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
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.