KCNN4-mediated potassium ion efflux maintains mitochondrial functions leading to platelet biogenesis.

Background: Potassium ions (K⁺) are essential for platelet function, yet their role in thrombopoiesis-particularly through specific K⁺ channels-remains poorly understood. This gap is especially relevant in the context of in vitro platelet production from induced pluripotent stem cell (iPSC)-derived immortalized megakaryocyte progenitor cell lines (imMKCLs), which we have developed for clinical-grade platelet manufacturing.

Objective: We aimed to elucidate how K⁺ channels contribute to platelet biogenesis, focusing specifically on the calcium ion (Ca²⁺)-activated K⁺ channel KCNN4 (also known as K_Ca3.1).

Methods: Using imMKCLs and human cord blood-derived megakaryocytes (CB-megakaryocytes), we analyzed intracellular cation dynamics during platelet production. RNA sequencing profiling was conducted to identify K⁺ channel gene expression changes, focusing on KCNN4. Its role in proplatelet formation and platelet release was examined using pharmacological inhibitors and gene knockdown (KD). We further investigated the link between KCNN4 and microtubule organization, mitochondrial function, and reactive oxygen species (ROS) levels.

Results: A progressive decline in intracellular K⁺ concentration ([K⁺]ᵢ) was observed during the six-day maturation period of imMKCLs. KCNN4 was upregulated at onset of platelet generation, and its inhibition or KD led to impaired proplatelet formation and reduced platelet yield in both imMKCLs and CB-megakaryocytes. These effects were accompanied by decreased [K⁺]ᵢ, reduced mitochondrial membrane potential, and increased ROS accumulation.

Conclusions: Our findings reveal that the KCNN4-mediated reduction in [K⁺]ᵢ is a crucial mechanism linking cytoskeletal reorganization, mitochondrial function, and ROS homeostasis to effective thrombopoiesis. This study provides new insights into platelet biogenesis and offers potential avenues to optimize ex vivo platelet production.