Release of soluble CD40 ligand after platelet activation: studies on the solubilization phase

sCD40L is released from platelets as a soluble, proteolyzed form of CD40 ligand (CD40L; CD154) which is exposed on the surface after platelet activation. Ethylenediaminetetraacetate (EDTA), the CD40-blocking antibody G28-5, and GPIIb-IIIa antagonists are known to inhibit the solubilization when added prior to activation. It is assumed that the surface expression of CD40L is a result of a separate fast process and that the solubilization is secondary to this. The release of sCD40L in this solubilization phase has been studied; that is, inhibitory substances were added to platelet-rich plasma (PRP) 10 min after addition of the activation agonist (100 microM SFLLRN), at which time the secretion phase was over as tested with beta-thromboglobulin (beta-TG). G28-5 (10 microg/ml) and EDTA (5 mM) inhibited the solubilization phase which did not require the presence of an activation agonist. Prostaglandin E1 (PGE1; 20 microM) and cytochalasin D (C8273; 60 and 100 microM), which exert their effects intracellularly, inhibited the solubilization even in the presence of abciximab (ReoPro; 40 microg/ml). The intracellular effect was not related to CD40L-containing microparticles as demonstrated by ultracentrifugation. Intracellular alkalinization by preincubation of PRP with 20 mM NH4Cl for 60 min resulted in a small but reproducible reduction in the amount of extracellular sCD40L. SFLLRN induced solubilization of CD40L also from the platelets of a Glanzmann's thrombasthenia patient lacking GPIIb-IIIa, albeit at a lower rate than from normal platelets, and fibrinogen enhanced the solubilization from washed normal platelets. The data show that the solubilization of CD40L not only depends on reactions on the platelet surface but also that intracellular structures are engaged even during the solubilization phase.