Platelet CD40 ligand (CD40L) - subcellular localization, regulation of expression, and inhibition by clopidogrel

This study compares the subcellular localization and the regulation of expr ession of the platelet activation markers CD62P and CD63 with CD30 ligand ( CD40L) on the surface of washed human platelets, CD40L was expressed upon s timulation with a wide range of platelet activators, However, quantitative flow cytometry demonstrated that, as compared with CD62P and CD63, CD40L ex pression was low. Upon stimulation with thrombin receptor-activating peptid e (TRAP-6), all activation markers were expressed, In contrast, upon stimul ation with low concentrations of collagen (1-3 mug/ml), CD40L, but not the granule proteins (CD62P, CD63), mere expressed. Using immunofluorescence mi croscopy, a cytoplasmic staining was observed for CD40L, and cytoplasmic lo calization of CD40L was verified by Western blotting of subcellular platele t fractions, The staining of CD40L was different from that of filamentous a ctin and only little association of CD40L with platelet cytoskeleton was fo und, Surface expression of CD40L was dependent on internal Ca2+ stores and protein kinase C, white the mitogen-activated protein kinases (ERK, p38) or tyrosine kinases were not involved. ADP (30 muM)-induced CD40L expression was not inhibited by aspirin. In contrast, clopidogrel treatment completely abolished ADP-induced expression of CD40L. Finally, the expression level o f CD40L was shown to be upregulated by phorbol myristate acetate (PMA) in t he promegakaryocytic cell line MEG-01.