The localization of the relaxed form of plasminogen activator inhibitor type 2 in human gingival tissues

The plasminogen activating system is important in extracellular proteolysis. Plasmin degrades tissues and activates proteases. Plasminogen activators (tissue type; t-PA and urokinase type; u-PA) and plasminogen activator inhibitors (PAI-1, PAI-2) are found in high concentrations in gingival crevicular fluid (GCF). Previous findings indicate the significance of PAI-2 in gingival inflammation. When PAI-2 inhibits a plasminogen activator its conformation relaxes and neoepitopes can be detected with a monoclonal antibody (#2H5). Our aim was to study if and where in the gingival region PAI-2 has acted as an inhibitor. Methodological studies were performed on GCF with western blotting. Frozen sections of human gingiva were studied immunohistochemically. The methodological studies showed that our antibody #2H5 selectively detects relaxed low molecular weight non-glycosylated PAI-2. Total PAI-2 and relaxed PAI-2 were found in all gingival epithelia with a honeycomb-like staining. Relaxed PAI-2 showed the most pronounced staining in the cell layers near the surface of the epithelium and no staining in the suprabasal layers, while total PAI-2 was found throughout the epithelium, often more pronounced suprabasally. The results showed that PAI-2 indeed has acted as an inhibitor of a protease in gingival tissues, primarily in the epithelia. The results also suggest primarily an intracellular localization and thus the interaction of PAI-2 with a protein other than t-PA.     

Fibrinolytic components in nasal mucosa and nasal secretion

 We evaluated a possible role for fibrinolytic components in nasal secretion by tissue localization with immunohistochemical techniques and by measuring their antigen concentrations in nasal discharge by means of ELISA and fibrin autography. Nasal mucosa was obtained surgically from the inferior turbinate. Urokinase-type plasminogen activator (u-PA) specific staining was observed in pseudostratified ciliated epithelium and was predominant in mucous cells of the seromucinous gland, while serous cells were almost devoid of stain. The pattern of staining of plasminogen activator inhibitor-2 was similar to that of u-PA. In contrast, plasminogen activator inhibitor-1(PAI-1) immunoreactive material was localized exclusively in serous cells of seromucinous glands. Positive staining for tissue-type plasminogen activator (t-PA) was observed in endothelial cells and basal cells, which differentiate into either ciliated or goblet cells. Nasal secretions were partially fractionated by immunospecific antibody-immobilized Sepharose. Subsequent fibrin autography patterns indicated the presence of u-PA, PAI-1, and t-PA. After methacholine provocation, the level of t-PA increased transiently but decreased rapidly with subsequent challenges. These differential stainings of fibrinolytic components and the existence of PAs and PAI-1 in the nasal discharge suggest that the fibrinolytic system may play a role in the movement and fluidity of nasal secretion. Accepted: 25 May 1998     

Variations of components of the plasminogen activation system with the cell cycle in benign prostate tissue and prostate cancer.

Background: Components of the fibrinolytic system are involved in tumor cell invasion and metastasis. Previous investigations suggested a cell cycle-dependent expression of urokinase-type plasminogen activator (u-PA) in epithelial cells. In order to determine a correlation of cell cycle phases with the fibrinolytic system, we investigated the expression of u-PA, tissue-type plasminogen activator (t-PA), and plasminogen activator inhibitor type 1 (PAI-1) in normal and tumor-containing prostate extracts and analyzed a possible relationship with flow cytometry-determined proliferative activity of the samples. Cell cycle phases were correlated with fibrinolytic parameters in prostate tissue. Methods: Samples were obtained from patients undergoing radical prostatectomy for prostate cancer and separated into two portions for DNA analysis and the detection of u-PA, t-PA, and PAI-1. Flow cytometric analysis was performed according to the Vindelov technique. The concentrations of u-PA, t-PA, and PAI-1 were determined from tissue extracts after homogenization by an enzyme-linked immunosorbent assay (ELISA) technique. Results: Correlations of u-PA and t-PA expression with the frequency of G0/G1, S, G2M, S-phase fraction (SPF), and proliferation index (PI) for normal prostate and prostate cancer revealed no significant correlation. The only significant finding was observed in normal tissue revealing a positive correlation between PAI-1 expression and G0/G1 and a negative correlation with S-phase, SPF, and PI. No dependence of PAI-1 expression on different cell phases was found in prostate cancer. Furthermore, no significant correlation of u-PA, t-PA, and PAI-1 with cell cycles in organ-confined ( or = pT3a) tumors was found. No significant correlation in prostate cancer of components of the fibrinolytic system differentiated according to tumor grade or perineural tumor infiltration and cell cycle analysis was found. Only in highly differentiated G1 (Gleason 2-4) cancer, u-PA had a significant positive correlation with G2M-phase. Conclusion: Absence of a correlation between levels of components of the fibrinolytic system and cell cycle phases suggests that the reported association between increases of some of these components and aggressive biological behavior of prostate cancer is secondary to non-cell cycle-related mechanisms.     

Plasma levels of t-PA and PAI-1 correlate with the formation of experimental post-surgical peritoneal adhesions

This study has evaluated whether systemic changes of plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) parallel the adhesions development and whether they could be used as predictors of adhesion risk. This has been studied in an animal model of post-surgical peritoneal adhesion by monitoring for 10 days the plasma and tissue levels of t-PA and PAI-1. The results showed that both tissular and plasmatic levels of t-PA were decreased in concomitance with the development of peritoneal adhesions. In contrast, PAI-1 was found increased into the tissue and into the plasma samples of the rats taken at 5 and 10 days time points. Inflammatory mediators such as ICAM-1, VCAM-1, and IL-6 within the peritoneal lavage fluid also correlated with the adhesion formation process. In conclusion, post-surgical peritoneal adhesions provide alterations of local inflammatory components and local and systemic fibrinolytic components, possibly with PAI-1 quenching t-PA. This may have potential for the identification of high-risk patients.     

Epistatic effects of polymorphisms in genes from the renin-angiotensin, bradykinin, and fibrinolytic systems on plasma t-PA and PAI-1 levels

Tissue plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1) directly influence thrombus formation and degradation and thereby risk for arterial thrombosis. Activation of the renin-angiotensin system has been linked to the production of PAI-1 expression via the angiotensin II type 1 receptor (AT1R). In addition, bradykinin can induce the release of t-PA through a B2 receptor mechanism. In the present study, we aimed to investigate the epistatic effects of polymorphisms in genes from the renin-angiotensin, bradykinin, and fibrinolytic systems on plasma t-PA and PAI-1 levels in a large population-based sample (n=2527). We demonstrated a strong significant interaction within genetic variations of the bradykinin B2 gene (P=0.002) and between ACE and bradykinin B2 (p=0.003) polymorphisms on t-PA levels in females. In males, polymorphisms in the bradykinin B2 and AT1R gene showed the most strong effect on t-PA levels (P=0.006). In both females and males, the bradykinin B2 gene interacted with AT1R gene on plasma PAI-1 levels (P=0.026 and P=0.039, respectively). In addition, the current study found a borderline significant interaction between PAI 4G5G and ACE I/D on plasma t-PA and PAI-1 levels. These results support the idea that the interplay between the renin-angiotensin, bradykinin, and fibrinolytic systems might play an important role in t-PA and PAI-1 biology.     

Epistatic interactions in genetic regulation of t-PA and PAI-1 levels in a Ghanaian population

The proteins, tissue plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1), act in concert to balance thrombus formation and degradation, thereby modulating the development of arterial thrombosis and excessive bleeding. PAI-1 is upregulated by the renin-angiotensin system (RAS), specifically by angiotensin II, the product of angiotensin converting enzyme (ACE) cleavage of angiotensin I, which is produced by the cleavage of angiotensinogen (AGT) by renin (REN). ACE indirectly stimulates the release of t-PA which, in turn, activates the corresponding fibrinolytic system. Single polymorphisms in these pathways have been shown to significantly impact plasma levels of t-PA and PAI-1 differently in Ghanaian males and females. Here we explore the involvement of epistatic interactions between the same polymorphisms in central genes of the RAS and fibrinolytic systems on plasma t-PA and PAI-1 levels within the same population (n = 992). Statistical modeling of pairwise interactions was done using two-way ANOVA between polymorphisms in the ETNK2, RENIN, ACE, PAI-1, t-PA, and AGT genes. The most significant interactions that associated with t-PA levels were between the ETNK2 A6135G and the REN T9435C polymorphisms in females (p = 0.006) and the REN T9435C and the TPA I/D polymorphisms (p = 0.005) in males. The most significant interactions for PAI-1 levels were with REN T9435C and the TPA I/D polymorphisms (p = 0.001) in females, and the association of REN G6567T with the TPA I/D polymorphisms (p = 0.032) in males. Our results provide evidence for multiple genetic effects that may not be detected using single SNP analysis. Because t-PA and PAI-1 have been implicated in cardiovascular disease these results support the idea that the genetic architecture of cardiovascular disease is complex. Therefore, it is necessary to consider the relationship between interacting polymorphisms of pathway specific genes that predict t-PA and PAI-1 levels.     

Plasminogen activation by tissue plasminogen activator on fibrin clots with different surface structures

Transformation of fibrinogen into fibrin with consequent formation of the fibrin clot trimeric structure is one of the final steps in the blood coagulation system. The plasminogen activation by the tissue plasminogen activator (t-PA) is one of the fibrinolysis system key reactions. The effect of different factors on transformation of plasminogen into plasmin is capable to change essentially the equilibrium between coagulation and fibrinolytic sections of haemostasis system. We have studied the plasminogen activation by tissue plasminogen activator on fibrin clots surface formed on the interface between two phases and in presence of one phase. The t-PA plasminogen activation rate on fibrin clots both with film and without it the latter has been analyzed. These data allow to assume that the changes of fibrin clot structure depend on its formations, as well as are capable to influence essentially on plasminogen activation process by means of its tissue activating agent.     

Plasminogen activator inhibitor-type 1 in Lewis lung carcinoma

Plasminogen activator inhibitor-type 1 (PAI-1) was identified in extracts of Lewis lung carcinoma, and its immunohistochemical localization was studied together with that of urokinase-type (u-PA) and tissue-type (t-PA) plasminogen activators. All primary tumors (n = 11) contained heterogeneously distributed immunoreactivity against each of the three components. Most often, areas that contained u-PA immunoreactivity also contained PAI-1 immunoreactivity. However, several areas showed a strong u-PA immunoreactivity, but no or low PAI-1 immunoreactivity. The latter staining pattern was only found in peripheral areas, and usually in areas with histological signs of tissue destruction. Lung metastases always contained u-PA immunoreactivity, while PAI-1 immunoreactivity was found in most, but not all, metastases. t-PA immunoreactivity was found in a few scattered tumor cells, in primary carcinomas as well as metastases. Controls that included absorption with highly purified antigen preparations and immunoblotting, indicated that all the immunoreactivity represented genuine PAI-1, u-PA and t-PA, respectively. The results are consistent with an assumption that the plasminogen activation system, and particularly u-PA and PAI-1, plays a role in regulation of breakdown of extracellular matrix proteins during invasive growth in this carcinoma.     

Plasminogen activator inhibitor-type 1 in Lewis lung carcinoma

Summary Plasminogen activator inhibitor-type 1 (PAI-1) was identified in extracts of Lewis lung carcinoma, and its immunohistochemical localization was studied together with that of urokinase-type (u-PA) and tissue-type (t-PA) plasminogen activators. All primary tumors (n=11) contained heterogeneously distributed immunoreactivity against each of the three components. Most often, areas that contained u-PA immunoreactivity also contained PAI-1 immunoreactivity. However, several areas showed a strong u-PA immunoreactivity, but no or low PAI-1 immunoreactivity. The latter staining pattern was only found in periferal areas, and usually in areas with histological signs of tissue destruction. Lung metastases always contained u-PA immunoreactivity, while PAI-1 immunoreactivity was found in most, but not all, metastases. t-PA immunoreactivity was found in a few scattered tumor cells, in primary carcinomas as well as metastases. Controls that included absorption with highly purified antigen preparations and immunoblotting, indicated that all the immunoreactivity represented genuine PAI-1, u-PA and t-PA, respectively. The results are consistent with an assumption that the plasminogen activation system, and particularly u-PA and PAI-1, plays a role in regulation of breakdown of extracellular matrix proteins during invasive growth in this carcinoma.     

IL-8 induces imbalances between nitric oxide and endothelin-1, and also between plasminogen activator inhibitor-1 and tissue-type plasminogen activator in cultured endothelial cells

Interleukin-8 (IL-8), a member of the CXC chemokine family, plays an important role in the modulation of multiple biological functions in endothelial cells containing the receptors CXCR1 and CXCR2. It has previously been shown that IL-8 directly enhances endothelial cell survival, and stimulates the production of matrix metalloproteinases, which in turn regulates angiogenesis. However, its role in the regulation of the production of vasoactive substances in endothelial cells is less well defined. In this study, we investigate the effects of IL-8 on the proliferation of human umbilical vein endothelial cells (HUVECs). In addition, we also study the effects of IL-8 on the production of vasodilator, vasoconstrictor and fibrinolytic factors in these cells. The results show that recombinant IL-8 (50-200ng/ml) induces neither HUVEC proliferation nor nitric oxide (NO) release. However, it significantly increases the production of endothelin-1 (ET-1) in a concentration-dependent manner. Furthermore, incubation of endothelial cells with IL-8 (200ng/ml) up-regulates the plasminogen activator inhibitor-1 (PAI-1) in HUVECs, while it down-regulates the tissue plasminogen activator (t-PA). These findings suggest that IL-8 offsets the balance between endothelial vasoconstrictors and vasodilators. Furthermore, IL-8 also leads to an imbalance between PAI-1 and t-PA, which causes the ECs to become procoagulative and hypofibrinolytic.     

Characterization of interaction of active-site serine mutants of tissue-type plasminogen activator with plasminogen activator inhibitor-1

To define determinants of interactions of tissue-type plasminogen activator (t-PA) with plasminogen activator inhibitor type-1 (PAI-1), we utilized site-directed mutagenesis to substitute either threonine or glycine for the active-site serine of tissue-type plasminogen activator. Assays of conditioned media of transfected cells demonstrated that the threonine substitution markedly decreased but did not entirely abolish plasminogen activating activity. In contrast, the glycine substitution yielded a mutant with absolutely no detectable plasminogen activating activity. Wild-type t-PA formed stable complexes with PAI-1. However, even when exogenous inhibitor was present in the medium or purified mutant was added to plasma that had been rendered PAI-1-rich in vivo, the mutants were present in the free form exclusively judging from results of fibrin autography and Western blot analysis. Thus, despite maintenance of some residual plasminogen-activating activity associated with preservation of the hydroxyl group at the active site, the threonine mutant did not form stable complexes with inhibitor. The glycine mutant, developed so that steric hindrance or other unfavorable interactions at the modified active site would be minimal, was similarly incapable of forming complexes with PAI-1. These results show that the presence of an active site serine residue is necessary for formation of stable complexes between t-PA and PAI-1.     

Vitronectin governs the interaction between plasminogen activator inhibitor 1 and tissue-type plasminogen activator.

The "serpin" plasminogen activator inhibitor 1 (PAI-1) is the fast acting inhibitor of plasminogen activators (tissue-type (t-PA) and urokinase type-PA) and is an essential regulatory protein of the fibrinolytic system. Its P1-P1' reactive center (R346 M347) acts as a "bait" for tight binding to t-PA/urokinase-type PA. In vivo, PAI-1 is encountered in complex with vitronectin, an interaction known to stabilize its activity but not to affect the second-order association rate constant (k1) between PAI-1 and t-PA. Nevertheless, by using PAI-1 reactive site variants (R346M, M347S, and R346M M347S), we show that the binding of vitronectin to the PAI-1 mutant proteins improves plasminogen activator inhibition. In the absence of vitronectin the PAI-1 R346M mutants are virtually inactive toward t-PA (k1 less than 1 x 10(3) M-1 s-1). In contrast, in the presence of vitronectin the rate of association increases about 1,000-fold (k1 of 6-8 x 10(5) M-1 s-1). This inhibition coincides with the formation of serpin-typical, sodium dodecyl sulfide-stable t-PA.PAI-1 R346M (R346M M347S) complexes. As evidenced by amino acid sequence analysis, the newly created M346-M/S347 peptide bond is susceptible to attack by t-PA, similar to the wild-type R346-M347 peptide bond, indicating that in the presence of vitronectin M346 functions as an efficient P1 residue. In addition, we show that the inhibition of t-PA and urokinase-type PA by PAI-1 mutant proteins is accelerated by the presence of the nonprotease A chains of the plasminogen activators.     

Plasminogen activator inhibitor (type-1) in rat adrenal medulla

Plasminogen activator inhibitor type-1 (PAI-1) was identified in extracts of rat adrenal medulla, and its immunohistochemical localization was studied together with that of tissue-type plasminogen activator (t-PA). By staining of adjacent sections and by double-staining of the same section we demonstrate that the same cells of the adrenal medulla contain both PAI-1 and t-PA immunoreactivity in the cytoplasm. In addition a few ganglion cells of the adrenal medulla were found to contain PAI-1 but not t-PA. Neither of the components were found in the adrenal cortex. Analysis of extracts from isolated adrenal medulla using reverse zymography showed the presence of a plasminogen activator inhibitor with Mr approximately 46,000. The inhibitory activity disappeared when the extract was passed through a column with sepharose-coupled anti-PAI-1 IgG, while the run-through from a similar column coupled with preimmune IgG still contained the inhibitor. The present findings suggest that PAI-1 could play a role in the regulation of t-PA activity in the rat adrenal gland medullary cells.     

Fibrinolytic activity is not dependent upon exercise mode in post-myocardial infarction patients

In this study we investigated possible differences in fibrinolytic activity in cardiac patients while they performed treadmill and cycle ergometry. Thirteen post-myocardial infarction patients completed two maximal exercise tests on treadmill and cycle ergometers. Blood was collected before and after each exercise test and was analyzed for the fibrinolytic variables, tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) activity, and lactate. Maximal oxygen uptake, heart rate, and ventilation were greater (P < 0.05) on the treadmill than during cycle ergometry, however, blood lactate was similar between modes. t-PA activity significantly increased with exercise (P < 0.05) and there was a trend toward a reduction in PAI-1 activity with exercise, but this did not reach statistical significance. The fibrinolytic responses to maximal exercise did not differ between the two modes of exercise studied. Therefore, exercise intensity, but not the mode of exercise, appeared to be the primary determinant of the fibrinolytic response to acute exercise in these patients. Accepted: 29 January 1998     

Profibrinolytic Effect of the Epigenetic Modifier Valproic Acid in Man

Aims

The aim of the study was to test if pharmacological intervention by valproic acid (VPA) treatment can modulate the fibrinolytic system in man, by means of increased acute release capacity of tissue plasminogen activator (t-PA) as well as an altered t-PA/Plasminogen activator inhibitor -1 (PAI-1) balance. Recent data from in vitro research demonstrate that the fibrinolytic system is epigenetically regulated mainly by histone deacetylase (HDAC) inhibitors. HDAC inhibitors, including VPA markedly upregulate t-PA gene expression in vitro.

Methods and Results

The trial had a cross-over design where healthy men (n = 10), were treated with VPA (Ergenyl Retard) 500 mg depot tablets twice daily for 2 weeks. Capacity for stimulated t-PA release was assessed in the perfused-forearm model using intra-brachial Substance P infusion and venous occlusion plethysmography. Each subject was investigated twice, untreated and after VPA treatment, with 5 weeks wash-out in-between. VPA treatment resulted in considerably decreased levels of circulating PAI-1 antigen from 22.2 (4.6) to 10.8 (2.1) ng/ml (p<0.05). It slightly decreased the levels of circulating venous t-PA antigen (p<0.05), and the t-PA:PAI-1 antigen ratio increased (p<0.01). Substance P infusion resulted in an increase in forearm blood flow (FBF) on both occasions (p<0.0001 for both). The acute t-PA release in response to Substance P was not affected by VPA (p = ns).

Conclusion

Valproic acid treatment lowers plasma PAI-1 antigen levels and changes the fibrinolytic balance measured as t-PA/PAI-1 ratio in a profibrinolytic direction. This may in part explain the reduction in incidence of myocardial infarctions by VPA treatment observed in recent pharmacoepidemiological studies.

Trial Registration

The EU Clinical Trials Register 2009-011723-31     

The effects of polymorphisms in genes from the renin–angiotensin, bradykinin, and fibrinolytic systems on plasma t-PA and PAI-1 levels are dependent on environmental context

Thrombosis is a key factor in the pathophysiology of cardiovascular disease. Important biochemical constituents of the fibrinolytic system, affecting thrombosis, include tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1). Both t-PA and PAI-1 are determined by multiple genetic and environmental factors. We aimed to investigate whether the effects of polymorphism in genes from the renin–angiotensin, bradykinin, and fibrinolytic systems on t-PA or PAI-1 levels are dependent on environmental factors in a large population-based sample from the PREVEND study in Groningen, The Netherlands (n = 2,527). We found strong evidence (P ≤ 0.02) for interaction effects of polymorphisms from the bradykinin receptor (BDKRB2) gene and alcohol consumption on t-PA in females and males and on PAI-1 in males. Only suggestive evidence (P ≤ 0.10) was present for an interaction effect of the BDKRB2 gene and alcohol consumption on PAI-1 levels in females. Another consistent finding was evidence for an interaction between bradykinin receptor (BDKRB2) gene polymorphisms and body size as measured by body mass index and/or waist–hip-ratio. For each gender and for both t-PA and PAI-1 there was at least one BDKRB2–body size combination that exhibited suggestive (P ≤ 0.10), significant (P ≤ 0.04) and/or strong evidence (P ≤ 0.02) for interaction. In conclusion, the genetic architecture of t-PA and PAI-1 is dependent on the environmental context such as body size and alcohol use. The present study emphasizes the importance of including environmental factors in genetic analyses to fully comprehend the genetic architecture of a specific trait. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Restoration of serine protease-inhibitor interaction by protein engineering

Tissue-type plasminogen activator (t-PA) catalyzes the rate-limiting step in the fibrinolytic cascade: conversion of plasminogen to plasmin. Plasma contains several inhibitors of t-PA that limit its activity and prevent systemic activation of plasminogen. The most important of these is endothelial cell plasminogen activator inhibitor (PAI-1), a member of the serine protease inhibitor (serpin) gene family. We have previously demonstrated that mutation of arginine 304 of t-PA to a glutamic acid residue drastically reduces the rate of interaction between the enzyme and its suicide substrate, PAI-1, without affecting the reactivity of the enzyme toward its normal substrate, plasminogen (Madison, E. L., Goldsmith, E. J., Gerard, R.D., Gething, M.J., and Sambrook, J.F. (1989) Nature 339, 721-724). We report here the use of protein modeling to design a compensatory mutation in PAI-1 (glutamic acid 350 to arginine) and create a molecule that rapidly inhibits this "serpin-resistant" variant of t-PA.