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Evidence for a novel binding protein to urokinase-type plasminogen
activator in platelet membranes
Y Jiang, R Pannell, JN Liu and V Gurewich
Institute for the Prevention of Cardiovascular Disease, Deaconess Hospital,
Harvard Medical School, Boston, MA, USA.
Endogenous urokinase-type plasminogen activator (u-PA) has been identified
in platelet membrane, and platelets have been shown to take up exogenous
high molecular weight u-PA from the ambient medium. In this report, the
mechanism of the association of u-PA with platelets was investigated using
recombinant, single chain u-PA. When gel filtered human platelets were
incubated with radiolabeled u-PA, the u- PA was found to specifically and
saturably bind to the resting platelets in a dose-dependent manner.
Unlabeled u-PA and the amino terminal fragment of u-PA inhibited 125I-u-PA
binding to platelets with a mean IC50 of 65 and 58 nmol/L, respectively. A
single saturable binding site in intact resting platelets was found with a
mean kd of 43 +/- 25 nmol/L and 2263 +/- 809 sites per platelet. In
contrast to resting platelets, 125I-u-PA did not bind to thrombin-induced
platelets. Western blotting studies, using a monoclonal or a polyclonal
antibody specific for the u-PA cell-surface receptor (u- PAR), failed to
show evidence of u-PAR in resting platelets, whereas, u-PAR was found at
approximately 54 and approximately 48 kD on U937 monocytes, which served as
a positive control. Ligand blotting of platelet membrane and of U937 cell
proteins with 125I-u-PA revealed a u-PA binding protein of approximately 70
kD in the platelets and one of approximately 54 kD in the U937 cells.
Complexion of u-PA with a platelet membrane protein was also shown by gel
filtration of a mixture of u-PA and platelet membrane proteins. A u-PA
complex was further shown by enzyme-linked immunosorbent assay when
microtiter plates were coated with platelet membrane proteins, and this
complex formation was shown to be dose-dependent and saturable with an
apparent kd of 17 nmol/L. It was concluded that platelet membrane contains
a specific, high affinity u-PA-binding protein that is distinct from u-PAR.
Volume 87,
Issue 7,
pp. 2775-2781,
04/01/1996
Copyright © 1996 by The American Society of Hematology
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