A classification of the fibrin network structures formed from the hereditary dysfibrinogens.

OBJECTIVE: The main objective was to study the relationships of the molecular defects in 38 dysfibrinogens with their fibrin networks. METHODS AND RESULTS: Scanning electron microscopic analyses revealed that all the fibrins formed under the same conditions had networks composed of either normal thickness fibers or thin fibers, accompanied by a variety of alterations in the network structure and characteristics. We classified these fibrin networks into five classes, designated normal, less-ordered, porous A, porous B and lace-like networks. The dysfibrinogens with defects in fibrinopeptide A release or the E:D binding sites formed normal or less-ordered networks, while those with defects in the D:D association formed porous A networks composed of many tapered terminating fibers, despite having fibers of normal width, and containing many pores or spaces. The porous B and lace-like networks were composed of highly branched thin fibers because of defects in the lateral association among protofibrils, and the major difference between them was the porosity of the porous B networks. All the porous B networks were easily damaged by mechanical stress, whereas the lace-like networks retained high resistance to such stress, indicating that the network strength was not dependent on the fiber width, but on the porosity that led to fragility of the network. CONCLUSION: Impairment of the D:D association is the major disturbing factor that leads to the formation of porous fibrin networks. The porosity may be introduced by severe impairment of the D:D association, as well as the lateral association, as has often been observed by extra glycosylation or defects in Ca2+ binding.

Induction of immune tolerance by neonatal intravenous injection of human factor VIII in murine hemophilia A.

Inhibitory antibody formation is the most serious complication of factor (F)VIII replacement therapy in hemophilia A patients. FVIII-deficient mice were used to study new approaches for induction of immune tolerance. Neither antiFVIII inhibitory antibodies nor antiFVIII IgGs were observed in 13 of 14 adult mice that received 0.05 U g(-1) body weight of human FVIII intravenously within 24 h after birth and repeated injections as adults. In contrast, high FVIII antibody titers (>50 Bethesda Units mL(-1)) developed in seven of 13 mice injected on day 3 postpartum and in all adult mice not treated neonatally. One of nine mice and three of 17 mice developed high-titer antiFVIII inhibitory antibody when they were treated initially with 2-fold (0.1 U g(-1) body weight) and 10-fold higher doses (0.5 U g(-1) body weight) FVIII on day 0, respectively. A human FVIII-specific T-cell proliferative response was absent in splenocytes from neonatally treated mice. The tolerance was FVIII specific because antitoxoid antibodies developed after immunization with tetanus toxoid. Splenocytes failed to proliferate or produce interferon (IFN)-gamma in response to FVIII stimulation, yet still secreted interleukin-2. A proliferative response was restored with exogenous IFN-gamma or interleukin-12, suggesting that lack of inhibitor to FVIII was due to IFN-gamma-dependent anergy. Thus, exposure on day 0 to physiological levels of FVIII antigen might be important for induction of immune tolerance. This immune tolerance model may provide a basis for new approaches to prevention of FVIII inhibitors during replacement therapy.

Hypofibrinogenemia caused by a nonsense mutation in the fibrinogen Bbeta chain gene.

Congenital hypofibrinogenemia, fibrinogen Tottori II, caused by a nonsense mutation in the fibrinogen Bbeta chain gene, was found in a 68-year-old Japanese female. The plasma fibrinogen level was 99.2 mg dL(-1) as determined by the thrombin time method. No overt molecular abnormalities were observed in purified patient fibrinogen by SDS-PAGE analysis. After sequencing all exons and exon-intron boundaries of three fibrinogen genes, we found a heterozygous single point mutation of T-->G at position 3356 of the patient fibrinogen Bbeta chain gene. This nucleotide mutation results in a nonsense mutation (TAT sequence for Bbeta 41Tyr to TAG sequence for a translation termination signal). The mutation was confirmed by polymerase chain reaction-restriction fragment length polymorphism analysis, since this nucleotide mutation results in a new NheI recognition sequence at this position. These data indicated that the nonsense mutation of the fibrinogen Bbeta chain gene caused a truncated fibrinogen Bbeta chain, which may not be assembled in the fibrinogen molecule.

Concentration of 17beta-estradiol using an immunoaffinity porous hollow-fiber membrane.

We describe attempts to achieve high throughput of 17beta-estradiol (E2) analysis, including the development of an immunocleanup membrane using polyclonal antibodies and an enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies. An epoxy-group-containing monomer, glycidyl methacrylate (GMA), was graft-polymerized onto a porous hollow-fiber membrane. Subsequently, anti-estrogen (ES) antibody, as a ligand, was coupled with the epoxy group. The ligand density ranged from 3.1 to 5.8 mg/g of the GMA-grafted porous hollow-fiber membrane. A 1.0 microg/L E2 solution was forced to permeate through pores rimmed by the anti-ES-antibody-immobilized polymer chains, at a constant permeation rate. A breakthrough curve, that is, the change in the E2 concentration of the effluent penetrating the outside of the hollow fiber with a change of the effluent volume, was determined. Bound E2 in amounts ranging from 0.42 to 0.80 microg was quantitatively eluted with 3-5 mL of methanol in the permeation mode. The higher permeation rate of the E2 solution resulted in the higher overall binding rate of E2 to the anti-ES-antibody-immobilized porous hollow-fiber membrane because of the negligible diffusional mass-transfer resistance of E2 to the antibody.

S-Adenosyl-L-homocysteine hydrolase is sequestered into actin rods in Dictyostelium discoideum spores.

Here we show evidence that S-adenosyl-L-homocysteine hydrolase (SAHH) is linked to the actin cytoskeleton. Actin rods formed in Dictyostelium discoideum spores during the final stage of development are structurally composed of novel bundles of actin filaments. SAHH only accumulates with actin at this stage of development in the life cycle of D. discoideum. Recently SAHH is believed to be a target for antiviral chemotherapy and the suppression of T cells. Our finding may contribute to designing novel antiviral and immunosuppressive drugs.

High conversion in asymmetric hydrolysis during permeation through enzyme-multilayered porous hollow-fiber membranes.

We describe a novel porous hollow-fiber support for immobilizing aminoacylase in multilayers. Epoxy-group-containing polymer chains were grafted onto a porous hollow-fiber membrane by radiation-induced graft polymerization of glycidyl methacrylate, and subsequently a diethylamino group as an anion-exchange group was introduced into the graft chain. Aminoacylase was adsorbed in multilayers by allowing the amioacylase buffer solution to permeate through the pores across the hollow fiber; the graft chains provided three-dimensional space for the enzymes because of their electrostatic repulsion. The adsorbed enzyme at a degree of multilayer binding of 15 was cross-linked with glutaraldehyde to prevent leakage. An acetyl-DL-methionine solution was allowed to permeate through the pores surrounded by the aminoacylase-immobilized graft chain. Production of L-methionine was observed at a 4.1 mol/h per L of the fiber for a space velocity of 200 h(-1), defined as the flow rate of the effluent penetrating the outside surface of the hollow fiber divided by the membrane volume including the lumen.

Purification of docosahexaenoic acid ethyl ester using a silver-ion-immobilized porous hollow-fiber membrane module.

Docosahexaenoic acid ethyl ester (DHA-Et) was purified by adsorption on Ag-ion-immobilized membranes via selective interaction between silver ion and carbon-carbon double bonds of DHA-Et. Silver ions were immobilized onto sulfonic-acid-group-containing porous hollow-fiber membranes at an Ag ion density of 1.4 mol/kg of membrane, and 30 membranes were housed in one module (inner diameter = 18 mm and effective length = 80 mm). The adsorption isotherms of DHA-Et in various organic solvents revealed that DHA-Et was adsorbed on the immobilized Ag ions with a DHA-Et/Ag ion molar binding ratio of 1/5 in methanol, and that acetonitrile was the solvent of choice for the elution of the adsorbed DHA-Et. Permeation of bonito oil ethyl ester solution in methanol through the Ag-ion-immobilized hollow-fiber membrane module demonstrated that the displacement adsorption of other lower unsaturated fatty-acid ethyl esters by DHA-Et proceeded along the membrane thickness. The purity of DHA-Et was improved to 99 wt % by permeating first bonito oil ethyl ester containing 95 wt % DHA-Et and then acetonitrile through the module.

Comparison of L-tryptophan binding capacity of BSA captured by a polymer brush with that of BSA adsorbed onto a gel network.

A polymer brush containing a diethylamino group as an anion-exchange group was appended onto a polymer substrate by radiation-induced graft polymerization and subsequent chemical modifications. Bovine serum albumin as a chiral ligand for L-tryptophan was bound to the polymer brush at a density ranging from 17 to 150 g BSA/l. For comparison, BSA was adsorbed onto the gel network containing a diethylaminoethyl group. The molar binding ratio of L-tryptophan to BSA on the polymer brush was 1.7-fold higher than that to BSA on the gel network.

Mode of perturbation of Asahi fibrin assembly by the extra oligosaccharides.

Steric hindrance by the backbone of extra oligosaccharides at gamma-Asn 308 may cause the repulsive force to widen the junction at the D:D interface, and thus, interfere with the longitudinal elongation and lateral association of protofibrils.

Hereditary disorders of fibrinogen.

Fibrinogen, a 340-kDa plasma protein, is composed of two identical molecular halves each consisting of three non-identical A alpha-, B beta- and gamma-chain subunits held together by multiple disulfide bonds. Fibrinogen is shown to have a trinodular structure; that is, one central nodule, the E domain, and two identical outer nodules, the D-domains, linked by two coiled-coil regions. After activation with thrombin, a pair of binding sites comprising Gly-Pro-Arg is exposed in the central nodule and combines with its complementary binding site a in the outer nodule of another molecules. By using crystallographic analysis, the alpha-amino group of alpha Gly-1 is shown to be juxtaposed between gamma Asp-364 and gamma Asp-330, and guanidino group of alpha Arg-3 between the carboxyl group of gamma Asp-364 and gamma Gln-329 in the a site. Half molecule-staggered, double-stranded protofibrils are thus formed. Upon abutment of two adjacent D domains on the same strand, D-D self association takes place involving Arg-275, Tyr-280, and Ser-300 of the gamma-chain on the surface of the abutting two D domains. Thereafter, carboxyl-terminal regions of the alpha-chains are untethered and interact with those of other protofibrils leading to the formation of thick fibrin bundles and networks. Although many enigmas still remain concerning the exact mechanisms of these molecular interactions, fibrin assembly proceeds in a highly ordered fashion. In this review, these molecular interactions of fibrinogen and fibrin are discussed on the basis of the data provided by hereditary dysfibrinogens on introducing representative molecules at each step of fibrin clot formation.

Autoantibody against prothrombin aberrantly alters the proenzyme to facilitate formation of a complex with its physiological inhibitor antithrombin III without thrombin conversion.

Acquired coagulation factor inhibitors include pathologic immunoglobulins that specifically bind to coagulation factors and either neutralize their procoagulant activity, accelerate their clearance from the circulation, or have proteolytic activity to degrade them into inactive polypeptides. Here, an autoantibody against prothrombin is described in a patient with serious hemorrhagic diatheses. The autoantibody exerts its influence by a previously unknown mechanism in which it inhibits coagulation through aberrant activation of the proenzyme in a catalytic manner. The antibody-bound prothrombin formed a stable stoichiometric complex with antithrombin III, consisting of intact prothrombin and an antithrombin III molecule cleaved at the (393)Arg-(394)Ser bond. The antibody dissociated from prothrombin after the complex formation with antithrombin III. Although the bound antibody elicited protease activity from prothrombin, the complex was not able to convert fibrinogen to fibrin or to activate protein C. Thus, this is the first description of an autoantibody that induces protease-like activity from a human proenzyme, permitting subsequent neutralization by its physiological inhibitor. (Blood. 2001;97:3783-3789)

Cloning of a novel G protein-coupled receptor, SLT, a subtype of the melanin-concentrating hormone receptor.

A DNA fragment encoding an amino acid sequence possessing common features to the G protein-coupled receptor (GPCR) superfamily was found in the human genomic sequence, and from this information, the full-length cDNA of a novel GPCR, designated SLT, was cloned from the human hippocampus cDNA library. SLT showed the highest homology to the melanin-concentrating hormone (MCH) receptor, SLC-1 (31.5% identity), and to a lesser extent, to the somatostatin (SST) receptor subtypes. MCH exhibited agonistic behavior when applied to the SLT-expressing CHO cells at subnanomolar doses whereas more than 200 known peptides, including SST and cortistatin, did not. These results indicated that MCH is the cognate ligand of the SLT receptor and that this newly cloned GPCR is the second subtype of the MCH receptor. Quantitative polymerase chain reaction analysis of the SLT gene expression in human tissues showed that the SLT receptor is expressed mainly in brain areas including the cerebral cortex, amygdala, hippocampus, and corpus callosum, as well as in a limited number of peripheral tissues. The distribution of the SLT nearly overlapped that of SLC-1, suggesting that some of the neural functions of MCH may be mediated by both of these receptor subtypes.

End-linked homodimers in fibrinogen Osaka VI with a B beta-chain extension lead to fragile clot structure.

The authors have identified a 12-residue carboxyl-terminal extension of Lys-Ser-Pro-Met-Arg-Arg-Phe-Leu-Leu-Phe-Cys-Met in a dysfibrinogen derived from a woman heterozygotic for this abnormality and associated with severe bleeding. This extension is due to a T-to-A mutation that creates AAG encoding Lys at the stop (TAG) codon, thus translating 36 base pairs in the noncoding region of the Bbeta gene. The extra Cys residues appear to be involved in 1 or 2 disulfide bonds between 2 adjacent abnormal fibrinogen molecules, forming a fibrinogen homodimer as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Indeed, about half of the fibrinogen molecules exist as end-linked dimers oriented in parallel or with an angle, as observed by transmission electron microscopy. These end-linked dimers may well alter the conformations of D and DD regions on fibrin assembly, leading to increased fiber branching at their sites in the growing protofibrils. By scanning electron microscopy, the Osaka VI fibrin network appears to have a lacelike structure composed of highly branched, thinner fibers than the normal fibrin architecture. Such fibrin networks may be easily damaged to form large pores when fluids are allowed to pass through the gels. The fragility of Osaka VI fibrin clots, further confirmed by permeation and compaction studies, may account for the massive bleeding observed in this patient. (Blood. 2000;96:3779-3785)

High-speed recovery of germanium in a convection-aided mode using functional porous hollow-fiber membranes.

A porous hollow-fiber membrane capable of recovery of germanium from a liquid stream was prepared by radiation-induced graft polymerization of an epoxy-group-containing vinyl monomer, glycidyl methacrylate, and subsequent functionalization with 2,2'-iminodiethanol, di-2-propanolamine, N-methylglucamine, and 3-amino-1,2-propanediol. The functional group density was as high as 1.4 mol per kg of the resultant hollow fiber. The polymer chains containing functional groups surrounding the pores enabled a high-speed recovery of germanium during permeation of a germanium oxide (GeO2) solution through the pores of the hollow fiber. Because of a negligible diffusional mass-transfer resistance, germanium concentration changes with the effluent volume, i.e., breakthrough curves, overlapped irrespective of the residence time of the solution, which ranged from 0.37 to 3.7 s across the hollow fiber. After repeated use of adsorption and elution, the adsorption capacity did not deteriorate.

Multilayer binding of proteins to polymer chains grafted onto porous hollow-fiber membranes containing different anion-exchange groups.

Various anion-exchange groups were introduced into the polymer chains grafted onto a porous hollow-fiber membrane for protein recovery by radiation-induced graft polymerization and subsequent functionalization of a monomer containing an epoxy group. The graft chains extended from the pore surface toward the pore interior, resulting in the multilayer binding of proteins to the graft chains. Combinations of three anion-exchange groups, namely, amino (AM), ethylamino (EA), and diethylamino (DEA) groups, and three proteins, namely, beta-lactoglobulin, bovine serum albumin, and urease, were examined to evaluate the degree of multilayer binding of protein to the graft chains in the permeation mode. Multilayer binding was observed for hollow-fiber membranes containing EA and DEA groups, with conversions of epoxy groups to EA or DEA groups of higher than 80%. The amount of adsorbed protein remained constant irrespective of the conversion for the hollow-fiber membrane containing an AM group. The dependence of the flux on the conversion was consistent with that of the degree of multilayer binding to the graft chains.

A monoclonal antibody specific to the granulocyte-derived elastase-fragment D species of human fibrinogen and fibrin: its application to the measurement of granulocyte-derived elastase digests in plasma.

When granulocytes are stimulated under certain clinical conditions, elastase is released therefrom and digests fibrin(ogen) independently of the plasmin system, which may also be mobilized simultaneously. Thus, discrimination of these 2 systems becomes urgent for the diagnosis and treatment of the underlying diseases. Using as immunogen a 97-kd granulocyte-elastase digest of human fibrinogen, we raised an antibody IF-123 that specifically recognizes elastase digests of human fibrin(ogen). The 97-kd elastase fragment resembles plasmic fragment D(1), and the epitope of this antibody is located on the Aalpha (196-204) residue segment. This segment appears to be masked in fibrin(ogen) but exposed when the Aalpha Leu 204-Ile 205 peptide bond is cleaved by elastase. Cathepsin G concomitantly released from granulocytes failed to expose the epitope. By an enzyme immunoassay using IF-123 as the capture antibody, the elastase digests of fibrin(ogen) can be measured in plasma samples without interference by abundantly coexisting fibrinogen. Indeed, we found that the elastase digests were mostly elevated in patients with inflammation or malignant tumors, but remained in a normal range in patients with a benign gastrointestinal tract disease such as duodenal ulcer and polyps in the gallbladder or the colon. Like the plasmic D-dimer, the elastase digests predominantly consisted of the DD/E complex and DD/E-containing high-molecular weight derivatives apparently corresponding to the phase-3 plasmic digests of cross-linked fibrin. (Blood. 2000;95:1721-1728)

Structure and function of fibrinogen: insights from dysfibrinogens.

The structure-function relationships of dysfibrinogens and their clinical implications are discussed on the basis of the data provided from representative molecules.

Urotensin II is the endogenous ligand of a G-protein-coupled orphan receptor, SENR (GPR14).

Two molecular species of urotensin II (UII) were isolated from porcine spinal cords and identified as the endogenous ligands of a G-protein-coupled orphan receptor, SENR (sensory epithelium neuropeptide-like receptor), which is identical to GPR14. We established a CHO cell line stably expressing the rat SENR and investigated several tissue extracts to evoke the response mediated by the SENR. Extract from porcine spinal cords showed an activity of arachidonic acid metabolites release from SENR-expressing cells and was purified using HPLC. Two active substances were isolated and their sequences were determined as GPTSECFWKYCV and GPPSECFWKYCV, which were revealed to be porcine UII. Synthetic UII peptides caused arachidonic acid metabolites release activity in the rat SENR-expressing cells with an EC(50) value of 1 nM. Three cDNAs encoding the precursor proteins of porcine UII were cloned from a porcine spinal cord cDNA library; 2 consist of 121 amino acid residues and the other, which seemed to be a splicing variant, consist of 85 residues.

Fibrinogen Niigata with impaired fibrin assembly: an inherited dysfibrinogen with a Bbeta Asn-160 to Ser substitution associated with extra glycosylation at Bbeta Asn-158.

A novel BbetaAsn-160 (TAA) to Ser (TGA) substitution has been identified in fibrinogen Niigata derived from a 64-year-old asymptomatic woman, who is heterozygotic for this abnormality. The mutation creates an Asn-X-Ser-type glycosylation sequence, and a partially sialylated biantennary oligosaccharide was linked to the BbetaAsn-158 residue. The functional abnormality was attributed to delayed lateral association of normally formed double-stranded protofibrils based on normal cross-linking of fibrin gamma-chains and tissue-type plasminogen activator-catalyzed plasmin generation by polymerizing fibrin monomers. Enzymatic removal of all the N-linked oligosaccharides from fibrinogen Niigata accelerated fibrin monomer polymerization that reached the level of untreated normal fibrin monomers, but the thrombin time was prolonged from 18.2 seconds to 113 seconds (normal: 11.2 seconds to 8.9 seconds). By scanning electron micrographic analysis, Niigata fibrin fibers were found to be more curvilinear than normal fibrin fibers. After deglycosylation, Niigata fibers became straight being similar to untreated normal fibrin fibers, whereas normal deglycosylated fibrin appeared to be less-branched than untreated normal or deglycosylated Niigata fibrin. Although normal and Niigata fibrins were similar to each other in permeation and compaction studies, deglycosylated normal and Niigata fibrins had much higher permeability and compaction values, indicating that deglycosylation had brought about the formation of more porous networks. The enzymatic deglycosylation necessitates an Asn to Asp change at position Bbeta-158 that is responsible for reducing the fiber thickness because of either local repulsive forces or steric hindrance in the coiled-coil region.

Isolation and identification of melanin-concentrating hormone as the endogenous ligand of the SLC-1 receptor.

Melanin-concentrating hormone (MCH), which is an orexigenic peptide, was isolated and identified as the endogenous ligand of the SLC-1 receptor. We established a CHO cell line expressing the rat SLC-1 receptor to search for its endogenous ligand. The extract of rat whole brain showed inhibition of intracellular forskolin-induced cAMP accumulation in rat SLC-1-expressing CHO cells and was purified. Using HPLC purification, we isolated and identified MCH as the endogenous ligand of the SLC-1 receptor. The authentic MCH demonstrated a dose-dependent inhibitory effect on cAMP accumulation in forskolin-stimulated rat and human SLC-1-expressing CHO cells with an EC(50) value of 0.2 nM for both the rat and human SLC-1 receptors. This is the first description of the functional receptor for MCH.