Endogenous Arteriolar Fibrinolysis Requires Urokinase Plasminogen Activator and Leukocyte Urokinase Plasminogen Activator Receptor-1.

Abstract

Background: In venous thrombi, urokinase associated with leukocytes activates plasminogen to initiate endogenous fibrinolysis. Leukocytes also interact with arteriolar thrombi.Objectives: We hypothesized that urokinase plasminogen activator (uPA) activity would also be delivered to arteriolar thrombi by leukocytes.Methods: Using established techniques of high-speed intravital fluorescence microscopy to observe the cremaster muscle of mice, we measured platelet and fibrin accumulation in thrombi generated by a laser injury to arterioles. The accumulation of platelets in a thrombus is quantitated by measuring the fluorescence from anti-CD41 antibody tagged with a fluorescent secondary antibody and the presence of fibrin is detected using a fluorescence-tagged anti-fibrin antibody (T2G1).Results: In C57BL/6 wild type mice, fibrin increased to a maximum at about three minutes after thrombus formation and then decreased, such that by eight minutes after thrombus formation fibrin is present at 34% of its maximal value. When wild type mice are pretreated with epsilon-aminocaproic acid (16 mg/kg IV), the decrease in thrombus fibrin content was less (to 54% of maximal 8 minutes after thrombus formation), implying that the fibrin loss is at least partially the result of plasmin activity. Thrombus fibrin accumulation in tissue-plasminogen activator deficient mice (Plat) was greater, peaking at 229% the amount found in wild type mice, but the amount of fibrin present at eight minutes after thrombus formation was not dissimilar from wild type mice (45% ± 5% v. 34% ± 3%, P=0.09). In uPA-deficient mice (Plau) and uPA-receptor-1-deficient mice (Plaur), the amount of maximal thrombus fibrin was 189% and 273% that of fibrin content in wild type mice. Also, although thrombus fibrin did decrease over time in Plau and Plaur mice, it decreased to only 51% and 48% of maximal (each were P < 0.005 v. wild type) at eight minutes after thrombus formation. When wild type leukocytes, isolated from blood by differential centrifugation and sucrose density gradient, were transfused into Plaur mice, thrombus fibrin loss was restored to wild type levels (38% of maximal at 8 minutes after thrombus formation, P=0.36 vs. wild type, P=0.016 vs. Plaur), implying that transfused wild type leukocytes, which express uPA-receptor-1, can rescue fibrinolysis in uPA-receptor-1-deficient mice.Conclusions: These results confirm that endogenous fibrinolytic activity in vivo occurs soon after arteriolar thrombus formation. The plasminogen activators that are required for this fibrinolysis to occur are uPA, and the uPA-receptor-1 on leukocytes. These findings are compatible with the hypothesis that leukocytes can deliver plasminogen activator activity to arteriolar thrombi to initiate endogenous fibrinolysis.