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

BACKGROUND: 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. OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Functional analysis of recombinant Bbeta15C and Bbeta15A fibrinogens demonstrates that Bbeta15G residue plays important roles in FPB release and in lateral aggregation of protofibrils.

BACKGROUND AND OBJECTIVES: Analysis of dysfibrinogens has improved our understanding of molecular defects and their effects on the function of intact fibrinogen. To eliminate the influence of plasma heterozygous molecules, we synthesized and analyzed recombinant-variant fibrinogens. METHODS: We synthesized two recombinant-variant fibrinogens with a single amino acid substitution at the 15Gly residue in the Bbeta-chain: namely, Bbeta15Cys and Bbeta15Ala. RESULTS: Western blotting analysis of purified fibrinogen revealed the existence of a small amount of a dimeric form only for Bbeta15Cys fibrinogen. For Bbeta15Cys fibrinogen, functional analysis indicated (a) no thrombin-catalyzed fibrinopeptide B (FPB) release and (b) markedly impaired lateral aggregation in thrombin- and reptilase-catalyzed fibrin polymerizations. For Bbeta15Ala fibrinogen, such analysis indicated slight impairments of both thrombin-catalyzed FPB release and lateral aggregation in thrombin-catalyzed fibrin polymerization, but nearly normal lateral aggregation in reptilase-catalyzed fibrin polymerization. These impaired lateral aggregations were accompanied by thinner fibrin fiber diameters (determined by scanning electron microscopy of the corresponding fibrin clots). CONCLUSION: We conclude that a region adjacent to Bbeta15Gly plays important roles in lateral aggregation not only in desA fibrin polymerization, but also in desAB fibrin polymerization, and we speculate that the marked functional differences between Bbeta15A and Bbeta15C fibrinogens in FPB release and fibrin polymerization might not only be due to the presence of a substituted cysteine residue in Bbeta15C fibrinogen, but also to the existence of disulfide-bonded forms. Finally, our data indicate that the Bbeta15Gly residue plays important roles in FPB release and lateral aggregation of protofibrils.

Recombinant fibrinogen, gamma275Arg-->Cys, exhibits formation of disulfide bond with cysteine and severely impaired D:D interactions.

BACKGROUND AND OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Comparison of thrombin-catalyzed fibrin polymerization and factor XIIIa-catalyzed cross-linking of fibrin among three recombinant variant fibrinogens, gamma 275C, gamma 275H, and gamma 275A.

BACKGROUND AND OBJECTIVES: 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. METHODS: 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. RESULTS: 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). CONCLUSION: 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.

Fibrinogens Kosai and Ogasa: Bbeta15Gly-->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.

Fibrinogen Matsumoto III: a variant with gamma275 Arg-->Cys (CGC-->TGC)--comparison of fibrin polymerization properties with those of Matsumoto I (gamma364 Asp-->His) and Matsumoto II (gamma308 Asn-->Lys).

Fibrinogen Matsumoto III (M-III) is a dysfibrinogen identified in a 66-year-old woman with rectal cancer. The fibrinogen level determined by the thrombin-time method was markedly decreased in preoperative coagulation tests of her plasma. Three fibrinogen polypeptide-chain gene fragments from the proposita were amplified by the polymerase chain reaction method, then sequenced. The triplet CGC encoding the amino acid residue gamma275 was replaced by TGC, resulting in the substitution of Arg->Cys. There have been previous reports of nine families with the same alteration, nine families with an Arg->His variant and one family with an Arg->Ser variant in this residue, which has been shown to be one of the most important amino acids in the 'D:D' interaction site. In addition, there are three silent mutations in the Aalpha-chain gene and two mutations in the intron of the Bbeta-chain and the gamma-chain gene. However, none of these mutations is thought to be the cause of the dysfunctional fibrinogen. The thrombin-catalyzed fibrin polymerization in the presence of 1 mM Ca ions was markedly delayed in purified M-III. Its lag period was longer than those of Matsumoto II (M-II; gamma308Asn->Lys) and Matsumoto I (M-I; gamma364Asp-His). gamma364Asp is one of the most important residues in the polymerization pocket of the 'D:E' interaction site and gamma308Asn is located in the vicinity of a high affinity Ca2+ binding site in the D-domain, gamma311-336. The maximum slope of the polymerization curve for M-III was about 4-fold steeper than that for M-1 but less steep than that for M-II. These results may suggest that the tertiary structure of the polymerization pocket plays a more important role in the lateral aggregation of protofibrils than that of the 'D:D' interaction site.

Fibrinogen Matsumoto V: a variant with Aalpha19 Arg-->Gly (AGG-->GGG). Comparison between fibrin polymerization stimulated by thrombin or reptilase and fibrin monomer polymerization.

Fibrinogen Matsumoto V (M-V) is a dysfibrinogen identified in a 52-year-old woman with systemic lupus erythematous. The triplet AGG encoding the amino acid residue Aalpha19 was replaced by GGG, resulting in the substitution of Arg-->Gly. Residue Aalpha19 has been shown to be one of the most important amino acids in the so-called 'A' site or alpha-chain knob. The thrombin-catalyzed release of fibrinopeptide A from M-V fibrinogen was only slightly delayed yet release of fibrinopeptide B was significantly delayed. Both thrombin-catalyzed fibrin polymerization and fibrin monomer polymerization were markedly impaired compared to normal fibrinogen. In addition, reptilase-catalyzed fibrin polymerization of M-V was much more impaired than thrombin-catalyzed fibrin polymerization. These results indicate 'B' and/or 'b' site of M-V fibrinogen play a more important role in thrombin-catalyzed fibrin polymerization than that of normal control fibrinogen.

Fibrinogen Matsumoto I: a gamma 364 Asp-->His (GAT-->CAT) substitution associated with defective fibrin polymerization.

Fibrinogen Matsumoto I is a novel hereditary dysfibrinogen identified in a 1-year-old boy with Down's syndrome. Though he showed no apparent bleeding or thrombotic tendency, he had a congenital heart disease. Preoperative coagulation tests of his plasma revealed a prolonged thrombin time and the fibrinogen level determined by the thrombin time method was markedly decreased. Molecular weight of fibrinogen chains showed apparently normal A alpha-, B beta-, and gamma-chains. The rate of fibrinopeptide release was normal, whereas fibrin polymerization was delayed. Fibrinogen gamma-chain gene fragments from the propositus were amplified by polymerase chain reaction then sequenced. The triplet GAT, coding for the amino acid residue gamma 364, was replaced by CAT, resulting in the substitution of Asp-->His. This residue is adjacent to the Tyr-363 that is demonstrated to be the primary site for fibrin polymerization. Our results indicate that the residue gamma 364 Asp is essential for normal polymerization of fibrin monomer.

A functional assay suggests that heterodimers exist in two C-terminal gamma-chain dysfibrinogens: Matsumoto I and Vlissingen/Frankfurt IV.

Because it contains three pairs of polypeptides, fibrinogen isolated from heterozygous individuals is expected to be a mixture of homodimers and heterodimers. Nevertheless, heterozygous individuals with only homodimers have been identified. We synthesized two recombinant fibrinogens with the mutations from fibrinogen Vlissingen/ Frankfurt IV (gamma(delta)319, 320) and Matsumoto I (gammaD364H), both identified in heterozygous individuals. We found that polymerization of these fibrinogens was undetectable in 30 min; polymerization of a 1:1 mixture of variant and normal fibrinogen was the same as polymerization of a 1:1 mixture of buffer and normal fibrinogen; polymerization of either plasma fibrinogen was markedly impaired when compared to the 1:1 mixture of the respective variant and normal fibrinogens. We conclude that each plasma fibrinogen is a mix of homodimers and heterodimers, such that the incorporation of heterodimers into the fibrin clot impairs polymerization. We suggest that incorporation of heterodimers can induce clinical symptoms.

Hypofibrinogenemia associated with a heterozygous C-->T nucleotide substitution at position -1138 BP of the 5'-flanking region of the fibrinogen A alpha-chain gene.

We found a novel genetic abnormality, heterozygous C-->T nucleotide substitution at position -1138 bp in the 5'-flanking region of the fibrinogen A alpha gene, in patients with hypofibrinogenemia. Luciferase reporter assay using the pGL3-basic vector and CHO cells indicates that the transcriptional activity of a vector incorporated with -1138T was reduced to one-third that of a vector incorporated with -1138C. These results suggest that the region adjacent to the -1138C bp of the 5'-flanking region of the fibrinogen A alpha gene is one of the most crucial sites for the transcription of the fibrinogen A alpha gene.

Identification of a dysfibrinogen, the substitution of gamma308Asn(AAT) to Lys(AAG), using coagulation tests, immunoblot analysis, and allele-specific polymerase chain reaction.

During the past 3 years, we encountered 12 new cases suspected of being dysfibrinogenemias, via plasma coagulation screening tests, which included determination of fibrinogen concentration both by thrombin time and immunologic methods. We performed sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions and immunoblot analysis for these plasma fibrinogens. We identified two cases that were characterized by two distinct gamma-chain bands, similar to previous observations with Matsumoto-II (the substitution of gamma308Asn to Lys). Therefore, in order to identify the gamma308Lys variant easily and rapidly, we established an allele-specific polymerase chain reaction (AS-PCR). AS-PCR results indicated that the two cases were indeed heterozygous for the gamma308Lys variant; ten other cases were negative for this mutation. In conclusion, the ratio of fibrinogen concentrations determined by functional and antigenic methods in combination with the immunoblot analysis made these cases attractive for identifying the gamma308Lys mutation. The AS-PCR method proved to be a useful procedure to identify the gamma308K mutation.

The prevalence of Toxoplasma gondii antibodies in wild and captive cetaceans from Japan.

The seroprevalence of Toxoplasma gondii was investigated in wild and captive cetaceans from Japan. Antibodies against T. gondii were examined by both latex agglutination test (LAT) and indirect hemagglutination test (IHAT) for 77 serum or plasma samples obtained from 59 individuals of 6 species, including 2 hybrids. Antibody titers greater than 1:64 in LAT and greater than 1:640 in IHAT, indicative of the presence of T. gondii, were found in 11.9% of 59 individuals. In 7 samples that showed a positive reaction by IHAT, T. gondii titers were examined for each immunoglobulin (Ig) fraction separated by sucrose gradient centrifugation. The antibody peaks in each fraction were divided into 3 types, thought to be a reaction of IgM (type 1), IgG (type 2), and IgM with IgG (type 3). Type 1 was found in serum from a bottle-nosed dolphin (Tursiops truncatus) and a killer whale (Orcinus orca) sampled soon after capture off the Japanese coast in 1988; it was concluded that infection in the wild had occurred less than 15 yr before the study was performed. The prevalence of putative IgM and IgG antibodies from a captive-bred T. truncatus suggested that T. gondii infection also occurred in the aquarium.

Effects of dietary casein and soy-protein on metabolic conversion of eicosapentaenoic acid to docosahexaenoic acid in the liver of rat.

The differential effects of dietary proteins on the metabolic process of eicosapentaenoic acid (EPA, 20:5n-3) via docosapentaenoic acid (DPA, 22:5n-3) to docosahexaenoic acid (DHA, 22:6n-3) were studied using parameters calculated from the proportion of n-3 polyenoic acid in liver phospholipid (PL) of rats. Rats were given casein or soy-protein isolate (SPI) diet containing 3% EPA with or without methionine (Met) supplementation for 3 weeks. The (22:5 + 22:6)/20:5 and 22:5/20:5 ratios of the PL fractions in the liver of rats given SPI were evidently elevated compared with those given casein. These ratios were also elevated when linoleic acid (LA, 18:2n-6) was added to the diet. There were no significant differences in the 22:6/22:5 ratio of the same PL fractions between the casein and SPI groups without the addition of LA, but the ratio was lowered by the addition of LA. It was found that these parameters for metabolic conversion were not affected by supplementation of Met or by a decrease in body weight. The above results suggest that the elongation step from EPA to DPA is affected by the type of protein, i.e., it is accelerated by SPI and is not affected by the presence of LA, while the metabolic process from DPA to DHA is not affected by the type of proteins without the addition of LA but is suppressed when LA is added.

[Comparison of coagulation screening test results in two cases having dysfibrinogen].

We compared coagulation screening test [prothrombin time (PT), activated partial prothrombin time (APTT), fibrinogen level determined by thrombin time method] results in two cases of dysfibrinogenemia which were named Matsumoto I (MI) and II (MII), respectively. Amino acid substitution in MI, gamma 364Asp-->His, and that in MII, gamma 308 Asn-->Lys, were deduced by sequencing analysis of PCR amplified products from each genomic DNA. The ratios of functional fibrinogen levels determined by the thrombin time method to immunological levels determined by the latex photometric assay were markedly decreased in both cases. Thrombin time (TT) in the absence of Ca2+ and fibrin aggregation test were also decreased. It was noted that the above described abnormalities were more prominent in MI than MII. We thought these results reflected the extent of abnormality in fibrin monomer polymerization. Furthermore, the mutation at residue gamma 364Asp in MI is adjacent to the gamma 363Tyr which is within the primary polymerization site of fibrin monomer, whereas the mutation at the residue gamma 308Asn in MII is outside the primary site. Since fibrin monomer polymerization is promoted in lower ionic strength and in higher concentration of Ca2+, the data of PT, APTT, and TT, that were tested in the presence of higher concentration of Ca2+ and fibrin monomer, were similar in MI and MII. In addition, it would be speculated that mutant fibrinogen interferes the function of normal fibrinogen existed in heterozygous dysfibrinogenemia.

[Changes in coagulation parameters during the clinical courses of recipients of living-related partial liver transplantation].

We had rare opportunities to examine changes in fibrin degradation products (FDP)-D-dimer (DD), thrombin-antithrombin III complex (TAT), plasmin-alpha 2-plasmin inhibitor complex (PIC) and other coagulation parameters during the clinical courses of living-related partial liver transplantation (LRPLT). In seven out of eight recipients without severe rejection and/or disseminated intravascular coagulation (DIC), FDP-DD values reached their maximum at 5 to 10 days after transplantation, then gradually decreased. On the other hand, TAT values rose to the maximum at anhepatic or reperfusion phase of liver transplantation. These data represent hypercoagulation in consequence of tissue thromboplastin activation after extensive operation. Changes in PIC, tissue-type plasminogen activator, and plasminogen activator inhibitor-1 (PAI-1) in the clinical course of case 1 suggested that fibrinolysis was suppressed by relatively elevated level of PAI-1 around the operation, but thereafter was adversely accelerated by relatively lower levels of PAI-1. In comparison with patients with DIC, TAT was much higher but PIC was significantly lower in recipients of LRPLT. These findings indicated that marked hypercoagulation and mild to moderate hyperfibrinolysis occurred in recipients of LRPLT.

[Studies on the blood coagulation factor XIII in patients with increased levels of FDP D-dimer].

Antigen levels of blood coagulation factor XIII (XIII) were determined in plasmas from patients with increased levels of fibrin degradation products-D-dimer (FDP-DD), including disseminated intravascular coagulation (DIC), by latex photometric immunoassay using polyclonal anti-XIII a subunit antibody-coated latex reagent. Since stable fibrin is directly degradated by plasmin and FDP-DD is produced, plasma FDP-DD levels correlate with plasmin-alpha 2-plasmin inhibitor complex levels, but not with thrombin-antithrombin III complex (TAT) or XIII levels. In order to clarify other causes of discordant relationships among the related three parameters, we studied the changes in plasma XIII, TAT and FDP-DD levels in fourteen DIC patients induced by various primary disorders. Only in two cases, XIII levels changed up and down irrelevant to the fluctuating levels of TAT and FDP-DD. In seven cases, plasma XIII levels remained low during the clinical courses, indicating possibilities that elevated condition of XIII consumption continued and/or production of XIII was low. On the other hand, in four patients, including two patients with nephrosis, XIII might be produced at higher rate than that of consumption. Same phenomenon was observed in one of eight recipients with living-related liver transplantation who showed remarkably increased levels of FDP-DD without DIC. In conclusion, plasma XIII level in patients with elevated FDP-DD may be influenced by the balance between consumption of XIII by unstable fibrin and/or surgical stress and the following tissue recovery etc. and production of XIII in liver, megakaryocytes and monocytes.