Plasminogen Activator Inhibitor Type 1 Activity Research Articles

Eccentric, but not concentric muscle contraction induce inflammation and impairs fibrinolysis in healthy young men.

Different types of muscle contraction can cause different damage to the musculature and differences in inflammatory responses. Acute increases in circulatory inflammation markers can influence the crosstalk between coagulation and fibrinolysis processes, increasing the risk of thrombus formation and detrimental cardiovascular events. The aim of this study was to analyze the effects of concentric and eccentric exercise on hemostasis markers, C-reactive protein (CRP), and the relationship between these variables. Eleven healthy subjects with a mean age of 25.4±2.8, non-smokers, with no history of cardiovascular disease and blood type O, randomly performed an isokinetic exercise protocol consisting of 75 concentric (CP) or eccentric (EP) contractions of knee extension, divided into five sets of 15 repetitions combined with 30-s rest. Blood samples for analysis of FVIII, von Willebrand factor, tissue plasminogen activator (t-PA), plasminogen activator inhibitor type-1 (PAI-1), and CRP were collected pre, post, 24 h, and 48 h after each protocol. Increased levels of CRP at 48 h in EP versus CP (p=0.002), increased PAI-1 activity 48 h in EP versus CP (p=0.044), and a reduction in t-PA at 48 h when compared with post-protocol in both protocols (p=0.001). A correlation was found between CRP and PAI-1 at 48 h of PE (r2=0.69; p=0.02). This study showed that both EP and CP increase the clotting process, albeit only the exercise performed eccentrically induces inhibition of fibrinolysis. This is possibly due to the increase in PAI-1 48 h after the protocol, which correlates with the increase in inflammation as demonstrated by the CRP levels.

Tissue Plasminogen Activator as a Possible Indicator of Breast Cancer Relapse: A Preliminary, Prospective Study.

Simple SummaryBreast cancer is the most common malignant neoplasm and the leading cause of cancer death in women worldwide. The heterogeneity of breast cancer is a significant challenge facing modern medicine, especially at the stage of diagnosis. In recent years, a substantial role of fibrinolytic biomarkers in the pathogenesis of breast cancer has been proved as the primary cause capable of initiating local recurrence and metastasis to other organs. Due to its proteolytic properties, fibrinolytic protein activity may lead to subendothelial layer degradation and promote neoplastic cell motility. Thus, we examined the fibrinolytic profile markers’ prognostic value in predicting the disease’s relapse. Our studies indicate that the t-PA antigen and PAI-1 activity are biomarkers with high prognostic potential. We showed that a lower baseline plasma concentration of t-PA antigen and higher PAI-1 activity might be strong predictors for distant metastases and independent prognostic markers in breast cancer patients.(1) Background: The fundamental causes of breast cancer mortality are the cancer spread and hypercoagulability state. The study aimed to evaluate the prognostic efficacy of the fibrinolytic profile concerning 5-year follow-up. (2) Methods: We investigated the predictive potential of the plasma activity of urokinase plasminogen activator (u-PA) and plasminogen activator inhibitor type 1 (PAI-1) as well as antigen of tissue plasminogen activator (t-PA), u-PA, PAI-1, and PAI-1/t-PA and PAI-1/u-PA complexes in 41 breast cancer patients. The median follow-up was 66 months, with full evidence of the first event. (3) Results: A significantly lower level of PAI-1 antigen was noted in IBrC patients with lymph node involvement (N1) than in patients with free lymph node metastases (N0). According to ROC curve analysis, a t-PA antigen was the strongest predictor of disease relapse (the area under the curve, AUC = 0.799; p < 0.0006). Patients with PAI-1 activity < 3.04 U/mL had significantly better disease-free survival (DFS) compared to those with PAI-1 activity > 3.04 U/mL. Patients with both t-PA antigen lower than 1.41 ng/mL (cut-off according to median value) and lower than 1.37 ng/mL (cut-off according to ROC curve) had significantly shorter DFS (p = 0.0086; p = 0.0029). (4) Conclusions: The results suggest that a higher plasma t-PA antigen level or lower PAI-1 activity are linked to better outcomes in breast cancer patients.

Plasminogen Activator Inhibitor Type 1 (PAI-1) and Thrombophilia: A 10 Year Retrospective Review of Clinical Data at a Large Children's Hospital

Introduction: Plasminogen activator inhibitor type 1 (PAI-1) is the most important regulator of fibrinolysis. Elevated PAI-1 levels are reported to be prothrombotic and have been associated with cardiovascular disease, obesity and cancer. PAI-1 synthesis is regulated by insulin, glucocorticoids and cytokines like tumor necrosis factor-α. The single base pair guanine deletion 4G PAI-1 genetic polymorphism has been linked to elevation of PAI-1 antigen and/or activity levels. Genotype frequencies in Caucasian populations have been estimated at 0.25 (5G/5G), 0.49 (4G/5G) and 0.26 (4G/4G). Increased thrombosis risk has been reported in patients with the combination of PAI-1 4G and factor V Leiden (FVL) genetic polymorphisms. Our pediatric specialty laboratory offers PAI-1 genotyping, PAI-1 antigen and PAI-1 activity assays as part of thrombophilia testing. The laboratory at the children's hospital is also a reference lab for the community. We retrospectively reviewed the results of PAI-1 testing over the last 10 years.We hypothesized that the PAI-1 4G polymorphism would be more prevalent in young patients with thrombosis. We also hypothesized that there would be an additive thrombophilia risk from the combination of PAI-1 4G and FVL in young patients with thrombosis.Methods: After IRB approval, using information from Cerner and EPIC, we created a deidentified thrombophilia database of 11,459 unique patients who had thrombophilia testing between March 8, 2007 and June 6, 2017. We identified 1,896 who had PAI-1 genotyping and 2,573 who had antigen and activity testing. Removing those <3 months of age for whom normal ranges for PAI-1 have not been established, we identified 1,418 subjects who had genotype, antigen and activity levels. We also evaluated a subset of 510 pediatric patients with thrombosis, of whom 338 had all three PAI-1 tests and were 3 months or older, and 292 who had both PAI-1 and FVL genotyping. Local genotype frequency was established by the clinical laboratory on 100 controls and was in agreement with published estimates. All comparisons were made using two-sided Fisher's Exact tests. Significance was assessed at the 0.05 level.Results: Ages for subjects tested for PAI-1 ranged from 0.1 - 89.7 years with a mean of 14.3 years, and 41% were male. Compared to the local genotype frequency, PAI-1 4G was not significantly altered in either the thrombophilia cohort or in the subset with pediatric thrombosis (Table 1). This was true even when we re-evaluated by gender or by selecting for those with thrombophilia testing <22 years of age. Among 53 pediatric thrombosis patients with cancer, genotype frequencies for PAI-1 4G did not differ strongly from control group (p=0.14), but numbers were very small. Of the 1,418 thrombophilia testing subjects who had PAI-1 genotype, antigen and activity, correlation between abnormal antigen (p=0.07) or activity (p=0.28) levels and the 4G polymorphism did not reach statistical significance, though it did for those with both abnormal antigen and activity (p=0.05). Among 292 pediatric thrombosis patients with testing (Table 2), there was a suggestion of an effect from the combination of FVL and PAI-1 4G polymorphisms and thrombosis (p=0.06).Conclusions: Abnormal PAI-1 antigen and activity levels are not solely dependent upon the 4G genotype and are likely influenced by other congenital and acquired factors. Among pediatric patients with thrombosis and particularly those with cancer and thrombosis, a possible effect from the PAI-1 4G polymorphism alone or in combination of FVL should be validated in a larger cohort. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Plasminogen activator inhibitor type-1 is a negative regulator of hematopoietic regeneration in the adipocyte-rich bone marrow microenvironment

Bone marrow adipocytes (BMAs) have recently been recognized as a niche component with a suppressive function. Obese individuals with abundant BMAs exhibit impaired hematopoietic regeneration after hematopoietic stem cell transplantation (HSCT). We hypothesized that plasminogen activator inhibitor type-1 (PAI-1), an adipokine that regulates the fibrinolytic system, contributes to impaired hematopoiesis in bone marrow (BM) microenvironment with abundant BMAs. We demonstrated that BMAs differentiated in vitro could secrete PAI-1 and were positive for PAI-1 in vivo. In addition, the abundance of BMAs was associated with high levels of PAI-1 expression. The BMA-rich microenvironment exhibited impaired hematopoietic regeneration after HSCT when compared with a BMA-less microenvironment. The impaired hematopoietic regeneration in BMA-rich microenvironment was significantly alleviated by PAI-1 knockout or PAI-1 inhibitor treatment. Obese mice with abundant BMAs, compared with normal-weight mice, exhibited higher bone marrow PAI-1 concentrations, increased fibrinolytic system suppression, and lower stem cell factor (SCF) concentrations after HSCT. PAI-1 inhibitor administration significantly activated the fibrinolytic system in obese mice, contributing to the higher SCF concentration. Moreover, PAI-1 inhibitor treatment significantly alleviated the impaired hematopoietic regeneration in obese mice both after 5-fluorouracil injection and HSCT. These results indicate that PAI-1 hinders hematopoietic regeneration in BMA-rich microenvironments. The blockade of PAI-1 activity could be a novel therapeutic means of facilitating hematopoietic reconstitution in BMA-rich patients.

Effect of water-extractive components from funazushi, a fermented crucian carp, on the activity of fibrinolytic factors.

Japanese fermented foods, including funazushi, have have been studied insufficiently. Related research into fermented products has led to the hope that they might have positive effects on blood circulation, including anti-thrombosis effects. The possible antithrombotic effects of funazushi on the fibrinolytic system were examined. The administration of extracts from funazushi increased the activity of plasmin and tissue plasminogen activators in the fibrinolytic system but decreased the activity of plasminogen activator inhibitor type-1 (PAI-1). This decrease was positively correlated with the decreased plasma triglyceride levels. Funazushi extract directly inhibited PAI-1 activity in vitro despite alimentary enzyme digestion, although direct PAI-1 inhibition was not observed in an extract from salted crucian carp. These results suggest that funazushi extracts are closely involved in the antithrombotic effects of the fibrinolytic system, and that they exert their effect through a reduction in PAI-1 activity. The findings also indicate that fermentation processing is necessary to achieve the antithrombotic effects of funazushi. © 2020 Society of Chemical Industry.

Effects of 17β-Estradiol on the Plasminogen Activator System in Vascular Smooth Muscle Cells Treated with Lysophophatidylcholine.

ObjectivesWhen administered soon after menopause, hormone therapy can prevent coronary heart diseases in women. To explore the mechanism underlying the cardioprotective actions of estrogen, we investigated the effects of 17β-estradiol (17β-E2) on the plasminogen activator system using cultured vascular smooth muscle cells (VSMCs).MethodsVSMCs were isolated from rat aortas. Protein expression of plasminogen activator inhibitor type 1 (PAI-1) and tissue-type plasminogen activator (t-PA) were evaluated using Western blotting and enzyme-linked immunosorbent assay, respectively. The enzyme activity of PAI-1 in a conditioned medium was assessed via reverse fibrin overlay zymography and that of t-PA was assessed via fibrin overlay zymography. Gene expression was quantified using real-time reverse transcription-polymerase chain reaction.ResultsFollowing pre-treatment for 24 hours, 17β-E2 suppressed both protein expression and enzyme activity of PAI-1 stimulated by lysophosphatidylcholine (lysoPC) in a significant and dose-dependent manner at a near physiological concentration. Moreover, 17β-E2 (10−7 M) inhibited PAI-1 gene expression, and ICI 182,780—a specific estrogen receptor antagonist—blocked the effects of 17β-E2 on the PAI-1 protein. 17β-E2 did not affect t-PA secretion but significantly enhanced free t-PA activity through reduced binding to PAI-1. Furthermore, 17β-E2 suppressed intracellular reactive oxygen species production and nuclear factor-κB-mediated transcription.ConclusionsIn VSMCs stimulated with lysoPC, 17β-E2 reduced PAI-1 expression through a non-receptor-mediated mechanism via antioxidant activity as well as a receptor-mediated mechanism; however, it did not alter t-PA secretion. Of note, 17β-E2 suppressed PAI-1 activity and concurrently enhanced t-PA activity, suggesting a beneficial influence on fibrinolysis.

Bone Marrow Adipocyte-Derived PAI-1 As a Negative Regulator of Hematopoietic Regeneration

Introduction: Bone marrow adipocytes (BMAs) reportedly act as negative regulators of hematopoietic regeneration. This can be observed in obese or elderly patients, both of whom exhibit BMA-rich microenvironment with poor hematopoietic regeneration. Though this relationship is known, the mechanism(s) underlying the suppressive effects of BMAs on hematopoietic regulation are yet to be elucidated. Recently, in our previous study, we demonstrated that blocking plasminogen activator inhibitor type-1 (PAI-1) enhances hematopoietic regeneration through the activation of the fibrinolytic pathways in the bone marrow. As PAI-1 is known as one of the major adipokines, we reasoned that PAI-1 produced from BMAs could have a causative role in the impairment of hematopoietic regeneration in the BMA-rich microenvironment. Methods: C57B6/J and PAI-1-deficient mice with a C57B6/J background were used. Diet-induced obesity (DIO) C57B6/J mice were fed a 60 kcal% fat diet for 12 weeks prior to performing experiments. After syngeneic hematopoietic stem cell transplantation (HSCT), either TM5614 (a specific inhibitor of PAI-1 molecules) or vehicle (distilled water) were administered daily via oral gavage using a feeding needle for 14 consecutive days. Results: We compared the hematopoietic regeneration after syngeneic HSCT between femurs (microenvironment with a low BMA content) and tail vertebrae (which consist of abundant BMAs). As expected, the microenvironment in tail vertebrae was significantly associated with higher PAI-1 expression in comparison with that in femurs. Hematopoietic regeneration was less efficient in the tail vertebrae, with a reduced number of total donor hematopoietic and CD34-Lin-Sca1+c-kit+ cells observed than in femurs at 1 and 4 weeks following syngeneic HSCT. This impaired regeneration was significantly alleviated in PAI-1-knockout (Figure a) and PAI-1 inhibitor-treated mice (Figure b). Following this, we examined the effects of PAI-1 inhibitor on hematopoietic regeneration after syngeneic HSCT in DIO mice, which had BMA-rich bone marrow microenvironment associated with higher PAI-1 expression compared with that in normal-weight mice. The comparable homing activity of donor cell was observed in normal-weight and DIO mice by using PKH26 staining. However, DIO mice, in comparison to normal-weight mice, exhibited impaired hematopoietic regeneration, with a reduced number of total donor hematopoietic, CD34-Lin-Sca1+c-kit+, and CD48-CD150+Lin-Sca1+c-kit+ SLAM cells at 1 week following HSCT. The impaired hematopoietic regeneration was completely restored by PAI-1 inhibitor administration in these mice (Figure c and d). Furthermore, the number of total donor hematopoietic and CD34-Lin-Sca1+c-kit+ cells at 3 weeks following HSCT was significantly higher in PAI-1 inhibitor treated DIO mice than in normal-weight mice. Conclusion: Our results indicated that PAI-1 hampers hematopoietic regeneration in the BMA-rich microenvironment and the blockade of PAI-1 activity could be a novel therapeutic approach to facilitate hematopoietic regeneration after HSCT in BMA-rich patients. Figure Disclosures Onizuka: Sumitomo Dainippon Pharma: Research Funding; Astellas: Research Funding; Novartis: Research Funding; pfizer: Research Funding; Chugai Pharma: Research Funding; Bristol-Myers Squibb: Research Funding. Ando:Eisai: Research Funding.

Upregulation of P2Y2R, Active uPA, and PAI-1 Are Essential Components of Hantavirus Cardiopulmonary Syndrome.

Sin Nombre virus (SNV) causes hantavirus cardiopulmonary pulmonary syndrome (HCPS) with the loss of pulmonary vascular endothelial integrity, and pulmonary edema without causing cytopathic effects on the vascular endothelium. HCPS is associated primarily with a dysregulated immune response. We previously found occult signs of hemostatic imbalance in the form of a sharp >30–100 fold increase in the expression of plasminogen activator inhibitor type 1 (PAI-1), in serial blood plasma draws of terminal stage-patients. However, the mechanism of the increase in PAI-1 remains unclear. PAI-1 is a primary inhibitor of fibrinolysis caused by tissue plasminogen activator (tPA) and urokinase plasminogen activator plasma (uPA). Here, we investigate factors that contribute to PAI-1 upregulation during HCPS. Using zymography, we found evidence of PAI-1-refractory uPA activity and no tPA activity in plasma samples drawn from HCPS patients. The sole prevalence of uPA activity suggested that severe inflammation drove PAI-1 activity. We have recently reported that the P2Y2 receptor (P2Y2R) mediates SNV infectivity by interacting in cis with β3 integrins, which activates the latter during infection. P2Y2R is a known effector for several biological processes relevant to HCPS pathogenesis, such as upregulation of tissue factor (TF), a primary initiator of the coagulation cascade, stimulating vascular permeability and leukocyte homing to sites of infection. As P2Y2R is prone to upregulation under conditions of inflammation, we compared the expression level of P2Y2R in formalin fixed tissues of HCPS decedents using a TaqMan assay and immunohistochemistry. Our TaqMan results show that the expression of P2Y2R is upregulated significantly in HCPS cases compared to non- HCPS controls (P < 0.001). Immunohistochemistry showed that lung macrophages were the primary reservoir of high and coincident localization of P2Y2R, uPA, PAI-1, and TF antigens. We also observed increased staining for SNV antigens in the same tissue segments where P2Y2R expression was upregulated. Conversely, sections of low P2Y2R expression showed weak manifestations of macrophages, SNV, PAI-1, and TF. Coincident localization of P2Y2R and PAI-1 on macrophage deposits suggests an inflammation-dependent mechanism of increasing pro-coagulant activity in HCPS in the absence of tissue injury.

The Role of Plasminogen Activator Inhibitor Type-1 in Fibrosis.

Extracellular matrix (ECM) deposition during wound healing is a physiological response to an insult. Wound healing becomes deregulated in the setting of chronic injury or long-standing metabolic disease, leading to the accumulation of ECM components and fibrosis. Matrix protein turnover is determined by the rate of synthesis as well as the rate of proteolytic degradation and clearance by matrix metalloproteinases (MMPs). The persistent activation of interstitial myofibroblasts, coupled with defects in matrix proteolysis, ultimately disrupts tissue architecture and leads to biochemical and mechanical organ dysfunction with eventual organ failure. Plasminogen activator inhibitor type-1 (PAI-1) regulates tissue homeostasis and wound healing by inhibiting plasmin-mediated MMP activation. Multiple reports using models of liver, lung, and kidney fibrosis suggest that PAI-1 deficiency or inhibition of PAI-1 activity attenuates fibrosis. The disinhibition of plasmin-mediated MMP activation leads to collagen degradation and its diminished accumulation, resulting in the reduction of fibrotic matrix deposition in these organs. Paradoxically, homozygous deficiency of PAI-1 promotes age-dependent spontaneous cardiac fibrosis, suggesting a protective role for PAI-1 in the heart. It remains unclear whether PAI-1-deficient cardiac fibroblasts have increased proliferative, migratory, or differentiation capabilities, that allow them to overcome increased plasmin and MMP activity and matrix clearance. In this review, we examine the specific roles of PAI-1 in fibrosis of different organs including the lung, liver, kidney, and cardiovascular system.

Copper(II) Ions Increase Plasminogen Activator Inhibitor Type 1 Dynamics in Key Structural Regions That Govern Stability.

Plasminogen activator inhibitor type 1 (PAI-1) regulates the fibrinolysis pathway by inhibiting the protease activity of plasminogen activators. PAI-1 works in concert with vitronectin (VN), an extracellular protein that aids in localization of active PAI-1 to tissues. The Peterson laboratory demonstrated that Cu(II) and other transition metals modulate the stability of PAI-1, exhibiting effects that are dependent on the presence or absence of the somatomedin B (SMB) domain of VN. The study presented here dissects the changes in molecular dynamics underlying the destabilizing effects of Cu(II) on PAI-1. We utilize backbone amide hydrogen/deuterium exchange monitored by mass spectrometry to assess PAI-1 dynamics in the presence and absence of Cu(II) ions with and without the SMB domain of VN. We show that Cu(II) produces an increase in dynamics in regions important for the function and overall stability of PAI-1, while the SMB domain elicits virtually the opposite effect. A mutant form of PAI-1 lacking two N-terminal histidine residues at positions 2 and 3 exhibits similar increases in dynamics upon Cu(II) binding compared to that of active wild-type PAI-1, indicating that the observed structural effects are not a result of coordination of Cu(II) to these histidine residues. Finally, addition of Cu(II) results in an acceleration of the local unfolding kinetics of PAI-1 presumed to be on pathway to the latency conversion. The effect of ligands on the dynamics of PAI-1 adds another intriguing dimension to the mechanisms for regulation of PAI-1 stability and function.

Bidirectional functions of thrombin on fibrinolysis: Evidence of thrombin-dependent enhancement of fibrinolysis provided by spontaneous plasma clot lysis

Besides procoagulant activity, thrombin exhibits anticoagulant and profibrinolytic activities. We demonstrated that the euglobulin clot lysis time (ECLT) was shortened by endogenously generated thrombin as a result of the inactivation of plasminogen activator inhibitor type 1 (PAI-1). In contrast, thrombin suppressed fibrinolytic activity through the activation of thrombin activatable fibrinolysis inhibitor (TAFI). Here, using three different clot lysis assays of the ECLT, the tissue plasminogen activator supplemented plasma clot lysis time (tPA-PCLT) and the spontaneous plasma clot lysis time (s-PCLT), we analyzed how the coagulation process modifies fibrinolysis. The ECLT was shortened by exogenously supplemented thrombin in a dose-dependent manner in the absence of calcium ion (Ca++), whereas this shortening was not observed in the presence of Ca++ where endogenous prothrombin was effectively activated to thrombin. This shortening was also not observed for the tPA-PCLT, in which tPA is supplemented in excess and PAI-1 activity is mostly lost. On the contrary, thrombin dose-dependently prolonged the tPA-PCLT, which was mostly abolished by inhibitors of carboxypeptidase and activated FXIII, suggesting that the prolongation is TAFI- and Factor XIII-dependent. The s-PCLT was shortened when thrombin generation was boosted by supplementing tissue factor and phosphatidylserine together with Ca++, which was more apparent in the presence of inhibitors of activated FXIII and activated TAFI. Thus, thrombin appeared to express its enhancing effect on fibrinolysis even in plasma, in addition to its inhibiting effect. These bidirectional functions of thrombin on fibrinolysis seem to take place on demand under different environments to maintain adequate vascular blood flow.

Inhibitory effect of water extractive components from heshiko and narezushi on plasma PAI-1 activity in rats

The effect of water-extractive components solutions (extracts) from heshiko and narezushi, fermented mackerel products, on plasminogen activator inhibitor type-1 (PAI-1) was investigated in rats. Administration of heshiko and narezushi extracts tended to suppress the increase in both PAI-1 activity and total content associated with high-fat diet administration in rats. PAI-1 activity but not total content showed a relation to plasma triglyceride level and a reverse relation to tPA activity before and after administration of heshiko and narezushi extracts. The extracts directly inhibited PAI-1 activity in vitro, and the inhibitory activity was not lost after alimentary enzyme digestion of the extracts. The PAI-1 inhibitory activity of heshiko extract was tended to be stronger than that of narezushi extract. These results suggest that heshiko and narezushi extracts are closely involved in the promotion of fibrinolytic activity via direct PAI-1 inhibition as well as indirect suppression by decreasing the plasma triglyceride level.

Decreased expression of the plasminogen activator inhibitor type 1 is involved in degradation of extracellular matrix surrounding cervical cancer stem cells.

The plasminogen activator (PA) system consists of plasminogen activator inhibitor type 1 (PAI-1), urokinase-type plasminogen activator and its receptor (uPA and uPAR). PAI-1 inhibits the activation of uPA (which converts plasminogen to plasmin), and is involved in cancer invasion and metastasis, by remodeling the extracellular matrix (ECM) through regulating plasmin. Cancer stem cells (CSCs) are a small subset of cells within tumors, and are thought to be involved in tumor recurrence and metastasis. Considering these facts, we investigated the relationship between PAI-1 and cervical CSCs. We used ALDH1 as a marker of cervical CSCs. First, we demonstrated that culturing ALDH1-high cells and ALDH-low cells on collagen IV-coted plates increased their expression of active PAI-1 (ELISA), and these increases were suggested to be at mRNA expression levels (RT-qPCR). Secondly, we demonstrated PAI-1 was indeed involved in the ECM maintenance. With gelatin zymography assays, we found that ALDH1-high cells and ALDH-low cells expressed pro-matrix metalloproteinase-2 (pro-MMP-2) irrespective of their coatings. With gelatinase/collagenase assay kit, we confirmed that collagenase activity was increased when ALDH1-low cells were exposed to TM5275, a small molecule inhibitor of PAI-1. Putting the data together, we hypothesized that cancer cells adhered to basal membrane secrete abundant PAI-1, on the other hand, cancer cells (especially CSCs rather than non-CSCs) distant from basal membrane secrete less PAI-1, which makes the ECM surrounding CSCs more susceptible to degradation. Our study could be an explanation of conflicting reports, where some researchers found negative impacts of PAI-1 expression on clinical outcomes and others not, by considering the concept of CSCs.

Increased incorporation of antiplasmin into the fibrin network in patients with type 1 diabetes.

Diabetes is associated with various vascular complications and is suggested to induce a prothrombotic state. In the current study, we characterized antiplasmin incorporation into fibrin in relation to other fibrinolytic compounds in patients with type 1 diabetes. A total of 236 patients with type 1 diabetes and 78 control subjects were investigated. The incorporation of antiplasmin into the fibrin network and the plasma levels of plasminogen activator inhibitor type 1 (PAI-1) activity, tissue plasminogen activator (tPA) activity, tPA/PAI-1 complex, plasmin-antiplasmin complex, antiplasmin, factor XIII, and d-dimer were measured. In addition, we used global assays to study fibrinolysis. The incorporation of antiplasmin into the fibrin network was significantly higher in patients with type 1 diabetes than in control subjects without diabetes (1.65 ± 0.25 vs. 1.35 ± 0.18 mg/L, respectively; P < 0.0001). The patients also had lower PAI-1 activity (2.19 units/mL [interquartile range 0.96-5.42] vs. 4.25 units/mL [1.95-9.0]; P = 0.0012) and antiplasmin level in plasma (78.5 ± 13.3 vs. 83.2 ± 15.4 mg/L; P < 0.05), resulting in a higher fibrinolytic capacity (shorter clot lysis time; P = 0.0090). We did not find any important sex differences regarding fibrinolysis in the patients or in the control subjects. Patients with type 1 diabetes incorporate more antiplasmin into the fibrin network than control subjects without diabetes do and have a reduced PAI-1 activity and a shorter clot lysis time. These results suggest that patients with type 1 diabetes produce a fibrin clot that is more resistant to fibrinolysis, which, however, may be counteracted by an increased fibrinolytic potential in plasma.

Systemic inhibition and liver‐specific over‐expression of PAI‐1 failed to improve survival in all‐inclusive populations or homogenous cohorts of CLP mice

The role of plasminogen activator inhibitor type-1 (PAI-1) in abdominal sepsis remains elusive. To study the influence of inhibition and over-expression of PAI-1 upon survival in cecal ligation and puncture (CLP) sepsis. (i) Mice underwent moderate CLP and received 10 mg kg(-1) of either monoclonal anti-PAI-1 (MA-MP6H6) or control (MA-Control) antibody intravenously at 0, 18 or 30 h post-CLP. The 30-h treatment group was additionally stratified into mice predicted to survive (P-SUR) or die (P-DIE) based on IL 6 measured at 24 h post-CLP. (ii) PAI-1 expression was induced with pLIVE.PAI-1 plasmid administered 72 h pre-CLP. Blood was sampled for 5 days and survival was monitored for 28 days. MA-MP6H6 effectively neutralized active PAI-1 and fully restored fibrinolysis while PAI-1 over-expression was liver-specific and correlated with PAI-1 increase in the blood. Without stratification, MA-MP6H6 co-/post-treatment conferred no survival benefit. Prospective stratification (IL-6 cut-off: 14 ng mL(-1) ) suggested increased mortality by MA-MP6H6 treatment in P-SUR that reached 30% difference (vs. MA-Control; P < 0.05) after a retrospective cut-off readjustment to 3.3 ng mL(-1) for better P-SUR homogeneity. Subsequent prospective anti-PAI-1 treatment in P-SUR mice with 3.3 ng mL(-1) cut-off demonstrated a negative but statistically insignificant effect: mortality was higher by 17% after MA-MP6H6 vs. MA-Control. Over-expression of PAI 1 did not alter post-CLP survival. Neither PAI-1 inhibition nor over-expression meaningfully modified inflammatory response and/or organ function. Restoration of fibrinolysis in early abdominal sepsis was not beneficial and it may prove detrimental in subjects with the lowest risk of death, while preemptive PAI-1 up-regulation at the current magnitude was not protective.

The association between plasminogen activator inhibitor type 1 (PAI-1) levels, PAI-1 4G/5G polymorphism, and myocardial infarction: a Mendelian randomization meta-analysis

The circulating levels of plasminogen activator inhibitor type 1 (PAI-1) are increased in individuals carrying the 4G allele at position -675 of the PAI-1 gene. In turn, overexpression of PAI-1 has been found to affect both atheroma and thrombosis. However, the association between PAI-1 levels and the incidence of myocardial infarction (MI) is complicated by the potentially confounding effects of well-known cardiovascular risk factors. The current study tried to investigate in parallel the association of PAI-1 activity with the PAI-1 4G/5G polymorphism, with MI, and some components of metabolic syndrome (MetS). Using meta-analytical Mendelian randomization approaches, genotype-disease and genotype-phenotype associations were modeled simultaneously. According to an additive model of inheritance and the Mendelian randomization approach, the MI-related odd ratio for individuals carrying the 4G allele was 1.088 with 95% confidence interval (CI) 1.007, 1.175. Moreover, the 4G carriers had, on average, higher PAI-1 activity than 5G carriers by 1.136 units (95% CI 0.738, 1.533). The meta-regression analyses showed that the levels of triglycerides (p=0.005), cholesterol (p=0.037) and PAI-1 (p=0.021) in controls were associated with the MI risk conferred by the 4G carriers. The Mendelian randomization meta-analysis confirmed previous knowledge that the PAI-1 4G allele slightly increases the risk for MI. In addition, it supports the notion that PAI-1 activity and established cardiovascular determinants, such as cholesterol and triglyceride levels, could lie in the etiological pathway from PAI-1 4G allele to the occurrence of MI. Further research is warranted to elucidate these interactions.

Carbamylated LDL induces a pro-thrombotic state via the LOX-1 receptor and arterial thrombus formation: a novel mechanism of cardiovascular events in end-stage renal disease

Background: Carbamylation of low-density lipoproteins (cLDL) alters protein structure and is thought to promote vascular inflammation and dysfunction during end-stage renal disease (ESRD). However, it remains unknown whether cLDL exerts prothrombotic effects in vascular cells and platelets and whether cLDL affects arterial thrombus formation in vivo. Methods: LDL was isolated from healthy subjects or patients with ESRD by sequential ultra-centrifugation. Ex vivo carbamylation of LDL (cLDL) from healthy subjects was induced with potassium cyanate. Arterial thrombus formation was analysed in a murine carotid artery photochemical injury model. Analysis of tissue factor (TF) and plasminogen activator inhibitor type-1 (PAI-1) expression was performed by Western blot and real-time PCR analysis. Platelet aggregation was analysed by impedance aggregometry. Results: Intravenous administration of cLDL (2mg/kg body weight) accelerated arterial thrombus formation in a murine photochemical carotid artery injury model as compared to treatment with native LDL (2mg/kg body weight) or vehicle (PBS) (n=8; p<0.05 versus nLDL and control). Analyses of tissue lysates demonstrated that cLDL induced the expression of TF and PAI-1 mRNA levels in carotid arteries (n=5, p<0.01). In human aortic smooth muscle cells (AoSMC) cLDL (10-300 μg/ml) induced TF and PAI-1 expression by 3.5- and 3-fold, as compared with nLDL (n=4; p<0.01). cLDL also enhanced basal and TNF-α induced expression of TF and PAI-1 in human aortic endothelial cells (n=4; p<0.02). In contrast, native LDL (nLDL) had no effect on endothelial expression or activity of TF and PAI-1. In both cell types, cLDL activated the mitogen-activated protein kinases p38 and ERK (n=3; p<0.05) as well as the transcription factor NFkB (n=4, p<0.05). Silencing of lectin-like oxidized LDL receptor-1 (LOX-1) using specific siRNA prevented the induction of TF and PAI-1 expression in vascular cells. In platelets cLDL activated the p38 MAP kinase and induced platelet aggregation. Finally, LDL isolated from patients with ESRD mimicked the prothrombotic effects of cLDL on vascular cells and platelets. Conclusions: These data demonstrate that cLDL at physiologically relevant concentrations,via activation of the LOX-1 receptor induces potent pro-thrombotic effects in vascular cells and platelets leading to enhanced thrombus formation in vivo. This observation may explain the markedly increased incidence of fatal acute thrombotic events in patients with end-stage renal disease and lead to the development of novel LDL targeting strategies in this population.

PI3K/p110alpha inhibition selectively interferes with arterial thrombosis and neointima formation, but not re-endothelialization: a new concept in drug-eluting stent design

Background: Impaired re-endothelialization and stent thrombosis are a safety concern associated with drug-eluting stents (DES) as mTOR inhibitors reduce restenosis rates, but impair endothelialization and facilitate thrombus formation. PI3K/p110α controls cellular wound healing pathways, thereby representing an emerging drug target to modulate vascular homeostasis after injury. However, its effect on arterial thrombosis and neointima formation remains unknown. Methods: PI3K/p110α was inhibited by treatment with the small molecule inhibitor PIK75 or a specific siRNA. Arterial thrombosis, neointima formation and re-endothelialization studies were performed in a murine carotid artery injury model. Proliferation and migration of VSMC and EC were assessed by cell number and Boyden chamber, respectively. Endothelial senescence was evaluated by β-galactosidase assay, endothelial dysfunction by organ chambers for isometric tension. Tissue factor (TF), plasminogen activator inhibitor type 1 (PAI-1), and eNOS expression was determined by Western blot and real-time PCR analysis. Results: Male C57Bl6 mice were either treated with PIK75 (10 mg/kg/d for 7 days) or vehicle. Arterial thrombus formation was delayed in mice treated with PIK75 as compared to controls (n=8; p<0.005). PIK 75 impaired arterial expression and activity of TF and PAI-1 as well as NFκB activity (n=8; p<0.05); in contrast, plasma clotting and tail bleeding times did not differ (n=8; p=NS). In human vascular smooth muscle and endothelial cells, PIK75 inhibited expression and activity of TF and PAI-1 (n=4; p<0.01). These effects occurred at the transcriptional level via the RhoA signaling cascade and the transcription factor NFkB. Furthermore, inhibition of PI3K/p110α with PIK75 or a specific siRNA selectively impaired proliferation and migration of VSMC while sparing EC completely. In line with this observation, treatment with PIK75 selectively inhibited neointima formation without affecting re-endothelialization following vascular injury. In contrast to rapamycin and paclitaxel, treatment with PIK75 did not induce endothelial senescence nor inhibit eNOS expression or endothelium-dependent relaxations. Conclusions: Following vascular injury, PI3K/p110α inhibition selectively interferes with arterial thrombosis and neointima formation, but not reendothelialization. Hence, PI3K/p110α represents an attractive new target in DES design.

Effects of black cohosh on the plasminogen activator system in vascular smooth muscle cells

ObjectiveThe rhizome of the Cimicifuga racemosa plant (commonly known as black cohosh) has been used for menopausal complaints. Studies regarding the cardiovascular effects of black cohosh are lacking. We investigated the effect of black cohosh on the plasminogen activator system in cultured vascular smooth muscle cells (VSMCs). MethodsVSMCs were isolated from rat aortae. Expression of plasminogen activator inhibitor type 1 (PAI-1) and tissue-type plasminogen activator (t-PA) proteins were evaluated by Western blot analysis and enzyme-linked immunosorbent assay, respectively. The activities of PAI-1 and t-PA in the conditioned media were assessed by fibrin overlay zymography. A 40% 2-propanol extract of black cohosh was used. ResultsBlack cohosh extract (BcEx) stimulated the protein expression of PAI-1, but it did not affect that of t-PA. Vitamin E, a potent antioxidant, inhibited the BcEx-induced increase in PAI-1 expression, while ICI 182,780, an estrogen receptor antagonist, had no effect. Fibrin overlay zymography revealed that BcEx increased the activity of PAI-1 in the conditioned media, while concurrently decreasing that of free t-PA by inducing a binding to PAI-1. ConclusionsBcEx induces PAI-1 protein expression in the VSMCs likely via an oxidant mechanism. It also stimulates the enzyme activity of PAI-1 and reduces that of free t-PA. These findings suggest that black cohosh might exert a negative influence on fibrinolysis.

SAT0240 The levels of plasminogen and plasminogen activator inhibitor type 1 in antiphospholipid syndrome

BackgroundA disturbance in the fibrinolytic system may cause thrombosis in patients with antiphospholipid syndrome (APS).ObjectivesTo determine the levels of plasminogen (Pg) and plasminogen activator inhibitor type 1 (PAI-1) in patients...