Journal
JOURNAL OF THORACIC AND CARDIOVASCULAR SURGERY
Volume 131, Issue 1, Pages 21-27Publisher
MOSBY-ELSEVIER
DOI: 10.1016/j.jtcvs.2005.08.050
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Objective: Thrombin is generated in significant quantities during cardiopulmonary bypass and mediates adverse events, such as platelet aggregation and proinflammatory responses, through activation of the high-affinity thrombin receptor protease-activated receptor 1, which is expressed on platelets and endothelium. Thus antagonism of protease-activated receptor 1 might have broad therapeutic significance. Aprotinin, used clinically to reduce transfusion requirements and the inflammatory response to bypass, has been shown to inhibit protease-activated receptor 1 on platelets in vitro and in vivo. Here we have examined whether aprotinin inhibits endothelial protease-activated receptor 1 activation and resulting proinflammatory responses induced by thrombin. Methods: Protease-activated receptor 1 expression and function were examined ill cultured human umbilical vein endothelial cells after treatment with a-thrombin at 0.02 to 0.15 U/mL in the presence or absence of aprotinin (200-1600 kallikrein inhibitory units/mL). Protease-activated receptor 1 activation was assessed by using an antibody, SPAN-12, which detects only the unactivated receptor, and thrombin-mediated calcium fluxes. Other thrombin-dependent inflammatory pathways investigated were phosphorylation of the p42/44 mitogen-activated protein kinase, upregulation of the early growth response 1 transcription factor, and production of the proinflammatory cytokine interleukin 6. Results: Pretreatment of cultured endothelial cells with aprotinin significantly spared protease-activated receptor 1 receptor cleavage (P < .0001) and abrogated calcium fluxes caused by thrombin. Aprotinin inhibited intracellular signaling through p42/44 mitogen-activated protein kinase (P < .05) and early growth response 1 transcription factor (P < .05), as well as interleukin 6 secretion caused by thrombin (P < .005). Conclusions: This study demonstrates that endothelial cell activation by thrombin and downstream inflammatory responses can be inhibited by aprotinin in vitro through blockade of protease-activated receptor 1. Our results provide a new molecular 41 basis to help explain the anti-inflammatory properties of aprotinin reported clinically.
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