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The general hypothesis for the biological function of beta 2- glycoprotein I is that it neutralizes all negatively charged macromolecules that might enter the bloodstream and diminishes unwanted activation of the blood coagulation. In the present study we report that beta 2-glycoprotein I inhibits the activation of the contact phase system of the intrinsic pathway of blood coagulation. Activation was accomplished by an ellagic acid-phospholipid suspension (Cephotest) and measured by the appearance of amidolytic activity using the chromogenic substrate H-D-Pro-Phe-Arg-p-nitroanilide (S-2302). This inhibitory effect of beta 2-glycoprotein I was observed both when Cephotest was preincubated with beta 2-glycoprotein I and when the amount of beta 2- glycoprotein I in plasma was increased by addition of beta 2- glycoprotein I to either normal or beta 2-glycoprotein I-deficient plasma. The inhibitory effect of beta 2-glycoprotein I on the contact phase activation could be one of the physiological functions of this protein.

c-Aminobutyric acid (GABA) synthesis from glutamate is catalyzed by glutamate decarboxylase (GAD) of which two isoforms, GAD65 and GAD67, have been identified. The GAD65 has repeatedly been shown to be important during intensified synaptic activity. To specifically elucidate the significance of GAD65 for maintenance of the highly compartmentalized intracellular and intercellular GABA homeostasis, GAD65 knockout and corresponding wild-type mice were injected with [1-13 C]glucose and the astrocyte-specific substrate [1,2-13 C]acetate. Synthesis of GABA from glutamine in the GABAergic synapses was further investigated in GAD65 knockout and wild-type mice using [1,2-13 C]acetate and in some cases c-vinylGABA (GVG, Vigabatrin), an inhibitor of GABA degradation. A detailed metabolic mapping was obtained by nuclear magnetic resonance (NMR) spectroscopic analysis of tissue extracts of cerebral cortex and hippocampus. The GABA content in both brain regions was reduced by B20%. Moreover, it was revealed that GAD65 is crucial for maintenance of biosynthesis of synaptic GABA particularly by direct synthesis from astrocytic glutamine via glutamate. The GAD67 was found to be important for synthesis of GABA from glutamine both via direct synthesis and via a pathway involving mitochondrial metabolism. Furthermore, a severe neuronal hypometabolism, involving glycolysis and tricarboxylic acid (TCA) cycle activity, was observed in cerebral cortex of GAD65 knockout mice. Blood Flow & Metabolism (2011) 31, 494-503; doi:10.1038/jcbfm.2010; published online 28 July 2010 Journal of Cerebral Keywords:13 C isotopes; g-vinylGABA (GVG, vigabatrin); glutamate decarboxylase; hypometabolism; neuronal-glial trafficking; nuclear magnetic resonance IntroductionThe most abundant neurotransmitters in the brain are glutamate and g-aminobutyric acid (GABA) for excitatory and inhibitory transmission, respectively.A tight regulation of the synthesis and degradation of these two compounds is therefore crucial. Disturbances in this regulation are likely involved in GABA-glutamate imbalances characteristic for a number of neurodegenerative and psychiatric disorders (Sonnewald and Kondziella, 2003). The GABA synthesis from glutamate is catalyzed by the enzyme glutamate decarboxylase (GAD, EC 4.1.1.15) of which two isoforms, GAD65 and GAD67, have been identified. These isoforms are encoded for by separate genes and differ with regard to regulation and intracellular localization (Erlander et al, 1991;Kaufman et al, 1991;Esclapez et al, 1994). The GAD65 appears to exist predominantly as a dormant apoenzyme, being rapidly activated on binding of pyridoxal phosphate. In contrast, GAD67 is present as the active holoenzyme having the coenzyme bound . The GAD67 is localized throughout the cytosol of GABAergic neurons, Correspondence: Dr HS Waagepetersen, Department of Pharmacology and Pharmacotherapy, Faculty of Pharmaceutical Sciences, University of Copenhagen, 2 Universitetsparken, DK-2100 Copenhagen, Denmark. E-mail: hsw@farma.ku.dk 5 The major part of the wo...

Screening of serum by using a surface plasmon resonance analysis assay identified ␤ 2 -glycoprotein-I/apolipoprotein H as a plasma component binding to the renal epithelial endocytic receptor megalin. A calcium-dependent megalin-mediated ␤ 2 -glycoprotein-I endocytosis was subsequently demonstrated by ligand blotting of rabbit renal cortex and uptake analysis in megalin-expressing cells. Immunohistochemical and immunoelectron microscopic examination of kidneys and the presence of high concentrations of ␤ 2 -glycoprotein-I in urine of mice with disrupted megalin gene established that megalin is the renal clearance receptor for ␤ 2 -glycoprotein-I. A significant increase in functional affinity for purified megalin was observed when ␤ 2 -glycoprotein-I was bound to the acidic phospholipids, phosphatidylserine and cardiolipin. The binding of ␤ 2 -glycoprotein-I and ␤ 2 -glycoprotein-Iphospholipid complexes to megalin was completely blocked by receptor-associated protein.In conclusion, we have demonstrated a novel receptor recognition feature of ␤ 2 -glycoprotein-I. In addition to explaining the high urinary excretion of

The dependency of concentrations of Zn2+ and the negatively charged surfaces, phosphatidylinositol phosphate (PtdInsP), sulfatide and dextran sulfate, on the autoactivation of human factor XII, has been studied. While the autoactivation induced by sulfatide, and low concentrations of dextran sulfate, was unaffected by the presence of Zn2+, that induced by PtdInsP and higher concentrations of dextran sulfate was completely dependent on Zn2+ : the excess of Zn2+ needed to induce maximal activity with PtdInsP was 12-fold the concentration of factor XII, while with dextran sulfate it was 40-fold. Determination of the Znz+-binding properties of factor XI1 revealed that a total of four zinc ions could bind to each factor XI1 molecule. The first bound zinc ions (Kd 0.1 pM) induced an increase in the intrinsic tryptophan fluorescence of factor XII, while further titration up to a 40-fold surplus resulted in a quenching of the fluorescence. Binding of the zinc ions that caused the quenching had an average Kd of approximately 1 pM, independent of whether it was determined from the fluorescence changes or by equilibrium filtration. Low concentrations of both sulfatide and PtdInsP induced a fluorescence increase similar to that at low concentrations of Zn2+ but, in contrast to sulfatide, higher concentrations of PtdInsP did not induce a quenching in fluorescence. As the Zn2+-independent activating surface (sulfatide) induced quenching in the fluorescence intensity, while the Zn*+-dependent activating surface (PtdInsP) did not, the quenching, whether it was caused by sulfatide or zinc ions, was assigned to a change in the conformation which resulted in a molecular structure of factor XI1 that could be autoactivated. Association of factor XI1 in this conformation on the activating surface was suggested to be responsible for the autoactivation.Keywords: human factor XI1 ; zinc ; autoactivation; negatively charged surfaces ; conformational changes.Factor XI1 (FXII) circulates in normal plasma at a concentration of 30 pg/ml and constitutes, together with prekallikrein and high-molecular-mass kininogen the plasma contact activation system (for reviews see [I-41). It is a monomeric zymogen (80 kDa) that, by a single proteolytic cleavage of the Arg373-Val374 peptide bond within a disulfide loop, is transformed into the serine protease a-factor XIIa (FXIIa). In addition to its function in the contact activation system, it plays a role in the activation of the classical complement pathway as well as in the fibrinolytic system, in the production of kinins and in the initiation of cell-mediated inflammatory responses. By limited proteolysis, FXIIa activates prekallikrein and factor XI1 (autoactivation) and is connected to the intrinsic pathway of coagulation by activation of factor XI. Kallikrein can, in turn, reciprocally activate FXII. In the absence of a procoagulant surface, however, the kallikrein-dependent activation of FXII is slow and FXII autoactivation is practically not detectable [ 5 ] . Negatively charged surfaces stimulat...

The general hypothesis for the biological function of beta 2- glycoprotein I is that it neutralizes all negatively charged macromolecules that might enter the bloodstream and diminishes unwanted activation of the blood coagulation. In the present study we report that beta 2-glycoprotein I inhibits the activation of the contact phase system of the intrinsic pathway of blood coagulation. Activation was accomplished by an ellagic acid-phospholipid suspension (Cephotest) and measured by the appearance of amidolytic activity using the chromogenic substrate H-D-Pro-Phe-Arg-p-nitroanilide (S-2302). This inhibitory effect of beta 2-glycoprotein I was observed both when Cephotest was preincubated with beta 2-glycoprotein I and when the amount of beta 2- glycoprotein I in plasma was increased by addition of beta 2- glycoprotein I to either normal or beta 2-glycoprotein I-deficient plasma. The inhibitory effect of beta 2-glycoprotein I on the contact phase activation could be one of the physiological functions of this protein.

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Glutamatergic neurotransmission accounts for a considerable part of energy consumption related to signaling in the brain. Chemical energy is provided by adenosine triphosphate (ATP) formed in glycolysis and tricarboxylic acid (TCA) cycle combined with oxidative phosphorylation. It is not clear whether ATP generated in these pathways is equivalent in relation to fueling of the energy-requiring processes, i.e., vesicle filling, transport, and enzymatic processing in the glutamatergic tripartite synapse (the astrocyte and pre- and postsynapse). The role of astrocytic glycogenolysis in maintaining theses processes also has not been fully elucidated. Cultured astrocytes and neurons were utilized to monitor these processes related to glutamatergic neurotransmission. Inhibitors of glycolysis and TCA cycle in combination with pathway-selective substrates were used to study glutamate uptake and release monitored with D-aspartate. Western blotting of glyceraldehyde-3-P dehydrogenase (GAPDH) and phosphoglycerate kinase (PGK) was performed to determine whether these enzymes are associated with the cell membrane. We show that ATP formed in glycolysis is superior to that generated by oxidative phosphorylation in providing energy for glutamate uptake both in astrocytes and in neurons. The neuronal vesicular glutamate release was less dependent on glycolytic ATP. Dependence of glutamate uptake on glycolytic ATP may be at least partially explained by a close association in the membrane of GAPDH and PGK and the glutamate transporters. It may be suggested that these enzymes form a complex with the transporters and the Na(+) /K(+) -ATPase, the latter providing the sodium gradient required for the transport process.