Low-density lipoprotein (LDL) binds to a G-protein coupled receptor in human platelets. Evidence that the proaggregatory effect induced by LDL is modulated by down-regulation of binding sites and desensitization of its mediated signaling.
We present evidence of a link between low-density lipoprotein (LDL) receptor binding and activation of a platelet G-coupled protein. LDL stimulation induced cytosolic [Ca2+]i mobilization, increase in inositol 1,4,5-triphosphate (IP3) formation and a rapid cytosol-to-membrane translocation of protein kinase C (PKC) enzymatic activity. Pertussis toxin inhibited all the stimulatory effects, whereas cholera toxin had no effect. Using ligand-binding assays, we demonstrated that exposing platelet LDL receptors to high concentrations of LDL (1.5 g/l) caused a rapid down-regulation and desensitization, as shown by the reduction in the Bmax, intracellular [Ca2+]i mobilization and IP3 formation to 65, 73 and 63%, respectively. The inhibitory effects were reversible and dose and time dependent. Furthermore, VLDL (0.2 g/l) and IDL (0.07 g/l) induced similar desensitization effects. However, HDL3 (up to 1.5 g/l), chylomicrons (up to 0.5 g/l) and cyclohexandione-modified LDL (which does not bind to platelets) had no significant effects. Protein kinase C inhibitors (150 nmol/l staurosporine, 100 micromol/l H-7, and 10 nmol/l bisindolylmaleimide) inhibited desensitization to 71%, on average. Sequestration blocking agents (0.30 g/l, concanavalin A) had no significant effect if phosphorylation was operative. However, there was a complete blockade with the concurrent inhibition of both pathways. In contrast, cAMP-dependent protein kinase inhibitors (PKI, 1 micromol/l) or beta2-adrenergic receptor kinase inhibitors (100 nmol/l, heparin), had no effect. Overall results indicate that LDL binds to a pertussis sensitive G-protein coupled receptor and that high levels of lipoproteins down-regulate the number of receptors and desensitize its mediated response by a mechanism that involves PKC-phosphorylation and sequestration of binding sites. This new regulatory mechanism may have implications for the thrombogenicity in hyperlipidemia and for effects of lipid lowering therapy.
Unitat de Recerca en Lipids i Arteriosclerosi, Facultat de Medicina, Universitat Rovira i Virgili, Sant Llorenç 21 Reus, 43201, Tarragona, Spain. jp@fmcs.urv.es
J
J
Pedreño
E
E
Hurt-Camejo
O
O
Wiklund
L
L
Badimón
L
L
Masana
D001665:Binding Sites; D001792:Blood Platelets / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002772:Cholera Toxin / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D015536:Down-Regulation; D020555:GTP-Binding Protein Regulators / Q000502:physiology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D008074:Lipoproteins / Q000494:pharmacology; D053419:Lipoproteins, IDL; D008077:Lipoproteins, LDL / Q000378:metabolism* / Q000494:pharmacology*; D008079:Lipoproteins, VLDL / Q000494:pharmacology; D037342:Pertussis Toxin; D010974:Platelet Aggregation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011973:Receptors, LDL / Q000502:physiology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D020555:GTP-Binding Protein Regulators; D008074:Lipoproteins; D053419:Lipoproteins, IDL; D008077:Lipoproteins, LDL; D008079:Lipoproteins, VLDL; D011956:Receptors, Cell Surface; D011973:Receptors, LDL; D010566:Virulence Factors, Bordetella; D015544:Inositol 1,4,5-Trisphosphate; D002772:Cholera Toxin; D037342:Pertussis Toxin; D011493:Protein Kinase C; D019204:GTP-Binding Proteins; D002118:Calcium