Figure 5. Platelet aquaporin-1 mediates rapid swelling kinetics after hypotonic shock.

(A) Platelets adherent to serum-coated dishes were challenged with a Δ-70 mOsm osmotic change, cell swelling was monitored by phase-contrast microscopy, and images at indicated time points are shown. The box-and-whisker plot shows AQP1^+/+ and AQP1^–/– platelet diameter after 3 minutes of hypotonic shock. The diameter versus time graph shows the time course of change in diameter (blue symbols indicate AQP1^–/– platelets; magenta symbols indicate AQP1^+/+ platelets). (B) Using an optical aggregometer, light transmittance was determined in platelet suspensions in modified Tyrode solutions. Baseline light transmittance was recorded before and after AQP1^+/+ and AQP1^–/– platelet suspensions were exposed to hypotonic shock (Δ-40, Δ-70 and Δ-100-mOsm osmotic change). Percentage light transmission values were recorded over time and plotted as shown in representative graphs. The first-order rate constants of these plots were derived for the first 20 seconds after hypotonic shock and plotted as shown in the box-and-whisker plot. (C) Time courses of cytosolic calcium signals in untreated platelets adherent to bovine serum-coated surfaces and exposed to normotonic or a 70-mOsm osmotic gradient stimulus; here, normotonic and hypotonic Tyrode were supplemented with 1 mM calcium. Arrows in the F/F₀ graph indicate time points for induction of hypotonic stimulus. In addition, the box-and-whisker plot of peak calcium mobilized in platelets is shown. From left to right, representative images of AQP1^+/+ and AQP1^–/– platelets at basal and at peak cytosolic calcium signal are shown. (D) As in C, but platelets were monitored as they adhered to collagen fibers. The box-and-whisker plot shows peak calcium mobilized in platelets. Arrows in time-course graph indicate the time point of platelet adherence to collagen fiber. Data analysis was performed by Wilcoxon signed-rank test. *P < 0.05, ** P < 0.01 were considered significant. Scale bar: 1 μm (A and C). Data were from 4 independent experiments.