Fibrinogen Seattle II: congenital dysfibrinogenemia with an Arg (A alpha 16)----his substitution

Incubation of fibrinogen Seattle II with thrombin (17 mu/ml) resulted in the release of two forms of fibrinopeptide A (FpA) which were resolved by high-performance liquid chromatography. Amino acid analysis disclosed that the abnormal FpA contained histidine in place of arginine. At lower, approximately physiologic thrombin concentrations only half the normal amount of FpA was released, and fibrinopeptide B (FpB) release was delayed. The effect of this substitution on the time course of fibrinopeptide release is consistent with conclusions drawn from other studies on the kinetics of fibrinopeptide release, viz., that prior removal of FpA is not required before FpB hydrolysis by thrombin, and that optimal rates of FpB release occur after formation of fibrin I polymer.     

Fibrinogen Hershey IV: a novel dysfibrinogen with a gammaV411I mutation in the integrin alpha(IIb)beta(3) binding site

The carboxyl terminal segment of the fibrinogen gamma chain from gamma408-411 plays a crucial role in platelet aggregation via interactions with the platelet receptor alpha(IIb)beta(3). We describe here the first naturally-occurring fibrinogen point mutation affecting this region and demonstrate its effects on platelet interactions. DNA sequencing was used to sequence the proband DNA, and platelet aggregation and direct binding assays were used to quantitate the biological effects of fibrinogen Hershey IV. The Hershey IV proband was found to be heterozygous for two mutations, gammaV411I and gammaR275C. Little difference in aggregation was seen when fibrinogen Hershey IV was compared to normal fibrinogen. However, less aggregation inhibition was observed using a competing synthetic dodecapeptide containing the V411I mutation as compared to the wild-type dodecapeptide. Purified fibrinogen Hershey IV also bound to purified platelet alpha(IIb)beta(3) with a lower affinity than wild-type fibrinogen. These findings show that the gammaV411I mutation results in a decreased ability to bind platelets. In the heterozygous state, however, the available wild-type fibrinogen appears to be sufficient to support normal platelet aggregation.     

Platelet adhesion to type I collagen and alpha 1 (I)3 trimers: involvement of the C-terminal alpha 1 (I) CB6A peptide

The adhesion of human platelets to calf-skin acid-soluble and pepsin extracted type I and type III collagens has been determined. In fibrillar form, all three preparations were equiactive. Modification of their charge pattern (methylation, deamidation) diminished or abolished platelet adhesiveness. Alpha 1(I) and alpha 2 chains, isolated from type I collagen by preparative PAGE followed by CMC chromatography, did not induce the adhesion of platelets. After reassociation as fibrillar trimers, (alpha 1(I))3 fibrils were active while (alpha 2)3 were not, suggesting that in the ordered structure required for platelet adhesion to type I collagen, the alpha 1 chains are essential. Platelets adhered significantly to alpha 1(I) CB6 peptide obtained by the cyanogen bromide cleavage of alpha 1(I) chains : the adhesive properties of type I collagen therefore seems to be associated with the C terminal end of the alpha 1 chains.     

Fibrinogen Magdeburg I: a novel variant of human fibrinogen with an amino acid exchange in the fibrinopeptide A (Aalpha 9, Leu-->Pro)

INTRODUCTION: The exchange of Aalpha 16, Arg for Cys or His is the most common molecular defect in dysfibrinogenemia directly affecting the thrombin cleavage site involved in fibrinopeptide A (FPA) release. Other amino acid exchanges within the fibrinopeptide A have been only rarely reported. MATERIALS AND METHODS: In clinically asymptomatic dysfibrinogenemic patients with low functional plasma fibrinogen (Fg) levels and prolonged thrombin time but normal or slightly prolonged batroxobin (reptilase) time, mutation analysis was carried out by direct sequencing of the coding regions of the three fibrinogen genes. Isolated fibrinogen was functionally characterized for thrombin- or batroxobin-induced fibrinopeptide release and fibrin formation. Fibrinogen and fibrinopeptides were structurally studied by electrophoretic techniques or high-performance liquid chromatography. RESULTS AND CONCLUSIONS: Molecular analysis revealed heterozygosity for a novel missense mutation T1182C in the FGA gene causing the amino acid exchange Aalpha 9, Leu-->Pro. Fibrin generation induced by thrombin was moderately impaired, whereas batroxobin-induced fibrin formation was almost normal. Release of the abnormal fibrinopeptide A by thrombin was delayed but fibrin monomer aggregation was almost normal. Cleavage of Aalpha chains by batroxobin was only slightly delayed. Fibrinopeptides A of the patient fibrinogen did not show any gross abnormality in chromatographic behaviour. This new molecular variant designated fibrinogen Magdeburg I supports the view that amino acid residue Leu-9 in the Aalpha chain as part of a small hydrophobic cluster is involved in the interaction with an apolar binding site of thrombin, thus adding to our understanding of the thrombin-fibrinogen interaction crucial in coagulation.     

The platelet reactivity of the alpha 2(I)-chain of type I collagen: platelet aggregation induced by polymers of the molecule [alpha 2(I)]3

Polymers of the collagenous species alpha 2(I) trimer, a molecule of which contains three alpha 2(I) chains derived from type I collagen, have been shown to induce the aggregation of platelets when tested at a temperature low enough to avoid loss of the tertiary structure of the molecule. Under these conditions, the alpha 2(I) chain appears to possess greater platelet reactivity than the corresponding type I collagen-derived alpha 1(I) chain. In contrast to previous reports of its lack of reactivity, our results indicate that the alpha 2(I) chain must contribute importantly to the overall platelet reactivity of collagen type I in vivo. Our findings furthermore support the concept that any collagen-like structure may be expected to interact with platelets provided due regard is given to tertiary and quaternary structural requirements.     

Identification of residues within GABA(A) receptor alpha subunits that mediate specific assembly with receptor beta subunits.

GABA(A) receptors can be constructed from a range of differing subunit isoforms: alpha, beta, gamma, delta, and epsilon. Expression studies have revealed that production of GABA-gated channels is achieved after coexpression of alpha and beta subunits. The expression of a gamma subunit isoform is essential to confer benzodiazepine sensitivity on the expressed receptor. However, how the specificity of subunit interactions is controlled during receptor assembly remains unknown. Here we demonstrate that residues 58-67 within alpha subunit isoforms are important in the assembly of receptors comprised of alphabeta and alphabetagamma subunits. Deletion of these residues from the alpha1 or alpha6 subunits results in retention of either alpha subunit isoform in the endoplasmic reticulum on coexpression with the beta3, or beta3 and gamma2 subunits. Immunoprecipitation revealed that residues 58-67 mediated oligomerization of the alpha1 and beta3 subunits, but were without affect on the production of alpha/gamma complexes. Within this domain, glutamine 67 was of central importance in mediating the production of functional alpha1beta3 receptors. Mutation of this residue resulted in a drastic decrease in the cell surface expression of alpha1beta3 receptors and the resulting expression of beta3 homomers. Sucrose density gradient centrifugation revealed that this residue was important for the production of a 9S alpha1beta3 complex representing functional GABA(A) receptors. Therefore, our studies detail residues that specify GABA(A) receptor alphabeta subunit interactions. This domain, which is conserved in all alpha subunit isoforms, will therefore play a critical role in the assembly of GABA(A) receptors composed of alphabeta and alphabetagamma subunits.     

GABA(A) receptor subtype-selective efficacy: TPA023, an alpha2/alpha3 selective non-sedating anxiolytic and alpha5IA, an alpha5 selective cognition enhancer

TPA023 and α5IA are structurally related compounds that selectively modulate certain GABA_A receptor subtypes. Hence, TPA023 has weak partial agonist efficacy at the α2 and α3 subtypes whereas α5IA has inverse agonist efficacy at the α5 subtype. These efficacy characteristics translate into novel pharmacological profiles in preclinical species with TPA023 being a nonsedating anxiolytic in rats and primates whereas α5IA enhanced cognition in rats but was devoid of the proconvulsant or kindling liabilities associated with nonselective inverse agonists. In vitro and in vivo metabolic studies showed that TPA023 was metabolized via CYP3A4-mediated t -butyl hydroxylation and N -deethylation whereas α5IA was metabolized to produce the hydroxymethyl isoxazole, the latter of which was highly insoluble and caused renal toxicity in preclinical species. In humans, TPA023 had a half-life in the region of 6–7 h whereas the half-life of α5IA was 2–2.5 h. TPA023 was clearly differentiated from the nonselective agonist lorazepam in terms of saccadic eye movement and unlike lorazepam, it did not impair either postural stability, as judged by body sway, or cognition. The occurrence of the hydroxymethyl isoxazole metabolite of α5IA in human urine precluded the use of α5IA in prolonged dosing studies. Nevertheless, α5IA was evaluated in an alcohol-induced cognitive impairment model in healthy normal volunteers and was found to reverse the memory-impairing effects of alcohol. To date, however, no efficacy data for either TPA023 or α5IA in patient populations has been reported, although at the very least, the preclinical and limited clinical data with TPA023 and α5IA validate the approach of targeting specific GABA_A receptors through subtype-selective efficacy.     

The adaptive behavior scale-residential and community (part I): towards the development of a short form

A potential 24-item short form (SABS) of the 73-item Adaptive Behavior Scale-Residential and Community (Part I) (ABS-RC2; Nihira et al., 1993a, b) was developed, based on data from two diverse UK samples of adults with intellectual disabilities living in residential services (n = 560 and 254). SABS factor and total scores showed good internal reliability in both samples (alpha 0.89-0.98), and were highly correlated with their full ABS-RC2 Part I equivalents (r = 0.97-0.99). Regression equations were calculated for SABS factor and total scores against their full ABS-RC2 Part I equivalents. Levels of agreement between predicted quartile scores (derived from the regression equations) and actual full ABS-RC2 Part I quartile scores were high (kappa 0.75-0.89; percentage agreement 82%-92%). It is concluded that the SABS is a potentially useful research tool, although further work is clearly needed to establish the reliability and cross-cultural validity of the instrument.     

Increase in expression of the GABA(A) receptor alpha(4) subunit gene induced by withdrawal of, but not by long-term treatment with, benzodiazepine full or partial agonists

The effects of long-term exposure to, and subsequent withdrawal of, diazepam or imidazenil (full and partial agonists of the benzodiazepine receptor, respectively) on the abundance of GABA(A) receptor subunit mRNAs and peptides were investigated in rat cerebellar granule cells in culture. Exposure of cells to 10 microM diazepam for 5 days significantly reduced the amounts of alpha(1) and gamma(2) subunit mRNAs, and had no effect on the amount of alpha(4) mRNA. These effects were accompanied by a decrease in the levels of alpha(1) and gamma(2) protein and by a reduction in the efficacy of diazepam with regard to potentiation of GABA-evoked Cl- current. Similar long-term treatment with 10 microM imidazenil significantly reduced the abundance of only the gamma(2)S subunit mRNA and had no effect on GABA(A) receptor function. Withdrawal of diazepam or imidazenil induced a marked increase in the amount of alpha(4) mRNA; withdrawal of imidazenil also reduced the amounts of alpha(1) and gamma(2) mRNAs. In addition, withdrawal of diazepam or imidazenil was associated with a reduced ability of diazepam to potentiate GABA action. These data give new insights into the different molecular events related to GABA(A) receptor gene expression and function produced by chronic treatment and withdrawal of benzodiazepines with full or partial agonist properties.     

Comparison of thrombin-antithrombin complex (TAT) levels and fibrinopeptide A following thrombin incubation of human plasma using hirudin as an inhibitor of TAT formation

Following incubation of citrated plasma with human thrombin, the interaction of thrombin with antithrombin III was measured as thrombin-antithrombin complex (TAT) concentration. Comparison was made to thrombin activity on fibrinogen, assayed as fibrinopeptide A (FPA). Light scattering studies were included to evaluate polymerization of fibrin. Hirudin, at a final concentration of 100 U/ml, effectively inhibited TAT generation at final thrombin concentrations below 0.4 NIH U/ml. Hirudin by itself did not affect the TAT ELISA analysis. TAT and FPA values correlated closely (r = 0.83, p less than 0.001) and may equally well represent in vitro thrombin activity.     

A special report I. Prion protein (PrP)--amyloid plaques in the transmissible spongiform encephalopathies, or prion diseases revisited

We present a retrospective analysis of PrP-amyloid plaques encountered in CJD and GSS. In human TSEs (kuru, CJD and GSS) several PrP-immunopositive plaques and plaque-like deposits were detected. In kuru, plaques were typical "kuru" plaques--stellate structures deposited mostly in the granular- and Purkinje-cell layer of the cerebellum. Many smaller or larger clusters were visible but, in contrast to GSS, they never merged together to form multicentric plaques. In all cases of GSS, plaques were localised in the granular- and Purkinje-cell layer and the molecular cell layer. There were many different forms of plaques: from kuru plaques (unicentric stellate plaques) to clusters of unicentric plaques, which by merging eventually formed "multicentric plaques". The latter are the hallmark of this disease. By electron microscopy, several types of amyloid plaques, which corresponded to those seen by PrP immunohistochemistry, were observed. The first type, unicentric "kuru" plaque, consisted of stellate arrangements (stars or cores) of amyloid bundles emanating from a densely interwoven centre. Amyloid stars were surrounded by astrocytic processes and invaded by microglial cells but dystrophic neurites were only rarely seen. In contrast, multicentric plaques were often surrounded by dystrophic neurites. The rarest type of plaque were neuritic plaques. In 263K- and 22C-H scrapie-infected hamster brains, by light microscopy and semi-thin (1 microm) sections, discrete PrP-immunopositive plaques were observed in the subependymal region but not in the deep brain neuroparenchyma. These plaques were not discernible by routine H & E staining. Ultrastructurally, plaques were recognised as areas of low electron density containing haphazardly-oriented fibrils and not as stellate compact structures typical of plaques in human cases of CJD and GSS. These plaques were located beneath the basal border of the ependymal cells and adjacent blood vessels. Occasional dystrophic neurites containing electron-dense inclusion bodies were seen within the plaque perimeter, which always remained PrP-negative.     

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.     

Compartmentation of alpha 1 and alpha 2 GABA(A) receptor subunits within rat extended amygdala: implications for benzodiazepine action

The extended amygdala, a morphological and functional entity within the basal forebrain, is a neuronal substrate for emotional states like fear and anxiety. Anxiety disorders are commonly treated by benzodiazepines that mediate their action via GABA(A) receptors. The binding properties and action of benzodiazepines depend on the alpha-subunit profile of the hetero-pentameric receptors: whereas the alpha1 subunit is associated with benzodiazepine type I pharmacology and reportedly mediates sedative as well as amnesic actions of benzodiazepines, the alpha2 subunit confers benzodiazepine type II pharmacology and mediates the anxiolytic actions of benzodiazepines. We determined the localization of alpha1 and alpha2 subunits within the extended amygdala, identified by secretoneurin immunostaining, to define the morphological substrates for the diverse benzodiazepine actions. A moderate expression of the alpha1 subunit could be detected in compartments of the medial subdivision and a strong expression of the alpha2 subunit throughout the central subdivision. It is concluded that the alpha1 and alpha2 subunits are differentially expressed within the extended amygdala, indicating that this structure is compartmentalized with respect to function and benzodiazepine action.     

A pilot study of glioblastoma multiforme in elderly patients: Treatments,O-6-methylguanine-DNA methyltransferase (MGMT) methylation status and survival

Objectives

Elderly Glioblastoma multiforme (GBM) patients have a worse prognosis and receive variable treatments. MGMT gene promoter methylation is linked with improved survival in GBM. We examined treatments administered and survival including in relation to MGMT methylation status in elderly GBM patients.

Patients and methods

Patients ≥65 years with diagnosed GBM between 1/01/2007 and 30/04/2009 and undergoing either a biopsy, subtotal (STR) or gross total resection (GTR) were included. The collected information included MGMT status [methylated (ME) vs. unmethylated (UN)] and survival data. p < 0.05 was considered significant.

Results

59 patients were identified with median age at diagnosis being 72.68 years (65.72–85.04). Treatment included surgery (25 GTR, 8 STR, 26 biopsy), chemoradiation (22) and radiotherapy alone (20). Overall median overall survival (MOS) was 219 days. MOS with chemoradiation was 316 days vs. 143 days without it (p = 0.011). 47 patients had definite MGMT status (28 ME, 19 UN). In ME patients, 9/28 received temozolamide compared to 10/19 in UN category. Temozolamide administration in patients with definite MGMT status was based on WHO performance status (p = 0.007). MOS in UN group was 308 days vs. 167 days in ME group (p = 0.068). In a multivariate Cox model including use of temozolamide, WHO score and methylation status, only temozolamide use was significantly associated with a reduced risk for death (HR 0.443, 95% CI 0.200–0.982, p = 0.045).

Conclusions

In this small cohort of patients, chemoradiation in suitable elderly GBM patients seemed to afford a survival benefit. MGMT methylation was not associated with an improved survival with temozolamide being the only factor leading to a better survival. Temozolamide use should be considered irrespective of MGMT status in this population with future large prospective studies needed to elucidate this further.     

Delayed upregulation of GABA(A) alpha1 receptor subunit mRNA in somatosensory cortex of mice following learning-dependent plasticity of cortical representations.

Experience-dependent modifications of cortical representational maps are accompanied by changes in several components of GABAergic inhibitory neurotransmission system. We examined with in situ hybridization to 35S-labeled oligoprobe changes of expression of GABA(A) receptor alpha1 subunit mRNA in the barrel cortex of mice after sensory conditioning training. One day and 5 days after the end of short lasting (3 daily sessions) training an increased expression of GABA(A) alpha1 mRNA was observed at the cortical site where the plastic changes were previously found. Learning associated activation of the cerebral cortex increases expression of GABA(A) receptor mRNA after a short post-training delays.