Summary
Phosphatidylserine (PS) is normally restricted to the inner leaflet of the plasma
membrane of cells (including blood platelets). Upon cell activation PS may become
exposed to the outer surface of the cell. Cell membranes with surface exposed PS at
the outside form a catalytic surface for coagulation reactions. When platelets are
activated with ionophore or with thrombin in combination with thapsigargi, calcium
induced scrambling of phospholipids takes place, resulting in PS exposure. Concomitant
with PS exposition structural changes take place. On resting and activated platelets
we combined the immunocytochemical detection of surface exposed PS with (ultra)structural
information. Blood platelets were activated in the presence of annexin V, a protein
which binds to PS in the presence of Ca2+. Annexin V was found to bind to lipid bilayers containing more than 5 mole % PS as
estimated by binding of fluorescent-labelled annexin V to liposomes with varying PS
concentrations. After vitrification, freeze-substitution and embedding of the platelets,
annexin V was located on ultra thin sections, as detected by an anti-annexin V antibody
and gold labelled protein A. Upon activation, the platelets show two different forms;
irregular platelets with unchanged cytoplasm and round cells with apparently diluted
cytoplasm. Activation with ionophore initially resulted in both forms, but after ten
minutes only round platelets with diluted cytoplasm were observed. Both forms of these
platelets as well as the microvesicles were found to be annexin V positive. However
upon activation with thrombin in combination with thapsigargin, only the round cells
with diluted cytoplasm and microvesicles were annexin V positive, whereas platelets
with unchanged cytoplasm, even when microvesicles are present, are negative for annexin
V.
