Extracellular chloride is required for efficient platelet aggregation

Figures & data

Figure 1. Extracellular chloride is required for efficient platelet activation. (a) Washed platelets were stimulated by increasing concentrations of thrombin or 1 µg mL⁻¹ CRP-XL in the presence of 151 mM (black) or 1 mM (gray) [Cl⁻]_o. Representative aggregation traces are shown for each condition (ai). Maximum aggregation (aii) and the initial rate of aggregation (aiii) were calculated for each condition. (b) In the presence of 2 mM [Ca²⁺]_o, [Cl⁻]_o substitution continued to reduce the initial rate of thrombin-evoked aggregation. (c) The sensitivity of thrombin-evoked (0.1 U mL⁻¹) aggregation to Cl⁻ was assessed by increasing [Cl⁻]_o from 1 to 151 mM in 30 mM increments. Representative traces (ci), maximum (cii) and initial rate of thrombin-induced (0.1 U mL⁻¹) aggregation (ciii) for each [Cl⁻]_o are shown. The rate of aggregation increased across the concentration range with an EC₅₀ = 46.5 ± 23.3 mM. Data are representative of a minimum of four independent experiments. Thrombin and CRP-XL data sets were analyzed by two-way ANOVA and Student’s t test, respectively.

Figure 2. Role for secondary signaling during Cl^–-dependent platelet aggregation. (a) Washed platelets were preincubated with 100 µM aspirin (cyclooxygenase inhibitor) and 5 U mL⁻¹ apyrase (ectonucleotidase) prior to performing aggregometry in the presence of 151 mM (black) or 1 mM (gray) [Cl⁻]_o. Representative traces (ai), maximum (aii), and initial rate (aiii) of thrombin-induced (0.1 U mL⁻¹) aggregation are shown in the presence of vehicle control (0.1% ethanol) or aspirin plus apyrase. (b) Summary data for maximum (bi) and initial rate of thrombin-evoked (0.1 U mL⁻¹) platelet aggregation (bii) in the presence of vehicle control (H₂O) or 1 µM Ar-C66096 (P2Y12 inhibitor). (c) Alpha (i) and dense (ii) granule release before (unstimulated) and after 0.1 U mL⁻¹ thrombin stimulation was assessed by flow cytometry using fluorescently labeled CD62P and CD63 antibodies, respectively. Data are representative of a minimum of four independent experiments and data were analyzed by two-way ANOVA.