To investigate the roles and signaling pathways of CD40L and CD40 in platelet–platelet interactions and thrombus formation under conditions relevant for atherothrombosis.

Platelets from mice prone to atherosclerosis lacking CD40L (Cd40lg^−/−Apoe^−/−) showed diminished α_IIbβ₃ activation and α-granule secretion in response to glycoprotein VI stimulation, whereas these responses of CD40-deficient platelets (Cd40^−/−Apoe^−/−) were not decreased. Using blood from Cd40lg^−/−Apoe^−/− and Cd40^−/−Apoe^−/− mice, the glycoprotein VI-dependent formation of dense thrombi was impaired on atherosclerotic plaque material or on collagen, in comparison with Apoe^−/− blood. In all genotypes, addition of CD40L to the blood enhanced the growth of dense thrombi on plaques and collagen. Similarly, CD40L enhanced glycoprotein VI–induced platelet aggregation, even with platelets deficient in CD40. This potentiation was antagonized in Pik3cb^R/R platelets or by inhibiting phosphatidylinositol 3-kinase β (PI3Kβ). Addition of CD40L also enhanced collagen-induced Akt phosphorylation, which was again antagonized by absence or inhibition of PI3Kβ. Finally, platelets from Chuk1^A/AApoe^−/− mice deficient in IκB kinase α (IKKα), implicated in CD40 signaling to nuclear factor (NF) κB, showed unchanged responses to CD40L in aggregation or thrombus formation.

Under atherogenic conditions, CD40L enhances collagen-induced platelet–platelet interactions by supporting integrin α_IIbβ₃ activation, secretion and thrombus growth via PI3Kβ, but not via CD40 and IKKα/NFκB. This role of CD40L exceeds the no more than modest role of CD40 in thrombus formation.