Analysis of out-of-context targets reveals Rab GTPases as systematic targets in GPVI mediated platelet function. (A) STRING-derived interactome of 3723 platelet proteins in supplemental Table 1, grouped by Reactome pathways, including protein expression, secretion, metabolism, signaling, and others. Yellow diamonds represent proteins with significant phosphorylation changes that did not place into CausalPath models previously discussed. (B) Reactome pathway analysis of 1234 out-of-context targets reveals significant enrichment in signaling by Rho GTPases (Reactome Pathway identifier R-HSA-194315), Membrane trafficking (R-HSA-199991), Rab regulation of trafficking (R-HSA-9007101), and other pathways. (C) Rab system proteins with significant phosphorylation modifications identified in conditions 1 and 2 experiments (selected). (D) Rab7A S₇₂ phosphorylation reporter ion intensity for condition 2 experiments (n = 5). (E) Replicate samples (n = 3) of washed human platelets (5 × 10⁸/mL) were incubated with inhibitors against PKCα (Ro 31-8220), PKA (H89), LRRK2 (MLi-2, 1 μM), TAK1 ((5Z)-7-oxozeaenol, 10 μM), or vehicle alone (0.1% dimethyl sulfoxide) before stimulation with CRP-XL (10 minutes, 37°C) and lysis in Laemmli sample buffer. Lysates were separated by Phos-tag sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by western blot (WB) for Rab7 phosphorylation. Empty circle indicates migration position of phosphorylated Rab7. Filled circle indicates migration position of nonphosphorylated Rab7. Results representative of n = 3 experiments. (F) Superresolution structured illumination microscopy of Rab7 (green) localization in resting vs activated platelets. Wide field scale bar = 10 μm. Magnified scale bar = 2 μm.