Ischemic stroke in PAR1 KO mice: Decreased brain plasmin and thrombin activity along with decreased infarct volume

Abstract

Background: Ischemic stroke is a common and debilitating disease with limited treatment options. Protease activated receptor 1 (PAR1) is a fundamental cell signaling mediator in the central nervous system (CNS). It can be activated by many proteases including thrombin and plasmin, with various down-stream effects, following brain ischemia.

Methods: A permanent middle cerebral artery occlusion (PMCAo) model was used in PAR1 KO and WT C57BL/6J male mice. Mice were evaluated for neurological deficits (neurological severity score, NSS), infarct volume (Tetrazolium Chloride, TTC), and for plasmin and thrombin activity in brain slices.

Results: Significantly low levels of plasmin and thrombin activities were found in PAR1 KO compared to WT (1.6±0.4 vs. 3.2±0.6 ng/μl, p<0.05 and 17.2±1.0 vs. 21.2±1.0 mu/ml, p<0.01, respectively) along with a decreased infarct volume (178.9±14.3, 134.4±13.3 mm3, p<0.05).

Conclusions: PAR1 KO mice have smaller infarcts, with lower thrombin and plasmin activity levels. These findings may suggest that modulation of PAR1 is a potential target for future pharmacological treatment of ischemic stroke.

Fig 1. Preserved NSS at 2 and at 24 hours following ischemia and decreased infarct volume in PAR1 KO mice 24 hours following ischemic stroke.

(A) There was no significant difference between NSS scores in WT (black circles) compared to PAR1 KO (gray circles) mice at 2- and 24-hours following ischemia (mixed model analysis, with Sidak’s post-hoc analysis. 2 hours: WT = 25, PAR1 KO = 23, 24 hours: WT = 25, PAR1 KO = 22). (B) Representative images of TCC staining brain slices of WT (left panel) and PAR1 KO (right panel), including slices 3#-8#. (C) Total infarct volume is significantly lower in PAR1 KO mice compared to control (t-test, WT = 6, PAR1 KO = 6). (D) Infarct volume decreases as a function of distance from the ischemic core (slices #5&6). Infarct volume is lower in all PAR KO slices compared to WT mice (two-way ANOVA with Sidak’s post-hoc analysis, WT = 6, PAR1 KO = 6). Results are presented as dot plot individual values and mean±SEM, *p<0.05.

Fig 2. Decreased plasmin activity levels in PAR1 KO mice at 24 hours following ischemia.

(A) Total plasmin activity levels were significantly lower in the ischemic hemisphere of PAR KO mice compared to WT (t-test). (B) Plasmin activity measurements by slice number show an elevation closer to the infarct core in WT and PAR1 KO mice (two-way ANOVA with Sidak’s post-hoc analysis). (C) Total plasmin activity levels in the non-ischemic hemisphere were undetectable in PAR1 KO mice compared to WT (t-test). (D) Spatial dispersion was present in the non-ischemic hemisphere as well (two-way ANOVA with Sidak’s post-hoc analysis). N = 6, results are presented as dot plot individual values and mean±SEM, *p<0.05, **p<0.01.

Fig 3. Decreased thrombin activity levels in PAR KO following 24 hours following ischemia.

(A) Total thrombin activity is significantly decreased in PAR1 KO compared to WT mice (t-test). (B) Thrombin activity elevation in the ischemic hemisphere of both groups demonstrated per slice. Levels are significantly higher in WT mice in slice #3 and in slices #5 and #6 representing the ischemic core (two-way ANOVA with Sidak’s post-hoc analysis). (C) A non-significant trend towards lower thrombin activity levels in PAR1 KO mice non-ischemic hemisphere (t-test). (D) Similar higher levels of thrombin activity with spatial dispersion in WT compared to PAR1 KO mice (two-way ANOVA with Sidak’s post-hoc analysis). PAR1 KO = 3, WT = 4, results are presented as dot plot individual values and mean ± SEM, *p<0.05, **p<0.01.