Apolipoprotein E isoforms and their Cys-thiol modifications impact LRP1-mediated metabolism of triglyceride-rich lipoproteins.

The low-density lipoprotein (LDL) receptor-related protein (LRP)1 participates in the metabolism of apolipoprotein (apo) E-containing lipoproteins (apoE-LP). We investigated the effects of modifications of cysteine (Cys)-thiol of apoE on LRP1-mediated metabolism. Among the three isoforms, apoE2-LP exhibited the lowest affinity for LRP1 but was significantly catabolized, whereas apoE4-LP was sufficiently bound to LRP1 but showed the lowest catabolic capability. The reduction enhanced the binding and suppressed the catabolism of apoE3-LP, but had no effect on apoE2-LP. The formation of disulfide-linked complexes with apoAII suppressed binding, but enhanced the catabolism of apoE2-LP. Redox modifications of apoE-Cys-thiol may modulate the LRP1-mediated metabolism of apoE2- or apoE3-LP, but not apoE4-LP. The failure of this function may be involved in the pathophysiology of dyslipidemia.

First Detection of Chimeric ß-Lactamase CTX-M-64-Producing Salmonella Typhimurium from a Domestic Source in Japan.

Salmonella enterica subsp. enterica serovar Typhimurium has recently emerged worldwide as a producer of extended-spectrum ß-lactamase (ESBL). However, drug-resistant clinical isolates are rare in Japan. The common types of ESBLs found are the CTX-M-type ß-lactamases, including novel ß-lactamases such as CTX-M-64. CTX-M-64 has a chimeric structure comprising a combination of the CTX-M-1 and CTX-M-9 groups. In 2017, S. Typhimurium was isolated from stool, blood, and urine cultures of an 82-year-old man. Herein, we describe the discovery of a clinical isolate of S. Typhimurium in Japan. Antimicrobial susceptibility testing revealed that the isolate was resistant to third- and fourth-generation cephalosporins, including ceftazidime and monobactam. The minimum inhibitory concentrations of ceftazidime and ceftriaxone were restored by administration of clavulanic acid. Whole-genome sequencing analysis revealed that the isolate harbored the blaCTX-M-64 gene on an IncHI2/IncHI2A-type plasmid, with an assembly length of 174,477 bp. The genetic structure of the region surrounding the blaCTX-M-64 gene, ISKpn26-ΔISEcp1-blaCTX-M-64-orf477, was shared only with the chromosome sequence of S. Typhimurium detected in food-producing chickens in Guangdong, China. Although rare, S. Typhimurium can induce bloodstream infections and produce ESBL. To our knowledge, this is the first report of a CTX-M-64-producing Enterobacterales clinical isolate of domestic origin in Japan.

A novel variant fibrinogen, AαE11del, demonstrating the importance of AαE11 residue in thrombin binding.

INTRODUCTION: We identified a novel heterozygous AαE11del variant in a patient with congenital dysfibrinogenemia. This mutation is located in fibrinopeptide A (FpA). We analyzed the effect of AαE11del on the catalyzation of thrombin and batroxobin and simulated the stability of the complex structure between the FpA fragment (AαG6-V20) peptide and thrombin. MATERIALS AND METHODS: We performed fibrin polymerization and examined the kinetics of FpA release catalyzed by thrombin and batroxobin using purified plasma fibrinogen. To clarify the association between the AαE11 residue and thrombin, we calculated binding free energy using molecular dynamics simulation trajectories. RESULTS: Increasing the thrombin concentration improved release of FpA from the patient's fibrinogen to approximately 90%, compared to the previous 50% of that of normal fibrinogen. Fibrin polymerization of variant fibrinogen also improved. In addition, greater impairment of variant FpA release from the patient's fibrinogen was observed with thrombin than with batroxobin. Moreover, the calculated binding free energy showed that the FpA fragment-thrombin complex became unstable due to the missing AαE11 residue. CONCLUSIONS: Our findings indicate that the AαE11 residue is involved in FpA release in thrombin catalyzation more than in batroxobin catalyzation, and that the AαE11 residue stabilizes FpA fragment-thrombin complex formation.

Automated screening procedure for the phenotypes of congenital fibrinogen disorders using novel parameters, |min1|c and Ac/|min1|c, obtained from clot waveform analysis using the Clauss method.

INTRODUCTION: Fibrinogen activity (Ac) is widely measured, but fibrinogen antigen (Ag) is measured only in specialized laboratories, so it is difficult to discriminate congenital fibrinogen disorders (CFDs) from acquired hypofibrinogenemia (aHypo). In this study, to screen for CFD phenotypes we adopted novel parameters, |min1|c and Ac/ |min1|c, and compared these with validated Ac, Ag, and Ac/Ag, and previously proposed Ac/dH and Ac/|min1|. MATERIALS AND METHODS: We calibrated |min1| using a CN-6000 instrument and investigated the correlation between Ag and |min1|c for aHypo (n = 131) and CFD [18 dysfibrinogenemia (Dys), two hypodysfibrinogenemia (Hypodys) and four hypofibrinpogenemia (Hypo)]. Furthermore, we proposed a schema for screening CFD phenotypes using |min1|c and Ac/|min1|c. RESULTS: The |min1|c correlated well with Ag in aHypo, and Ac/|min1|c was a better parameter for screening Dys and Hypodys than Ac/dH and Ac/|min1|. With the combination of |min1|c and Ac/|min1|c parameters, 15 Dys, 2 Hypodys and four Hypo were categorized in agreement with the phenotype determined using Ag and Ac/Ag; conversely three Dys were classified as one Hypodys (AαR16C) and two Hypo (BßG15C). CONCLUSION: We demonstrated that |min1|c and Ac/|min1|c are valuable parameters for screening CFD patients and phenotypes in laboratories that do not measure Ag or perform genetic analysis.

Novel variant fibrinogen γp.C352R produced hypodysfibrinogenemia leading to a bleeding episode and failure of infertility treatment.

INTRODUCTION: We identified a patient with a novel heterozygous variant fibrinogen, γp.C352R (Niigata II; N-II), who had a bleeding episode and failed infertility treatment and was suspected to have hypodysfibrinogenemia based on low and discordant fibrinogen levels (functional assay 0.33 g/L, immunological assay 0.91 g/L). We analyzed the mechanism of this rare phenotype of a congenital fibrinogen disorder. MATERIALS AND METHODS: Patient plasma fibrinogen was purified and protein characterization and thrombin-catalyzed fibrin polymerization performed. Recombinant fibrinogen-producing Chinese hamster ovary (CHO) cells were established and the assembly and secretion of variant fibrinogen analyzed by ELISA and western blotting. RESULTS: Purified N-II plasma fibrinogen had a small lower molecular weight band below the normal γ-chain and slightly reduced fibrin polymerization. A limited proportion of p.C352R fibrinogen was secreted into the culture medium of established CHO cell lines, but the γ-chain of p.C352R was synthesized and variant fibrinogen was assembled inside the cells. CONCLUSION: We demonstrated that fibrinogen N-II, γp.C352R was associated with markedly reduced secretion of variant fibrinogen from CHO cells, that fibrin polymerization of purified plasma fibrinogen was only slightly affected, and that fibrinogen N-II produces hypodysfibrinogenemia in plasma.

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.

Screening method for congenital dysfibrinogenemia using clot waveform analysis with the Clauss method.

INTRODUCTION: Congenital fibrinogen disorders (CFDs) are classified as afibrinogenemia or hypofibrinogenemia (Hypo), dysfibrinogenemia (Dys), or hypodysfibrinogenemia (Hypodys), according to functional and antigenic fibrinogen concentrations. However, in routine laboratory tests, plasma fibrinogen levels are mostly measured using the functional Clauss method and not as an antigenic level. Therefore, it is difficult to discriminate CFD from acquired hypofibrinogenemia (aHypo). To establish a screening method for CFD, we investigated the parameters of clot waveform analysis (CWA) from the Clauss method. METHODS: We compared fibrinogen concentrations determined using Clauss and prothrombin time (PT)-derived methods for 67 aHypo and CFD cases (19 Dys, 4 Hypodys, and 1 Hypo determined using antigen levels and DNA sequence analysis) with a CS-2400 instrument, and the CWA parameters, dH and Min1, were analyzed automatically with an on-board algorithm. dH and Min1 are the maximum change in transmittance at the end of coagulation and the maximum velocity of transmittance change during coagulation, respectively. RESULTS: Clauss/PT-derived ratios detected 18 cases of Dys and Hypodys but no Hypo cases, whereas Clauss/dH plus Clauss/Min1 ratios were calculated from fibrinogen concentration using the Clauss method and CWA parameters detected 21 cases of Dys and Hypodys and one Hypo case. Moreover, the Clauss/PT-derived ratio and Clauss/dH plus Clauss/Min1 ratio detected 22 cases of Dys and Hypodys cases and one Hypo case. CONCLUSION: This report demonstrates that CWA parameters of the Clauss method, Clauss/dH plus Clauss/Min1 ratio, screened Dys patients with a higher rate, whereas Clauss/PT-derived ratios did not.

A Novel Amino Acid Substitution, Fibrinogen Bßp.Pro234Leu, Associated with Hypofibrinogenemia Causing Impairment of Fibrinogen Assembly and Secretion.

We identified a novel heterozygous variant, Bßp.Pro234Leu (fibrinogen Tokorozawa), which was suspected to be associated with hypofibrinogenemia. Therefore, we analyzed the assembly and secretion of this fibrinogen using Chinese hamster ovary (CHO) cells. To determine the impact on the synthesis and secretion of fibrinogen of the Bßp.P234L and γp.G242E substitutions, we established recombinant variant fibrinogen-producing CHO cell lines. Synthesis and secretion analyses were performed using an enzyme-linked immunosorbent assay (ELISA) and immunoblotting analysis with the established cell lines. In addition, we performed fibrin polymerization using purified plasma fibrinogen and in-silico analysis. Both Bßp.P234L and γp.G242E impaired the secretion and synthesis of fibrinogen. Moreover, immunoblotting analysis elucidated the mobility migration of the Bßγ complex in Bßp.P234L. On the other hand, the fibrin polymerization of fibrinogen Tokorozawa was similar to that of normal fibrinogen. In-silico analysis revealed that the Bßp.P234 residue is located in the contact region between the Bß and γ chains and contacts γp.G242 residue. The present study demonstrated that the Bßp.P234L substitution resulted in hypofibrinogenemia by decreasing the assembly and secretion of fibrinogen. Therefore, there is a possibility that substitutions in the contact region between the Bß and γ chains impact the assembly and secretion of fibrinogen.

Comparison of molecular structure and fibrin polymerization between two Bß-chain N-terminal region fibrinogen variants, Bßp.G45C and Bßp.R74C.

We identified two heterozygous dysfibrinogenemias, Bßp.Gly45Cys (Kyoto VII; K-VII) and Bßp.Arg74Cys (Iida II; I-II). The impairment of polymerization of Bßp.G45C has been well analyzed; however, that of Bßp.R74C has not. Thus, we compared fibrin polymerization between these variants. To determine the structural and functional characterization of purified fibrinogens, we performed immunoblotting analysis, kinetic analyses of fibrinopeptide A and B release, and thrombin- or batroxobin-catalyzed fibrin or fibrin monomer polymerization. Immunoblotting analysis showed that both variant fibrinogens had variant fibrinogen-albumin complexes and variant fibrinogen multimers, and the amounts of fibrinogen-albumin complexes with fibrinogen K-VII was more than with fibrinogen I-II. Moreover, fibrinopeptide B release from fibrinogen K-VII was about 50% of the control, whereas the others were normal. The maximum slopes of polymerization for variant fibrinogens were reduced, but fibrinogen K-VII was reduced more than fibrinogen I-II. The present study demonstrated that both Bßp.G45C and Bßp.R74C variants showed the presence of variant fibrinogen-albumin complexes and variant fibrinogen multimers, and polymerization of Bßp.G45C was impaired more than Bßp.R74C. Our study and several previous reports concerning the clinical phenotype of both variants suggested the risks of bleeding for patients with Bßp.G45C and thrombosis for patients with Bßp.R74C.

Acquired dysfibrinogenemia: monoclonal λ-type IgA binding to fibrinogen caused lower functional plasma fibrinogen level and abnormal clot formation.

We report a case of acquired dysfibrinogenemia with monoclonal gammopathy of undetermined significance presenting λ-type IgA M protein. The patient showed lower functional (0.4 g/dL) and normal immunological fibrinogen (2.9 g/dL). To examine the cause of the false lower value of fibrinogen, we performed experiments using the patient's purified fibrinogen and IgA. Fibrinogen was purified from the patient's plasma; IgA was purified from plasma or serum by immunoaffinity chromatography. We performed thrombin-catalyzed fibrin polymerization, scanning electron microscopy (SEM), immunoblotting analysis, and enzyme-linked immunosorbent assays (ELISAs). Fibrin polymerization in the patient's plasma was markedly reduced and SEM showed no fiber bundles or sponge-like structures. Purified IgA did not influence polymerization, whereas immunoprecipitated plasma with an anti-IgA (α-chain) antibody indicated normalization of polymerization and clot structure. Western blotting analysis revealed the presence of monoclonal λ-type IgA-bound fibrinogen, the proportion of which was significantly higher than normal control plasma using ELISA. Our results suggest that IgA M protein-bound fibrinogen is not normally converted into fibrin, but rather leads to formation of an aberrantly structured fragile clot. The patient's reduced plasma fibrinogen level was caused by the presence of IgA M protein-bound fibrinogen, not by IgA M protein alone.

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.

γD318Y fibrinogen shows no fibrin polymerization due to defective "A-a" and "B-b" interactions, whereas that of γK321E fibrinogen is nearly normal.

BACKGROUND: The fibrinogen γ-module has several functional sites and plays a role in dysfibrinogenemia, which is characterized by impaired fibrin polymerization. Variants, including γD318Y and γΔN319D320, have been reported at the high affinity Ca2+-binding site, and analyses using recombinant fibrinogen revealed the importance of this site for fibrinogen functions and secretion. We examined the polymerization abilities of the recombinant fibrinogen variants, γD318Y and γK321E. MATERIALS AND METHODS: γD318Y and γK321E were produced using CHO cells and fibrinogen functions were examined using thrombin- or batroxobin-catalyzed polymerization, gel chromatography, protection against plasmin degradation, and factor XIIIa cross-linking. RESULTS: γD318Y did not show any polymerization by thrombin or batroxobin, similar to γΔN319D320, whereas γK321E had slightly impaired polymerization. The functions of Ca2+ binding, hole 'a', and the "D-D" interaction were markedly reduced in γD318Y, and gel chromatography suggested altered protofibril formation. In silico analyses revealed that structural changes in the γ-module of these variants were inconsistent with polymerization results. The degree of structural changes in γD318Y was moderate relative to those in γD318A and γD320A, which had markedly impaired polymerization, and γK321E, which showed slightly impaired polymerization. CONCLUSION: Our results suggest that no polymerization of γD318Y or γΔN319D320 was due to the loss of both "A-a" and "B-b" interactions. Previous studies demonstrated that "B-b" interaction alone causes polymerization of neighboring γD318A and γD320A fibrinogen, which is subsequently decreased. Marked changes in the tertiary structure of the γD318Y γ-module influenced the location and/or orientation of the adjacent ß-module, which led to impaired "B-b" interactions.