Fig. 5.
FcαRI-mediated TF pre-mRNA splicing and protein synthesis in platelets. (A) Cross-linking of platelet FcαRI induced mature TF mRNA production. Purified human platelets (5×108) were stimulated with anti-FcαRI mAb A59 F(ab′)2 plus goat anti-mouse IgG F(ab′)2 (Anti FcαRI-marked lanes), with fibrinogen plus thrombin (Fib+Thr-marked lanes), or with hIgA plus goat anti-hIgA F(ab′)2 (hIgA-marked lanes) for various periods of time (in min). Nonstimulated platelets were labeled as Non. Total RNA was isolated from platelet pellets and used for RT-PCR. The lower panel shows RT-PCR results of GAPDH (internal controls for equal amount of total RNA) at each time-point. The results were representative of at least four independent experiments. (B) Induction of TF protein production by clustering of FcαRI. The platelets (5×108) were stimulated in 24-well tissue-culture plates coated with hIgA, anti-FcαRI mAb A59 F(ab′)2 (Anti FcαRI), and fibrinogen plus thrombin, respectively. The platelets were incubated at 37°C in 5% CO2 for various periods of time (hours). Platelets in culture media were collected by centrifugation at various points of time (0, 1, 5, and 20 h) and lysed in 1× radioimmunoprecipitation assay buffer. Proteins in platelet lysates were separated by SDS-PAGE, and the production of TF was analyzed on Western blots. The results were representative of three independent experiments.