Urokinase-Type Plasminogen Activator Production by Cultured Megakaryocytes and Blood Outgrowth Endothelial Cells in the Quebec Platelet Disorder.

The Quebec Platelet Disorder (QPD) is an unusual α-granule protein disorder, associated with increased expression and storage of urokinase-type plasminogen activator (u-PA) in platelets and platelet-dependent, accelerated clot lysis. The increased u-PA in QPD platelets, and normal to increased levels in QPD plasma, led us to investigate the production of u-PA by cultured QPD megakaryocytes (MKs) and by QPD blood outgrowth endothelial cells (BOECs). Our objectives were to 1) characterize production of u-PA and other platelet proteins by normal and QPD MKs at different stages of differentiation, and 2) determine if the abnormal production of u-PA in the QPD extended to endothelial cells. Thrombopoietin-stimulated MKs were obtained by culturing CD34⁺ progenitor cells isolated from peripheral blood. BOECs were obtained from fibronectin-adherent peripheral blood mononuclear cells. Cell lysates and culture media were harvested from MKs on days 7 and 13, and from BOECs on day 11. Proteins were quantified by ELISA. MK differentiation was evaluated by flow cytometry for α_IIbβ₃ expression. For BOECs, endothelial lineage differentiation was assessed by immunostaining for acetylated low-density lipoprotein uptake and Ulex europaeus agglutinin binding. QPD and normal control BOECs were indistinguishable in morphology and differentiation during culture, and their content of u-PA was low (ng u-PA/mg cellular protein, mean ± SD for n = 9: QPD 3.5 ± 1.1, control 3.1 ± 1.5, p = 0.54), without detectable u-PA-PAI-1 (plasminogen activator inhibitor 1) complexes. QPD BOECs also produced normal amounts of the control secretory proteins PAI-1, multimerin 1 and von Willebrand factor (VWF). The morphology, viability, proliferation and differentiation of QPD and control MKs grown in culture were indistinguishable. By 7 days of MK culture, QPD and control cells had secreted similar, low quantities of u-PA into the culture media (ng u-PA/10⁶cells, mean ± SD for n = 4: QPD 0.3 ± 0.1, control 0.4 ± 0.1; p = 0.18). However, by day 13 of MK culture (when most cells expressed α_IIbβ₃), QPD cultures contained significantly more u-PA in their culture media (ng u-PA/10⁶ cells: QPD 20.5 ± 9.8, control 0.2 ± 0.1, p < 0.05 compared to controls and to day 7 QPD samples). The increased production of u-PA in late stage QPD MK cultures was associated with increased intracellular u-PA (ng u-PA/10⁶ cells: QPD 9.6 ± 6.3, control 0.03 ± 0.02, p < 0.05) and increases in intracellular and secreted u-PA-PAI-1 complexes (p < 0.05). In contrast, the levels of PAI-1, VWF, thrombospondin-1 and platelet factor 4 were normal in day 13 QPD MK cultures. Taken together, these data indicate that low levels of u-PA are normally produced by MKs and BOECs isolated from peripheral blood progenitors. The increased u-PA in differentiating QPD MKs and peripheral blood platelets, and normal levels of u-PA in QPD BOECs and early MK progenitors, indicate that the molecular defect in this disorder induces a cell-type restricted defect in u-PA regulation that is manifested during the later stages of MK differentiation. The molecular nature of this congenital defect in u-PA regulation is under investigation.