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

Citation
Hh. Chang et al., Recombinant rhodostomin substrates induce transformation and active calcium oscillation in human platelets, EXP CELL RE, 250(2), 1999, pp. 387-400
Citations number
61
Categorie Soggetti
Cell & Developmental Biology
Journal title
EXPERIMENTAL CELL RESEARCH
ISSN journal
00144827 → ACNP
Volume
250
Issue
2
Year of publication
1999
Pages
387 - 400
Database
ISI
SICI code
0014-4827(19990801)250:2<387:RRSITA>2.0.ZU;2-K
Abstract
Platelet activation has been a focus of numerous studies in normal and abno rmal 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 in dividual platelets upon activation have not yet been reported. In this stud y, we first examined the ability of a recombinant fusion protein of rhodost omin (GST-rhodostomin), a snake disintegrin containing an Arg-Gly-Asp (RGD) motif, to activate platelets when GST-rhodostomin served as a substrate. F our aspects of platelet activities induced by immobilized GST-rhodostomin a nd fibrinogen were analyzed in parallel. Examinations of (1) translocation of P-selectin from intracellular compartments to the plasma membrane, (2) p latelet adhesion to and spreading on substrates, (3) platelet contact patte rn on substrates, and (4) the degree of phosphorylation of focal adhesion k inase in platelets indicated that GST-rhodostomin was a better substrate fo r platelet activation than fibrinogen. Analysis of the rate of platelet spr eading on GST-rhodostomin was examined by time-lapsed video microscopy. The spreading rate averaged 0.43 mu m/minute, while cell spreading averaged 0. 22 mu m/minute when platelets were plated on fibrinogen and treated with th rombin. A newly developed method, using time-lapsed microscopy and the Meta morph program, was used to analyze calcium signals within platelets. We fou nd that platelets on GST-rhodostomin evoked calcium oscillation at a freque ncy 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 t he determination of the rate of platelet spreading and the frequency of cal cium oscillation within platelets performed in this study provides more qua ntitative parameters for measuring platelet activities. Our results also su ggest that GST-rhodostomin might potentially be used as a probe to dissect the molecular mechanisms underlying the kinetic processes of platelet activ ation. (C) 1999 Academic Press.