by John C. Kermode, Qi Zheng, and Elizabeth P. Milner Marked Temperature Dependence of the Platelet Calcium Signal Induced by Human von Willebrand Factor by John C. Kermode, Qi Zheng, and Elizabeth P. Milner Blood Volume 94(1):199-207 July 1, 1999 ©1999 by American Society of Hematology

Optical configuration and stirring arrangement for simultaneous assessment of platelet aggregation and [Ca2+]i signaling. Optical configuration and stirring arrangement for simultaneous assessment of platelet aggregation and [Ca2+]i signaling. Measurements are conducted in a cylindrical glass aggregometer cuvette (8-mm diameter) in a reconfigured dual-emission spectrofluorometer. The platelet suspension is stirred with a novel stirrer, comprising an opaque Teflon cylinder with a bar magnet at its base and a UV-grade methacrylate stirring vane at its top. This stirrer protrudes into the excitation beam of the spectrofluorometer to ensure that platelet aggregates cannot settle below its detection zone.17 Platelet aggregation is monitored through measurement of transmitted light intensity, with the lower part of the transmitted light beam blocked so that the stirrer does not interfere with these measurements. Platelet [Ca2+]i is monitored through fluorescence measurements perpendicular to the excitation beam. Reflections from the curved surfaces of the cuvette are eliminated by use of a vertical polarizer in the excitation beam and a horizontal one in the emission beam. This arrangement provides efficient stirring of the platelet suspension and ensures that the fluorescence signal is representative of the entire population of platelets regardless of the extent of aggregation. John C. Kermode et al. Blood 1999;94:199-207 ©1999 by American Society of Hematology

Temperature dependence of the [Ca2+]i signal induced by ristocetin-mediated binding of vWF. 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. John C. Kermode et al. Blood 1999;94:199-207 ©1999 by American Society of Hematology

Effect of GP IIb-IIIa blockade on the vWF-induced [Ca2+]i signal. 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. John C. Kermode et al. Blood 1999;94:199-207 ©1999 by American Society of Hematology

Temperature dependence of platelet aggregation and agglutination by ristocetin-mediated binding of vWF. Temperature dependence of platelet aggregation and agglutination by ristocetin-mediated binding of vWF. (A and B) Aggregation of live platelets was monitored simultaneously with platelet [Ca2+]i. The platelet suspension (50,000 cells/μL) was incubated at 20°C (A) or 37°C (B) in the presence of 1 mmol/L CaCl2. Aggregation was deduced from the transmitted light intensity (during the 380 nm fluorescence excitation phase). Aggregation patterns at 25°C and 30°C (not shown) were indistinguishable from those at 20°C and 37°C. The results illustrated were acquired in parallel with the [Ca2+]i measurements shown in Fig 2; these aggregation data are typical of four such studies. (C and D) Agglutination of paraformaldehyde-fixed platelets (50,000 cells/μL) was evaluated at 20°C (C) and 37°C (D) in an analogous manner. Agglutination patterns at 30°C (not shown) were comparable to those at 37°C. These data are typical of three such studies. Panels A through D present the responses on incubation either with 1 mg/mL ristocetin and 5 μg/mL multimeric human vWF (solid traces) or with 1 mg/mL ristocetin alone (dotted traces). John C. Kermode et al. Blood 1999;94:199-207 ©1999 by American Society of Hematology

Temperature dependence of the [Ca2+]i signals induced by U-46619, -thrombin, and ADP. The [Ca2+]itransient in a stirred suspension of human platelets (50,000 cells/μL) was monitored with Fura-PE3. Temperature dependence of the [Ca2+]i signals induced by U-46619, -thrombin, and ADP. The [Ca2+]itransient in a stirred suspension of human platelets (50,000 cells/μL) was monitored with Fura-PE3. The platelets were stimulated at 20°C (dotted traces) or 37°C (solid traces) by 0.1 μmol/L U-46619 (A), 0.02 U/mL human -thrombin (B), or 5 μmol/L ADP (C) in the presence of extracellular calcium (1 mmol/L CaCl2). Platelet [Ca2+]i was calculated from the fluorescence excitation ratio (340:380 nm) using the kd value for Fura-PE3 at the corresponding temperature. Observations at 25°C and 30°C (not shown) were intermediate between those at 20°C and 37°C. The results shown are from a typical one of six such studies. John C. Kermode et al. Blood 1999;94:199-207 ©1999 by American Society of Hematology

Temperature dependence of the [Ca2+]i signal induced by exogenous arachidonic acid. Temperature dependence of the [Ca2+]i signal induced by exogenous arachidonic acid. The [Ca2+]i transient in a stirred suspension of human platelets (50,000 cells/μL) was monitored with Fura-PE3. The platelets were stimulated at 20°C (A), 30°C (B), or 37°C (C) by 20 μmol/L arachidonic acid (solid traces) in the presence of extracellular calcium (1 mmol/L CaCl2). Data obtained in parallel control studies with 0.2% (wt/vol) dimethylsulfoxide (vehicle for arachidonic acid; dotted traces) are also displayed. Observations at 25°C (not shown) were indistinguishable from those at 20°C. The results illustrated are from a typical one of seven such studies. John C. Kermode et al. Blood 1999;94:199-207 ©1999 by American Society of Hematology

Temperature dependence of thromboxane A2production induced by exogenous arachidonic acid and vWF. Temperature dependence of thromboxane A2production induced by exogenous arachidonic acid and vWF. A stirred suspension of human platelets (500 μL at 50,000 cells/μL) was stimulated with either 20 μmol/L arachidonic acid (A) or a combination of 1 mg/mL ristocetin and 5 μg/mL multimeric human vWF (B) at 20°C, 25°C, 30°C, or 37°C (as indicated). Platelet thromboxane A2 production was assessed by enzyme immunoassay of its stable breakdown product, thromboxane B2. Control samples were incubated with dimethylsulfoxide (vehicle for arachidonic acid) or 1 mg/mL ristocetin alone; thromboxane A2 production in the control (typically, 0.4 ng in both cases) was subtracted from that in the corresponding experimental sample. Data are presented as a Tukey box plot: the central line in the box shows the median value for the agonist-induced increment in thromboxane A2 production from seven studies, the lower and upper limits of the box designate the quartiles, and the error bars extending below and above the box represent the 10th and 90th percentiles, respectively. Statistical analysis was based on Friedman’s test with a nonparametric Student-Newman-Keuls posthoc test; significant differences (P < .01) from measurements at the higher temperatures are designated: †compared with 37°C, and ‡compared with 30°C. John C. Kermode et al. Blood 1999;94:199-207 ©1999 by American Society of Hematology

Effect of cyclo-oxygenase inhibition on the vWF-induced [Ca2+]i signal and thromboxane A2 production. Effect of cyclo-oxygenase inhibition on the vWF-induced [Ca2+]i signal and thromboxane A2 production. Human platelets (50,000 cells/μL, loaded with Fura-PE3) were preincubated (5 minutes at 37°C) with 0.1% dimethylsulfoxide (vehicle) or 0.2 mmol/L aspirin. The platelets were then stimulated at 37°C by 1 mg/mL ristocetin and 5 μg/mL multimeric human vWF in the presence of 1 mmol/L CaCl2. Parallel control studies were undertaken with 1 mg/mL ristocetin alone. (A) The results from a typical one of nine such studies are shown. The solid trace denotes the vWF-induced [Ca2+]isignal in vehicle-treated platelets, and the dotted trace that after aspirin treatment. (B) The amplitude of the vWF-induced [Ca2+]i increment, relative to ristocetin alone, in the whole set of studies is presented as a Tukey box plot. (C) Thromboxane A2 production was assessed in parallel. The vWF-induced increment in thromboxane A2 production, relative to ristocetin alone (typically, 0.3 ng), is displayed as an analogous Tukey box plot. The hatched bars in panels B and C summarize data for platelets preincubated with vehicle and the open bars data for aspirin-treated platelets. John C. Kermode et al. Blood 1999;94:199-207 ©1999 by American Society of Hematology