Increased brain plasmin levels following experimental ischemic stroke in male mice.

Abstract

Many coagulation factor proteases are increased in the brain during ischemic stroke. One of these proteases is plasmin. In this study we established a novel method for direct quantitative measurement of plasmin activity in male mouse brain slices using a sensitive fluorescent substrate in the presence of specific protease inhibitors. In both the ischemic and contralateral hemispheres, plasmin activity increased 3, 6, and 24hr following stroke in comparison to healthy mice (F(3, 72)=39.5, p<0.0001, repeated measures ANOVA) after the induction of permanent middle cerebral artery occlusion (PMCAo). Plasmin activity was higher in the ischemic hemisphere (F(1,36)=9.1, p=0.005) and there was a significant interaction between time and ischemic hemisphere (F(3,36)=4.4, p=0.009). Plasmin activity was correlated with infarct volume (R2 =0.5289, p=0.0009 by Spearman). The specificity of the assay was verified utilizing tissue-type plasminogen activator (tPA)-deficient mice which, as expected, had significantly lower levels of plasmin 24hr following ischemia compared to wild-type mice (ischemic (0.6±0.23 and 1.94±0.5, respectively), p=0.049 and contralateral hemispheres (0.13±0.14 and 0.75±0.10, respectively), p=0.018 by t test). There is a time-dependent increase in plasmin levels and an association of higher levels of plasmin with larger infarct volumes in an experimental stroke model. This suggests caution in the use of recombinant tPA (rtPA) and that plasmin inhibition in the brain may be a therapeutic target in acute ischemic stroke.