TSP-1 enhances platelet adhesion by antagonizing NO and 8Br-cGMP signaling via Rap1 and blocks VASP-Ser²³⁹ phosphorylation by inhibiting cGK. Platelets preincubated in the presence or absence of TSP-1 (2.2 nM) were treated with DEA/NO (10 μM) or 8Br-cGMP (100 μM) 2 minutes prior to stimulation with 0.5 U/mL thrombin (A). Rap1 activation was analyzed by affinity purification using GST-RalGDS-RBD fusion protein immobilized on Glutathione-Sepharose beads. Platelets incubated at 37°C in the presence or absence of GGTI-298 (10 μM for 30 minutes) before addition of 1 U/mL thrombin for 1 minute, or incubated with TSP1 (2.2 nM for 15 minutes), were lysed and subjected to a Rap activation assay (B). Fresh, washed human platelets were used directly or preincubated in the presence of GGTI-298 for 30 minutes, added to 35-mm plastic dishes precoated with either type I collagen (3 μg/mL) or fibrinogen (5 μg/mL) (C,D), and incubated in Tyrode buffer and the indicated treatment agents for 1 hour at 37°C. Wells were washed and platelets fixed, stained, and counted. Washed human platelets in Tyrode buffer (2 × 10⁵ platelets/μL) were incubated in the presence of thrombin (0.2 U/mL) and exogenous NO (DEA/NO 10 μM) plus or minus TSP1 (2.2 nM) for 5 minutes or preincubated with GGTI-298 for 30 minutes and treated as described earlier for 5 minutes; aggregation was determined under high shear (E). Washed human platelets, either untreated or treated with thrombin (0.1 U/mL), 8Br-cGMP (100 μM for 2 minutes), or 2.2 nM TSP1 followed by 8Br-cGMP, were lysed, resolved on SDS gels, blotted, and probed with a polyclonal antiserum against Ser²³⁹-phosphorylated VASP (F). Washed human platelets were preincubated with TSP1 (2.2 nM) or Rp-8pCPT-cGMP (5 μM) for 15 minutes prior to treatment with NO (DEA-NO 10 μM) for 5 minutes (G) or 8Br-cGMP (100 μM) for 1 minute (H). The platelets were chilled to terminate the reaction, washed, lysed, and centrifuged. Lysate supernatants containing equal amounts of protein (100 μg) were assayed for phosphorylation of the cGK-I–selective substrate Arg-Lys-Arg-Ser-Arg-Ala-Glu. Data are representative of at least 3 experiments (A,B, E-H). Results are the mean (± SD) of at least 3 experiments (C,D).