Alboaggregin-A induces platelet aggregation, release of 5-HT and an increase in cytosolic calcium upon binding to GP Ib. For 5-HT release, studies platelets were preloaded with [³H]-5-HT and stimulated with various concentrations of alboaggregin-A (A, D) or 3.5 μg/mL alboaggregin-A (B, C) for 1 minute. Collagen (100 μg/mL; 2 minutes) was used for comparison and as a negative control (B, C). Release of 5-HT is presented as a percentage of total 5-HT content, and a dose-response relationship was determined in (A), showing an EC₅₀ of 3.5 μg/mL. Platelet aggregation is presented as the decrease in optical density induced by agonist as a percentage of the maximal possible decrease (B). Platelets were pretreated with MoAb 6D1 (9 μg/mL) for 6 minutes, or EGTA (1 mmol/L) for 10 minutes, before stimulation with agonist (B, C). For assessment of changes in cytosolic calcium (D), platelets were preloaded with the calcium indicator dye Fura-2 and fluorescence measurements were made at emission wavelength 510 nm. Data are presented as the ratio of fluorescence measurements at excitation wavelengths 340 and 380 nm. Alboaggregin-A is added at the time point indicated by the arrow, at concentrations indicated on the right of the graphs. Data presented are means ± SEM for 3 experiments (A, B, C) or are representative of 3 separate experiments (D).