Thrombin receptor-dependent assembly of the IQGAP2/arp2/3-actin scaffold.
(A-B) PRP (5 × 107 platelets/lane corresponding to 100 μg) supplemented with 3 mM GPRP (thrombin activation only) were stimulated with 10 nM α-thrombin (1 U/mL) (A) or 20 μM PAR142-47 (B) for indicated time points, and total soluble lysates were immunoprecipitated with the anti-IQGAP2 mAb, followed by immunoblot detection for IQGAP2, arp3a, arp2, actin, and gelsolin. Note the immediate and specific assembly of arp2/3 and actin without any evidence for gelsolin involvement (for PAR1,42-47 arp2 was maximally detected at 30 seconds with low-level detection at 120 and 600 seconds). Ctrl indicates 5 μg SDS-solubilized platelet lysate control. Comparable results were seen using GFP solubilized in HBMT/1% Triton-X (not shown). (C) Percentage of platelet actin immunoprecipitated at individual time points was determined by densitometric comparison to 5 μg SDS-solubilized platelets. (D) F-actin content was monitored as described in “Materials and methods.” Results represent one complete set of experiments duplicated on 2 occasions.
