GPVI stimulation in human whole blood decreases cytokine production but increases platelet–leukocyte complex formation, leukocyte activation, and Klebsiella phagocytosis. (A-B) Heparin human whole blood with collagen-related peptide (CRP-XLl 1 µg/mL) or vehicle control was incubated with UV-irradiated K pneumoniae (106 CFUs) for 4 hours, and plasma was stored for cytokine measurements (A, IL-6; B, TNF-α). (C-D) Human whole blood was stimulated with the GPVI agonist CRP-XL for 30 minutes in the presence or absence of the BTK inhibitor ibrutinib, and neutrophil (C) or monocyte (D) activation was assessed by CD11b expression. (E) Isolated neutrophils were stimulated with CRP-XL for 30 minutes, and neutrophil activation was assessed by CD11b. (F-G) Human whole blood was stimulated with CRP-XL for 30 minutes in the presence or absence of the BTK inhibitor ibrutinib, and platelet–neutrophil (F) or platelet–monocyte (G) complexes were determined by flow cytometry. (H-J) Human whole blood was stimulated with CRP-XL for 30 minutes with Fab9012 or vehicle control, and platelet activation (H) and platelet–neutrophil (I) or platelet–monocyte (J) complex formation was assessed by flow cytometry. (K) Human whole blood was stimulated with TRAP for 30 minutes in the presence of Fab9012 (blocking GPVI), anti-PSGL-1, or vehicle control, and platelet–neutrophil complex formation was assessed. (L-M) Human whole blood with or without CRP-XL was incubated at 37°C with fluorescein isothiocyanate–labeled heat-inactivated Klebsiella for 20 minutes, after which phagocytosis was determined in neutrophils (L) or monocytes (M). Data show representative results for 4 out of 6 donors (n = 8 replicates per group for cytokines, and n = 4 replicates per group for fluorescence-activated cell sorting readouts). Experiments were performed at least twice with different donors. *P < .05 for CRP-XL vs unstimulated, CRP-XL vs CRP-XL+Fab9012, and TRAP vs TRAP+aPSGL-1; **P < .01 and ****P < .0001 vs control.
