The plasminogen activation system, i.e., the fibrinolytic system, is one of the major plasma proteolytic pathways. The proteolytic conversion of the zymogen plasminogen to the active serine protease plasmin is on the cell surface catalyzed by the serine protease urokinase-type plasminogen activator (urokinase, uPA). Upon binding to the urokinase receptor (uPAR, CD87), single-chain pro-uPA is processed to double-chain uPA which in turn specifically converts cell-bound plasminogen to plasmin. Plasmin is harnessed in many physiological processes, e.g., blood clots' resolution, or proteolytic activation of growth factors. Plasmin is essential also for migratory cells, for instance, activated immune cells; however, malignant cells hijack plasmin for invasion as well. The activation of plasminogen to plasmin is thus at the physiological level tightly controlled. One of the negative regulators of plasminogen activation has been identified in the cation-independent mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R, CIMPR, CD222). M6P/IGF2R is a multifunctional receptor involved in protein sorting, internalization, and degradation, being considered a tumor suppressor. M6P/IGF2R binds both plasminogen and uPAR and facilitates in this way the proteolytic cleavage of uPAR resulting in the loss of the uPA binding on the cell surface. Hence, this molecular device contributes to the negative feedback loop in regulation of pericellular plasminogen activation and cell invasion.In this chapter, we describe the experimental approach, i.e., biotin-chasing assay, to evaluate uPAR stability and cleavage on the surface of cells.
Keywords: Cell migration; Fibrinolysis; Pericellular proteolysis; Plasminogen; Urokinase.