De novo synthesis of TF by platelets and PBMCs. Platelets or PBMCs with or without pretreatment with puromycin, actinomycin D, DRB, or tunicamycin were incubated with [³⁵S]-methionine, and activated with TRAP or LPS. The platelet membranes before and after stimulation were immunoprecipitated with anti-TF antibodies and subjected to SDS-PAGE electrophoresis, protein transfer, and autoradiography. (A) TF immunoprecipitated from platelet membranes with polyclonal α-TF Ab. Nonstimulated platelets reveal a dominant [³⁵S]-TF band of approximately 47 kDa (lane 1). This pattern is almost identical to that of lane 3 (1-hour preincubation with 10 ng/mL tunicamycin). TRAP activation induces a new band (approximately 60 kDa), indicated by the asterisk, that is also unaffected by tunicamycin (lanes 2 and 4). When the incubation with tunicamycin was extended for 2 hours in nonactivated (lane 7) and activated (lane 8) platelets, the same results shown in lanes 3 and 4 were obtained. Puromycin abolishes all neosynthesis in TRAP-activated platelets (lane 6). In nonstimulated samples, puromycin induces a marked inhibition of the approximately 47-kDa TF, and abolishes all the remaining bands (lane 5). (B) the membrane TF of nonstimulated and TRAP-activated platelets was now immunoprecipitated with the α-TF MoAb. Lane 1 shows a modest incorporation of [³⁵S]-methionine into TF in nonstimulated platelets, and the puromycin effect is shown in lane 2. Lane 3 shows the enhancement of the approximately 47-kDa TF neosynthesis after 30 minutes of TRAP activation, with a new approximately 60-kDa immunoreactive TF species. This band was abolished by puromycin, and the approximately 47-kDa band is considerably reduced (lane 4). (C) PBMC preparations were incubated with [³⁵S]-methionine while activated with LPS for 5 hours. The flow cytometry of this sample showed that 17% and 10% of all events were CD14⁺ and CD61⁺, respectively. After cell lysis and centrifugation (18 000g for 15 minutes) the membrane (lanes 1-3) and supernatant (lanes 4-6) fractions were immunoprecipitated. With this prolonged stimulation, most of the radioactivity is recovered in the membrane fraction. The predominant bands in nonstimulated cells are of approximately 47 kDa and approximately 35 kDa, which were enhanced after LPS (lane 3) and suppressed by puromycin (lane 2). In the supernatant, the unstimulated PBMCs exhibit a predominant band of approximately 47 kDa (lane 4) which, upon stimulation, is slightly amplified with appearance of new bands, predominantly 1 of approximately 35 kDa (lane 5). All the bands are abolished by puromycin (lane 6).