HYAL2 becomes surface expressed upon thrombin receptor–mediated platelet activation. (A) Freshly isolated human platelets were treated without or with TRAP (15 µM) for 10 minutes before microparticles were isolated. Immunoblot analysis showed that, upon activation, HYAL2 was associated with platelets (750-g pellet) and not with microparticles (104-g pellet) or platelet releasate (104 g supernatant). (B) Immunoblot analysis showing that HYAL2 remains associated with platelets (750-g pellet) and not with microparticles or releasate (REL) even after 30 minutes incubation with TRAP. (C) Freshly isolated human platelets were treated without or with TRAP (25 µM) for 1 minute before they were fixed with 3.5% paraformaldehyde for 30 minutes. Fixed platelets were spun down onto slides coated with poly-l-lysine and stained for P-selectin (red) and HYAL2 (green). Similar to P-selectin, HYAL2 was detected only on the surfaces of activated platelets. Image and software details: Leica upright microscope DM5500 B (Leica), HCX PLAN APO ×63/1.32NA oil immersion objective, QImaging Retiga cooled CCD camera, QCapture Suite Software (QImaging). Scale bar: 20 µm. (D-F) Freshly isolated platelets were treated without or with TRAP (25 µM) for 1 minute at room temperature before they were fixed in 1% paraformaldehyde for 2 hours at room temperature. Fixed platelets were stained for HYAL2 and P-selectin and analyzed by using flow cytometry. (D) Representative histograms of surface P-selectin and HYAL2 staining in unstimulated and TRAP-stimulated platelets. The P-selectin–negative population in unstimulated platelets demonstrated no surface HYAL2 expression, whereas the P-selectin–positive population in the TRAP-stimulated sample showed significant surface HYAL2 expression. PE, phycoerythrin. (E) Percentage of surface HYAL2-positive platelets measured by flow cytometry. As a result, 37.6% ± 6.4% (±SE) of TRAP-activated platelets expressed surface HYAL2 compared with only 6.8% ± 1.3% of nonactivated (P-selectin negative) platelets. n = 9; ***P < .001. (F) Fold-increase in surface HYAL2 MFI of TRAP-activated platelets in comparison with nonactivated (P-selectin negative) platelets. Activated platelets demonstrated a threefold increase in surface HYAL2 expression compared with nonactivated platelets. n = 9; ***P < .001.
