Effect of GP IIb-IIIa blockade on the vWF-induced [Ca²⁺]_i signal. (A) The ability of the 10E5 antibody to block GP IIb-IIIa was evaluated by aggregometry. Washed human platelets (50,000 cells/μL, suspended in Tyrode’s solution with 1 mmol/L CaCl₂) were preincubated (5 minutes at 37°C) with isotype-matched control IgG (solid trace) or with 10E5 IgG (dotted trace). Each IgG was essentially azide-free and was used at a final concentration of 10 μg/mL. Platelets were stimulated with 0.1 μmol/L U-46619 and aggregation was assessed at 37°C in the presence of 5 μg/mL multimeric human vWF. (B and C) The effect of the 10E5 antibody on the platelet [Ca²⁺]_isignal was evaluated. Human platelets (50,000 cells/μL, loaded with Fura-PE3) were preincubated with 10 μg/mL control IgG (B) or 10E5 IgG (C). The platelets were then stimulated at 37°C by 1 mg/mL ristocetin and 5 μg/mL vWF (solid traces) in the presence of 1 mmol/L CaCl₂. Data obtained in parallel control studies with 1 mg/mL ristocetin alone (dotted traces) are also displayed. Fluorescence measurements were performed in a cylindrical aggregometer cuvette, with the platelet suspension stirred by the novel stirrer. Platelet [Ca²⁺]_i was calculated from the fluorescence excitation ratio (340:380 nm). Results of a typical one of four such studies are illustrated. Aggregation and platelet [Ca²⁺]_i were also assessed in the absence of either IgG (not shown); results were indistinguishable from those in the presence of the control IgG.