Vasudevan J, Humphries J E, Gonias S L
Department of Biochemistry, University of Virginia Health Sciences Center, Charlottesville 22908.
Thromb Haemost. 1993 Apr 1;69(4):370-4.
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 degrees C in solution 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 120 min; epsilon-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 as 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 but 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.
通过在Superose - 12上进行色谱分离，从Eminase中纯化了茴香酰化赖氨酸 - 纤溶酶原链激酶激活剂复合物（APSAC）。通过水解D - 缬氨酸 - L - 亮氨酸 - L - 赖氨酸 - 对硝基苯胺盐酸盐（S - 2251）测定，纯化的APSAC在4℃溶液中4小时内不会显著脱酰基。在37℃时，120分钟内酰胺酶活性达到最大值；ε - 氨基 - n - 己酸（EACA）不影响APSAC脱酰基的表观速率，但稳定了形成的链激酶 - 纤溶酶（原）复合物（SkPl）。APSAC与培养中的C6胶质瘤细胞和人脐静脉内皮细胞（HUVECs）结合。EACA完全抑制了这种结合，表明纤溶酶原kringle结构域起着至关重要的作用。细胞相关的APSAC脱酰基形成活性SkPl，其水解S - 2251和D - 缬氨酸 - 亮氨酸 - 赖氨酸 - 7 - 氨基 - 4 - 甲基香豆素。当APSAC与细胞相关时，其脱酰基速率增加。最初与C6细胞或HUVECs结合的APSAC也激活了125I - 纤溶酶原。这种活性可能反映了细胞相关的APSAC或解离到溶液中的APSAC。纤溶酶在细胞结合状态和溶液中均被回收。这些研究表明APSAC与细胞表面结合并保留活性。在循环中，细胞表面可能为静脉注射的APSAC提供一个重要的药理学区域。
