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Figure 2 (previous page). Release of pro-collagen by activated platelets and processing by thrombin. (A) Procollagen released from platelets from healthy donors or polycystic ovary syndrome (PCOS) patients treated with solvent (Sol), thrombin (Thr) (1 U/mL, 10 minutes) or the thromboxane A2 analog U46619 (U46, 1 mmol/L, 10 minutes); n=4-5 (ANOVA and Tukey’s multiple comparisons post test, ***P<0.001). (B) Pro-collagen I (red), and Gp1b (green) expression in non-permeabilized (-TX-100) and permeabilized (+TX-100) platelets from healthy donors, treated with Sol or Thr (1U/mL, 10 minutes); bar = 5 mm. Comparable results were obtained in five additional experiments. (C) Representative blots (left panel) and quantification (right panel) showing collagen I in platelets from healthy donors stimulated with either Sol or Thr in the presence or in the absence of the BMP inhibitor UK383367 (UK), n=6 (ANOVA and Tukey’s multiple comparisons post test, ***P<0.001). (D) Representative blot (upper panel) and quan- tification (lower panel) showing pro-collagen (PC) and its cleavage product (ΔPC) in platelets treated with Sol, Thr (1U/mL) or U46619 (U46, 1 mmol/L) for up to 60 minutes. The bar graph shows the quantification of cleaved pro-collagen (ΔPC) after 60 minutes, n=8 (ANOVA and Tukey’s mul- tiple comparisons post test, ***P<0.001). (E) Representative silver gel (upper panel) and quantification (lower panel) showing the cleavage of a 80 kD recombinant pro-collagen (rPC) by Thr) (1U/mL, 30 minutes) in the absence or in the presence of hirudin (Hir) (1U/mL), n=4 independent exper- iments (ANOVA and Tukey’s multiple comparisons post-test, ***P<0.001).
haematologica | 2021; 106(3)