This study compares the subcellular localization and the regulation of expression of the platelet activation markers CD62P and CD63 with CD40 ligand (CD40L) on the surface of washed human platelets. CD40L was expressed upon stimulation with a wide range of platelet activators. However, quantitative flow cytometry demonstrated that, as compared with CD62P and CD63, CD40L expression was low. Upon stimulation with thrombin receptor-activating peptide (TRAP-6), all activation markers were expressed. In contrast, upon stimulation with low concentrations of collagen (1-3 microg/ml), CD40L, but not the granule proteins (CD62P, CD63), were expressed. Using immunofluorescence microscopy, a cytoplasmic staining was observed for CD40L, and cytoplasmic localization of CD40L was verified by Western blotting of subcellular platelet fractions. The staining of CD40L was different from that of filamentous actin and only little association of CD40L with platelet cytoskeleton was found. Surface expression of CD40L was dependent on internal Ca2+ stores and protein kinase C, while the mitogen-activated protein kinases (ERK, p38) or tyrosine kinases were not involved. ADP (30 microM)-induced CD40L expression was not inhibited by aspirin. In contrast, clopidogrel treatment completely abolished ADP-induced expression of CD40L. Finally, the expression level of CD40L was shown to be upregulated by phorbol myristate acetate (PMA) in the promegakaryocytic cell line MEG-01.