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 I-125-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 k(d) of 43 +/- 25 nmol/L and 2263 +/- 809 sites per platelet. In contrast to resting platelets, I-125-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 similar to 54 and similar to 48 kD on U937 monocytes, which served as a positive control. Ligand blotting of platelet membrane and of U937 cell proteins with I-125-u-pA revealed a u-PA binding protein of similar to 70 kD in the platelets and one of similar to 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 k(d) 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. (C) 1996 by The American Society of Hematology.