Platelet membrane glycoprotein IIb heavy chain forms a complex with glycoprotein IIIa that binds Arg-Gly-Asp peptides

Platelet membrane GPIIb is comprised of a disulfide-linked heavy chain (GPIIb(H)) and light chain (GPIIb(L)). We have examined the role of the two chains of GPIIb in the maintenance of the GPIIb-IIIa heterodimer and Arg-Gly-Asp (RGD) peptide-binding function. Lysates of surface radioiodinated platelets were treated with 1% 2-mercaptoethanol for 18 hours at 4 degrees C. Reduction of the interchain disulfide in GPIIb was followed by immunoprecipitation with antipeptide antibodies specific for GPIIb(H) or GPIIb(L). In addition to the GPIIb-IIIa complex, a polypeptide of 120 Kd was precipitated by anti-GPIIb(H) and a polypeptide of 23 Kd was precipitated by anti-GPIIb(L) from reduced platelet lysates. To determine whether GPIIb(H) or GPIIb(L) remained complexed with GPIIIa, reduced platelet lysates were immunoprecipitated with AP3, a monoclonal anti-GPIIIa antibody, resulting in the coimmunoprecipitation of GPIIb(H) but not GPIIb(L). Conversely, the monoclonal anti-GPIIb(H) antibody PMI-1 immunoprecipitated GPIIIa with GPIIb(H). Thus GPIIb(H) maintains its association with GPIIIa. Furthermore, the GPIIb(H)-IIIa complex retains its reactivity with AP2, a monoclonal antibody (MoAb) specific for the nondissociated GPIIb-IIIa complex. Affinity chromatography of reduced platelet lysates on immobilized KYGRGDS resulted in binding and specific elution of the GPIIb(H)-IIIa complex. These findings indicate that GPIIb(H) contains sufficient information for maintenance of a complex with GPIIIa and support of the binding of the heterodimer to RGD peptides.     

Thrombin receptors activate potassium and chloride channels

We used DAMI human megakaryocytic leukemia cells to study transmembrane ion currents activated through the G-protein-coupled thrombin receptor pathway. When the cells were stimulated by thrombin receptor-activating peptide, an increase in cytosolic Ca2+ ([Ca2+]i) developed as predicted by the known effect that thrombin exerts in the platelet. We then monitored the membrane potentials of individual DAMI cells during this response and observed complex, triphasic changes that could not be accounted for by Ca2+ fluxes alone. These consisted of rapid hyperpolarization, followed by depolarization to values more positive than the resting potential and then by slow repolarization. For the purpose of this study, we focused on the hyperpolarizing current that developed immediately after thrombin receptor activation. This proved to be composed of (1) a Ca(2+)-independent, outwardly rectifying Cl- current and (2) a strongly hyperpolarizing, inwardly rectifying, Ba(2+)- sensitive K+ current that required an increase of [Ca2+]i for activation. By analogy with their functions in other cell systems, it is logical to conclude that these prominent K+ and Cl- conductances may serve to regulate the complex volume changes that accompany thrombin receptor activation and/or to increase the electromotive drive that supports Ca2+ influx under these conditions through hyperpolarization of the cell membrane.     

Interleukin-10 is an autocrine growth factor for acquired immunodeficiency syndrome-related B-cell lymphoma [see comments]

Interleukin-10 (IL-10) is an acid-sensitive protein of 35 kD that has pleiotropic effects including inhibition of cytotoxic T-cell response, induction of major histocompatibility complex type II in B lymphocytes, induction of B-cell growth and differentiation, and autocrine growth factor activity in monocytes. We and others have shown that IL-10 is produced spontaneously by blood mononuclear cells from human immunodeficiency virus-seropositive patients. In an attempt to ascertain the potential role of IL-10 in acquired immunodeficiency syndrome (AIDS)-related B-cell lymphoma, we evaluated the expression of human IL-10 in both tumor-derived B-cell lines and primary tumor cells. Expression of human IL-10 (hIL-10) mRNA and protein was detected in four of five cell lines examined. An IL-10 antisense oligonucleotide inhibited IL-10 mRNA expression and IL-10 protein production. The proliferation of all B-cell lines was inhibited by an antisense oligonucleotide in a dose-dependent manner that was abrogated by the addition of recombinant hIL-10 protein. No effect of antisense oligonucleotide was observed in the B-cell line not producing hIL-10. Evaluation of primary tumor cells from patients with AIDS-lymphoma cells showed similar production and response to IL-10. These data suggest an autocrine growth mechanism for IL-10 in AIDS-related lymphoma cells and that IL-10 may be important in its pathogenesis.     

Determination of adhesion force between single cell pairs generated by activated GpIIb-IIIa receptors

A biophysical approach was used to directly determine the avidity of the junction between two Chinese hamster ovary (CHO) cells bearing recombinant GpIIb-IIIa in the presence and absence of fibrinogen. Micromanipulation was used to induce conjugation of the cell pairs with or without activating the GpIIb-IIIa molecules with monoclonal antibody (MoAb) 62. Activation of GpIIb-IIIa caused an increase in the force required to separate the conjugates. The molecular bonding force between cells bearing activated GpIIb-IIIa and fibrinogen molecules was found to be 2.1 x 10(-7) dyne, which is 3.7 times higher than that between nonactivated GpIIb-IIIa and fibrinogen (5.7 x 10(-8) dyne). The results provide a quantitative assessment of the molecular bonding force between fibrinogen and the GpIIb-IIIa expressed on cell surface. The findings indicate that the activation of GpIIb-IIIa leads to an increase in the adhesive force in CHO cell aggregation by increasing the strength of the GpIIb-IIIa-fibrinogen bonds rather than the number of these bonds.     

Normalization of markers of coagulation activation with a purified protein C concentrate in adults with homozygous protein C deficiency

Homozygous or double heterozygous protein-C deficiency can present at birth with purpura fulminans or later in life with venous thrombosis. Two homozygous patients who had previously sustained thrombotic episodes were investigated at a time when they were asymptomatic and not receiving antithrombotic therapy. The plasma levels of protein-C antigen and activity in both individuals were approximately 20% of normal. We administered a highly purified plasma-derived protein C concentrate to these individuals and monitored levels of several markers of in vivo coagulation activation. Assays for protein-C activation (activated protein C and protein C activation peptide) showed a sustained increase from reduced baseline levels, whereas thrombin generation (as measured by prothrombin fragment F1 + 2) gradually decreased over about 24 hours into the normal range. These investigations provide direct evidence that protein C is converted to activated protein C in vivo, and that the protein-C anticoagulant pathway is a tonically active mechanism in the regulation of hemostatic system activation in humans.     

Reduced Symptoms of Inattention after Dietary Omega-3 Fatty Acid Supplementation in Boys with and without Attention Deficit/Hyperactivity Disorder

Attention deficit/hyperactivity disorder (ADHD) is one of the most common child psychiatric disorders, and is often treated with stimulant medication. Nonpharmacological treatments include dietary supplementation with omega-3 fatty acids, although their effectiveness remains to be shown conclusively. In this study, we investigated the effects of dietary omega-3 fatty acid supplementation on ADHD symptoms and cognitive control in young boys with and without ADHD. A total of 40 boys with ADHD, aged 8–14 years, and 39 matched, typically developing controls participated in a 16-week double-blind randomized placebo-controlled trial. Participants consumed 10 g of margarine daily, enriched with either 650 mg of eicosapentaenoic acid (EPA)/docosahexaenoic acid (DHA) each or placebo. Baseline and follow-up assessments addressed ADHD symptoms, fMRI of cognitive control, urine homovanillic acid, and cheek cell phospholipid sampling. EPA/DHA supplementation improved parent-rated attention in both children with ADHD and typically developing children. Phospholipid DHA level at follow-up was higher for children receiving EPA/DHA supplements than placebo. There was no effect of EPA/DHA supplementation on cognitive control or on fMRI measures of brain activity. This study shows that dietary supplementation with omega-3 fatty acids reduces symptoms of ADHD, both for individuals with ADHD and typically developing children. This effect does not appear to be mediated by cognitive control systems in the brain, as no effect of supplementation was found here. Nonetheless, this study offers support that omega-3 supplementation may be an effective augmentation for pharmacological treatments of ADHD ({"type":"clinical-trial","attrs":{"text":"NCT01554462","term_id":"NCT01554462"}}NCT01554462: The Effects of EPA/DHA Supplementation on Cognitive Control in Children with ADHD; http://clinicaltrials.gov/show/{"type":"clinical-trial","attrs":{"text":"NCT01554462","term_id":"NCT01554462"}}NCT01554462).     

CD5-CD56+ T-cell receptor silent peripheral T-cell lymphomas are natural killer cell lymphomas [see comments]

Non-Hodgkin's lymphomas are divided into B- and T-cell neoplasms. The existence and the clinical relevance of lymphomas derived from the third lymphocyte lineage, ie, natural killer (NK) cells are still controversial. NK cells are lymphocytes that mediate cytotoxicity without prior sensitization. NK cells also have phenotypic and genotypic characteristics: they express the NK-related antigen CD56, T- cell markers such as CD2 and CD7, but do not express CD5 and T-cell receptor (TCR) proteins, and their TCR locus is not rearranged. Therefore, if NK cell lymphomas exist, they should express some T-cell markers, but not alpha beta or gamma delta TCR proteins. Such lymphomas are actually called TCR silent peripheral T cell lymphomas (PTCL). To detect and characterize NK cell lymphomas, we investigated the immunophenotype and immunogenotype of 35 patients with TCR silent PTCL. The first group included 16 patients with a lymphoma of CD5-CD56+ phenotype, which is identical to normal NK cells. These patients had either a nasal/nasopharyngeal lymphoma (11 cases) or a lymphoma with predominant non-nasal/non-nodal initial involvement (five cases). Eight of the nine cases for which immunogenotypic data were available lacked clonal rearrangement of the TCR gamma genes. Thus, these tumors are likely to be NK cell lymphomas. The second group of 15 cases had a CD5+ phenotype (14 were CD56-, and 1 was CD56+) and clonal rearrangement of TCR gamma genes, indicating that they were true PTCL with unproductive TCR rearrangement. The four remaining cases were CD5- CD56- lymphomas and disclosed either a clonal (two cases) or no clonal (two cases) rearrangements of the TCR gamma genes. Altogether these findings show that CD5-CD56+ so-called "TCR silent PTCL" bear the immunophenotype and immunogenotype of normal NK cells and display peculiar clinical features distinct from true PTCL.