title: Soluble CD40 ligand induces beta3 integrin tyrosine phosphorylation and triggers platelet activation by outside-in signaling.
abstract: We earlier reported that the soluble form of the CD40 ligand (sCD40L), is involved in thrombosis by stabilizing platelet thrombi. In this article, we have determined the mechanism by which this protein affects platelet biology. Addition of sCD40L to washed platelets was found to activate the receptor function of alphaIIbbeta3 as measured by the induction of fibrinogen binding and the formation of platelet microparticles. Mutation in the KGD sequence (D117E) of sCD40L, the alphaIIbbeta3-binding domain in the N terminus of the protein resulted in a loss of the platelet-stimulatory activity of this protein. Integrilin, a alphaIIbbeta3 antagonist, but not an antibody to CD40 that blocked the ligand-binding activity, inhibited these platelet-stimulatory events. CD40-/- platelets bound fibrinogen and formed microparticles similar to WT platelets, again indicating that CD40 is not involved in sCD40L-induced platelet activation. Exposure of platelets to sCD40L, but not D117E-sCD40L-coated surfaces, induced platelet thrombi formation under arterial shear rate. sCD40L-induced platelet stimulation resulted in the phosphorylation of tyrosine-759 in the cytoplasmic domain of beta3. Platelets from the diYF mouse strain, expressing beta3 in which both cytoplasmic tyrosines are mutated to phenylalanine, were defective in sCD40L-induced platelet stimulation. These data indicate that sCD40L is a primary platelet agonist and that platelet stimulation is induced by the binding of the KGD domain of sCD40L to alphaIIbbeta3, triggering outside-in signaling by tyrosine phosphorylation of beta3.
authors.affiliation: Department of Cardiovascular Biology, Millennium Pharmaceuticals, Inc., 256 East Grand Avenue, South San Francisco, CA 94080, USA.
authors.forename: K S Srinivasa
authors.initials: KS
authors.lastname: Prasad
authors.forename: Patrick
authors.initials: P
authors.lastname: Andre
authors.forename: Ming
authors.initials: M
authors.lastname: He
authors.forename: Ming
authors.initials: M
authors.lastname: Bao
authors.forename: Jeanne
authors.initials: J
authors.lastname: Manganello
authors.forename: David R
authors.initials: DR
authors.lastname: Phillips
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000235:genetics; D019013:CD40 Antigens / Q000235:genetics / Q000378:metabolism; D023201:CD40 Ligand / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D005260:Female; D006801:Humans; D066298:In Vitro Techniques; D039661:Integrin beta3 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D015539:Platelet Activation / Q000187:drug effects* / Q000502:physiology; D040201:Platelet Membrane Glycoprotein IIb / Q000378:metabolism; D015398:Signal Transduction; D012995:Solubility; D014443:Tyrosine / Q000737:chemistry
chemical_list: D019013:CD40 Antigens; D039661:Integrin beta3; D040201:Platelet Membrane Glycoprotein IIb; D023201:CD40 Ligand; D014443:Tyrosine