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  • Title: The interaction of recombinant tissue type plasminogen activator and recombinant plasminogen activator (r-PA/BM 06.022) with human endothelial cells.
    Author: Mulder M, Kohnert U, Fischer S, van Hinsbergh VW, Verheijen JH.
    Journal: Blood Coagul Fibrinolysis; 1997 Mar; 8(2):124-33. PubMed ID: 9518044.
    Abstract:
    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.
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