Recombinant γY278H Fibrinogen Showed Normal Secretion from CHO Cells, but a Corresponding Heterozygous Patient Showed Hypofibrinogenemia.

We identified a novel heterozygous hypofibrinogenemia, γY278H (Hiroshima). To demonstrate the cause of reduced plasma fibrinogen levels (functional level: 1.12 g/L and antigenic level: 1.16 g/L), we established γY278H fibrinogen-producing Chinese hamster ovary (CHO) cells. An enzyme-linked immunosorbent assay demonstrated that synthesis of γY278H fibrinogen inside CHO cells and secretion into the culture media were not reduced. Then, we established an additional five variant fibrinogen-producing CHO cell lines (γL276P, γT277P, γT277R, γA279D, and γY280C) and conducted further investigations. We have already established 33 γ-module variant fibrinogen-producing CHO cell lines, including 6 cell lines in this study, but only the γY278H and γT277R cell lines showed disagreement, namely, recombinant fibrinogen production was not reduced but the patients' plasma fibrinogen level was reduced. Finally, we performed fibrinogen degradation assays and demonstrated that the γY278H and γT277R fibrinogens were easily cleaved by plasmin whereas their polymerization in the presence of Ca2+ and "D:D" interaction was normal. In conclusion, our investigation suggested that patient γY278H showed hypofibrinogenemia because γY278H fibrinogen was secreted normally from the patient's hepatocytes but then underwent accelerated degradation by plasmin in the circulation.

Thermal shift assay to probe melting of thrombin, fibrinogen, fibrin monomer, and fibrin: Gly-Pro-Arg-Pro induces a fibrin monomer-like state in fibrinogen.

BACKGROUND: Thrombin activates fibrinogen and binds the fibrin E-domain (Kd ~ 2.8 µM) and the splice variant γ'-domain (Kd ~ 0.1 µM). We investigated if the loading of D-Phe-Pro-Arg-chloromethylketone inhibited thrombin (PPACK-thrombin) onto fibrin could enhance fibrin stability. METHODS: A 384-well plate thermal shift assay (TSA) with SYPRO-orange provided melting temperatures (Tm) of thrombin, PPACK-thrombin, fibrinogen, fibrin monomer, and fibrin. RESULTS: Large increases in Tm indicated that calcium led to protein stabilization (0 vs. 2 mM Ca2+) for fibrinogen (54.0 vs. 62.3 °C) and fibrin (62.3 vs. 72.2 °C). Additionally, active site inhibition with PPACK dramatically increased the Tm of thrombin (58.3 vs. 78.3 °C). Treatment of fibrinogen with fibrin polymerization inhibitor GPRP increased fibrinogen stability by ΔTm = 9.3 °C, similar to the ΔTm when fibrinogen was converted to fibrin monomer (ΔTm = 8.8 °C) or to fibrin (ΔTm = 10.4 °C). Addition of PPACK-thrombin at high 5:1 M ratio to fibrin(ogen) had little effect on fibrin(ogen) Tm values, indicating that thrombin binding does not detectably stabilize fibrin via a putative bivalent E-domain to γ'-domain interaction. CONCLUSIONS: TSA was a sensitive assay of protein stability and detected: (1) the effects of calcium-stabilization, (2) thrombin active site labeling, (3) fibrinogen conversion to fibrin, and (4) GPRP induced changes in fibrinogen stability being essentially equivalent to that of fibrin monomer or polymerized fibrin. SIGNIFICANCE: The low volume, high throughput assay has potential for use in understanding interactions with rare or mutant fibrin(ogen) variants.

Mutations Causing Mild or No Structural Damage in Interfaces of Multimerization of the Fibrinogen γ-Module More Likely Confer Negative Dominant Behaviors.

Different pathogenic variants in the same protein or even within the same domain of a protein may differ in their patterns of disease inheritance, with some of the variants behaving as negative dominant and others as autosomal recessive mutations. Here is presented a structural analysis and comparison of the molecular characteristics of the sites in fibrinogen γ-module, a fibrinogen component critical in multimerization processes, targeted by pathogenic variants (HGMD database) and by variants found in the healthy population (gnomAD database). The main result of this study is the identification of the molecular pathogenic mechanisms defining which pattern of disease inheritance is selected by mutations at the crossroad of autosomal recessive and negative dominant modalities. The observations in this analysis also warn about the possibility that several variants reported in the non-pathogenic gnomAD database might indeed be a hidden source of diseases with autosomal recessive inheritance or requiring a combination with other disease-causing mutations. Disease presentation might remain mostly unrevealed simply because the very low variant frequency rarely results in biallelic pathogenic mutations or the coupling with mutations in other genes contributing to the same disease. The results here presented provide hints for a deeper search of pathogenic mechanisms and modalities of disease inheritance for protein mutants participating in multimerization phenomena.

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

INTRODUCTION: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Fibrinogen Columbus II: A novel c.1075G>T mutation in the FGG gene causing hypodysfibrinogenemia and thrombosis in an adolescent male.

Hypodysfibrinogenemia, the least frequently reported congenital fibrinogen disorder is characterized by low circulating levels of a dysfunctional protein, and is associated with phenotypic features of both hypo- and dysfibrinogenemia. Herein, we report an adolescent male with unprovoked venous thromboembolism and hypodysfibrinogenemia. Patient had recurrent, progressive thrombosis despite therapeutic anticoagulation with both low molecular weight heparin and warfarin. He had clinical and radiological improvement after transition to a direct thrombin inhibitor. Sequencing of the FGG gene identified a novel heterozygous mutation, c.1075G>T. Structural visualization of the identified variant was pursued and suggested that the mutation likely destabilizes the Ca2+ -binding site of fibrinogen resulting in pathogenicity.

Impact of γ'γ' fibrinogen interaction with red blood cells on fibrin clots.

AIM: γ' fibrinogen has been associated with thrombosis. Here the interactions between γ'γ' or γAγA fibrinogen and red blood cells (RBCs), and their role on fibrin clot properties were studied. MATERIALS & METHODS: Atomic Force microscopy (AFM)-based force spectroscopy, rheological, electron and confocal microscopy, and computational approaches were conducted for both fibrinogen variants. RESULTS & CONCLUSION: AFM shows that the recombinant human (rh)γ'γ' fibrinogen increases the binding force and the frequency of the binding to RBCs compared with rhγAγA, promoting cell aggregation. Structural changes in rhγ'γ' fibrin clots, displaying a nonuniform fibrin network were shown by microscopy approaches. The presence of RBCs decreases the fibrinolysis rate and increases viscosity of rhγ'γ' fibrin clots. The full length of the γ' chain structure, revealed by computational analysis, occupies a much wider surface and is more flexible, allowing an increase of the binding between γ' fibers, and eventually with RBCs.

Fibrinogen splice variation and cross-linking: Effects on fibrin structure/function and role of fibrinogen γ' as thrombomobulin II.

Fibrin is an important matrix protein that provides the backbone to the blood clot, promoting tissue repair and wound healing. Its precursor fibrinogen is one of the most heterogeneous proteins, with an estimated 1 million different forms due to alterations in glycosylation, oxidation, single nucleotide polymorphisms, splice variation and other variations. Furthermore, ligation by transglutaminase factor XIII (cross-linking) adds to the complexity of the fibrin network. The structure and function of the fibrin network is in part determined by this natural variation in the fibrinogen molecule, with major effects from splice variation and cross-linking. This mini-review will discuss the direct effects of fibrinogen αEC and fibrinogen γ' splice variation on clot structure and function and also discuss the additional role of fibrinogen γ' as thrombomodulin II. Furthermore, the effects of cross-linking on clot function will be described. Splice variation and cross-linking are major determinants of the structure and function of fibrin and may therefore impact on diseases affecting bleeding, thrombosis and tissue repair.

Genetic analyses of novel compound heterozygous hypodysfibrinogenemia, Tsukuba I: FGG c.1129+62_65 del AATA and FGG c.1299+4 del A.

INTRODUCTION: We found a novel hypodysfibrinogenemia designated Tsukuba I caused by compound heterozygous nucleotide deletions with FGG c.1129+62_65 del AATA and FGG c.1299+4 del A on different alleles. The former was deep in intron 8 of FGG (IVS-8 deletion) and the latter in exon 9 of FGG (Ex-9 deletion), which is translated for the γ'-chain, but not the γA-chain. A Western blot analysis of plasma fibrinogen from our patient revealed an aberrant γ-chain that migrated slightly faster than the normal Bß-chain. MATERIALS AND METHODS: To clarify the complex genetic mechanism underlying Tsukuba I's hypodysfibrinogenemia induced by nucleotide deletions in two regions, we generated two minigenes incorporating each deletion region, transfected them into Chinese Hamster Ovary (CHO) cells, and analyzed RT-PCR products. We also established CHO cells producing the recombinant variant fibrinogen, γ'409ΔA (Ex-9 deletion). RESULTS AND CONCLUSIONS: Minigene I incorporating the IVS-8 deletion showed two products: a normal splicing product and the unspliced product. Minigene II incorporating the Ex-9 deletion only produced the unspliced product. The established γ'409ΔA-CHO cells secreted variant fibrinogen more effectively than normal fibrinogen. Therefore, the aberrant splicing products derived from the IVS-8 deletion cause hypofibrinogenemia most likely due to nonsense-mediated mRNA decay and the partial production of normal γA- and γ'-chains; moreover, the Ex-9 deletion causes hypodysfibrinogenemia due to the absence of normal γA- and γ'-chain production (hypofibrinogenemia) and augmented aberrant γ'-chain production (dysfibrinogenemia).

Diagnosis of congenital fibrinogen disorders.

Congenital fibrinogen disorders comprise quantitative disorders defined by a complete absence (afibrinogenemia) or by a decreased level (hypofibrinogenemia) of circulating fibrinogen and qualitative disorders characterized by a discrepancy between the activity and the antigenic levels of fibrinogen (dysfibrinogenemia and hypodysfibrinogenemia). The biological diagnosis is based on a standard haemostasis assessment. All the coagulation tests that depend on the formation of fibrin as the end point are affected; although in dysfibrinogenemia the specificity and sensitivity of routine test depend on reagent and techniques. A genetic exploration permits to confirm the diagnosis and may enhance the prediction of the patient's phenotype. Homozygous or composite heterozygous null mutations are most often responsible for afibrinogenemia while hypofibrinogenemic patients are mainly heterozygous carrier of an afibrinogenemic allele. Heterozygous missense mutations are prevalent in dysfibrinogenemia, with two hot spot localized in exon 2 of the FGA and in the exon 8 of the FGG. The correlation between phenotype and genotype has been identified in some fibrinogen variants, including six mutations clustered in exons 8 and 9 of the FGG leading to hypofibrinogenemia with hepatic inclusions of abnormal fibrinogen aggregates as well as a few mutations associated with an increase risk of thrombotic events. A familial screening and additional functional assays should be carried out when possible.

Hepatic fibrinogen storage disease: identification of two novel mutations (p.Asp316Asn, fibrinogen Pisa and p.Gly366Ser, fibrinogen Beograd) impacting on the fibrinogen γ-module.

BACKGROUND: Quantitative fibrinogen deficiencies (hypofibrinogenemia and afibrinogenemia) are rare congenital disorders characterized by low/unmeasurable plasma fibrinogen antigen levels. Their genetic basis is invariably represented by mutations within the fibrinogen genes (FGA, FGB and FGG coding for the Aα, Bß and γ chains). Currently, only four mutations (p.Gly284Arg, p.Arg375Trp, delGVYYQ 346-350, p.Thr314Pro), all affecting the fibrinogen γ chain, have been reported to cause fibrinogen storage disease (FSD), a disorder characterized by protein aggregation, endoplasmic reticulum retention and hypofibrinogenemia. OBJECTIVES: To investigate the genetic basis of FSD in two hypofibrinogenemic patients. METHODS: The mutational screening of the fibrinogen genes was performed by direct DNA sequencing. The impact of identified mutations on fibrinogen structure was investigated by in-silico molecular modeling. Liver histology was evaluated by light microscopy, electron microscopy and immunocytochemistry. RESULTS: Here, we describe two hypofibrinogenemic children with persistent abnormal liver function parameters. Direct sequencing of the coding portion of fibrinogen genes disclosed two novel FGG missense variants (p.Asp316Asn, fibrinogen Pisa; p.Gly366Ser, fibrinogen Beograd), both present in the heterozygous state and affecting residues located in the fibrinogen C-terminal γ-module. Liver sections derived from biopsies of the two patients were examined by immunocytochemical analyses, revealing hepatocyte cytoplasmic inclusions immunoreactive to anti-fibrinogen antibodies. CONCLUSIONS: Our work strongly confirms the clustering of mutations causing FSD in the fibrinogen γ chain between residues 284 and 375. Based on an in-depth structural analysis of all FSD-causing mutations and on their resemblance to mutations leading to serpinopathies, we also comment on a possible mechanism explaining fibrinogen polymerization within hepatocytes.

The fibrinogen γA/γ' isoform does not promote acute arterial thrombosis in mice.

BACKGROUND: Elevated plasma fibrinogen is associated with arterial thrombosis in humans and promotes thrombosis in mice by increasing fibrin formation and thrombus fibrin content. Fibrinogen is composed of six polypeptide chains: (Aα, Bß, and γ)2. Alternative splicing of the γ chain leads to a dominant form (γA/γA) and a minor species (γA/γ'). Epidemiological studies have detected elevated γA/γ' fibrinogen in patients with arterial thrombosis, suggesting that this isoform promotes thrombosis. However, in vitro data show that γA/γ' is anticoagulant due to its ability to sequester thrombin and suggest its expression is upregulated in response to inflammatory processes. OBJECTIVE: To determine whether γA/γ' fibrinogen is prothrombotic in vivo. METHODS: We separated γA/γA and γA/γ' fibrinogen from human plasma-purified fibrinogen and determined the effects on in vitro plasma clot formation and on in vivo thrombus formation and circulating thrombin-antithrombin complexes in mice. RESULTS AND CONCLUSIONS: Both γA/γA and γA/γ' fibrinogen were cleaved by murine and human thrombin and were incorporated into murine and human clots. When γA/γA or γA/γ' was spiked into plasma, γA/γA increased the fibrin formation rate to a greater extent than γA/γ'. In mice, compared to controls, γA/γA infusion shortened the time to carotid artery occlusion, whereas γA/γ' infusion did not. Additionally, γA/γ' infusion led to lower levels of plasma thrombin-antithrombin complexes following arterial injury, whereas γA/γA infusion did not. These data suggest that γA/γ' binds thrombin in vivo and decreases prothrombotic activity. Together, these findings indicate that elevated levels of γA/γA fibrinogen promote arterial thrombosis in vivo, whereas γA/γ' does not.

Recombinant γT305A fibrinogen indicates severely impaired fibrin polymerization due to the aberrant function of hole 'A' and calcium binding sites.

INTRODUCTION: We examined a 6-month-old girl with inherited fibrinogen abnormality and no history of bleeding or thrombosis. Routine coagulation screening tests showed a markedly low level of plasma fibrinogen determined by functional measurement and also a low level by antigenic measurement (functional/antigenic ratio=0.295), suggesting hypodysfibrinogenemia. MATERIALS AND METHODS: DNA sequence analysis was performed, and γT305A fibrinogen was synthesized in Chinese hamster ovary cells based on the results. We then functionally analyzed and compared with that of nearby recombinant γN308K fibrinogen. RESULTS: DNA sequence analysis revealed a heterozygous γT305A substitution (mature protein residue number). The γT305A fibrinogen indicated markedly impaired thrombin-catalyzed fibrin polymerization both in the presence or absence of 1mM calcium ion compared with that of γN308K fibrinogen. Protection of plasmin degradation in the presence of calcium ion or Gly-Pro-Arg-Pro peptide (analogue for so-called knob 'A') and factor XIIIa-catalyzed fibrinogen crosslinking demonstrated that the calcium binding sites, hole 'a' and D:D interaction sites were all markedly impaired, whereas γN308Kwas impaired at the latter two sites. Molecular modeling demonstrated that γT305 is localized at a shorter distance than γN308 from the high affinity calcium binding site and hole 'a'. CONCLUSION: Our findings suggest that γT305 might be important for construction of the overall structure of the γ module of fibrinogen. Substitution of γT305A leads to both dysfibrinogenemic and hypofibrinogenemic characterization, namely hypodysfibrinogenemia. We have already reported that recombinant γT305A fibrinogen was synthesized normally and secreted slightly, but was significantly reduced.

Inhibition of thrombin-mediated factor V activation contributes to the anticoagulant activity of fibrinogen γ'.

BACKGROUND: Besides its role in blood clotting, fibrinogen exerts a poorly understood anticoagulant function by binding thrombin and modulating its activity. In particular, the γA/γ' fibrinogen isoform binds with high affinity to thrombin exosite II through the anionic carboxyl-terminal end of the γ' chain. This interaction down-regulates thrombin-mediated factor VIII (FVIII) activation, but its effect on FV activation is unknown. OBJECTIVES: To investigate the overall anticoagulant activity of fibrinogen and particularly of fibrinogen γ' in plasma, and to verify whether the fibrinogen γ' carboxyl-terminal peptide affects thrombin-mediated FV activation. METHODS: Thrombin generation was measured by calibrated automated thrombography in whole and defibrinated plasma and in plasma supplemented with the (sulfated) fibrinogen γ' carboxyl-terminal peptide (0-500 µmol L(-1) ). The effect of the peptide on thrombin-mediated FV activation was studied in model systems and in plasma. RESULTS: Total fibrinogen prolonged the lag time of thrombin generation at low tissue factor (TF) concentrations. The fibrinogen γ' peptide dose-dependently prolonged the lag time and decreased the peak height of thrombin generation at low TF, whereas a scrambled control peptide was ineffective. These effects persisted in the presence of an anti-FVIII antibody, suggesting that the peptide may also inhibit thrombin-mediated activation of FV. This was confirmed in model systems and in plasma. CONCLUSIONS: Total fibrinogen and the fibrinogen γ' peptide have an overall anticoagulant effect on thrombin generation determined at low TF. Inhibition of thrombin-mediated FV activation by the fibrinogen γ' peptide is a novel mechanism of the anticoagulant activity of fibrinogen γ'.

An engineered fibrinogen variant AαQ328,366P does not polymerise normally, but retains the ability to form α cross-links.

A fibrin clot is stabilised through the formation of factor XIIIa-catalysed intermolecular ε-lysyl-γ-glutamyl covalent cross-links between α chains to form α polymers and between γ chains to form γ dimers. In a previous study we characterised fibrinogen Seoul II, a heterozygous dysfibrinogen in which a cross-linking acceptor site in Aα chain, Gln328, was replaced with Pro (AαQ328P). Following on the previous study, we investigated whether the alteration of Gln residues Aα328 and Aα366 affects fibrin polymerisation and α chain cross-linking. We have expressed three recombinant fibrinogens: AαQ328P, AαQ366P, and AαQ328,366P in Chinese hamster ovary cells, purified these fibrinogens from the culture media and performed biochemical tests to see how the introduced changes affect fibrin polymerisation and α chain cross-linking. Thrombin-catalysed fibrin polymerisation of all variants was impaired with the double mutation being the most impaired. In contrast, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis showed α polymer formation with all three engineered proteins. This study demonstrates that AαQ328 and AαQ366 are important for normal fibrin clot formation and in the absence of residues AαQ328 and AαQ366, other Gln residues in the α chain can support FXIIIa-catalysed fibrin cross-linking.

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.

Role of electrostatic interactions in binding of thrombin to the fibrinogen γ' chain.

Thrombin binds to the highly anionic fibrinogen γ' chain through anion-binding exosite II. This binding profoundly alters thrombin's ability to cleave substrates, including fibrinogen, factor VIII, and PAR1. However, it is unknown whether this interaction is due mainly to general electrostatic complementarity between the γ' chain and exosite II or if there are critical charged γ' chain residues involved. We therefore systematically determined the contribution of negatively charged amino acids in the γ' chain, both individually and collectively, to thrombin binding affinity. Surface plasmon resonance binding experiments were performed using immobilized γ' chain peptides with charged-to-uncharged amino acid substitutions, i.e., Asp to Asn, Glu to Gln, and pTyr to Tyr. Individually, the substitution of uncharged for charged amino acids resulted in only minor changes in binding affinity, with a maximal change in K(d) from 0.440 to 0.705 µM for the Asp419Asn substitution. However, substitution of all three charged amino acids in a conserved ß-turn that is predicted to contact thrombin, pTyr418Tyr, Asp419Asn, and pTyr422Tyr, resulted in the loss of measurable binding, as did substitution of all the flanking charged amino acids. In addition, the binding of the γ' chain to thrombin was weakened in a dose-dependent manner with increasing NaCl concentration, resulting in a net loss of three or four ion pairs between thrombin and the γ' chain. Therefore, although each of the individual charges in the γ' chain contributes only incrementally to the overall binding affinity, the ensemble of the combined charges plays a profound role in the thrombin-γ' chain interactions.