S-Nitrosylation and subsequent oxidation of protein thiol in the prodomain of MMP-9 can thus lead to enzyme activation, and homolo-

fonic acid modification, as we were able to observe these derivatives in our peptide analysis of proMMP-9. To confirm the pathophysiological relevance of these findings, we performed the same ischemia and reperfusion experiments after nNOS inhibition with 3br7NI, which is known to be neuroprotective and decrease stroke size. Under these conditions with NO formation blocked, the sulfinic and sulfonic acid oxidation products of activated MMP-9 were not observed in our MALDI-TOF analysis (Fig. 4C). One caveat with these findings is that nNOS deletion or NOS inhibition diminishes stroke damage, and hence one could argue that other stroke-related processes responsible for MMP activation would be reduced. Nonetheless, taken together with the data demonstrating S-nitrosylation of MMPs and our finding that MMPs activated in this manner cause neuronal apoptosis in vitro, it is likely that NO activation of MMPs participates in neuronal injury in vivo. S-Nitrosylation and subsequent oxidation of protein thiol in the prodomain of MMP-9 can thus lead to enzyme activation, and homologous MMPs may be activated in a similar manner. This series of reactions confers responsiveness of the extracellular matrix to nitrosative and oxidative stress. Such insults are relevant to a number of pathophysiological conditions, including cerebral ischemia and neurodegenerative diseases. Extracellular proteolytic cascades triggered by MMPs can disrupt the extracellular matrix, contribute to cell detachment, and lead to a form of apoptotic cell death known as anoikis, similar to that observed in our neuronal cultures (27). The elucidation of an extracellular signaling pathway to neuronal apoptosis involving NO-activated MMPs may contribute to the development of new therapies for stroke and other disorders associated with nitrosative and oxidative stress.