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PubMed2004

Enhancement of urokinase-type plasminogen activator (uPA) secretion, but not that of substrate plasminogen (PGn), by rat microglia stimulated with neuronal conditioned medium.

Assuming the presence of intercellular interactions between injured motoneurons and microglia in the axotomized facial nucleus, we investigated the effects of neuronal conditioned medium (NCM) on the release of urokinase-type plasminogen activator (uPA) from microglia. Zymography revealed that NCM markedly enhanced the release of uPA from microglia, although NCM itself did not contain a significant amount of uPA. In contrast, the secretion of plasminogen (PGn), a substrate of uPA, was not promoted by the NCM treatment. The specific effect of NCM was found to be quite distinct from that of microglial activators, interferon-gamma (IFN-gamma) or lipopolysaccharide (LPS), which reduce uPA release from microglia. In summary, Neuron-derived soluble molecules appear to stimulate microglia to enhance the production/release of uPA, but not that of PGn, by a mechanism independent of the activation reaction by IFN-gamma or LPS.

Animals↗
PubMed2003

Secretion of tissue plasminogen activator and plasminogen activator inhibitor 1 during cardiopulmonary bypass.

INTRODUCTION: Cardiopulmonary bypass (CPB) is associated with elevated tissue plasminogen activator (t-PA) levels during CPB and increased plasminogen activator inhibitor 1 (PAI-1) levels post-operatively. The goal of this study was to estimate the rate of t-PA and PAI-1 secretion in vivo, before, during and after CPB. MATERIALS AND METHODS: Estimated rates of t-PA and PAI-1 secretion were based on measured levels of active and total t-PA, and active and total PAI-1, obtained before, during and after CPB from nine males, combined with a computer model of each patient's vascular system that continuously accounted for secretion, clearance, hemodilution, blood loss and transfusion. RESULTS AND CONCLUSIONS: At baseline, the average t-PA and PAI-1 secretion rates were 0.74+/-0.33 and 1.28+/-0.74 pmol/s, respectively. Within 5 min of CPB initiation, t-PA secretion increased six-fold to 4.41+/-2.58 pmol/s, while PAI-1 secretion was unchanged, resulting in a six-fold increase in active t-PA levels. t-PA secretion remained elevated throughout CPB and into the early post-operative period. Average PAI-1 secretion did not start to increase until the end of CPB. By 2 h after surgery, average PAI-1 secretion had increased 15-fold to 19.60+/-17.10 pmol/s, resulting in reduced levels of active t-PA even though t-PA secretion was still elevated. We conclude that CPB induces an immediate sustained increase in t-PA secretion followed by a delayed progressive increase in PAI-1 production. Variations in the level of active t-PA are a function of the relative rates of t-PA versus PAI-1 secretion at different times during and after surgery.

Blood Chemical Analysis↗
PubMed1997

Plasma levels of tissue plasminogen activator and plasminogen activator inhibitor-1 are correlated with the presence of transplant coronary artery disease in cardiac transplant recipients.

Hemostatic factors are involved in the pathogenesis of native coronary artery disease. However, their role in transplant coronary artery disease is less established. To assess the role of hemostatic factors in transplant coronary artery disease we studied 52 consecutive cardiac transplant patients. The presence of transplant coronary artery disease was determined by angiography. Plasma levels of tissue plasminogen activator (t-PA), plasminogen activator inhibitor-1 (PAI-1), von Willebrand Factor (vWF), and fibrin D-dimer were determined by enzyme-linked immunosorbent assays. Serum lipids were measured by enzymatic methods. Patients with transplant coronary artery disease had higher circulating t-PA (8.6 +/- 0.8 vs. 5.4 +/- 0.6 ng/ml, p = 0.021) and PAI-1 antigen concentrations (38.0 +/- 3.4 vs 25.8 +/- 2.2 ng/ml, p = 0.037). t-PA and PAI-1 antigen concentrations correlated with the severity of angiographic disease (R = 0.34; p = 0.014 for t-PA, and R = 0.45; p = 0.001 for PAI-1). Serum cholesterol levels were higher in patients with transplant coronary artery disease (221 +/- 7.6 vs 191 +/- 9.2 mg/dl, p = 0.039). Serum triglycerides were also higher in patients with transplant coronary artery disease by angiography (246 +/- 38.3 vs 139 +/- 20.8 mg/dl, p = 0.050). Multivariate analysis identified t-PA antigen (p = 0.003) and triglyceride levels (p = 0.038) as independent predictors for the presence of transplant coronary artery disease. We conclude that cardiac transplant patients with evidence of transplant coronary artery disease on coronary angiography have altered hemostatic function which is reflected by elevated levels of circulating t-PA and PAI-1 antigens. The interaction of the hemostatic system and serum lipids in the development of transplant coronary artery disease warrants further study.

Cholesterol↗
PubMed2001

Increasing expression of tissue plasminogen activator and plasminogen activator inhibitor type 2 in dog gingival tissues with progressive inflammation.

Urokinase and tissue-type plasminogen activators (u--PA and t--PA) are serine proteases that convert plasminogen into plasmin, which degrades matrix proteins and activates metalloproteinases. The PAs are balanced by specific inhibitors (PAI--1 and PAI--2). Local production of t--PA and PAI--2 was recently demonstrated in human gingival tissues. The aim now was to investigate the production and localization of t--PA and PAI--2 in gingival tissues from dogs in three well-defined periodontal conditions; clinically healthy gingiva, chronic gingivitis and an initial stage of ligature-induced loss of attachment. At the start of the experiment the gingiva showed clear signs of inflammation. Clinically healthy gingiva were obtained after 21 days period of intense oral hygiene. Attachment loss was induced by placing rubber ligatures around the neck of some teeth. Biopsies were taken from areas representing the different conditions and prepared for in situ hybridization and immunohistochemistry. In clinically healthy gingiva both t--PA mRNA and antigen were expressed in a thin outer layer of the sulcular and junctional epithelia. No t--PA signals or staining were seen in connective tissue. Both mRNA signaling and immunostaining for t--PA were stronger in chronic gingivitis. In areas with loss of attachment, t--PA mRNA as well as antigen were found in the sulcular and junctional epithelia to a similar degree as in gingivitis. Occasionally the connective tissue was involved, especially in connection with vessels. PAI--2 mRNA was seen in a thin outer layer of the sulcular and junctional epithelia in clinically healthy gingiva, but no signals were seen in connective tissue. PAI--2 antigen was found primarily in the outer layer of the sulcular and junctional epithelia. Some cells in the connective tissue were stained. In gingivitis, PAI--2 signals were mainly found in the same locations, but more intense and extending towards the connective tissue. Immunostaining was seen in the outer half of the sulcular and junctional epithelia as well as in the upper part of the connective tissue, close to the sulcular epithelium. In sites with loss of attachment, PAI--2 mRNA was found throughout the sulcular and junctional epithelia, as was the antigen, which stained intensely. No PAI--2 mRNA was seen in connective tissue; the antigen was found scattered, especially near vessels. This study shows that the expression of both t--PA and PAI--2 increases with experimental gingival inflammation in the dog, and furthermore, the two techniques demonstrate a strong correlation between the topographical distribution of the site of protein synthesis and the tissue location of the antigens for both t--PA and PAI--2. The distribution correlates well with previous findings in humans.

Animals↗
PubMed2000

Overexpression of a dominant negative CREB protein in HT-1080 cells selectively disrupts plasminogen activator inhibitor type 2 but not tissue-type plasminogen activator gene expression.

The tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor type 2 (PAI-2) genes are differentially regulated by 12-phorbol 13-myristate acetate (PMA) in HT-1080 fibrosarcoma cells. PMA transcriptionally down-regulates the t-PA gene in HT-1080 cells, while the PAI-2 gene is simultaneously induced by this agonist. The t-PA and PAI-2 gene promoters harbour a cAMP-response element (CRE) which influences the expression of both genes. We have compared the binding activity of nuclear factors that recognise these CRE sites. We show that CREB (CRE binding protein) recognises each CRE and that the degree of constitutive Ser119-phosphorylated t-PA CRE-bound CREB was greater than for PAI-2 CRE bound CREB. Stable transfection of HT-1080 cells with a plasmid containing a CREB that could not be phosphorylated on Ser119 (pCI-CREB(ala119)) did not influence PMA-mediated suppression of t-PA mRNA, but markedly impaired PMA-mediated induction of PAI-2 mRNA. Our results demonstrate that the Ser119 residue of CREB plays a crucial role in PMA-mediated induction of PAI-2 gene expression, whereas PMA-mediated suppression of t-PA in HT-1080 cells requires a different process.

Cyclic AMP Response Element Modulator↗
PubMed1996

No influence of proinsulin and insulin on plasma levels of plasminogen activator inhibitor type 1 and tissue plasminogen activator in young women before and during intake of contraceptive steroids.

Clinical observations in patients predisposed to cardiovascular disorders and recent experimental observations suggest that proinsulin and insulin participate in the regulation of fibrinolysis in vivo. In the present study, we examined if proinsulin and insulin affect the constitutive (fasting) secretion of plasminogen activator inhibitor type 1 (PAI-1) and tissue plasminogen activator (t-PA) in young healthy women (N = 17). We also measured the antigen concentrations of PAI-1 and t-PA during slow and fast changes in proinsulin and insulin levels induced by oral (OGTT) and intravenous (IVGTT) glucose tolerance tests. The assessments were performed before and after 6 months of treatment with contraceptive steroids, which have a well-defined influence on the fibrinolytic variables. We observed no consistent correlations between fasting values of proinsulin, insulin, PAI-1, and t-PA either before or during hormonal treatment. Before hormonal treatment, PAI-1 and t-PA antigen levels decreased (P < .05) during the hyperproinsulinemia and hyperinsulinemia induced by the OGTT and IVGTT. After hormonal intake for 6 months, a decrease only in t-PA concentrations during the OGTT was observed despite similar proinsulin and insulin responses to the glucose loads. Our findings suggest that proinsulin and insulin have no influence on the regulation of plasma levels of PAI-1 and t-PA in young healthy women, irrespective of intake of contraceptive steroids.

Adult↗
PubMed1997

Epitopes on plasminogen activator inhibitor type-1 important for binding to tissue plasminogen activator.

The molecular details of the rapid complex formation between tissue plasminogen activator (tPA, E.C. 3.4.21.68) and plasminogen activator inhibitor type-1 (PAI-1) are still not fully elucidated. We have used surface plasmon resonance (SPR), the BIAcore, to characterize the binding of a large panel of monoclonal antibodies to four forms of recombinant human PAI-1, including active and latent PAI-1 as well as the complex between PAI-1 and recombinant human tc tPA or the protease part of tPA, the B-chain. Antibodies that discriminate between these different forms of PAI-1 have been identified, which is reflected by differences in k(a), k(d) as well as in Kd. In addition, in a chromogenic assay with PAI-1 and tPA we determined the IC50-values for these antibodies, i.e., studied their ability to inhibit the decrease in tPA-activity caused by PAI-1. In a competition assay using SPR, we have also been able to study whether concurrent binding of these antibodies to PAI-1 was possible. We could thereby assign the antibodies to five groups according to their binding areas. Furthermore, by using this technique, we have for the first time been able to identify three distinct epitopes on PAI-1, which are all of importance for the interaction and complex-formation with tPA. Since the antibodies that bind to one of these areas all have very poor affinity for the complex between PAI-1 and tPA, we suggest that this not previously described epitope must be located near the final binding site for tPA in this complex. Altogether, this also supports the theory of a multistep reaction between PAI-1 and tPA, in which tPA interacts with different parts of the PAI-1-molecule.

Antibodies, Monoclonal↗
PubMed1999

Protein movement during complex-formation between tissue plasminogen activator and plasminogen activator inhibitor-1.

Plasminogen activator inhibitor-1 (PAI-1) rapidly inactivates tissue plasminogen activator (tPA). After initial binding and cleavage of the reactive-centre loop of PAI-1, this complex is believed to undergo a major rearrangement. Using surface plasmon resonance and SDS-PAGE, we have studied the influence of a panel of monoclonal antibodies on the reaction leading to the final covalent complex. On the basis of these data, we suggest the mechanisms for the action of different classes of inhibitory antibodies. We propose that the antibodies which convert PAI-1 into a substrate for tPA do this by means of preventing the conversion of the initial PAI-1/tPA complex into the final complex by sterical intervention. Moreover, the localisation of the binding epitopes on free PAI-1, as well as on the PAI-1/tPA complex, suggests that tPA in the final complex cannot be located near helices E and F, as has previously been proposed.

Antibodies, Monoclonal↗
PubMed1997

mRNAs encoding urokinase-type plasminogen activator and plasminogen activator inhibitor-1 are elevated in the mouse brain following kainate-mediated excitation.

Urokinase-type plasminogen activator (uPA) is an inducible extracellular serine protease implicated in fibrinolysis and in tissue remodeling. Recently, we have localized uPA mRNA strictly in limbic structures and the parietal cortex of the adult mouse brain. Here, we tested whether the systemic treatment of mice with kainic acid (KA), an amino acid inducing limbic seizures, could elevate in the brain mRNAs encoding uPA and its specific inhibitor, plasminogen activator inhibitor-1 (PAI-1), a major antifibrinolytic agent. Brain sections encompassing the hippocampus were tested through in situ hybridization using radiolabeled riboprobes specific for the two mRNA species. The results showed that KA greatly enhanced both mRNA species in sites of limbic structures and cortex. However, in the hypothalamus and brain blood vessels only PAI-1 mRNA was elevated. Those were also the only two locations where PAI-1 mRNA was detected in the non-treated control brain, although at a low level. For both mRNAs, KA enhancement was first evident 2-4 h after treatment, and it was most prolonged in the hippocampal area, where prominent hybridization signals persisted for three days. Here, both mRNAs were initially elevated in the hilar region of the dentate gyrus and in the molecular and oriens layers; however, PAI-1 mRNA became evident throughout the area, while uPA mRNA became especially pronounced in the CA3/CA4 subfield. In the cortex both mRNA types were induced, but only uPA mRNA was elevated in the retrosplenial cortex, and also in the subiculum. In the amygdaloid complex, uPA mRNA was restricted to the basolateral nucleus, whereas PAI-1 mRNA was seen throughout the structure, however, excluding this nucleus. These data show that seizure activity enhances the expression of uPA and PAI-1 genes in the brain; the patterns of enhancement suggest that the protease and its inhibitor may act in brain plasticity in synchrony, however, also independently of each other. Furthermore, the results suggest that by elevating PAI-1 mRNA in brain blood vessels, limbic seizures generate a risk for stroke.

Animals↗
PubMed1996

Fibrinogen, coagulation factor VII, tissue plasminogen activator, plasminogen activator inhibitor-1, and lipid as cardiovascular risk factors in chronic hemodialysis and continuous ambulatory peritoneal dialysis patients.

Mortality rates associated with cardiovascular disease (CVD) are high in long-term dialysis patients. Increased levels of plasma fibrinogen (FBG), coagulation factor VII (FVII), tissue plasminogen activator (t-PA), and plasminogen activator inhibitor-1 (PAI-1) as well as hyperlipidemia are regarded as important risk factors for CVD. To investigate whether there are differences in the risk of CVD between chronic hemodialysis (HD) and continuous ambulatory peritoneal dialysis (CAPD) patients, serum lipid levels and plasma FBG, FVII, t-PA, and PAI-1 levels were measured in 17 patients on HD and 17 patients on CAPD. FBG was measured by the thrombin time method, FVII activity (FVIIc) by the chromogenic prothrombin time method, and t-PA and PAI-1 activity by the chromogenic substrate assay. No difference was found in body mass index (BMI) between HD and CAPD patients. Total cholesterol (TC), TC/high-density lipoprotein (HDL)-C ratio, low-density lipoprotein (LDL)-C, and triglycerides (TG) were significantly increased, and HDL-C was significantly decreased in CAPD patients compared with HD patients. FBG and FVIIc were significantly elevated in CAPD patients compared with controls or HD patients. T-PA activities were significantly higher in HD and CAPD patients than in controls. CAPD patients showed significantly higher PAI-1 activities than controls or HD patients. Significant positive correlations were found between FBG or FVIIc and TC, between FBG and LDL-C or TG, and between FVIIc and LDL-C in these patients. T-PA showed significant negative correlations with FBG, PAI-1, TC, LDL-C, and TG. There was a significant positive correlation between PAI-1 and TG and a significant negative correlation between PAI-1 and HDL-C. We conclude that CAPD patients may have a greater risk of CVD than do HD patients, and that coagulation and fibrinolytic activity are correlated with lipid disorders in these patients.

Adult↗
PubMed1998

Agonist activity of antiestrogen-receptor complexes to regulate urokinase plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI-1) endogenous gene expression in breast cancer cells.

We have shown that 4-hydroxytamoxifen (4-OHT) has estrogen-like effects on induction of TGFalpha mRNA in estrogen receptor (ER)-negative MDA-MB-231 human breast cancer cells, transfected with either wildtype (S30 cells) or a codon 351asp-->tyr mutant ER (BC-2 cells). The mutant receptor used to produce the stable transfectants was identified in a tamoxifen-stimulated human breast tumor. We have also demonstrated that raloxifene exhibits a gene-specific estrogen-like effect with mutant ER (BC-2 cells) but not with wildtype ER (S30 cells) (Levenson, A.S., Catherino, W.H. and Jordan, V.C. (1997) Estrogenic activity is increased for an antiestrogen by a natural mutation of the estrogen receptor. J. Steroid Biochem. Mol. Biol., 60, 261-268). We now describe the regulation of urokinase plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI-1) endogenous gene expression by estradiol (E2) and different antiestrogens in BC-2 cells. Northern blot analyses revealed that 4-OHT and raloxifene have concentration-dependent agonistic (E2-like) effects on the regulation of these genes. In contrast, the pure antiestrogen ICI 182780 alone had no effect but could block the action of E2, 4-OHT and raloxifene. The E2-like effects of non-steroidal antiestrogens in this model system cannot be explained by the mutation in the ER alone because 4-OHT acts as an agonist with wildtype receptor as well. We propose that the clear cut biological expression of estrogen-like qualities with different antiestrogens will in the future serve as an important model to dissect the signal transduction pathway.

Breast Neoplasms↗
PubMed2000

Plasminogen enhances the secretion of plasminogen activator inhibitor-1 from cultured rat astrocytes.

To investigate the physiological significance of microglia-derived plasminogen (PGn) in the central nervous system, we determined the effects of rat PGn on the secretion of plasminogen activator inhibitor (PAI) in cultured rat astrocytes by reverse zymography and Western blotting. The cultured astrocytes normally produce only a limited amount of PAI-1. After stimulation with PGn, however, the amount of active PAI-1 in the astrocyte-conditioned medium was significantly increased in a time-dependent manner. PGn was also proved to activate p38 MAP kinase and c-Jun N-terminal kinase in these astrocytes. Taken together, these results suggest that microglia-derived PGn increases the secretion of PAI-1 in astrocytes through the activation of MAP kinases, and that enhanced PAI-1 regulates various physiological phenomena including tissue remodeling, neuronal plasticity and neurotoxicity.

Animals↗
PubMed1996

Expression of tissue-type plasminogen activator, plasminogen activator inhibitor and von Willebrand factor in the supernatant of endothelial cell cultures in response to the seeding of adenocarcinoma cell line HRT-18.

Plasminogen activator inhibitor (PAI-1), tissue type plasminogen activator (tPA) and von Willebrand factor (vWF) concentrations were measured by ELISA in the supernatant of the following cultures: endothelial cells from human umbilical vein (HUVEC); human colon cancer cells (HRT-18); and co-culture cells of HUVEC + HRT-18. No measurable amount of the three substances was found in the supernatant of HRT-18 cell culture. Compared to the value in the HUVEC supernatant, in the UVEC/HRT-18 co-cultures, tPA concentration was significantly lower (P = 0.0047), PAI-1 significantly higher (P = 0.026) and vWF also significantly higher (P = 0.0048). These data indicate that HRT-18 tumor cells do not produce tPA, PAI-1 and vWF; however, these tumor cells induce endothelial cells to change the production of these substances. As a consequence, the interaction between tumor and endothelial cells in vivo may lead to depression of fibrinolysis and enhancement of platelet adhesion.

Adenocarcinoma↗
PubMed1998

Urokinase-type plasminogen activator and plasminogen activator inhibitors (PAI-1 and PAI-2) in extracts of invasive cervical carcinoma and precursor lesions.

In a previous study we reported a direct correlation between the degree of total proteolytic activity and the natural history of cervical carcinoma. The present work examined whether an increase in the urokinase-type plasminogen activator (uPA) and the plasminogen activator inhibitors (PAIs) is correlated with the natural history of cervical carcinoma. We measured uPA and PAI-1 activities and uPA, PAI-1 and PAI-2 antigen concentrations in cervical extracts from normal, squamous intraepithelial lesions (SIL) or invasive carcinoma patients. The uPA activity in invasive carcinoma extracts was 8.46 times that of normal extracts and 4.9 times that of SIL extracts. The PAI-1 activity in invasive carcinoma extracts was 1.3 times that of normal extracts and 1.24 times that of SIL extracts. uPA, PAI-1 and PAI-2 amounts were 25.7-, 12.1- and 7.9-fold higher, respectively, in invasive carcinoma than in SIL, and 39.1-, 21.38- and 27.3-fold higher, respectively, than in normal extracts. uPA and PAI-1 activities were 2.02- and 1.42-fold higher in extracts from patients with stages II-IV than those from stage I extracts, respectively. uPA, PAI-1 and PAI-2 amounts were 3.06-, 4.2- and 1.4-fold higher in extracts from patients with stages II-IV than those from stage I extracts, respectively. The increase in uPA activity and the antigen levels of uPA and PAIs (PAI-1 and PAI-2) in stages II-IV of invasive carcinoma of the cervix suggests that these components play an important role in invasion and metastasis in advanced stages of this tumour.

Adult↗
PubMed1998

Urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI-1) in tissue and serum of head and neck squamous cell carcinoma patients.

The aim of this study was to determine urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI-1) concentrations in tumour and adjacent normal tissue samples from 58 patients, and in serum samples from 40 of 58 patients with squamous cell carcinoma of the head and neck obtained at diagnosis and after completion of therapy. uPA and PAI-1 serum concentrations were also measured in 28 healthy volunteers who served as controls. Measurements were made using enzyme-linked immunosorbent assay (ELISA) techniques. For both uPA and PAI-1, significantly elevated concentrations were measured in tumour tissue as compared with normal tissue (uPA: 8.89 versus 0.41 ng/mg total protein (mgp), P < 0.0001; PAI-1: 23.9 versus 1.47 ng/mgp, P < 0.0001). A statistically significant difference in uPA concentrations was found between normal laryngeal and nonlaryngeal tissue (0.52 versus 0.3 ng/mgp, P = 0.008), and in PAI-1 concentrations between T1 + 2 and T3 + 4 stage of disease (17.32 versus 35.63 ng/mgp, P = 0.04). The uPA concentrations positively correlated with those of PAI-1 measured in both tumour (Rs = 0.62, P < 0.0001) and normal tissue (Rs = 0.30, P = 0.02). In serum samples, lower concentrations of PAI-1 were measured in the control group than in patients with cancer (412.0 versus 680.5 ng/ml serum (mls), P = 0.0006). The time of collection of the serum sample did not influence uPA and PAI-1 concentrations, and no association was observed between their concentrations and any clinical and histopathological prognostic factors tested. Our results indicate that both uPA and PAI-1 may play a specific role in the process of invasion and metastasis, and might also be of prognostic value in squamous cell carcinoma of the head and neck.

Adult↗
PubMedDate not supplied

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.

Adult↗
PubMed2001

Binding of plasminogen and tissue plasminogen activator to plasmin-modulated factor X and factor Xa.

Previous work in our laboratory has suggested that the fibrinolytic enzyme plasmin (Pn) inactivates coagulation factors X (FX) and Xa (FXa) in the presence of Ca(2+) and anionic phospholipid (aPL), producing fragments which bind plasminogen (Pg) and accelerate tissue plasminogen activator (t-PA). Our goals here were to determine if the Pn-mediated fragments of FX or FXa remain associated, whether they directly bind t-PA, and to quantify their interaction with Pg. Binding to aPL, benzamidine-Sepharose, or the active-site inhibitor dansyl-Glu-Gly-Arg-chloromethyl ketone demonstrated that Pn cleavage yielded noncovalent heterodimers of a fragment containing the aPL-binding domain (FXgamma(47) or FXagamma(33)) and a 13-kDa fragment (FXgamma(13) or FXagamma(13)). Both ligand blotting and surface plasmon resonance (SPR) showed that Pn-cleaved FX and FXa bound t-PA directly when Pn-treatment was effected in the presence of aPL and Ca(2+). Using SPR, apparent K(d) values of 1-3 microM and 0.3-0.4 microM were measured directly and by competition for the FXgamma(47/13)-Pg and FXagamma(33/13)-Pg interactions, respectively. For the first time, Pg-binding to a receptor was shown to be Ca(2+) enhanced, although primarily mediated by C-terminal lysine residues. Mathematical modeling of kinetic data suggesting two Pg per FXgamma(47/13) or FXagamma(33/13) was consistent with our conclusion that each subunit of FXgamma(47/13) or FXagamma(33/13) contains a C-terminal lysine. Earlier X-ray structures show that these Lys residues are distal from each other and the membrane, supporting the model where each interacts with a separate Pg. t-PA acceleration by FXgamma(47/13) or FXagamma(33/13) may therefore involve simultaneous presentation of two substrate molecules.

Anions↗
PubMed1989

Platelet plasminogen activator inhibitor: purification and characterization of interaction with plasminogen activators and activated protein C.

Plasminogen activator inhibitor (PAI) was purified in active form from porcine platelets under nondenaturing conditions. The purified inhibitor (Mr 47,000) reacts with tissue-type plasminogen activator (t-PA), urokinase (UK), and activated protein C (APC) to yield both SDS-stable complexes and a modified PAI of slightly reduced molecular weight. The second-order rate constants for the inhibition of t-PA and UK by PAI are 3.5 X 10(7) and 3.4 X 10(7) M-1 s-1, respectively. Activated protein C reacts with PAI with a second-order rate constant of 1.1 X 10(4) M-1 s-1. This rate is not accelerated by protein S, phospholipid, and calcium, or heparin. It is concluded that (1) PAI can function as both inhibitor and substrate of its target proteases, (2) if APC promotes fibrinolysis via inactivation of PAI, then APC must be present in concentrations several orders of magnitude greater than t-PA, or the interaction of APC and PAI must be accelerated by presently unknown mechanisms, and (3) in the absence of heparin, platelet PAI is the most rapid inhibitor of APC yet described.

Animals↗