NADPH oxidase mediates tissue factor-dependent surface procoagulant activity by thrombin in human vascular smooth muscle cells.
BACKGROUND
Tissue factor (TF) initiates the extrinsic coagulation cascade leading to thrombin formation. Thrombin induces TF mRNA in vascular smooth muscle cells (VSMCs), thereby contributing to the prolonged procoagulant activity and enhanced thrombogenicity at sites of vascular injury. However, the signaling mechanisms mediating this thrombogenic cycle are unclear. Characteristically, vascular injury promotes the generation of reactive oxygen species (ROS). Because ROS exert signaling functions, we investigated whether the NADPH oxidase, an important source of ROS in VSMCs, contributes to upregulation of TF by thrombin.


RESULTS
Thrombin not only stimulated TF mRNA expression, but also TF-dependent surface procoagulant activity in cultured human VSMCs. This response was attenuated by antioxidants; the flavin inhibitor diphenylene-iodonium, Clostridium difficile toxin B, which inhibits Rho GTPases, p22phox antisense oligonucleotides, or the dominant-negative RacT17N mutant. Inhibitors of p38 MAP kinase and phosphatidylinositol (PI) 3-kinase also prevented thrombin-stimulated TF mRNA expression. Furthermore, thrombin stimulated the phosphorylation of the PI 3-kinase target protein kinase B/Akt in a redox-sensitive and NADPH oxidase-dependent manner.


CONCLUSIONS
These findings indicate that the NADPH oxidase is essentially involved in the redox-sensitive induction of TF mRNA expression and surface procoagulant activity by thrombin. This response is mediated by NADPH oxidase-dependent activation of p38 MAP kinase and the PI 3-kinase/protein kinase B/Akt pathway. Given that active TF promotes thrombin formation, the NADPH oxidase may play a crucial role in perpetuating the thrombogenic cycle in the injured vessel wall.
Herkert
Olaf
O
Institut für Kardiovaskuläre Physiologie, Klinikum der Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany.
Diebold
Isabel
I
Brandes
Ralf P
RP
Hess
John
J
Busse
Rudi
R
Görlach
Agnes
A
D001426:Bacterial Proteins; D001427:Bacterial Toxins / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D003029:Coagulants / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D026901:Membrane Transport Proteins; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D009252:NADPH Dehydrogenase / Q000235:genetics; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000502:physiology*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D010750:Phosphoproteins / Q000235:genetics; D047428:Protein Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D012333:RNA, Messenger / Q000096:biosynthesis; D017382:Reactive Oxygen Species / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology*; D013925:Thromboplastin / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation / Q000187:drug effects; D048051:p38 Mitogen-Activated Protein Kinases; D020744:rac GTP-Binding Proteins / Q000235:genetics; D020741:rho GTP-Binding Proteins / Q000037:antagonists & inhibitors
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D003029:Coagulants; D004791:Enzyme Inhibitors; D026901:Membrane Transport Proteins; D016376:Oligonucleotides, Antisense; D010750:Phosphoproteins; D047428:Protein Kinase Inhibitors; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; C031815:toxB protein, Clostridium difficile; D013925:Thromboplastin; D019255:NADPH Oxidases; C086986:CYBA protein, human; D009252:NADPH Dehydrogenase; C494918:AKT1 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D013917:Thrombin; D020744:rac GTP-Binding Proteins; D020741:rho GTP-Binding Proteins