Platelet aggregation inhibitors from Philippine marine invertebrate samples screened in a new microplate assay

A new microplate assay for Ca(2+)-induced platelet aggregation as detected by Giemsa dye was used to screen marine invertebrate samples from the Philippines for inhibitors of human platelet aggregation. Out of 261 crude methanol extracts of marine sponges and tunicates, 25 inhibited aggregation at 2 mg/ml. Inhibition of agonist-induced aggregation in an aggregometer was used to confirm results of the microplate assay and to determine the specific mode of inhibition of 2 samples. The marine sponge Xestospongia sp. yielded a xestospongin/araguspongine-type molecule that inhibited collagen-induced aggregation by 87% at 2 micro g/ml, and epinephrine-induced aggregation by 78% at 20 micro g/ml, while the marine sponge Aplysina sp. yielded 5,6-dibromotryptamine, which inhibited epinephrine-induced aggregation by 51% at 20 micro g/ml. In this study we have found that the microplate assay is a simple, inexpensive, yet useful preliminary tool to qualitatively screen a large number of marine samples for antiplatelet aggregation activity.