Electrospray ionization mass spectrometry identification of fibrinogen Banks Peninsula (gamma280Tyr-->Cys): a new variant with defective polymerization

cis-Diamminedichloroplatinum (cisplatin) is an important anticancer drug used to treat solid tumors. The nephrotoxicity of cisplatin is recognized as the most important dose-limiting factor, but high doses of cisplatin also produce hepatotoxicity. However, little is known about cisplatin-induced liver injury and the role of metallothionein, a cysteine-rich, metal-binding protein, in modulating its hepatotoxicity. This study was designed to examine cisplatin hepatotoxicity in control and metallothionein-I/II knockout (MT-null) mice. Animals were given a single injection of cisplatin (50-200 mumol/kg i.p.), and liver injury was evaluated 3-16 h later. Cisplatin produced dose- and time-dependent liver injury, as evidenced by increased serum activity of alanine aminotransferase (ALT), as well as by histopathology. Apoptosis, rather than necrosis, predominates in cisplatin-induced liver injury, as indicated by increased numbers of apoptotic cells (hematoxylin and eosin staining), in situ apoptotic DNA detection, and DNA fragmentation on agarose gel electrophoresis. MT-null mice were more sensitive than controls to cisplatin-induced hepatotoxicity. Cisplatin (200 mumol/kg) was lethal to 12% of control mice, but 60% of MT-null mice died within 16 h. At the dose of 150 mumol/kg, serum ALT activities were increased 2-fold in control mice compared to 6.5-fold in MT-null mice. Apoptotic lesions were more pronounced in MT-null than in control mice. MT-null mice were also more susceptible than controls to cisplatin-induced nephrotoxicity, as evidenced by having higher blood urea nitrogen concentrations. Furthermore, cultured MT-null hepatocytes were more sensitive than control cells to the cytotoxicity of cisplatin (50-200 microM), as indicated by lactate dehydrogenase leakage into the medium. These results demonstrate that (1) high doses of cisplatin produce hepatotoxicity, with apoptosis as the major lesion, and (2) MT protects against cisplatin-induced liver injury.     

Plasma factor XIII binds specifically to fibrinogen molecules containing gamma chains

The difference between peak 1 and peak 2 fibrinogen lies in their gamma chains. Peak 1 molecules contain 2 gamma A chains; peak 2 molecules contain 1 gamma A and 1 gamma chain, the latter of which contains a 20 amino acid extension (gamma 408-427) replacing the carboxyl-terminal 4 amino acids of the gamma A chain (gamma A 408-411). While the existence of gamma chains in plasma fibrinogen molecules has been known for many years, their function remains unknown. When fibrinogen is purified from plasma, the factor XIII zymogen (A2B2) copurifies with it and is found only in the peak 2 fibrinogen when this fraction is separated from peak 1 fibrinogen by ion-exchange chromatography on DEAE-cellulose. Factor XIII alone applied to the same DEAE column elutes at a position between peak 1 and peak 2. When mixtures of peak 1 fibrinogen plus factor XIII or peak 2 fibrinogen plus factor XIII are applied to DEAE columns, the peak 1/factor XIII mixture elutes in two peaks, whereas the peak 2/factor XIII mixture elutes in the peak 2 fibrinogen position. Gel sieving on Superose 6 of peak 1/factor XIII mixtures results in two protein peaks, the first of which contains the fibrinogen. Most factor XIII activity elutes in the second peak with a small amount of activity emerging with the trailing end of the fibrinogen peak. Gel sieving of mixtures of peak 2 and factor XIII results in a single protein peak with all factor XIII activity emerging with the leading edge of the fibrinogen peak. The interaction between peak 2 fibrinogen and plasma factor XIII appears to be through binding to the B subunit of factor XIII since placental or platelet factor XIII (A2), which does not contain B subunits, elutes independently from peak 2 fibrinogen on DEAE-cellulose chromatography. The results indicate that peak 2 fibrinogen gamma chains have a physiologically significant affinity for the B subunits of plasma factor XIII and that through this interaction fibrinogen serves as a carrier for the plasma zymogen in circulating blood.     

Fibrinogen coating density affects the conformation of immobilized fibrinogen: implications for platelet adhesion and spreading

Adhesion of platelets to immobilized fibrinogen appears to play an important role in a variety of physiologic and pathologic phenomena. We previously observed that the fibrinogen concentration used to coat polystyrene wells affected the morphology and distribution of GP IIb/IIIa receptors on the surface of platelets adherent to the fibrinogen. One possible explanation for these differences is that fibrinogen immobilized at high density adopts a different conformation than fibrinogen immobilized at low density. To address this possibility, we studied the binding of a panel of anti-fibrinogen monoclonal antibodies (mAbs) to fibrinogen immobilized at different coating densities. Three different patterns of binding were observed: 1) a linear increase in binding to wells coated with 1-10 microg/ml fibrinogen, followed by a lesser increase or plateau at higher fibrinogen concentrations (mAbs Fd4-4E1, Fd4-7B3, 1D4, 4-2); 2) minimal reactivity at all fibrinogen concentrations (mAbs GC4-1A12, 2C34); 3) a biphasic response, with a linear increase up to 10 microg/ml fibrinogen and then a significant decline in binding at higher fibrinogen concentrations (mAbs 311, 31A9, FPA 19/7, 9C3, 1C5-A5/2, 44-3). The patterns of mAb binding to fibrinogen immobilized from plasma were similar. Most mAbs that demonstrated a biphasic response bound poorly or not at all to soluble fibrinogen, while mAbs that demonstrated a linear/plateau response were able to bind soluble fibrinogen. At equal surface densities, mAbs that bound biphasically, particularly mAb 1C5-A5/2, were more reactive to urea-denatured than native fibrinogen. mAbs 1C5-A5/2 and 44-3 are specific for gamma 1-78 and 95-265, respectively, suggesting that the fibrinogen gamma-chain may be sensitive to changes in conformation induced by immobilization. In summary, these data suggest that fibrinogen immobilized at 1-10 microg/ml adopts a conformation unlike soluble fibrinogen, while fibrinogen immobilized at > 30 microg/ml adopts a more solution-like conformation. These differences in fibrinogen conformation may partially account for the ability of platelets to bind to immobilized fibrinogen without the addition of agonist, as well as the differences in spreading and GPIIb/IIIa distribution on platelets adherent to high- versus low-density immobilized fibrinogen.     

Carboxy-terminal amino acid sequence of a human fibrinogen gamma-chain variant (gamma')

A normal human fibrinogen gamma-chain variant, termed gamma', is larger than the gamma chain (51 500 vs. 49 500) due to an extended COOH-terminal sequence. The extended COOH-terminal cyanogen bromide peptide (CNBr e') was isolated by high-pressure liquid chromatography, and its amino acid sequence was determined. Comparison with the corresponding COOH-terminal gamma-chain peptide (CNBr e) showed that the last four amino acids of the gamma chain were replaced in gamma' chains by a 20-residue fragment rich in aspartic and glutamic acids, having the sequence Val-Tyr-Pro-Glu-His-Pro-Ala-Glx-Thr-Glx-Tyr-Asx-Ser-Leu-Arg-Pro-Glx-Asx-Asx-Leu . Mutant gamma chains (gamma Paris I) from a congenitally dysfunctional fibrinogen molecule (fibrinogen Paris 1) express both gamma and gamma' features, suggesting that both gamma and gamma' chains are produced from a single gene. If this suggestion is correct, the observed differences in amino acid sequence could be explained by the existence of different mRNAs for gamma and gamma' chains, respectively, which are transcribed from one gene by differential RNA splicing.     

The fibrinogen RIBS-I epitope (gamma373-385) appears proximate to the gamma408-411 adhesive domain but is not involved in interaction between receptor-bound or surface-adsorbed fibrinogen and platelet GPIIbIIIa

The carboxyl terminus of the fibrinogen (Fg) gamma chain (gamma400-411) is necessary and sufficient to support platelet aggregation and adhesion. However, a monoclonal antibody (mAb) to the Fg RIBS-I epitope (gamma373-385), the anti-Fg-RIBS-I, which binds only to platelet-bound or surface-adsorbed Fg but not soluble Fg, inhibits platelet aggregation. In this study, we showed that this same antibody also inhibits the adhesion of platelets to Fg-coated polystyrene beads. We then investigated the mechanisms by which the anti-Fg-RIBS-I antibody inhibits platelet aggregation and adhesion. The Fg RIBS-I epitope does not interact with platelet GPIIbIIIa, since recombinant Fg missing the last four amino acids, the Ala-Gly-Asp-Val, on the carboxyl terminus of its gamma chains supports neither platelet aggregation nor adhesion to surfaces, nor GPIIbIIIa binding, while it binds anti-Fg-RIBS-I normally. Purified, soluble GPIIbIIIa (265 kDa) inhibits the binding of both the anti-Fg-RIBS-I and 4A5 (a mAb specific to gamma408-411 of Fg), however, peptide G13 (1.5 kDa), corresponding to the Fg gamma chain binding domain on GPIIba (GPIIb300-312), only inhibits the binding of 4A5, and does not affect the binding of the anti-Fg-RIBS-I to Fg. The anti-Fg-RIBS-I reduces the on-rate of the 4A5 binding to Fg with no measurable changes in the dissociation of the Fg-bound 4A5. These data indicate that the inhibition of platelet aggregation and adhesion by the anti-Fg-RIBS-I antibody is due to the steric hindrance of the Fg gamma400-411 to platelet GPIIbIIIa. Thus the Fg RIBS-I epitope (gamma373-385) does not appear to be involved in direct interaction with platelet GPIIbIIIa, leaving the gamma408-411 of Fg as the sole domain mediating platelet aggregation and adhesion.     

Activation of the fibrinogen binding site on platelets isolated from a patient with the Strasbourg I variant of Glanzmann's thrombasthenia

One proposed ligand binding site on platelet integrin alpha IIb beta 3 is the region of the beta 3 subunit encompassing amino acids 211-221. However, we recently showed that synthetic peptides corresponding to amino acids 211-221 inhibit fibrinogen binding to alpha IIb beta 3 by binding to alpha IIb beta 3 and not to fibrinogen. In this study, we show that AP6, a monoclonal antibody (MoAb) directed against amino acids 214-221 of beta 3, bound to immobilized active alpha IIb beta 3 but did not inhibit fibrinogen binding to the complex. We then determined whether nonfunctional alpha IIb beta 3 on platelets with a beta 3 Arg-214-->Trp mutation (Strasbourg I variant of Glanzmann's thrombasthenia or GTV) could be induced to aggregate after treatment with dithiothreitol (DTT). DTT has been shown to expose the fibrinogen receptor on normal platelets. DTT treatment of GTV platelets did result in the formation of the fibrinogen binding site as indicated by the binding of pI-55, an MoAb that only binds to the activated form of alpha IIb beta 3. Furthermore, DTT-treated GTV platelets aggregated in the presence of fibrinogen and divalent cations. This aggregation was inhibited by EDTA, RGDS, and the selective alpha IIb beta 3 antagonist, Ro 43-5054. These data show that Arg-214 of beta 3 is not required for fibrinogen binding or for platelet aggregation. However, this amino acid appears to be critical for the formation and for the maintenance of the correct tertiary structure of the fibrinogen binding site on alpha IIb beta 3.     

Reactivity of fibrinogen and fibrinopeptide A containing fibrinogen fragments with antisera to fibrinopeptide A

Two antisera used in the radioimmunoassay for human fibrinopeptide A (FPA) which appear to have different immunochemical specificities have been tested for cross-reactivity with fibrinogen and with three fragments of fibrinogen which contain the FPA sequence. These fragments were the three-chain, NH2-terminal disulfide knot (N-DSK) produced by CNBr cleavage of fibrinogen, the reduced, carboxymethyl Aalpha chain portion of the N-DSK, and fragment E produced by plasmin digestion of fibrinogen. One antiserum (R-2) showed high specificity for free FPA and less than 2% cross-reactivity with fibrinogen or the FPA-containing fragments. The other antiserum (R-33) possessed a much higher degree of cross-reactivity with the FPA-containing fragments. Synthetic and native fibrinopeptides were found to be indistinguishable in the assay system with either antiserum. As a result of these studies, an hypothesis has been developed concerning the nature of the antigenic determinants on FPA which favor measurement of free FPA and limit cross-reactivity with larger, FPA-containing peptides.     

Severely impaired polymerization of recombinant fibrinogen gamma-364 Asp --> His, the substitution discovered in a heterozygous individual

During blood coagulation, soluble fibrinogen is converted to fibrin monomers that polymerize to form an insoluble clot. Polymerization has been described as a two-step process: the formation of double-stranded protofibrils and the subsequent lateral aggregation of protofibrils into fibers. Previous studies have shown that gamma chain residues Tyr-363 and Asp-364 have a significant role in polymerization, most likely in protofibril formation. To better define the role of these residues, we synthesized three fibrinogens with single substitutions at these two positions: Tyr-363 --> Ala, Asp-364 --> Ala, and Asp-364 --> His. We found that the release of fibrinopeptides A and B was the same for these variants and normal recombinant fibrinogen, showing that all variants had normal fibrin formation. In contrast, we found that polymerization was significantly delayed for both Ala variants and was almost nonexistent for the His variant. Clottability for the Ala variants was only slightly reduced, and fibrin gels were formed. Surprisingly, clottability of the His variant was substantially reduced, and fibrin gels were not formed. Our data suggest that both protofibril formation and lateral aggregation were altered by these substitutions, indicating that the C-terminal domain of the gamma chain has a role in both polymerization steps.     

Improved biodistribution, tumor targeting, and reduced immunogenicity in mice with a gamma 4 variant of Campath-1H

Radiolabeled antibodies have shown promise for the treatment of lymphoma and for solid tumor targeting. Campath-1H is a humanized monoclonal antibody that reacts with the CD52 antigen present on human lymphoid and myeloid cells. Campath-1H is a gamma1 (G1) isotype that induces lymphopenia via an Fc-mediated mechanism(s). Isotype switches were engineered, and the resulting antibodies were expressed in NS0 mouse myeloma cells and biosynthetically radiolabeled with [35S]methionine. The forms included G1, G4, and a G4 variant that contained alanine substitutions at (EU numbering) Leu-235, Gly-237, and Glu-318. All isotypes bound antigen equivalently as assessed by target cell binding in vitro. The G4 variant had a greatly reduced capacity to interact with Fc receptor by virtue of reduced binding to THP-1 human myeloid cells and by a 1000-fold increase in EC50 to intermediate antibody-dependent cellular cytotoxicity. The pharmacokinetics of the isotypes were compared in CD-1 (nu/nu) mice bearing an experimental antigen-expressing tumor. The plasma half-life and tumor uptake were increased for the G4 variant. The G4 variant showed significantly less spleen, liver, and bone uptake but similar uptake in the lung, kidney, and stomach and lower tissue-to-blood ratios. Immunogenicity was assessed after repeated monthly administrations of unlabeled antibody in BALB/c mice. A 50% reduction in the incidence of anti-globulin response was observed for the G4 variant. These properties suggest that antibodies with reduced Fc receptor interaction merit additional study as potential targeting vehicles relative to other isotypes for radioimmunotherapy or situations where diminished normal tissue binding contributes to efficacy.     

Simultaneous communication with autistic and other severely dysfunctional nonverbal children

Despite its widespread application, the teaching of speech does not appear to be effective with all autistic and other severely dysfunctional, nonverbal children. A body of recent evidence points to the peculiarities in the information processing of these children and to the importance of gestures as aids or alternatives to speech. The present study reports on the use of simultaneous communication (gestures plus speech) with five such children. The fact that four of the five children showed marked gains in their ability to communicate over a 5-week period raises some theoretical and methodological issues pertinent to the treatment of autistic and autistic-like children and to our general understanding of the processes involved in nonverbal communication.     

A new variant of Bernard-Soulier syndrome characterized by dysfunctional glycoprotein (GP) Ib and severely reduced amounts of GPIX and GPV

We describe a new variant of Bernard-Soulier syndrome characterized by almost normal amounts of GPIb and severely reduced GPIX and GPV. Despite surface expression, GPIbalpha failed to support ristocetin-induced platelet agglutination and to bind two conformation-dependent monoclonal antibodies, suggesting a qualitative defect. Sequence analysis of the gene coding for GPIX revealed a T-to-C substitution at base 1811, leading to a Leu40Pro conversion, whereas no defects were found in the coding region of the GPIbalpha gene. Allele-specific restriction enzyme analysis showed that the propositus and one of his sisters. both with severe bleeding diathesis. were homozygous for the GPIX mutation: the members of the family with mild bleeding diathesis and/or giant platelets in the peripheral blood were heterozygous, whereas the healthy ones were homozygous for the normal allele. Infusion of 1-desamino-8-D-arginine vasopressin normalized bleeding time in the two severely affected patients, although it did not modify ristocetin-induced platelet agglutination or membrane expression of GPIbalpha, GPIX, GPIIb-IIIa and GMP-140. Moreover, in one patient, normalization of bleeding time and rise of von Willebrand factor plasma concentration did not seem to be directly related.     

A dysfunctional desmin mutation in a patient with severe generalized myopathy

Mice lacking desmin produce muscle fibers with Z disks and normal sarcomeric organization. However, the muscles are mechanically fragile and degenerate upon repeated contractions. We report here a human patient with severe generalized myopathy and aberrant intrasarcoplasmic accumulation of desmin intermediate filaments. Muscle tissue from this patient lacks the wild-type desmin allele and has a desmin gene mutation encoding a 7-aa deletion within the coiled-coil segment of the protein. We show that recombinant desmin harboring this deletion cannot form proper desmin intermediate filament networks in cultured cells, nor is it able to assemble into 10-nm filaments in vitro. These findings provide direct evidence that a mutation in desmin can cause human myopathies.     

Interaction of heparin with fibrinogen using surface plasmon resonance technology: investigation of heparin binding site on fibrinogen

Persistence of the Lyme disease spirochete, Borrelia burgdorferi, in the presence of an active immune response has been well documented. Evidence from the past year indicates that modulation of surface antigens by the spirochete may be a major mechanism for evading the immune response.     

Depressed patients with dysfunctional families: Description and course of illness.

68 depressed patients were subdivided according to their family's level of family functioning into functional and dysfunctional groups. Patients from dysfunctional families did not differ from those of functional families on measures of severity of depression, chronicity of depression, depression subtypes, other nonaffective psychiatric diagnoses, history of depression, or neuroendocrine functioning. Ss from dysfunctional families did have significantly higher levels of neuroticism. A 12-mo follow-up of these patients indicated that depressed patients with dysfunctional families had significantly poorer course of illness, as manifested by higher levels of depression, lower levels of overall adjustment, and a lower proportion of recovered patients. Thus, impaired family functioning appears to be an important prognostic factor in major depression. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

A late onset familial amyloidotic polyneuropathy (FAP) with a novel variant transthyretin characterized by a basic-for-acidic amino acid substitution (Glu61-->Lys)

A 64-year-old man has suffered from intractable diarrhea since January 1990. He noticed numbness and weakness in the distal portion of four extremities in the following several months. His symptoms were gradually progressive. In June 1992, neurological examination revealed mild muscular atrophy and weakness in the proximal and distal portions of four extremities. There were paresthesia and severe impairment of superficial sensations in the lower limbs, lower half of the trunk and upper limbs. All deep tendon reflexes were reduced or absent. Autonomic dysfunctions such as orthostatic hypotension, impotence and diarrhea were evident. On sural nerve biopsy, myelinated fibers showing axonal degeneration were predominantly seen, and densities of both myelinated and unmyelinated fibers were markedly decreased. No amyloid deposits were found in the endoneurium. Amyloid deposition was identified in the gastric mucosa by Congo red staining and immunostaining with anti-transthyretin (TTR) antibody. Edman degradation showed one amino acid substitution of Lys for Glu at position 61 in the TTR-peptides from the serum. Direct DNA sequencing revealed a new point mutation in the 61st codon of TTR gene. The same point mutation of TTR gene was identified in the DNAs from his 67-year-old brother and 63-year-old sister and one of the paternal cousins, a 64-year-old woman, although their clinical symptoms and signs were negative. Clinical features such as late onset of the symptoms and signs and presence of carriers in their sixties in this family are unique and atypical as compared with those of more frequent Val30-->Met FAP families. A variant TTR, characterized by a Glu61-->Lys substitution (a basic-for-acidic amino acid substitution) found in this family, has not been reported in the literature. In the case of the examination of the patients with autonomic and sensory symptoms and signs of unknown etiology, amyloidotic polyneuropathies, including FAP even in the absence of the family history, should be differentiated. When FAP is highly suspected, the combination of family study and DNA analysis of a possible variant TTR is indispensable for the establishment of the diagnosis.