[A Case of Secondary Cryofibrinogenemia with Cholangiocarcinoma and Deep Venous Thrombosis].

Cryofibrinogen (CF) is a type of cryoprotein (CP) that can precipitate in cooled plasma but not in serum, and resolves upon warming. We identified a case of secondary cryofibrinogenemia with cholangiocarcinoma and deep venous thrombosis. The patient's cryocrit measured using a Wintrobe tube was 19% in sodium citrate plasma stored for 7 days at 4 degrees C. We performed quantitative analysis of plasma proteins (fibrinogen, IgG, IgA, IgM, C3, C4, α1-antitrypsin, and C-reactive protein) before and after precipitation for 12 hours at 4 degrees C. The plasma fibrinogen concentration decreased by 16.7% (120 mg/dL --> 100 mg/dL), whereas the others were unaffected by precipitation. The CP purified from the patient's plasma was washed three times with saline and subjected to Western blot and Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) analyses. Western blot analysis indicated that the purified CP was composed of not only fibrinogen but also fibronectin, α1-antitrypsin, α2-macroglobulin, coagulation factor VIII, and IgG, IgA, and IgM. Interestingly, SDS-PAGE analysis showed that the molecular weight of the patient's CF differed from that of purified normal fibrinogen (340 KDa) and consisted of several low-molecular-weight bands (50-250 KDa). From these results, we speculated that CF found in this case was a mixture of degradated fibrinogen and some plasma proteins. In summary, cryofibrinogenemia is a rare and under-recognized disease. Sample information in routine clinical practice is valuable to diagnose this disease.

[Polymorphism frequency of fibrinogen Bß-chain 448Arg and Lys, and the differences of plasma fibrinogen level and clotting function with three genotypes in Japanese].

The 448th residue in the fibrinogen Bß-chain molecule is known to be polymorphic (Arg/Lys, R/K), and its allele frequency was previously estimated to be R: 0.85 and K: 0.15 in the US. In the present study, we collected blood samples from 64 healthy individuals and examined the frequency of the fibrinogen Bß-chain 448 polymorphism in the Japanese population as well as the relationship between polymorphic types and the function and levels of fibrinogen. The polymorphic site was confirmed by MnlI restriction analysis and direct sequencing analysis for amplified 860 bp PCR products containing the Bß 448 residue. Fibrinogen plasma levels were estimated based on functional and immunological methods. Functional analyses were performed on the R/R, R/K, and K/K types using thrombin-catalyzed fibrin polymerization. The R/R type was detected in 48 out of 64 subjects, R/K in 15, and K/K in one. Therefore, the allele frequency was found to be R: 0.87 and K: 0.13 for the Bß 448 site, which was similar to that reported previously in the US. The polymorphism did not affect fibrinogen plasma levels. The results of the analysis on fibrin polymerization of the three types suggested that lateral aggregation may be significantly slower in the fibrinogen Bß-chain 448R/K and K/K types than in the R/R type. (Original).

Successful living-related kidney transplantation in a boy with inherited dysfibrinogenemia.

In kidney transplantation, it is essential to avoid acute vascular complications, such as hemorrhage and renal vascular thrombosis, which may often lead to allograft loss. Inherited dysfibrinogenemia is a rare coagulation disorder with a wide spectrum of clinical manifestations, such as excessive bleeding and thrombosis. A 12-yr-old boy, previously diagnosed with renal hypodysplasia, was found to have reduced fibrinogen concentrations. Coagulation tests assessing surgical risk during kidney transplantation showed a discrepancy between functional and immunologic fibrinogen concentrations. Gene analysis confirmed inherited dysfibrinogenemia, with a heterozygous mutation in FGA (Aα Arg16His) in the patient and his mother. Based on the molecular and functional properties of the mutation, and a familial phenotype, in which his aunt had experienced a previous bleeding episode, the patient was considered at greater risk of bleeding than of thrombosis. The patient was administered fibrinogen concentrate before surgery, and kidney transplantation was performed with his father as the organ donor. The patient received additional prophylactic infusions of fibrinogen concentrate postoperatively, and his postoperative course was uneventful. Accurate diagnosis of dysfibrinogenemia, including gene analysis, is important for correctly managing patients with this coagulation disorder who are undergoing kidney transplantation.

[Comparison of fibrinogen synthesis and secretion between novel variant fibrinogen, nagakute (gamma305Thr --> Ala), and other variants located in gamma305-308 residues].

We found and identified a novel heterozygous dysfibrinogenemia with gammaT305A (ACA --> GCA) mutation in a 6-month old boy. Since his plasma antigenic concentration of fibrinogen was 1.12g/l and less than the lower limit of the reference interval, we guessed that the production of a variant fibrinogen might be a partial defect. To clarify this speculation, we altered the gamma-chain expression vector, transfected it into Chinese Hamster Ovary(CHO) cells, and synthesized recombinant gammaT305A fibrinogen alongside three other variant fibrinogens, gammaS306P, gammaH307Y, and gammaN308K, and the wild type (gammaN) fibrinogen. Fibrinogen concentration ratio of culture media/cell lysates decreased in the order of gammaT305A-, gammaS306P-, gammaH307Y-CHO cells, all three being lower in comparison to the gammaN-CHO cells. Western blotting analyses indicated that all of variant gamma-chains were assembled into fibrinogen molecules in the cells. These data indicate the possibility that secretion of gamma T305A-fibrinogen is slightly impaired and variant fibrinogen is accumulated in the cell. Of interest, the secretion of gammaH307Y-fibrinogen was decreased the most, whereas that of the gammaN308K-CHO cells was not affected. The tertiary structure of the yC nodule indicated that gamma305T-gamma307H residues are located in the inside of the nodule. In contrast, that of gamma308N is located on surface of the nodule. In conclusion, our results showed the variant fibrinogen, gammaT305A, has characteristics not only of dysfibrinogenemia, but also might be hypofibrinogenemia, namely, hypo/dysfibrinogenemia. Furthermore, gamma306S-gamma307H residues of the gammaC nodule play crucial roles for protein synthesis and fibrin polymerization.

[Functional analysis for dysfibrinogenemias, Toyama and Adachi, which have a mutation of Aalpha16Arg-->His (CGT-->CAT) with aberrant fibrinopeptide A release].

We found and identified four heterozygous dysfibrinogenemias with AalphaR16H(CGT-->CAT) mutation in two families by coagulation tests and direct sequence analysis for PCR-amplified DNA fragments. Two dysfibrinogens were designated as fibrinogen Toyama and Adachi, according to the place of residence of proposituses, respectively. Patients' fibrinogen purified from plasma using immunoaffinity-chromatography was subjected to thrombin- or batroxobin-catalyzed fibrin polymerization, fibrinopeptide A (FPA) release, and clottability test. AalphaR16H-fibrinogen showed impaired thrombin or batroxobin-catalyzed fibrin polymerization in comparison with normal control fibrinogen. It is interesting that the period of protofibril formation of Toyama propositus was longest in those of four affected people. The clottability of AalphaR16H-fibrinogen was 66-70% with thrombin and higher than with batroxobin, 35-50%. In the same condition with fibrin polymerization, thrombin and batroxobin did not cleave the Aalpha16H-17G peptide-bonding, resulting in no release of variant FPA. From these results, we speculated that elongation of the two-stranded protofibril formation would be terminated by participation of the heterodimer fibrinogen molecules composed with a normal and an aberrant Aalpha-chain, and it would result in a decrease in fibrin polymerization. We speculated that the difference in the extent of impairment of fibrin polymerization among the patients might be caused by the different amount of heterodimers. Moreover, we also speculated that batroxobin-induced clottability was lower than thrombin-induced clottability, because batroxobin cannot induce the so-called "B-knob-b-hole" interaction, which enhances fibrin formation.

[SNP-specific mismatched PCR for embryonic DNA detection using blood from pregnant mothers].

BACKGROUND: Although prenatal diagnoses are performed using amniotic fluid cells, chorionic villus, or cord blood, these methods are hazardous for pregnant woman and the fetus. To determine a procedure for safe prenatal diagnosis, we have developed a sensitive method to analyze SNPs and we evaluated the possibility to detect fetal DNA in the maternal blood. METHODS: GeneScan analysis was performed by using mismatched specific primers for 5 SNP types and fluorescein amidite (FAM) labeled primers, and Real-time PCR analysis was also performed by using mismatched specific primers for the same SNP type and probes labeled with FAM and black hole quencher. DNA from healthy volunteers' blood was used for a primary examination to establish procedures, and DNA from 200 microl of blood from pregnant women, their partners and children were used for detection of fetal DNA and/or typing and selection of SNPs. RESULTS: To evaluate the sensitivity of this method, mixing tests of DNA containing a SNP nucleotide and its counterpart indicated the sensitivities were 10(-1)-10(-3) for GeneScan analysis and 10(-1)-10(-4) for Real-time PCR analysis. Fetal DNA (rs3769393-A and G) was detected in blood from pregnant women (GG-type mother: 2 out of 2, AA-type mother: 1 out of 2) only at 18 and/or 28 gestation weeks by Real-time PCR analysis. However, 4 SNPs measured by Real-time PCR analysis and 5 SNPs examined by GeneScan analysis were not detected in all women in different gestation periods. CONCLUSIONS: We established a SNP detection method using GeneScan and Real-time PCR analysis. The latter detected fetal DNA in 200 microl of blood in only some pregnant women. We speculate that using a large amount of DNA from nucleated erythrocytes in 20 ml of blood will improve the detection rate of fetal DNA.

[Role of fibrinogen Bbeta-chain D-region 454-458 residues for assembly and secretion of intact fibrinogen].

BACKGROUND: To examine the role of fibrinogen Bbeta-chain D region in the assembly and/or secretion of multichain protein, we synthesized eight variant fibrinogens with truncated Bbeta-chains in the C terminal region, terminating with 454, 455, 456 or 458 residues, and with substitution at Bbeta-455Arg by Lys, Ile, Ala or Asp in Chinese hamster ovary (CHO) cells. METHODS: A fibrinogen Bbeta-chain expression vector was altered and transfected into CHO cells that expressed normal human fibrinogen Aalpha- and gamma-chains. Expressed fibrinogens of cell lysates and culture media of the established cell lines were subjected to ELISA and immunoblot analysis. RESULTS: The CHO cells synthesized eight variant Bbeta-chains and assembled these into fibrinogen except for Bbeta-454 and Bbeta-455Asp. However, in the cell lysates, concentrations of these variant fibrinogens were lower than that in wild type cells. These assembled variant fibrinogens were secreted into the culture medium, and the levels in culture media were also lower than that in the medium of wild type cells. Significant differences in the mean ratios of fibrinogen concentration in medium to that in cell lysate were not observed between the variant type cells and the wild type cells. CONCLUSIONS: Residues of the Bbeta-chain D domain are essential for fibrinogen assembly, especially the Bbeta-455 residue was critical. The present study indicated that the structure of the fibrinogen Bbeta-chain C terminal D region is necessary for fibrinogen assembly, but not for secretion.

Changes in serum citrullinated fibrinogen concentration associated with the phase of bacteremia patients.

BACKGROUND: Citrullinated fibrinogen (C-Fbg) has been detected in rheumatoid arthritis; however, few studies have reported the role of C-Fbg in other inflammatory diseases. This study aimed to clarify the changes in serum C-Fbg associated with the bacteremia phase. METHODS: We measured serum C-Fbg concentration in bacteremia patients. C-Fbg levels at each phase of bacteremia, classified by white blood cell (WBC) count and neutrophil left shift change, were compared with those of healthy control (HC). The correlation between C-Fbg concentration and certain inflammatory markers, or citrullinated histone H3 concentration was assessed. Multiple linear regression (MLR) analysis was used to examine the association of log C-Fbg with certain inflammatory markers. RESULT: Serum C-Fbg levels were significantly higher in bacteremia patients than in HC (p < 0.001) and positively correlated with WBC and neutrophil count. Further, C-Fbg levels were significantly higher in phases III and IV of bacteremia than in HC (p < 0.001). MLR analysis indicated that log C-Fbg had a stronger relationship with log neutrophil counts than other certain inflammatory markers (p < 0.01). CONCLUSION: Serum C-Fbg levels increased in bacteremia patients, and this was consistent with an influx of neutrophils into the blood stream in accordance with the bacteremia phase.

[Assembly and secretion of mutant fibrinogens with variant gamma-chain C terminal region (gamma313-gamma345)].

BACKGROUND: It has been reported that the structure of the fibrinogen gamma-chain C terminal (D) region (140-411 residues) has important functions in fibrinogen assembly and/or secretion. Variant fibrinogens, gamma313S>N, gamma336M>I, gamma341A>D, and gamma345N>D have been reported as hypofibrinogenemias or dysfibrinogenemias. To study the assembly and secretion of the variant fibrinogens containing aberrant D regions, we established CHO cells producing these four fibrinogens. METHODS: A fibrinogen gamma-chain expression vector was altered and transfected into CHO cells that expressed normal human fibrinogen Aalpha and Bbeta-chains. Cell lysates and culture media of the selected cell lines were subjected to ELISA and immunoblot analysis. RESULTS: The CHO cells synthesized mutant gamma-chains and assembled these into fibrinogen, although these variant fibrinogens were barely secreted into the culture media. In the cell lysates, however, concentrations of these variant fibrinogens were higher than the normal levels. CONCLUSIONS: The present study indicated that the tertiary structure of the fibrinogen gamma-chain C terminal region between 313 and 345 is necessary for fibrinogen secretion. These findings suggest that reduced levels of fibrinogen secretion lead to the hypofibrinogen in the patients and secreted fibrinogens might show dysfibrinogenemia.

[Functional analysis of heterozygous plasma dysfibrinogens derived from two families of gammaArg275Cys and three families of gammaArg275His, and haplotype analysis for these families].

We have identified five heterozygous dysfibrinogenemias, two families with variant fibrinogen gammaArg275Cys (CGC > TGC; Matsumoto III and Sendai) and three families with gammaArg275His (CGC > CAC; Otsu II, Iida, and Shizuoka), from PCR-amplified DNA fragments and direct sequence analysis. gammaArg275 is the most important residue in fibrinogen for the so-called "D-D interface" in protofibril elongation. We compared the functions of plasma fibrinogen purified from affected family members with gammaArg275Cys and gammaArg275His. Both fibrinogens showed markedly impaired thrombin-catalyzed fibrin polymerization in comparison with normal controls. The degree of impairment of gammaArg275Cys fibrinogen was greater than that of gammaArg275His. These results were consistent with the fibrinogen concentration ratio (thrombin time method/immunological method). That is, the ratio of gammaArg275Cys was significantly lower than that of gammaArg275His. Moreover, scanning electron microscopy indicated significantly thicker fibers in fibrin clots made from gammaArg275Cys than in those of normal controls or gammaArg275His, and abnormal bundles with tapered ends. Factor XIIIa-catalyzed cross-linking of the fibrinogen gamma-chain (in the absence of thrombin) showed a similar delay for gammaArg275Cys and gammaArg275His. We report markedly impaired fibrin polymerization of gammaArg275Cys compared to gammaArg275His, and speculate that the difference is due to the disulfide-linked Cys in gammaArg275Cys, as we have already demonstrated for plasma and recombinant mutant fibrinogens. These results also indicate that an amino acid substitution of gammaArg275 disrupts D:D interactions in fibrin fiber formation. Furthermore haplotype analysis for three families with gammaArg275His suggested that founder of Iida family might be different from that of Otsu II or Shizuoka family.

[Analysis of antibody reactivity for FDP D-dimer fragments by Western blotting].

We evaluated three test kits for fibrin degradation products (FDP) D-dimer. We found that six of 217 plasma sample values obtained by Nanopia test were markedly higher than the values obtained using the other two kits. The regression equation for 211 samples (excluding six) was y=0.64x+3.05 (y: Nanopia, x: LIAS AUTO) and the correlation coefficient was 0.915. Therefore, we classified these samples into three categories, namely correlated(y< 1.0x), incompatible (y= 1.0x-2.9x) and markedly incompatible (y> or =3.0x). Selected samples, eight correlated, four incompatible and four markedly incompatible, were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western blotting(WB). WB analysis using anti-fibrinogen antibody showed that both high molecular weight fragments of cross-linked fibrin (HMW-XDP) and DD/E fragments were present in the correlated samples, but there was less HMW-XDP than DD/E in the incompatible samples and mostly DD/E (HMW-XDP was significantly less than DD/E) in the markedly incompatible samples. These data suggest that plasma FDP samples that contain mostly DD/E and little HMW-XDP demonstrated markedly incompatible values using the three D-dimer test kits. These data was reflected by markedly elevated plasmin alpha2-plasmin inhibitor complex values in the incompatible and markedly incompatible samples. Unfortunately, we did not directly demonstrate these phenomena by WB analysis with two anti-D-dimer antibodies used Nanopia or LPIA reagent. In the near future, we expect that standardization of FDP D-dimer assay will be accomplished.

[Analysis of hypofibrinogenemias found on routine coagulation screening tests and identification of heterozygous dysfibrinogenemia or fibrinogen deficiency].

We analyzed the clinical factors resulting in hypofibrinogenemia, which is defined as less than 100mg/dl of plasma fibrinogen values determined by a procedure based on the Thrombin-time method. Within a 12-month period, we assayed 5,746 patients (19,309 plasmas) and found 113 patients (1.97%) with hypofibrinogenemia. We categorized these patients as having decreased synthesis of fibrinogen (less than 3.0g/dl of albumin, 140 IU/l of Cholinesterase, and/or 50% on Hepaplastin Test), increased consumption of fibrinogen (more than 10 microg/ml of FDP D-dimer), known side effect of L-asparaginase administration, or other causes. Details are follows: 1) decreased synthesis: 26 patients, suspected of decreased synthesis (albumin: 3.1-3.4 g/dl): 4 patients, 2) increased consumption: 15 patients, suspected of increased consumption (FDP D-dimer: 5.0-9.9 g/dl): 1 case, 3) decreased synthesis combined with increased consumption: 24 patients, suspected of decreased synthesis and/or suspected of increased consumption: 14 patients, 4) side-effect of L-asparaginase administration: 24 patients, 5) heterozygous dysfibrinogenemia: 1 patient, 6) heterozygous fibrinogen deficiency: 1 patient, suspected of heterozygous fibrinogen deficiency: 1 patient, 7) unidentified: 2 patients with West syndrome treated with a combination of ACTH and valproic acid. Three patients with dysfibrinogenemia or fibrinogen deficiency showed normal or slightly prolonged PT values and normal APTT values. These data and our previous reports suggest that heterozygous patients with dysfibrinogenemia or fibrinogen deficiency do not demonstrate markedly prolonged PT and APTT values, differing from patients with afibrinogenemia.

Importance of cystatin C and uric acid levels in the association of cardiometabolic risk factors in Japanese junior high school students.

BACKGROUND: Serum cystatin C (CysC), a novel marker of renal function, is associated with the components of metabolic syndrome in adults. Little is known about the utility of CysC and its association with cardiometabolic risks in young subjects. METHODS AND RESULTS: In a cohort of 454 Japanese junior high school students, the distribution of serum CysC levels and associated variables were analyzed. CysC levels were significantly higher in boys than in girls (0.92±0.10mg/L vs. 0.77±0.08mg/L, p<0.001). CysC was significantly correlated with serum creatinine (r=0.473, p<0.001), and serum uric acid (SUA) (r=0.546, p<0.001). Multivariable regression analysis revealed significant associations between CysC and SUA in all subjects (ß=0.241, p<0.001), and in boys and girls separately (ß=0.264 and 0.240, respectively, both p<0.001). Importantly, subjects with elevation of both serum CysC and SUA levels had the highest ratio of triglyceride to high-density lipoprotein cholesterol. CONCLUSIONS: CysC had significant associations with both creatinine and SUA in Japanese junior high school students. The concomitant elevation of serum CysC and SUA levels was associated with subclinical lipid metabolism dysregulation, and suggested the presence of cardiometabolic risk accumulation.

In vitro fibrin clot formation and fibrinolysis using heterozygous plasma fibrinogen from gammaAsn319, Asp320 deletion dysfibrinogen, Otsu I.

INTRODUCTION: We have reported a heterozygous dysfibrinogenemia, fibrinogen Otsu I, caused by the deletion of gammaAsn319 and gammaAsp320, which was originally identified in the dysfibrinogen Vlissingen/Frankfurt IV (V/FIV) associated with thrombosis. Unlike the V/FIV family, the Otsu propositus showed no thrombotic tendencies. To analyze the relationship between thrombosis and the heterozygous plasma variant fibrinogen, we used purified plasma fibrinogen from the Otsu patient and compared it with a normal control. MATERIALS AND METHODS: Thrombin-induced fibrin clot formation and clot structure were observed by fibrin polymerization and scanning electron microscopy, respectively. For in vitro observation of fibrinolysis, plasmin generation and clot lysis assays were performed by the addition of tissue type plasminogen activation (tPA) and plasminogen. RESULTS AND CONCLUSIONS: Polymerization of Otsu was markedly impaired, while fibrin fibers were much thicker and the density of the bundles of fibrin fibers was less and porous compared with normal. Lysis of the Otsu clot was not significantly different from normal when a tPA and plasminogen mixture was overlaid onto the clots. For Otsu, the penetration of the tPA/plasminogen mixture into the clot was much faster than normal and the protection against plasmin cleavage was impaired; however, tPA-induced plasmin activation of the Otsu fibrin was slower than that of normal fibrin, resulting in a clot lysis of Otsu similar to normal.

A novel variant fibrinogen, deletion of Bbeta111Ser in coiled-coil region, affecting fibrin lateral aggregation.

BACKGROUND: Functional fibrinogen concentration of a male infant showed <0.50 g/l and we speculated this patient as a dysfibrinogenemia or hypofibrinogenemia. METHODS: We analyzed propositus and his parent by DNA sequencing and by thrombin-catalyzed fibrin polymerization for purified plasma fibrinogen. RESULTS: Although functional fibrinogen determinations based on Clauss method showed the marked discrepancy of values among 3 sets of reagent and analyzer, we found a novel heterozygous variant fibrinogen, Kyoto IV, caused by 3-bp deletion in Bbeta-chain gene corresponding to the deletion of 111Ser located in coiled-coil region. We suggested that the discrepancy of fibrinogen values among 3 assays was caused by the difference in NaCl concentration in reagents for determination and analyzed the polymerization under the conditions of various NaCl concentrations. Although under normal physiological conditions Kyoto IV fibrinogen augmented the polymerization as compared with normal control, in 0.21 mol/l NaCl Kyoto IV fibrinogen showed abruptly impaired polymerization curve compared with normal control. CONCLUSION: Variant fibrinogen, BbetaDelta111Ser, showed augmented lateral aggregation under normal physiological conditions and the residue located in coiled-coil region, Bbeta111Ser, plays an important role in the lateral aggregation.

An immunoglobulin A1 that inhibits lactate dehydrogenase activity, with reversal of inhibition by addition of NADH.

We discovered a patient with low serum lactate dehydrogenase (LD) activity and an abnormal LD isozyme pattern. We analyzed the patient's LD inhibitor using electrophoresis, affinity chromatography, and immunochemical technologies. The LD activity of the patient's serum was inhibited more strongly at 4 degrees C than at 37 degrees C. The decrease of LD activity was more marked in a mixture of the patient's serum with purified LD5 than in that with purified LD1. The immunoglobulin responsible for LD inhibition was an IgA1-lambda. The LD inhibition by the patient's IgA1 was blocked by reduction and alkylation and by NADH. Polymerization of the patient's IgA1 might play an important role in its interaction with LD. Moreover, the possibility exists that part of the patient's IgA1 molecule fits into a pocket of LD in instead of NADH. This is the first report of NADH reversing such LD inhibition.

Neuregulin receptor ErbB2 localization at T-tubule in cardiac and skeletal muscle.

Previous studies have indicated that ErbB receptors for neuregulins play an important role in cardiac development and muscle spindle formation during embryogenesis; however, little is known about their functions in adulthood. Recent reports indicate that breast cancer therapy with humanized monoclonal ErbB2 antibody induces cardiomyopathy, suggesting that ErbB2 serves as a crucial signaling receptor, even in the adult heart. Here, we examine ErbB2 expression and localization in both cardiac and skeletal muscle of adult mice via immunoblot and immunohistochemistry. ErbB2 was detected as a band approximately 185 kD molecular mass in each cardiac and skeletal muscle extraction. Confocal images of double labeling showed that ErbB2 was colocalized with caveolin-3 in cardiac muscle and with dihydropyridine receptor in skeletal muscle, suggesting that ErbB2 was localized at the T-tubule. In addition, immunoelectron micrographs clearly demonstrated that ErbB2 was located at the T-tubule in both types of muscle. Taken together, the results of the present study suggest that neuregulin-ErbB2 signaling plays a role in the physiological function of cardiac and skeletal muscle, even in adulthood.

In vitro expression demonstrates impaired secretion of the gammaAsn319, Asp320 deletion variant fibrinogen.

The hypodysfibrinogenemia Otsu is caused by the two-residue deletion, gammaAsn319 and gammaAsp320. Analysis of plasma or purified fibrinogen from the heterozygous propositus revealed that the amount of variant gamma-chain was lower than that of normal gamma-chain. In order to examine the basis for this difference, we transfected Chinese hamster ovary cells and established stable cell lines that expressed both chains, gammaDelta/gammaN, only the normal chain, gammaN, and only the variant chain, gammaDelta. We measured fibrinogen concentration of confluent cultures by ELISA. We found the ratios of the concentrations in the media to the concentrations in the cell lysates of gammaDelta, gammaDelta/gammaN, and gammaN-cells were 0.42, 0.60, and 1.00, respectively. We measured the concentrations of the gammaDelta and gammaN chains by densitometric analysis of samples following separation by SDS-PAGE and found the fraction of gammaDelta-chains in cell lysates was always greater than the fraction in the respective culture media. We examined the kinetics of fibrinogen synthesis, assembly and secretion in pulse-chase experiments, and found that the gammaDelta-chain was assembled into intact fibrinogen at a rate similar to assembly of the gammaN-chain into normal fibrinogen, but was secreted into the medium at a slightly slower rate than normal fibrinogen. Considered together, these experiments indicate secretion of the variant fibrinogen was slightly impaired. These results suggest that the reduced level of gammaDelta319,320 fibrinogen in the plasma of the Otsu patient arises from modestly impaired secretion of this variant fibrinogen.

Residue gamma153Cys is essential for the formation of the complexes Aalphagamma and Bbetagamma, assembly intermediates for the AalphaBbetagamma complex and intact fibrinogen.

BACKGROUND: Fibrinogen Matsumoto IV was found in a hypofibrinogenemia caused by a heterozygous missense mutation, i.e., the substitution of the fibrinogen gamma-chain residue Cys153 by Arg. METHODS: To examine the precise basis for the fibrinogen deficiency, mixtures of any two vectors, the fibrinogen Aalpha-, Bbeta-, gamma- (153Cys) or gammam-(153Ala) chain were transfected into Chinese hamster ovary cells (CHO-Aalpha/gamma, -Aalpha/gammam, -Bbeta/gamma, -Bbeta/gammam). Expression and constitution of each of two chains and their complexes in the individual CHO cell lines were identified by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and immunoblot analysis using polypeptide specific antibodies and two-dimensional electrophoresis (2-DE). RESULTS: In the CHO-Aalpha/gamma and -Bbeta/gamma, the Aalpha/gamma- or Bbeta/gamma-complex was formed, whereas in the CHO-Aalpha/gammam and -Bbeta/gammam, no Aalpha/gammam- or Bbeta/gammam-complex was observed. These results demonstrate that gamma153Ala cannot assemble with the Aalpha- and Bbeta-chains, leading to impaired fibrinogen assembly and secretion. CONCLUSION: gamma153Cys is an essential residue for the fibrinogen assembly which is dependent on Aalpha/gamma- and Bbeta/gamma-complex formation.

Serotonin receptor antagonist inhibits monocrotaline-induced pulmonary hypertension and prolongs survival in rats.

OBJECTIVES: It has been reported that serotonin (5-HT) is involved in the development of pulmonary arterial hypertension (PAH) with pulmonary vascular remodeling. The purpose of the present study was to examine the role of a 5-HT2A receptor antagonist, sarpogrelate hydrochroride, in preventing or reversing monocrotaline (MCT)-induced PAH in rats. METHODS: Rats were injected with 40 mg/kg of MCT subcutaneously and randomized to either sarpogrelate (50 mg/kg, intraperitoneally) or placebo for 3 weeks. Animals treated with MCT and survived for 3 weeks were assigned to either sarpogrelate (50 mg/kg, intraperitoneally) or placebo for next 3 weeks. The animals had pressure measurement of the pulmonary artery, and then underwent histologic, immunohistochemical, and Western blot analyses of the lung tissue. Survival rate was also assessed after treatment. RESULTS: Sarpogrelate immediately following MCT injection suppressed PAH with severe pulmonary vascular remodeling and right-sided heart failure. The survival rate was significantly increased in the sarpogrelate-treated group compared with the placebo group (71% vs. 44%, p<0.05). Intense expression of P-selectin was found on the endothelium of the pulmonary arteries in the placebo group, and it was markedly attenuated in the sarpogrelate-treated group. The numbers of the CD45-positive cells and those of the proliferating cell nuclear antigen (PCNA)-positive cells in the lung tissue were significantly increased in the placebo group, and the increases in these cells were prevented by sarpogrelate. Endothelial nitric oxide synthase (eNOS) expression in the lung tissue was markedly decreased in the placebo group, but it was prevented by sarpogrelate (p<0.001). In contrast, late treatment with sarpogrelate failed to reverse established PAH. CONCLUSIONS: Specific 5-HT2A receptor blockade with sarpogrelate immediately after MCT inhibited PAH and prolongs survival in rats. These effects were accompanied by anti-inflammatory and anti-proliferative effects in the lung tissue and marked improvement of pulmonary vascular endothelial dysfunction and activation.