TLT-1 promotes neutrophil release from platelets during transmigration. (A-B) In transwell plates, WT or treml1^−/− neutrophils were exposed to resting or thrombin-activated (0.05 U/mL) WT platelets (A) or treml1^−/− platelets (B), and transmigration of neutrophils was measured by flow cytometry. Shown are representative flow cytometry plots. Neutrophils were measured by Gr1fitc (y-axis), and platelets were identified by Gp1b Alexa 647 (x-axis). (C) Measurement of neutrophil cell death plotted as the number of neutrophils (gated on the red boxes in panels A and B) that stained positive for the cell impermeable PI dye (n = 3; **P ≤ .001). Quantification of the transmigrated neutrophils (D-E) and platelet-neutrophil conjugates (F-G) from panels A and B. (H) Flow cytometric analysis of WT neutrophils incubated with WT platelets in the presence of nonimmune antiserum (i) or anti-TLT-1 (ii). (I) Analysis of treml1^−/− neutrophils incubated with treml1^−/− platelets in the presence of supernatant from mock transfected cells (i) or supernatant from sTLT-1 transfected cells (ii, n = 4). Quantifications of transmigration from panels H and I of transmigrated WT (J) or treml1^−/− (K) neutrophils. Quantifications of platelet-neutrophil aggregates from panels H and I from WT (L) or treml1^−/− cells (M). *P ≤ .05, **P ≤ .01; Student t test. All cytometric quantifications were done using BD Accuri C6 software (BD Biosciences).