Mulder M, Kohnert U, Fischer S, van Hinsbergh V W, Verheijen J H
Gaubius Laboratory, TNO-PG, Leiden, The Netherlands.
Blood Coagul Fibrinolysis. 1997 Mar;8(2):124-33. doi: 10.1097/00001721-199703000-00007.
The Escherichia coli-expressed recombinant plasminogen activator (r-PA) comprising the kringle 2 and protease domains of human tissue-type plasminogen activator (t-PA) has a four-fold longer half-life time in the circulation than t-PA, possibly resulting in an increased opportunity for r-PA to interact with the endothelial lining. In the present study we investigated the interaction of r-PA and t-PA with human umbilical vein endothelial cells (HUVEC). Specific binding of 125I-t-PA and 125I-r-PA were similar at 4 degrees C (Kd 6 nmol/l; Bmax about 120 fmol/mg cell protein). About half of the specific binding sites were shared by t-PA and r-PA, because unlabeled t-PA and r-PA competed equally with 125I-labeled t-PA and r-PA for binding to HUVEC. The low affinity interaction of 125I-t-PA was several-fold higher than that of 125I-r-PA. When PA binding was studied at 37 degrees C, HUVEC bound more t-PA than r-PA to both specific and non-specific binding sites. Both t-PA and r-PA were internalized and degraded, but t-PA internalization proceeded more efficiently than that of r-PA. In the presence of 100 microM chloroquine, the degradation of t-PA and r-PA was inhibited by 75% and 40%, respectively, indicating lysosomal degradation. When the active sites of t-PA and r-PA were blocked by PPACK, part of the cell association and most of the degradation of both t-PA and r-PA were inhibited. This points to plasminogen activator inhibitor-1 (PAI-1) as one of the specific binding sites. A possible role of LDL-receptor related protein (LRP) or related members of this receptor family was investigated by using the 39 kD receptor associated protein (RAP) which prevents interaction of ligands with these receptors. RAP reduced the association of 125I-t-PA by 25% and the degradation of 125I-t-PA and 125I-r-PA by 65% and 50%, respectively. Our data show that both t-PA and r-PA bind to HUVEC and can subsequently be internalized and degraded. However, r-PA interacts less effectively with HUVEC than t-PA. This indicates that binding to the endothelium does not prevent the clearance of r-PA and is not the cause of its long half-life.
包含人组织型纤溶酶原激活剂（t-PA）kringle 2和蛋白酶结构域的大肠杆菌表达重组纤溶酶原激活剂（r-PA）在循环中的半衰期比t-PA长四倍，这可能使r-PA与血管内皮的相互作用机会增加。在本研究中，我们研究了r-PA和t-PA与人脐静脉内皮细胞（HUVEC）的相互作用。125I-t-PA和125I-r-PA在4℃时的特异性结合相似（解离常数6 nmol/l；最大结合量约120 fmol/mg细胞蛋白）。约一半的特异性结合位点由t-PA和r-PA共享，因为未标记的t-PA和r-PA与125I标记的t-PA和r-PA竞争结合HUVEC的能力相同。125I-t-PA的低亲和力相互作用比125I-r-PA高几倍。当在37℃研究纤溶酶原激活剂（PA）结合时，HUVEC与t-PA结合到特异性和非特异性结合位点上的量均多于r-PA。t-PA和r-PA均被内化并降解，但t-PA的内化比r-PA更有效。在存在100μM氯喹的情况下，t-PA和r-PA的降解分别被抑制75%和40%，表明是溶酶体降解。当t-PA和r-PA的活性位点被PPACK阻断时，t-PA和r-PA的部分细胞结合及大部分降解均被抑制。这表明纤溶酶原激活剂抑制剂-1（PAI-1）是特异性结合位点之一。通过使用39 kD受体相关蛋白（RAP）研究了低密度脂蛋白受体相关蛋白（LRP）或该受体家族相关成员的可能作用，RAP可阻止配体与这些受体相互作用。RAP使125I-t-PA的结合减少25%，使125I-t-PA和125I-r-PA的降解分别减少65%和50%。我们的数据表明，t-PA和r-PA均与HUVEC结合，随后均可被内化并降解。然而，r-PA与HUVEC的相互作用比t-PA低效。这表明与内皮细胞的结合并不阻止r-PA的清除，也不是其长半衰期的原因。
