Benefits of pravastatin on cardiovascular events and mortality in older patients with coronary heart disease are equal to or exceed those seen in younger patients: Results from the LIPID trial.

BACKGROUND: The effect of cholesterol-lowering therapy on death from coronary heart disease in older patients with previous coronary heart disease and average cholesterol levels is uncertain. OBJECTIVE: To compare the relative and absolute effects of pravastatin on cardiovascular disease outcomes in patients with coronary heart disease who are 65 years of age or older with those in patients 31 to 64 years of age. DESIGN: Subgroup analysis of a randomized, placebo-controlled trial. SETTING: 87 centers in Australia and New Zealand. PATIENTS: 3514 patients 65 to 75 years of age, chosen from among 9014 patients with previous myocardial infarction or unstable angina and a baseline plasma cholesterol level of 4.0 to 7.0 mmol/L (155 to 271 mg/dL). INTERVENTION: Pravastatin, 40 mg/d, or placebo. MEASUREMENTS: Major cardiovascular disease events over 6 years. RESULTS: Older patients were at greater risk than younger patients (31 to 64 years of age) for death (20.6% vs. 9.8%), myocardial infarction (11.4% vs. 9.5%), unstable angina (26.7% vs. 23.2%), and stroke (6.7% vs. 3.1%) (all P < 0.001). Pravastatin reduced the risk for all cardiovascular disease events, and similar relative effects were observed in older and younger patients. In patients 65 to 75 years of age, pravastatin therapy reduced mortality by 21% (CI, 7% to 32%), death from coronary heart disease by 24% (CI, 7% to 38%), coronary heart disease death or nonfatal myocardial infarction by 22% (CI, 9% to 34%), myocardial infarction by 26% (CI, 9% to 40%), and stroke by 12% (CI, -15% to 32%). For every 1000 older patients treated over 6 years, pravastatin prevented 45 deaths, 33 myocardial infarctions, 32 unstable angina events, 34 coronary revascularization procedures, 13 strokes, or 133 major cardiovascular events, compared with 22 deaths and 107 major cardiovascular events per 1000 younger patients. Among older patients, the numbers needed to treat were 22 (CI, 17 to 36) to prevent one death from any cause, 35 (CI, 24 to 67) to prevent one death from coronary heart disease, and 21 (CI, 17 to 31) to prevent one coronary heart disease death or nonfatal myocardial infarction. CONCLUSIONS: In older patients with coronary heart disease and average or moderately elevated cholesterol levels, pravastatin therapy reduced the risk for all major cardiovascular events and all-cause mortality. Since older patients are at greater risk than younger patients for these events, the absolute benefit of treatment is significantly greater in older patients.

Characterization of the soluble form of the low density lipoprotein receptor-related protein (LRP).

We report characterization of the soluble form of the low density lipoprotein receptor-related protein (sLRP) which circulates in human plasma. Amino acid sequence analysis confirmed that sLRP isolated from human plasma contains the alpha-chain of LRP1. In addition, Western blot analysis identified a truncated beta-chain noncovalently associated with the purified alpha-chain. The molecular size (M(r) 55K) of the peptide portion of the truncated beta-chain indicates that the subunit comprises the extracellular portion of the beta-chain and terminates in a membrane-proximal region. We investigated the mechanism by which sLRP may be generated using the trophoblast cell line, BeWo, which releases sLRP in culture. Cell surface labeling experiments indicate that LRP is released from BeWo cells following expression at the cell surface. Incubation of BeWo cells in the presence of a metalloproteinase inhibitor, INH-3855-PI, results in a dose-dependent inhibition of LRP shedding. The metalloproteinase responsible for the shedding of LRP by BeWo cells is not up-regulated by phorbol ester and is not dependent on serine proteases, such as plasmin, for activity. The BeWo cell line is derived from a human gestational choriocarcinoma and preliminary studies suggest that LRP may be shed within the placenta during gestation. Increased levels of sLRP were detected in cord blood. In term placenta, LRP is expressed in the syncytium, which comprises the maternal-fetal interface. Increased levels of sLRP in cord blood may reflect cellular dysfunction and increased metalloproteinase activity at this important interface.

Evolutionary conservation of circulating soluble low density lipoprotein receptor-related protein-like ("LRP-like") molecules.

Low density lipoprotein receptor family members characteristically bind 39-kDa receptor associated protein (RAP). Soluble forms of these receptors have been described in humans including the 515/85-kDa dimeric receptor, low density lipoprotein receptor-related protein (LRP/alpha2MR), which is involved in multiple processes including lipoprotein and protease metabolism. Here we demonstrate evolutionary conservation in the generation of these soluble RAP-binding proteins of high molecular weight, by identifying their presence in mammalian, avian, and reptilian sera as well as in the circulating haemolymph of a mollusc. Sera extracted on immobilized RAP, produced bands at approximately 500 kDa in radiolabeled ligand blots by using the LRP/alpha2MR-specific ligand, Pseudomonas exotoxin A (PEA). These findings suggest that circulating RAP-binding proteins with high molecular weight in vertebrates share features of LRP/alpha2MR (LRP-like molecules). RAP-binding molecules in the mammalian serum extracts were further characterized as LRP/alpha2MR homologues in Western blots by using antibodies against the 515-kDa alpha-chain of LRP/alpha2MR. Western blots of mammalian serum extracts using two monoclonal antibodies recognizing the 85-kDa transmembrane beta-chain suggested that a portion of the beta-chain's ectodomain remains associated with the alpha-chain, but the beta-chain's intracellular carboxy terminus is absent. These results are consistent with evolutionary conservation in the generation, composition, and ligand-binding ability of soluble LRP-like receptors and suggest that their presence is a necessary aspect of the receptor's function.

Natural rubber latex hypersensitivity: incidence and prevalence of type I allergy in the dental professional.

The authors investigated the prevalence of immediate (Type I) hypersensitivity to gloves made from natural rubber latex, or NRL, by performing skin-prick tests on 2,166 dental workers over the course of a two-year period (with two one-year intervals). The investigator used two separate eluents made from different brands of natural rubber latex gloves. The study, conducted in 1994 and 1995 as part of the American Dental Association's Annual Health Screening Program, found that 6.2 percent of the participants (dentists, hygienists and assistants) tested positive for Type I hypersensitivity to NRL proteins.

Soluble low-density lipoprotein receptor-related protein.

Soluble forms of receptors can influence the activity of their membrane-bound counterparts by affecting their interactions with ligands. Low density lipoprotein (LDL) receptor-related protein (LRP), a member of the LDL receptor family, binds multiple classes of ligands and has been implicated in a broad range of normal and disease processes involving lipid metabolism, protease clearance, and cell migration. We recently identified a soluble form of LRP (sLRP) in human plasma and showed that it retains LRP-ligand binding ability. These findings open potentially important additional aspects in the biology of this multifunctional receptor. This review summarizes characteristics of soluble LRP and relates these to the membrane-bound form of the receptor.

Soluble low density lipoprotein receptor-related protein (LRP) circulates in human plasma.

Our studies have identified a soluble molecule in normal human plasma and serum with the characteristics of the alpha-chain of the low density lipoprotein receptor-related protein (LRP). LRP is a large multifunctional receptor mediating the clearance of diverse ligands, including selected lipoproteins, various protease inhibitor complexes, and thrombospondin. A soluble molecule (sLRP) has been isolated from plasma using an affinity matrix coupled with methylamine-activated alpha2-macroglobulin, the ligand uniquely recognized by LRP, and eluted with EDTA. This eluate contains a protein that co-migrates on SDS-polyacrylamide gel electrophoresis with authentic human placental LRP alpha-chain, is recognized by anti-LRP alpha-chain monoclonal antibodies, and binds the 39-kDa receptor-associated protein (RAP) and tissue plasminogen activator-inhibitor complexes. A similar RAP-binding molecule was detected in medium conditioned for 24 h by primary cultures of rat hepatocytes, suggesting that the liver may be the in vivo source of sLRP. In contrast, immunoprecipitation experiments failed to detect the production of sLRP by cultured HepG2 hepatoma and primary human fibroblast cells. Addition of a soluble form of LRP to cultured HepG2 cells resulted in a significant inhibition of capacity of these cells to degrade tPA, a process that has been demonstrated to be mediated by cell surface LRP. Preliminary data indicate that the concentration of sLRP is altered in the plasma of patients with liver disease. Increased levels of sLRP may antagonize the clearance of ligands by cell bound LRP perturbing diverse processes including lipid metabolism, cell migration and extracellular proteinase activity.

Low density lipoprotein receptor-related protein (LRP) expression varies among Hep G2 cell lines.

The multiligand receptor, low density lipoprotein receptor-related protein (LRP), is implicated in processes such as atherosclerosis and fibrinolysis through its mediation of the catabolism of lipoproteins, proteases, and protease inhibitor complexes. The hepatoma cell line Hep G2 expresses LRP and has been used widely to investigate the catabolism of LRP ligands including tissue-type plasminogen activator (tPA). However, the mechanism and degree by which tPA interacts with Hep G2 has been reported with some inconsistencies which may reflect variation in their level of LRP expression. To address this possibility we characterized, antigenically and functionally, LRP expression in high and low passage Hep G2 cells both from the parental line (ATCC sourced) and a cloned subline, a16. The LRP contribution to 125I-tPA binding varied from 65% for high passage a16 cells, to 20% for low passage parent cells as quantified by inhibition in the presence of 39-kD receptor associated protein (RAP) which prevents binding of all known LRP ligands. The same trend in LRP expression among Hep G2 sublines was further evident in their ability to degrade 125I-tPA and survive Pseudomonas exotoxin A challenge. These results imply wide variability in basal LRP expression among Hep G2 lines dependent on cell lineage and long-term culture conditions.

Urokinase binding and catabolism by Hep G2 cells is plasminogen activator inhibitor-1 dependent, analogous to interactions of tissue-type plasminogen activator with these cells.

The adherent human hepatoma cell line Hep G2 exhibits receptor mediated endocytosis and catabolism of tissue-type plasminogen activator.plasminogen activator inhibitor type-1 (t-PA.PAI-1) complexes formed when exogenous t-PA combines with endogenous PAI-1 in the extracellular matrix. To determine whether the other major PA, urokinase (u-PA), which also complexes with PAI-1, is metabolised via the same mechanism, 125I-labelled high (hmw) and low (lmw) molecular weight forms of u-PA were incubated with Hep G2 cells at 4 degrees C for 2 hr in the absence and presence of a 100-fold excess of unlabelled ligand in order to detect specific binding. Both hmw and lmw 125I-u-PA formed complexes with PAI-1 and these bound specifically and with high affinity (apparent Kd 3.9 and 4.1 nM, with Bmax 78 x 10(3) and 83 x 10(3) binding sites/cell respectively). Binding by each form of radiolabelled u-PA was inhibited in a dose-dependent fashion by unlabelled t-PA, hmw-u-PA, lmw-u-PA, and by monoclonal anti-PAI-1 antibody. At 37 degrees C, bound hmw and lmw 125I-u-PA.PAI-1 complexes were internalised and degraded rapidly. These findings indicate that the specificity of the previously described receptor which mediates PAI-1 dependent catabolism of t-PA by Hep G2 cells extends to complexes of u-PA with this inhibitor.

Thrombospondin 1 is a tight-binding competitive inhibitor of neutrophil cathepsin G. Determination of the kinetic mechanism of inhibition and localization of cathepsin G binding to the thrombospondin 1 type 3 repeats.

Thrombospondin 1 was recently shown to bind to and inhibit the activity of neutrophil elastase (Hogg, P. J., Owensby, D. A., Mosher, D. F., Misenheimer, T. M., and Chesterman, C. N. (1993) J. Biol. Chem. 268, 7139-7146). This finding led us to question whether thrombospondin 1 also binds and inhibits the other major serine proteinase of neutrophils, cathepsin G. In a competitive binding assay, cathepsin G bound to thrombospondin 1 reversibly and saturably with a dissociation constant in the low nanomolar range. The kinetic mechanism of inhibition of cathepsin G activity by thrombospondin 1 was determined using the synthetic cathepsin G substrate, Suc-Ala-Ala-Pro-Phe-p-nitroanilide, and is consistent with hyperbolic tight-binding inhibition in which thrombospondin 1 binds cathepsin G and the Michaelis cathepsin G-substrate complex and weakens, but does not abolish, the efficiency of hydrolysis of Suc-Ala-Ala-Pro-Phe-p-nitroanilide. In the presence of 2 mM calcium ions, 2.9 +/- 0.4 mol of cathepsin G interacted with 1 mol of thrombospondin 1 trimer with a site-binding constant of 7.0 +/- 3.5 nM, which reduced the efficiency of hydrolysis of Suc-Ala-Ala-Pro-Phe-p-nitroanilide 8.5 +/- 1.4-fold. A lower limit for the on rate constant of 5 x 10(6) M-1 S-1 was established. The affinity of binding and stoichiometry for the interaction between cathepsin G and thrombospondin 1 was enhanced in the absence of calcium ions. In the presence of EDTA, 5.3 +/- 0.5 mol of cathepsin G interacted with 1 mol of thrombospondin 1 with a site-binding constant of 2.1 +/- 1.6 nM, implying the existence of two binding sites for cathepsin G on each subunit of thrombospondin 1, one or both of which is variably exposed and sensitive to calcium ions. Thrombospondin 1 protected fibronectin from cleavage by cathepsin G and blocked cathepsin G-mediated platelet aggregation. In summary, the binding of cathepsin G to thrombospondin 1 is tight, reversible, and close enough to the active site of cathepsin G to perturb the interactions of a small synthetic substrate and exclude a macromolecular protein substrate and platelets. Using defined proteolytic fragments and different conformers of thrombospondin 1, the binding sites for cathepsin G have been localized to the thrombospondin 1 type 3 repeats.

Thrombospondin is a tight-binding competitive inhibitor of neutrophil elastase.

Thrombospondin, a glycoprotein of three identical disulfide-bonded subunits, is a constituent of platelet alpha-granules and a variety of normal and transformed cells and binds to cell surfaces and becomes incorporated into extracellular matrix. It has been implicated in processes such as wound healing and tumor growth and metastasis. In addition, thrombospondin was shown recently to be an inhibitor of the fibrinolytic enzyme, plasmin. In the cause of studying the effects of thrombospondin on other serine proteinases, we found that thrombospondin binds neutrophil elastase in an active-site-dependent manner and competitively inhibits the activity of the enzyme. In a competitive binding assay, neutrophil elastase bound to thrombospondin with a dissociation constant of 17 +/- 7 nM, expressed per mole of thrombospondin trimer, or 52 +/- 20 nM, expressed per mole of thrombospondin subunit. In kinetic studies of the inhibition of the amidolytic activity of neutrophil elastase by thrombospondin, 2.7 +/- 0.3 mol of elastase interacted with 1 mol of thrombospondin trimer with a site-binding constant of 57 +/- 13 nM. Lower limits for the on rate constant of 5 x 10(6) M-1 s-1 and off rate constant of 0.27 s-1 were established. Affinity of binding of neutrophil elastase to thrombospondin was sensitive to ionic strength and calcium ions. Thrombospondin was cleaved by neutrophil elastase, but the site(s) of the limited cleavage are independent of the competitive inhibition of elastase activity by thrombospondin. Neutrophil elastase inactivated with phenylmethylsulfonyl fluoride did not compete with active elastase for binding to thrombospondin, implying that a functional active site is important for the interaction of elastase with thrombospondin. Thrombospondin protected fibronectin from cleavage by neutrophil elastase. In summary, the binding of neutrophil elastase to thrombospondin is tight, reversible, and close enough to the active site of elastase to exclude small synthetic tripeptidyl p-nitroanilide substrates and macromolecular protein substrates. Two potential reactive centers that may be involved in binding elastase have been identified in the calcium-binding type 3 domains of thrombospondin. Neutrophil elastase is the enzyme primarily responsible for degrading and solubilizing connective tissue during inflammatory processes. These findings suggest a previously unsuspected mechanism for regulation of elastase activity at inflammatory sites.

Endocytosis and lysosomal delivery of tissue plasminogen activator-inhibitor 1 complexes in Hep G2 cells.

Receptor-mediated endocytosis of tissue-type plasminogen activator (t-PA)-plasminogen activator inhibitor type 1 (PAI-1) complexes results in their clearance by Hep G2 cells. After complexes are internalized, the t-PA component is degraded. However, neither the locus of intracellular catabolism nor the fate of PAI-1 has been elucidated. To characterize these aspects of t-PA-PAI-1 catabolism, the subcellular distribution of a prebound cohort of ligand molecules was delineated after internalization at 37 degrees C. 125I-t-PA.PAI-1 and t-PA.125I-PAI-1 were compared in separate experiments. After ligand uptake, intracellular vesicles were separated on density gradients. Internalized 125I-t-PA.PAI-1 concentrated initially in endosomes. After 20 minutes of uptake, the complex began to appear in lysosomes. Subsequently, low molecular weight labeled ligand fragments were detected in culture media. A panel of lysosomotropic agents, including primaquine, chloroquine, ammonium chloride, and a combination of leupeptin and pepstatin A, inhibited degradation. When t-PA.125I-PAI-1 rather than 125I-t-PA.PAI-1 was internalized, strikingly different results were observed. Although the kinetics of internalization and the intracellular itinerary were indistinguishable for the differently labeled complexes, the 125I-PAI-1 component of t-PA.125I-PAI-1 resisted rapid degradation. After a rapid loss of t-PA, the 125I-PAI-1 moiety persisted in lysosomes for up to 180 minutes. Thus, internalized t-PA.PAI-1 is targeted to lysosomes in which PAI-1 is relatively more stable than t-PA.

A tyrosinated peptide representing the alternatively spliced exon of the platelet-derived growth factor A-chain binds specifically to cultured cells and interferes with binding of several growth factors.

Platelet-derived growth factor (PDGF) is a potent mitogen for cells of mesenchymal origin. Alternative exon splicing is responsible for two forms of the PDGF A-chain which differ at the carboxyl terminus by a highly basic region consisting of 18 amino acids. To clarify the function of the region, we synthesized an octadecapeptide corresponding to this extension (A194-211), incorporated a tyrosine residue at the amino terminus, and used the radioiodinated construct in binding studies with Balb/c3T3 cells and a variety of human cell lines. 125I-(Y)A194-211 bound specifically, reversibly, saturably, and with low affinity to a large population of binding sites on these cells. In addition, (Y)A194-211 markedly reduced the binding of its parent protein, 125I-PDGF-AAL, to its receptor. (Y)A194-211 also attenuated the binding of epidermal growth factor and several other isoforms of PDGF, but did not interfere with the binding of transferrin to its receptor. These observations were not due to competitive binding of peptide directly to known receptors for the respective growth factors, but was likely due to interaction of (Y)A194-211 with extracellular glycosaminoglycan. Thus, A194-211 may represent an additional heparin binding domain on mature PDGF-AAL, and as an isolate, is capable of modulating interactions between several potent growth factors and their respective receptors.

A crossreactive antipeptide monoclonal antibody with specificity for lysyl-lysine.

Synthetic peptides meeting certain guidelines have been used as immunogens to generate antibodies with predefined specificity. We have raised and characterized using established methods a monoclonal antibody against a synthetic peptide corresponding to the 18-amino acid carboxyterminal sequence (A194-211) of the platelet-derived growth factor (PDGF) A chain expressed by the U343 human glioma cell line. This antibody was generated in order to carry out structure-function studies on this region of PDGF whose biological significance is not yet clear. Anti-PDGF-A194-211 was found to be a low titre, IgM kappa molecule, with a Kd of 2.8 x 10(-7) M. When antibody reactivity was tested with parent PDGF-AAL (A chain homodimer containing a carboxyterminal extension) significant binding was observed. Surprisingly, 125I-PDGF-AAS, consisting of truncated A chains but lacking the extension was also bound. Moreover, poly-L-lysine, beta-thromboglobulin, PDGF-A194-211, and myoglobin competed dose-dependently with 125I-PDGF-AAL for antibody. 125I-bovine serum albumin was also bound. Examination of the primary sequence of proteins and peptides bound by the antibody revealed only one shared structural motif: a lysyl-lysine moiety. Selected small synthetic peptides containing this and other sequences were used as potential competitors of 125I-PDGF-A194-211 in antibody binding. Lysyl-lysyl-glycyl-glutamic acid [corrected] and lysyl-lysine competed, whereas lysyl-leucine did not. These results suggest that as few as two amino acid residues constitute a functional antigenic determinant and contrast with most previous estimates of the minimum number of residues required. Furthermore, we show that guidelines governing the design of synthetic peptides for their use as antigens to produce monoclonal antibodies of predetermined specificity may be unreliable.

Binding of plasminogen activator inhibitor type-1 to extracellular matrix of Hep G2 cells. Evidence that the binding protein is vitronectin.

Catabolism of plasminogen activators by Hep G2 cells is mediated by a specific receptor which recognizes complexes of these serine proteases with their physiological inhibitor, plasminogen activator inhibitor type-1 (PAI-1). This catabolic process is initiated by interaction of exogenous plasminogen activators with bioactive PAI-1, which is secreted and localizes in an active form to the extracellular matrix (ECM) of Hep G2 cells. We now report that vitronectin (VN) mediates the specific binding of PAI-1 to the ECM of these cells. Purified bovine or human VN competes for specific binding of PAI-1 to Hep G2 ECM, and ligand blotting reveals specific binding of PAI-1 to ECM-associated VN. Hep G2 cells secrete both VN and PAI-1, and pulse-chase studies strongly suggest that these proteins associate only following secretion. Although Hep G2 cell-derived VN does not significantly bind to ECM in vitro, 30-40% of endogenous PAI-1 binds to the ECM, even in the presence of human serum, suggesting that ECM-associated VN is entirely derived from bovine serum. PAI-1 was localized by indirect immunofluorescence to ECM beneath cells and at cell margins, whereas VN exhibited a uniform distribution throughout the growth substratum. VN associated with the ECM may confer retention and bioactivity to PAI-1, potentially facilitating both pericellular regulation of plasmin generation and the rapid hepatic clearance of plasminogen activators.

Identification of determinants involved in binding of tissue-type plasminogen activator-plasminogen activator inhibitor type 1 complexes to HepG2 cells.

Complexes between tissue-type plasminogen activator (t-PA) and its rapidly acting inhibitor plasminogen activator inhibitor type 1 (PAI-1) are bound, internalized, and degraded by HepG2 cells. The mechanism involves endocytosis mediated by a specific high-affinity receptor. However, the particular domains of the complex that are recognized by the receptor have not been elucidated. To identify the determinants involved in ligand binding to the receptor, several variants of t-PA were assessed for their ability to form complexes with PAI-1 and thereby to inhibit specific cellular binding of complexes between structurally unmodified 125I-t-PA and PAI-1. Catalytically active variants lacking selected structural domains form complexes with PAI-1 and inhibit 125I-t-PA.PAI-1 binding to HepG2 cells. In addition, several forms of the plasminogen activator urokinase (u-PA), which shares partial structural homology with t-PA, were evaluated as competitors of cellular binding. The catalytically active two-chain forms of u-PA, but not the inactive proenzyme single-chain form, complex with PAI-1 and inhibit specific binding of 125I-t-PA.PAI-1, suggesting that the serine protease domain, rather than other domains, may confer the determinants required for cellular binding. However, a mutant t-PA with markedly reduced catalytic activity, resulting from replacement of the active site serine with threonine, not only forms complexes with PAI-1 but also inhibits specific cellular binding of unmodified 125I-t-PA.PAI-1. These data indicate that specific binding of t-PA.PAI-1 to HepG2 cells does not require a serine-containing catalytic site in the protease domain. To determine whether binding of the complex is mediated through other components of t-PA or through structural elements of PAI-1, both t-PA and PAI-1 were examined separately for capacity to bind directly to HepG2 cells. To exclude potential interactions with components of the extracellular matrix which contains binding sites for PAI-1, ligand binding to HepG2 cells in suspension was assessed. Although neither t-PA nor PAI-1 alone binds specifically to HepG2 cells, the preformed t-PA.PAI-1 complexes do. These findings suggest that specific binding of t-PA.PAI-1 requires elements of the PAI-1 moiety and/or parts of the protease domain of t-PA.

Interactions between tissue-type plasminogen activator and extracellular matrix-associated plasminogen activator inhibitor type 1 in the human hepatoma cell line HepG2.

Hepatic parenchymal cells contribute to the clearance of circulating tissue-type plasminogen activator (t-PA) in vivo. The hepatocyte extracellular matrix is interposed between the endothelial-lined sinusoids and the parenchymal cell surface and thus may influence t-PA clearance. To test this hypothesis, the well differentiated human hepatoma cell line HepG2 was used to characterize the role of extracellular matrix in t-PA clearance in vitro. Previous studies with these cells demonstrated their capacity for specific catabolism of t-PA in a system modulated by plasminogen activator inhibitor type 1 (PAI-1). In the present study the extracellular matrix growth substratum of HepG2 cells is shown to contain active PAI-1. PAI-1 is distributed in a punctuate pattern throughout the substratum. Components of the substratum confer stability to active PAI-1 for intervals of at least 24 h. Exposing substratum to 125I-t-PA leads rapidly to the formation and release of a sodium dodecyl sulfate-stable 95-kDa 125I-t-PA.PAI-1 complex. In comparison, cell monolayers have the additional capacity for specific binding of the complex. However, PAI-1 is not detected at the surface of HepG2 cells in suspension, suggesting that 125I-t-PA.PAI-1 complexes form in substratum and subsequently bind to cells. Specific binding of performed 125I-t-PA.PAI-1, but not 125I-t-PA, was demonstrated for HepG2 cells in suspension. These results suggest that components of extracellular matrix participate in the clearance of t-PA by hepatocytes.

Catabolism of tissue-type plasminogen activator by the human hepatoma cell line Hep G2. Modulation by plasminogen activator inhibitor type 1.

Catalytic activity of tissue-type plasminogen activator (t-PA) in plasma is regulated in part by formation of complexes with specific inhibitors as well as by hepatic clearance. Potential interaction of these two regulatory mechanisms was examined in the human hepatoma cell line Hep G2. These cells secrete plasminogen activator inhibitor type-1 (PAI-1) and initiate catabolism of exogenous t-PA by receptor-mediated endocytosis. Specific binding of 125I-t-PA to cells at 4 degrees C results in dose-dependent formation of a 95-kDa species recognized by monospecific anti-PAI-1 and anti-t-PA antibodies and stable in the presence of low (0.2%) concentrations of sodium dodecyl sulfate (SDS). Specific binding of 125I-t-PA and formation of the 95-kDa SDS-stable species are inhibited in a concentration-dependent manner following preincubation of cells with anti-PAI-1 antibodies. High and low molecular weight forms of urokinase plasminogen activator (u-PA) capable of forming specific complexes with PAI-1 complete for 125I-t-PA binding sites. However, the proenzyme form of u-PA (scu-PA), incapable of forming complexes with PAI-1, does not compete for 125I-t-PA binding sites. The role of the serine protease active site of t-PA in mediating both interaction with PAI-1 and specific binding was examined using 125I-t-PA that had been functionally inactivated with D-phenylalanyl-L-propyl-L-arginyl-chloromethyl ketone (PPACK). 125I-t-PA-PPACK, despite a 6-fold lower affinity than active 125I-t-PA, exhibited specific binding to cells without detectable formation of SDS-stable complexes with PAI-1. Both surface-bound 125I-t-PA and 125I-t-PA-PPACK are internalized and degraded by cells at 37 degrees C. 125I-t-PA is internalized as a stable complex with PAI-1, whereas 125I-t-PA-PPACK is internalized with similar kinetics but without the presence of an SDS-stable complex. Thus, PAI-1 appears capable of modulating t-PA catabolism in the human hepatocyte.

Receptor-mediated endocytosis of tissue-type plasminogen activator by the human hepatoma cell line Hep G2.

Receptor-mediated endocytosis of tissue-type plasminogen activator (t-PA) was characterized with the human hepatoma cell line Hep G2. At 4 degrees C binding of 125I-t-PA to Hep G2 cells is rapid, specific, saturable, and reflective of a homogeneous population of 76,000 high-affinity surface sites per cell (Kd = 3.7 nM). The kinetics of 125I-t-PA binding to its receptor are characterized by rate constants for association (k1 = 1.2 x 10(6) min-1 M-1) and dissociation (k-1 = 0.001 min-1). A specific glycosylation pattern does not appear to be required for binding. Binding does not appear to be mediated by other recognized hepatic receptor systems. At 37 degrees C a single cohort of bound 125I-t-PA molecules disappears rapidly from the cell surface. Ligand then accumulates intracellularly. Thereafter, the intracellular concentration of ligand declines simultaneously with the release of ligand degradation products into the media. In the continued presence of 125I-t-PA at 37 degrees C the concentration of cell-associated ligand plateaus after 30 min with the concomitant appearance of low molecular weight 125I-labeled fragments in the media. Cumulative degradation then increases linearly with time. Under steady state conditions half-maximal ligand uptake and degradation is 26.6 nM and maximal rate of catabolism is 1.2 pmol/10(6) cells/h. At saturating ligand concentrations uptake and degradation by Hep G2 cells continue linearly for at least 6 h even in the absence of protein synthesis. During this period the cumulative ligand uptake exceeds the total cellular capacity of binding sites, consistent with receptor recycling. We conclude that t-PA clearance in human Hep G2 cells involves ligand binding, uptake, and degradation mediated by a novel high-capacity, high-affinity specific receptor system.

Intramuscular administration of human tissue-type plasminogen activator in rabbits and dogs and its implications for coronary thrombolysis.

To determine whether sustained plasma concentration of human tissue-type plasminogen activator (t-PA) can be induced promptly after intramuscular injection with enhancers of absorption devoid of deleterious local and systemic effects, we studied 250 rabbits and 13 dogs. In rabbits with t-PA injected directly into exposed muscle followed by local electrical stimulation at the site, early absorption was increased markedly by addition of 0.63M methylamine plus 0.079M hydroxylamine to the excipient. Elevations peaked within 5 min and increased with dose of t-PA, concentration of methylamine, and volume of injection medium. The enhancers were effective with percutaneous injections in the absence of local electrical stimulation as well. They did not elicit any obviously deleterious local or systemic effects. In separate experiments in rats, intramuscular injections of 0.63M methylamine plus 0.079M hydroxylamine induced local egress of intravascular radiolabeled albumin within the injection site and endothelial gaps in venules detected with colloidal carbon--changes consistent with direct effects on vascular permeability. In dogs, percutaneous intramuscular injection of t-PA in excipient without enhancers did not lead to early elevations of human t-PA in plasma, although late elevations were seen. When the enhancers were used, early elevations occurred as well, with functional activity documented by fibrin plate assays of serially obtained plasma samples and by sequential coronary angiography delineating thrombolysis after experimentally induced coronary thrombosis. The results indicate that intramuscular administration of t-PA with selected enhancers of absorption is a feasible approach for rapid induction of fibrinolysis.