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Heterogeneity in filamentous actin content among individual human blood
platelets
A Oda, JF Daley, C Cabral, JH Kang, M Smith and EW Salzman
Department of Surgery, Beth Israel Hospital, Boston, MA 02215.
The content of filamentous actin in individual platelets was measured by
flow cytometry, using a fluorescent probe specific for filamentous actin
(F-actin), 7-nitrobenz-2-oxa-1,3-phallacidin (NBD-phallacidin).
NBD-phallacidin binding to fixed platelets was specific in that either
pretreatment of platelets with unlabeled phallacidin or absorption of
NBD-phallacidin by rabbit skeletal F-actin, but not globular actin (G-
actin), resulted in a significant loss in the bound fluorescent probe. Mean
NBD-phallacidin binding to fixed platelets varied with the agonist and
paralleled the changes in F-actin reported with the DNAse I inhibition
assay. (1) NBD-phallacidin binding increased with stimulation by ADP,
U46619 (a prostaglandin H2 analogue), or collagen and paralleled shape
change. (2) Epinephrine did not increase NBD- phallacidin binding. (3)
Platelets treated at 4 degrees C contained more F-actin than did platelets
kept at 37 degrees C. (4) Cytochalasin D (10 mumol/L) inhibited the
increase of phallacidin binding to individual platelets stimulated by
either ADP or U46619. In measurements of cytosolic free calcium
concentration ([Ca2+]i) by flow cytometry in Indo-1-loaded platelets, ADP's
dose-response for actin polymerization was similar to that for calcium
mobilization. As shown by flow cytometry, a tail population that had a
minimal increase in F- actin upon stimulation with ADP or U46619 also
contained the platelets with the least forward and right angle light
scattering, which are functions of platelet size and shape. When platelets
treated with NBD- phallacidin were incubated with S12-murine monoclonal
antibody (a marker of alpha-granule secretion detected by
phycoerythrin-conjugated antimouse IgG second antibody), phallacidin
fluorescence paralleled S12 binding. Thus, human blood platelets are
heterogeneous in regard to actin polymerization at rest and in association
with platelet activation; different degrees of phallacidin binding may
identify functionally different platelet populations.
Volume 79,
Issue 4,
pp. 920-927,
02/15/1992
Copyright © 1992 by The American Society of Hematology
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