human integrin αvβ3 (Millipore, Temecula, CA) were employed. Single-molecule atomic force microscopy (AFM) adhesion maps and force-distance curves were obtained using an AsylumMFP-3D-BIO (Asylum Research, Santa Barbara, CA) AFM equipped with a “liquid cell” setup. Baseline measurements were performed in PBS solutions at room temperature. Epinephrine-mediated experiments were performed in a 10 nM epinephrine solution at room temperature. Unless otherwise specified, all force measurements were recorded with a loading rate of 24000 pN/s, calculated by multiplying the tip retraction velocity (nm/s) by the spring constant of the cantilever (pN/nm). The nominal spring constant kc of the employed cantilever was 30 pN/nm, as provided by the manufacturer. Exact values for the cantilever spring constants were obtained via a thermal noise based method implemented by the manufacturer and were used in all calculations. The approach and retraction velocities were held constant at 800 nm/s. Probes had nominal tip radii of 20 nm and nominal angle of 20°, as provided by the manufacturer. RESULTS To test the hypothesis that an increase in epinephrine levels results in an increase in the magnitude of the adhesive force associated with BCAM/Lu-laminin and ICAM-4-αvβ3, as well as an increase in the frequency of adhesive interactions, we employed an approach in which the AFM probe was functionalized with LAMA5 or αvβ3 to measure the interaction between the α5 chain of laminin and BCAM/Lu receptor expressed on the tested RBC, or the interaction between integrin αvβ3 and ICAM-4 receptor expressed on the tested RBC. Treatment of normal RBCs with 10 nM epinephrine, a physiologically relevant level, induced a significant increase from 6.0% to 10.2% (p < 0.02) in the frequency of BCAM/Lu-laminin binding events (n = 6), and an insignificant increase from 5.3% to 7.6% (p > 0.05) in the frequency of ICAM-4-αvβ3 binding events (n = 7). There was no notable increase in the bond strength of either pair with epinephrine stimulation. Treatment of sickle trait RBCs (n = 7) with epinephrine induced an increase in the frequency of BCAM/Lu-laminin binding events of from 9.3% to 14.0% (p < 0.04), as well as a significant increase (p < 0.002) in the magnitude of the BCAM/Lulaminin interaction. Treatment of sickle trait RBCs (n = 7) with epinephrine induced an increase in the frequency of ICAM-4αvβ3 binding events from 7.9% to 13.1% (p < 0.05), as well as an increase (p < 0.02) in the magnitude of the ICAM-4-αvβ3 interaction. Notably, 500 nm x 500 nm spatially-resolved adhesion maps revealed that the BCAM/Lu-mediated laminin binding activity is not homogeneously distributed over the SCT RBC surface, but concentrated on nanodomains. These nanodomains, often attributed to membrane rafts, are only observed on SCT RBCs (n = 7) in the presence of epinephrine Figure 1. Distribution of BCAM/Lu and ICAM-4 on the surface of normal RBCs and sickle cell trait RBCs using different tips and cell preparations. Adhesion force maps were created via a MATLAB script from data obtained from force-distance curves obtained via AFM. The figure was then compiled using CorelDRAW X4. (A,B) Adhesion force maps (gray scale 0100 pN) recorded with LAMA5 tips over normal RBCs (A) and sickle cell trait RBCs (B) in PBS. (C,D) Adhesion force maps recorded with LAMA5 tips over normal RBCs (C) and sickle cell trait RBCs (D) in 10 nM epinephrine. (E,F) Adhesion force maps recorded with αvβ3 tips over normal RBCs (E) and sickle cell trait RBCs (F) in PBS. (G,H) Adhesion force maps recorded with αvβ3 tips over normal RBCs (G) and sickle cell trait RBCs (H) in 10 nM epinephrine. Green arrows indicate nanodomains. Scale bars, 100 nm. 160
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