Activation of platelets stimulates binding of plasminogen. Platelets (2 × 108/ml) were stimulated with thrombin (100 nM) + CVX (100 ng/ml) or TRAP-6 (15 µM) + CVX. After 40 minutes stimulation, either (A) fibrinogen-AF647 (16.7 µg/ml) or (B) plasminogen-DL633 (0.27 µM) was added for 5 minutes before the addition of Annexin A5–FITC (1/20 dilution), followed by HEPES buffer (pH 7.45) containing 2 mM CaCl2. Platelets were then analyzed by flow cytometry. (A) Percentage of fibrinogen–AF647-positive platelets. (B) Representative contour plots showing side scatter (SSC-A) against plasminogen-DL633, gated on unstained platelets (left). Plasminogen-DL633 binding quantified as MFI (middle) and the percentage of plasminogen-positive platelets that are PS-exposing (open bars) or negative (closed bars) (as indicated by Annexin A5–FITC) are shown (right). (C) Representative contour plots of thrombin/CVX-stimulated platelets in the presence of GPRP (5 mM) or tirofiban (1 µg/ml) (left). Plasminogen-DL633 binding quantified as MFI (middle) and the percentage of plasminogen-positive platelets that are PS-exposing (open bars) or negative (closed bars) are shown (right). Expressed as mean ± SEM. *P < .05; **P < .01; ***P < .001; ****P < .0001; n = 3.
