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Blood, 15 June 2002, Vol. 99, No. 12, pp. 4466-4474
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Arp2/3 complex is required for actin polymerization during
platelet shape change
Zhi Li,
Eric S. Kim, and
Elaine L. Bearer
From the Department of Pathology and Laboratory
Medicine, Brown University, Providence, RI.
Platelets undergo a series of actin-dependent morphologic
changes when activated by thrombin receptor activating peptide (TRAP) or when spreading on glass. Polymerization of actin results in the
sequential formation of filopodia, lamellipodia, and stress fibers, but
the molecular mechanisms regulating this polymerization are unknown.
The Arp2/3 complex nucleates actin polymerization in vitro and could
perform this function inside cells as well. To test whether Arp2/3
regulated platelet actin polymerization, we used recombinant Arp2
protein (rArp2) to generate Arp2-specific antibodies ( Arp2). Intact
and Fab fragments of Arp2 inhibited TRAP-stimulated
actin-polymerizing activity in platelet extracts as measured by the
pyrene assay. Inhibition was reversed by the addition of rArp2 protein.
To test the effect of Arp2/3 inhibition on the formation of specific
actin structures, we designed a new method to permeabilize resting
platelets while preserving their ability to adhere and to form
filopodia and lamellipodia on exposure to glass. Inhibition of Arp2/3
froze platelets at the rounded, early stage of activation, before the
formation of filopodia and lamellipodia. By morphometric analysis, the
proportion of platelets in the rounded stage rose from 2.85% in
untreated to 63% after treatment with Arp2. This effect was also
seen with Fab fragments and was reversed by the addition of rArp2
protein. By immunofluorescence of platelets at various stages of
spreading, the Arp2/3 complex was found in filopodia and lamellipodia.
These results suggest that activation of the Arp2/3 complex at the
cortex by TRAP stimulation initiates an explosive polymerization of
actin filaments that is required for all subsequent actin-dependent events.
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