Figure 3.
Figure 3. PKCδ negatively regulates filopodia formation in human platelets. (A) Washed human platelets were preincubated with secramine A at the concentrations indicated for 15 minutes. (i) Platelet aggregation in response to collagen (30 mg/mL) was monitored by turbidimetric aggregometry. Traces shown are representative of 3 independent experiments. (ii) Platelets were added to collagen-coated coverslips and allowed to settle on the surface and adhere to collagen fibers under static conditions. Images shown are of single representative platelets from at least 3 independent experiments, either prior to adhesion (suspension) or at time points of activation in contact with collagen either at an early stage where filopodia are predominant or at a late stage where lamellipodia are predominant. (iii) The activity of secramine A was investigated by cdc42 pull-down using GST-tagged PAK-PBD protein beads. Washed human platelets were either not stimulated or stimulated with collagen (30 mg/mL) in the absence or presence of secramine A (3 or 30 μM, 15 minutes of preincubation). Cells were lysed and pull-downs performed as described in “Materials and methods,” and the amount of GTP-loaded cdc42 pulled down was assessed by immunoblot. As a positive control, platelet extracts were treated with GTPγS (200 μM), while platelets treated with GDP (1 mM) were used as negative control. Images shown are representative of 3 independent experiments. (B) Washed human platelets were preincubated for 15 minutes with either rottlerin (5 μM) or DMSO as control (vehicle) as indicated. In some experiments washed human platelets were also preincubated for 15 minutes with apyrase (0.2 U/mL), A3P5P (1 mM), and AR-C69931MX (1 μM) as indicated (ADP inhib.). Platelets were added to collagen-coated coverslips and allowed to settle on the surface and adhere to collagen fibers under static conditions. (i) Images shown are for platelets allowed to adhere to the collagen-coated surface for 40 minutes in the presence or absence of the various inhibitors as indicated. These images are representative of 5 independent experiments. The perimeter (ii) and the number (iii) of adherent platelets were measured 0, 10, 20, 30, or 40 minutes after being dispensed onto the collagen-coated surface. Rottlerin induced a statistically significant increase in the perimeter of adherent platelets (P < .01), while no statistically significant difference was detected (P = .86) for the number of adherent platelets. Incubation with ADP receptor antagonists did not influence the platelet perimeter (P > .01) but significantly reduced the number of adherent platelets (P < .01). Data shown are mean ± SEM from at least 3 independent experiments.

PKCδ negatively regulates filopodia formation in human platelets. (A) Washed human platelets were preincubated with secramine A at the concentrations indicated for 15 minutes. (i) Platelet aggregation in response to collagen (30 mg/mL) was monitored by turbidimetric aggregometry. Traces shown are representative of 3 independent experiments. (ii) Platelets were added to collagen-coated coverslips and allowed to settle on the surface and adhere to collagen fibers under static conditions. Images shown are of single representative platelets from at least 3 independent experiments, either prior to adhesion (suspension) or at time points of activation in contact with collagen either at an early stage where filopodia are predominant or at a late stage where lamellipodia are predominant. (iii) The activity of secramine A was investigated by cdc42 pull-down using GST-tagged PAK-PBD protein beads. Washed human platelets were either not stimulated or stimulated with collagen (30 mg/mL) in the absence or presence of secramine A (3 or 30 μM, 15 minutes of preincubation). Cells were lysed and pull-downs performed as described in “Materials and methods,” and the amount of GTP-loaded cdc42 pulled down was assessed by immunoblot. As a positive control, platelet extracts were treated with GTPγS (200 μM), while platelets treated with GDP (1 mM) were used as negative control. Images shown are representative of 3 independent experiments. (B) Washed human platelets were preincubated for 15 minutes with either rottlerin (5 μM) or DMSO as control (vehicle) as indicated. In some experiments washed human platelets were also preincubated for 15 minutes with apyrase (0.2 U/mL), A3P5P (1 mM), and AR-C69931MX (1 μM) as indicated (ADP inhib.). Platelets were added to collagen-coated coverslips and allowed to settle on the surface and adhere to collagen fibers under static conditions. (i) Images shown are for platelets allowed to adhere to the collagen-coated surface for 40 minutes in the presence or absence of the various inhibitors as indicated. These images are representative of 5 independent experiments. The perimeter (ii) and the number (iii) of adherent platelets were measured 0, 10, 20, 30, or 40 minutes after being dispensed onto the collagen-coated surface. Rottlerin induced a statistically significant increase in the perimeter of adherent platelets (P < .01), while no statistically significant difference was detected (P = .86) for the number of adherent platelets. Incubation with ADP receptor antagonists did not influence the platelet perimeter (P > .01) but significantly reduced the number of adherent platelets (P < .01). Data shown are mean ± SEM from at least 3 independent experiments.

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