Priming of platelet responses to collagen by TPO is accompanied by a PI 3-kinase–dependent Ca⁺⁺ increase.

Fura-2–loaded platelets (10⁸/mL) were stimulated in a plastic cuvette with stirring at 37°C in the presence of 1 mmol/L extracellular Ca⁺⁺. Fluorescence emission was recorded at 510 nm for an excitation ratio 340/380 nm. In (A) experiments were performed as follows: (i) collagen (2 μg/mL) or TPO (100 ng/mL) for 3 minutes and then collagen (2 μg/mL); (ii) wortmannin (Wt) (100 nmol/L) for 3 minutes, followed by TPO for 3 minutes and then collagen (2 μg/mL), or TPO (100 ng/mL) for 3 minutes, followed by Wt (100 nmol/L) for 3 minutes and then collagen (2 μg/mL). In (B) experiments were performed as follows: (i) collagen (5 μg/mL), or TPO (100 ng/mL) for 3 minutes, followed by collagen (5 μg/mL); (ii) same as (i) but in the presence of apyrase (1 U/mL). The addition of collagen is indicated by an arrow. Results are from 1 experiment made in triplicate representative of 4.