GPVI-dependent signaling contributes to platelet secretion and prevention of bleeding during the cutaneous rpA reaction. (A) Plasma serotonin levels in control (100% platelets), thrombocytopenic (<3% platelets), and GPVI^−/− mice that were subjected or not to the cutaneous rpA reaction. n = 5-8 mice per group. # indicates a significant difference (P < .002) as compared with plasma serotonin level in unchallenged control wild-type mice. (B) GPVI^−/− mice were transfused with 9 × 10⁶ green-fluorescent calcein-AM–labeled wild-type platelets that were treated or not with the Syk inhibitor R406 and the Btk inhibitor ibrutinib, as indicated. The cutaneous rpA reaction was then elicited, and the recruitment of transfused platelets to the reaction site was quantified by measurement of green fluorescence intensity in extracts from skin biopsy specimens harvested after 4 hours of reaction. (C) Hemoglobin content in skin biopsy specimens from control and rpA reaction areas from GPVI^−/− mice and from GPVI^−/− mice that were transfused with control or Syk/Btk-inhibitor–treated wild-type platelets. n = 4-8 mice per group. # indicates a statistically significant difference (P < .05) in hemoglobin content as compared with control spots from GPVI^−/− mice transfused with control wild-type platelets. Representative pictures of the skin at the site of the rpA reaction are shown below. NS, not significant; WT, wild-type.