A Role for Plasma Urokinase Plasminogen Activator Receptor (uPAR) in Thrombosis in Paroxysmal Nocturnal Hemoglobinuria (PNH).

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Venous thrombotic events are responsible for serious morbidity and the most frequent cause of death in patients with paroxysmal nocturnal hemoglobinuia (PNH). Urokinase plasminogen activator receptor (uPAR) is a glycosylphosphatidylinositol (GPI)-linked protein which is expressed on neutrophils and endothelial cells and mediates endogenous thrombolysis through a urokinase plasminogen activator (uPA)-dependent mechanism. On the cell surface, pro-uPA binds to uPAR via its growth factor domain, promoting the conversion of pro-uPA to active uPA. Active uPA in turn cleaves the proenzyme plasminogen, yielding active plasmin. Previous work in PNH patients has shown that the membrane form of uPAR is decreased or absent on granulocytes, and soluble uPAR levels are increased in patient's plasma. In the current study of 45 patients with PNH and 20 normal controls, we demonstrate that plasma suPAR concentrations correlated directly with the proportion of neutrophils lacking CD16, another GPI-anchored protein (R2=0.9), and that levels are 4-10-fold greater in PNH than in controls. Patients with a history of thrombosis had higher soluble uPAR levels (in stored samples pre-dating the thrombotic event) than did patients without a history of thrombosis (mean 2.2 ng/mL compared to 0.5 ng/mL; p=0.0001). suPAR levels returned to normal in all of five patients undergoing successful bone marrow transplantation. In normal persons soluble uPAR may be generated by alternative splicing or proteolytic cleavage. In vitro we demonstrated release of suPAR from PNH hematopoietic cells: supernatants obtained from PNH CD34 cells cultured in liquid media with standard growth factor in three experiments showed suPAR levels 20 fold greater than those obtained from normal CD34 cells. Platelets also appeared to be a substantial source of suPAR: uPAR was expressed on the surface of ADP-activated CD59-positive but not CD59-negative platelets from PNH patients. By an ELISA specific for suPAR and by immunoblot, we showed that substantial quantities of suPAR were released following collagen-activation of PNH but not normal platelets. Plasma from PNH patients contained much greater quantitites of suPAR than did peripheral blood cells from those patients. Theoretically, suPAR might bind to plasma uPA, preventing its interaction with the active membrane-anchored form. In vitro, we demonstrated dose-dependent decreases in the activity of single-chain urokinase following the addition of recombinant suPAR, with loss of 25% of the activity after the addition of 0.45nM suPAR. The absence of leukocyte and platelet plasma membrane associated uPAR and high levels of soluble uPAR in the circulation may be useful in predicting thrombosis and suggest mechanisms of anticoagulation in these unusual patients.