Fig. 3

BML-111 prevents adhesion and spreading of platelets on immobilised fibrinogen and clot retraction. Human isolated platelets (2 × 10⁷ cells/ml) were incubated with modified-Tyrode’s HEPES buffer as vehicle control or different concentrations of BML-111 (12.5, 25, and 50 µM) for 5 min at room temperature and then dispensed onto glass coverslips coated with fibrinogen (100 µg/ml) and incubated at 37 °C for 1 h. Samples were fixed with 0.2% (w/v) formyl saline and then permeabilised with 0.2% (v/v) TritonTM x-100. Adhered platelets were stained with Alexa-Fluor 488 Phalloidin for 1 h in the dark and mounted onto glass slides. Platelets were then visualised using confocal microscopy, objective (100x). Multiple images were taken randomly for each slide. (a) Representative image of platelet adhesion and spreading on fibrinogen. (b) An average number of platelets adhered. (c) Spreading of platelets was divided into 3 types: adhered, filopodia and lamelliopodia. (d) PRP was incubated with modified-Tyrode’s HEPES buffer or different concentrations of BML-111 (3.125, 6.25, 12.5, 25 and 50 µM) along with 10 µl red blood cells in glass test tubes for 15 min at room temperature. Thrombin (1 U/ml) was added to initiate clot formation. The clot was retracted around a sealed glass capillary tube placed in the middle of the tube. Clots were observed over 90 min. (e) The clot retraction was calculated by measuring the clot weights. The cumulative data were used to show clot weight. Data represent mean ± SEM (n = 3). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 and ****P ≤ 0.0001values were as calculated by One-way ANOVA.