International Society on Antiphospholipid Antibodies, Tours, France, 12-16 September 2000.
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Animals↗
Biomedical subjects
T Exner
Publications and source records attributed to T Exner.
Conceptions and misconceptions in testing for lupus anticoagulants.
The aim of the following presentation is to review certain technical issues complicating clotting tests for lupus anticoagulants (LA). This is a field riddled with incorrect and misleading studies which often make progress difficult and which are difficult to retract. Inconsistent test sensitivity comparisons are sometimes due to incorrect methods being used but are more often due to contamination with platelets and instrument effects. Time dependence of LA is often due to pH drift during incubation and probably all LA are immediate acting (at least in our hands). The use of platelets in confirmatory tests is dangerous and can lead to false-positive results in patients with anti-factor V and heparin-like inhibitors.
Blood Coagulation Tests↗
Effect of instruments on lupus anticoagulant testing.
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Biological Assay↗
Posttranslational modification of Galphao1 generates Galphao3, an abundant G protein in brain.
Galphao, the most abundant G protein in mammalian brain, occurs at least in two subforms, i.e., Galphao1 and Galphao2, derived by alternative splicing of the mRNA. A third Galphao1-related isoform, Galphao3, has been purified, representing about 30% of total Go in brain. Initial studies revealed distinct biochemical properties of Galphao3 as compared with other Galphao isoforms. In matrix-assisted laser desorption/ionization peptide mass mapping of gel-isolated Galphao1 and Galphao3, C-terminal peptides showed a difference of +1 Da for Galphao3. Nanoelectrospray tandem mass spectrometry sequencing revealed an Asp instead of an Asn at position 346 of Galphao3. Gel electrophoretic analysis of recombinant Galphao3 showed the same mobility as native Galphao3 but distinct to Galphao1. The conversion of 346Asn-->Asp changed the signaling properties, including the velocity of the basal guanine nucleotide-exchange reaction, which points to the involvement of the C terminus in basal guanosine 5'-[gamma-thio]triphosphate binding. No cDNA coding for Galphao3 was detected, suggesting an enzymatic deamidation of Galphao1 by a yet-unidentified activity. Therefore, Galpha heterogeneity is generated not only at the DNA or RNA levels, but also at the protein level. The relative amount of Galphao1 and Galphao3 differed from cell type to cell type, indicating an additional principle of G protein regulation.
Amino Acid Sequence↗
Nonselective coupling of the human mu-opioid receptor to multiple inhibitory G-protein isoforms.
The human mu-opioid receptor was expressed in Saccharomyces cerevisiae. Binding of [3H]diprenorphine to yeast spheroplasts was specific and saturable (Kd = 1 nm, Bmax = 0.2-1 pmol x mg-1 of membrane proteins). Inhibition of [3H]diprenorphine binding by antagonists and agonists with varying opioid selectivities (mu, delta and kappa) occurred with the same order of potency as in mammalian tissues. Affinities of antagonists were the same with yeast spheroplasts as in reference tissues whereas those of agonists, except etorphine and buprenorphine, were 10-fold to 100-fold lower. Addition of heterotrimeric Gi,o-proteins purified from bovine brain shifted the mu-opioid receptor into a high-affinity state for agonists. Using individually purified Galpha-subunits re-associated with betagamma-dimers, we showed that alphao1, alphao2, alphai1, alphai2 and alphai3 reconstituted high-affinity agonist binding with equal efficiency. This suggests that the structural determinants of the mu-opioid receptor responsible for G-protein coupling are not able to confer a high degree of specificity towards any member of the Gi,o family. The selective effects of opioid observed in specialized tissues upon opioid stimulation may be a result of regulation of G-protein activity by cell-specific factors which should conveniently be analysed using the reconstitution assay described here.
Binding Sites↗
Gbetagamma dimers stimulate vascular L-type Ca2+ channels via phosphoinositide 3-kinase.
We have previously reported that, in venous myocytes, Gbetagamma scavengers inhibit angiotensin AT1A receptor-induced stimulation of L-type Ca2+ channels (1). Here, we demonstrate that intracellular infusion of purified Gbetagamma complexes stimulates the L-type Ca2+ channel current in a concentration-dependent manner. Additional intracellular dialysis of GDP-bound inactive Galphao or of a peptide corresponding to the Gbetagamma binding region of the beta-adrenergic receptor kinase completely inhibited the Gbetagamma-induced stimulation of Ca2+ channel currents. The gating properties of the channel were not affected by intracellular application of Gbetagamma, suggesting that Gbetagamma increased the whole-cell calcium conductance. In addition, both the angiotensin AT1A receptor- and the Gbetagamma-induced stimulation of L-type Ca2+ channels were blocked by pretreatment of the cells with wortmannin, at nanomolar concentrations. Correspondingly, intracellular infusion of an enzymatically active purified recombinant Gbetagamma-sensitive phosphoinositide 3-kinase, PI3Kgamma, mimicked Gbetagamma-induced stimulation of Ca2+ channels. Both Gbetagamma- and PI3Kgamma-induced stimulations of Ca2+ channel currents were reduced by protein kinase C inhibitors suggesting that the Gbetagamma/PI3Kgamma-activated transduction pathway involves a protein kinase C. These results indicate for the first time that Gbetagamma dimers stimulate the vascular L-type Ca2+ channels through a Gbetagamma-sensitive PI3K.
Animals↗
A monoclonal antibody against prothrombin fragment 1 behaves like a lupus anticoagulant.
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Animals↗
Detecting APC-resistant factor V: a functional method without plasma dilution.
A method for detecting activated protein C (APC)-resistant factor V, especially factor V Leiden, is described, which uses reagents containing two unfractionated snake venoms. The procedure can be used for testing plasma samples from patients receiving oral anticoagulant therapy, heparin therapy and patients with lupus anticoagulant, and does not require the use of factor-V-deficient plasma. The sample plasma is first incubated with dilute venom from Agkistrodon contortrix contortrix (Southern Copperhead) which activates the endogenous protein C and then a dilute Russell's viper venom time test is performed. In individuals with APC-resistant factor V, especially factor V Leiden, a marginal prolongation of dilute Russell's viper venom time was noted [1.14 +/- 0.14 s (n = 16)]. Non-carriers were easily discriminated in each patient group, with a prolongation of 2.69 +/- 0.30 s for normal blood donors (n = 127), 2.61 +/- 0.38 s for patients taking oral anticoagulants (n = 102), 2.41 +/- 0.45 s for patients taking heparin (n = 96), and 2.38 +/- 0.41 s for patients with lupus anticoagulant (n = 22). Patients taking oral anticoagulants with moderate prolongation (between 1.5- and 2.0-fold) may have low levels of functional protein C and this might additionally indicate a subgroup of such patients at higher than normal thrombotic risk.
Activated Protein C Resistance↗
Immuno- and gold staining of a single western blot.
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Animals↗
Gbetagamma stimulates phosphoinositide 3-kinase-gamma by direct interaction with two domains of the catalytic p110 subunit.
Class I phosphoinositide 3-kinases (PI3Ks) regulate important cellular processes such as mitogenesis, apoptosis, and cytoskeletal functions. They include PI3Kalpha, -beta, and -delta isoforms coupled to receptor tyrosine kinases and a PI3Kgamma isoform activated by receptor-stimulated G proteins. This study examines the direct interaction of purified recombinant PI3Kgamma catalytic subunit (p110gamma) and Gbetagamma complexes. When phosphatidylinositol was used as a substrate, Gbetagamma stimulated p110gamma lipid kinase activity more than 60-fold (EC50, approximately 20 nM). Stimulation was inhibited by Galphao-GDP or wortmannin in a concentration-dependent fashion. Stoichiometric binding of a monoclonal antibody to the putative pleckstrin homology domain of p110gamma did not affect Gbetagamma-mediated enzymatic stimulation, whereas incubation of Gbetagamma with a synthetic peptide resembling a predicted Gbetagamma effector domain of type 2 adenylyl cyclase selectively inhibited activation of p110gamma. Gbetagamma complexes bound to N- as well as C-terminal deletion mutants of p110gamma. Correspondingly, these enzymatically inactive N- and C-terminal mutants inhibited Gbetagamma-mediated activation of wild type p110gamma. Our data suggest that (i) p110gamma directly interacts with Gbetagamma, (ii) the pleckstrin homology domain is not the only region important for Gbetagamma-mediated activation of the lipid kinase, and (iii) Gbetagamma binds to at least two contact sites of p110gamma, one of which is close to or within the catalytic core of the enzyme.
Adenylyl Cyclases↗
The heterotrimeric G protein Go2 regulates catecholamine uptake by secretory vesicles.
Secretory vesicles store neurotransmitters that are released by exocytosis. Their membrane contains transporters responsible for transmitter loading that are driven by an electrochemical proton gradient across the vesicle membrane. We have now examined whether uptake of noradrenaline is regulated by heterotrimeric G proteins. In streptolysin O-permeabilized PC 12 cells, GTP-analogues and AlF4- inhibited noradrenaline uptake, an effect that was sensitive to treatment with pertussis toxin. Inhibition of uptake was prevented by Galphao-specific antibodies and mimicked by purified activated Galphao2. No effect was seen when Galphao2 in its inactive GDP-bound form or purified activated Galphao1, Galphai1 and Galphai2 were tested. Down-regulation of uptake remained unchanged when exocytosis was inhibited by the light chain of tetanus toxin. Vesicular acidification was not affected whereas binding of [3H]reserpine was reduced by GTPgammaS and Galphao2. These data suggest that the monoamine transporter rather than the vacuolar ATPase is affected. We conclude that catecholamine uptake is controlled by Galphao2, suggesting a novel function for heterotrimeric G proteins in the control of neurotransmitter storage.
Animals↗
Methods for subtyping lupus anticoagulants.
Two simple procedures were tested for their potential to identify beta2-glycoprotein 1 (beta2GP1) or prothrombin cofactor dependence among lupus anticoagulants (LA). The first comprised mixing test plasma 1:4 with beta2GP1-deficient plasma instead of with normal plasma. beta2GP1 deficiency decreased, but did not abolish most LA detectable in KCT, DRVVT and APTT clotting tests. Mixing 1:4 with bovine plasma was evaluated in a second test based on the KCT in the expectation that prothrombin-dependent LA would be preferentially shortened. Bovine plasma had a similar correcting effect on LA in all three tests considered here. Conversely, a prothrombin antibody was found to have similar prolonging effect on all three of these tests. LA patient plasmas displayed considerable heterogeneity when analysed using a combination of these two tests. The clinical significance of these tests remains uncertain. DRVVT and KCT tests do not appear to discriminate beta2GP1-dependent from prothrombin-dependent LA.
Adult↗
Molecular modeling information transfer with VRML: from small molecules to large systems in bioscience.
The suitability of the Virtual Reality Modeling Language (VRML) for the communication of scientists via the internet is demonstrated with recent results from computer assisted cancer research: I. Substrate channels in cytochrome P450 enzymes. II. Binding properties of the wild type and mutated p53 tumor suppressor protein. Complex 3D molecular models were used to visualize new insights in the active site access of cytochrome P450 enzymes and in the p53 protein-DNA binding achieved by the use of computational methods. These 3D models of biomolecular systems were transferred into VRML scenarios. Additional implemented features allow users to receive related information interactively. With these examples it is shown that VRML provides an efficient method for scientific information exchange by the use of complex 3D molecular models.
Binding Sites↗
Alkyl-substituted amino acid amides and analogous di- and triamines: new non-peptide G protein activators.
Synthesis and pharmacological properties of new potent direct activators of heterotrimeric G proteins are described. Compounds were synthesized from protected amino acids with alkylamines using coupling reagents (CDI, DCC, and EDC). Alkyl-substituted amino acid amides and their corresponding di- and triamines were subjected to structure-activity analysis. All compounds activated membrane-bound HL-60 GTPases in a pertussis toxin-sensitive fashion. This suggests a specific effect of compounds on the carboxy terminus of a defined subclass of heterotrimeric G proteins, i.e., members of the G alpha i subfamily. Elongation of the alkyl chain and increasing the number of amino groups enhanced the potency of compounds on HL-60 membrane-bound GTPase. N-(2,5-Diaminopentyl)dodecylamine (21) was selected to study its mode of action employing purified pertussis toxin-sensitive G proteins. It stimulated G alpha subunits by inducing the release of bound GDP. In contrast to receptors G beta gamma complexes were not required for 21-mediated activation of G alpha. Moderate isoform selectivity of its action was observed within a group of highly homologous members of the Gi subfamily with G alpha o1 being activated at lowest concentrations, whereas higher concentrations were necessary for the stimulation of G alpha i1 or transducin. We conclude that these compounds represent important tools for studying G protein-dependent cellular functions.
Amides↗
Distinct biochemical properties of the native members of the G12 G-protein subfamily. Characterization of G alpha 12 purified from rat brain.
G12 and G13 are insufficiently characterized pertussis toxin-insensitive G-proteins. Here, we describe the isolation of G alpha 12 from rat brain membranes. G alpha 12 was purified to apparent homogeneity by three steps of conventional chromatography, followed by two cycles of subunit-exchange chromatography on immobilized G subunits. Purified G alpha 12 bound guanosine 5'-[gamma-thio]triphosphate slowly and substoichiometrically. For isolation of functionally active G alpha 12, it was mandatory to use sucrose monolaurate as a detergent. Comparative studies of both rat-brain-derived members of the G12 subfamily revealed differences in the affinity of G alpha 12 and G alpha 13 for G beta gamma. G alpha 12 required a higher Mg2+ concentration for AlF4- -induced dissociation from immobilized G beta gamma than did G alpha 13. In addition, the G12 subfamily members differed in their sedimentation velocities, as determined by sucrose-density-gradient centrifugation. Analysis of sedimentation coefficients revealed a higher tendency of G12 to form supramolecular structures in comparison to G13 and other G-proteins. These G13 structures were stabilized by sucrose monolaurate, which in turn may explain the necessity for this detergent for purification of functionally active G alpha 12. Despite these distinct biochemical characteristics of G12 and G13, both purified G-proteins coupled to a recombinant thromboxane A2 (TXA2) receptor reconstituted into phospholipid vesicles. These data indicate, (1) significant differences in the biochemical properties of native members of the G12 subfamily, and (2) their specific coupling to TXA2 receptors.
15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
Species- and tissue-dependent diversity of G-protein beta subunit phosphorylation: evidence for a cofactor.
We previously reported that, in the membranes of HL-60 cells during activation of G-proteins, a phosphate transfer reaction occurs which involves transient G-protein beta subunit (G beta) phosphorylation [Wieland, Nürnberg, Ulibarri, Kaldenberg-Stasch, Schultz and Jakobs (1993) J. Biol. Chem. 268, 18111-18118]. Here, the generality of this phenomenon is evaluated by studying membranes of various tissues obtained from different mammalian species. All membranes tested expressed at least G beta 1 and G beta 2 subunits. Cell membranes from bovine and porcine brain and liver, rat brain and human blood cells exhibited predominantly G beta 1 or both subtypes at roughly equal concentrations. In contrast, significantly more G beta 2 immunoreactivity was detected in membranes from human placenta. Bovine and porcine liver membranes exhibited weak, G beta-specific immunoreactive signals. Conversely, these membranes showed the highest levels of G beta phosphorylation after incubation with [gamma-32P]GTP or 35S-labelled guanosine 5'-[gamma-thio]triphosphate. Interestingly, G beta-specific phosphorylation of membranes from human erythrocytes and platelets was very weak. G beta phosphorylation was confirmed by immunoprecipitation with G beta-specific antibodies, and the target amino acid was identified as histidine. On SDS/PAGE, phosphorylated or thiophosphorylated G beta-proteins differed in their apparent molecular size from unmodified G beta-proteins. Moreover, phosphorylated G beta-proteins differed in a species-dependent fashion in their electrophoretic mobility. Solubilization of membrane proteins with detergent did not abolish G beta phosphorylation. In contrast, reconstituted purified Gi/Go proteins showed no G beta phosphorylation. From these experiments we conclude that: (i) G beta phosphorylation represents a general phenomenon occurring in the cells of various species to different degrees, (ii) phosphorylated G beta-proteins exhibit species-dependent diverse electrophoretic mobilities, and (iii) G beta phosphorylation requires a membrane-associated cofactor(s) which is lost during routine G-protein purification.
Animals↗
Diagnostic methodologies for circulating anticoagulants.
Various types of circulating anticoagulant are encountered in coagulation testing laboratories. Those associated with bleeding often cause problems in diagnosis. The most common type of acquired coagulation inhibitor not associated with bleeding is the so-called lupus anticoagulant (LA). Differing from SLE which occurs predominantly in women, primary LA occurs both in females and males. LA are now frequently sought in patients with recurrent foetal losses and acquired thrombotic problems as a causative factor, whereas in the past they were regarded as a laboratory nuisance. Due to the complicating effect of inhibitors on clotting tests, diagnosis of various coagulation inhibitors remains difficult. There may also be significant overlap between different types of inhibitors. With the recent interest shown in LA, almost all non-specific inhibitors tend to be classed as LA. LA are defined as phospholipid-interfering antibodies. Current criteria have recently been confirmed and include screening with phospholipid-responsive tests, abnormal mixing studies and correction with phospholipids. However it is becoming clear that even LA as defined may be heterogeneous. Most LA are not directed at negatively-charged phospholipids alone as originally suggested, but rather at complexes of either beta-2-glycoprotein 1 or prothrombin with such phospholipids. There may also be other lipid-associated antigens involved. Although earlier work suggested that all LA functioned through a similar mechanism, there is now some preliminary evidence suggesting that various subclasses of LA may account for discrepant results sometimes obtained with different clotting tests. A variety of improvements to the basic screening tests for LA (APTT, KCT, DTTI and DRVVT) have recently been suggested.(ABSTRACT TRUNCATED AT 250 WORDS)
Adolescent↗
von Willebrand's disease: use of collagen binding assay provides potential improvement to laboratory monitoring of desmopressin (DDAVP) therapy.
This report describes studies investigating the use of a collagen binding assay to improve the laboratory monitoring of desmopressin (DDAVP) therapy in patients with von Willebrand's disease (vWD). We evaluated the response of seven patients with vWD (four type I, three type IIA) to DDAVP, administered using a standard protocol, by assessing levels of von Willebrand factor (vWF) and factor VIII, as well as performing skin bleeding times (SBT) prior to, and at sequential time points following, DDAVP administration. The study employed the following assays: von Willebrand factor antigen assay (vWF:Ag; determined by ELISA); a novel functionally based collagen binding assay (CBA; determined by ELISA); ristocetin cofactor assay (RCof; determined by platelet aggregometry); von Willebrand factor multimer analysis (using SDS-agarose gels); factor VIII coagulant (FVIIIC; determined by clotting assay); and factor VIII antigen (FVIIICAG; determined by ELISA). All patients showed an initial incremental increase in vWF/FVIII levels using all assays above, and some showed some correction in SBT. Although the absolute levels of vWF/FVIII antigen or activity varied between patients, the CBA was found to provide consistently the greatest proportional incremental increases (i.e., -fold) compared to baseline (pre-DDAVP) levels. Accordingly, we consistently observed an increase in the CBA to vWF:Ag ratio for all patients evaluated. This supplements previous findings that have suggested a unique ability of our CBA procedure to bind preferentially to higher molecular weight (i.e., more functionally active) forms of vWF. We therefore propose that the use of the above test combination (e.g., vWF:Ag plus CBA) may provide the basis for more accurate estimation of a patient's functional responsiveness to DDAVP therapy in future studies.
Biopolymers↗