The role of plasminogen activator inhibitor type 1 in the clearance of tissue-type plasminogen activator by rat hepatoma cells.

The role of plasminogen activator inhibitor type 1 (PAI-1) in the clearance of tissue-type plasminogen activator (t-PA) by hepatocyte-like cells was studied. Rat (Novikoff) hepatoma cells were able to bind and degrade t-PA in a PAI-1 independent fashion, but PAI-1 markedly increased the rate of degradation and t-PA/PAI-1 was a more efficient inhibitor of 125I-t-PA or of 125I-t-PA/PAI-1 degradation than free t-PA. Competition studies revealed that the effect of PAI-1 is unlikely to involve determinants located on the PAI-1 part of the complex: 1) an excess of free PAI had no effect on the rate of degradation of 125I-t-PA/PAI-1.2) Complexes of PAI-1 with urokinase-type PA or with a t-PA mutant lacking the finger and growth factor domains were unable to compete for the binding and degradation of free or PAI-1-complexed 125I-t-PA.3) t-PA KHRR296-299AAAA, a mutant which reacts 2 orders of magnitude slower with PAI-1 than wild type t-PA, behaved similar to wild type t-PA. The clearance via both the PAI-1-dependent and the PAI-1-independent mechanisms was inhibited by the receptor-associated protein, a general inhibitor of clearance mediated by the LDL receptor-related protein. We conclude that t-PA can be cleared by rat hepatoma cells in a PAI-1 independent fashion, but after complex formation with PAI-1, binding of t-PA to the cells is increased and clearance accelerated. Both mechanisms seem to involve the same receptor

Plasminogen activator inhibitor 1 and plasminogen activator inhibitor 2 in various disease states.

The association of increased PA-inhibitor (PAI) activity and of PAI-1 and PAI-2 antigen levels with different pathological conditions was studied in a collective of over 300 patients. PAI-1 and PAI-2 levels were measured by specific radioimmunoassays. A good correlation was observed of PAI activity with PAI-1 antigen (r = 0.718; p less than 0.0001) but not with PAI-2 (r = 0.070; n.s.). Both in the controls and in the patients, PAI activity and PAI-1 antigen showed an extremely large range of values. PAI activity ranged from 0.5 to 68 U/ml and PAI-1 antigen from 6 to 600 ng/ml. Increased PAI activity and PAI-1 antigen was observed in patients with malignant tumors, cardiovascular or thromboembolic disease, in the postoperative phase, with hepatic insufficiency, after trauma and after extracorporeal circulation. The large spectrum of disease states with increased PAI activity and PAI-1 antigen reinforces previous suggestions that PAI-1 is an acute phase reactant. After extracorporeal circulation, PAI activity and PAI-1 concentrations strongly increased within one hour, remained elevated for at least one week and returned to preoperation values within 7 days. PAI-2 values ranged from below detection limit (15 ng/ml), observed in half of the plasmas, to 485 ng/ml in a pregnant woman. High values of PAI-2 were only observed in pregnancy

Expression of LDL receptor-related protein/alpha 2-macroglobulin receptor in human normal and atherosclerotic arteries.

Low-density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor (LRP/alpha 2-MR) is a multifunctional cell-surface receptor that is responsible for the clearance of lipoprotein remnants, proteases, or cytokines/growth factors in complex with alpha 2-macroglobulin as well as of plasminogen activators complexed with inhibitors. We investigated the expression of LRP/alpha 2-MR in healthy and atherosclerotic human arteries by in situ hybridization using an LRP/alpha 2-MR mRNA-specific riboprobe and immunocytochemistry using specific monoclonal antibodies. The cell types expressing LRP/alpha 2-MR were identified by immunolabeling of antigens specific for endothelial cells, smooth muscle cells, and macrophages. In normal arteries, LRP/alpha 2-MR mRNA and protein were found in smooth muscle cells of the media and vasa vasorum and in adventitial fibroblasts. Endothelial cells were negative for LRP/alpha 2-MR protein but positive for its mRNA. Atherosclerotic arteries exhibited a strong labeling for LRP/alpha 2-MR mRNA and protein that was observed in intimal smooth muscle cells exhibiting normal or foam cell characteristics and in lipid-laden cells positive for macrophage markers. A particularly high expression was detected in macrophages located in the cap of the lipid-rich necrotic core. These results suggest that cellular components of the atherosclerotic plaque express LRP/alpha 2-MR. This receptor may play an important local scavenger role for lipoprotein remnants, for growth factors/cytokines, and for extracellular protease-inhibitor complexes

Short-term infusions of insulin, triacylglycerol and glucose do not cause acute increases in plasminogen activator inhibitor-1 concentrations in man.

1. To investigate the acute effects of insulin and triacylglycerol ('triglyceride') on circulating plasminogen activator inhibitor-1 concentrations, seven healthy volunteers were studied during hyperinsulinaemic clamps in the presence of euglycemia (mean glucose concentration 5 mmol/l) and hyperglycaemia (mean glucose concentration 9 mmol/l) with and without triacylglycerol infusions. 2. During euglycemia, plasma insulin levels rose from baseline values [median (range)] of 13 (6.6-20.6) m-units/l to 89 (74-105) m-units/l and 99 (74-109) m-units/l after 1 and 2 h of insulin infusion, respectively. Concentrations of plasminogen activator inhibitor-1 fell from 27.5 (10-47) ng/ml to 25.0 (14.5-55) ng/ml and 15.5 (11.5-28.5) ng/ml (P less than 0.02) over the same time. 3. During hyperglycaemia, plasma insulin concentrations were 12.1 (9.3-17.1) m-units/l at the run-in period and rose to 87 (73-112) m-units/l and 91 (84-97) m-units/l after 1 and 2 h of insulin infusion, respectively. Concentrations of plasminogen activator inhibitor-1 again showed a gradual fall from 24.7 (22-50) ng/ml to 14 (8.3-25.5) ng/ml and 13 (6.0-35.0) ng/ml (P less than 0.02) over the same period. 4. Infusion of Intralipid in the presence of hyperinsulinaemia with either euglycemia or hyperglycaemia was associated with a similar fall in concentrations of plasminogen activator inhibitor-1 over the study period. 5. The results from this study indicate that short-term increases in insulin, glucose or triacylglycerol do not cause acute increases in plasma concentrations of plasminogen activator inhibitor-1

Hypofibrinolysis in patients with a history of idiopathic deep vein thrombosis and/or pulmonary embolism.

An impaired fibrinolytic activity after a venous occlusion test is the most common abnormality associated with thomboembolic disease. To better characterize the causes of abnormal responses we have measured different fibrinolytic parameters, before and after 10 and 20 min of venous occlusion, in 77 patients with a history of idiopathic deep vein thrombosis and/or pulmonary embolism and in 38 healthy volunteers. The patients had a lower mean fibrinolytic response to venous occlusion than the controls and higher antigen levels of tissue-type plasminogen activator (t-PA:Ag) and plasminogen activator inhibitor type 1 (PAI-1:Ag). Before venous occlusion, PAI-1 levels were at a molar excess over those of t-PA in all patients and controls. After 20 min of venous occlusion, the release of t-PA from the vascular endothelium resulted in a molar excess of t-PA over PAI-1 in the majority of controls (72%) but only in a minority of patients (39%). To identify patients with fibrinolytic abnormalities, reference intervals (RI) for fibrinolytic activity, t-PA:Ag and PAI-1:Ag were established in healthy controls. None of the patients had low levels of t-PA:Ag, but 17 (22%) had elevated PAI-1:Ag levels before venous occlusion and 12 (16%) exhibited low fibrinolytic activity after 20 min of venous occlusion. Ten of these were among the 17 subjects with high PAI-1:Ag levels before venous occlusion. Thus, the measurement of PAI-1:Ag levels before venous occlusion (i.e. in samples taken without any stimulation) is a sensitive (83%) and specific (89%) assay for the detection of patients with an impaired fibrinolytic response to venous occlusion

Localization and production of plasminogen activator inhibitor-1 in human healthy and atherosclerotic arteries.

High plasma levels of plasminogen activator inhibitor type-1 (PAI-1), the principal inhibitor of the fibrinolytic system, have been associated with thrombotic and arterial disease. To study PAI-1 expression in healthy and atherosclerotic human arteries, a detailed analysis was made by light and electron microscopy immunocytochemistry and by in situ hybridization. In healthy arteries PAI-1 was found both at the level of endothelial cells and of smooth muscle cells (SMCs) of the arterial media. In early atherosclerotic lesions PAI-1 was also detected in intimal SMCs and in extracellular areas in association with vitronectin. Immunogold analysis by electron microscopy revealed PAI-1 in vesicular structures in endothelial cells and in SMCs with normal or foam cell characteristics. In advanced atheromatous plaques, PAI-1 mRNA expression in SMCs within the fibrous cap was increased compared with SMCs located in the adjacent media or in normal arterial tissue. PAI-1 mRNA was also detected in macrophages located at the periphery of the necrotic core. The increased synthesis of PAI-1 by cellular components of the atherosclerotic plaque and the extracellular accumulation of PAI-1 may contribute to the thrombotic complications associated with plaque rupture and possibly play a role in the accumulation of extracellular matrix deposits

Fibrinolysis in pregnancy: a study of plasminogen activator inhibitors.

During pregnancy the plasma concentration of two different inhibitors of plasminogen activators (PAIs) increases. The only one found in the plasma of nonpregnant women (PAI1) is immunologically related to a PAI of endothelial cells; its plasma activity, as deduced from the inhibition of single-chain tissue-type plasminogen activator (t-PA), increased from 3.4 +/- 2.3 U/mL (mean +/- 95% confidence limits) in the plasma of nonpregnant women to 29 +/- 7 U/mL at term, and its antigen level, measured by a radioimmunoassay, increased from 54 +/- 17 ng/mL to 144 +/- 25 ng/mL. In pregnancy plasma a second PAI (PAI 2) related to a PAI found in placenta extracts was observed. Its level, quantified with a radioimmunoassay, increased from below the detection limit (approximately 10 ng/mL) in normal plasma to 260 ng/mL at term. One hour after delivery, PAI 1 activities and antigen decreased sharply, but the PAI 2 antigen levels remained constant. Three days later, the PAI 1 antigen levels had fallen to normal levels, but the PAI 2 antigen levels were still at least eightfold above the nonpregnant values. During pregnancy, the t-PA and prourokinase (u-PA) antigen concentrations increased 50% and 200%, respectively, whereas the plasminogen and alpha 2-antiplasmin levels remained constant. Despite the large variations in the levels of PAs and PAIs, the overall fibrinolytic activity as measured in diluted plasma by a radioiodinated fibrin plate assay did not change significantly. Just after delivery, a great increase in the t-PA antigen levels was observed. Three to five days after delivery most parameters of the fibrinolytic system were normal again. Our results demonstrate that during pregnancy and in the puerperium profound alterations of the fibrinolytic system occur that are characterized by increases in PAs and their inhibitors, but these alterations do not affect the overall fibrinolytic activity

Characterization of Human Late Outgrowth Endothelial Progenitor-Derived Cells under Various Flow Conditions.

Background: Endothelial progenitor-derived cells (EPC) are a cell therapy tool in peripheral arterial disease and for re-endothelialization of bypasses and stents. Objective: To assess EPC behavior under flow conditions normally found in vivo. Results: EPC were isolated from human cord blood, cultured on compliant tubes and exposed in an in vitro flow system mimicking hemodynamic environments normally found in medium and large arteries. EPC exposed for 24 h to unidirectional (0.3 ± 0.1 or 6 ± 3 dynes/cm(2)) shear stress oriented along flow direction, while those exposed to bidirectional shear stress (0.3 ± 3 dynes/cm(2)) or static conditions had random orientation. Under bidirectional flow, tissue factor (TF) activity and mRNA expression were significantly increased (2.5- and 7.0-fold) compared to static conditions. Under low shear unidirectional flow TF mRNA increased 4.9 ± 0.5-fold. Similar flow-induced increases were observed for TF in mature umbilical vein-derived endothelial cells. Expression of tissue-type plasminogen activator (t-PA), urokinase (u-PA) and monocyte chemotactic protein 1 (MCP1) were reduced by 40-60% in late outgrowth endothelial progenitor-derived cells (LO-EPC) exposed to any flow environment, while MCP1, but not t-PA or u-PA, was decreased in HUVEC. Conclusions: Flow, in particular bidirectional, modifies the hemostatic balance in LO-EPC with increased TF and decreased plasminogen activator expression