Objective: Matrix metalloproteinases (MMPs) cause extracellular matrix degradation and may be involved in the rupture of atherosclerotic plaques by degrading fibrous cap, resulting in the intravascular thrombus formation. Here we examined whether local overexpression of MMP-9 alters the characteristics of arteriosclerotic vascular lesions and promotes thrombosis after balloon injury in porcine coronary arteries in vivo. Methods and results: Balloon angioplasty was performed in the left coronary arteries followed by injection of adenovirus vector solution encoding either MMP-9 or β-galactosidase (β-gal) gene into the injured coronary arteries. Three weeks after the gene transfer, histological examination demonstrated that macroscopic intravascular thrombus formation was noted at the MMP-9-transfected site but not at the β-gal-transfected site. Microscopic intramural thrombus area was significantly larger at the MMP-9-transfected site as compared to the β-gal-transfected site. Co-transfection of tissue inhibitor of metalloproteinase-1 (TIMP-1) with MMP-9 prevented the intravascular thrombus formation in vivo. Western blot analysis revealed the reduced expression of intact tissue factor pathway inhibitor-1 and the increased tissue factor (TF) expression at the MMP-9-transfected sites. Conclusion: These results provide the first in vivo evidence that overexpression of MMP-9 promotes intravascular thrombus formation after balloon injury due in part to the activation of TF-mediated coagulation cascade.