The effect of combined G_i and G_z signaling on thromboxane generation in human and mouse platelets. (A) Thromboxane B₂ (TxB2) was generated from washed human platelets in the presence of 1 mg/mL fibrinogen and stopped after 3.5 minutes of stimulation. Levels of TxB2 were obtained using a 96-well competitive ELISA kit as noted in the “Materials and methods” section. (B) Samples were preincubated with varying doses of PP2 for 10 minutes at 37°C and then stimulated with combined G_i and G_z signaling. (C) The effect of combined G_i and G_z stimulation on thromboxane generation in P2Y₁-deficient mouse platelets. Where noted, samples were preincubated with 10 μM PP2 for 10 minutes at 37°C. (D) The effect of GPIIb/IIIa antagonism on thromboxane generation induced by combined P2Y₁₂ and α_2A adrenergic stimulation. The GPIIb/IIIa antagonist, 10 μM SC57101A, was added 1 minute prior to the addition of agonists (where noted). Similarly, 1 mg/mL fibrinogen was added to samples 1 minute prior to stimulation (where noted). In A-D, the bars are representative of the average thromboxane A₂ generated (± standard error) from 3 different donors on 3 days of experiments. (E) Final model depicting G_i- and G_z-induced platelet aggregation. The agonists 2-MeSADP and ADP both activate the P2Y₁ and P2Y₁₂ receptors. MRS2179 and AR-C 69931MX antagonize the P2Y₁ and P2Y₁₂ receptors, respectively (noted by hatch marks). Epinephrine is an agonist for the α_2A adrenergic receptor and is blocked by the antagonist yohimbine. The combined signaling through the P2Y₁₂ and α_2A adrenergic receptors mediates Src family tyrosine kinase activity, which modulates integrin activation.