American Society of Hematology

Figure 5

$Figure 5. HYAL2 is stored in a distinct subset of α-granules in resting platelets. (A) Immmunoblot analysis for the presence of CD42b and HYAL2 of different platelet subcellular fractions obtained by differential centrifugation. HYAL2 was detected exclusively in the fraction containing granules, lysosomes, and mitochondria (19 000-g pellet), whereas CD42b was detected in the membranes fraction (100 000-g pellet). (B) Platelets were sonicated and applied on top of a linear sucrose density gradient before the gradient was centrifuged at 100 000g for 90 minutes. Nine fractions were collected from the top of the gradient. Equal volumes of fractions were analyzed by immunoblotting for the presence of CD42b (plasma membrane marker), LAMP-1 (lysosomal marker), P-selectin (α-granule marker), and HYAL2. HYAL2 and P-selectin were both enriched in fraction 5, the α-granules fraction, whereas CD42b and LAMP-1 were enriched in fractions 2 and 4, respectively. (C) Confocal microscopy images (maximum projection) of platelets stained for HYAL2 (green) and one of the following proteins: CD42b (plasma membrane marker), LAMP-2 (dense granule marker), protein disulfide isomerase (T-granule marker), KDEL (dense tubular system marker), LAMP-1 and NEU1 (lysosomal markers), P-selectin (α-granule marker), vWF (α-granule marker), and fibrinogen (α-granule marker). HYAL2 did not colocalize with any of the tested markers. Pearson’s correlation coefficients were obtained by analyzing individual images (layers) of the Z-stack using Image-Pro Plus software (Media Cybernetics, Rockville, MD). Image, detection, and software details: Leica TCS SP5 II confocal/multiphoton high-speed upright microscope (Leica), HCX PL APO 63Χ/1.4NA oil immersion objective, Leica HyD system detector, Leica LAS AF software (Leica). Scale bar: 1 µm. (D) Immunoelectron microscopy images showing resting and activated platelets. Washed human platelets were fixed, sectioned, and mounted on formvar-coated nickel grids. Ultrathin platelet sections were probed for HYAL2, and the bound antibody was labeled with immunogold (10 nm). In resting platelets, HYAL2 appeared to be localized within platelet α-granules (black arrows), whereas platelet surface appeared to be devoid of any HYAL2. However, in activated platelets, HYAL2 was clearly detected on the surface. Image and detection details: FEI Tecnai G2 Spirit BioTWIN Transmission Electron Microscope (FEI Company, Hillsboro, OR), Orius 832 CCD 11-megapixel camera, Digitalmicrograph (Gatan, Inc., Pleasanton, CA). Scale bars: 200 nm.$

HYAL2 is stored in a distinct subset of α-granules in resting platelets. (A) Immmunoblot analysis for the presence of CD42b and HYAL2 of different platelet subcellular fractions obtained by differential centrifugation. HYAL2 was detected exclusively in the fraction containing granules, lysosomes, and mitochondria (19 000-g pellet), whereas CD42b was detected in the membranes fraction (100 000-g pellet). (B) Platelets were sonicated and applied on top of a linear sucrose density gradient before the gradient was centrifuged at 100 000g for 90 minutes. Nine fractions were collected from the top of the gradient. Equal volumes of fractions were analyzed by immunoblotting for the presence of CD42b (plasma membrane marker), LAMP-1 (lysosomal marker), P-selectin (α-granule marker), and HYAL2. HYAL2 and P-selectin were both enriched in fraction 5, the α-granules fraction, whereas CD42b and LAMP-1 were enriched in fractions 2 and 4, respectively. (C) Confocal microscopy images (maximum projection) of platelets stained for HYAL2 (green) and one of the following proteins: CD42b (plasma membrane marker), LAMP-2 (dense granule marker), protein disulfide isomerase (T-granule marker), KDEL (dense tubular system marker), LAMP-1 and NEU1 (lysosomal markers), P-selectin (α-granule marker), vWF (α-granule marker), and fibrinogen (α-granule marker). HYAL2 did not colocalize with any of the tested markers. Pearson’s correlation coefficients were obtained by analyzing individual images (layers) of the Z-stack using Image-Pro Plus software (Media Cybernetics, Rockville, MD). Image, detection, and software details: Leica TCS SP5 II confocal/multiphoton high-speed upright microscope (Leica), HCX PL APO 63Χ/1.4NA oil immersion objective, Leica HyD system detector, Leica LAS AF software (Leica). Scale bar: 1 µm. (D) Immunoelectron microscopy images showing resting and activated platelets. Washed human platelets were fixed, sectioned, and mounted on formvar-coated nickel grids. Ultrathin platelet sections were probed for HYAL2, and the bound antibody was labeled with immunogold (10 nm). In resting platelets, HYAL2 appeared to be localized within platelet α-granules (black arrows), whereas platelet surface appeared to be devoid of any HYAL2. However, in activated platelets, HYAL2 was clearly detected on the surface. Image and detection details: FEI Tecnai G2 Spirit BioTWIN Transmission Electron Microscope (FEI Company, Hillsboro, OR), Orius 832 CCD 11-megapixel camera, Digitalmicrograph (Gatan, Inc., Pleasanton, CA). Scale bars: 200 nm.

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