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Thrombin-induced GPIb-IX centralization on the platelet surface requires
actin assembly and myosin II activation
TJ Kovacsovics and JH Hartwig
Department of Medicine, Brigham and Women's Hospital, Boston, MA 02115,
USA.
In resting platelets, the GPIb-IX complex, the receptor for the von
Willebrand factor (vWF), is linked to underlying actin filaments by
actin-binding protein (ABP-280). Thrombin stimulation of human platelets
leads to a decrease in the surface expression of the GPIb-IX complex, which
is redistributed from the platelet surface into the open canalicular system
(OCS). Because the centralization of GPIb-IX is inhibited by cytochalasin,
it is believed to be linked to actin cytoskeletal rearrangements that take
place during platelet activation. We have further characterized the
mechanism of GPIb-IX centralization in platelets in suspension. Following
thrombin stimulation, GPIb-IX shifts from the membrane skeleton of the
resting cell to the cytoskeleton of the activated cell in a reaction
sensitive to cytochalasin B. The cytoskeletal association of GPIb-IX
involves ABP- 280, as it correlates with the incorporation of ABP-280 into
the activated cytoskeleton and because no dissociation of the ABP-280/GPIb-
IX complexes is detected after thrombin activation. However, the
incorporation of GPIb-IX into the cytoskeleton is complete within 1 minute,
whereas GPIb-IX centralization requires 5 to 10 minutes for completion. The
movement of GPIb-IX to the cytoskeleton of activated platelets is therefore
necessary, but not sufficient for GPIb-IX centralization. Blockage of
cytosolic calcium increases induced by thrombin by loading with the cell
permeant calcium chelator Quin-2 AM inhibited GPIb-IX centralization by
70%, but did not prevent its association with the activated cytoskeleton.
Quin-2 loading did, however, decrease the incorporation of myosin II into
the activated cytoskeleton. The role of myosin II was further probed using
the myosin light chain kinase (MLCK) inhibitor wortmannin. Wortmannin
prevents myosin II association to the activated cytoskeleton and inhibits
GPIb- IX centralization by 50%, without affecting actin assembly or the
association of GPIb-IX to the cytoskeleton. Only micromolar concentrations
of wortmannin, high enough to inhibit MLCK, prevent GPIb- IX
centralization. These results indicate that thrombin-induced GPIb-IX
centralization requires a minimum of two steps, one associating GPIb-IX to
the activated cytoskeleton and the second requiring myosin II activation.
The involvement of myosin II implies that GPIb-IX/ABP-280 complexes, linked
to actin filaments, are pulled into the cell center, and that platelets may
exert contractile tension on vWF bound to its receptor.
Volume 87,
Issue 2,
pp. 618-629,
01/15/1996
Copyright © 1996 by The American Society of Hematology
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