Purification, identification, and characterization of NAP-2 as the major platelet-derived chemokine inducing neutrophil shape change and polarization. (A) Platelet releasate (3.42 mg, 4,000 units activity) was applied to a Heparin HP Column, bound proteins (gray line)and eluted with an NaCl gradient (0-2 M; green line); active fractions (shaded area) were identified by using the neutrophil shape change assay. Eighty-three percent of the original activity was recovered in two fractions co-eluted with NAP-2 as demonstrated by immunoblot analysis using anti–NAP-2/CTAP-III and anti-PF4 Abs. (B) Platelet releasate was subjected to 2 rounds of NAP-2 immunodepletion by using the anti–NAP-2/CTAP-III Ab, as detailed in supplemental Methods. The activity in the starting material (SM) and NAP-2–depleted releasate (first and second IP) was assessed as described above (mean ± SEM; n = 3; P < .01). Samples were also subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blotting analysis for NAP-2 (inset). (C) Neutrophils (2 × 10⁶/mL) were first treated for 10 minutes with buffer (Control), serine protease inhibitor leupeptin (10 μg/mL), His-EB22.4 (10 μg/mL), or phenylmethanesulfonylfluoride (PMSF; 1 mM), and then incubated with platelet releasate (15 μg/mL) for 20 minutes at 37°C. The percentage of cells undergoing shape change was analyzed by DIC microscopy (percentage of total cells per field; mean ± SEM; n = 3). (D) Dose-dependent effects of recombinant NAP-2 (NAP-2_Rec) on neutrophil shape change. (E-F) Regulation of Mac-1 activation through platelet–leukocyte cross-talk. (E) Relative potency of intact activated platelets (activated platelet + releaseate) versus MP-free releasate or MP-rich platelet releasate at inducing increased Mac-1 expression on the surface of isolated neutrophils. Intact activated platelets, MP-rich releasates, and MP-free releasates were prepared as described in supplemental Methods, equalized for volume, and incubated with washed neutrophils (2 × 10⁶/mL) at the indicated platelet:neutrophil ratios for 30 minutes at 37°C. Mac-1 expression was assessed by FACS using an anti-human CD11b (ICRF44) Ab (mean ± SEM; n = 3). Representative FACS histogram profiles of Mac-1 expression at a platelet:neutrophil ratio of 30:1 are depicted on the right. (F) Neutrophils (2 × 10⁶/mL) were treated with dimethylsulfoxide (Control), CXCR1/2 antagonist MSGA 8-73 (5 μM; CXCR1/2 Antag), or PAF antagonist CV-3988 (10 μM; PAF Antag) for 10 minutes at 37°C. The effect of PAF and CXCR1/2 antagonists on activated platelets induced Mac-1 activation and was assessed by using the CBRM1/5-FITC Ab (platelet:neutrophil ratio of 10:1) (mean ± SEM; n = 3). ns, not significant, P > .05; **P < .01; ***P < .001.