Outside-in signaling initiated by platelet interaction with immobilized fibrinogen activates the FcγRIIa signal transduction pathway. (A) Schematic showing use of the FcγRIIa signaling pathway downstream of αIIbβ3 engagement. Note the importance of physical approximation of the integrin with FcγRIIa, allowing integrin-associated Src family members to function as ITAM kinases, thus initiating the outside-in signaling cascade. (B,C) Washed platelets were plated onto 8-chamber glass tissue-culture slides coated with either BSA or fibrinogen (Fg), and allowed to spread for 45 minutes in the presence or absence of 10 μg/mL IV.3 Fab Immunoprecipitation and Western blot analysis (B) reveals strong activation of FcγRIIa, Syk, and PLCγ2 after platelet binding to immobilized fibrinogen, and inhibition of this outside-in signaling pathway by Fab fragments of mAb IV.3. (C) Effect of IV.3 on platelet spreading. Preincubation of platelets with IV.3 Fab markedly inhibits platelet spreading on immobilized fibrinogen, shown in panels A through C, with quantitative analysis of the pixel area of spread platelets in panel. Data shown are the mean plus or minus SEM from one of 4 representative experiments using 3 different platelet donors.