Thrombin promotes activation of matrix metalloproteinase-2 produced by cultured vascular smooth muscle cells.
Thrombin generated at sites of vascular injury not only participates in the coagulation cascade but can signal other events related to development and complication of atherosclerotic plaques. We investigated here a novel non-thrombotic action of thrombin: the possibility that this protease influences the expression or activation of matrix metalloproteinases (MMPs) produced by vascular smooth muscle cells (SMCs). Matrix-degrading proteinases likely contribute to several aspects of vascular lesion development. Vascular SMCs constitutively elaborate the zymogen form of gelatinase A (MMP-2), found in cell supernatants complexed with its inhibitor, the tissue inhibitor of metalloproteinases (TIMP)-2. When activated, MMP-2 digests collagens and elastin and may thus promote cell migration and vascular remodeling. Analysis of culture supernatants harvested from either human or rabbit vascular SMCs by gelatin zymography revealed that compared with supernatants of unstimulated SMCs, media conditioned by thrombin-stimulated cells contained increased amounts of proteolytically processed MMP-2, suggesting activation of this MMP. Further experiments tested whether thrombin directly activates MMP-2. In cell-free experiments, when added to medium harvested from unstimulated SMCs, alpha-thrombin increased in a dose- and time-dependent manner the amount of proteolytically processed MMP-2, as shown by zymography and by Western blotting with specific antibodies. Thrombin cleaved pro-MMP-2 within 4 hours, even when the gelatinase was bound with its inhibitor, TIMP-2. Thrombin treatment rendered culture media of unstimulated SMCs able to degrade collagen type IV, consistent with generation of active MMP-2. Addition of inhibitors of either thrombin or MMPs decreased this type IV collagenolytic activity, but thrombin in the absence of SMC-conditioned medium containing pro-MMP-2 exhibited only minimal collagenolysis. Our results suggest that at sites of vascular injury, thrombin may activate locally produced MMP-2 and thereby facilitate cell migration and proliferation. In the case of complicated atherosclerotic plaques, episodes of intraplaque hemorrhage or plaque disruption with thrombosis may promote plaque instability by increasing local matrix-degrading activity.
Galis
Z S
ZS
Emory University School of Medicine, Cardiology Division, Atlanta, GA 30322, USA. zgalis@emory.edu
Kranzhöfer
R
R
Fenton
J W
JW
Libby
P
P
D000818:Animals; D002478:Cells, Cultured; D004789:Enzyme Activation / Q000187:drug effects; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D006801:Humans; D008666:Metalloendopeptidases / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000201:enzymology*; D011817:Rabbits; D013917:Thrombin / Q000494:pharmacology*
D013917:Thrombin; D008666:Metalloendopeptidases