Synergistic Effect of Thrombin and CD40 Ligand on Endothelial Matrix Metalloproteinase-10 Expression and Microparticle Generation In Vitro and In Vivo

Abstract

Thrombin induces CD40 ligand (CD40L) and matrix metalloproteinases (MMPs) under inflammatory/prothrombotic conditions. Thrombin and CD40L could modulate endothelial MMP-10 expression in vitro and in vivo. Human endothelial cells were stimulated with thrombin (0.1-10 U/mL), CD40L (0.25-1 μg/mL), or their combination (thrombin/CD40L) to assess MMP-10 expression and microparticle generation. Thrombin/CD40L elicited higher MMP-10 mRNA (5-fold; P<0.001) and protein levels (4.5-fold; P<0.001) than either stimulus alone. This effect was mimicked by a protease-activated receptor-1 agonist and antagonized by hirudin, a-protease-activated receptor-1, α-CD40L, and α-CD40 antibodies. The synergistic effect was dependent on p38 mitogen-activated protein kinase and c-Jun N-terminal kinase-1 pathways. Thrombin also upregulated the expression of CD40 in endothelial cell surface increasing its availability, thereby favoring its synergistic effects with CD40L. In mice, thrombin/CD40L further increased the aortic MMP-10 expression. Septic patients with systemic inflammation and enhanced thrombin generation (n=60) exhibited increased MMP-10 and soluble CD40L levels associated with adverse clinical outcome. Endothelial and systemic activation by thrombin/CD40L and lipopolysaccharide also increased microparticles harboring MMP-10 and CD40L. Thrombin/CD40L elicited a strong synergistic effect on endothelial MMP-10 expression and microparticles containing MMP-10 in vitro and in vivo, which may represent a new link between inflammation/thrombosis with prognostic implications.