Plasma-driven broad-spectrum protein synthesis in human platelets. (A) Platelet-rich plasma was treated with vehicle (−) or azidohomoalanine (AHA) in the absence (−) or presence (+) of puromycin (Puro, 10 μg/mL), and platelets were isolated at the indicated times, washed, and lysed. NSPs were labeled with biotin-alkyne and detected with streptavidin conjugate, and membranes were counter-stained with β-actin antibodies, as shown. (B) Washed human white blood cell/red blood cell–depleted platelets were either resuspended in autologous plasma–EDTA (platelet-poor plasma [PPP]; EDTA added to inhibit platelet activation) or SB followed by feedback with AHA and plasma or buffer as indicated, then harvested and processed for NSP detection as described earlier. (C) AHA (0.5 mg/g) with/without Puro (20 mg/kg) was injected into tail veins of wild-type (WT) mice, and blood was extracted after 1 hour for platelet isolation and platelet NSP detection. (D) Puromycin was administered as in panel C in the absence of AHA, platelets were collected at the indicated times, and platelet lysates were subject to immunoblotting with puromycin-specific antibodies. Each panel representative of 3 independent experiments.