Activator Inhibitor-1 Complex Research Articles

The expression of the LDL receptor-related protein (LRP) correlates with the differentiation of human neuroblastoma cells

The low density lipoprotein receptor-related protein (LRP) has been localized in human brain at the level of neurons, astrocytes and along capillary membranes. It is a multifunctional receptor responsible for binding and internalization of lipoproteins enriched with apoliprotein E, lipoprotein lipase, protease-α 2macroglobulin complexes and plasminogen activator-inhibitor complexes. LRP expression is observed in cells involved in Alzheimer's disease, neoplastic transformation and tissue repair. Moreover, its synthesis is modulated during brain development. In this study we used the SK-N-AS human neuroblastoma cell line as a model system to study LRP expression during cellular differentiation induced by phorbol esters, retinoic acid and interferonγ. Since LRP plays a major role in the regulation of lipid metabolism, the decreased levels of LRP measured by immunofluorescence, western blot and PCR on differentiated neuroblastoma cells may be the consequence of the lower requirements of cholesterol and lipids of differentiated cells in relation to their reduced mitotic index.

THE EFFECT OF PHYSICAL CONDITIONING SUGGESTS ADAPTATION IN PROCOAGULANT AND FIBRINOLYTIC POTENTIAL

Acute exercise evokes a transient increase in procoagulant activity. We evaluated the effect of physical conditioning on the activation of the coagulation and fibrinolytic systems. Two groups of subjects of different aerobic endurance levels (athletes and controls with maximal oxygen uptake ( VO 2 max ) 68,4 and 52,6 ml·kg −1·min −1, respectively), were tested at rest and after standardized exercise at 80 % of their individual VO 2 max . There was a significant increase in prothrombinfragment 1+2 (F1+2) level among controls in response to standardized exercise (p<0.05), whereas no significant difference in the level of F1+2 between athletes and controls at rest or in response to exercise was demonstrated. Tissue plasminogen activator (tPA) antigen level at rest was significantly lower in athletes compared to controls (p<0.03). A significant increase was found in the tPA level after standardized exercise in both groups (p<0.02), which was lower in athletes compared to controls (p<0.05). There were no significant differences between athletes and controls in plasminogen activator inhibitor-1 (PAI-1) and thrombin antithrombin complex (TAT) levels at rest. Athletes had a significantly lower PAI-1 level than controls after exercise (p<0.05). In conclusion, the present study suggests an increased activation of the coagulation system in response to exercise in controls only. It also suggests adaptive changes in fibrinolytic potential induced by physical conditioning, as demonstrated by the lower level of tPA at rest and the lower levels of tPA and PAI-1 after exercise in athletes compared to controls. © 1997 Elsevier Science Ltd

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.

Comparison of extradural and general anaesthesia on the fibrinolytic response to total knee arthroplasty

Extradural anaesthesia is associated with lower incidences of deep vein thrombosis after total knee arthroplasty. It is not known if the type of anaesthesia influences thrombogenesis or fibrinolysis during knee surgery performed under tourniquet. We studied 31 patients allocated randomly to receive either extradural or general anaesthesia for primary unilateral total knee arthroplasty performed under tourniquet. Radial artery blood samples were obtained before surgery, during surgery with the tourniquet inflated and on deflation of the tourniquet. Plasma samples were assayed for markers of thrombin generation and fibrinolysis. Two of the circulating indices of thrombin generation, fibrinopeptide A and thrombin-antithrombin complexes, increased to a similar degree in the perioperative period in both groups. Fibrinolytic activity was similar in both groups, as measured by tissue plasminogen activator (t-PA) antigen, t-PA activity, t-PA-plasminogen activator inhibitor complexes, alpha 2-plasmin inhibitor-plasmin complexes and D-dimer. Extradural and general anaesthesia did not result in significant differences in either thrombin generation or fibrinolytic activity during total knee arthroplasty performed under tourniquet.

Do Tissue Plasminogen Activator-Plasminogen Activator Inhibitor-1 Complexes Relate to the Complications of Insulin-Dependent Diabetes Mellitus? Pittsburgh Epidemiology of Diabetes Complications Study

The purpose of this study was to examine the potential relationship of tissue plasminogen activator-plasminogen activator inhibitor-1 (tPA-PAI-1) complexes and diabetic complications in individuals with insulin-dependent diabetes mellitus (IDDM). To address this issue, data from the third follow-up visit of participants in the Epidemiology of Diabetes Complications (EDC) study were examined. There were 454 participants, aged 32 ± 8 years, with duration of IDDM of 23 ± 8 years. Higher levels of tPA-PAI-1 complexes were seen for both men and women with IDDM complications. Specifically, statistically significant differences were seen in men with neuropathy (1.81 ± 0.9 versus 1.42 ± 0.8 ng/mL, p < 0.01), microalbuminuria (1.77 ± 1.1 versus 1.35 ± 0.6 ng/mL, p < 0.01), retinopathy (1.67 ± 0.9 versus 1.43 ± 0.8 ng/mL, p < 0.05), and lower extremity arterial disease (1.93 ± 0.7 versus 1.50 ± 0.9 ng/mL, p < 0.05) versus men without the particular complication. In women, higher complex levels were shown for those with retinopathy (1.51 ± 0.8 versus 1.29 ± 1.1 ng/mL, p < 0.01). Potential mechanisms for the relationship of higher complex levels and diabetic complications include an altered fibrinolytic response and/or insulin resistance. Because the results are cross sectional, it cannot be established whether the higher concentration of complexes is a result of the presence of complications or are antecedent. Prospective follow-up will be required to determine if tPA-PAI-1 complexes are predictive of the development of IDDM complications.

Increased tissue factor pathway inhibitor in patients with acute myocardial infarction.

We examined hemostatic abnormalities in 23 patients with acute myocardial infarction (AMI), 10 with pulmonary embolism (PE), and 10 with deep vein thrombosis (DVT). At the onset of AMI, plasma levels of tissue-type plasminogen activator (t-PA), PA inhibitor-I (PAI-I), fibrin-D-dimer, thrombin-antithrombin complex (TAT), and plasmin-plasmin inhibitor complex (PPIC) were significantly increased. Both the plasma total TFPI and free-TFPI levels in the AMI patients were significantly higher than those in the healthy volunteers, PE patients, and DVT patients. There was no significant difference in total TFPI or free-TFPI among patients with PE, those with DVT, and healthy volunteers. One hour after percutaneous transluminal coronary angioplasty (PTCA) in the AMI group, the total TFPI level was further increased, and it was significantly reduced 24 hr after PTCA, to a level similar to that in healthy volunteers. Free-TFPI showed a pattern similar to that of total TFPI. The ratio of free-TFPI/total TFPI was highest 1 hr after PTCA. Increased TFPI in AMI patients might be released from ischemic tissues.

25. Coagulation factor XI: rather a fibrinolytic inhibitor than a coagulation factor

Post-cardiac arrest syndrome (PCAS) is often associated with disseminated intravascular coagulation (DIC), thus leading to the development of multiple organ dysfunction syndrome (MODS). The aim of this study was to examine the pathophysiological relationships between coagulation, fibrinolysis and fibrinolytic shutdown by evaluating the levels of coagulofibrinolytic markers, including soluble fibrin, thrombin-activatable fibrinolysis inhibitor (TAFI), tissue plasminogen activator-plasminogen activator inhibitor-1 complex (tPAIC), plasmin-alpha2 plasmin inhibitor complex (PPIC), neutrophil elastase and fibrin degradation product by neutrophil elastase (EXDP).Fifty-two resuscitated patients were divided into two groups: 22 DIC and 30 non-DIC patients.The levels of soluble fibrin, PPIC, tPAIC, EXDP and neutrophil elastase in the DIC patients with PCAS were significantly higher than those observed in the non-DIC patients. The values of the tPAIC and JAAM DIC scores were found to be independent predictors of increased SOFA scores in the DIC patients. The MODS patients demonstrated significantly higher levels of soluble fibrin and tPAIC; however, the levels of TAFI and EXDP were identical between the patients with and without MODS. In addition, positive correlations were observed between the levels of tPAIC and EXDP in the patients with non-MODS; however, no correlations were observed between these markers in the MODS patients.Thrombin activation and fibrinolytic shutdown play important roles in the development of organ dysfunction in PCAS patients. Neutrophil elastase-mediated fibrinolysis cannot overcome the fibrinolytic shutdown that occurs in DIC patients with PCAS, thus resulting in the development of MODS.

399 A novel mechanism for paracrine stimulation of endometrial angiogenesis: Urokinase plasminogen activator (u-PA) / plasminogen activator inhibitor-1 (PAI-1) complex, released from stromal cells stimulated with TGFβ 1, stimulates endothelial cell migration

399 A novel mechanism for paracrine stimulation of endometrial angiogenesis: Urokinase plasminogen activator (u-PA) / plasminogen activator inhibitor-1 (PAI-1) complex, released from stromal cells stimulated with TGFβ 1, stimulates endothelial cell migration

Influence of an ultramarathon race at moderate altitude on coagulation and fibrinolysis

Summary Acute physical exercise induces a temporary increase in blood coagulability as well as in fibrinolysis. Only few data have been reported concerning the hemostatic system after long lasting strenuous exercise. In order to evaluate the consequences of prolonged strenuous endurance exercise on the hemostatic system, 11 athletes were examined before, immediately after as well as 24 h after the start of a 67 km mountain run. Immediately after the race we found significant increases in thrombin-antithrombin III complexes (TAT; 1.4-fold), in D-dimer (1.4-fold), in plasmin-α2-antiplasmin complexes (PAP; 3.9-fold), in tissue type plasminogen activator (t-PA; 3.7-fold) and in t-PA-plasminogen activator inhibitor-1 complexes (t-PA-PAI-1; 7.6-fold). Antithrombin III as well as PAI-1 remained unchanged. One day after the start of the race only PAP complexes were significantly elevated (1.7-fold). This study demonstrates that extreme long lasting endurance exercise activates both, the coagulatory as well as the fibrinolytic system. As compared with the activation of fibrinolysis the activation of the coagulation is small. Thus, we conclude that prolonged strenuous endurance exercise seems not to be associated with a high prothrombotic potential.

Molecular characterization of plasminogen activators in human gingival crevicular fluid

Plasminogen activators (PAs), a family of serine proteases, and their inhibitors (PAIs) are important in fibrinolysis, wound healing and tissue remodelling. Previous studies revealed differences in the localization of PA activity between healthy and diseased gingival tissues, suggesting that PAs and PAIs could play a part in periodontal homeostasis and disease. PAs and PAIs are synthesized by most of the cell types making up the periodontium and can be identified in gingival crevicular fluid (GCF). These studies sought to characterize the molecular species of PAs and their inhibitors in GCF collected from clinically healthy sites. PA enzymatic activity in GCF samples demonstrated by fibrin zymography revealed the presence of only tissue-type PA (tPA) activity. No urokinase-type PA (uPA) enzymatic activity was detected. tPA enzymatic activity appeared predominantly as an uncomplexed 70-kDa species, although some samples contained enzyme-inhibitor complexes. Quantitation of total tPA by enzyme immunoassay showed a mean concentration of 1.6ng/μl. Analysis of GCF samples for uPA by immunoblotting and enzyme immunoassay disclosed the presence of small amounts of uPA (0.2 ng/μl), which were present predominantly in activator-inhibitor complexes. Immunoblotting showed specific PAI-2 immunoreactivity bands in high molecular-weight complexes and low molecular-weight degradation products, but less than nanogram amounts of free PAI-2 molecules. Enzyme immunoassay revealed that PAI-2 was present in an at least a seven times greater amount than PAI-1. These observations support the hypothesis that PA-generated proteolysis and its regulation by endogenous inhibitors has a role in the diverse biochemical mechanisms underlying periodontal physiology and pathology including host-microbial interaction, polymorphonuclear leucocyte infiltration, turnover and migration of epithelial cells, connective tissue degradation and remodelling, fibrinolysis and wound healing.

Very Low Density Lipoprotein Receptor Binds and Mediates Endocytosis of Urokinase-type Plasminogen Activator-Type-1 Plasminogen Activator Inhibitor Complex

Very low density lipoprotein receptor (VLDL-R) was found to be expressed in bovine mammary gland and the human breast carcinoma cell line MCF-7 as an M(r) 105,000 variant, and in Chinese hamster ovary (CHO) cells transfected with human VLDL-R cDNA as an M(r) 130,000 variant. The receptor was purified by ligand affinity chromatography with immobilized M(r) 40,000 receptor-associated protein (RAP). The purified receptor was found to bind urokinase-type plasminogen activator-type-1 plasminogen activator inhibitor complex (u-PA.PAI-1), while there was no or very weak binding of active site blocked u-PA (DFP-u-PA), PAI-1 or u-PA-type-2 plasminogen activator inhibitor complex. The binding of u-PA.PAI-1 was blocked by RAP. The transfected CHO cells had an efficient, RAP-sensitive endocytosis of u-PA.PAI-1, severalfold higher than non-transfected parental CHO cells. u-PA.PAI-1 endocytosis was partially inhibited by DFP-u-PA, which blocks binding of the complex to the u-PA receptor. RAP and DFP-u-PA sensitive u-PA.PAI-1 endocytosis was also observed in MCF-7 cells, which were without detectable levels of other RAP-binding endocytosis receptors. These results show that VLDL-R represents a novel endocytosis mechanism for u-PA receptor-bound u-PA.PAI-1.

Cathepsin D-like aspartyl protease activity mediates the degradation of tissue-type plasminogen activator/plasminogen activator inhibitor-1 complexes in human monocytes

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 purifed. lysosomal aspartyl protease, cathepsin D, is capable of digesting t-PA ( t 1 2 15 min ), active PAI-1 ( t 1 2 2 h ), and t-PA/PAI-1 complex ( t 1 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 PAM exhibiting resistance to proteolysis and > 10-fold prolongation in t 1 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 PAM and latent PAI-1 are relatively resistant to degradation by cathepsin D, which may be of importance in complex physiological environments.

Gp330 on type II pneumocytes mediates endocytosis leading to degradation of pro-urokinase, plasminogen activator inhibitor-1 and urokinase-plasminogen activator inhibitor-1 complex.

Glycoprotein 330 (gp330) is a member of a family of receptors related to the low density lipoprotein receptor (LDLR). Although several ligands have been shown to bind gp330 in solid-phase assays, the ability of gp330 to mediate ligand endocytosis has not been demonstrated. To develop a cellular model for gp330 function we screened a variety of cultured cell lines and identified several that expressed this protein, including immortalized rat type II pneumocytes and a human and two rodent tumor cell lines. Using type II pneumocytes, endocytosis of a previously described gp330 ligand, urokinase (uPA) complexed with plasminogen activator inhibitor-1 (uPA:PAI-1) and two new ligands, PAI-1 and pro-uPA, was demonstrated. RAP, the 39 kDa receptor-associated protein known to antagonize ligand binding to gp330 in solid-phase binding assays, completely inhibited both internalization and degradation of the radiolabeled ligands by type II pneumocytes. This suggested that the clearance of these ligands was dependent on either gp330 or the LDLR-related protein (LRP), which shares several ligand-binding characteristics with gp330. By using polyclonal antibodies to gp330, the cellular internalization and degradation of the ligands were inhibited by 30-50%; remaining ligand internalization and degradation activity could be partially inhibited by polyclonal antibodies against LRP. These findings indicate that gp330, like other LDLR family members, mediates endocytosis of its ligands. In addition, gp330 acts in concert with LRP in type II pneumocytes to mediate clearance of a variety of proteins involved in plasminogen activation, including uPA:PAI-1 complexes PAI-1 and pro-uPA.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of the binding of pro-urokinase and urokinase-plasminogen activator inhibitor-1 complex to the low density lipoprotein receptor-related protein using a Fab fragment selected from a phage-displayed Fab library.

The low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor (LRP) mediates endocytosis of a number of structurally unrelated ligands, including complexes of plasminogen activator inhibitor type 1 (PAI-1) and tissue-type plasminogen activator (t-PA) or urokinase plasminogen activator (u-PA), free t-PA, single-chain urokinase (pro-u-PA), alpha 2-macroglobulin-protease complexes, and lipoprotein lipase. So far, all ligands have in common the fact that they bind to the receptor in a Ca(2+)-dependent way and the fact that binding to the receptor can be inhibited by a 39-40-kDa protein, termed the receptor-associated protein. To obtain inhibitory antibodies for the analysis of the structure and function of the receptor we applied the combinatorial immunoglobulin repertoire cloning technique in order to specifically select monoclonal Fab fragments directed against Ca(2+)-dependent epitopes. In this report we describe the isolation of a Fab fragment (Fab A8) showing a high relative affinity for the receptor (0.5 nM). The binding of this Fab fragment to purified LRP is inhibited in the presence of 5 mM EDTA, receptor-associated protein, and lipoprotein lipase (IC50 values of 1.4 and 31 nM, respectively). By immunoblotting of CNBr-digested LRP it is shown that Fab A8 binds to a fragment that harbors the second cluster of cysteine-rich complement-type repeats flanked by epidermal growth factor repeats. Binding studies using 125I-labeled ligands and immobilized receptor show that Fab A8 partially inhibits the binding of [125I]u-PA.PAI-1 complexes (IC50 = 1.1 nM) and completely inhibits the binding of [125I]pro-u-PA to the receptor (IC50 = 2.2 nM). No inhibition was observed for the binding of 125I-labeled methylamine-activated alpha 2-macroglobulin or [125I]t-PA.PAI-1 to LRP. Degradation of [125I]u-PA.PAI-1 complexes by COS-1 cells was also partially (43%) inhibited by Fab A8. Our results provide evidence for the presence of an interaction site for pro-u-PA localized in the second cluster of cysteine-rich repeats that is unrelated to the t-PA.PAI-1 or methylamine-activated alpha 2-macroglobulin interaction sites.

Human tissue plasminogen activator-human plasminogen Activator inhibitor complex immunoassay and kit therefor : Hasegawa et al. Teijin Ltd. US 5352583; October 4, 1994

Human tissue plasminogen activator-human plasminogen Activator inhibitor complex immunoassay and kit therefor : Hasegawa et al. Teijin Ltd. US 5352583; October 4, 1994

High factor-VII activity in the elderly is related to vascular disease through activation of the coagulation system.

High factor-VII activity has recently been recognized as an independent risk factor for coronary artery disease. The aim of the present study was to determine whether high levels of factor-VII activity in elderly patients are caused by endothelial cell injury and are related to vascular disease through activation of the coagulation system. We studies 27 asymptomatic healthy elderly men with high factor-VII activity (155-206%) and 22 healthy elderly men with low factor-VII activity (96-122%) selected from 246 men over 60 years of age attending annual health examinations. We also studied 36 patients with vascular disease (22 with coronary artery disease and 14 with chronic cerebral infarction). The levels of endothelium-derived markers (thrombomodulin, tissue plasminogen activator-plasminogen activator inhibitor-1 complex, active plasminogen activator inhibitor 1 and tissue factor pathway inhibitor) did not differ between the subjects with high and those with low factor-VII activity. We therefore conclude that high factor-VII activity is not primarily caused by endothelial cell injury. The high-factor-VII group had higher levels of coagulation inhibitors (protein C, antithrombin III and heparin cofactor II), coagulation activation markers (prothrombin fragment 1 + 2, D-dimer and plasmin-alpha2- plasmin-inhibitor complex), lipoprotein (a) and total cholesterol than the low-factor-VII group. The coagulation and lipid profiles of the group with vascular disease were closer to those of the high-factor-VII group than to those of the low-factor-VII group; the same was true of elevated active plasminogen activator inhibitor-1 levels. High factor-VII activity in the elderly is accompanied by hyperlipidaemia, including increased levels of lipoprotein (a), and leads to coagulation activation in vivo. Thus, elderly people with high factor-VII activity are predisposed to vascular disease.

Low density lipoprotein receptor-related protein is necessary for the internalization of both tissue-type plasminogen activator-inhibitor complexes and free tissue-type plasminogen activator.

Tissue-type plasminogen activator (t-PA) is used as a thrombolytic agent in treatment of myocardial infarction. However, large doses of this agent must be administered in treatment to maintain a thrombolytic state because t-PA is cleared rapidly from circulation. We designed specific ligands to distinguish between two major mechanisms by which t-PA is taken into cells and degraded. One of these mechanisms involves internalization of complexes between t-PA and its cognate inhibitor plasminogen activator inhibitor type-1 (PAI-1); the other mechanism is independent of PAI-1. Using specific inhibitors for low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor (LRP), we show that the degradation by hepatocytes of both free t-PA and t-PA.PAI-1 complexes involve the receptor LRP. We demonstrate that fibroblasts degrade both free t-PA (PAI-1-independent) and t-PA complexed with its specific inhibitor PAI-1 (PAI-1-dependent), whereas genetically altered fibroblasts that do not express LRP neither internalize nor degrade these ligands. We also show that a PAI-1-independent, t-PA ligand can inhibit the degradation of both free t-PA and t-PA.PAI-1 complexes. We propose LRP is the receptor for both PAI-1-independent and PAI-1-dependent t-PA ligands.

Molecular dissection of ligand binding sites on the low density lipoprotein receptor-related protein.

The low density lipoprotein receptor-related protein (LRP) is a large multifunctional receptor that is involved in the cellular uptake of a number of functionally diverse ligands including apoE-rich remnant lipoproteins, lipoprotein lipase, alpha 2-macroglobulin-protease complexes, plasminogen activator-inhibitor complexes, and the active protease tissue-type plasminogen activator. Ligand binding and competition experiments suggest that most LRP ligands bind to specific, independent sites on the large 515-kDa subunit of the receptor. In a previous study (Moestrup, S.K., Holtet, T.L., Etzerodt, M., Thøgersen, H.C., Nykjaer, A., Andreasen, P.A., Rasmussen, H.H., Sottrup-Jensen, L., and Gliemann, J. (1993) J. Biol. Chem. 268, 13691-13696), ligand blotting was used to localize the binding sites for urokinase-type plasminogen activator-plasminogen activator inhibitor-1 (PAI-1) complexes and for alpha 1-macroglobulin to a proteolytic fragment of LRP containing the second cluster of complement-type cysteine-rich repeats. Here, we have used a recombinant DNA approach to express functionally restricted chimeric "LRP-minireceptors" containing two different regions of the extracellular domain of the receptor in cultured cells. Receptor-associated protein, a negative modulator of LRP activity, is bound and internalized by cells transfected with either construct. A minireceptor containing the cluster of eight complement-type cysteine-rich repeats followed by four epidermal growth factor precursor homologous domains binds and internalizes 125I-labeled plasminogen activator-PAI-1 complexes. It also mediates the cellular uptake of the uncomplexed protease tissue-type plasminogen activator (tPA), suggesting that the tPA and PAI-1 binding sites on LRP are in close vicinity and might promote cooperative binding of tPA-PAI-1 complexes. However, alpha 2-macroglobulin is not internalized by this minireceptor suggesting that this ligand requires the presence of a single epidermal growth factor-repeat which is contained in the previously studied proteolytic fragment but is absent from the minireceptor.

Tissue plasminogen activator, plasminogen activator inhibitors, and activator-inhibitor complex in liver disease.

To identify the relative contribution of plasminogen activators, particularly tissue plasminogen activator (t-PA) and specific plasminogen activator inhibitors (PAI-1, PAI-2), to the fibrinolytic changes associated with various types of liver disease or severe chemical and physical damage to the liver. Platelet rich (PRP) and platelet poor plasma (PFP) from patients with alcoholic cirrhosis, primary biliary cirrhosis, hepatic malignancy, or paracetamol overdose, or who were undergoing partial hepatectomy or liver transplantation, were assayed for t-PA, PAI-1, t-PA-PAI-1 complex and PAI-2 antigen values using specific enzyme linked immunosorbent assays (ELISAs) developed in this laboratory. Appreciable increases in the plasma concentration of t-PA, PAI-1, and t-PA-PAI-1 were seen in patients with alcoholic cirrhosis, primary biliary cirrhosis, and hepatic malignancy. Liver damage due to paracetamol overdose and partial hepatectomy both resulted in a striking increase in plasma PAI-1 concentration, although concentrations of t-PA and t-PA-PAI-1 complex were less affected. Concentrations of t-PA, PAI-1, and t-PA-PAI-1 complex returned to near normal values after successful liver transplantation in a patient with chronic active hepatitis. PAI-2 was also detected in several patients with chronic liver disorders. Haemorrhage due to fibrinolytic bleeding is commonly associated with liver disease. The patients studied here all had appreciable increases in circulating t-PA antigen concentrations. This was associated with increased concentrations of PAI-1 antigen and t-PA-PAI-1 complex and the balance between activator and inhibitor did not result in systemic plasmin generation. Reduced PAI-1 activity in cirrhosis or a critical difference in the ratio of t-PA to PAI-1 concentrations may explain the enhanced plasminogen activator activity previously noted in cirrhosis but not metastatic disease. Reduced hepatic clearance of t-PA and t-PA-PAI-1 complex due to impaired liver function may account for increased concentrations of free and complexed t-PA.

LDL Receptor-related Proteinの動脈硬化巣での発現I

LRP (LDL receptor-related protein) is considered to be one of the remnant receptors. Amino acid sequence analysis indicates that LRP is identical to the alpha2 macroglobulin receptor, which has been shown to bind and internalize following ligands; beta-VLDL with apoprotein E or lipoprotein lipase, alpha2 macroglobulin-protease complex, and plasminogen activator-inhibitor complexes. These characteristics indicate that LRP/alpha2 macro globulin receptor might be responsible in atherosclerotic lesions for foam-cell formation and regulation of protease activity. In order to investigate the role of LRP in atherogensis, we used the RT-PCR method to examine the expression of LRP mRNA in human atherosclerotic lesions.Atherosclerotic lesions were divided into three groups: normal intima, raised lesion, and calcified lesions. From these fractions, total RNA were extracted by the AGPC method and RT-PCR was performed.The results of PCR showed that expression of LRP mRNA was more abundant in atherosclerotic lesions than in normal intima or media, suggesting that LRP might play a role in atherosclerosis.