Temperature dependence of the [Ca²⁺]_i signal induced by ristocetin-mediated binding of vWF. Human platelets were loaded with 5 μmol/L Fura-PE3/AM. Measurements were performed in an aggregometer cuvette, with the platelet suspension (50,000 cells/μL) stirred by the novel stirrer. The platelets were stimulated at 20°C (A), 30°C (B), or 37°C (C) by adding 1 mg/mL ristocetin and 5 μg/mL multimeric human vWF (solid traces) in the presence of extracellular calcium (1 mmol/L CaCl₂). Data obtained in parallel control studies with 1 mg/mL ristocetin alone (dotted traces) are also displayed. The fluorescence intensity at 510-nm emission wavelength was measured with excitation alternating between 340 and 380 nm. Platelet [Ca²⁺]_i was calculated from the fluorescence ratio (340:380 nm), using the kd value for Fura-PE3 at the corresponding temperature. Observations at 25°C (not shown) were indistinguishable from those at 20°C. The results illustrated are from a typical one of eight such studies.