Thrombin receptor-mediated platelet activation results in increased platelet hyaluronidase activity. (A) PolyI:C-stimulated human M-SMCs were coincubated with decreasing concentrations of either untreated freshly isolated platelets or TRAP-activated platelets for 2 hours at 37°C. The amount of HA released into the media was measured by ELISA-like assay. Activated platelets caused a significantly higher increase in HA released into the media than nonactivated platelets. At a concentration of 30 × 106 platelets per well, nonactivated platelets appeared to have no hyaluronidase activity compared with activated platelets, which still released significant amounts of HA into media. n = 3; **P < .01. (B) Freshly isolated untreated or TRAP-activated or lysed platelets (3 × 108 platelets per well) were incubated with purified HA immobilized to wells of a 96-well plate for 18 hours at 37°C (pH 3.5). Platelets and digested HA were then washed, and the HA remaining on the wells was detected colorimetrically. Activated platelets demonstrated significantly higher hyaluronidase activity (80% maximum) than nonactivated platelets (10% maximum) and platelet lysate (50% maximum). n = 3; ***P < .001. Maximum activity was achieved by HAase.
