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The mechanisms for regulating platelet HDL3 binding sites were investigated. HDL3 binding was rapid (T1/2 association=4 minutes) and completely reversible (T1/2 dissociation=14.5 minutes) at 4°C, 22°C, and 37°C, and kinetic analysis yielded forward and reverse constants of 7.3×10−4×s−1 and 7.13×103×s−1×M−1, respectively. Nevertheless, neither inhibitors of binding sites recycling or of pinocytosis, such as ammonium chloride, chloroquine, monensin, colchicine, and sodium azide, modified the binding characteristics. Moreover, when platelets were loaded with cholesterol, binding sites were not regulated (up or down). However, when exposed to high concentrations of HDL3 (1.5 g/L), apoE-free HDL (1.5 g/L), HDL2 (0.5 g/L), apoE-rich HDL (0.5 g/L), and VLDL (0.3 g/L) there was rapid downregulation of the number of binding sites in isolated permeabilized platelets, as shown by the reduction of Bmax to 66%, 58%, 45%, 53%, and 51%, respectively. Downregulation was rapid, reversible, and dose and time dependent. In contrast, LDL (up to 2.0 g/L), IDL (up to 0.1 g/L), and chylomicrons (up to 0.5 g/L) had no effect. Protein kinase C inhibitors (150 nmol/L staurosporine, 100 μmol/L H-7, and 10 nmol/L bisindolylmaleimide) inhibited downregulation up to 62% (as average value). The role of the PKC activation in regulating the activity of HDL3 binding sites also was analyzed by determining the cytosol-to-membrane translocation of enzymatic activity. Downregulation mediated by HDL3 rapidly translocated PKC activity (21%±11 of total PKC activity was membrane-associated in control platelets vs. 55±8% in downregulated platelets, mean±SEM, n=3 ). However, agents that block sequestration (0.30 g/L, concanavalin A), and other protein kinase inhibitors, such a cAMP-dependent protein kinase inhibitors (1 μmol/L, PKI), and β2-adrenergic receptor kinase inhibitors (100 nmol/L, heparin) had no effect. The results show that neither endocytotic response nor cholesterol-dependent mechanisms participate in the modulation of platelet HDL3 binding sites. However, a new regulatory mechanism that involves PKC-dependent downregulation of the number of binding sites may be an important pathway to regulate the thrombogenicity of lipoproteins and their effects on platelet reactivity. |
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