Retroviral β3 expression restores bidirectional signaling in β3-deficient platelets. (A) Schematic representation of the retroviral vector used to express both integrin β3 and GFP in β3-deficient hematopoietic cells. LTR indicates long terminal repeat; itgβ3, coding region of the mouse β3 cDNA; IRES, internal ribosomal entry site; GFP, green fluorescent protein; pA, SV40 polyadenylation signal. (B) Retroviral expression of β3 integrins. β3-deficient fetal liver cells exposed to the retroviral vector shown in panel A or a control vector expressing only GFP were used to reconstitute lethally irradiated mice. Shown is the expression of β3 integrins in β3-deficient (β3 null) platelets, wild-type (WT) platelets, platelets from β3-deficient cells exposed to vector expressing GFP only (β3 null + vector), and platelets from β3-deficient cells exposed to vector expressing wild-type β3 and GFP (β3 null + β3). Dot plots show GFP vsersus β3 expression in individual platelets. The histograms show β3 expression in wild-type and β3 null platelets and in GFP-positive platelets from reconstituted animals. MFI indicates mean fluorescent intensity. (C) Retroviral expression of β3 integrins restores inside-out signaling in β3-deficient platelets. Fibrinogen binding was measured in the platelets described in panel B with (blue lines) and without (red lines) ADP stimulation and binding by soluble Alexa-Fluor 647–conjugated fibrinogen was measured. (D) Retroviral expression of β3 integrins restores outside-in signaling in β3-deficient platelets. The platelets described in panel B were exposed to immobilized fibrinogen and platelet spreading observed at 45 minutes. Platelets were visualized using Alexa-Fluor 594–conjugated phalloidin to detect cellular actin and GFP to detect platelets with retroviral expression of β3 integrins.