Summary
Anisoylated Lys-plasminogen streptokinase activator complex (APSAC) was purified from
Eminase® by chromatography on Superose-12. Purified APSAC did not significantly deacylate
within 4 h at 4° C in solution as determined by hydrolysis of D-Val-L-leu-L-lys-p-nitroanilide
HCl (S-2251). At 37° C, maximum amidase activity developed in 120 min; ε-amino-n-caproic
acid (EACA) did not affect the apparent rate of APSAC deacylation but stabilized the
streptokinase-plasmin(ogen) complex (SkPl) which formed. APSAC bound to C6 glioma
cells and human umbilical vein endothelial cells (HUVECs) in culture. Binding was
completely inhibited by EACA suggesting an essential role for the plasminogen kringle
domains. Cell-associated APSAC deacylated to form active SkPl which hydrolyzed S-2251
and D-Val-Leu-Lys-7-amino-4-methyl coumarin. 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 125I-plasminogen. This activity may have reflected cell-associated APSAC or APSAC that
dissociated into solution. Plasmin was 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 compartment
for intravenously administered APSAC.
