339Cys demonstrated markedly impaired secretion of assembled fibrinogen

Background To study the functions of residues γ326Cys and γ339Cys in the assembly and/or secretion of fibrinogen, recombinant fibrinogens were synthesized to replicate naturally occurring γ326Tyr and γ326Ser variants, along with γ326Ala and γ339Ala variants. Methods A fibrinogen γ-chain expression vector was altered and transfected into Chinese hamster ovary (CHO) cells. Cell lysates and culture media of the established cell lines were subjected to ELISA and immunoblotting analysis. In addition, pulse-chase analysis was performed. Results The CHO cells synthesized mutant γ-chains and assembled these into fibrinogen in the cells, although these variant fibrinogens were barely secreted into the culture media. Pulse-chase analysis indicated that the rates of both assembly and secretion of the variant fibrinogens were lower than that of normal fibrinogen. Conclusions The present study indicated that the 326-339 intrachain disulfide bond has a crucial role in maintaining the tertiary structure of the C-terminal domain of the γ-module, which is necessary for fibrinogen assembly and specifically secretion. A combination of the present results and observations from naturally occurring heterozygous cases of γ326Tyr and γ326Ser suggest that heterozygous fibrinogen molecules containing variant γ-chains might be secreted into plasma and show impaired fibrin polymerization, resulting in a phenotype of hypodysfibrinogenemia.

B:b interactions are essential for polymerization of variant fibrinogens with impaired holes 'a'.

Fibrin polymerization is mediated by interactions between knobs 'A' and 'B' exposed by thrombin cleavage, and holes 'a' and 'b' always present in fibrinogen. The role of A:a interactions is well established, but the roles of knob:hole interactions A:b, B:b or B:a remain ambiguous. To determine whether A:b or B:b interactions have a role in thrombin-catalyzed polymerization, we examined a series of fibrinogen variants with substitutions altering holes 'a': gamma364Ala, gamma364His or gamma364Val. We examined thrombin- and reptilase-catalyzed fibrinopeptide release by high-performance liquid chromatography, fibrin clot formation by turbidity, fibrin clot structure by scanning electron microscopy (SEM) and factor (F) XIIIa-catalyzed crosslinking by sodium dodecylsulfate polyacrylamide gel electrophoresis. Thrombin-catalyzed fibrinopeptide A release was normal, but fibrinopeptide B release was delayed for all variants. The variant fibrinogens all showed markedly impaired thrombin-catalyzed polymerization; polymerization of gamma364Val and gamma364His were more delayed than gamma364Ala. There was absolutely no polymerization of any variant with reptilase, which exposed only knobs 'A'. SEM showed that the variant clots formed after 24 h had uniform, ordered fibers that were thicker than normal. Polymerization of the variant fibrinogens was inhibited dose-dependently by the addition of either Gly-Pro-Arg-Pro (GPRP) or Gly-His-Arg-Pro (GHRP), peptides that specifically block holes 'a' and 'b', respectively. FXIIIa-catalyzed crosslinking between gamma-chains was markedly delayed for all the variants. These results demonstrate that B:b interactions are critical for polymerization of variant fibrinogens with impaired holes 'a'. Based on these data, we propose a model wherein B:b interactions participate in protofibril formation.

Mutant Fibrinogen Cleared from the Endoplasmic Reticulum via Endoplasmic Reticulum-Associated Protein Degradation and Autophagy: An Explanation for Liver Disease

The endoplasmic reticulum (ER) quality control processes recognize and remove aberrant proteins from the secretory pathway. Several variants of the plasma protein fibrinogen are recognized as aberrant and degraded by ER-associated protein degradation (ERAD), thus leading to hypofibrinogenemia. A subset of patients with hypofibrinogenemia exhibit hepatic ER accumulation of the variant fibrinogens and develop liver cirrhosis. One such variant named Aguadilla has a substitution of Arg375 to Trp in the gamma-chain. To understand the cellular mechanisms behind clearance of the aberrant Aguadilla gamma-chain, we expressed the mutant gammaD domain in yeast and found that it was cleared from the ER via ERAD. In addition, we discovered that when ERAD was saturated, aggregated Aguadilla gammaD accumulated within the ER while a soluble form of the polypeptide transited the secretory pathway to the trans-Golgi network where it was targeted to the vacuole for degradation. Examination of Aguadilla gammaD in an autophagy-deficient yeast strain showed stabilization of the aggregated ER form, indicating that these aggregates are normally cleared from the ER via the autophagic pathway. These findings have clinical relevance in the understanding of and treatment for ER storage diseases.

Comparison of thrombin‐catalyzed fibrin polymerization and Factor XIIIa‐catalyzed cross‐linking of fibrin among three recombinant variant fibrinogens, γ275C, γ275H, and γ275A

We have previously reported that recombinant gamma 275Cys fibrinogen exhibits a marked impairment of functions as well as aberrant fibrin clot and bundle structures, as compared with wild-type, gamma 275Arg, and plasma fibrinogen from a heterozygous proband. Since gamma Arg275His mutations have also been reported in 10 families, we synthesized recombinant gamma 275His fibrinogen and gamma 275Ala fibrinogen (as a control) and analyzed and compared them with gamma 275Cys and gamma 275Arg. A variant gamma-chain expression plasmid was transfected into Chinese hamster ovary cells expressing normal human fibrinogen A alpha- and B beta-chains. After purification of the recombinant variant fibrinogens, we performed functional analyzes for thrombin-catalyzed fibrin polymerization and factor XIIIa (FXIIIa)-catalyzed gamma-gamma dimer formation from fibrin or fibrinogen and also ultrastructural analysis of fibrin clots and bundles. By comparison with both gamma 275His and gamma 275Ala fibrinogens, recombinant gamma 275Cys fibrinogen exhibited a more impaired gamma-gamma dimer formation from fibrin or fibrinogen, a more aberrant fibrin clot structure, and thicker fibers in fibrin bundles. In 1 : 1 mixtures of gamma 275Arg and gamma 275Cys fibrinogens or gamma 275Arg and gamma 275His fibrinogens, thrombin-catalyzed fibrin polymerization and both fibrin clot and fiber structures showed some compensation (as compared with gamma 275Cys or gamma 275His alone). These results strongly suggest that an amino acid substitution of gamma 275Arg alone disrupts D:D interactions in thrombin-catalyzed fibrin polymerization and the formation of fibrin bundles and fibrin clots. Moreover, the existence of a subsequent disulfide-linked Cys in gamma 275C fibrinogen augments the impairment caused by a His or Ala substitution.

Substitution of the γ-chain Asn308 disturbs the D:D interface affecting fibrin polymerization, fibrinopeptide B release, and FXIIIa-catalyzed cross-linking

AbstractCrystallographic structures indicate that γ-chain residue Asn308 participates in D:D interactions and indeed substitutions of γAsn308 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: γAsn308 changed to lysine (γN308K), isoleucine (γN308I), and alanine (γN308A). 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 γN308K 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 γN308K. Factor XIIIa–catalyzed γ-γ dimer formation was delayed with fibrinogen (in absence of thrombin), whereas with fibrin (in presence of thrombin) γ-γ dimer formation of only γN308K 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.

Recombinant fibrinogen, γ275Arg→Cys, exhibits formation of disulfide bond with cysteine and severely impaired D:D interactions

Analysis of dysfibrinogens has provided useful information aiding our understanding of molecular defects in fibrin polymerization. We have already reported impaired fibrin polymerization in a variant fibrinogen (gammaArg275Cys), the Cys being located in the D:D interface. Since this substitution occurred in a heterozygous individual, interpretation of the functional analysis was complicated. We tried to resolve this complication by synthesizing a recombinant variant fibrinogen. A variant gamma-chain expression plasmid was transfected into Chinese hamster ovary cells expressing normal human fibrinogen Aalpha- and Bbeta-chains. The recombinant variant fibrinogen (gamma275C) was purified using an immunoaffinity column, and we compared its structure and functions with those of normal recombinant fibrinogen (gamma275R) and plasma variant fibrinogen. Mass analyses showed the existence of disulfide-linked Cys in both patient and recombinant variant fibrinogens. Functional analyses indicated that both fibrin polymerization and gamma-gamma dimer formation were markedly impaired in the variant fibrinogen. The impairments were much more pronounced in gamma275C than in plasma variant fibrinogen. In addition, scanning electron microscopic observation of fibrin clots made from gamma275C revealed less dense fibrin fiber bundles and larger fiber diameter than in those made from gamma275R, and also the existence of many aberrant fibrin fibers with tapered ends. These results indicate that gammaArg275 has an important residue affecting the structure and function of the gamma-chain C-terminal domain. However, the variant D:D interface can interact with that of the normal fibrinogen existing in a heterozygous patient with dysfibrinogenemia.

Fibrinogens Kosai and Ogasa: Bβ15Gly→Cys (GGT→TGT) substitution associated with impairment of fibrinopeptide B release and lateral aggregation

We found two heterozygous dysfibrinogenemias, designated fibrinogen Kosai and fibrinogen Ogasa. Kosai was associated with arteriosclerosis obliterans but Ogasa showed no bleeding or thrombotic tendencies. The plasma fibrinogen concentrations from the two propositi (Ogasa and Kosai) were much lower when determined by the thrombin-time method (0.94 and 1.06 g L(-1), respectively) than when determined by the immunological method (2.87 and 2.72 g L(-1), respectively). We performed DNA sequencing and functional analyses to clarify the relationship between the structural and functional abnormalities. Genetic analysis of PCR-amplified DNA from the propositi identified the heterozygous substitution Bbeta15Gly-->Cys (GGT-->TGT). Western blotting analysis of purified fibrinogen revealed the existence of albumin-fibrinogen complexes. Functional analyses indicated that compared with the normal control, the propositi's fibrinogen released only half the normal amount of fibrinopeptide B and showed markedly impaired polymerization. In addition, the observation of thinner fibers in fibrin clots (by scanning electron microscopy) indicated markedly defective lateral aggregation in the variant fibrinogens. The impaired functions may be due to the substitution of Cys for Bbetao15Gly plus the existence of some additional disulfide-bonded forms.

Analysis of fibrinogen γ-chain truncations shows the C-terminus, particularly γIle387, is essential for assembly and secretion of this multichain protein

To examine the role of the fibrinogen gamma chain in the assembly and secretion of this multichain protein, we synthesized a series of fibrinogen variants with truncated gamma chains, terminating between residues gamma379 and the C-terminus, gamma411. The variant fibrinogens were synthesized from altered gamma-chain complementary DNAs in cultured Chinese hamster ovary cells. Immunoassays of the culture media demonstrated that only those variants with gamma chain longer than 386 residues were secreted and that the concentration of fibrinogen decreased with the length of the gamma chain, from 1.4 microg/mL for normal fibrinogen to 0.39 microg/mL for gamma 387 fibrinogen. Immunoassays of cell lysates showed that all variant gamma chains were synthesized, although the levels varied significantly. For variants longer than 386 residues, levels decreased with length but remained near normal. In contrast, expression of the 4 variants with 386 residues or less was about 20-fold reduced. Quantitative reverse transcription-polymerase chain reaction demonstrated that the gamma-chain messenger RNA level was independent from chain length. Western blot analyses showed that lysates expressing variants with 387 residues or more contained species comparable to the known intermediates in fibrinogen assembly, including half-molecules. For shorter variants, these intermediates were not evident. We conclude that residues near the C-terminus of the gamma chain are essential for fibrinogen assembly, and more specifically, that gamma387 is critical. We propose that the loss of residue gamma387 destabilized the structure of gamma chain, preventing assembly of alphagamma and betagamma dimers, essential intermediates in the assembly of normal fibrinogen.

Analysis of engineered fibrinogen variants suggests that an additional site mediates platelet aggregation and that "B-b" interactions have a role in protofibril formation.

The C-terminal domain of the fibrinogen gamma-chain includes multiple functional sites that have been defined in high-resolution structures and biochemical assays. Calcium binds to this domain through the side chains of gammaD318 and gammaD320 and the backbone carbonyls of gammaF322 and gammaG324. We have examined variant fibrinogens with alanine at position gamma318 and/or gamma320 and found that calcium binding, fibrin polymerization, and fibrinogen-mediated platelet aggregation, but not FXIIIa-catalyzed cross-linking, were abnormal. When measured by turbidity, thrombin-catalyzed polymerization was severely reduced, and batroxobin-catalyzed polymerization was completely obliterated. Moreover, thrombin-catalyzed polymerization was abolished by the peptide GHRP, which binds to the polymerization site in the beta-chain but does not inhibit polymerization of normal fibrinogen. ADP-induced platelet aggregation was also severely impaired. In contrast, as measured by SDS-PAGE, FXIIIa introduced cross-links between gamma-chains for all three variants, as expected if the gamma-chain C-terminal sites were normal. In addition, binding of the monoclonal antibody 4A5, which recognizes the C-terminal residues, was not different from normal. These data suggest two specific conclusions: (1) a site in the gamma-module other than the C-terminus is critical for platelet aggregation and (2) "B-b" interactions have a role in protofibril formation.

Clot lysis of variant recombinant fibrinogens confirms that fiber diameter is a major determinant of lysis rate.

Previous studies have suggested that clots with thinner fiber diameter lyse at slower rates than clots with thicker fiber diameter. We examined lysis of fibrin clots formed from three variant fibrinogens, each with substitutions in the N-terminal region of the B beta chain. When we measured lysis as the rate of decrease in turbidity at 350 nm, we found that the rate of lysis was slower than normal for clots with thinner fibers. We noted, however, that the time to complete lysis was the same for all clots. Thus, when the data were considered as the percent of lysis with time, we found that the curves were the same as normal. We suggest that a complete and accurate characterization of clot dissolution requires comparison of normalized lysis rates.

Polymerization site a function dependence on structural integrity of its nearby calcium binding site.

To explore the functional relationship between the polymerization site a and the nearby high affinity calcium binding site, we analyzed four variant fibrinogens with substitutions at these sites: gamma D364A in the a site and gamma D318A, gamma D320A, and gamma D318 + gamma D320A in the Ca2+ site. In all cases fibrinopeptide A release was normal and thrombin catalyzed polymerization was markedly impaired (unpublished observations). We examined the functional connection between the Ca2+ site and the a site by testing for plasmin protection in the presence of Ca2+ or the a site peptide ligand GPRP. SDS-PAGE analysis of the products showed that gamma D364A fibrinogen was protected from plasmin cleavage by Ca2+ but not by the GPRP peptide. In contrast, neither Ca2+ nor the GPRP peptide protected gamma D318A, gamma D320A, or gamma D318 + gamma D320A fibrinogens from complete plasmin cleavage. These results suggest that the structural integrity of the calcium binding site is required for expression of the a site. In contrast, the structural integrity of the a site has no functional consequence on Ca2+ binding to this high affinity site.