Progression of GPVI-mediated platelet responses. (A) Replicate samples (n = 3) of washed human platelets (2 × 10⁸/mL) were incubated with apyrase (2 U/mL), indomethacin (10 μM), Integrilin (20 μg/mL), and a combination thereof (“+all”) or vehicle alone (“veh”). Following the addition of the GPVI-specific agonist crosslinked collagen-related peptide (CRP-XL, 10 μg/mL) at t = 0 (arrow), platelet suspensions were monitored by Born light transmission aggregometry under stirring conditions at 37°C. Representative aggregation traces are shown (left) and quantified for percent aggregation relative to control (right, *P < .01). (B) Replicate samples (n = 3) of washed human platelets (2 × 10⁸/mL) were incubated with apyrase, indomethacin, Integrilin, or vehicle alone before stimulation with 10 μg/mL CRP-XL in the presence of fluorescently labeled antisera against the active conformation of human integrin α_IIbβ₃ (FITC-PAC-1). After 30 minutes, samples were analyzed by flow cytometry. The percentage of FITC-PAC-1⁺, platelet-gated events per condition is shown (**P < .001; *P < .05; for indomethacin, P = .0850). (C) Platelets as prepared above were fixed in paraformaldehyde, permeabilized, and stained for filamentous actin with TRITC-phalloidin before flow cytometry analysis. Mean fluorescence intensity (MFI) for TRITC-phalloidin stained platelet-gated events are shown. (*P < .001). (D) Replicate samples (n = 3) of washed human platelets (2×10⁸/mL) were incubated with P2Y1 and P2Y12 inhibitors (“+P2Yi”; 10 μM MRS 2179 and 10 μM AR-C 66096), indomethacin, or a combination thereof (“+all”) or vehicle alone. Following the addition of Chrono-Lume reagent and CRP-XL, ATP-dependent luciferase luminescence (lumin.) was measured as an indicator of platelet ADP release and δ-granule secretion (*P < .0001). a.u., arbitrary units. (E) Platelets were prepared as previously, before stimulation with 10 μg/mL CRP-XL in the presence of anti-CD62P-APC. After 30 minutes, samples were analyzed by flow cytometry. The percentage of CD62P⁺ platelet-gated events per condition quantifies platelet α-granule secretion (*P < .01). (F) Replicate samples of washed human platelets (5 × 10⁸/mL) were prepared as described before stimulation with CRP-XL. After 5 minutes, platelets were collected into Laemmli sample buffer, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and analyzed for phosphorylation of consensus tyrosine kinase, PKC, Akt, and MAPK substrates motifs by western blot. Blot lane pixel intensities as measured in Image J are graphed above respective lanes as fold change relative to control. Significance (P) of intensity measurements are shown for analysis of variance test for trend. Tick marks on right side of each blot panel show positions of molecular weight (MW) markers for 4G10 (top to bottom: 150, 75, 60 kD), PKC substrates (180, 130, 100, 75, 50 kD), Akt substrates (75, 63, 45, 35 kD), and MAPK substrates (150, 100, 80 kD). Additional experiments also supported roles for ADP release, thromboxane generation, and integrin activation in GPVI-mediated platelet adhesion (supplemental Figure 3). Although platelet thromboxane generation was eliminated by indomethacin, neither apyrase nor Integrilin significantly affected platelet TxB₂ levels following 10 μg/mL CRP-XL treatment, as determined by enzyme-linked immunosorbent assay (supplemental Figure 4).