BINDING OF ANISOYLATED LYS-PLASMINOGEN STREPTOKINASE ACTIVATOR COMPLEX TO CELLS IN CULTURE

Anisoylated Lys-plasminogen streptokinase activator complex (APSAC) wa s purified from Eminase(R) by chromatography on Superose-12. Purified APSAC did not significantly deacylate within 4 h at 4-degrees-C in sol ution as determined by hydrolysis of D-Val-L-leu-L-lys-p-nitroanilide HCl (S-2251). At 37-degrees-C, maximum amidase activity developed in 1 20 min; epsilon-amino-n-caproic acid (EACA) did not affect the apparen t rate of APSAC deacylation but stabilized the streptokinase-plasmin(o gen) complex (SkPl) which formed. APSAC bound to C6 glioma cells and h uman umbilical vein endothelial cells (HUVECs) in culture. Binding was completely inhibited by EACA suggesting an essential role for the pla sminogen kringle domains. Cell-associated APSAC deacylated to form act ive SkPl which hydrolyzed S-2251 and D-Val-Leu-Lys-7-amino-4-methyl co umarin. The rate of APSAC deacylation was increased when the APSAC was cell-associated. APSAC that was initially bound to C6 cells or HUVECs also activated I-125-plasminogen. This activity may have reflected ce ll-associated APSAC or APSAC that dissociated into solution. Plasmin w as recovered bound to cells and in solution. These studies demonstrate that APSAC associates with cell-surfaces and retains activity. In the circulation, cell-surfaces may provide a significant pharmacologic co mpartment for intravenously administered APSAC.