Figure 4.

Cell-derived polyP triggers TG in mouse whole blood. (A) PolyP was isolated from 1.1 × 10⁶ HEK293 cells by anion exchange chromatography. Purified polyanions (50 ng/lane) were resolved by denaturating urea-PAGE [7 M urea/15% (w/v) polyacrylamide gels] and visualized using negative DAPI-staining. 20 ng/lane of synthetic polyP (P42–P68) was loaded as size and mass standard. Chain length of synthetic polyP - P 42: ≥ 650 mers, P30: ≥ 400 mers, P75: ≤ 650 mers, P70: ≤ 400 mers, P68: ≤ 150 mers. (B) Real-time TG in mouse whole blood was stimulated with cell-purified polyP (7.1 and 10 μg/ml, respectively) in presence or absence of recombinant polyP inhibitor PPX_Δ12 (500 μg/ml). Representative TG curves of n = 3 individual experiments measured in triplicates each. Corresponding time that elapses until TG is initiated (lag time) in (B) is shown in (C). Mean ± SD are indicated in bars. Statistical analysis was performed using one-way ANOVA followed by Dunnett's multiple comparisons test. PolyP-stimulated TG was compared to buffer. P > 0.05 (ns, not significant), P < 0.05 (*).