Induction of Diabetes Abolishes the Antithrombotic Effect of Clopidogrel in Apolipoprotein E–Deficient Mice

Figures

• 

  Fig. 1. Effects of clopidogrel on the patency of carotid arteries. Clopidogrel (10 mg/kg/d, orally) or vehicle was administered once daily for 5 days. Carotid artery occlusion was initiated by application of FeCl 3 and blood flow was determined for 1 hour. Individual patency data are shown for vehicle-treated WT (1), clopidogrel-treated WT (2), vehicle-treated apoE-deficient (3), clopidogrel-treated apoE-deficient (4), vehicle-treated diabetic apoE-deficient (5), and clopidogrel-treated diabetic apoE-deficient groups (6). Open and filled bars indicate patent and occluded arteries, respectively. The number on the left side of each column represents animal ID number. apoE, apolipoprotein E; WT, wild-type.

• 

  Fig. 2. Effect of clopidogrel on time to first occlusion. Clopidogrel (C) at 10 mg/kg/d (orally) or vehicle (V) was administered once daily for 5 days to wild-type (WT), apoE-deficient, and diabetic apoE-deficient mice ( n = 10 per group). Carotid artery occlusion was initiated by application of FeCl 3 2 hours after the last dose of clopidogrel, arterial blood flow was monitored, and the time in seconds to first occlusion was determined. The results are presented as the mean + SE ( n = 10). *** p < 0.001 versus each vehicle group, † p < 0.05 versus the vehicle group of WT mice, # p < 0.05 (Student's t -test).

• 

  Fig. 3. Hematoxylin–eosin-stained cross-sections of injured carotid arteries. ( A ) Vehicle-treated wild-type (WT) (animal ID no. 105 in Fig. 1 ); ( B ) clopidogrel-treated WT (no. 202); ( C ) vehicle-treated apoE-deficient (no. 308); ( D ) clopidogrel-treated apoE-deficient (no. 406); ( E ) vehicle-treated diabetic apoE-deficient (no. 501); and ( F ) clopidogrel-treated diabetic apoE-deficient mice (no. 607). Scale bar = 100 μm.

• 

  Fig. 4. Effects of clopidogrel on morphometric parameters of injured carotid arteries, thrombus area ( A ) and lumen stenosis ( B ). Clopidogrel (C) at 10 mg/kg/d (orally) or vehicle (V) was administered once daily for 5 days to wild-type (WT), apoE-deficient, and diabetic apoE-deficient mice ( n = 10 per group) and arterial occlusion was induced 2 hours after the last dose of clopidogrel. After completing blood flow monitoring, mice were euthanized. The FeCl 3 -injured regions of the carotid arteries were collected, further fixed in 10% neutral-buffered formalin and paraffin sections prepared, and stained. The lumen and thrombotic areas were measured using an image analysis software (WinROOF 2013), and lumen stenosis (100 × thrombus area/lumen area) was calculated. Results are presented as the mean + SE ( n = 10). *** p < 0.001 versus each vehicle group, ### p < 0.001 (Student's t -test).

• 

  Fig. 5. Effects of clopidogrel on platelet expression of ADP-induced activated GPIIb/IIIa expression. Clopidogrel (10 mg/kg/d) or vehicle was administered orally once daily for 5 days to wild-type (WT), apoE-deficient, and streptozotocin-induced diabetic apoE-deficient mice, and blood collected 2 hours after the last dose. Ex vivo expression of activated GPIIb/IIIa on platelets induced by 5 and 20 μM ADP was assessed using a flow cytometer. In the vehicle groups, ADP stimulation resulted in a significant increase in the expression of activated platelet GPIIb/IIIa compared with no agonist ( p < 0.01 for diabetic apoE-deficient mice, p < 0.001 for other groups). The results are presented as mean ± SE ( n = 10). ** p < 0.01 versus corresponding vehicle group (Student's t -test).

• 

  Fig. 6. Effects of clopidogrel on PAR4 TRAP-induced expression of platelet P-selectin. Clopidogrel (10 mg/kg/d) or vehicle was administered orally once daily for 5 days to wild-type (WT), apoE-deficient, and diabetic apoE-deficient mice, and blood collected 2 hours after the last dose. Ex vivo expression of P-selectin on platelets induced by 100 and 300 µM PAR4 TRAP was assessed using a flow cytometer. In the vehicle groups, PAR4 TRAP stimulation resulted in a significant increase in platelet P-selectin expression compared with no agonist ( p < 0.001, all groups). The results are presented as the mean ± SE ( n = 10). * p < 0.05, ** p < 0.01 versus corresponding vehicle group; # p < 0.05 versus the vehicle group of diabetic apoE-deficient mice; †† p < 0.01 versus clopidogrel group of diabetic apoE-deficient mice (Student's t -test).