Much of the controversy surrounding the binding of plasminogen activator inhibitor-1 (PAI-1) to the low density lipoprotein receptor-related protein (LRP) may be due to the labile structure of PAI-1 and the distinct conformations that it can adopt. To examine this possibility and to test the hypothesis that PAI-1 contains a specific high affinity binding site for LRP, a sensitive and quantitative assay for PAI-1 binding to LRP was developed. This assay utilizes a unique PAI-1 mutant that was constructed with a hexapeptide tag at the NH 2 terminus, which is recognized by the protein kinase, heart muscle kinase and can be specifically labeled with 32 P. Our results show that only 32 P-PAI-1 in complex with a proteinase binds LRP with high affinity and is efficiently endocytosed by cells, indicating that a high affinity site for LRP is generated on PAI-1 only when in complex with a proteinase. In addition, PAI-1 in complex with different proteinases is shown to cross-compete for LRP binding, demonstrating that the binding site is independent of the proteinase and therefore must reside on the PAI-1 portion of the complex. Finally, mutagenesis of PAI-1 results in loss of LRP binding, confirming that the high affinity binding site is located on PAI-1 and suggesting that the LRP binding site lays within a region of PAI-1 previously shown to contain the heparin binding domain. The serine proteinase inhibitor (serpin)1 PAI-1 is the most efficient in vivo inhibitor known of both tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) (1). Under normal physiological conditions PAI-1 expression is primarily limited to smooth muscle cells and megacaryocytes. However, many cells induce PAI-1 expression in response to cytokines associated with defensive processes such as inflammation (2). The active form of PAI-1 is thought to have its reactive center loop (RCL) fully exposed and thus available for interaction with proteinases (3, 4). However, the active conformation is unstable and decays to an inactive latent conformation which in turn can be partially reactivated by treatment with denaturants (5). Cleaved PAI-1 can be generated either by deacylation of the covalent enzyme-inhibitor complex (3, 6) or by reaction with a non-target protease such as elastase, which cleaves the RCL at a site other than the P1-P1Ј reactive center bond (7). In the latent and cleaved forms of PAI-1, the RCL is fully inserted into ␤-sheet A of the inhibitor, making these forms of PAI-1 inactive against target proteinases (8, 9). Upon complex formation with a target proteinase the RCL is cleaved at the P1-P1Ј reactive center bond and the aminoterminal portion of the RCL is inserted into ␤-sheet A (3, 10). Although the precise extent of RCL insertion in the stable complex is not known, recent data suggest that the insertion may be less than that of the latent and cleaved forms of PAI-1 (4, 11, 12). The integration of the RCL into ␤-sheet A is an essential step in the serpin inhibitory mechanism and results in a conversio...

Basolateral apoB secretion decreased. Using the same expression system, fulllength human apoA-IV (376 amino acids); a "pig-like" human apoA-IV, lacking the C-terminal EQQQ repeats (361 amino acids); and a "chicken-like" apoA-IV, further truncated to 343 amino acids, were expressed in IPEC-1 cells. With increasing protein secretion, cells expressing the full-length human apoA-IV displayed a 2-fold increase in TG secretion; in sharp contrast, cells expressing the piglike human apoA-IV displayed a 25-fold increase in TG secretion and a 27-fold increase in lipoprotein diameter. When human apoA-IV was further truncated to yield a chicken-like protein, TG secretion was inhibited. We conclude that overexpression of swine apoA-IV enhances basolateral TG secretion in a dose-dependent manner by increasing the size of secreted lipoproteins. These data suggest that the region in the human apoA-IV protein from residues 344 to 354 is critical to its ability to enhance lipid secretion, perhaps by enabling the packaging of additional core TG into chylomicron particles. The EQQQ-rich region may play an inhibitory or modulatory role in chylomicron packaging in humans.

Evaluation of lungs from GalTKO.hCD46 pigs, genetically modified to lack the galactose-α(1,3)-galactose epitope (GalTKO) and to express human CD46, a complement regulatory protein, has not previously been described. Physiologic, hematologic and biochemical parameters during perfusion with heparinized fresh human blood were measured for 33 GalTKO.hCD46, GalTKO (n=16), and wild type pig lungs (n=16), and 12 pig lungs perfused with autologous pig blood. Median GalTKO.hCD46 lung survival was 171 minutes compared to 120 for GalTKO (p=0.27) and 10 for wild type lungs (p<0.001). Complement activation, platelet activation, and histamine elaboration were significantly reduced during the first 2h of perfusion in GalTKO.hCD46 lungs compared to GalTKO (ΔC3a at 120′ 812±230 vs. 1412±1047, p=0.02; ΔCD62P at 120′ 9.8±7.2 vs. 25.4±18.2, p<0.01; Δhistamine at 60′ 97±62 vs. 189±194, p=0.03). We conclude that, in addition to significant down-modulation of complement activation, hCD46 expression in GalTKO lungs diminished platelet and coagulation cascade activation, neutrophil sequestration and histamine release. Because GalTKO.hCD46 lung failure kinetics correlated directly with platelet and neutrophil sequestration, coagulation cascade activation, and a rise in histamine levels within the first hour of perfusion, further progress will likely depend upon improved control of these pathways, by rationally targeted additional modifications to pigs and pharmacologic interventions.