Injured neurons act as a fibrin-like plasminogen-activating cofactor. (A) Neuronal cultures were subjected to OGD for 72 hours. After this, tPA, plasminogen, and S2251 (a plasmin-sensitive substrate) were added to the cultures and S2251 hydrolysis was monitored over time. The reaction velocities from 4 independent experiments were collated. Data represent average ± SEM. *P < .05 by Student t test. Note, no S2251 hydrolysis occurred when only exogenous tPA was bath-applied, suggesting a lack of endogenous plasminogen expression in neuronal culture. Also note, a limited amount of S2251 hydrolysis occurred when plasminogen was bath-applied alone, suggesting a low level of endogenous tPA expression in neuronal culture (which is significantly decreased by OGD injury). The ability of uninjured cultures to drive high rates of plasminogen activation upon bath application of both exogenous tPA and plasminogen reflects the existence of cofactor molecules (eg, annexin II³⁷ ) on the surface of viable neurons and the unavoidable presence of a low number of nonviable cells. As OGD dramatically increased the number of nonviable cells, bath application of both tPA and plasminogen to injured cultures achieved even higher rates of plasmin generation. (B) Using the same concentrations of exogenous tPA, plasminogen, and S2251, the rates of plasmin generation in the presence of a preformed fibrin clot were determined. Inclusion of 1 mM TXA in the reaction inhibited the cofactor activity of fibrin and thereby significantly attenuated the rate of plasminogen activation. The reaction velocities from 3 independent experiments were collated. Data represent average ± SEM. *P < .05 by Student t test.