Using an agonist panel and aggregation to assess antiplatelet therapy in mice

Abstract

Abstract Platelets play a crucial role in hemostasis. For cardiovascular and stroke prophylaxis, patients are commonly placed on either single or dual antiplatelet drugs, such as aspirin and/or clopidogrel. Here we outline a mouse model examining antiplatelet therapy and the resulting changes in platelet activation and aggregation. Using commercially available transgenic mice with platelets expressing green fluorescent protein (GFP) or red fluorescent protein (RFP), endogenous platelets were inhibited with a single, dual, or control antiplatelet treatment. We used 100µg aspirin/g mouse or 50µg clopidogrel/g mouse for a single dose; dual antiplatelet therapy was a combined dose of 100µg aspirin/g mouse and 50µg clopidogrel/g; control treatment was DMSO alone. One hour after treatment, whole blood (WB) was collected by aseptic cardiac puncture and mixed in the following groups: aspirin treated RFP and GFP WB, clopidogrel treated RFP and GFP WB, aspirin and clopidogrel treated RFP and GFP WB, control treated RFP and GFP WB. Each combined sample was then treated with a panel of agonists: no agonist, 0.5U/mL thrombin, 20µM ADP, and 500µg/mL arachidonic acid. Thrombin samples were activated for 2 minutes at room temperature while ADP and arachidonic acid samples were activated for 10 minutes at 37°C. Platelets were labeled with anti-CD41a to label all platelets and anti-CD62P to label activated platelets. Aggregation is measured as double-colored GFP and RFP platelet events. Compared to control mice, mice treated with antiplatelet agents demonstrated statistically significant decreases in activation following thrombin administration (ranging from 90% to 83% activated); there was no significant difference in platelet aggregation following thrombin among the samples from different antiplatelet therapies. Following ADP activation, clopidogrel treatment decreased platelet activation (1% activation) compared to control (3% activation) and aspirin-treated (5% activation) mice with no additional decrease in dual treatment (1% activation); unexpectedly, aspirin treatment alone or dual therapy decreased platelet aggregation with ADP (15% and 17% aggregated, respectively) compared to control and clopidogrel (23% and 27% aggregated, respectively). Finally, after arachidonic acid activation, aspirin treatment significantly decreased activation (7% activated) compared to control (20% activated), while clopidogrel decreased activation more than that of aspirin (3% activated), with no additional decrease from dual therapy (3% activated). Though not significant, aspirin treatment (1.4% aggregation) and dual therapy (1.2%) demonstrated a trend with decreased platelet aggregation following arachidonic acid activation compared to control (1.8% aggregation) and clopidogrel-treated platelets (3.2%). These data presented here show a novel application of platelet activation and aggregation of GFP and RFP platelet populations in a mouse model following aspirin and/or clopidogrel treatment. This model can be applied generally to other drugs and disease models for platelet dysfunction using additional markers for platelet activation. Additionally, these experiments can be used to further investigate the function of transfused murine platelets.