Safety and efficacy of targeting platelet proteinase-activated receptors in combination with existing anti-platelet drugs as antithrombotics in mice

Abstract

Background and purpose: Developing novel anti-platelet strategies is fundamental to reducing the impact of thrombotic diseases. Thrombin activates platelets via proteinase-activated receptors (PARs), and PAR antagonists are being evaluated in clinical trials for prevention of arterial thrombosis. However, one such trial was recently suspended due to increased bleeding in patients receiving a PAR₁ antagonist in addition to anti-platelet drugs that most often included both aspirin and clopidogrel. Therefore, it remains unclear how to best manipulate PARs for safe antithrombotic activity. To address this, we have examined potential interactions between existing anti-platelet drugs and strategies that target PARs.

Experimental approach: We used in vivo mouse models in which interactions between various anti-platelet strategies could be evaluated. We examined the effects on thrombosis and haemostasis in PAR₄ -/- mice (platelets unresponsive to thrombin) treated with therapeutic doses of either aspirin or clopidogrel.

Key results: Using a model in which occlusive thrombosis occurred in PAR₄ -/- mice or wild-type mice treated with aspirin or clopidogrel, PAR₄ -/- mice treated with either anti-platelet agent showed marked protection against thrombosis. This antithrombotic effect occurred without any effect on haemostasis with aspirin, but not clopidogrel. Furthermore, specifically targeting thrombin-induced platelet activation (via PARs) improved the therapeutic window of non-specifically inhibiting thrombin functions (via anticoagulants).

Conclusions and implications: Our results indicate that PAR antagonists used in combination with aspirin provide a potent yet safe antithrombotic strategy in mice and provide insights into the safety and efficacy of using PAR antagonists for the prevention of acute coronary syndromes in humans.

Figure 1

PAR₄-deficiency, aspirin or clopidogrel provide marked protection against in vivo thrombosis in mouse carotid arteries. In vivo thrombosis in PAR₄+/+ mice in the absence and presence of the anti-platelet drugs aspirin (200 mg·kg⁻¹) or clopidogrel (3 mg·kg⁻¹) as well as PAR₄−/− mice. Electrolytic injury of carotid arteries was induced under stasis by a current of 18 mA for 2 min. (A) Body weight-adjusted blood flow rates were continuously recorded from 5 min before to 30 min after injury. (B) Body weight-adjusted total blood flow over the 30 min post-injury period. Data are mean ± SEM; *P < 0.05 (one-way anova, with Dunnett's test for multiple comparisons vs. vehicle-treated PAR₄+/+ mice). (C,D) Aggregation of platelets isolated from PAR₄+/+ mice treated with either clopidogrel (3 or 20 mg·kg⁻¹) or aspirin (200 mg·kg⁻¹) induced by ADP (20 µM), arachidonic acid (AA, 200 µM) or a PAR₄-activating peptide (AYPGKF, 100 µM; PAR₄-AP). The maximum response induced by each treatment is presented. Data are mean ± SEM; n = 3; ***P < 0.001 (one-way anova with Dunnett's test for multiple comparisons vs. vehicle-treated PAR₄+/+ mice).

Figure 2

Development of an in vivo thrombosis model resistant to PAR₄ deficiency or to pretreatment with aspirin or clopidogrel. In vivo thrombosis in (A) PAR₄+/+ versus PAR₄−/− mice and (B) PAR₄+/+ mice in the absence and presence of the anti-platelet drugs aspirin (200 mg·kg⁻¹) or clopidogrel (3 mg·kg⁻¹). Electrolytic injury was induced under stasis by a current of 18 mA for 2 min in arteries with a stenosis in which blood flow was reduced to ∼50% of control levels. (A,C) Body weight-adjusted blood flow rate and (B,D) body weight-adjusted total blood flow. Vehicle is saline. Data are mean ± SEM; N.S., not significant.

Figure 3

Aspirin and clopidogrel act synergistically with PAR₄ deficiency to provide robust antithrombotic activity. In vivo thrombosis in (A) PAR₄−/− and (B) PAR₄+/+ mice in the absence and presence of the anti-platelet drugs aspirin (200 mg·kg⁻¹) and/or clopidogrel (3 mg·kg⁻¹). Electrolytic injury was induced under stasis by a current of 18 mA for 2 min in arteries with a stenosis in which blood flow was reduced to ∼50% of control levels. (A,C) Body weight-adjusted blood flow rate and (B,D) body weight-adjusted total blood flow. Vehicle is saline. Data are mean ± SEM; *P < 0.05; **P < 0.01 (one-way anova with Dunnett's test in (B), Student's unpaired t-test in (D)).

Figure 4

Clopidogrel, but not aspirin, affects haemostasis when in combination with PAR₄-deficiency. (A) Tail bleeding times in PAR₄+/+ and PAR₄−/− mice treated with vehicle (saline), aspirin (200 mg·kg⁻¹) or clopidogrel (3 mg·kg⁻¹), and/or (B) hirudin (5 or 10 mg·kg⁻¹), as determined using the template method. Individual data points are shown. (Note partial obstruction of n = 8 data points in the clustered group of PAR₄−/− mice treated with clopidogrel.) Bars are mean ± SEM. Note the similar bleeding time in untreated PAR₄−/− mice (A), aspirin-treated PAR₄−/− mice (A), and aspirin- and hirudin-treated PAR₄+/+ mice (B) (arrows). In vivo thrombosis was examined in these three groups in Figure 5.

Figure 5

Increased therapeutic window in PAR₄−/− mice treated with aspirin compared with mice treated with hirudin and aspirin. In vivo thrombosis in aspirin-treated PAR₄−/− mice versus aspirin- and hirudin-treated PAR₄+/+ mice. Electrolytic injury was induced under stasis by a current of 18 mA for 2 min in arteries with a stenosis in which blood flow was reduced to ∼50% of control levels. (A) Body weight-adjusted blood flow rate and (B) body weight-adjusted total blood flow. (C) Surgical bleeding scores. 0 = no/limited bleeding; 1 = bleeding requiring intervention in order for the experiment to proceed; 2 = bleeding causing death. Vehicle is saline. Data are mean ± SEM. N.S., not significant. ***P < 0.001 (Student's two-way, unpaired t-test).