Figure 5.

Platelet spreading on fibrinogen is inhibited by S1PR₄ and S1PR₅ agonists and SPHK inhibitor. Washed platelets (2 × 10⁷/mL) were incubated with vehicle control (0.2% methanol), S1P receptor subtype specific agonists or SphK inhibitor DMS for 10 min. Platelets were then left to adhere onto a 13 mm glass coverslip coated with 100 μg/mL fibrinogen (60 min, 30 °C). Samples were fixed (4% PFA) before permeabilization (0.1% triton) and staining with ActinGreen 488 ReadyProbes reagent. (a) Representative images of basal (no fibrinogen), vehicle control, 1 μM FTY720 phosphate, 1 μM SEW2871,1 μM CYM50260, 1 μM CYM50358 hydrochloride, 1 μM A971432, 1 μM DMS. (b) Bar graphs of the (i) total number of platelets and (ii) average platelet cell area of platelets adhered in the absence and presence of S1P receptor subtype specific agonists or SphK inhibitor. N = 5. Three images were taken from each well. Average cell area was measured per 1000 μm², data are plotted as mean ± SEM. Statistical analysis: One-way ANOVA with Dunnett’s test. ns = not significant, **P < 0.01, ****P < 0.0001.