Abstract

In this study, we examined 2-aminoethoxydiphenyl borate (2APB) as an inhibitor of Ca²⁺ influx in human platelets. 2APB was found to inhibit thrombin-mediated intracellular Ca²⁺mobilization rapidly in platelets incubated in the absence of extracellular Ca²⁺. This result supports an intracellular action of 2APB on inositol 1,4,5-trisphosphate (IP₃)-receptor Ca²⁺ channels. 2APB was without effect on the ability of thapsigargin to mobilize intracellular Ca²⁺. This result suggests that the efflux of Ca²⁺ from the endoplasmic reticulum mediated by thapsigargin is not via IP₃ Ca²⁺ channels. However, 2APB was able to prevent the entry of Ca²⁺ and Sr²⁺ through thapsigargin-activated, store-operated Ca²⁺ channels (SOCC). This result supports a direct inhibitory effect of 2APB on SOCC. 2APB was also able to block the entry of Sr²⁺, Ba²⁺, and Mn²⁺ entry into unstimulated platelets, which suggests that 2APB was inhibiting the Ca²⁺ influx channels directly. The capacity of 2APB to prevent Ca²⁺ influx and Sr²⁺ influx was rapid because it occurred immediately upon addition to the platelets. The inhibition of Ca²⁺ and Sr²⁺ influx by 2APB was similar to that seen with the cell-impermeable nonselective Ca²⁺-channel blocker La³⁺ or the Ca²⁺ chelator EGTA. Diphenylboronic anhydride and 2,2-diphenyltetrahydrofuran, two compounds that are structurally similar to 2APB, also inhibited Ca²⁺ influx. It was concluded that 2APB was a rapid and effective direct inhibitor of SOCC in human platelets; as such, it cannot be used to support the involvement of IP₃ receptors in the activation of SOCC.