Evidence for a novel binding protein to urokinase-type plasminogen activator in platelet membranes.
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.
Jiang
Y
Y
Institute for the Prevention of Cardiovascular Disease, Deaconess Hospital, Harvard Medical School, Boston, MA, USA.
Pannell
R
R
Liu
J N
JN
Gurewich
V
V
D001665:Binding Sites; D001792:Blood Platelets / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D006801:Humans; D011994:Recombinant Proteins / Q000378:metabolism; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism*
D011994:Recombinant Proteins; D014568:Urokinase-Type Plasminogen Activator