Validation of platelet GPVI signaling relations and functional responses. (A) Replicate samples (n = 3) of washed human platelets (5 × 10⁸/mL) were incubated with inhibitors (10 μM, unless noted) against Src family kinases SFKs (PP2), Syk (entospletinib), BTK (acalabrutinib), PKCα (Ro 31-8220), PKA (H89), p38 (SB202190, 2 μM), ERK1/2 (LY3214996), and GSK3 (CHIR 99021). After stimulation with CRP-XL (10 μg/mL, 37°C, 5 minutes), lysates were examined by western blot for phosphorylation of sites of interest (see supplemental Table 3 for additional details regarding inhibitor specificities). (B) Quantitative summary of effects of inhibitors on site-specific phosphorylation as determined by western blot above. Color legend indicates fold-change (f.c.) relative to +CRP-XL condition. Gray dots indicate false discovery rate (FDR) by size relative to +CRP-XL condition. (C) Summary of effects of pathway node and effector inhibition on GPVI-mediated platelet adhesion, ADP secretion, α-granule secretion, and integrin activation, as well as platelet spreading on fibrinogen (supplemental Figure 14). (D) Summary of validated platelet GPVI signaling relations in platelet function following from biochemical and physiological experiments previous. From all combinations of inhibitor-antibody pairs, 16 of 22 inferred relations were directly validated (green arrows). Other validated pairs can also be explained by a multistep path in the inferred model (supplemental Figure 15). Shaded circles indicate supporting experimental evidence for specific pathway nodes in GPVI-mediated platelet adhesion (blue), secretion (red), and integrin activation (green), and combinations thereof, colored according to function (gray = associated with all functions tested).