Figure 2. TMAO augments stimulus-dependent release of Ca²⁺ from intracellular stores in human platelets.

(A and B) Thrombin evoked changes in intracellular calcium concentration [Ca²⁺]_i in Fura 2-AM loaded washed human platelets incubated with the indicated amounts of TMAO. (A) Representative fluorescent signal from a single subject’s platelet preparation; and (B) mean ± SEM results from n=4 subjects are shown (*, p<0.05 for comparison with vehicle). (C and D) Fluorescence ratio of Fura-2 loaded platelets incubated in medium supplemented with TMAO or vehicle. Cells were first stimulated at the indicated time with a sub-maximal thrombin dose in a Ca²⁺-free medium, and then where indicated, Ca²⁺ was added to the medium. Representative (C) fluorescent signal and (D) mean fluorescence ± SEM (n=4 donors) are shown. (E and F) Washed human platelets pre-incubated (30 min, 22°C) with the indicated concentrations of TMAO wer e stimulated with sub-maximal levels of either (E) thrombin or (F) ADP, and then platelet IP₁ concentrations determined. Data shown represent mean ± SEM (n=4 donors each). Linear mixed effect models were used to analyze the repeated measure data. A Wilcoxon Signed-Rank Test was used to conduct pairwise comparisons. See also Figure S2.