(A) Experimental flowchart of plasma injection assay. (B) Scratching bouts from WT mice injected with either vehicle- or ANIT-treated plasma from WT and BVR KO animals. For the vehicle plasma cohort: n = 7. For cholestatic ANIT-treated plasma: WT n = 10 and BVR KO n = 8. (C) Scratching bouts from either WT or A1 KO mice injected with control donor plasma. Ctrl 1, WT n = 5, A1 KO n = 5. Ctrl 2, WT n = 5, A1 KO n = 5. Ctrl 3, WT n = 5, A1 KO n = 4. Ctrl 4, WT n = 4, A1 KO n = 5. Ctrl 5, WT n = 4, A1 KO n = 4. Ctrl 6, WT n = 4, A1 KO n = 4. (D) Scratching bouts from either WT or A1 KO mice injected with either cholestatic patient plasma. Patient 1, WT n = 7, A1 KO n = 9. Patient 2, WT n = 8, A1 KO n = 5. Patient 3, WT n = 7, A1 KO n = 8. Patient 4, WT n = 6, A1 KO n = 8. Patient 5, WT n = 5, A1 KO n = 6. Patient 6, WT n = 7, A1 KO n = 5. (E) Scratching bouts from either WT or A1 KO mice injected with plasma collected from all tested control donor samples and all tested cholestatic patient samples. For Control: WT n = 27, A1 KO n = 27. For cholestatic patient: WT n = 41, A1 KO n = 41. (F–G) Biochemical characteristics of patient and control plasma. (F) Age, sex, and bilirubin levels of control plasma collected. All control plasma donors did not suffer from any chronic itch condition. (G) 5D itch questionnaire was administered at time of plasma collection. Asterisk denotes patients taking anti-pruritic medication at time of plasma collection and questionnaire administration. Patient one was taking sertraline (100 mg QD) and Patient four was taking Gabapentin (800 mg TID). A score of 25 represents the maximum level of itchiness. (H) Scratching bouts from mice injected with either untreated (NT) control human plasma, FeCl3-treated control human plasma, NT cholestatic patient 1 and 4 plasma (same data from Patient WT data in (D), or FeCl3-treated patient plasma. For control plasma, NT n = 6 and FeCl3n = 5. For Patient one plasma, NT n = 7 and FeCl3n = 7. For Patient four plasma, NT n = 7 and FeCl3n = 6. (I) Scratching bouts from mice injected with either normal rabbit IgG –treated patient plasma or anti-bilirubin IgG – treated patient plasma. For Patient 1, Normal IgG n = 5, Anti-bilirubin n = 7. For Patient 4, Normal IgG n = 5, Anti-bilirubin n = 6. (B–I) Mean ±s.e.m. depicted. Open circles represent independent data points. n.s., not significant; *, p<0.05; **, p<0.01; ***, p<0.001 by unpaired two-tailed Student’s t-test.