Measurement of Platelet Function in an Experimental Stroke Model With Aspirin and Clopidogrel Treatment

Figure 1. (A) Timeline diagram of the experimental procedures: platelet function in control and ASA+CPG mice was tested in vitro using flow cytometry at the end of 3 days pretreatment. Mice were subjected to 2 h MCAO and tPA administration. Twenty-two hours later, tail bleeding tests and a second flow cytometry analysis were performed, followed by sacrifice and histological assessment of hemorrhage. (B–D) Gating strategy: Ungated whole blood (B). Gated cells scored high for platelet identification marker FITC-CD41 (C) and low for the platelet activation marker PE-CD62P (D), consistent with the properties of resting platelets (Analysis performed with FlowJo).

Figure 2. (A) Platelet activation rates in unstimulated samples: Control: 3.6 ± 2.0%, n = 12; ASA+CPG: 2.0 ± 0.7%, n = 15, p = 0.3649 with selected example images of fluorescence intensity for platelet activation marker anti-CD62P-PE in unstimulated blood samples (FACSDiva software). (B) Platelet activation rates in thrombin stimulated samples: Controls: 88.0 ± 0.6%, n = 12; ASA+CPG: 47.5 ± 3.8%, n = 15, p < 0.0001, images of anti-CD62P-PE fluorescence intensity after in vitro thrombin stimulation. (C) Blood volumes in post-stroke tail bleeding testing: Controls: 1.4 ± 0.2 μl/3 min, n = 4; ASA+CPG: 5.4 ± 0.9 μl/3 min, n = 8, p = 0.004. (D) Correlation of in vitro platelet function with in vivo tail bleeding volumes: Linear regression: R2=0.5518, y = −0.08175 (95% CI: −0.134 to −0.03)*X+8.846). Correlation: r = −0.7902, p = 0.0033, n = 12. **p < 0.01; ****p < 0.0001.

Figure 3. (A) Hemorrhage area measurements in brain sections: Platelet activation rate >80% CD62P expression: 0.9 ± 0.3 mm2, n = 7; Platelet activation rate <80% CD62P expression: 10.8 ± 4.5 mm2, n = 11, p = 0.0335. (B) Morphological classification of the bleeding types: 0/7 control mice were rated HI-2, compared to 6/11 mice in the ASA+CPG (p = 0.0125). (C) Platelet activation rates in thrombin stimulated samples (post-stroke): Controls: 91.9 ± 2.1%, n = 5; ASA+CPG: 71.6 ± 4.1%, n = 3, p = 0.0026. (D) Platelet activation following thrombin stimulation pre- and post-stroke in ASA+CPG pretreated mice: Platelet activation rates increased significantly by 22.9 ± 8.9% (p = 0.0204) within 24 h between the two measurements. *p < 0.05; **p < 0.01.