title: Aprotinin inhibits proinflammatory activation of endothelial cells by thrombin through the protease-activated receptor 1.
abstract: 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 in cultured human umbilical vein endothelial cells after treatment with alpha-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 basis to help explain the anti-inflammatory properties of aprotinin reported clinically.
authors.affiliation: Eric Bywaters Centre, Imperial College London, Faculty of Medicine, Hammersmith Hospital, London, United Kingdom.
authors.forename: Jonathan R S
authors.initials: JR
authors.lastname: Day
authors.forename: Kenneth M
authors.initials: KM
authors.lastname: Taylor
authors.forename: Elaine A
authors.initials: EA
authors.lastname: Lidington
authors.forename: Justin C
authors.initials: JC
authors.lastname: Mason
authors.forename: Dorian O
authors.initials: DO
authors.lastname: Haskard
authors.forename: Anna M
authors.initials: AM
authors.lastname: Randi
authors.forename: R Clive
authors.initials: RC
authors.lastname: Landis
mesh_terms: D007611:Aprotinin / Q000494:pharmacology* / Q000627:therapeutic use; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects* / Q000276:immunology*; D006801:Humans; D007249:Inflammation / Q000517:prevention & control; D013917:Thrombin / Q000502:physiology*; D010975:Platelet Aggregation Inhibitors
chemical_list: D007611:Aprotinin; D013917:Thrombin; D010975:Platelet Aggregation Inhibitors