Figure 1
Figure 1. Fibrinogen binding to αIIbβ3 promotes activation of cPLA2α in platelets. (A-C) Values on the y-axis represent the average cPLA2α activity of each sample minus the baseline cPLA2α activity determined for reaction buffer alone. (A) Washed human platelets were preincubated with DMSO vehicle or with 25 or 50 μM of pyrrophenone before incubation at 37°C for 30 minutes in the presence or absence of the indicated agonists (300 μg/mL of fibrinogen (Fg) + 1 mM of MnCl2, 20 μM of ADP, or 1 U/mL of thrombin). cPLA2α activity in platelet lysates was measured as described in “Methods.” Data represent the mean plus or minus SEM of 2 independent experiments, each performed in triplicate. (B) cPLA2α activity was measured in platelet lysates from cPLA2α−/− and wild-type cPLA2α+/+ littermates. Platelets were treated while in suspension with saline (no agonist), 100 μg/mL fibrinogen + 1 mM MnCl2, or 1 U/mL of thrombin. Data represent mean plus or minus SEM of triplicate determinations carried out in individual mice (2 mice for each genetic background) on 2 separate occasions. (C) cPLA2α activity was measured in platelets from either wild-type or cPLA2α−/− mice. The assay was performed on lysates from platelets adherent to either recombinant murine VWF A1A2 domain or human fibrinogen. As a further control, platelets incubated on immobilized bovine serum albumin failed to adhere. Data represent mean plus or minus SEM of triplicate determinations carried out in individual mice (2 mice for each genetic background) on 2 separate occasions. (D) Lysates from human platelets treated with either 300 μg/mL of fibrinogen + 1 mM of MnCl2 or 1 U/mL of thrombin in the presence or absence of 50 μM of pyrrophenone were immunoprecipitated with anti-β3 SSA6 antibody or a nonimmune murine IgG1 control. Immunoprecipitates were incubated with 200 ng of the PLA2 substrate β-BODIPY FL C5-HPC for 3 hours at 37°C. The presence of αIIbβ3-associated PLA2 activity was monitored by thin layer chromatography detection of a cleaved product of β-BODIPY FL C5-HPC, an assay strictly qualitative in nature. This experiment was performed twice with identical results.

Fibrinogen binding to αIIbβ3 promotes activation of cPLA2α in platelets. (A-C) Values on the y-axis represent the average cPLA2α activity of each sample minus the baseline cPLA2α activity determined for reaction buffer alone. (A) Washed human platelets were preincubated with DMSO vehicle or with 25 or 50 μM of pyrrophenone before incubation at 37°C for 30 minutes in the presence or absence of the indicated agonists (300 μg/mL of fibrinogen (Fg) + 1 mM of MnCl2, 20 μM of ADP, or 1 U/mL of thrombin). cPLA2α activity in platelet lysates was measured as described in “Methods.” Data represent the mean plus or minus SEM of 2 independent experiments, each performed in triplicate. (B) cPLA2α activity was measured in platelet lysates from cPLA2α−/− and wild-type cPLA2α+/+ littermates. Platelets were treated while in suspension with saline (no agonist), 100 μg/mL fibrinogen + 1 mM MnCl2, or 1 U/mL of thrombin. Data represent mean plus or minus SEM of triplicate determinations carried out in individual mice (2 mice for each genetic background) on 2 separate occasions. (C) cPLA2α activity was measured in platelets from either wild-type or cPLA2α−/− mice. The assay was performed on lysates from platelets adherent to either recombinant murine VWF A1A2 domain or human fibrinogen. As a further control, platelets incubated on immobilized bovine serum albumin failed to adhere. Data represent mean plus or minus SEM of triplicate determinations carried out in individual mice (2 mice for each genetic background) on 2 separate occasions. (D) Lysates from human platelets treated with either 300 μg/mL of fibrinogen + 1 mM of MnCl2 or 1 U/mL of thrombin in the presence or absence of 50 μM of pyrrophenone were immunoprecipitated with anti-β3 SSA6 antibody or a nonimmune murine IgG1 control. Immunoprecipitates were incubated with 200 ng of the PLA2 substrate β-BODIPY FL C5-HPC for 3 hours at 37°C. The presence of αIIbβ3-associated PLA2 activity was monitored by thin layer chromatography detection of a cleaved product of β-BODIPY FL C5-HPC, an assay strictly qualitative in nature. This experiment was performed twice with identical results.

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