We tested the hypothesis that CD40 ligand(CD40L)induces a prothrombotic state by enhancing oxidative stress. Patients with hypercholesterolemia show an ongoing prothrombotic state, but the underlying mechanism is still unclear. Circulating levels of the soluble form of CD40L(sCD40L), prothrombin fragment(F1+2, a marker of thrombin generation), and 8-hydroxy-2-deoxyguanosine(8-OHdG, a marker of oxidative stress)were measured in 40 patients with hypercholesterolemia and in 20 age-and gender-matched healthy subjects. Patients with hypercholesterolemia showed significantly higher levels of sCD40L(p< 0.005), 8-OHdG(p< 0.005), and prothrombin F1+2(p< 0.005), as compared with control subjects. Soluble CD40L significantly correlated with 8-OHdG(r=0.85, p < 0.0001)and prothrombin F1+2(r=0.83, p< 0.0001); a significant correlation between 8-OHdG and prothrombin F1+2 was also observed(r=0.64, p< 0.0001). An in vitro study demonstrated that CD40L-stimulated monocytes from patients with hypercholesterolemia expressed more tissue factor(TF)and prothrombin F1+2 than monocytes from controls; co-incubation of monocyteswith either an inhibitor of NADPH oxidase or an inhibitor of phosphatidylinositol-3-kinase signifi cantly reduced CD40L-mediated clotting activation. A marked inhibition of CD40L-mediated clotting activation was also observed in two male patients with hereditary deficiency of gp91 phox, the central core of nicotinamide adenine dinucleotide phosphate(NADPH)oxidase. Finally, we demonstrated that CD40L-mediated clotting activation was significantly inhibited by vitamin C, a known antioxidant. This study indicates that in patients with hypercholesterolemia, CD40L over-expresses TF and increases the thrombin generation rate by an oxidative stress-mediated mechanism that requires the activation of NADPH oxidase.
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