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LDL Binding Sites on Platelets Differ From the "Classical" Receptor of Nucleated Cells Javier Pedreno, Conxita de Castellarnau, Cristina Cullare, Jose Sanchez, Juan Gomez-Gerique, Jordi Ordonez-Llanos, and Francesc Gonzalez-Sastre Washed human platelets bound radioiodinated low density lipoprotein (125I-LDL) to a class of saturable binding sites; they numbered 1,348±126 per platelet, and the dissociation constant (KD) was 50.7±9 nM. I25 I-LDL binding to platelets was reversible, and apparent equilibrium was attained within 25 minutes at 22°C and was characterized by forward and reverse rate constants of 1.47xlO4Xsec"'xM"1 and 8xlO~4xsec~' xM"1, respectively. Such binding was largely unaltered by temperature, divalent ions, and chelating agents. In addition, neither did receptor regulation (up or down) occur when platelets were loaded with cholesterol, nor did prostaglandin E, (PGE,) increase the binding of 12SI-LDL to platelets. On the other hand, the specificity of LDL binding was not typical of the LDL receptor of nucleated cells. Lipoproteins competed for the occupancy of LDL binding sites in platelets with the following order of potency: very low density >> intermediate density > high density subfraction 2. High density lipoprotein subfraction 3, heparin, and PGE, had no effect on this binding. 125I-LDL binding to lymphocytes and fibroblasts and proteolytic degradation of 125I-LDL by lymphocytes was inhibited by the monoclonal antibody IgG-C7 directed against the LDL receptor to 88%, 85%, and 85% (p 1.210 g/ml) were obtained from pooled sera of normolipidemic blood donors and isolated by sequential ultracentrifugation20 in a Centrikon T 2060 ultracentrifuge (Kontron Instruments, Milano, Italy). Lipoproteins were washed during flotation with a potassium bromide solution of the respective lipoprotein density. The lipoproteins were exhaustively dialyzed against phosphate-buffered saline (PBS), pH 7.4, containing 10 mM EDTA. The concentration of lipoproteins was expressed in terms of protein content, as determined by the method of Bradford,21 with BSA as the standard. The apoprotein (apo) composition of each lipoprotein class was routinely assessed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. The specific mobility of each lipoprotein was confirmed by acrylamideagarose gel electrophoresis. lodination of Lipoproteins Freshly isolated LDL from a pool of normolipidemic blood donor serum was labeled with 125I by the IodoGen method.22 Unbound iodine was removed by chromatography with a Sephadex PD-10 column previously equilibrated with PBS buffer, pH 7.4, containing 10 mM EDTA. The protein fractions were pooled and dialyzed against the same buffer for 24 hours and used immedi-
ately. Final specific radioactivity was 2±0.2 Bq/ng protein. In all cases, labeling efficiencies >80% were achieved, and
LDL Binding Sites on Platelets Differ From the "Classical" Receptor of Nucleated Cells Javier Pedreno, Conxita de Castellarnau, Cristina Cullare, Jose Sanchez, Juan Gomez-Gerique, Jordi Ordonez-Llanos, and Francesc Gonzalez-Sastre Washed human platelets bound radioiodinated low density lipoprotein (125I-LDL) to a class of saturable binding sites; they numbered 1,348±126 per platelet, and the dissociation constant (KD) was 50.7±9 nM. I25 I-LDL binding to platelets was reversible, and apparent equilibrium was attained within 25 minutes at 22°C and was characterized by forward and reverse rate constants of 1.47xlO4Xsec"'xM"1 and 8xlO~4xsec~' xM"1, respectively. Such binding was largely unaltered by temperature, divalent ions, and chelating agents. In addition, neither did receptor regulation (up or down) occur when platelets were loaded with cholesterol, nor did prostaglandin E, (PGE,) increase the binding of 12SI-LDL to platelets. On the other hand, the specificity of LDL binding was not typical of the LDL receptor of nucleated cells. Lipoproteins competed for the occupancy of LDL binding sites in platelets with the following order of potency: very low density >> intermediate density > high density subfraction 2. High density lipoprotein subfraction 3, heparin, and PGE, had no effect on this binding. 125I-LDL binding to lymphocytes and fibroblasts and proteolytic degradation of 125I-LDL by lymphocytes was inhibited by the monoclonal antibody IgG-C7 directed against the LDL receptor to 88%, 85%, and 85% (p 1.210 g/ml) were obtained from pooled sera of normolipidemic blood donors and isolated by sequential ultracentrifugation20 in a Centrikon T 2060 ultracentrifuge (Kontron Instruments, Milano, Italy). Lipoproteins were washed during flotation with a potassium bromide solution of the respective lipoprotein density. The lipoproteins were exhaustively dialyzed against phosphate-buffered saline (PBS), pH 7.4, containing 10 mM EDTA. The concentration of lipoproteins was expressed in terms of protein content, as determined by the method of Bradford,21 with BSA as the standard. The apoprotein (apo) composition of each lipoprotein class was routinely assessed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. The specific mobility of each lipoprotein was confirmed by acrylamideagarose gel electrophoresis. lodination of Lipoproteins Freshly isolated LDL from a pool of normolipidemic blood donor serum was labeled with 125I by the IodoGen method.22 Unbound iodine was removed by chromatography with a Sephadex PD-10 column previously equilibrated with PBS buffer, pH 7.4, containing 10 mM EDTA. The protein fractions were pooled and dialyzed against the same buffer for 24 hours and used immedi-
ately. Final specific radioactivity was 2±0.2 Bq/ng protein. In all cases, labeling efficiencies >80% were achieved, and
