Recombinant rhodostomin substrates induce transformation and active calcium oscillation in human platelets

Platelet activation has been a focus of numerous studies in normal and abnormal states. Morphological changes and calcium signals found with activated platelets in vitro have been well characterized. However, the rate of cell spreading on substrates and the frequency of calcium oscillation within individual platelets upon activation have not yet been reported. In this study, we first examined the ability of a recombinant fusion protein of rhodostomin (GST-rhodostomin), a snake disintegrin containing an Arg-Gly-Asp (RGD) motif, to activate platelets when GST-rhodostomin served as a substrate. Four aspects of platelet activities induced by immobilized GST-rhodostomin and fibrinogen were analyzed in parallel. Examinations of (1) translocation of P-selectin from intracellular compartments to the plasma membrane, (2) platelet adhesion to and spreading on substrates, (3) platelet contact pattern on substrates, and (4) the degree of phosphorylation of focal adhesion kinase in platelets indicated that GST-rhodostomin was a better substrate for platelet activation than fibrinogen. Analysis of the rate of platelet spreading on GST-rhodostomin was examined by time-lapsed video microscopy. The spreading rate averaged 0.43 micrometer/minute, while cell spreading averaged 0.22 micrometer/minute when platelets were plated on fibrinogen and treated with thrombin. A newly developed method, using time-lapsed microscopy and the Metamorph program, was used to analyze calcium signals within platelets. We found that platelets on GST-rhodostomin evoked calcium oscillation at a frequency of 4.77 spike/cell/minute vs 2.76 spike/cell/minute on fibrinogen. The results of cell spreading and calcium oscillation were consistent with the results of microscopic and biochemical assays. We therefore conclude that the determination of the rate of platelet spreading and the frequency of calcium oscillation within platelets performed in this study provides more quantitative parameters for measuring platelet activities. Our results also suggest that GST-rhodostomin might potentially be used as a probe to dissect the molecular mechanisms underlying the kinetic processes of platelet activation.