Figure 3.

Platelet adhesion on different ECM proteins measured by BCECF-AM. Coating of 96-well microplates with ECM proteins (50 µL) was performed for 1 h at 37 °C. Different proteins and concentrations of ECM were used: fibrinogen (0.06 to 8 mg/mL); fibronectin (0.3 to 40 µg/mL); non-fibrillar collagen-I (0.06 to 8 µg/mL); fibrillar collagen-I (0.06 to 8 µg/mL); collagen-III (0.5 to 64 µg/mL); collagen-IV (0.125 to 16 µg/mL); laminin-411 (2.5 to 15 µg/mL); laminin-511 (2.5 to 15 µg/mL); collagen-related peptide (CRP) (0.15 to 20 µg/mL); vitronectin (0.15 to 20 µg/mL). In the wells without coating distillated water was added during the coating incubation time. After blocking the wells with BSA (0.03%), platelets (8 × 10⁴/µL in 100µL) were added, followed by incubation for 1 h at 37 °C. Next, non-adherent platelets were removed, and adherent platelets were incubated with BCECF-AM (4 µg/mL in 50 µL) for 30 min at 37 °C. Fluorescence intensity was measured using a plate reader (VictorX, PerkinElmer, Waltham, MA, USA) and images were taken using a fluorescence microscopy (Eclipse Ti2, Nikon, Tokyo, Japan) with a 20× objective (scale bar 10 µM). Values were compared with the non-coated control condition by one-way ANOVA followed by Dunnett’s post hoc test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, data are mean ± SEM; n = 4).