Holvoet P, Laroche Y, Lijnen H R, Van Hoef B, Brouwers E, De Cock F, Lauwereys M, Gansemans Y, Collen D
Center for Thrombosis and Vascular Research, University of Leuven, Belgium.
Eur J Biochem. 1992 Dec 15;210(3):945-52. doi: 10.1111/j.1432-1033.1992.tb17499.x.
K12G0S32 is a 57-kDa recombinant single-chain chimeric plasminogen activator consisting of scFv-K12Go, a single-chain variable-region antigen-binding fragment (Fv) of the monoclonal antibody MA-15C5, which is specific for fragment D-dimer of human cross-linked fibrin, and a low-molecular-mass (33 kDa) urokinase-type plasminogen activator (u-PA-33k) containing amino acids Ala132-Leu411 (Holvoet, P., Laroche, Y., Lijnen, H. R., Van Cauwenberghe, R., Demarsin, E., Brouwers, E., Matthyssens, G. & Collen D. (1991) J. Biol. Chem. 266, 19717-19724). In addition, the Arg156-Phe157 thrombin-cleavage site in the u-PA moiety of K12G0S32 is removed by substitution of Phe157 with Asp. In the present study, the fibrinolytic potency of K12G0S32, determined in a system composed of a 125I-fibrin-labeled human plasma clot submerged in citrated plasma, was found to be only twofold higher than that of intact single-chain u-Pa (rscu-PA), but 17-fold higher than that of rscu-PA(M), a variant of rscu-PA in which the thrombin-cleavage site was removed by substitution of Phe157 with Asp. The fibrinolytic potency of K12G0S32T, with an intact thrombin-cleavage site, was 6-15-fold higher than that of rscu-PA. Conversion of 1 microM single-chain K12G0S32 or rscu-PA(M) into their two-chain derivatives with plasmin occurred at a rate of 1.0 +/- 0.15 nmol.min-1.nmol plasmin-1 and 0.85 +/- 0.074 nmol.min-1.nmol plasmin-1, compared to 14 +/- 2.3 nmol.min-1.nmol plasmin-1 and 18 +/- 2.6 nM.min-1.nmol plasmin-1 for K12G0S32T and rscu-PA, respectively. Purified fragment D-dimer of human cross-linked fibrin inhibited the fibrinolytic potency of single-chain K12G0S32T, but not of two-chain K12G0S32T, in a dose-dependent manner. Furthermore, the fibrinolytic potencies of two-chain K12G0S32 and K12G0S32T were not significantly higher than those of recombinant two-chain u-PA (rtcu-PA) or of rtcu-PA(M). These findings suggest that the 59-fold increase in fibrinolytic potency of K12G0S32T, relative to that of rscu-PA(M), is due both to targeting of the activator to the clot via the single-chain Fv fragment (sixfold increase) and to a more efficient conversion of single-chain K12G0S32T to its two-chain derivative (eightfold increase). Thus, targeting to clots by means of fibrin-specific antibodies results in a significant increase of the fibrinolytic potency of single-chain but not of two-chain u-PA.(ABSTRACT TRUNCATED AT 400 WORDS)
K12G0S32是一种57千道尔顿的重组单链嵌合纤溶酶原激活剂，由scFv-K12Go组成，scFv-K12Go是单克隆抗体MA-15C5的单链可变区抗原结合片段（Fv），该抗体对人交联纤维蛋白的D-二聚体片段具有特异性，以及一种低分子量（33千道尔顿）的尿激酶型纤溶酶原激活剂（u-PA-33k），其包含氨基酸Ala132-Leu411（霍尔沃特，P.，拉罗什，Y.，利嫩，H. R.，范考温伯格，R.，德马尔辛，E.，布劳wers，E.，马蒂斯森斯，G.和科伦，D.（1991年）《生物化学杂志》266，19717 - 19724）。此外，通过将Phe157替换为Asp，去除了K12G0S32的u-PA部分中的Arg156 - Phe157凝血酶切割位点。在本研究中，在由浸泡于枸橼酸盐血浆中的125I - 纤维蛋白标记的人血浆凝块组成的系统中测定发现，K12G0S32的纤溶活性仅比完整单链u-Pa（rscu-PA）高两倍，但比rscu-PA（M）高17倍，rscu-PA（M）是rscu-PA的一种变体，其中通过将Phe157替换为Asp去除了凝血酶切割位点。具有完整凝血酶切割位点的K12G0S32T的纤溶活性比rscu-PA高6 - 15倍。1微摩尔单链K12G0S32或rscu-PA（M）与纤溶酶转化为其二链衍生物的速率分别为1.0±0.15纳摩尔·分钟-1·纳摩尔纤溶酶-1和0.85±0.074纳摩尔·分钟-1·纳摩尔纤溶酶-1，而K12G0S32T和rscu-PA的速率分别为14±2.3纳摩尔·分钟-1·纳摩尔纤溶酶-1和18±2.6纳摩尔·分钟-1·纳摩尔纤溶酶-1。纯化的人交联纤维蛋白D-二聚体片段以剂量依赖性方式抑制单链K12G0S32T的纤溶活性，但不抑制二链K12G0S32T的纤溶活性。此外，二链K12G0S32和K12G0S32T的纤溶活性并不显著高于重组二链u-PA（rtcu-PA）或rtcu-PA（M）的纤溶活性。这些发现表明，相对于rscu-PA（M），K12G0S32T的纤溶活性增加59倍，这既归因于激活剂通过单链Fv片段靶向凝块（增加6倍），也归因于单链K12G0S32T更有效地转化为其二链衍生物（增加8倍）。因此，通过纤维蛋白特异性抗体靶向凝块导致单链而非二链u-PA的纤溶活性显著增加。（摘要截短至400字）
