Background: Despite the safety of pulsed field ablation (PFA), periprocedural thromboembolic events and arrhythmia recurrence remain clinical concerns. Both are partly driven by post-ablation tissue remodeling mediated by inflammation, oxidative stress, and endothelial dysfunction. Moreover, clinical reports of procedure-related strokes and silent cerebral lesions suggest that PFA may directly activate platelets. Current PFA systems employ microsecond pulses (µsPEF), whereas nanosecond pulses (nsPEF) have only recently entered clinical trials. Our previous studies demonstrated that both µsPEF and nsPEF induce substantial endothelial injury at electric field strengths needed for effective ablation. We therefore hypothesized that endothelial disruption, together with direct platelet activation, contributes to the thrombogenic effects associated with PFA. In the present study, we investigated the mechanisms underlying PFA-induced platelet activation and explored the potential for its pharmacological mitigation.
Methods: Platelets isolated from healthy donors were electroporated in suspension using 1 mm cuvettes. Electric field strengths for nsPEF (200 x 300 nanosecond pulses at 10 Hz) and µsPEF (20 x 100 microsecond pulses at 1 Hz) protocols were adjusted to ED₅₀ values—defined as the electric field strength causing 50% cardiomyocyte death—validated in vitro and ex vivo. Platelet activation was quantified by flow cytometry using CD42b (platelet identification), Annexin V (phosphatidylserine exposure) and CD62P (P-selectin, α-granule release) staining. To assess whether pharmacological stimulation of the NO–GC–cGMP pathway could attenuate platelet activation, samples were incubated with the NO–GC stimulator riociguat prior to pulsed electric field exposure.
Results: Both nsPEF and µsPEF directly activated human platelets in vitro, significantly increasing the proportion of procoagulant Annexin V⁺/CD62P⁺/CD42b⁺ platelets compared with sham controls (p < 0.01). Detailed analysis of platelet subpopulations revealed that nsPEF produced fewer apoptotic platelets, less aggregates and preserved a larger population of resting platelets, indicating a more favorable thrombogenic profile. Importantly, pre-incubation with riociguat significantly reduced the proportion of procoagulant following nsPEF exposure (p < 0.05), whereas no comparable protective effect was observed after µsPEF.
Conclusion: Pulsed electric fields directly activate platelets, with µsPEF inducing stronger apoptotic and procoagulant responses than nsPEF. Pharmacological activation of the NO–GC–cGMP pathway with riociguat effectively attenuates nsPEF-induced platelet activation, indicating its translational potential as a peri-procedural protective agent. Understanding and modulating thrombogenic mechanisms of cardiac electroporation may improve the safety and long-term outcomes of pulsed field ablation.
