Apolipoprotein E alters metabolism of AbetaPP in cells engaged in beta-amyloidosis.

Canine smooth muscle cells (SMCs), cultured from amyloid-affected brain blood vessels accumulate Alzheimer amyloid-beta peptide (Abeta) intracellularly, either spontaneously or after treatment with apolipoprotein E (apoE). ApoE is codeposited with Abeta, which suggests that apoE participates in Abeta accumulation. We tested the hypothesis that apoE-induced accumulation of Abeta in SMCs is caused by an increased production of amyloid-beta precursor protein (AbetaPP) and/or its altered metabolism. We found that 24 hours of treatment with apoE3 or apoE4 induced intracellular accumulation of Abeta-immunoreactive deposits in SMCs but did not influence AbetaPP production and processing. The treatment with apoE3 or E4 for 3 days resulted in the following: increased Abeta-accumulation; reduced levels of secreted Abeta; increased production and cellular retention of mature AbetaPP770; and reduced culture growth, cell proliferation, and viability. ApoE4, but not apoE3, increased cellular levels of mRNA AbetaPP 770 (the main form produced in SMCs) about ninefold. ApoE3 stimulated production and cellular retention of endogenous apoE. We hypothesize that Abeta accumulation is triggered by apoE, which may bind and immobilize soluble Abeta produced in SMCs. The newly formed Abeta deposits may further accelerate Abeta accumulation by altering metabolism of AbetaPP.

Changes in platelet response to adenosine 5'-diphosphate caused by verapamil.

Verapamil is widely used in the treatment of patients with coronary artery disease. The effect of verapamil on vascular smooth muscle cells is well documented. This effect is mediated by the inhibition of calcium fluxes across plasma membranes. Some data suggest that verapamil may affect platelet functions in thrombosis, but those observations were made for much higher verapamil concentration than could be achieved in vivo. Our current investigations are focused on an influence of low doses of verapamil (0.1-1.0 microM) on platelet response to ADP. We have found that verapamil at concentration of 0.1 microM can inhibit platelet aggregation (by 10%) evoked in PRP by 1.0-1.5 microM ADP. Moreover, the inhibitory effect is potentiated by prolonged time of platelet preincubation with verapamil. On the other hand, we have found a significant reduction in the number of fibrinogen receptors exposed on the platelet surface of patients (n = 21) treated with therapeutic doses (240 mg/day) of verapamil during two weeks of drug administration. The mean number of exposed receptors was reduced from 75,000 to 40,000 per platelet, with significance p < 0.0001. In vitro platelet preincubation with verapamil, even in much higher concentrations, did not affect fibrinogen binding to ADP activated platelets. It suggests, that in vitro exposure of platelets to verapamil for a short time has no effect on the expression of fibrinogen receptors on platelets, but prolonged in vivo interaction of this drug with platelets results in reduction of the fibrinogen receptor exposition. Thus, observed inhibition of platelet aggregation does not relay on a simple reduction of the number of exposed receptors, but intraplatelet signalling has to be affected. In fact, we have observed, in platelets pretreated with low doses of verapamil, significantly reduced release of calcium ions upon activation by ADP, whereas the calcium influx under such conditions does not seem to be affected.

Expression of fibrinogen receptors and GPIIb molecules on uraemic platelets: effect of recombinant human erythropoietin therapy.

In 14 chronic uraemic patients on regular haemodialysis treatment mean numbers of fibrinogen (Fg) receptors and glycoprotein IIb (GPIIb) molecules on one blood platelet were 8370 +/- 1282 and 15,694 +/- 2102 respectively, and they were significantly lowered in comparison with those found in 14 healthy persons (37,826 +/- 966 and 57,994 +/- 6824 respectively). A high positive correlation was found between numbers of GPIIb molecules and those of Fg receptors both in chronic uraemic patients and healthy subjects. Four-month treatment of haemodialysis patients with recombinant human erythropoietin (rHuEpo) induced significant rises in haematocrit from 22.3 +/- 1.2 to 31.2 +/- 1.7%, blood platelet count from 164,000 +/- 27,000/microliters to 206,000 +/- 16,000/microliters and mean platelet volume from 7.08 +/- 0.29 fl to 8.26 +/- 0.22 fl. However, in haemodialysis patients numbers of the investigated surface platelet receptors, measured at 8 and 16 week of rHuEpo treatment, remained unchanged.

Increased platelet-fibrinogen interaction in patients with hypercholesterolemia and hypertriglyceridemia.

Binding of fibrinogen to platelets washed from the blood of patients with hypercholesterolemia and hypertriglyceridemia (n = 25) and control donors (n = 12) was compared. In addition, the content of platelet glycoprotein IIb was determined by radioimmunoassay. Fibrinogen was bound in significantly higher amounts (P < 0.02) to hyperlipidaemic platelets activated by ADP than to control ones (107,112 +/- 16,371 and 45,612 +/- 6495 molecules per platelet, respectively). The mean content of GPIIb was the same in hyperlipidaemic and in control platelets (2.06 +/- 0.16 and 1.94 +/- 0.21 micrograms/10(8) platelets, respectively). The amount of fibrinogen bound to the activated hyperlipidaemic platelets showed a positive correlation with total plasma cholesterol and LDL (r = 0.45 and 0.47, respectively) whereas a negative correlation with plasma HDL was found (r = -0.50). The increased expression of fibrinogen binding sites similar to that of hyperlipidaemic platelets could be produced by preincubation of normal platelets with palmitic acid. This was evidenced by a significant increase of fibrinogen binding sites in control platelets. This suggests that either palmitoylation of the receptor or microenvironment changes in the membrane lipid bilayer may be responsible for the enhanced platelet receptor capacity to bind fibrinogen.

[The number and affinity of fibrinogen receptors in blood platelets of patients with ischemic stroke]. / Liczba i powinowactwo receptorów dla fibrynogenu w krwinkach plytkowych chorych na niedokrwienny udar mózgu.

Parameters of fibrinogen binding with blood platelets (number of receptors and their affinity) have been studied in patients with ischemic stroke. Due to the increased platelet ability to aggregate in the ischemic diseases such studies seem helpful. The studies involved 13 patients with ischemic stroke. Blood platelets collected from younger patients (under 50 years) possessed significantly higher number of receptors binding fibrinogen than blood platelets of healthy individuals (p less than 0.02). These receptors significantly more strongly bound ligand than those in the control group (p less than 0.05), and in the group of older patients with stroke (less than 0.05). Fibrinogen binding to blood platelets in patients over 50 years of age did not differ significantly from that in the control group. These results may indicate, that the increased platelet aggregation might be a significant pathogenic factor of the stroke in younger patients.

Palmitylation of the glycoprotein IIb-IIIa complex in human blood platelets.

The presence of covalently bound palmitic acid in fibrinogen receptors, glycoproteins (GP) IIb and IIIa, has been explored in human blood platelets. Membrane fractions were isolated from fresh blood platelets labeled with [9,10-3H]palmitic acid and then analyzed for radioactive proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein bands were visualized by staining with Coomassie Brilliant Blue, excised, and counted in a liquid scintillation counter. The results indicate that membrane proteins with electrophoretic mobility corresponding to glycoproteins IIb and IIIa incorporate [9,10-3H]palmitic acid. The palmitylated glycoproteins IIb and IIIa were immunoprecipitated by specific anti-GP IIb and GP IIIa antisera. It is interesting to note that the palmitylation of these glycoproteins occurred rapidly in platelets activated with 0.5 unit of thrombin or 30 microM ADP. At the concentration used (100 micrograms/ml), cycloheximide did not inhibit incorporation of [3H]palmitate into the glycoproteins showing that this process is not dependent upon protein synthesis. The acyl moiety was resistant to denaturating detergents, delipidation with organic solvents, and hydrolyzable with hydroxylamine. In the case of membrane protein with the electrophoretic mobility of GP IIb, the radioactive label was significantly decreased after reduction with 2-mercaptoethanol. Final identification of GP IIIa as an acylated product in human platelets incubated with [9,10-3H]palmitic acid was provided by two-dimensional polyacrylamide gel electrophoresis. In contrast to GP IIb alpha, GP IIIa isolated by this method showed the presence of attached radioactive palmitic acid residues. Analysis by high performance liquid chromatography after methanolysis of the [3H]palmitate-labeled glycoproteins confirmed the fatty acid nature of the label. Palmitylation is a newly identified post-translational modification of the fibrinogen receptor which may play an important role in its interaction with the membrane and/or its biological function.