Release of RGS10 from binding sites on spinophilin leads to a rise in free RGS10 levels in platelets incubated with agonists or PGI2. (A) Human platelets were incubated for 3 minutes with a PAR1 agonist peptide (SFLLRN 50 µM), a TxA2 mimetic (U46619 10 µM), ADP (10 µM), or collagen (10 µg/mL) in the absence or presence of 100 μM aspirin (ASA) and 1 U/mL apyrase (APY), as indicated. Lysates were precipitated with anti-RGS10 and probed for spinophilin before reprobing with anti-RGS10 (data are mean ± SEM, N = 3). (B) Human platelets were incubated for 3 minutes with ADP (10 µM) in the absence or presence of 100 μM aspirin (ASA). Lysates were precipitated with anti-RGS10 and probed for spinophilin (SPL) before reprobing with anti-RGS10 (data are mean ± SEM, N = 4). (C) Human platelets were incubated with 20 µM forskolin (Forsk) or 15 µM PGI2, with or without 1 µM okadaic acid, as indicated. Proteins were precipitated with anti-RGS10 or nonimmune immunoglobulin (Ig) and then probed with anti-spinophilin before reprobing with anti-RGS10 (data mean ± SEM, N = 4). P values are relative to resting platelets. (D) Lysates were prepared from resting platelets and from platelets incubated with PGI2 (15 µM) or the PAR1 agonist peptide SFLLRN (50 µM). The lysates were then incubated with GST-Gi2α coupled to glutathione beads in the presence of GDP plus AlF4− or GDP alone, as indicated. Bound proteins were subjected to electrophoresis and probed with anti-RGS10 and Gi2α antibodies to detect RGS10 and GST–Gi2α fusion protein, respectively (upper panels). Summary of 3 experiments expressed as the percentage of the result obtained with resting platelets (data are mean ± SEM) (lower panel). P values are relative to resting platelets.
