Figure 4

Pxn-KD platelets exhibit augmented outside-in signaling. (A) Control and Pxn-KD platelets treated with 50 ng/mL convulxin were allowed to adhere on immobilized fibrinogen for 30 min. The platelets were then fixed and stained with an anti-GFP antibody (green; left panel) and rhodamine-conjugated phalloidin (red; middle panel). The merged images show colocalization of GFP and actin staining (yellow; right panel). Original magnification, ×600; Bar, 5 μm. Data are representative of three independent experiments. (B) Platelets treated with or without 50 ng/mL convulxin were incubated in dishes coated with 400 μg/mL fibrinogen for 30 min. The area of cell spreading was quantified by ImageJ software. The mean platelet size on BSA was subtracted from the total spread area on fibrinogen to determine the actual increase in platelet spreading. The horizontal bar denotes the mean, and each symbol denotes an individual cell (n = 281–394 cells). (C) Clot retraction of platelet-rich plasma consisting of diluted human plasma and control or Pxn-KD platelets was initiated by 0.1 U/mL thrombin and then photographed at 0, 45, 90, and 120 min. (D) Clot retraction was quantified by measuring serum formation extruded by clot retraction. Columns and error bars represent the mean ± s.d. (n = 3). Open bars: control platelets; black bars: Pxn-KD platelets. Statistical significance was determined using Student’s t-test. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. control.