Binding of human platelet glycoprotein Ib and actin to fragments of actin-binding protein.

Actin-binding protein (ABP) is degraded into fragments of 190 and 90 kDa by calpain. A monoclonal antibody (MAb TI10) against the 90 kDa fragment of ABP coprecipitated with the glycoprotein Ib (GP Ib) peak observed on crossed immunoelectrophoresis of Triton X-100 extracts of platelets prepared without calpain inhibitors. MAb PM6/317 against the 190 kDa fragment was not coprecipitated with the GP Ib peak under such conditions. The 90 kDa fragment was adsorbed on protein A agarose from extracts that had been preincubated with antibodies to GP Ib. This supports the idea that the GP Ib-ABP interaction resides in the 90 kDa region of ABP. GP Ib was sedimented with the Triton-insoluble actin filaments in trace amounts only, and only after high speed centrifugation (100,000 x g, 3 h). Both the 190 kDa and the 90 kDa fragments of ABP were sedimented with the Triton-insoluble actin filaments.

Effects of toluene on platelet membrane glycoprotein Ib and actin-binding protein.

Effects of the organic solvent toluene on the platelet membrane receptor glycoprotein Ib (GP Ib) and the cytoskeletal protein, actin-binding protein (ABP), were studied and related to the effects of the local anesthetic dibucaine. The glycocalicin-region of GP Ib contains the binding site for von Willebrand factor; intracellularly GP Ib is linked to the cytoskeleton via ABP. Both GP Ib and ABP are substrates for a calcium-dependent protease, calpain. Washed platelets were incubated with toluene or dibucaine. The toluene concentration in the platelet suspension was analysed by gas chromatography. Using 1.5-2.8 mmol/L toluene, calpain was activated, leading to degradation of ABP and release of glycocalicin from GP Ib. The latter phenomenon was paralleled by a reduced von Willebrand factor-induced platelet agglutination. At lower toluene concentrations (0.3-1.4 mmol/L), degradation of ABP was not detected but an initial increased agglutination that declined to the control level with time was observed. These effects of toluene on the GP Ib-ABP complex are similar to those observed with 1 mmol/L dibucaine. The lowest toluene concentrations used correspond to those that have been found in blood from toluene abusers ("sniffers").

A pharmacoeconomic evaluation of escitalopram, a new selective serotonin reuptake inhibitor. Comparison of cost-effectiveness between escitalopram, citalopram, fluoxetine, and venlafaxine for the treatment of depression in Norway.

This study compared the cost-effectiveness of escitalopram to that of citalopram,fluoxetine, and venlafaxine in the treatment of depression in Norway. A two-path decision analytic model with a 6-month horizon was used. Patients start at the primary path and are referred to specialist care in the secondary care path. Model inputs included drug-specific probabilities from comparative trial data, literature, and a panel of experts. The main outcome measure is success (remission), and costs of treatment (total and drug costs). Treatment with escitalopram yielded lower expected cost and greater effectiveness than citalopram, fluoxetine, and venlafaxine. The expected success rate was 64.2% with escitalopram,58.7% with citalopram, 58.7% with fluoxetine, and 62.1% with venlafaxine. Average expected total costs per patient were similar with escitalopram (19,661 Norwegian crowns) and venlafaxine (20,989) and somewhat higher with citalopram (22,379) and fluoxetine (22,558). Budgetary impact estimates a decrease in total health care budget of 72 million crowns. Escitalopram is therefore the most cost-effective alternative and its use would significantly reduce health care costs for the treatment of depression in Norway.

Studies on a patient with thrombocytopenia, giant platelets and a platelet membrane glycoprotein Ib with reduced amount of sialic acid.

A 70-year-old patient with a life-long bleeding tendency, giant platelets and thrombocytopenia (10-40 x 10(9) platelets/l) has been studied. This is a condition often associated with lack of platelet membrane glycoprotein Ib (GP Ib). Electron microscopy of fixed platelets incubated with monoclonal antibodies to GP Ib (AN 51, AP 1) and gold-labelled goat anti-mouse IgG, showed a distinct distribution of GP Ib on the patient's platelets, however. Crossed immunoelectrophoresis and SDS-PAGE demonstrated a reduced mobility of the patient's GP Ib which could be explained by absence of sialic acid. Blotting with peroxidase-conjugated peanut agglutinin confirmed this conclusion. This lectin binds to galactose-N-acetyl-galactosamine residues exposed terminally when sialic acid is absent from the carbohydrate side-chains. Such binding could be seen with normal GP Ib only after neuraminidase treatment. Fluorescence studies with FITC-conjugated peanut agglutinin showed binding of the lectin to intact patient platelets, indicating that lack of sialic acid was not introduced during the platelet isolation procedure. Neither could the lack of sialic acid be attributed to increased neuraminidase activity as studied in vitro. Platelets treated with neuraminidase in vivo or in vitro are rapidly cleared from the circulation. Therefore the patient's thrombocytopenia may be associated with the reduced amount of GP Ib sialic acid. As far as we know, similar cases have not been described previously.

The PhastSystem equipment used for crossed immunoelectrophoresis combined with immunoblotting of coprecipitated monoclonal antibodies as studied with platelet membrane receptor proteins.

The Pharmacia PhastSystem equipment has been used for crossed immunoelectrophoresis combined with a technique for immunoblotting with monoclonal antibodies. This miniaturized gel system is compared to the conventional approach using platelet membrane receptor proteins as a model. Whereas in the conventional system the electrophoretic procedure takes place within 20 h, 3 h are adequate for the small gel system. Because of the short second-dimensional electrophoresis, and only one overnight incubation, the total electrophoretic and blotting procedure could be reduced from about 48 h to 24 h. The amount of antiserum used during the second-dimensional electrophoresis could be reduced roughly by a factor of 5. The examples with electrophoresis and immunoblotting using platelet extracts in 1% Triton X-100 demonstrate that membrane receptor proteins can be studied even when present as noncovalent complexes. The immunoblotting can be used with monoclonal antibodies that do not function in Western blotting.

The use of PhastSystem crossed immunoelectrophoresis with immunoblotting to demonstrate a complex between glycoprotein Ib and the actin-binding protein (ABP) of human platelets.

The study shows how a technique described in an accompanying paper can be applied to solve a biological problem. The technique makes use of the observation that a monoclonal antibody that has been coprecipitated with its antigen during crossed immunoelectrophoresis can be transferred to a nitrocellulose membrane and visualized. Previous studies using crossed immunoelectrophoresis of Triton X-100 extracts of platelets have indicated that a particular immunoprecipitate (peak III) of the membrane receptor glycoprotein Ib (GP Ib) might contain a complex between the receptor and the actin-binding protein (filamin). When a monoclonal antibody (PM6/317) directed towards the actin-binding protein was added to a platelet extract prior to immunoelectrophoresis and blotting, this was visualized on the blot as a replica of the peak III immunoprecipitate. This demonstrates a colocalization of GP Ib and the actin-binding protein in the precipitate, and thus the existence of a complex between the membrane receptor and the cytoskeletal protein.