![Fig. 3. 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.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/99/1/10.1182_blood.v99.1.151/6/h80121959003.jpeg?Expires=1722871057&Signature=PHt5Mm5TM-T8wqVLbtrDR51j5NbdR4JZrE0Pme~8uuS462fv2vX2-5xjdeS7qsGEs5JKDqZUSgFrKNcF7rlNet2j4-CpHwV6o66DIOtlxEOnJjngjWa7MQrO0Bi~fghZPWODpbjMK2Dh7UvcG~~J-uxFSPXXTEqXPtaslGIXFW2zLDcQntiAGdAlegAcaE9mQjIrAhy44qJuE92kwinlnVG3Xiwg-STqz3ykLe2ZImUfiWtO6hqqr2MQBY9SbW83sClsvh8vBgjPiQtbnTn4n4G4WlBDJRRFhtqW4lSRl21w8PpWk7akbPbtPtx9JqgN5AYlkD~QTEFD9QUf5c3LWQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
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.
