Simon D I, Xu H, Vaughan D E
Department of Medicine, Brigham and Women's Hospital, Boston, MA, USA.
Biochim Biophys Acta. 1995 Aug 31;1268(2):143-51. doi: 10.1016/0167-4889(95)00063-x.
Plasminogen activator inhibitor-1 (PAI-1) is the most important inhibitor of tissue-type plasminogen activator (t-PA) in plasma and plays a major role in the regulation of fibrinolysis. Plasma t-PA/PAI-1 complexes are cleared via a receptor-dependent mechanism in hepatocytes, while the fate of complexes formed in the extracellular matrix and in thrombi is less well understood. In this study, the degradation of t-PA/PAI-1 complexes by monocytes was examined. THP-1 monocytoid cells and freshly isolated human monocytes internalize and degrade [125I]t-PA/PAI-1 complexes at rates of 11.4 +/- 5.9 (mean +/- S.D.) and 44.6 +/- 6.3 ng/10(6) cells/h, respectively. Degradation is blocked by receptor-associated protein (RAP), indicating a member of the low density lipoprotein (LDL) receptor family is involved in the uptake/degradation of t-PA/PAI-1 complexes by monocytes. Degradation of t-PA/PAI-1 complexes is also inhibited by chloroquine and by pepstatin A, suggesting that a lysosomal aspartyl protease is likely involved. SDS-PAGE and Western blotting demonstrated that the purified lysosomal aspartyl protease, cathepsin D, is capable of digesting t-PA (t1/2 15 min), active PAI-1 (t1/2 2 h), and t-PA/PAI-1 complex (t1/2 30 min). Cathepsin D sequentially cleaves PAI-1 after hydrophobic amino acids, yielding lower molecular weight fragments. PAI-1 conformation influences the degradative efficiency of cathepsin D, with vitronectin-bound PAI-1 and latent PAI-1 exhibiting resistance to proteolysis and > 10-fold prolongation in t1/2. These data provide evidence that t-PA/PAI-1 complexes are internalized by human monocytes via a member of the low density lipoprotein (LDL) receptor family, and identifies cathepsin D-like aspartyl protease activity as largely responsible for the degradation of these complexes. Furthermore, vitronectin-bound PAI-1 and latent PAI-1 are relatively resistant to degradation by cathepsin D, which may be of importance in complex physiological environments.
纤溶酶原激活物抑制剂-1（PAI-1）是血浆中组织型纤溶酶原激活物（t-PA）最重要的抑制剂，在纤维蛋白溶解调节中起主要作用。血浆t-PA/PAI-1复合物通过肝细胞中一种依赖受体的机制清除，而在细胞外基质和血栓中形成的复合物的命运则了解较少。在本研究中，检测了单核细胞对t-PA/PAI-1复合物的降解情况。THP-1单核细胞样细胞和新鲜分离的人单核细胞摄取并降解[125I]t-PA/PAI-1复合物的速率分别为11.4±5.9（平均值±标准差）和44.6±6.3 ng/10(6)个细胞/小时。降解被受体相关蛋白（RAP）阻断，表明低密度脂蛋白（LDL）受体家族的一个成员参与了单核细胞对t-PA/PAI-1复合物的摄取/降解。氯喹和胃蛋白酶抑制剂A也抑制t-PA/PAI-1复合物的降解，提示可能有溶酶体天冬氨酸蛋白酶参与。SDS-PAGE和蛋白质印迹法表明，纯化的溶酶体天冬氨酸蛋白酶组织蛋白酶D能够消化t-PA（半衰期15分钟）、活性PAI-1（半衰期2小时）和t-PA/PAI-1复合物（半衰期30分钟）。组织蛋白酶D依次在疏水氨基酸后切割PAI-1，产生分子量较低的片段。PAI-1的构象影响组织蛋白酶D的降解效率，与玻连蛋白结合的PAI-1和潜伏型PAI-1对蛋白水解有抗性，半衰期延长>10倍。这些数据证明t-PA/PAI-1复合物通过低密度脂蛋白（LDL）受体家族的一个成员被人单核细胞内化，并确定组织蛋白酶D样天冬氨酸蛋白酶活性是这些复合物降解的主要原因。此外，与玻连蛋白结合的PAI-1和潜伏型PAI-1对组织蛋白酶D的降解相对抗性，这在复杂的生理环境中可能很重要。
