Localization of PDI and granule release in WT and HPS6^−/− platelets. (A) Localization of PDI in platelet granules. (a) Unstimulated platelets isolated from WT and HPS6^−/− mice were sedimented by centrifugation. The pellet was solubilized with SDS lysis buffer to generate the lysate. Lysate of intact resting platelets from WT and HPS6^−/− mice was subjected to SDS-PAGE and blotted with anti-PDI antibodies DL-11. (b) Washed WT mouse resting platelets were fixed, frozen, and sectioned before mounting on Formvar carbon-coated copper grids. Ultrathin platelet sections were probed for PDI, and bound antibody labeled with Protein A-gold. Samples were examined by transmission electron microscopy and reveal distribution of PDI in platelet T granules. Bar represents 100 nm. (c) As per panel Ab, but HPS6^−/− mouse platelets were examined. (B) Decreased thrombin sensitivity of granule exocytosis in HPS6^−/− platelets. Comparing WT platelets and HPS6^−/− platelets, thrombin-induced granule exocytosis was studied in vitro to characterize α granule, T granule, and lysosomes. Mouse platelets in HTG buffer (250 × 10⁵ platelets per 100 μL of 5 mM d-glucose, 134 mM sodium chloride, 0.34 mM disodium phosphate, 2.9 mM potassium chloride, 12 mM sodium bicarbonate, 20 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid, and 1 mM magnesium chloride, pH 7.3) were incubated with mouse α-thrombin for 10 minutes at room temperature. Twenty microliters of thrombin-stimulated or resting platelets were incubated with fluorescein isothiocyanate–conjugated P-selectin, TLR9, or Alexa Fluor 488–labeled LAMP1 antibodies for 15 minutes. Surface expression of platelet granule markers was measured using CellQuest on a FACSCalibur flow cytometer (Becton Dickinson). Data are expressed as percent maximal release. Immunoflow cytometry of P-selectin monitored α granule exocytosis, whereas α granule content release, monitored by PF-4 secretion, was measured by enzyme-linked immunosorbent assay. T granule exocytosis was monitored by surface exposure of TLR9 using flow cytometry and lysosome exocytosis by the release of LAMP1. For PF-4 and PDI, hirudin (1 U/mL) was added to quench thrombin activity, then platelets were sedimented by centrifugation, the supernatant was collected and ultracentrifuged at 71 000g for 30 minutes, and the releasate was assayed. Platelets from WT or HPS6^−/− mice were activated with varying amounts of thrombin, and the markers for granule exocytosis were measured. Thrombin agonist concentrations were 0.007, 0.036, 0.072, 0.36, 0.72, 3.6, and 7.2 nM; the average of 5 measurements defines each point ± SD. (a) P-selectin (α granules); (b) PF-4 (α granules); (c) TLR9 (T granules); (d) LAMP 1 (lysosomes); **P < .01, ***P < .001. (e) Band densities of PDI antigen in releasates of thrombin-stimulated WT and HPS6^−/− platelets detected by SDS-PAGE, followed by immunoblotting with anti-PDI antibodies (DL-11; 1 μg/mL). Data represent mean ± SD (n = 2; **P < .01). (f) Thiol isomerase secretion after platelet activation with 0.72 or 7.2 nM thrombin. Thiol isomerase activity was monitored by the reduction of a di-E-GSSG as a substrate. The increase in fluorescence was measured at excitation/emission of 525/540 nm for 20 minutes at 25°C. **P < .01. WT platelets, closed bars; HPS6^−/− platelets, open bars. (C) Agonist-induced adenosine triphosphate release as a marker of dense granule release in WT and HPS6^−/− platelets as measured by luminometry. WT and HPS6^−/− platelets were activated with SFLLRN (SF; 252 μM), collagen (Col; 19 μg/mL), or buffer control (Control), and the kinetics of release of adenosine triphosphate was monitored as a function of time (0, 5, 10, 15, 20, 25, and 30 seconds, left to right) by luminometry. (D) Decreased collagen sensitivity of granule exocytosis from HPS6^−/− platelets. Band densities of PDI antigen in releasates of collagen-stimulated (0.1-5 μg/mL of type 1 equine collagen; 10 minutes) WT and HPS6^−/− platelets detected by SDS-PAGE, followed by immunoblotting with anti-PDI antibodies (DL-11; 1 μg/mL) (n = 3; mean ± SD; **P = .001, *P = .01). WT platelets, closed bars; HPS6^−/− platelets, open bars. SD, standard deviation; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis.