Figure 4
Figure 4. Platelet phagocytosis requires integrity and function of the actin-based cytoskeleton. Neutrophils were incubated in the presence or the absence of activated platelets for 5 minutes at 37°C or 4°C. Phagocytosis of platelets, assessed by flow cytometry as described in “Adhesion and phagocytosis,” occurred at 37°C and abated in conditions in which the assembly of the actin-based cytoskeleton was prevented by pretreatment with cytochalasin D (CCD) or by nonpermissive temperatures (4°C). In these conditions, degranulation was also inhibited, as revealed by MPO content of neutrophils. Consistent results were observed by confocal microscopy: glass-adhered platelets were activated with thrombin (0.5 U/mL) for 2 minutes, washed, and incubated in the presence of purified autologous neutrophils. Platelets were revealed by staining with antiplatelet glycoprotein Ib mAbs (red) and neutrophils with anti-CD66b mAb (green). At 4°C, sample washings removed neutrophils that did not firmly adhere; at 37°C neutrophils are evident, whereas extracellular platelets were not any more detectable; in the presence of cytochalasin D, few scattered neutrophils and extracellular platelets are evident.

Platelet phagocytosis requires integrity and function of the actin-based cytoskeleton. Neutrophils were incubated in the presence or the absence of activated platelets for 5 minutes at 37°C or 4°C. Phagocytosis of platelets, assessed by flow cytometry as described in “Adhesion and phagocytosis,” occurred at 37°C and abated in conditions in which the assembly of the actin-based cytoskeleton was prevented by pretreatment with cytochalasin D (CCD) or by nonpermissive temperatures (4°C). In these conditions, degranulation was also inhibited, as revealed by MPO content of neutrophils. Consistent results were observed by confocal microscopy: glass-adhered platelets were activated with thrombin (0.5 U/mL) for 2 minutes, washed, and incubated in the presence of purified autologous neutrophils. Platelets were revealed by staining with antiplatelet glycoprotein Ib mAbs (red) and neutrophils with anti-CD66b mAb (green). At 4°C, sample washings removed neutrophils that did not firmly adhere; at 37°C neutrophils are evident, whereas extracellular platelets were not any more detectable; in the presence of cytochalasin D, few scattered neutrophils and extracellular platelets are evident.

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