αArp2 inhibits TRAP-stimulated actin-polymerizing activity.

Platelets were stimulated with TRAP and were permeabilized with and without antibody as indicated. Actin-polymerizing activity was measured immediately after antibody addition. For each graph, all tracings are from the same preparations of platelets and actin preparation obtained on the same day. Results are presented in arbitrary units (au), as is standard in the field. Final fluorescence intensity with nonstimulated platelets (no TRAP) was defined as 10 au. Platelet extracts always produced at least a 20% increase over actin alone (not shown). (A) Effect of αArp2 on actin-polymerizing activity of TRAP-stimulated sonicated platelets. TRAP (top tracing) increased actin-polymerizing activity 5-fold over nonstimulated platelets (lowest tracing) in samples permeabilized by sonication. Affinity-purified αArp2 added to the TRAP-stimulated platelets decreased the initial rate and the extent of polymerization, whereas anti-kaptin had little to no effect at the same concentration (0.01 mg/mL, 0.7 μM) used for αArp2. (B) Effect of αArp2 and αp34 on actin-polymerizing activity of TRAP-stimulated Triton-permeabilized platelets. Platelets were permeabilized by either sonication, as in Figure 1A, or with Triton and antibody added as indicated (αArp2 and αp34 were at 15 μg/mL [0.1 μM]). A similar increase in fluorescence was obtained with TRAP stimulation, whether platelets were permeabilized by sonication (sonic) or Triton (Tx). The increase in the initial rate of polymerization after TRAP stimulation was blocked by αArp2 regardless of whether permeabilization was with sonication or Triton. Less of an effect on TRAP-stimulated activity was detected with αp34. (C) Effect of Fab fragments of αArp2 on actin-polymerizing activity of TRAP-stimulated platelets. Platelets stimulated with TRAP and permeabilized as in Figure 1A were treated with antibodies as indicated at the following concentrations: anti-kaptin (0.1 μM), αArp2 + rArp2 (0.1 and 0.5 μM, respectively), Fab fragments of αArp2 (0.12 and 0.44 μM), and intact αArp2 from a different rabbit (0.1 μM). (D) Effect of αArp2 on actin-polymerizing activity of nonstimulated platelets. αArp2 was added (as indicated) to nonstimulated platelets immediately after sonication. Note that the arbitrary units (au) on the y-axis of the graph was expanded to increase the sensitivity to demonstrate the effect of the antibody on the lower level of activity present in nonstimulated platelets. (E) Dose dependence of αArp2 inhibition. The final fluorescence of representative experiments treated with increasing amounts of αArp2 or preimmune IgG (pre-IgG) is compared. A similar graph could be drawn for either anti-p34 or anti-kaptin as control antibody (Table 1). Experiments were normalized by setting the final fluorescence of parallel preparations, measured in the absence of antibody, at 100%, which allowed comparison of the low level of activity in nonstimulated platelets with the much higher activity after TRAP. Thus, results from TRAP-stimulated and nonstimulated experiments could be superimposed on the same graph.