BACKGROUND: Platelet aggregation at sites of vascular injury is essential for normal hemostasis, but may also cause pathological vessel occlusion. Rho GTPases are molecular switches that regulate essential cellular processes and they have pivotal functions in the cardiovascular system. Rac1 is an important regulator of platelet cytoskeletal reorganizations and contributes to platelet activation. Rac1 inhibitors are thought to be beneficial in a wide range of therapeutic settings and therefore have been tested in vivo for a variety of disorders. Two small molecule inhibitors, NSC23766 and EHT1864, have been characterized in different cell types, demonstrating high specificity for Rac1 or Rac, respectively.

OBJECTIVES: We sought to analyze the specificity of NSC23766 and EHT1864.

METHODS: Platelet function was assessed in mouse wild-type and Rac1-deficient platelets by using flow cytometric analysis of cellular activation and aggregometry. Platelet spreading was analyzed by differential interference contrast microscopy and activation of effector molecules by biochemical approaches.

RESULTS: NSC23766 and EHT1864 exhibited strong and distinct Rac1-independent effects at 100 μM in platelet function tests. Both inhibitors induced a Rac1-specific inhibition of platelet spreading, but also markedly impaired agonist-induced activation of Rac1(-/-) platelets. Furthermore, GPIb-mediated signaling was dramatically inhibited by NSC23766 in both wild-type and Rac1-deficient platelets. Importantly, these inhibitors directly affected the activation of the Rac1 effectors PAK1/2.

CONCLUSIONS: Our results reveal critical off-target effects of NSC23766 and EHT1864 at 100 μM in mammalian cells, raising questions about their utility as specific Rac1/Rac inhibitors in biochemical studies at these concentrations and possibly as therapeutic agents. This article is protected by copyright. All rights reserved.