Urokinase plasminogen activator (uPA) is thought to exert its effects on cell growth, adhesion, and migration by mechanisms involving proteolysis and interaction with its cell surface receptor (uPAR). The functional properties of uPA and the significance of its various domains for chemotactic activity were analyzed using human airway smooth muscle cells (hAWSMC). The wild-type uPA (r-uPAwt), inactive urokinase with single mutation (His 204 to Gln) (r-uPA H/Q ), urokinase with mutation of His 204 to Gln together with a deletion of growth factor-like domain (r-uPA H/Q -GFD), the catalytic domain of urokinase (r-uPA LMW ), and its kringle domain (r-KD) were expressed in Escherichia coli. We demonstrate that glycosylated uPA, r-uPAwt, r-uPA H/Q , and r-uPA H/Q -GFD elicited similar chemotactic effects. Half-maximal chemotaxis (EC 50 ) were apparent at approximately 2 nM with all the uPA variants. The kringle domain induced cell migration with an EC 50 of about 6 nM, whereas the denaturated r-KD and r-uPA LMW were without effect. R-uPAwt-induced chemotaxis was dependent on an association with uPAR and a uPA-kringle domain-binding site, determined using a monoclonal uPAR antibody to prevent the uPA-uPAR interaction, and a monoclonal antibody to the uPA-kringle domain. The binding of iodinated r-uPAwt with hAWSMC was due to interaction with a high affinity binding site on the uPAR, and a lower affinity binding site on an unidentified cell surface target, which was mediated exclusively through the kringle domain of urokinase. Specific binding of r-uPA H/Q -GFD to hAWSMC involved an interaction with a single site whose characteristics were similar to those of the low affinity site of r-uPAwt binding to hAWSMC. uPAR-deficient HEK 293 cells specifically bound r-uPAwt and r-uPA H/Q -GFD via a single, similar type of binding site. These cells migrated when stimulated by r-uPA H/Q -GFD and uPAwt, but not r-uPA LMW . HEK 293 cells transfected with the uPAR cDNA expressed two classes of sites that bound r-uPAwt; however, only a single site was responsible for the binding of r-uPA H/Q -GFD. Together, these findings indicate that uPA-induced chemotaxis is dependent on the binding of the uPA-kringle to the membrane surface of cells and the association of uPA with uPAR.

uPA (urokinase-type plasminogen activator) stimulates cell migration through multiple pathways, including formation of plasmin and extracellular metalloproteinases, and binding to the uPAR (uPA receptor; also known as CD87), integrins and LRP1 (low-density lipoprotein receptor-related protein 1) which activate intracellular signalling pathways. In the present paper we report that uPA-mediated cell migration requires an interaction with fibulin-5. uPA stimulates migration of wild-type MEFs (mouse embryonic fibroblasts) (Fbln5+/+ MEFs), but has no effect on fibulin-5-deficient (Fbln5−/−) MEFs. Migration of MEFs in response to uPA requires an interaction of fibulin-5 with integrins, as MEFs expressing a mutant fibulin-5 incapable of binding integrins (FblnRGE/RGE MEFs) do not migrate in response to uPA. Moreover, a blocking anti-(human β1-integrin) antibody inhibited the migration of PASMCs (pulmonary arterial smooth muscle cells) in response to uPA. Binding of uPA to fibulin-5 generates plasmin, which excises the integrin-binding N-terminal cbEGF (Ca2+ -binding epidermal growth factor)-like domain, leading to loss of β1-integrin binding. We suggest that uPA promotes cell migration by binding to fibulin-5, initiating its cleavage by plasmin, which leads to its dissociation from β1-integrin and thereby unblocks the capacity of integrin to facilitate cell motility.

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Urokinase caused plasmin-dependent inhibition of platelet aggregation in platelet-rich plasma, while its proteolytically inactive form had no such effect. Both forms potentiated the increase in platelet calcium concentration induced by aggregation inductors and facilitated aggregation of washed platelets. In contrast to full-length urokinase molecule, its aminoterminal fragment inhibited platelet aggregation and the corresponding elevation of intracellular calcium. These data suggest that urokinase exerts a plasmin-independent effect on platelet activity. This effect depends on urokinase structure.