Quebec Platelet Disorder Is Associated With Greater Than Expected Increases In Urokinase Plasminogen Activator In Granulocytes and Monocytes

Abstract

Myeloid leukocytes produce urokinase plasminogen activator (uPA), an important activator of fibrinolysis. Stimuli such as lipopolysaccharide and formyl-methionyl-leucyl-phenylalanine (LPS/fMLP), increase the expression of PLAU by myeloid leukocytes. We postulated that the basal, and/or stimulus-induced, uPA production by myeloid leukocytes would be increased in Quebec platelet disorder (QPD), a congenital bleeding disorder caused by duplication of PLAU, the uPA gene. In QPD, plasma and urine uPA levels are within the expected range. However, overexpression of PLAU in QPD was found to emerge during megakaryopoiesis and QPD platelets to contain >100-fold increased uPA.We investigated PLAU expression by platelet-free, granulocytes and monocytes that were differentiated in culture, from peripheral blood CD34+ cells, without added thrombopoietin and erythropoietin. QPD and control (C) myeloid cells were harvested on day 7 or 14 of culture (n= 6 samples per subject for each endpoint). Granulocytes and monocytes were isolated by affinity purification. For some experiments, granulocytes from day 14 cultures were treated with: LPS/fMLP; complement and heat activated immunoglobulin (C5a/IgG); thrombin, thrombin plus fibrinogen, or thrombin plus plasma (IIa, IIa/Fg or IIa/plasma). PLAU expression was characterized by real time quantitative polymerase chain reaction and uPA was quantified by enzyme-linked immunosorbent assays. The lysis of plasma clots (optical density endpoint) that had been spiked with day 14 granulocytes or uPA, was assessed with or without added tranexamic acid. Data were expressed as means ± standard error (SEM) and analyzed by Mann-Whitney tests.Higher PLAU expression by QPD myeloid leukocytes was evident in day 7 (23-fold, p = 0.002) and day 14 (13-fold; p = 0.002) myeloid leukocyte cultures, accompanied by increased cellular uPA (day 7, 16-fold increase [pg/106 cells, QPD: 162 ± 11; C: 10.2 ± 0.3, p = 0.002]; day 14, 9-fold increase [pg/106 cells, QPD: 458 ± 8, C: 50.9 ± 2.5, p = 0.002]). In day 14 cultured cells, PLAU expression was higher in QPD granulocytes (57-fold) and monocytes (9-fold)(p values ≤ 0.005), and allele specific analysis confirmed overexpression of PLAU by the disease chromosome. The higher PLAU expression was associated with higher levels of uPA (pg/106 day 14 cultured cells) in QPD granulocytes (170-fold [QPD: 477 ± 33, C: 2.8 ± 0.4, p = 0.005]) and monocytes (5-fold increase [QPD: 208 ± 11; C: 41.5 ± 4.3; p = 0.002]). Like control granulocytes, QPD granulocytes expressed and secreted much more uPA after exposure to immune, infectious and procoagulant stimuli (Table 1). Interestingly, some of these stimuli reduced the differences between QPD and control granulocytes in PLAU expression and secreted uPA (Table 1). In fibrin clots, QPD granulocytes triggered more fibrinolysis than control granulocytes, and release higher levels of uPA. Tranexamic acid inhibited the fibrinolysis-induced by QPD and control granulocytes.Table1Effect of stimuli on PLAU expression and uPA secreted by granulocytesStimulus:noneLPS/fMLPC5a/IgGIIaFg + IIaPlasma + IIaPLAU expression (fold difference, QPD vs. C)773311143P value (QPD vs. C)0.0020.0020.0020.0020.0020.002uPA released (pg/106 cells ± SEM)C4.5 ± 1.28,250 ± 30011,050 ± 7501,300 ± 501,300 ± 1007,250 ± 550QPD33.5 ± 2.615,900 ± 130032,250 ± 90011,000 ± 35014,800 ± 75016,950 ± 800uPA release (fold difference, QPD vs. C)7238112P value (QPD vs. C)0.0020.0020.0020.0020.0020.002We conclude that the greater than expected effect of the QPD PLAU duplication on PLAU expression during myeloid differentiation extends to granulocytes and monocytes. In QPD, PLAU dysregulation was associated with much higher uPA levels in granulocytes than in monocytes. Based on changes to PLAU expression and uPA in response to procoagulant, immune or septic stimuli, we conclude that QPD has greater effects on the basal expression of PLAU by myeloid cells. The upregulation of PLAU expression by granulocytes exposed to thrombin, fibrin clots, immune and septic stimuli, may contribute to the increased fibrinolysis in some pathological conditions. Disclosures:No relevant conflicts of interest to declare.