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Annexin V as a probe of aminophospholipid exposure and platelet membrane
vesiculation: a flow cytometry study showing a role for free sulfhydryl
groups
J Dachary-Prigent, JM Freyssinet, JM Pasquet, JC Carron and AT Nurden
URA CNRS 1464, Hopital Cardiologique, Pessac, France.
Annexin V, a protein with a high affinity and a strict specificity for
aminophospholipids at physiologic calcium concentrations, was used to probe
platelet activation and the development of procoagulant activity. Platelet
secretion was studied in parallel using VH10, a murine monoclonal antibody
specific for GMP-140, an alpha-granule membrane glycoprotein. Both proteins
were labeled with fluorescein isothiocyanate and platelet activation was
assessed by flow cytometry. Microparticles, which are shed from the
platelet surface and also support procoagulant activity, were distinguished
from platelets according to their associated light scattering signal. The
relative ability of different inducers to trigger exposure of the
procoagulant surface and microparticle formation was: ionophore A23187 >
thrombin plus collagen > collagen > thrombin. The density of
aminophospholipid on microparticles was higher than on remnant platelets.
Platelet activation by these agonists was accompanied by GMP-140 exposure,
both on platelets and microparticles. Here, thrombin was the most efficient
agonist. The mechanisms responsible for the above processes were
investigated using E-64-d, a specific membrane-permeable inhibitor of
Ca(2+)-activated protease (calpain); tetracaine, an activator of calpain;
and N-ethylmaleimide and diamide, two sulfhydryl-reactive agents. These
agents were added to platelets alone or before stimulation by agonists.
Calpain activity was assessed by the hydrolysis of cytoskeletal proteins as
determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Results showed that calpain activity is not essential for aminophospholipid
translocation or for secretion. In contrast, although sulfhydryl-reactive
agents alone can trigger procoagulant activity, they inhibit microvesicle
formation and platelet secretion induced by the above agonists, suggesting
that different mechanisms account for these phenomena. The use of annexin V
in flow cytometry is a rapid method to assess procoagulant activity in
platelets and the loss of phospholipid asymmetry in cell membranes.
Volume 81,
Issue 10,
pp. 2554-2565,
05/15/1993
Copyright © 1993 by The American Society of Hematology
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