Abstract 620: Platelet CD40L: a Powerful Leukocyte and Endothelial Cell Activator in Atherosclerosis

Abstract

Here we investigated whether platelet CD40L plays a role in atherosclerosis. Platelets were obtained from donor ApoE−/− or CD40L−/− ApoE−/− mice, activated with thrombin, neutralized by hirudin and injected intravenously into n=30 ApoE−/− recipients (3E10 7 platelets) every 5 days. Infusion of activated CD40L−/− platelets in ApoE−/− mice from age 5–17wks caused a decrease in atherosclerosis (predominantly early lesions) in the aortic arch and its major branch points (ApoE−/− platelets 2.01E10 5 ± 3.74E10 4 μm 2 vs CD40L−/−ApoE−/− platelets 1.01E10 5 ± 1.57E10 4 μm 2 ; p<0.05) to levels that were comparable to the sham (NaCl) group (1.17E10 5 ± 1.61E10 4 μm 2 ). To induce advanced atherosclerosis, a silastic non-constrictive collar was placed around both carotid arteries in diet fed mice, and activated platelets were injected every 5 days during 5 wks. Infusion of activated CD40L−/−ApoE−/− platelets caused a 35.4% decrease in plaque volume (ApoE−/− platelets 2.08E10 8 ± 2.55E10 7 μm 3 vs CD40L−/−ApoE−/− platelets 1.36E10 8 ± 3.45E10 7 μm 3 , Sham 1.17E10 8 ± 2.71E10 7 μm 3 p<0.05), indicating an important role for activated platelet CD40L in both early and advanced atherosclerosis. Laminar flow assays showed that activated CD40L−/−ApoE−/− platelets lose their capacity of binding CD40L+/+ApoE−/− leukocytes, especially monocytes, with 30.1% compared to activated ApoE−/− platelets. Moreover, FACS analysis (CD45, CD11b,CD41) showed that activated ApoE−/−platelets bind 24.8% less to CD40−/−ApoE−/− leukocytes, and activated CD40L−/−ApoE−/−platelets bind 40.0% less to CD40−/−ApoE−/− leukocytes, thereby pointing out leukocyte CD40 as the receptor of platelet CD40L. Intravital microscopy in carotid arteries of mice, injected with activated CD40L−/− platelets, showed a 3-fold decrease in leukocyte adhesion. Even more, deficiency of CD40L on platelets causes a 58.4% decrease in thrombus formation compared to wild type. We demonstrate that CD40L on activated platelets plays an important role in atherosclerosis by promoting platelet aggregation, the formation of platelet-leukocyte aggregates, and leukocyte-endothelial interactions. This shows that platelet CD40L has a bridging function between the leukocyte and the endothelium.