PADGEM (Platelet Activation-Dependent Granule ⇒ External Membrane) glycoprotein, a
platelet alpha granule integral membrane protein with a molecular weight of 140,000,
is translocated to the plasma membrane during granule secretion. PADGEM protein is
expressed solely on activated platelets, but is not on the surface of resting platelets.
Because HEL cells contain platelet alpha granule-like organelles and proteins (e.g.
platelet factor 4, von Villebrands factor, β-thromboglobulin) and express certain
platelet membrane proteins (e.g. GP IIb/IIIa, GPIb), we evaluated induced and uninduced
HEL cells for the synthesis and expression of PADGEM protein. HEL cells were induced
with 1.25% DMSO for 3-4 days, then grown in the absence of DMSO for 1-3 weeks. After
eight cycles of DMSO exposure, the induced HEL cells were found to increase the expression
of PADGEM, in contrast to the uninduced cells. Intact fixed and unfixed induced HEL
cells were observed by immunofluorescence, utilizing KC4, a monoclonal anti-PADGEM
antibody, to express PADGEM while non-induced HFT. cells expressed low levels of PADGEM.
Both induced and uninduced HEL cells bound A2A9, an anti—GP Ilb/IIIa monoclonal antibody.
Quantitative analysis by fluorescence activated cell sorting demonstrated a 2.5—fold
increase in mean surface expression of PADGEM and 3.3—fold mean increase in GP IIb/IIIa
surface expression compared to uninduced cells. By fluoresence microscopy, 70% of
induced HEL cells expressed PADGEM protein versus 20% of the uninduced cells. GP-IIb/IIIa
expression inoreased from 40% in noninduced cells to 90% in induced cells. The induced
HEL cells contained PADGEM with a molecular weight identical to that of platelets,
as demonstrated by Western blotting using the KC4 antibody. Direct binding experiments
with 125I-KC4 antibody demonstrated that surface binding was specific, saturable, and time-dependent.
Surface expression of PADGEM protein was not increased with platelet agonists (thrombin,
epinephrine, ADP) nor cytokines (IL-1, IL-2, tissue necrosis factor). The surface
density of PADGEM protein on induced HEL cells and activated platelets appears similar.
HEL cells should provide a useful model to assist in the elucidation of the structure,
function and biology of PADGEM protein.
