![Fig. 6. Expression of scinderin in the MEG-01 cell line induced formation and release of platelet-like particles. / Cells transfected with either vector pcDNA3 (A,C) or vector (pcDNA3-Sc) carrying a full-length scinderin (Sc) cDNA insert (B, D) were cultured for 11 (A,B) and 23 (C,D) days and then were fixed and stained with Wright-Giemsa. After 23 days in culture, preparations of pcDNA3-Sc cells showed cytoplasmic areas smaller than those observed in panel B and many relatively uniform particles of dimensions similar to those of platelets. (E) Numerous cells in these cultures entered apoptosis as determined by the TUNEL assay. Bright green fluorescence indicates apoptotic nuclei and apoptotic bodies. Percentages of apoptosis after 4 and 23 days in culture are shown in panel F. Bars represent mean ± SEM of 5 different experiments (*P < .01). Platelet-like particles were purified as described in “Materials and methods” and were treated with 1 U thrombin/mL. This induced aggregation, as shown, after fixation and staining with Wright-Giemsa, before (G) and 6 minutes after (H, I) the addition of thrombin. (I) At-large magnification of the particle aggregate contained within the box shown in panel H. (J) Decrease in light absorbance of the same preparation. (K) Fibrinogen plus CaCl2 induced the aggregation of platelet-like particles pre-incubated for 5 minutes with 1 U thrombin/mL in the absence or presence of 40 μg PAC-1 antibody/mL. (L) Thrombin-induced aggregation was significantly inhibited in the presence of a CD41a (fibrinogen receptor) antibody, and (M) all particles present in the preparation showed intense fluorescence when stained with the same antibody. Tubulin antibody staining showing a similar array of microtubules in normal human platelets (N) and platelet-like particles (O). (P) Platelet-like particles were incubated with 1 μM serotonin for 60 minutes, fixed in glutaraldehyde, and processed for electron microscopy. The micrograph shows dense bodies within the cytoplasm. Platelet-like particles were also incubated with 10-8 M [3H]5-HT as indicated in “Materials and methods,” and serotonin uptake was measured in the absence or presence of 6 nM fluoxetine (Q). Bars represent mean ± SEM from 8 preparations (**P < .05). Treatment of [3H]5-HT-labeled particles with 1 U thrombin/mL for 2 minutes also induced the release of amines (R). Bars represent mean ± SEM of 8 preparations (*P < .001).](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/98/7/10.1182_blood.v98.7.2210/5/h81911604006.jpeg?Expires=1735522116&Signature=5Ma7Q4vV0Sj4A4QJWDZkwyMJC8sKQsmhjEKPvpHL7kCCXVbnscS4gLcpR8zkcosC2Eh0gzQgh6fXmOCbtlkFL6Pa0Jb7aTygdyXV7WGI9j-CfB9cGZZVRlsoHePVhERInpobHloPyz91X2WbEgRoC3RWAGRXnAvhSHbXzlRRCYNnQ744NJaSLuqfI-6bxGAahikEpYeKvJqGWFsekVDk-98BbpfjBbYrUMP0diwqmk8Ol7Y1Z~Xi-5qr59~vpnNAqNKE0m2hWn6JcR83DXSBmuYDTXwpUl4~E3QWZ2EygwIX5crpS5~lF5cYnNa-N1raj3rH42bQCYhNvx~Qvy~NNA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Expression of scinderin in the MEG-01 cell line induced formation and release of platelet-like particles.
Cells transfected with either vector pcDNA3 (A,C) or vector (pcDNA3-Sc) carrying a full-length scinderin (Sc) cDNA insert (B, D) were cultured for 11 (A,B) and 23 (C,D) days and then were fixed and stained with Wright-Giemsa. After 23 days in culture, preparations of pcDNA3-Sc cells showed cytoplasmic areas smaller than those observed in panel B and many relatively uniform particles of dimensions similar to those of platelets. (E) Numerous cells in these cultures entered apoptosis as determined by the TUNEL assay. Bright green fluorescence indicates apoptotic nuclei and apoptotic bodies. Percentages of apoptosis after 4 and 23 days in culture are shown in panel F. Bars represent mean ± SEM of 5 different experiments (*P < .01). Platelet-like particles were purified as described in “Materials and methods” and were treated with 1 U thrombin/mL. This induced aggregation, as shown, after fixation and staining with Wright-Giemsa, before (G) and 6 minutes after (H, I) the addition of thrombin. (I) At-large magnification of the particle aggregate contained within the box shown in panel H. (J) Decrease in light absorbance of the same preparation. (K) Fibrinogen plus CaCl2 induced the aggregation of platelet-like particles pre-incubated for 5 minutes with 1 U thrombin/mL in the absence or presence of 40 μg PAC-1 antibody/mL. (L) Thrombin-induced aggregation was significantly inhibited in the presence of a CD41a (fibrinogen receptor) antibody, and (M) all particles present in the preparation showed intense fluorescence when stained with the same antibody. Tubulin antibody staining showing a similar array of microtubules in normal human platelets (N) and platelet-like particles (O). (P) Platelet-like particles were incubated with 1 μM serotonin for 60 minutes, fixed in glutaraldehyde, and processed for electron microscopy. The micrograph shows dense bodies within the cytoplasm. Platelet-like particles were also incubated with 10-8 M [3H]5-HT as indicated in “Materials and methods,” and serotonin uptake was measured in the absence or presence of 6 nM fluoxetine (Q). Bars represent mean ± SEM from 8 preparations (**P < .05). Treatment of [3H]5-HT-labeled particles with 1 U thrombin/mL for 2 minutes also induced the release of amines (R). Bars represent mean ± SEM of 8 preparations (*P < .001).
