Platelets digest HA in the inflammatory matrix. PolyI:C-stimulated human M-SMCs were co-incubated with freshly isolated human platelets (PLTs) for 2 hours at 37°C. (A) Agarose gel electrophoretic analysis of HA purified from polyI:C-stimulated M-SMCs before (lane 1) and after (lane 2) coincubation with platelets. Polydisperse HA with a size range of 0.1 to 2 × 10⁶ Da associated with M-SMCs was no longer detected after co-incubation with platelets. Replicate samples were treated with HAase, an enzyme that specifically digests HA, to confirm that the stained bands were HA (lanes 3 and 4). HA ladder units: 10⁶ Da. (B) ELISA-like assay measurement of HA released into the media by M-SMCs. Coincubation of M-SMCs with platelets results in a significant increase of HA in culture media. Data represent mean ± standard error (SE) of 10 separate experiments. ***P < .001; N.S., not significant. (C) Histochemical staining of M-SMC-associated HA (green). M-SMCs were fixed in cold methanol, and HA was detected with biotinylated HA-binding protein and Alexa Fluor 488–conjugated streptavidin. M-SMCs responded to polyI:C treatment by producing high amounts of HA. The co-incubation of platelets with polyI:C-stimulated M-SMCs caused the removal of the M-SMC surface–associated HA. Image and capture details: Leica upright microscope DM5500 B (Leica, Wetzlar, Germany), HC PLAN APO ×20/0.7NA dry objective, QImaging Retiga cooled CCD camera, QCapture Suite Software (QImaging, Surrey, BC Canada). Scale bar: 100 µm. (D) Fold-increase in surface P-selectin and HYAL2 mean fluorescence intensity (MFI) of SMC-bound platelets in comparison with unbound platelets following culture as measured by flow cytometry. SMC-bound platelets demonstrated significantly higher surface P-selectin and HYAL2 compared with unbound platelets after culture. Platelets were defined by forward (FSC-A), and side scatter (SSC-A) characteristics.