The inhibitory effect of gadolinium-conjugated diflunisal on platelet function through regulating outside-in signaling of αIIbβ3 integrin

Abstract

The gadolinium-conjugated diflunisal derivative ETN102 has previously been shown to suppress cyclooxygenase-2 (COX-2) levels, reduce glial activation, and attenuate neuronal damage associated with stroke. In the present study, we investigated whether ETN102 modulates platelet aggregation and activation. ETN102 selectively inhibited thrombin-induced platelet aggregation while sparing platelet responses to other agonists, including collagen, adenosine diphosphate (ADP), and U46619. ETN102 also reduced adenosine triphosphate (ATP) secretion under aggregating conditions without altering thromboxane B2 (TXB2) generation, suggesting that its antiplatelet effect is independent of the COX-1-dependent thromboxane signaling. Flow cytometric and biochemical analyses further demonstrated that ETN102 suppressed thrombin-induced αIIbβ3 integrin activation, platelet spreading, clot retraction, and the phosphorylation of focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (Pyk2), without affecting granule secretion, intracellular Ca2+ mobilization, or phosphorylation of Syk, PI3K, and AKT signaling molecules. In vivo, ETN102 significantly prolonged carotid artery occlusion time in a ferric chloride (FeCl3)-induced thrombosis model and increased tail bleeding time and hemoglobin content. In addition, ETN102 reduced infarct volume and improved neuronal survival markers in a murine model of cerebral ischemia/reperfusion injury. These findings demonstrate that ETN102 is a thrombin-context selective modulator of platelet integrin αIIbβ3 outside-in signaling, with antithrombotic and neuroprotective potential.