Abstract Body: Aging is an independent risk factor for both atrial fibrillation (AF) and thrombogenesis. Our classical concept is: mechanical stasis during AF promotes thromboembolism formation. However, clinical evidence suggests that thromboembolism is not always preceded by AF. We recently identified a causal role of activated cardiac JNK2, but not JNK1, in aging-associated AF. Our pilot data showed a role of JNK2 in both AF and thrombogenesis. Here, we aimed to define the action of cardiac JNK2 (cJNK2) on platelet (PLT) hyperreactivity and thrombogenesis via a heart-platelet crosstalk.

CD62-P (PLT activity marker) flow cytometry, FeCl3-induced carotid artery thrombus formation time (TFT), and confocal imaging of thrombin-induced PLT calcium (Ca) release amplitude ([Ca]_i) were measured in aged wildtype (WT, 24 month (mo)) and cardiac-specific MKK7D (an upstream JNK2 activator) young (Yg, 5mo) mice with activated cJNK2. Cardiac-specific mouse models with either depleted or inhibited JNK2 (crossbred MKK7D with JNK2-flox (MKK-JNK2f) or overexpressed inactive JNK2 dominant negative (MKK-JNK2dn)) or activated JNK2 but depleted JNK1 (MKK-JNK1f) along with WT-littermates (WT-lits) were studied.

Aged mice showed shortened TFT (5.8±0.2 vs. 7.1±0.2 min, p<0.001, n=12,7), increased PLT CD62-P (hyperreactivity; 157±13% vs. 100±4%; p<0.001, n=23,16) vs. Yg controls. JNK2-specific inhibition using our well-characterized inhibitor JNK2I normalized these changes to levels seen in Yg controls. Moreover, MKK7D PLTs showed shortened TFT and increased CD62-P (149±13% vs. 100±5%, p<0.001; n=18,20), while [Ca]_i was elevated (4.2±0.3 vs. 2.5±0.1, p<0.001; n=32,94) vs. WT-lits. However, JNK2 depletion in MKK-JNK2f mice normalized [Ca]_i, CD62-P, and TFT to the levels seen in WT-lits. Either cJNK2 inhibition in MKK7-JNK2dn mice (in vivo) or JNK2I treatment in MKK7D PLTs (in vitro) led to normalized levels of PLT [Ca]_i and TFT seen in controls. In contrast, MKK7-JNK1f PLTs showed 1-fold increase in PLT [Ca]_i vs. WT-lits (4.6±0.3 vs. 2.5±0.1, p<0.001; n=41,94). These results support the critical role of cJNK2 in Ca-mediated PLT hyperreactivity and thrombogenesis. Finally, this heart-PLT crosstalk is implicated given that PLTs were found to uptake heart-origin JNK2-containing exosomes via a GPCR-mediated endocytosis, which leads to cJNK2-driven [Ca]_i elevation and PLT hyperreactivity, and enhanced thrombogenesis. Thus, JNK2 inhibition could be a novel anti-AF/thrombosis therapeutic approach.