Detection of MRP4 and MRP5 in platelet membranous fractions. (A) Immunoblot analysis of MRP4 (left panel) and MRP5 (right panel) in platelet crude membranes (Pl; 40 μg protein) detected by the SNG and AMF antisera directed against the carboxyl terminus of human MRP4 and MRP5, respectively. Crude membranes of human liver (Liv) or MRP5-transfected V79 cells (V79) were used as positive controls. (B) Platelet subcellular fractions were separated on a linear 30% to 60% sucrose gradient and visible bands were collected. According to Broekman,32 4 fractions of increasing density (1-4: 30%, 35%-40%; 50%-55%, 60% sucrose) enriched in (1) plasma membrane, (2) lysosomes, (3) α-granules, or (4) dense granules were further analyzed. MRP4 was detected by immunoblotting using the antiserum SNG (20 μg protein/lane). Blots were further incubated with specific antibodies against LAMP2 (lysosome-associated membrane glycoprotein 2), P-selectin, and the surface antigen GPIb (CD42b), as marker for lysosomes/dense granules, α-granules, and plasma membrane, respectively (left panels). The results were quantified by densitometric analysis and the relative optical density of the specific bands was plotted (middle panels). Right panels: ATP-dependent cGMP transport (cGMP tp) as well as mepacrine accumulation and β-glucuronidase (β-Gluc) activity were measured in the fractions as described in “Patients, materials, and methods” and plotted as relative specific activities (mean values ± SD, n = 3).
