RESUMEN
Essentials CpG oligodeoxynucleotide (ODN) immuotherapeutics cause undesired platelet activating effects. It is crucial to understand the mechanisms of these effects to identify protective strategies. CpG ODN-induced platelet activation depends on C-type lectin-like receptor 2 (CLEC-2) and P2Y12. Targeting CLEC-2 or P2Y12 fully prevents CpG ODN-induced platelet activation and thrombosis. SUMMARY: Background Synthetic phosphorothioate-modified CpG oligodeoxynucleotides (ODNs) show potent immunostimulatory properties that are widely exploited in clinical trials of anticancer treatment. Unexpectedly, a recent study indicated that CpG ODNs activate human platelets via the immunoreceptor tyrosine-based activation motif (ITAM)-coupled receptor glycoprotein VI. Objective To further analyze the mechanisms of CpG ODN-induced platelet activation and identify potential inhibitory strategies. Methods In vitro analyses were performed on human and mouse platelets, and on cell lines expressing platelet ITAM receptors. CpG ODN platelet-activating effects were evaluated in a mouse model of thrombosis. Results We demonstrated platelet uptake of CpG ODNs, resulting in platelet activation and aggregation. C-type lectin-like receptor 2 (CLEC-2) expressed in DT40 cells bound CpG ODNs. CpG ODN uptake did not occur in CLEC-2-deficient mouse platelets. Inhibition of human CLEC-2 with a blocking antibody inhibited CpG ODN-induced platelet aggregation. CpG ODNs caused CLEC-2 dimerization, and provoked its internalization. They induced dense granule release before the onset of aggregation. Accordingly, pretreating platelets with apyrase, or inhibiting P2Y12 with cangrelor or clopidogrel, prevented CpG ODN platelet-activating effect. In vivo, intravenously injected CpG ODN interacted with platelets adhered to mouse injured endothelium, and promoted thrombus growth, which was inhibited by CLEC-2 deficiency or by clopidogrel. Conclusions CLEC-2 and P2Y12 are required for CpG ODN-induced platelet activation and thrombosis, and might be targeted to prevent adverse events in patients at risk.
Asunto(s)
Anticuerpos/farmacología , Plaquetas/efectos de los fármacos , Lectinas Tipo C/metabolismo , Glicoproteínas de Membrana/metabolismo , Oligonucleótidos Fosforotioatos/toxicidad , Activación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/farmacología , Antagonistas del Receptor Purinérgico P2Y/farmacología , Receptores Purinérgicos P2Y12/efectos de los fármacos , Adyuvantes Inmunológicos/metabolismo , Adyuvantes Inmunológicos/toxicidad , Animales , Plaquetas/inmunología , Plaquetas/metabolismo , Línea Celular , Modelos Animales de Enfermedad , Humanos , Motivo de Activación del Inmunorreceptor Basado en Tirosina , Lectinas Tipo C/deficiencia , Lectinas Tipo C/inmunología , Masculino , Glicoproteínas de Membrana/deficiencia , Glicoproteínas de Membrana/inmunología , Ratones , Ratones Endogámicos C57BL , Oligonucleótidos Fosforotioatos/inmunología , Oligonucleótidos Fosforotioatos/metabolismo , Agregación Plaquetaria/efectos de los fármacos , Glicoproteínas de Membrana Plaquetaria/metabolismo , Unión Proteica , Receptores Purinérgicos P2Y12/metabolismo , Transducción de Señal/efectos de los fármacos , Trombosis/sangre , Trombosis/inmunología , Trombosis/prevención & control , Factores de TiempoRESUMEN
A series of 1H,3H-thiazolo[3,4-a]benzimidazoles were synthesized and tested for their in vitro antitumour activity against 60 human tumour cell lines. Some derivatives exhibited both tumour growth inhibition activity and cellular selectivity. In particular, compound 8c, the most active of the series, was very active towards all cell lines at concentrations ranging from 10(-7)-10(-5) M. Compound 4a, on the other hand, was highly selective against the CNS cancer cell line.