PAI-1 Activity Research Articles

Age-related characteristics of the content and activity of certain components of the blood fibrinolytic system in cases of uterine cancer

Uterine cancer (UC) is the third most common cancer in women in Russia. Knowledge of age-specific features of the fibrinolytic system in patients with UC can be useful in terms of improving its pathogenetic therapy. Aim of the study was to investigate the peculiarities of the content and activity of some components of the fibrinolytic system in the blood of UC patients of different age groups. Material and methods. 30 healthy women (donors) and 56 patients with uterine cancer T1a-2N0M0, adenocarcinoma G1–G3 were divided into subgroups according to age: reproductive, perimenopause and menopause. ELISA of urokinase (u-PA), its receptor (u-PAR) and its inhibitor (PAI-1), tissue-type plasminogen activator (t-PA) content were performed in blood collected before treatment. Results. In reproductive UC patients, inhibitor u-PA (PAI-1) activity increased 14-fold (hereinafter the difference is statistically significant, p < 0.05) and content increased 2.9-fold, while receptor u-PA (u-PAR) level decreased 1.7-fold compared to reproductive donors. Perimenopausal UC patients showed a 3.3-fold increase in PAI-1 content and a 6.3-fold increase in PAI-1 activity compared to perimenopausal donors, t-PA concentration decreased 1.3-fold relative to donors and was 1.9-fold lower than in reproductive UC patients. In menopausal patients with UC, the activity and content of PAI-1 enhanced 5.5-fold and 4.5-fold, respectively, compared to donors. Additionally, they were 2.1-fold and 1.2-fold lower than in reproductive UC patients. The activity of u-PA increased 2.6-fold, reaching the values of reproductive UC patients. The activity of t-PA was 1.3-fold higher than in donors, but did not differ from the activity in other RTM patients, while the level of t-PA occupied an intermediate position between the corresponding indices in young UC patients (it was 1.4-fold lower) and perimenopausal UC patients (it was 1.4-fold higher). Conclusions. The development of UC is accompanied by an imbalance of components of the fibrinolytic system in the blood, depending on the age of women, with a minimum spectrum of changes in the indicators in reproductive patients and maximum - in menopause patients, which indicates the pathognomonicity of these factors in PTM and requires an individual approach to the management of such patients.

Age-related features of some plasminogen activation system components content and activity in the blood of benign and malignant uterine body tumors

Purpose of the study. Is to determine the features in the content and activity of some components of the plasminogen activation system in the blood of patients of different ages with leiomyomas (LM) and uterine corpus endometrial cancer (UCEC). Patients and methods. The study was carried out in patients with LM (n = 35) and UCEC T1a-2N0M0 (n = 56) of reproductive, perimenopausal and postmenopausal ages. Using ELISA methods, the content and activity of urokinase (u-PA), tissue plasminogen activator (t-PA), their inhibitor PAI-1, as well as the content of the soluble form of the u-PA receptor (su-PAR) were determined in the blood of patients. The Student´s test was used for statistical processing. Results. Regardless of the nature of the uterus tumor, it has been noted a significant increase in the activity and blood level of PAI-1 (up to 8 times, p < 0.01), especially pronounced in patients with UCEC of reproductive age. It was combined with the absence of changes or with a decreasing in blood level of su-PAR by more than 40 % (p < 0.05). We observed a rise of u-PA blood level without changes in one´s activity in patients with LM of perimenopausal age. And in patients with LM and UCEC of postmenopausal age an increase in u-PA blood level as well as elevation of u-PA activity (up to 3.9 times, p < 0.01–0.05) were noted. There was an increase in the calculated t-PA index (activity per unit mass) by 1.4–2.8 times (p < 0.05) in the most patients. The indicators of LM patients were characterized by the minimum value of the ratio “t-PA activity/u-PA activity in the blood” among all studied subgroups of patients. Age-related features of the researched parameters were observed more often in the cases of LM than in the cases of UCEC. The most pronounced differences between LM and UCEC were observed in patients of reproductive and postmenopausal age, characterized by stable hormonal levels. Conclusion. The participation of the plasminogen activation system in the pathogenesis of tumor lesions of the uterine body has been shown. The system’s “response” to the development of tumors in the uterus has both general characteristics and features that depend on the nature of the tumors and on the age-specific hormonal regulation of the body, which are most pronounced in postmenopausal women. The results obtained can be used in research aimed at clarifying the targets of targeted therapy for LM and UCEC in accordance with the age of patients.

Reduced protein carbonylation on hormone therapy is associated with improved fibrinolysis in postmenopausal women: the impact of PAI-1 and TAFI activity.

Hormone therapy (HT) has been reported to reduce protein carbonylation (PC) in postmenopausal women, in whom fibrinolysis is impaired. We investigated whether PC affects fibrinolysis and if HT modulates this effect. We enrolled 150 women aged 55.5 ± 4.7years in a randomized interventional open-label study, including 50 on standard oral HT, 50 on ultra-low-dose HT, and 50 controls. PC, along with global fibrinolysis (clot lysis time, CLT), fibrinolysis proteins, and prothrombotic markers were determined at baseline and at 24weeks. Patients with the baseline top quartile PC (> 2.07nM/mg protein) had 10.3% longer CLT, higher activity (but not antigen) of TAFI (+ 19.9%) and PAI-1 (+ 68.1%) compared to the remainder. No differences were observed in thrombin generation, factor VIII, plasminogen or α2-antiplasmin. On-treatment PC decreased by 16.4% (p < 0.0001), without differences related to the type of HT, compared to baseline and by 30% compared to controls, in whom PC and fibrinolysis markers remained unchanged. Patients with PC > 2.07nM/mg had shortened CLT during HT compared to baseline, along with lower PAI-1 (-69%) and TAFI (-26%) activity. In this subgroup CLT was 5.8% shorter compared to controls with the highest PC. In postmenopausal women with increased PC, HT was accompanied by PC reduction and faster clot lysis together with decreased PAI-1 and TAFI activity.

Persistent hypofibrinolysis in severe COVID-19 associated with elevated fibrinolysis inhibitors activity.

Hypercoagulability and reduced fibrinolysis are well-established complications associated with COVID-19. However, the timelines for the onset and resolution of these complications remain unclear. The aim of this study was to evaluate, in a cohort of COVID-19 patients, changes in coagulation and fibrinolytic activity through ROTEM assay at different time points during the initial 30days following the onset of symptoms in both mild and severe cases. Blood samples were collected at five intervals after symptoms onset: 6-10days, 11-15days, 16-20days, 21-25days, and 26-30days. In addition, fibrinogen, plasminogen, PAI-1, and alpha 2-antiplasmin activities were determined. Out of 85 participants, 71% had mild COVID-19. Twenty uninfected individuals were evaluated as controls. ROTEM parameters showed a hypercoagulable state among mild COVID-19 patients beginning in the second week of symptoms onset, with a trend towards reversal after the third week of symptoms. In severe COVID-19 cases, hypercoagulability was observed since the first few days of symptoms, with a tendency towards reversal after the fourth week of symptoms onset. A hypofibrinolytic state was identified in severe COVID-19 patients from early stages and persisted even after 30days of symptoms. Elevated activity of PAI-1 and alpha 2-antiplasmin was also detected in severe COVID-19 patients. In conclusion, both mild and severe cases of COVID-19 exhibited transient hypercoagulability, reverted by the end of the first month. However, severe COVID-19 cases sustain hypofibrinolysis throughout the course of the disease, which is associated with elevated activity of fibrinolysis inhibitors. Persistent hypofibrinolysis could contribute to long COVID-19 manifestations.

A randomized, controlled clinical study of low-molecular-weight heparin improving pregnancy outcomes in PCOS women undergoing IVF: study protocol

BackgroundPolycystic ovary syndrome (PCOS), an incidence of 10–15% in women of reproductive age, shows sex hormone disorders, luteal insufficiency, and the tendency of placental villus space thrombus. The incidence of early pregnancy loss in women with PCOS is three to eight times higher than that in non-PCOS women. PCOS women were reported in a pre-thrombotic state, which was manifested by accelerated thrombin production, increased PAI-1 activity, and fibrinogen. Other research also found an over-activated state of women with PCOS in immune system. Therefore, changing the prethrombotic state of PCOS through anticoagulation may be a new way to improve the adverse pregnancy outcome of PCOS. Low-molecular-weight heparin (LMWH) is the most common used anticoagulant drug in pregnancy, and it also was proposed for the prevention of recurrent abortion, although the application of LMWH in PCOS population during early pregnancy has not been reported. The objective of this study is to investigate the effect of LMWH on pregnancy outcomes after invitro fertilization-frozen embryo transfer (IVF-FET) in patients with polycystic ovary syndrome.MethodsA total of 356 PCOS women aged between 20 and 38 years which prepared for IVF followed with FET will be enrolled in the study. The patients, from four different hospitals stratified by age and body mass index (BMI), will be randomly divided into the study group who will be treated with LMWH started on the day of progesterone transformation (hormone therapy) during FET cycle and the control group without additional medicine. Serum or urine hCG test will be given 14 days after embryo transfer to confirm biochemical pregnancy. If pregnancy is positive, LMWH+ hormone therapy/hormone therapy will be continued for another 2 weeks. Transvaginal ultrasonography will be performed 14 days later to confirm intrauterine pregnancy. The primary outcome is the ongoing pregnancy, which is defined as intrauterine live fetus with ultrasound after 12 weeks of gestation.DiscussionThis is the first study protocol to investigate the efficacy of LMWH as an adjuvant drug for IVF-FET outcomes in PCOS women, by comparing differences in ongoing pregnancy rate, clinical pregnancy rate, live birth rate, and early pregnancy loss rate between LMWH group and the control group.Trial registration ClinicalTrials.gov ChiCTR2000036527. Registered on August 24, 2020

Caveolin-1 promotes glioma proliferation and metastasis by enhancing EMT via mediating PAI-1 activation and its correlation with immune infiltrates

Glioma is typically characterized by a poor prognosis and is associated with a decline in the quality of life as the disease advances. However, the development of effective therapies for glioma has been inadequate. Caveolin-1 (CAV-1) is a membrane protein that plays a role in caveolae formation and interacts with numerous signaling proteins, compartmentalizing them in caveolae and frequently exerting direct control over their activity through binding to its scaffolding domain. Although CAV-1 is a vital regulator of tumour progression, its role in glioma remains unclear. Our findings indicated that the knockdown of CAV-1 significantly inhibits the proliferation and metastasis of glioma. Subsequent mechanistic investigations demonstrated that CAV-1 promotes proliferation and metastasis by activating the photoshatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway. Furthermore, we demonstrated that CAV-1 overexpression upregulates the expression of serpin peptidase inhibitor, class E, member 1 (SERPINE1, also known as PAI-1), which serves as a marker for the epithelial-mesenchymal transition (EMT) process. Further research showed that PAI-1 knockdown abolished the CAV-1 mediated activation of PI3K/Akt signaling pathway. In glioma tissues, CAV-1 expression exhibited a correlation with unfavorable prognosis and immune infiltration among glioma patients. In summary, our study provided evidence that CAV-1 activates the PI3K/Akt signaling pathway by upregulating PAI-1, thereby promoting the proliferation and metastasis of glioma through enhanced epithelial-mesenchymal transition (EMT) and angiogenesis, and CAV-1 is involved in the immune infiltration.

Abstract 12953: Targeting NEDD9 and SMAD3 in the Pathogenesis of Chronic Thromboembolic Pulmonary Hypertension

Background: Chronic thromboembolic pulmonary hypertension (CTEPH) is characterized by unresolved pulmonary thrombi due, in part, to impaired fibrinolysis. We showed previously that oxidation of the Cas protein NEDD9 at cysteine-18 (Cys 18 ) inhibits complex formation between NEDD9 and the anti-fibrinolytic transcription factor SMAD3 in human pulmonary artery endothelial cells (HPAECs). However, the effect of NEDD9-Cys 18 oxidation on SMAD3 bioactivity in HPAECs is not known. Hypothesis: NEDD9-Cys 18 oxidation increases bioavailable SMAD3, which upregulates SERPINE1 that encodes plasminogen activator inhibitor (PAI)-1 to inhibit fibrinolysis pathways in HPAECs. Methods/Results: To assess the effect of SMAD3 on SERPINE1 transcription, HPAECs were transfected with cDNA-SMAD3 (1 μg/μl) for 72 hr to induce SMAD3 overexpression. Compared to vehicle (V) control, cDNA-SMAD3 increased PAI-1 and SERPINE1 expression by 35% (P=0.004) by immunoblot and 70% (P=0.003) by real-time PCR, respectively (N=3). To determine the effect of NEDD9 inhibition on SMAD3 activation, we analyzed the ratio of P-SMAD-Ser423/425:SMAD3 by immunoblot. Compared to scrambled RNA (scRNA), transfection with siRNA against NEDD9 (siNEDD9) for 48 hr increased the relative quantity of activated SMAD3 by 1.4-fold (P=0.006) and upregulated PAI-1 (P=0.004) expression by 1.7-fold in HPAECs (N=4). Next, we monitored the effect of oxidant stress on SMAD3 bioactivity. Compared to V-control, the expression of NEDD9, total SMAD3, and P-SMAD3 was increased by 66% (P=0.04), 81% (P=0.03), and 29% (P=0.04), respectively, in HPAECs treated with hydrogen peroxide (H 2 O 2 ) (500 μM) for 20 min. Next, the effect of NEDD9 silencing on H 2 O 2 accumulation in HPAECs was measured by Amplex Red assay. HPAECs were transfected with scRNA or siNEDD9 for 48 hr followed by measurement of H 2 O 2 production. Compared to scRNA, NEDD9 silencing significantly decreased H 2 O 2 concentration by 56% (N=4, P=0.02). Conclusion: These data demonstrate that oxidant stress increases SMAD3 activity and SMAD3-dependent upregulation of PAI-1 through a mechanism involving redox regulation of NEDD9. Identifying prothrombotic control of SMAD3 by NEDD9 may have important therapeutic implications for CTEPH.

PLASMA PLASMINOGEN ACTIVATOR INHIBITOR 1 ASSOCIATES WITH HYPERTENSION IN PROSTATE CANCER

Objective: The aim was to determine the activity of serum PAI 1 in men with prostate cancer and its relationship with hypertension characteristics. Design and method: 39 men (median age 63.8 [52.7; 70.5]) with prostate cancer were enrolled. Serum PAI 1 activity levels were determined using a modified colorimetric method of plasminogen activator determination. We examined the association of plasma PAI 1 with incident hypertension by multivariate logistic regression, adjusting for age, obesity, lipids, fasting glucose, kidney function, inflammation, and follow-up years. Results: Arterial hypertension was detected in 26 (66.7 %) patients with prostate adenocarcinoma. The median PAI 1 activity level in patients with prostate cancer was 77.95 [64.72; 92.12] U/mL, in the control group – 64.54 [52.35;85.45] U/mL (p&lt; 0.05). The PAI 1 activity level correlated with diastolic blood pressure (R = 0.69, p &lt; 0.001), duration of prostate adenocarcinoma (R = 0.42, p &lt; 0.001), and testosterone level (R = 0.60, p &lt; 0.001). Higher level of log-transformed PAI 1 was associated with 1.29-fold increased risk of hypertension [odds ratio (OR) (95% confidence interval): 1.29 (1.09–1.53)]. Conclusions: Men with prostate cancer are characterized by increased levels of PAI 1 associated with hypertension. More research is needed to establish a relationship between PAI 1 and hypertension in men with prostate cancer.

Systematic review and meta-analysis of within-subject and between-subject biological variation data of coagulation and fibrinolytic measurands.

The diagnosis and monitoring of bleeding and thrombotic disorders depend on correct haemostatic measurements. The availability of high-quality biological variation (BV) data is important in this context. Many studies have reported BV data for these measurands, but results are varied. The present study aims to deliver global within-subject (CVI) and between-subject (CVG) BV estimates for haemostasis measurands by meta-analyses of eligible studies, by assessment with the Biological Variation Data Critical Appraisal Checklist (BIVAC). Relevant BV studies were graded by the BIVAC. Weighted estimates for CVI and CVG were obtained via meta-analysis of the BV data derived from BIVAC-compliant studies (graded A-C; whereby A represents optimal study design) performed in healthy adults. In 26 studies BV data were reported for 35 haemostasis measurands. For 9 measurands, only one eligible publication was identified and meta-analysis could not be performed. 74% of the publications were graded as BIVAC C. The CVI and CVG varied extensively between the haemostasis measurands. The highest estimates were observed for PAI-1 antigen (CVI 48.6%; CVG 59.8%) and activity (CVI 34.9%; CVG 90.2%), while the lowest were observed for activated protein C resistance ratio (CVI 1.5%; CVG 4.5%). This study provides updated BV estimates of CVI and CVG with 95% confidence intervals for a wide range of haemostasis measurands. These estimates can be used to form the basis for analytical performance specifications for haemostasis tests used in the diagnostic work-up required in bleeding- and thrombosis events and for risk assessment.

Role of insulin-like growth factor, pro-inflammatory and anti-inflammatory cytokines, transcription factors and adipokines in development of hepatocellular carcinoma in metabolic syndrome

Conflicting data on the role of the metabolic syndrome (MS) in the development of hepatocellular carcinoma (HCC) require studies on the influence of molecular factors that are important in the development of HCC in MS, which was the goal of our review. Publications (scientific articles and reviews) over the past 10 years were studied and analyzed using the databases Web of Science, Scopus, PubMed, RSCI. The terms used for the search were “metabolic syndrome and non-alcoholic fatty liver disease”, “metabolic syndrome and non-alcoholic steatohepatitis”, “metabolic syndrome and hepatocellular carcinoma”. The total number of publications studied in all databases exceeded 570 units, while the review presents the most significant results at the present stage. Insulin resistance and obesity, through the development of a systemic chronic inflammatory state, lead to increased inflammation and fibrosis in the liver, which are prodromal signs of hepatocarcinogenesis, increase the production of insulin-like growth factor-1 and disrupt the regulation of the insulin-like growth factor pathway. People with HCC have been shown to overexpress IGF-2. IGF-binding proteins, due to the reduced bioavailability of free IGF-1 and IGF-2 in the circulation, are able to inhibit the growth of HCC. In MS, a pro-inflammatory state is detected, which is caused by the production of cytokines by adipocytes (IL-6, IL-8, IL-1β, tumor necrosis factor α (TNFα), VEGF and chemokine ligands 2 and 5), which recruit immune cells, promoting angiogenesis and enhancing chronic inflammation. Transcription factors (PPAR) are involved in hepatocarcinogenesis, the significance of different factors is not fully understood. Leptin has a positive prognostic value in HCC, improving overall survival, and visfatin has a negative effect on hepatocarcinogenesis. Activation of PAI-1 inhibits the progression of HCC through PPARγ stimulation. Adiponectin may be a prognostic marker in HCC, with a lower serum concentration positively correlated with worse prognosis.

Laboratory evaluation of a new integrative assay to phenotype plasma fibrinolytic system

BackgroundThere is currently no universal and standardized test available to phenotype plasma fibrinolytic system.AimsOur main aims were to evaluate the performances of the ‘global fibrinolysis capacity’ assay (GFC) performed with the Lysis Timer® instrument, and to study the influence of some preanalytical conditions.MethodEuglobulin clot lysis time (ECLT) and GFC were performed under several preanalytical conditions.ResultsGFC showed satisfactory intra- and inter-run precision. Frozen controls and reagents showed stability over the studied period.There was no statistically significant difference between GFC assessed in plasma samples processed at 4 °C or at 20 °C. GFC assessed with frozen-thawed plasma samples was prolonged when compared to fresh samples (p = 0.014). The centrifugation scheme had no influence on PAI-1 activity levels, GFC and ECLT.Reference interval for GFC ranges from 29.3 (C I90% = 26.9–31.9) to 49.5 (90% CI = 45.9–52.2) minutes.In addition, a preliminary study in 40 healthy volunteers and 43 adult patients referred for investigation of a bleeding disorder was conducted to compare GFC and ECLT assays in their ability to classify samples with shortened or prolonged clot lysis times. Disagreements between ECLT and GFC were observed for 23 samples (out of 83), most of them minor.ConclusionGFC is suitable and convenient for a broad clinical use and can be performed with frozen-thawed plasma samples.Unlike ECLT, GFC is designed to take into account the balance between inhibitors and activators of the fibrinolytic system and could detect both hypo- and hyperfibrinolytic states. Whether it is as suitable as or even better than ECLT to detect a bleeding tendency due to a hyperactive fibrinolytic system deserves to be properly investigated.

Penggunaan Media Audio Visual dalam Meningkatkan Hasil Belajar Siswa Memahami Materi Iman kepada Hari Akhir di Kelas IX SMPIT Insan Harapan

The purpose of Classroom Action Research (CAR) using audio-visual media for this learning is to increase the activity and learning outcomes of PAI (Islamic Religious Education) about the End of Days for Class IX students of SMP. The time of the study was in November 2019. The subjects of this study were Class IXB students in Semester 1 of the 2019/2020 academic year which consisted of 35 children. The data in this study are learning activities and learning outcomes. Data collection tools using observation sheets and written tests. The research procedure uses a Cycle Model which consists of 4 stages, namely Planning, Action, Observation, and Reflection. The results of this study are (1) learning becomes concrete and interesting; (2) students learn actively and effectively by listening, seeing, analyzing, taking notes, doing individual assignments, asking questions and giving opinions; and (3) students achieving better learning outcomes by increasing average scores and completeness. In the initial conditions, learning completeness is only 28.6%. in this cycle the completeness reached 83.9%. The suggestion is that the teacher: (1) give individual assignments to students to answer questions in writing after watching the film so that students are not only interested in the appearance of the film, but actually observe and analyze the content of the film; (2) paying attention to the content of the film, taking notes and asking questions, so that mastery of the material is increasing; and (3) optimizing the function of the audio-visual room and the completeness of the facilities available as interesting and effective learning media, so that learning is more meaningful.

Abstract 13596: Effects of Plasminogen Activator Inhibitor-1 on the Circadian Clock in Liver

Introduction: Plasminogen activator inhibitor-1 (PAI-1, encoded by Serpine1 ) plays a major role in the mammalian fibrinolytic system, but elevated PAI-1 activity contributes to other biological processes including the metabolic syndrome. Multiple studies suggest that disruption of circadian rhythms also promotes features of the metabolic syndrome including obesity, hypertension, and type 2 diabetes. While it has been long known that PAI-1 plasma levels exhibit a marked circadian variation, and that the expression of PAI-1 is strongly influenced by the clock, a reciprocal role for PAI-1 in regulating components of the clock itself has never been described. Hypothesis: We hypothesized that changes in PAI-1 activity influence the expression of core circadian clock genes and clock-controlled genes in the liver. Methods: We used two mouse models of reduced PAI-1 activity: oral PAI-1 inhibitor TM5614 (20 mg/kg/day) versus vehicle in C57BL/6J wild-type mice (n = 5 per group), and Serpine +/- heterozygous mice versus wild-type littermate control mice (n = 3 per group). Mice were maintained on a 14:10 light:dark cycle. Livers were collected between 3 and 4 hours after lights-on, which corresponds to Zeitgeber Time (ZT) 3-4. RNA was isolated for RNA-seq and downstream analyses. Results: Between ZT 3-4, we found that reduced PAI-1 activity led to marked changes in circadian gene expression in the liver. Both PAI-1 inhibition with TM5614 and PAI-1 haploinsufficiency significantly increased expression of the core circadian clock components Arntl (aka Bmal1) and Clock while significantly reducing expression of core clock components Per1, Per2, Per3, and clock output genes. Furthermore, we found that PAI-1 inhibition in aged transgenic mice overexpressing the stable variant of human SERPINE1 rescued two markers of impaired circadian function: 1) the premature onset of activity prior to lights-off, and 2) reduced feeding during the lights-off period. Conclusions: Taken together, these data support a reciprocal role for PAI-1 in regulating components of the circadian clock. Elevated PAI-1 activity may contribute to the metabolic syndrome through effects on the circadian clock and may provide a mechanistic link between PAI-1, the circadian clock, and aging.

627 Inhibition of PAI-1 blocks PD-L1 endocytosis and improves the response of melanoma cells to immune checkpoint blockade

Immune checkpoint molecules, especially PD-1 and its ligand PD-L1, act as a major mechanism of cancer immune evasion. Although anti–PD-1/PD-L1 monotherapy increases therapeutic efficacy in melanoma treatment, only a subset of patients exhibits long-term tumor remission, and the underlying mechanism of resistance to PD-1/PD-L1 inhibitors remains unclear. In this study, we demonstrated that cell surface retention of PD-L1 is inversely correlated with PAI-1 expression in vitro, in vivo, and in clinical specimens. Moreover, extracellular PAI-1 induced the internalization of surface-expressed PD-L1 by triggering clathrin-mediated endocytosis. The endocytosed PD-L1 was transported to lysosomes for degradation by endolysosomal systems, resulting in the reduction of surface PD-L1. Notably, inhibition of PAI-1 by pharmacological inhibitor with tiplaxtinin led to elevated PD-L1 expression on the plasma membrane, both in vitro and in vivo. Strikingly, targeting PAI-1 by tiplaxtinin treatment synergizes with anti–PD-L1 immune checkpoint blockade therapy in a syngeneic murine model of melanoma. Our findings demonstrate a role for PAI-1 activity in immune checkpoint modulation by promoting surface PD-L1 for lysosomal degradation and provides an insight into the combination of PAI-1 inhibition and anti–PD-L1 immunotherapy as a promising therapeutic regimen for melanoma treatment.

Plasminogen Activator Inhibitor-1 in Acute Myeloid Leukemia: Is It Useful in Predicting Response to Induction Remission Therapy?

BACKGROUND: Coagulation and fibrinolytic abnormalities are common in patients with acute myeloblastic leukemia (AML). Plasminogen activator inhibitor (PAI) activity is believed to be elevated during the initial diagnosis and relapse of AML patients. AIM: This study aimed to evaluate the levels of plasma PAI-1 activity in AML patients before and after remission induction. METHODS: Thirty AML patients and 20 healthy volunteers were included in this study. The patients were classified according to AML-FAB subtypes. All patients received 7+3 induction chemotherapy. They were evaluated for complete remission after induction chemotherapy and followed up for 6 months. RESULTS: PAI-1 activity was measured by ELISA immunoassay. PAI-1 activity was significantly higher in AML patients than the control group (P=0.016), whereas there was insignificant difference among patients in various AML subgroups (P&gt;0.05). Before and after treatment, there was a significant difference in PAI-1 activity between patients with active disease and those at remission (P= 0.023 and &lt;0.0001, respectively). CONCLUSION: High PAI-1 activity in AML patients is found to be associated with poor response to treatment.

Cold-stored platelets are effective in an in vitro model of massive transfusion protocol assessed by rotational thromboelastometry.

Platelets are a key component of massive transfusion in treating actively bleeding patients. While optimized for prophylactic transfusions, the effectiveness of the current standard room temperature stored platelets (RPs) in treating actively bleeding patients is not clear. Cold-stored platelets (CPs) have been shown to have superior hemostatic functions and the potential to extend shelf life. In this study, we explored the effect of using CPs versus RPs in an in vitro transfusion model based on the massive transfusion protocol. RPs or CPs were combined with RBCs and plasma in a 1:1:1 volume ratio to make transfusion packages. Whole blood was collected and then either diluted to 20% hematocrit or mixed with tPA (8.8μg/ml). By volume, 70% of transfusion package was mixed with 30% whole blood to simulate massive transfusions and analyzed by rotational thromboelastometry. Transfusion package supernatant was analyzed for PAI-1 activity as well. Both transfusion packages restored the clot characteristics of hemodiluted or hyperfibrinolytic whole blood. Specifically, only transfusion packages made with CPs significantly reduced the maximum clot lysis of hyperfibrinolytic whole blood. PAI-1 activity in CPs transfusion packages were also significantly higher. Transfusion packages containing cold-stored platelets may be able to restore the blood hemostatic profile of bleeding patients. In addition, transfusion packages made from CPs may provide additional benefit of resisting hyperfibrinolysis in bleeding patients. In trauma where post-transfusion platelet recovery is less of a concern, CPs are a viable option to restore hemostasis.

Hemostatic Responses to Multiple Bouts of Firefighting Activity: Female vs. Male Differences in a High Demand, High Performance Occupation.

While the fire service has long been a male-dominated occupation, women’s participation in this strenuous, high risk, high performance activity has increased in recent years. Firefighting induces significant cardiovascular strain, including hemostatic disruption; however, the effect of sex on hemostatic responses has not been investigated despite evidence that there are sex-related differences in hemostatic variables at rest and following exercise. Thus, we investigated hemostatic responses in age- and BMI-matched male and female firefighters who performed 3–4 evolutions of firefighting drills over a 3 h period. Venous blood samples were collected before and after the firefighting training drills and hemostatic variables were assessed. Firefighting significantly increased platelet count and factor VIII, tissue plasminogen activator (t-PA) antigen, and t-PA activity, and decreased activated partial thromboplastin time and plasminogen activator inhibitor (PAI-1) activity. Females had lower values for epinephrine-induced platelet closure time, antithrombin III, PAI-1 activity, and PAI-1 antigen. There were no interactions between sex and time for any variables assessed. In conclusion, multiple bouts of firefighting activity resulted in a procoagulatory state. Although there were sex differences for several hemostatic variables, male and female firefighters did not differ in their hemostatic response to multiple bouts of firefighting.

Simulated in vitro hypoxic conditions from psoriatic arthritis cartilage change plasminogen activating system urokinase and serpine functionality. Reversal of antiapoptotic protection suggests common homeostatic buffering.

Rheumatoid and psoriatic arthritis are both characterised by synovial destruction associated with a higher turnover of the extracellular matrix. In both conditions, inflammatory processes create hypoxic environments which destabilise members of the plasminogen activating system. Comparing the effect of bioactive concentrations of urokinase (uPA) and serpine (PAI-1) on cellular survival of human fibroblast-like-synoviocytes (HFLS) in rich and hypoxic growth media. Monocultures of HFLS were exposed to bioactive uPA and PAI-1 concentrations in both media conditions for 24, 48 and 72 h. Cellular survival was evaluated with a cell viability assay by spectrum absorbance of formazan reduced WST-8. PAI-1 at 0.1 and 1 μg/ml was found to stimulate cell viability under hypoxic stress at 48 and 72 h of incubation, with the effect increasing from 48 to 72 h. uPA increased cell viability in rich medium at 48 and 72 h of incubation between 5 and 40 ng/l, but was found to reduce viability at 80 ng/l at 24 and 48 h. PAI-1 increased cell viability in the hypoxic stress model, while high concentrations of uPA decreased cell viability in rich medium. The alternative modes of function at extreme concentrations provide a novel description of PAI-1 and uPA activity based on their colocalization and mutual buffering capacity, helping to place these molecules more accurately in the context of arthritic synovial deterioration.

Anti-fibrotic effect of 6-bromo-indirubin-3'-oxime (6-BIO) via regulation of activator protein-1 (AP-1) and specificity protein-1 (SP-1) transcription factors in kidney cells.

PAI-1 and CTGF are overexpressed in kidney diseases and cause fibrosis of the lungs, liver, and kidneys. We used a rat model of unilateral ureteral obstruction (UUO) to investigate whether 6-BIO, a glycogen synthase kinase-3β inhibitor, attenuated fibrosis by inhibiting PAI-1 and CTGF in vivo. Additionally, TGFβ-induced cellular fibrosis was observed in vitro using the human kidney proximal tubular epithelial cells (HK-2), and rat interstitial fibroblasts (NRK49F). Expression of fibrosis-related proteins and signaling molecules such as PAI-1, CTGF, TGFβ, αSMA, SMAD, and MAPK were determined in HK-2 and NRK49F cells using immunoblotting. To identify the transcription factors that regulate the expression of PAI-1 and CTGF the promoter activities of AP-1 and SP-1 were analyzed using luciferase assays. Confocal microscopy was used to observe the co-localization of AP-1 and SP-1 to PAI-1 and CTGF. Expression of PAI-1, CTGF, TGFβ, and α-SMA increased in UUO model as well as in TGFβ-treated HK-2 and NRK49F cells. Furthermore, UUO and TGFβ treatment induced the activation of P-SMAD2/3, SMAD4, P-ERK 1/2, P-P38, and P-JNK MAPK signaling pathways. PAI-1, CTGF, AP-1 and SP-1 promoter activity increased in response to TGFβ treatment. However, treatment with 6-BIO decreased the expression of proteins and signaling pathways associated with fibrosis in UUO model as well as in TGFβ-treated HK-2 and NRK49F cells. Moreover, 6-BIO treatment attenuated the expression of PAI-1 and CTGF as well as the promoter activities of AP-1 and SP-1, thereby regulating the SMAD and MAPK signaling pathways, and subsequently exerting anti-fibrotic effects on kidney cells.

Comorbidity-associated glutamine deficiency is a predisposition to severe COVID-19.

SARS-CoV-2 vaccinations have greatly reduced COVID-19 cases, but we must continue to develop our understanding of the nature of the disease and its effects on human immunity. Previously, we suggested that a dysregulated STAT3 pathway following SARS-Co-2 infection ultimately leads to PAI-1 activation and cascades of pathologies. The major COVID-19-associated metabolic risks (old age, hypertension, cardiovascular diseases, diabetes, and obesity) share high PAI-1 levels and could predispose certain groups to severe COVID-19 complications. In this review article, we describe the common metabolic profile that is shared between all of these high-risk groups and COVID-19. This profile not only involves high levels of PAI-1 and STAT3 as previously described, but also includes low levels of glutamine and NAD+, coupled with overproduction of hyaluronan (HA). SARS-CoV-2 infection exacerbates this metabolic imbalance and predisposes these patients to the severe pathophysiologies of COVID-19, including the involvement of NETs (neutrophil extracellular traps) and HA overproduction in the lung. While hyperinflammation due to proinflammatory cytokine overproduction has been frequently documented, it is recently recognized that the immune response is markedly suppressed in some cases by the expansion and activity of MDSCs (myeloid-derived suppressor cells) and FoxP3+ Tregs (regulatory T cells). The metabolomics profiles of severe COVID-19 patients and patients with advanced cancer are similar, and in high-risk patients, SARS-CoV-2 infection leads to aberrant STAT3 activation, which promotes a cancer-like metabolism. We propose that glutamine deficiency and overproduced HA is the central metabolic characteristic of COVID-19 and its high-risk groups. We suggest the usage of glutamine supplementation and the repurposing of cancer drugs to prevent the development of severe COVID-19 pneumonia.