title: Protein kinase C-dependent desensitization of HDL3-activated phospholipase C in human platelets.
abstract: In isolated human platelets, exposure of subfraction 3 high-density lipoprotein (HDL3) binding sites to high concentrations of HDL3 (1 mg/mL) causes rapid desensitization of HDL3 (50 micrograms/mL)-stimulated breakdown of phosphatidylcholine, as shown in approximately a 70% depression of the maximal 1,2-diacylglycerol release activity by phospholipase C. This desensitization is HDL3 dose dependent (IC50, 150 +/- 20 micrograms/mL, n = 6) and time dependent (t1/2, < 30 seconds). It requires the binding of HDL3, as pretreatment of HDL3 by tetranitromethane does not cause the desensitization of HDL3-induced phospholipase C activity. Permeabilization of human platelets with 10 micrograms/mL digitonin, used to permit access of charged inhibitors to the cytosol, does not interfere with the pattern of HDL3 (1 mg/mL)-induced desensitization of HDL3 (50 micrograms/mL)-stimulated phospholipase C. Inhibitors of protein kinase C (100 mumol/L H-7 and 10 mumol/L staurosporine) markedly inhibit desensitization of HDL3-induced phospholipase C activity, whereas cAMP-dependent protein kinase inhibitor (1 mumol/L), heparin (100 nmol/L), or concanavalin A (0.25 mg/mL) were ineffective. HDL3-induced desensitization is accompanied at least by the phosphorylation of the 94- and 110-kD proteins. Inhibition of HDL3-induced desensitization by 100 mumol/L H-7 or 10 mumol/L staurosporine is characterized by a marked reduction of the phosphorylation state of these proteins in permeabilized platelets. Whereas protein kinase C inhibitors fully inhibited the phosphorylation of the 94- and 110-kD proteins, inhibitors of protein kinase A were less effective. These data establish that phosphorylation by protein kinase C represent a step in the desensitization of HDL3 binding sites in human platelets.
authors.affiliation: Serlia, Institut Pasteur, Lille, France.
authors.forename: H
authors.initials: H
authors.lastname: Nazih
authors.forename: F
authors.initials: F
authors.lastname: Nazih-Sanderson
authors.forename: V
authors.initials: V
authors.lastname: Magret
authors.forename: B
authors.initials: B
authors.lastname: Caron
authors.forename: J
authors.initials: J
authors.lastname: Goudemand
authors.forename: J C
authors.initials: JC
authors.lastname: Fruchart
authors.forename: C
authors.initials: C
authors.lastname: Delbart
mesh_terms: D001665:Binding Sites; D001792:Blood Platelets / Q000201:enzymology*; D015536:Down-Regulation / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D008075:Lipoproteins, HDL / Q000494:pharmacology*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000494:pharmacology / Q000502:physiology*; D015398:Signal Transduction; D010738:Type C Phospholipases / Q000097:blood*
chemical_list: D008075:Lipoproteins, HDL; D011493:Protein Kinase C; D010738:Type C Phospholipases