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Differential mechanisms targeting type 1 plasminogen activator inhibitor
and vitronectin into the storage granules of a human megakaryocytic cell
line
SA Hill, SG Shaughnessy, P Joshua, J Ribau, RC Austin and TJ Podor
Department of Pathology, McMaster University, Hamilton, Ontario, Canada.
Type 1 plasminogen activator inhibitor (PAI-1) and its cofactor vitronectin
(Vn) are stored within the alpha-granules of platelets. The two possible
sources for their biosynthetic origin are endogenous synthesis in
megakaryocytes or endocytosis from plasma. Using ultrastructural and
confocal laser scanning microscopic (CLSM) image analysis, we observed that
treatment of Dami cells, a human megakaryocytic cell line, with phorbol
myristate acetate (PMA) induces the accumulation of PAI-1 and Vn in
intracellular storage vacuoles that contain other alpha-granule proteins
such as von Willebrand factor. To examine evidence for biosynthesis of
PAI-1 and Vn by Dami cells, we immunoprecipitated PAI-1 and Vn from the
conditioned media of cells biosynthetically radiolabeled with
35S-methionine in the presence or absence of PMA. In contrast to Hep G2
cells, which synthesize both PAI- 1 and Vn, only 35S-PAI-1 was recovered
from PMA-treated Dami cells. Reverse transcription-PCR analysis of RNA
extracted from resting and PMA-treated Dami cells confirmed that PAI-1 mRNA
expression was detectable at low levels in resting cells and induced by PMA
treatment. In contrast, Vn mRNA was not detected. We examined binding and
internalization (endocytosis) of PAI-1 and Vn by Dami cells using
biotinylated analogs (b-PAI-1 and b-Vn). Flow cytometry analysis indicated
that the binding of b-Vn to Dami cells was dose-dependent, saturable, and
specific for multimeric forms of Vn. Cells were incubated at 4 degrees C or
37 degrees C and endocytosis of b-Vn was shown by probing
electrophoretically fractionated cell lysates with 125I-labeled
streptavidin. Only cells incubated at 37 degrees C internalized b-Vn. CLSM
image analysis confirmed that the b-Vn was internalized and that it
colocalized with PAI-1 in storage granules. The binding of b-Vn to cells
was inhibited by the presence of PAI-1, and there was no evidence of
specific b-PAI-1 binding or uptake to resting or PMA-treated cells. These
data suggest that accumulation of PAI-1 in Dami cell storage granules is
due to endogenous synthesis and that the accumulation of Vn is due to
endocytosis of serum-derived Vn.
Volume 87,
Issue 12,
pp. 5061-5073,
06/15/1996
Copyright © 1996 by The American Society of Hematology
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