Platelet granule release induces neutrophil polarization and motility. Neutrophils (2 × 106/mL) were incubated with platelet releasate or shed proteins generated by the indicated agonist. Neutrophils were then assessed for shape change and Mac-1 activation, as described in “Materials and methods.” (A) Representative DIC images demonstrating neutrophil shape change following incubation with platelet releasate (29 µg/mL) for 20 minutes at 37°C (red arrow, red blood cells; yellow arrow, neutrophils). (B-C) Relative potency of platelet releasate derived from ionophore A23187-stimulated platelets: (B) line graph demonstrating the dose-dependent effects of the releasate on neutrophil shape change and Mac-1 activation, and (C) representative fluorescence-activated cell sorter (FACS) profiles of Mac-1 activation for each indicated dose of releasate (protein per milliliter of neutrophils) and formyl methionyl leucyl phenylalanine (fMLP) (2 µM). (D) Neutrophils (2 × 106/mL) were incubated in the absence (Rest) or presence of platelet releasates generated by activating platelets with either ionophore (Iono, 2 μM), thrombin + collagen (Thr-collagen, 1 U/mL thrombin and 10 μg/mL collagen), thrombin alone (Thr, 1 U/mL), collagen alone (10 μg/mL), or adenosine diphosphate (ADP; 10 μM) for 20 minutes at 37°C as described in supplemental Methods. Platelet releasate-induced neutrophil shape change was quantified as described in “Materials and methods” (mean ± SEM; n = 3). (E) The neutrophil shape change activity in platelet releasate and surface-shed proteins was quantified as described in “Materials and methods” (mean ± SEM; n = 3). (F) The effect of shed proteins and platelet releasate on Mac-1 activation was also examined, as describe in supplemental Methods and compared with that achieved in response to fMLP (2 µM) (mean ± SEM; n = 3). FITC, fluorescein isothiocyanate; ns, not significant, P > .05; **P < .01; ***P < .001.
