Colocalization of thrombin, PAI-1, and vitronectin in the atherosclerotic vessel wall: A potential regulatory mechanism of thrombin activity by PAI-1/vitronectin complexes.
The serine protease thrombin is a mitogen for vascular smooth muscle cells. To that end, thrombin cleaves the surface-exposed, protease-activated receptor type 1 (PAR-1), resulting in signal transduction and ultimately, proliferation of these cells. Regulation of thrombin activity in the human atherosclerotic vessel wall has not been studied in great detail, conceivably because the traditional plasma thrombin inhibitor, anti-thrombin III, is not encountered at this location. By using immunofluorescence confocal microscopy, we demonstrate that the antigens of thrombin, plasminogen activator inhibitor 1 (PAI-1), and vitronectin (Vn) colocalize in human neointimal atherosclerotic arterial tissue. Furthermore, it is shown by in situ reverse zymography that these specimens harbor the active form of PAI-1, which is the only configuration of PAI-1 capable of complexing with Vn and inhibiting serine proteases, eg, thrombin. Two different criteria were used to establish that neointimal atherosclerotic material contains active alpha-thrombin, namely, its ability to bind to the thrombin inhibitor hirudin and to convert the thrombin-specific chromogenic substrate S2238. The latter activity could be fully prevented by preincubation with the thrombin-specific inhibitor, phenyl-prolyl-arginyl-chloromethyl ketone. The thrombin concentration measured by conversion of the chromogenic substrate was 7 to 12 nmol/L in the vascular specimens studied. This concentration range suffices to activate the PAR-1 receptor on vascular smooth muscle cells and to cause neointimal proliferation. It is concluded that the human atherosclerotic arterial vessel wall provides conditions that favor a regulatory mechanism of thrombin activity by PAI-1/Vn complexes.
Stoop
A A
AA
Department of Biochemistry, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands. a.a.stoop@amc.uva.nl
Lupu
F
F
Pannekoek
H
H
D001011:Aorta / Q000737:chemistry; D001158:Arteries / Q000737:chemistry*; D001161:Arteriosclerosis / Q000378:metabolism*; D002339:Carotid Arteries / Q000737:chemistry; D003331:Coronary Vessels / Q000737:chemistry; D005455:Fluorescent Antibody Technique; D006801:Humans; D018613:Microscopy, Confocal; D017395:Plasminogen Activator Inhibitor 1 / Q000032:analysis*; D012501:Saphenous Vein / Q000737:chemistry; D013917:Thrombin / Q000032:analysis*; D019096:Vitronectin / Q000032:analysis*
D017395:Plasminogen Activator Inhibitor 1; D019096:Vitronectin; D013917:Thrombin