A missense mutation (Tyr88 to Cys) in the platelet membrane glycoprotein Ibbeta gene affects GPIb/IX complex expression--Bernard-Soulier syndrome in the homozygous form and giant platelets in the heterozygous form

This study examined the molecular basis of a missense mutation of the platelet glycoprotein (GP) Ibbeta gene in two families. In the propositus with a novel form of Bernard-Soulier syndrome (BSS) from Family I, only GPIbalpha was detectable in reduced amounts on platelet surfaces by flow cytometry. There were no GPIX or GPIbbeta found by immunoblotting. DNA sequencing analysis showed a homozygous mutation in the GPIbbeta gene which changed Tyr (TAC) to Cys (TGC) at residue 88. Her parents were heterozygous for Tyr88Cys in the GPIbbeta gene. In transient transfection studies on 293T cells, both Tyr88Cys and Tyr88Ala mutations suppressed the expression of GPIb/IX complexes. In addition, Tyr88Cys GPIbbeta mutation was found to exert a dominant negative effect on the GPIbalpha expression. Five individuals from Family II, four of whom reported elsewhere as having giant platelet disorders with normal aggregation (BLOOD, 1997: 89: 2404) and one newly analyzed in this study, were heterozygous for Tyr88Cys in the GPIbbeta gene. Microsatellite analysis of chromosome 22 showed a common haplotype in 8 of the individuals with Tyr88Cys mutations in Families I and II. Tyr88 in the GPIbbeta gene plays a significant role in the GPIb/IX expression; the defect causes BSS in a homozygous form and possibly giant platelets in a heterozygous form.     

Critical role of the C-terminal segment in the maturation and export to the cell surface of the homopentameric alpha 7-5HT3A receptor

Many neurological pathologies are related to misfolded proteins. During folding and assembly in the endoplasmic reticulum, the nicotinic acetylcholine receptor (nAChR) subunits undergo several conformational changes to acquire the ability to bind ligands. After folding and maturation, by mechanisms largely unknown, receptors are exported to the cell surface. We investigated the maturational role of the extracellular C-terminal segment located at the boundary between the extracellular and the transmembrane domains. In the functional chimeric alpha7-5HT3A receptor used as a model system, amino acids from the C-terminal segment were successively deleted or mutated. Upon progressive shortening of the peptide we observed less and less alpha-bungarotoxin binding sites until no sites could be detected when the entire peptide had been deleted (chimera Del 5). Protein synthesis and pentameric assembly were not altered. In Del 5 transfected cells, pentameric receptors present in the endoplasmic reticulum were not detected on the cell surface where Del 5 proteins appeared as patches. With the Del 5 chimera, export of proteins to the cell surface diminished to about half that of wild-type. We propose that the C-terminal segment plays a double role: (i) through an interaction between the penultimate tyrosine residue of the C-terminal segment and the Cys loop of the N-terminal domain, it locks the receptor in a mature alpha-bungarotoxin binding conformation; (ii) this mature conformation, in turn, masks a retention signal present in the first transmembrane segment allowing properly assembled and matured receptors to escape to the cell surface.     

Tetraspanin-5 (Tm4sf9) mRNA expression parallels neuronal maturation in the cerebellum of normal and L7En-2 transgenic mice

Tetraspanin-5 (Tspan-5) mRNA was recently shown to be strongly expressed within the central nervous system. In order to address Tspan-5 function during nervous system development, we performed a detailed expression analysis in the postnatal FVB/N mouse cerebellum using in situ hybridizations. Tspan-5 mRNA was expressed within cerebellar Purkinje cells (PCs) throughout postnatal development. The expression level, however, changed significantly with ongoing development. At the day of birth (P0), Tspan-5 mRNA was expressed at very low levels in PCs. At this time, PCs of the FVB/N strain are postmitotic and bear axons, but no dendrites. At P7, Tspan-5 mRNA expression was visible in all PCs, but was more prominent in those of the posterior lobules as compared to those of the anterior lobules. After P7, high levels of Tspan-5 mRNA were seen in all PCs, which is when PCs elaborate and maintain their typical dendritic tree. This demonstrates that the level of Tspan-5 mRNA is related to the developmental status of PCs. Consistently, expression of Tspan-5 mRNA was specifically reduced in PCs of L7En-2 animals, which display a delay in PC maturation during postnatal cerebellar development. In addition, whereas no Tspan-5 mRNA signal could be detected in the proliferating granule cell layer, low levels could be found in postmitotic, premigratory granule cells and high levels in settled and differentiated granule cells. Thus, the level of Tspan-5 mRNA expression correlates very well with the differentiation status of particular neurons. The level of Tspan-5 expression might therefore be important for distinct phases of neuronal maturation.     

Association of C807T, Pl(A), and -5 C/T Kozak genotypes with density of glycoprotein receptors on platelet surface

This study investigates whether three platelet glycoprotein (GP) polymorphisms, C807T in GP Ia, Pl(A1/A2) in GP IIIa, and -5 T/C Kozak in GP Ibalpha gene, influence the density of the three important adhesion and activation receptors on the platelet surface. Fifty-four healthy donors were genotyped according to the three polymorphisms, and densities of the corresponding GPs were measured by flow cytometry. Our study confirmed the association between C807T polymorphism and platelet surface expression of GP Ia-IIa and GP Ia and demonstrated that the density of GP Ibalpha or GP IX is not associated with the Kozak polymorphism. Although the Pl(A1/A2) polymorphism did not affect the expression of GP IIb-IIIa and GP IIIa on the platelet surface, flow-cytometric analysis employing murine monoclonal antibody SZ21 against GP IIIa can be applied to distinguish Pl(A1/A1) and Pl(A1/A2) polymorphism.     

Differential in vitro effects of the platelet glycoprotein IIb/IIIa inhibitors abixicimab or SR121566A on platelet aggregation, fibrinogen binding and platelet secretory parameters

The aim of this study was to compare fibrinogen binding, inhibition of platelet aggregation and secretory potential of the MAb abciximab (0.5-5 microg/mL) and the peptidomimetic compound SR121566A (15-250 ng/mL) in vitro in whole blood. Fibrinogen binding was followed by flow cytometry; platelet function was evaluated by light transmittance and by impedance aggregometry. Secretory functions of platelets were evaluated using ATP as marker for early secretion by dense granulae and P-selectin (CD62) for alpha-granular secretion as well as CD63 for lysosomal degranulation. Results showed that fibrinogen binding induced by 5 microM TRAP was maximally inhibited greater than 80% at 3 microg/mL abciximab or at 250 ng/mL SR121566A. At these concentrations of antagonists, platelet aggregation induced by 5 microM ADP or 2 microg/mL collagen was inhibited completely. Expression of CD62 was reduced 34% with abciximab or 15% with SR121566A; CD63 expression was reduced 22% with both agents. With both agents, the EC50 for inhibition of CD62 and CD63 expressions was in similar magnitudes than the EC50 for fibrinogen binding inhibition. With 3 microg/mL abciximab, ATP secretion was maximally reduced to 50% of the control, whereas SR121566A at 250 ng/mL had no inhibitory effect on this parameter. A slight increase in ATP secretion was seen with 0.5 microg/mL abciximab and with SR121566A in concentrations of less than 45 ng/mL. The data suggest a discoupling between the anti-aggregatory and the antisecretory effects of IIb/IIIa antagonists. Because it is not established to what extend CD62 or CD63 expression can be reduced by any means, the reduction by 20-30% obtained by 3 microg/mL abciximab or 250 ng/mL SR121566A might already be the maximum possible inhibition by these agents.     

Short-term environmental enrichment exposure induces proliferation and maturation of doublecortin-positive cells in the prefrontal cortex

Previous studies have demonstrated that doublecortin-positive immature neurons exist pre- dominantly in the superficial layer of the cerebral cortex of adult mammals such as guinea pigs, and these neurons exhibit very weak properties of self-proliferation during adulthood under physiological conditions. To verify whether environmental enrichment has an impact on the proliferation and maturation of these immature neurons in the prefrontal cortex of adult guinea pigs, healthy adult guinea pigs were subjected to short-term environmental enrichment. Animals were allowed to play with various cognitive and physical stimulating objects over a period of 2 weeks, twice per day, for 60 minutes each. Immunofluorescence staining results indicated that the number of doublecortin-positive cells in layer II of the prefrontal cortex was significantly increased after short-term environmental enrichment exposure. In addition, these doublecortin-positive cells co-expressed 5-bromo-2-deoxyuridine （a marker of cell prolifera- tion）, c-Fos （a marker of cell viability） and NeuN （a marker of mature neurons）. Experimental findings showed that short-term environmental enrichment can induce proliferation, activation and maturation of doublecortin-positive cells in layer II of the prefrontal cortex of adult guinea pigs.     

Modulation of protein C inhibitor activity by histidine-rich glycoprotein and platelet factor 4: role of zinc and calcium ions in the heparin-neutralizing ability of histidine-rich glycoprotein.

Effects of zinc and calcium ions on the heparin-neutralizing abilities of histidine-rich glycoprotein (HRG) and platelet factor 4 (PF4) were examined. Both HRG and PF4 effectively neutralized the ability of heparin to accelerate the activated protein C (APC) and the thrombin inhibitions by protein C inhibitor (PCI). the heparin-neutralizing ability of HRG in the APC inhibition by PCI, however, was decreased in a Ca(2+)-dependent manner and apparently lost at 1 mM Ca2+, while it was enhanced by Zn2+ regardless of the presence or absence of Ca2+. The heparin-neutralizing ability of HRG in the thrombin inhibition by PCI was not affected by Ca2+. In contrast to HRG, there was no significant difference in the heparin-neutralizing ability of PF4 in the presence or absence of 1 mM Ca2+. These results strongly suggest additional physiological functions of HRG and PF4 as modulators of PCI.     

静脉滴注低温生理盐水降温对家兔的影响

目的 探讨静脉滴注低温生理盐水对家兔生命体征及血小板计数变化的影响。方法 24只家兔随机分成3组；低温盐水静滴组，对照组及体表降温组。观察各组动物生命体征，血小板计数变化及其他副反奕。结果 低温盐水静滴组及体表降温组体温均可降至35℃，维持亚低温20小时，低温盐水组其余生命体征及血小板计数未见异常改变，而体表降温组心率减慢。血压下降。结论 静脉滴注低温生理盐水可使家兔达到亚低温状态。且对家兔无不良影响，它可用来作为家兔亚低温实验研究的一种降温方法。     

Free and total platelet glycoprotein IIb/IIIa measurement in whole blood by quantitative flow cytometry during and after infusion of c7E3 Fab in patients undergoing PTCA.

A quantitative flow cytometry assay was used to evaluate the ex vivo kinetics of c7E3 Fab platelet effect in 16 patients undergoing PTCA treated with abciximab and compared with aggregometry assay. Immunolabeling of platelets was directly assessed on whole blood, using in parallel two monoclonal antibodies (Mabs) raised against GPIIIa, Mab1, the binding of which is inhibited by c7E3 Fab, and Mab2, the binding of which is not affected by c7E3 Fab. We found a severe and sustained inhibition of both GPIIb/IIIa receptors and platelet functions. The inter-individual variation in response to abciximab was low. A significant transient increase at H24 and H48 in the binding of Mab2 was found as an unexpected result, and confirmed in vitro. Results demonstrate that flow cytometry is a reliable method in agreement with aggregation. In addition, our results show that it is a standardized tool and a time-saving technique.     

The prevalence of the platelet glycoprotein IIIa Pl(A1/A2) polymorphism in three South African ethnic groups and its effect on platelet function

Introduction

In South Africa coronary artery disease (CAD) is less common in African than Indian or white subjects. Although the association between CAD and metabolic factors have been well documented, the role of genetic factors is as yet poorly understood. Specific polymorphisms in the platelet membrane glycoprotein (GP) IIIa gene Pl^A1/A2, have been implicated in the development of CAD.

Methods

The prevalence of platelet GPIIIa (Pl^A1/A2) polymorphisms and their effect on platelet function was determined in 313 Indian, 267 white and 227 African subjects with and without a history of CAD.

Results

In subjects without a history of CAD the frequency of the unfavourable Pl^A2 allele was 8.0%, 14.8% and 8.7% in the Indian, white and African populations respectively, with the frequency being significantly higher (p < 0.05) in the white than both other groups. The frequency of the Pl^A2 allele was higher in subjects with (23.0%) than without (10.0%; p < 0.0001) a history of CAD. Aggregation studies showed that platelets carrying the Pl^A2 allele were hypersensitive to the platelet aggregating agonists ADP and collagen and produced a higher amount of TXA₂ when stimulated with low concentrations of both these agonists.

Conclusions

The positive association observed between the platelet GPIIIa Pl^A1/A2 polymorphism and platelet function suggests that the GPIIIa Pl^A2 allele may be a genetic factor that contributes to the risk of sudden death from myocardial infarction in the absence of known risk factors but it does not explain ethnic differences in the prevalence of CAD.     

The impact of platelet membrane glycoprotein Ib alpha and Ia/IIa polymorphisms on the risk of thrombosis in the antiphospholipid syndrome

Background

Pathogenesis of thrombus formation in antiphospholipid syndrome (APS) is not clear. Platelet membrane glycoprotein (GP) receptors play important roles in development of thrombosis.

Objectives

We investigated the association between development of thrombosis in APS and polymorphisms of GPIb alpha variable number of tandem repeats (VNTR), Kozak, and GPIa C807T.Patients/MethodsSixty patients with APS (30 with proven thrombosis and 30 without thrombosis) and 63 controls were included. Presence of GPIa C807T polymorphism was determined with real-time PCR and GPIb alpha Kozak and VNTR polymorphisms by conventional PCR.

Results

Frequency of C807T TT genotype was significantly higher in APS with thrombosis than APS without thrombosis (p = 0.023) and also in APS with multiple thrombi compared to APS without thrombi (p = 0.023). Frequency of Kozak TC genotype was higher in APS with arterial thrombosis compared to APS with venous thrombosis, controls, and APS without thrombosis (p = 0.03, p = 0.0007, and p = 0.0024 respectively). D allele frequency and D allele carrier state for VNTR were significantly less in APS than controls (p = 0.0018 and p = 0.0046 respectively).

Conclusions

C807T TT genotype may confer a risk for thrombosis and Kozak TC genotype for arterial thrombosis. D allele of VNTR may protect from APS. No patients with C807T TT or Kozak TC genotypes carried the protective DD genotype of VNTR. These polymorphisms may increase risk for both arterial and venous thrombosis. The utility of prophylaxis with anti-platelet drugs in at least a subgroup of APS patients should be investigated with clinical trials.     

Weak platelet agonists and U46619 induce apoptosis-like events in platelets,in the absence of phosphatidylserine exposure

Platelets express apoptotic markers during storage, while aging and after stimulation with strong agonists thrombin and collagen. It is unknown if the weak agonists ADP and epinephrine or U46619, a thromboxane analog, induce the expression of apoptotic markers in platelets. To answer this question, we measured phosphatidylserine exposure, gelsolin cleavage and decrease in membrane mitochondrial potential after stimulation with these agonists. No phosphatidylserine exposure was evident, however, gelsolin cleavage and a platelet population with a decreased membrane mitochondrial potential appeared, suggesting that in platelets selective agonists can induce apoptosis in the absence of phosphatidylserine exposure. Interestingly, costimulation by thrombin plus collagen together with each of the other agonists increased the phosphatidylserine exposure induced by strong agonists. These findings may be of importance in platelet activation and apoptosis under pathophysiological conditions where multiple effectors are involved.     

The maturation of monoamine oxidase and 5-hydroxy-indole acetic acid in regions of the mouse brain

Monoamine oxidase (MAO) activity and 5-hydroxyindole acetic acid (5-HIAA) amount have been measured in 4 subdivisions of the mouse brain during various stages of postnatal maturation. Each region and each indoleamine pathway component (MAO and 5-HIAA) demonstrated an individual pattern of maturation. MAO increased rapidly from day 1 postpartum and reached adult-like specific activitity by 2 weeks postpartum except in the cerebellum where increase continued after week 6. 5-HIAA levels exceeded adult-like levels by day 3 postpartum, continued to rise during the first week and reached adult level by week 6. Comparison of these data to previously reported maturational patterns of 5-hydroxytryptamine (5-HT) and 5-hydroxytryptophan decar?ylase (5-HTPD), measured upon a similar regional basis, indicate that the components of the indoleamine pathway in the brain do not mature in a harmonious way.     

Anti-IL-12 antibody prevents the development and progression of multiple sclerosis-like relapsing--remitting demyelinating disease in NOD mice induced with myelin oligodendrocyte glycoprotein peptide

Treatment with monoclonal anti-IL-12 antibody injected on day 0, 7 and 10 after immunization with myelin oligodendrocyte glycoprotein (MOG) peptide 35-55 in NOD mice resulted in significant suppression of the development and the severity of the chronic relapsing-remitting experimental autoimmune encephalomyelitis (EAE) both clinically and histologically. The spleen cells from anti-IL-12 antibody treated mice displayed markedly inhibited MOG35-55 specific proliferation and IFN-gamma production. MOG35-55 specific antibody production was enhanced by anti-IL-12 antibody treatment. These results suggest that IL-12 is critically involved in the pathogenesis of MOG-induced EAE and that antibody to IL-12 could be an effective therapeutic agent in the clinical treatment of autoimmune demyelinating diseases such as multiple sclerosis (MS).     

The effect of neonatal exposure to chronic footshock on pain-responsiveness and sensitivity to morphine after maturation in the rat

Rat pups in 3 groups respectively were given daily footshock, exposure to a footshock apparatus without shock, or no handling from birth to 21 days of age and reared with no manipulation afterwards. After maturation (90-100 days of age), they were assessed for hot-plate paw-lick latency, morphine-induced analgesia and opiate receptor binding assay. In footshocked animals, a significant increase was found in paw-lick latency and in antinociceptive effects of morphine (1.25, 2.5, and 5.0 mg/kg) in comparison with two control groups. The antinociceptive effect of morphine in all 3 groups was antagonized by pretreatment with naloxone (2.0 mg/kg). No significant difference was found in binding activities (Bmax and Kd) for both [3H]naloxone and [3H]Dala2, D-Leu5-enkephalin between the 3 groups. These results suggest that exposure to footshock stress in the preweanling period has a long-term effect on the sensitivity of rats to painful events, probably due to chronic functional changes in endogenous opiate systems at presynaptic level rather than in postsynaptic opiate receptor binding activity.     

The fine specificity of the myelin oligodendrocyte glycoprotein autoantibody response in patients with multiple sclerosis and normal healthy controls

Antibodies directed against the extracellular immunoglobulin (Ig)-like domain of the myelin oligodendrocyte glycoprotein (MOG(Igd)) mediate demyelination in experimental autoimmune encephalomyelitis (EAE) and are implicated in the immunopathogenesis of multiple sclerosis (MS). In this study we investigated the epitope specificity of MOG(Igd)-specific autoantibodies immunopurified from MS patients (n=17) and normal healthy controls (HD; n=9). ELISA, using a panel of synthetic MOG(Igd) peptides, revealed that the epitope specificity of this response was heterogeneous in both groups. The most frequently recognised epitopes were located in amino acid sequences (a.a.) 1-26 (13/17) and 63-87 (15/17) in MS patients, and 14-39 (6/9) and 63-87 (6/9) in HDs, but there was no association between MS and any particular peptide specificity. We therefore investigated the ability of the immunopurified antibodies to recognise native MOG(Igd) expressed on at the membrane surface by FACS. Unexpectedly, antibodies fulfilling this essential criterion for a demyelinating antibody response were detected only in one of the MS samples. These results indicate that the epitope specificity of the human B cell response to MOG is not only heterogeneous, but may only mediate demyelination in a limited subset of MS patients.     

Acetylcholinesterase activity in the developing olfactory bulb: a biochemical study on normal maturation and the influence of peripheral and central connections

In the newborn rat olfactory bulb (OB), the specific activity of acetylcholinesterase (AChE) is about 20% of the adult value. During postnatal development, the specific activity remains unchanged until day 10; a growth spurt of 5X is observed between days 10 and 25, when adult values (90 nmoles ACh/min/mg protein; 6 nmol/min/mg wet wt.) are reached. However, total activity shows continuous increase slowly at first and rapidly between days 10 to 30, reaching a plateau by day 60. Between birth to day 60 total activity increases 65X. To determine the influence of peripheral and central connections on the development of AChE activity in the OB, rats were subjected to unilateral olfactory denervation and/or transection of olfactory peduncle, carried out either neonatally or at day 30; the bulbs were assayed a month later (days 30 and 60 respectively). It was found that in both neonatal and 30-day-old rats, denervation caused a 15% decrease in total activity, while transection led to more than 60% reduction. In the older rats, the reduction due to transection represented the degenerative loss of activity, but in the neonatally transected bulbs the growth of some cholinergic elements continued although very slowly. Between birth to day 30, total AChE activity increased only 12X in completely isolated OB, 25X in transected OB and 40X in denervated OB, compared to 45-50X in control OB. In the transected and isolated bulbs specific activity of AChE was also reduced significantly (25-50% depending on age and operation). These results suggest that while centrifugal fibers are the main source of cholinergic activity in the mature as well as the developing OB, the olfactory nerve and some intrabulbar sources such as cholinergic cells or cholinoceptive membranes also contribute to AChE activity in the OB. These intrinsic sources of AChE activity can persist and even show some growth in the developing olfactory bulb in the absence of the centrifugal fibers and/or the olfactory afferents.