CCP-224 inhibits platelet-neutrophil aggregation in SCD patient blood. (A-B) Human blood was flown through microfluidic channels presenting P-selectin, ICAM-1, and IL-8, and platelet-neutrophil interactions were assessed by using qMFM. qMFM images showing platelets (green circles) interacting with arrested neutrophils (purple polygons) in the blood of SCD patient 1 (A) and patient 2 (B) following treatment with 10 µg/mL of CCP-224 (top row) or control peptide (bottom row). Neutrophils were stained with Alexa Fluor 647 anti-human CD16 Ab (purple), and platelets were stained with fluorescein isothiocyanate anti-human CD49b Ab (green). qMFM images were recorded by using a Nikon Eclipse Ti inverted microscope equipped with a Zyla-5.5 sCMOS scientific camera and CFI Apochromat TIRF 60× oil objective (numerical aperture: 1.49). All microscope functions and image analyses were conducted by using NIS-Elements software. Borders of platelets are marked with green circles. The arrow indicates the direction of flow. Scale bars, 20 µm. The wall shear stress was 6 dyn/cm². See supplemental Methods for details. (C-D) Pre- and posttreatment paired analyses showing the effect of CCP-224 (C) or control peptide (D) treatment on total platelet-neutrophil interactions in SCD patient blood. (E) Platelet interactions per arrested neutrophil over a 2-minute observation period pre– and post–CCP-224 treatment in SCD human subject blood. (F-G) Pre- and posttreatment paired analyses showing the effect of CCP-224 (F) or control peptide (G) on control (African American as well as white) subject blood. (H) Platelet interactions per arrested neutrophil over a 2-minute observation period pre– and post–CCP-224 treatment in healthy control (African American as well as white) human subject blood. In panels E and H, 5 neutrophils were randomly selected per experiment, and the number of platelets interacting with each neutrophil were counted. Each data point in panels E and H corresponds to interactions with an individual neutrophil; mean ± SE. (I-J) Pre- and posttreatment paired analyses showing the effect of CCP-224 on the total number of arrested neutrophils in healthy control (I) and SCD (J) human blood. (K) Cumulative probability distribution of the lifetime of platelet-neutrophil interactions pre– and post–CCP-224 treatment in SCD human blood. The lifetime of 10 randomly selected platelet-neutrophil interactions were measured in each experiment. Pre- and posttreatment data points connected by a straight line in panels C-D, F-G, and I-J represent paired data from an individual experiment. Blood samples from 3 SCD and 3 control (2 African American and 1 white) human subjects were used. Two to 3 independent experiments were performed per subject. Closed circles, open circles, and open triangles in panels C-D and J represent independent experiments performed with SCD patient 1, 2, and 3, respectively. Closed circles, open circles, and open triangles in panels F-G and I represent independent experiments performed with control subject 1 (African American), 2 (African American), and 3 (white), respectively. ^#P < .05 post- vs pretreatment.