Essential role for phosphoinositide 3-kinase in shear-dependent signaling between platelet glycoprotein Ib/V/IX and integrin αIIbβ3 by Cindy L. Yap, Karen E. Anderson, Sascha C. Hughan, Sacha M. Dopheide, Hatem H. Salem, and Shaun P. Jackson Blood Volume 99(1):151-158 January 1, 2002 ©2002 by American Society of Hematology

The role of PI 3–kinase in regulating platelet adhesion and integrin αIIbβ3 activation under static conditions and the effect of apyrase on VWF-induced integrin αIIbβ3 activation.Washed platelets (1.5 × 108/mL) were incubated with vehicle alone (control) or... The role of PI 3–kinase in regulating platelet adhesion and integrin αIIbβ3 activation under static conditions and the effect of apyrase on VWF-induced integrin αIIbβ3 activation.Washed platelets (1.5 × 108/mL) were incubated with vehicle alone (control) or the indicated concentrations of LY294002 (0-25 μM) or wortmannin (0-100 nM) for 15 minutes or with apyrase (16 U/mL) for 30 minutes. Platelets were allowed to adhere and spread on immobilized human VWF (10 μg/mL) for 60 minutes under static conditions. In panel B, pretreated platelets were incubated with PAC-1 antibody, prior to adhesion and spreading. These results demonstrate (A) the effect of LY294002 or wortmannin on the level of platelet adhesion, and (B) the effect of LY294002, wortmannin, or apyrase on PAC-1 binding as visualized by confocal microscopy (× 63 W objective; bar = 10 μm). Statistical analysis of the results was performed using a t test and the P values are indicated where appropriate (P < .01**). Results are the mean ± SEM of 3 experiments, and images are from a single experiment representative of 3. Cindy L. Yap et al. Blood 2002;99:151-158 ©2002 by American Society of Hematology

The role of PI 3–kinase in regulating platelet spreading under static conditions.Washed platelets (1.5 × 108/mL for panels A and B or 1.5 × 107/mL for panel C) were incubated with vehicle alone (control) or the indicated concentrations of LY294002 (0-25 μM)... The role of PI 3–kinase in regulating platelet spreading under static conditions.Washed platelets (1.5 × 108/mL for panels A and B or 1.5 × 107/mL for panel C) were incubated with vehicle alone (control) or the indicated concentrations of LY294002 (0-25 μM) or wortmannin (0-100 nM) for 15 minutes. These results demonstrate (A) the mean surface area of adherent platelets, and (B) the morphology of spread platelets as determined by scanning electron microscopy (bar = 2 μm). Statistical analysis of the results was performed using a t test and the P values are indicated where appropriate (P < .05*). Results are the mean ± SEM of 3 experiments, and images are from a single experiment representative of 3. In panel C, platelet spreading was visualized in real time by differential interference contrast microscopy and recorded on video for off-line analysis (× 100 oil objective; bar = 5 μm). The images presented are typical of cells from a single experiment and are representative of 3 independent experiments. Cindy L. Yap et al. Blood 2002;99:151-158 ©2002 by American Society of Hematology

The effect of LY294002 and wortmannin on VWF-induced generation of PtdIns(3,4)P2 and on integrin αIIbβ3 activation and spreading on immobilized fibrinogen.Washed platelets (1.5 × 108/mL) were incubated with vehicle alone (control) or the indicated concentra... The effect of LY294002 and wortmannin on VWF-induced generation of PtdIns(3,4)P2 and on integrin αIIbβ3 activation and spreading on immobilized fibrinogen.Washed platelets (1.5 × 108/mL) were incubated with vehicle alone (control) or the indicated concentrations of LY294002 (0-25 μM) or wortmannin (0-100 nM) for 15 minutes. (A-C) Platelets were suspended in Tyrode buffer (A, resting) and allowed to spread on immobilized VWF (10 μg/mL) for 60 minutes under static conditions (A, spreading: control and LY294002). Platelets in suspension or adherent on the matrix were subsequently lysed with RIPA buffer, lipids extracted and analyzed by SAX HPLC. These results demonstrate: (A) the generation of the PI 3–kinase lipid product, PtdIns(3,4)P2 (PI[3,4]P2), in resting platelets and in spread platelets pretreated with vehicle (control) or LY294002 (25 μM); (B) the level of inhibition of PtdIns(3,4)P2 production in spreading platelets pretreated with LY294002 (0-25 μM; solid line) or wortmannin (0-100 nM; dotted line); and (C) the effect of LY294002 and wortmannin on the levels of PtdInsP (PI), PtdIns(4)P (PI[4]P), and PtdIns(4,5)P2(PI[4,5]P2) in spreading platelets. (D) Pretreated platelets were incubated with PAC-1 antibody prior to adhesion and spreading on immobilized fibrinogen (100 μg/mL) for 60 minutes. Adherent platelets were fixed and stained with a FITC-conjugated secondary antibody prior to visualization by confocal microscopy (× 63W objective). (A-D) Images and graphs are from a single experiment representative of 3. Cindy L. Yap et al. Blood 2002;99:151-158 ©2002 by American Society of Hematology

The role of PI 3–kinase in regulating platelet calcium responses under static conditions.Calcium indicator dye-loaded platelets were incubated with vehicle alone (control), LY294002 (25 μM), or wortmannin (100 nM) for 15 minutes prior to adhesion on immobil... The role of PI 3–kinase in regulating platelet calcium responses under static conditions.Calcium indicator dye-loaded platelets were incubated with vehicle alone (control), LY294002 (25 μM), or wortmannin (100 nM) for 15 minutes prior to adhesion on immobilized VWF (10 μg/mL). Adherent platelets were allowed to spread in the presence of extracellular Ca++ (1 mM) for up to 60 minutes under static conditions. Changes in the cytosolic calcium concentration of adherent cells were monitored at the indicated time points by confocal microscopy (× 63W objective) and fluorescence ratios quantified. The results presented in panel A demonstrate the percentage of platelets undergoing oscillatory calcium transients at the indicated time points. Results represent mean ± SEM from 3 to 5 independent experiments. The results presented in panel B show a representative calcium oscillation profile of individual vehicle-treated (control) and wortmannin-treated platelets at 0 minute (thin line) and after 15 minutes of spreading (bold line). Statistical analysis was performed using a ttest comparing control versus LY294002- or wortmannin-treated platelets (P < .05*; P < .01**). Cindy L. Yap et al. Blood 2002;99:151-158 ©2002 by American Society of Hematology

The role of PI 3–kinase in regulating platelet adhesion, spreading, and integrin αIIbβ3activation under flow conditions.Washed platelets (1.5 × 108/mL) were incubated with vehicle alone (control) or the indicated concentrations of LY294002 (0-25 μM) or wort... The role of PI 3–kinase in regulating platelet adhesion, spreading, and integrin αIIbβ3activation under flow conditions.Washed platelets (1.5 × 108/mL) were incubated with vehicle alone (control) or the indicated concentrations of LY294002 (0-25 μM) or wortmannin (0-100 nM) for 15 minutes. Pretreated platelets were either perfused immediately over immobilized VWF (100 μg/mL) at 150 s-1 or reconstituted with red blood cells prior to perfusion at 1800 s-1. In some experiments, tethered platelets were incubated with PAC-1 antibody prior to fixation and staining with a FITC-conjugated secondary antibody. These results demonstrate the effect of the PI 3–kinase inhibitors on (A) the level of stationary platelet adhesion and (B) the mean surface area of adherent platelets following perfusion at 150 s-1 or 1800 s-1; (C) the morphology of adherent platelets as visualized by scanning electron microscopy (bar = 2 μm); and (D) platelet spreading and PAC-1 binding (bar = 10 μm). (A,B) Results represent mean ± SEM from 4 independent experiments. Statistical analysis was performed using a t test comparing control versus LY294002- or wortmannin-treated platelets (P < .05*;P < .01**; P < .001***). (C,D) Images are from a single experiment representative of 3. Cindy L. Yap et al. Blood 2002;99:151-158 ©2002 by American Society of Hematology

The role of PI 3–kinase in regulating calcium mobilization under flow conditions.Calcium indicator dye-loaded platelets were incubated with vehicle alone (control) or wortmannin (100 nM) for 15 minutes prior to reconstitution with red blood cells and perfus... The role of PI 3–kinase in regulating calcium mobilization under flow conditions.Calcium indicator dye-loaded platelets were incubated with vehicle alone (control) or wortmannin (100 nM) for 15 minutes prior to reconstitution with red blood cells and perfusion over immobilized VWF (100 μg/mL) at 1800 s-1. Changes in the cytosolic calcium concentration of adherent cells were monitored by confocal microscopy (× 63W objective) and fluorescence ratios quantified. The results presented in panel A demonstrate the effect of wortmannin on the percentage of cells undergoing oscillatory calcium transients relative to untreated (control) platelets. The images and results presented in panels B and C show a representative calcium oscillation response (solid line) and the displacement (dotted line) of individual vehicle- (control) and wortmannin-treated platelets under shear conditions. Results and images are from a single experiment representative of 3. Bar = 10 μm. Cindy L. Yap et al. Blood 2002;99:151-158 ©2002 by American Society of Hematology

The role of PI 3–kinase in regulating platelet thrombus formation under flow conditions.Anticoagulated whole blood was incubated with vehicle alone (control) or the indicated concentrations of LY294002 (0-200 μM) for 15 minutes prior to perfusion over immob... The role of PI 3–kinase in regulating platelet thrombus formation under flow conditions.Anticoagulated whole blood was incubated with vehicle alone (control) or the indicated concentrations of LY294002 (0-200 μM) for 15 minutes prior to perfusion over immobilized VWF (100 μg/mL) at 1800 s-1. In panel A, thrombi were fixed and incubated with DiOC6. Platelet thrombi were reconstructed using a computer-assisted image analysis program. The upper panels represent an oblique view to demonstrate surface coverage; the lower panels represent a side-on view to demonstrate differences in thrombus height. The results in panel B demonstrate the effect of PI 3–kinase inhibition on thrombus formation. Cindy L. Yap et al. Blood 2002;99:151-158 ©2002 by American Society of Hematology