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Mechanism of shape change in chilled human platelets
R Winokur and JH Hartwig
Experimental Medicine Division, Brigham and Women's Hospital, Boston, MA
02115, USA.
The so-called cold activation of platelets that precludes refrigeration of
platelets for storage has long been recognized, but its mechanism has
remained a mystery. Cooling of discoid resting platelets to temperatures
below 15 degrees C causes shape distortions, and the chilled cells rewarmed
to above 25 degrees C are spheres rather than discs. As platelet shape
change responsive to receptor activation at normal temperatures requires
the remodeling of an actin scaffolding (Hartwig JH, 1992, J Cell Biol
118:1421-1442), we examined the role of actin in the morphologic changes
induced by cooling. The addition of actin monomers onto the fast-exchanging
(barbed) ends of actin filaments accompanies the initial physiologic
platelet shape changes, and a key control point in this growth is the
removal of proteins (caps) from the filament ends. This uncapping of actin
filament ends is mediated by polyphosphoinositide aggregates in vitro,
suggesting that cold-induced phase changes in membrane lipids might uncap
actin filaments and thereby account for actin assembly-mediated shape
alterations during cooling. Consistent with this hypothesis, reversible
inhibition of actin assembly with cytochalasin B prevented the distortions
in shape, although cooled platelets had increased actin nucleation sites
and became spherical. Another step in normal platelet shape changes
requires the severing of actin filaments that maintain the resting
platelet. The proteins that sever initially bind to the broken filament
ends, and uncapping of these fragmented filaments provides numerous
nucleation sites for growth of actin filaments to fill in spreading
filopodia and lamellae. Actin filament fragmentation requires a rise in
intracellular calcium, and we showed that chilling platelets from 37
degrees C to 4 degrees C increases free cytosolic calcium levels from 80
nmol/L to approximately 200 nmol/L in minutes, thus providing an
explanation for the spherical shape of cooled, rewarmed platelets. Blocking
the calcium transient with nanomolar concentrations of the permeant calcium
chelators Quin-2 and Fura-2 prevented the increase in nucleation sites and
the sphering, but not the other shape changes of chilled and rewarmed
platelets. However, a combination of micromolar cytochalasin B and
millimolar intracellular calcium chelators preserved the discoid shapes of
chilled and rewarmed platelets. After removal of cytochalasin B and
addition of sufficient extracellular calcium, these platelets responded
with normal morphologic alterations to glass and thrombin activation.
Volume 85,
Issue 7,
pp. 1796-1804,
04/01/1995
Copyright © 1995 by The American Society of Hematology
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