Analysis of a structural determinant in thrombin-protease nexin 1 complexes that mediates clearance by the low density lipoprotein receptor-related protein

We recently identified a synthetic peptide, pro(47)-Ile(58), derived from t he mature protease nexin 1 (PN1) sequence, that inhibited the low density l ipoprotein receptor-related protein (LRP)-mediated internalization of throm bin-PN1 (Th-PN1) complexes, Presently, we have analyzed this sequence in Th -PN1 complex catabolism using two independent approaches: 1) An antibody wa s generated against pro(47)-Ile(58), which inhibited complex degradation by 70% but had no effect on the binding of the complexes to cell surface hepa rins. This places the structural determinant in PN1 mediating complex inter nalization by the LRP outside of the heparin-binding site. 2) Site-directed genetic variants of PN1 with a single Ala substitution at His(48), or two Ala substitutions, one at His48 and another at Asp(49), Were expressed in S f9 insect cells. The catabolic rate of complexes formed between Th and the singly substituted and doubly substituted variants was lowered to 50 and 15 %, respectively, when compared with the catabolic rate of native Th-PN1 com plexes. This is the first analysis of a structural determinant in a serine protease inhibitor (SERPIN) required for LRP-mediated internalization and i n part may explain the cryptic nature of this site in the unreacted serine protease inhibitor.