Endorepellin enhances collagen platelet activation and aggregation. (A) Representative immunoblotting (n = 4 experiments) using anti-PTyr antibody (PY20) of total platelet lysate under various conditions as indicated. Washed human platelets were added to wells coated with BSA, ER, or collagen (same concentrations as in Figure 1A) with or without liquid-phase ER (20 μg/mL) or with or without PP1 (10 μM) for 60 minutes at 37°C, followed by removal of nonadherent platelets, lysis of adherent platelets with ice-cold RIPA buffer, and protein separation by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Control, unplated platelets in suspension are also shown. (B) Optical density quantification (ImageJ software, mean OD ± SE, n = 4) of several phosphorylated proteins as preliminarily identified with specific antibodies (not shown) and indicated in the bottom. (C) Representative superimposed platelet aggregation traces in response to fibrillar collagen with or without ER, or ER. (D) Quantification of percent of maximal aggregation response (as seen with fibrillar collagen 10 μg/mL). Mean ± SE, n = 4. (E) Representative superimposed platelet aggregation traces in response to acid-soluble collagen with or without ER, or with or without anti-α2β1-integrin antibody. (F) Quantification of percent difference in lag time to platelet activation (as compared to the lag time to aggregation obtained with acid-soluble collagen, trace ‘1′ in (E). Effects of a nonfunctional blocking α2β1 antibody (12F1) are also shown.