Characterization of a small molecule PAI-1 inhibitor, ZK4044

Plasminogen activator inhibitor-1 (PAI-1) is a key negative regulator of the fibrinolytic system. In animal studies, inhibition of PAI-1 activity prevents arterial and venous thrombosis, indicating that PAI-1 inhibitors may be used as a new class of antithrombotics. In this study, we characterize a small molecule PAI-1 inhibitor, ZK4044, which was identified by high throughput screening and chemically optimized. In a chromogenic substrate-based urokinse (uPA)/PAI-1 assay and a tissue-type plasminogen activator (tPA)-mediated clot lysis assay, ZK4044 inhibited human PAI-1 activity with IC50 values of 644+/-255 and 100+/-90 nM, respectively. ZK4044 had no detectable inhibitory activity toward other serpins such as antithrombin III, alpha1-antitrypsin and alpha2-antiplasmin, indicating that ZK4044 is a specific PAI-1 inhibitor. ZK4044 was shown to bind directly to PAI-1 and prevent the binding of PAI-1 to tPA in a dose-dependent manner in surface plasmon resonance Biacore-based experiments. ZK4044 also prevented PAI-1/tPA complex formation, as analyzed by SDS/PAGE. ZK4044 had little effect on elastase-mediated cleavage of active PAI-1, indicating that the primary mode of action of ZK4044 is most likely to directly block the PAI-1/tPA interaction rather than to convert active PAI-1 to latent PAI-1. In the chromogenic substrate-based uPA/PAI-1 assay, ZK4044 was approximately 2-fold less potent against a mutant PAI-1 (14B-1), which contains four mutations at N150H, K154T, Q319L and M354I, compared with wild-type PAI-1, suggesting that the ZK4044 binding site on the surface of PAI-1 is close to these mutant residues. Together, our data show that ZK4044 represents a new class of small molecule PAI-1 inhibitors with anti-thrombotic potential.     

Expression of plasminogen activator inhibitors type 1 and type 3 and urokinase plasminogen activator protein and mRNA in breast cancer

BACKGROUND: The urokinase plasminogen activator (uPA) system has been involved in cancer cell invasion and in metastasis. uPA activity is controlled by its principal inhibitor, the PA inhibitor type-1 (PAI-1), but it can also be inhibited by PAI-3. Increased levels of uPA and PAI-1 are known to be associated with a poor prognosis in breast cancer. To our knowledge this is the first study of the expression and role of PAI-3 in human breast cancer tissue. MATERIALS AND METHODS: Protein and mRNA levels were evaluated for uPA, PAI-1 and PAI-3 in breast cancer tissues from 70 different patients. The localization of antigen and mRNA of these proteins was studied by immunohistochemistry and in situ hybridization, respectively. RESULTS: No significant differences were observed for PAI-3 mRNA or protein levels between the nodal status groups or the different post-surgical tumor-node-metastasis (pTNM) stages. However, uPA and PAI-1 mRNA and antigen levels significantly increased at the pTNM stage and in node-positive patients. PAI-3 antigen levels were significantly higher in early relapse-free patients, whereas PAI-1 antigen levels were significantly higher in patients who suffered a relapse. PAI-3 protein and mRNA were localized in stromal cells. PAI-1 and uPA protein were detected in cancer, endothelial and stromal cells and their mRNA mainly in stromal cells. CONCLUSIONS: Our results indicate that PAI-3 is expressed in human breast cancer tissues, and that elevated levels of PAI-3 could be a positive prognostic factor in this disease. A potential mechanism for the contribution of PAI-3 to a positive long-term outcome may involve suppression of tumor invasion through protease inhibition in stroma.     

Urokinase‐type plasminogen activator modulates mammalian circadian clock phase regulation in tissue‐type plasminogen activator knockout mice

Glutamate phase shifts the circadian clock in the mammalian suprachiasmatic nucleus (SCN) by activating NMDA receptors. Tissue‐type plasminogen activator (tPA) gates phase shifts by activating plasmin to generate m(ature) BDNF, which binds TrkB receptors allowing clock phase shifts. Here, we investigate phase shifting in tPA knockout (tPA^?/?; B6.129S2‐Plat^tm1Mlg/J) mice, and identify urokinase‐type plasminogen activator (uPA) as an additional circadian clock regulator. Behavioral activity rhythms in tPA^?/? mice entrain to a light‐dark (LD) cycle and phase shift in response to nocturnal light pulses with no apparent loss in sensitivity. When the LD cycle is inverted, tPA^?/? mice take significantly longer to entrain than C57BL/6J wild‐type (WT) mice. SCN brain slices from tPA^?/? mice exhibit entrained neuronal activity rhythms and phase shift in response to nocturnal glutamate with no change in dose‐dependency. Pre‐treating slices with the tPA/uPA inhibitor, plasminogen activator inhibitor‐1 (PAI‐1), inhibits glutamate‐induced phase delays in tPA^?/? slices. Selective inhibition of uPA with UK122 prevents glutamate‐induced phase resetting in tPA^?/? but not WT SCN slices. tPA expression is higher at night than the day in WT SCN, while uPA expression remains constant in WT and tPA^?/? slices. Casein‐plasminogen zymography reveals that neither tPA nor uPA total proteolytic activity is under circadian control in WT or tPA^?/? SCN. Finally, tPA^?/? SCN tissue has lower mBDNF levels than WT tissue, while UK122 does not affect mBDNF levels in either strain. Together, these results suggest that either tPA or uPA can support photic/glutamatergic phase shifts of the SCN circadian clock, possibly acting through distinct mechanisms.     

TM5275 prolongs secreted tissue plasminogen activator retention and enhances fibrinolysis on vascular endothelial cells

Introduction

Elevated plasminogen activator inhibitor-1 (PAI-1) reduces fibrinolytic potential in plasma, contributing to thrombotic disease. Thus, inhibiting PAI-1 activity is clinically desirable. We recently demonstrated that tissue plasminogen activator (tPA) remains on the surface of vascular endothelial cells (VECs) after secretion in a heavy-chain dependent manner, which is essential for high fibrinolytic activity on the surface of VECs, and that PAI-1 dissociates retained tPA from the cell surface as a result of high-molecular weight complex formation. Based on the model whereby amounts of tPA and its equilibrium with PAI-1 dynamically change after exocytosis, we examined how TM5275, a newly synthesized small molecule PAI-1 inhibitor, modulated tPA retention and VEC surface-derived fibrinolytic activity using microscopic techniques.

Materials and methods

The effects of TM5275 on the kinetics of the secretion and retention of green fluorescent protein (GFP)-tagged tPA (tPA-GFP) on VECs were analyzed using total internal reflection fluorescence microscopy. The effects of TM5275 on the generation of plasmin activity were evaluated by both plasminogen accumulation and fibrin clot lysis on tPA-GFP-expressing VECs using confocal laser scanning microscopy.

Results

TM5275 at concentrations of 20 and 100 μM significantly prolonged the retention of tPA-GFP on VECs by inhibiting tPA-GFP-PAI-1 high-molecular-weight complex formation. TM5275 enhanced the time-dependent accumulation of plasminogen as well as the dissolution of fibrin clots on and around the tPA-GFP-expressing cells.

Conclusions

The profibrinolytic effects of TM5275 were clearly demonstrated by the prolongation of tPA retention and enhancement of plasmin generation on the VEC surface as a result of PAI-1 inhibition.     

Asymmetric dimethylarginine impairs fibrinolytic activity in human umbilical vein endothelial cells via p38 MAPK and NF-κB pathways

Introduction

Asymmetric dimethylarginine (ADMA) is a potent endogenous inhibitor of nitric oxide (NO) synthase. An increased synthesis and/or a reduced catabolism of ADMA might contribute to the onset and progression of thrombosis. The present study was designed to evaluate the effect of ADMA on fibrinolytic factors in endothelial cells, and to investigate the cellular mechanisms.

Materials and Methods

Human umbilical vein endothelial cells (HUVECs) were treated with different concentrations of ADMA for various periods; Then HUVECs were preincubated with NO precursor (L-arginine), MAPK inhibitors, or NF-κB inhibitor (PDTC) before ADMA treatment to repeat the experiment. Protein levels of tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1), and NF-κB activity were measured by ELISA; mRNA levels of tPA and PAI-1 were assayed by qRT-PCR; The activation of MAPK was characterized by western blot analysis.

Results

(1) ADMA decreased tPA antigen levels in time- and concentration-dependent manners, with the maximum effect of 30 μmol/L ADMA for 48 h (control 109.01 ± 4.15 ng/ml vs ADMA 86.76 ± 5.95 ng/ml, p < 0.01); (2) 30 μmol/L ADMA elevated antigen levels of PAI-1 in a time-dependent manner, with the maximum effect of 30 μmol/L ADMA for 48 h (control 2721.12 ± 278.02 ng/ml vs ADMA 3435.78 ± 22.33 ng/ml, p < 0.05); (3) ADMA reduced tPA mRNA levels and increased PAI-1 mRNA levels; (4) L-arginine, SB203580 (p38 MAPK inhibitor) and PDTC attenuated the effects of ADMA on tPA and PAI-1 significantly. (5) ADMA induced a rapid phosphorylation of p38 MAPK, and stimulated NF-κB activity greatly.

Conclusions

ADMA may accelerate thrombosis development by impairing fibrinolytic activity in vascular via inhibiting nitric oxide production and then activating its downstream p38 MAPK and NF-κB pathways.     

Astrocyte regulation of human brain capillary endothelial fibrinolysis

INTRODUCTION: Astrocytes are known to regulate a wide variety of brain endothelial cell functions. Prior work, using a mixed species co-culture system, has shown astrocyte regulation of brain capillary endothelial expression of key hemostasis factors tissue plasminogen activator (tPA) and its inhibitor, plasminogen activator inhibitor-1 (PAI-1). The purpose of this study is to define the fibrinolytic regulatory role of human astrocytes on human brain capillary endothelial cells. MATERIALS AND METHODS: We used a blood-brain barrier model consisting of human astrocytes grown on transwell membrane inserts and co-cultured with human brain capillary endothelial cells. Following 48 h co-culture, we analyzed both endothelial mono-cultures and astrocyte-endothelial co-cultures for expression of tPA and PAI-1 mRNA, protein, and activity. RESULTS AND CONCLUSIONS: There were significant changes for both tPA and PAI-1 mRNA:tPA mRNA levels were decreased in co-cultures (55+/-16% of mono-cultures, p<0.0005) and PAI-1 mRNA levels were increased 144+/-38%, compared to mono-cultures (p<0.005). Co-cultures produced a 54% reduction in tPA protein (12.7+/-3.8 vs. 27.5+/-7.1 ng/ml, p<0.005) and a 24% increase in PAI-1 protein (117.5+/-3.2 vs. 94.9+/-5.9 ng/ml, p<0.0005). TGF-beta neutralizing antibody attenuated the observed changes in both tPA and PAI-1. These data indicate that human astrocytes regulate human brain capillary fibrinolysis in vitro by inhibiting tPA and enhancing PAI-1 expression. This regulation is mediated, in part, by transforming growth factor-beta. Our findings provide further evidence for the role of astrocytes in brain-specific hemostasis regulation.     

水蛭提取液对培养的大鼠脑皮质微血管内皮细胞分泌组织型纤溶酶原激活物和纤溶酶原激活剂抑制物1的影响

目的探讨水蛭提取液(HEL)对培养的大鼠脑皮质微血管内皮细胞分泌组织型纤溶酶原激活物(tPA)、纤溶酶原激活剂抑制物1(PAI-1)的影响。方法建立大鼠大脑皮质微血管内皮细胞培养实验模型。MTT法筛选HEL的有效浓度。检测培养上清液的tPA、PAI-1含量与活性变化,RT-PCR检测经HEL治疗组与生理盐水对照组处理后的微血管内皮细胞tPA与PAI-1的表达,免疫组化检测两组微血管内皮细胞tPA的表达。结果 HEL在一定浓度范围内(0.25～1mg/μl)可促进微血管内皮细胞的生长,有剂量依赖关系(P<0.05)。HEL治疗组较生理盐水对照组能促进培养的大鼠脑皮质微血管内皮细胞分泌tPA,同时提高其活性,促进tPA mRNA的表达及tPA免疫活性表达,且呈剂量依赖性表达增强(P<0.01)。结论 HEL在体外能激活内源性纤溶系统。     

Possible role of plasminogen activator inhibitor 2 in the prevention of the metastasis of gastric cancer tissues

The concentrations of urinary type plasminogen activator (u-PA), plasminogen activator inhibitor 1 (PAI-1), and PAI-2 were measured in gastric cancer tissues and adjacent healthy mucosal tissues. Levels of u-PA, PAI-1 and PAI-2 were higher in cancer than in control tissues. PAI-1 levels were higher together with the progression of cancer however there were no differences in u-PA or PAI-2 levels. Tumors with higher PAI-1 and lower PAI-2 levels tend to metastasize to remote lymph nodes. When the numbers of involved lymph nodes were analyzed, tumors with the large number of metastatic lymph nodes showed higher PAI-1 and lower PAI-2 level. No difference was shown in u-PA levels among these groups. These tendencies were more significant in patients with progressed gastric cancer. These results suggest that tumor with higher PAI-2 levels tend to localize or have less tendency to metastasize to lymph nodes. On the other hand PAI-1 was generally higher in tumor with invasion into nearby tissue or with nodal metastasis.     

Multipotent mesenchymal stromal cells increase tPA expression and concomitantly decrease PAI-1 expression in astrocytes through the sonic hedgehog signaling pathway after stroke (in vitro study)

Multipotent mesenchymal stromal cells (MSCs) increase tissue plasminogen activator (tPA) activity in astrocytes of the ischemic boundary zone, leading to increased neurite outgrowth in the brain. To probe the mechanisms that underlie MSC-mediated activation of tPA, we investigated the morphogenetic gene, sonic hedgehog (Shh) pathway. In vitro oxygen and glucose deprivation and coculture of astrocytes and MSCs were used to mimic an in vivo ischemic condition. Both real-time-PCR and western blot showed that MSC coculture significantly increased the Shh level and concomitantly increased tPA and decreased plasminogen activator inhibitor 1 (PAI-1) levels in astrocytes. Inhibiting the Shh signaling pathway with cyclopamine blocked the increase of tPA and the decrease of PAI-1 expression in astrocytes subjected to MSC coculture or recombinant mouse Shh (rm-Shh) treatment. Both MSCs and rm-Shh decreased the transforming growth factor-β1 level in astrocytes, and the Shh pathway inhibitor cyclopamine reversed these decreases. Both Shh-small-interfering RNA (siRNA) and Glil-siRNA downregulated Shh and Gli1 (a key mediator of the Shh transduction pathway) expression in cultured astrocytes and concomitantly decreased tPA expression and increased PAI-1 expression in these astrocytes after MSC or rm-Shh treatment. Our data indicate that MSCs increase astrocytic Shh, which subsequently increases tPA expression and decreases PAI-1 expression after ischemia.     

Plasminogen activator inhibitor type 2 inhibits cell surface associated tissue plasminogen activator in vitro: potential receptor interactions

Regulation of cellular plasminogen activation is necessary for maintenance of tissue homeostasis. Despite increasing evidence for co-expression of tissue type plasminogen activator (tPA) and plasminogen activator inhibitor type-2 (PAI-2; SERPINB2) under patho/physiological conditions, the inhibition of cell-bound tPA mediated plasminogen activation by PAI-2 has not been addressed. Here we show that PAI-2 can inhibit cell-bound tPA activity in vitro and thus prevent plasmin formation. We also examined the potential involvement in this inhibition of the annexin II heterotetramer (AIIt), one of the many well characterized cell-surface co/receptors for tPA and plasminogen that efficiently promotes plasminogen activation. This receptor was of interest because AIIt has also been shown to directly bind PAI-2. Characterization of these potential interactions using purified protein systems revealed that PAI-2 directly bound AIIt via the p11 (S100A10) subunit. However, PAI-2 prevented AIIt/tPA-mediated plasminogen activation by its classic serpin inhibitory activity rather than through competition with tPA/plasminogen for binding. Further analysis showed that PAI-2 inhibited cell bound tPA-induced plasmin activity in both an AIIt-dependent and -independent manner. These data open new possibilities for further investigations regarding the regulation of cellular plasmin generation in vivo, especially in tissues where PAI-2 and tPA may be co-expressed.     

The kinetics of plasmin inhibition by aprotinin in vivo

INTRODUCTION: The purpose of this study was to estimate, in patients undergoing cardiopulmonary bypass (CPB), the in vivo rates of tissue plasminogen activator (tPA) and plasminogen activator inhibitor 1 (PAI-1) secretion, plasmin generation, fibrin degradation, and plasmin inhibition by aprotinin versus antiplasmin. MATERIALS AND METHODS: Estimates of in vivo rates were based on measured levels of tPA, PAI-1, antiplasmin, plasmin-antiplasmin complex (PAP), total aprotinin, plasmin-aprotinin complex and D-dimer, combined with a computer model of each patient's vascular system that continuously accounted for secretion, clearance, hemodilution, blood loss and transfusion. Plasmin regulation was studied in nine control patients undergoing CPB without aprotinin versus six patients treated with aprotinin. RESULTS: In controls, plasmin-antiplasmin levels rose from a baseline of 3.0+/-0.9 to a peak of 8.1+/-2.7 nmol/L after CPB due to an average 44-fold rise in the plasmin generation rate. This rise in plasmin generation during CPB lead to increased fibrin degradation causing D-dimer levels to increase from a baseline of 1.2+/-0.6 to a peak of 9.7+/-4.4 nmol/L due to an average 74-fold rise in the D-dimer generation rate. During CPB in the aprotinin group, plasmin-antiplasmin levels dropped, plasmin-aprotinin complex levels rose, while D-dimer levels remained unchanged from baseline. Compared to controls, the aprotinin group showed similar rates of plasmin generation during CPB, but an 11-fold faster plasmin inhibition rate and a 10-fold lower D-dimer generation rate. CONCLUSIONS: The rise in plasmin generation and fibrin degradation that occurs during standard CPB is suppressed by the addition of aprotinin, which returns the patient to near baseline fibrin degradation rates during CPB.     

Relationship between maternal and cord blood hemostatic disturbances in preeclamptic pregnancies

Endothelial cell activation or damage is believed to play a key role in preeclampsia (PE) and may underlie the hemostatic changes observed in this syndrome. The aim of this study was to evaluate a relationship between maternal and cord blood hemostatic disturbances in preeclamptic pregnancies. We measured the plasma levels of tissue plasminogen activator (tPA) antigen and of plasminogen activator inhibitor type 1 (PAI-1) antigen, both markers of hemostatic and endothelial function, and fibrin fragment D-dimer. Maternal blood from uncomplicated (n = 42) and PEc pregnancies (n = 44) were collected before delivery, and umbilical cord blood (UCB) immediately after delivery.In preeclamptic cases, UCB presented significantly higher tPA values and significantly lower PAI-1/tPA ratio. Preeclamptic women also presented significantly higher tPA, as well as PAI-1 values, when compared with normal pregnant women; no significant difference was found for D-dimer. In preeclamptic women, proteinuria (a marker of PE severity) correlated positively and significantly with tPA and PAI-1 antigen levels. An inverse relationship between maternal tPA antigen levels and fetal birth weigh in PE was also observed.Our data show that the hemostatic maternal disturbances observed in preeclamptic women have similarities with the UCB circulation, and that endothelial dysfunction is the most plausible underlying cause. Moreover, maternal hemostatic disturbances seem to be associated with the severity of PE. Further studies are needed to strength the values of tPA and PAI-1 as markers of severity in PE.     

Minimally oxidized low-density lipoprotein regulates hemostasis factors of brain capillary endothelial cells

Minimally oxidized low-density lipoprotein (MM-LDL) is a potent atherogenic lipoprotein. We analyzed the effects of MM-LDL on brain capillary endothelial expression of plasminogen activator inhibitor-1 (PAI-1), tissue-type plasminogen activator (tPA), and thrombomodulin (TM). Cultured bovine brain capillary endothelial cells (BEC) incubated with MM-LDL (25 microg/ml) for 24 h showed increased PAI-1 mRNA levels by approximately seven-fold, while tPA and TM mRNA levels were reduced by 84% and 75%, respectively. Moreover, PAI-1 protein levels increased two-fold (16.8+/-7.6 vs. 7.6+/-2.1 ng/ml, p<0.05), whereas tPA protein levels decreased by 45% (1.3+/-0.5 ng/ml vs. 2.3+/-0.7 ng/ml, p<0.05), and TM protein level decreased by 40%. Following incubation with MM-LDL, PAI-1 activity was increased 35% (18.4+/-5.0 vs. 24.8+/-5.2 AU/ml, p<0.05), while TM activity was decreased by 30%. MM-LDL therefore has substantial pro-thrombotic effects on brain capillary endothelial cells, reducing both endothelial fibrinolytic capacity (downregulating tPA while upregulating PAI-1) and anticoagulant function (downregulating TM). These results suggest that MM-LDL may contribute to thrombus formation in the brain.     

Plasminogen activator inhibitor-1 4G/5G polymorphism in breast cancer patients and its association with tissue PAI-1 levels and tumor severity

BACKGROUND: The plasminogen activator inhibitor type 1 (PAI-1) 4G/5G polymorphism may have significance for PAI-1 expression. High levels of PAI-1 in breast cancer patients are associated with a poor prognosis. In this study, we analyzed the influence of the PAI-1 4G/5G polymorphism on tissue PAI-1 levels and its association with tumor severity in women with breast cancer. MATERIAL AND METHODS: We studied 104 women with breast carcinoma (patient group) and 104 healthy age-matched women (control group). In patients and controls, the PAI-1 4G/5G polymorphism was determined by PCR amplification using allele-specific primers. In patients, PAI-1 levels were quantified in breast cancer tissue by using an ELISA. RESULTS: The frequency of the PAI-1 4G allele tended to be higher in patients than in controls (p=0.062). The presence of the 4G allele (4G/5G plus 4G/4G genotypes) was significantly higher among patients with histological grade 3 tumors than among those with grade 1 tumors (p=0.026). Furthermore, patients with the 4G/4G genotype had significantly higher tissue PAI-1 levels than those with the 5G/5G genotype. Moreover, tissue PAI-1 antigen levels were significantly and positively correlated with tumor severity (p=0.003) and tumor size (p=0.009). However, no significant differences in PAI-1 level were observed in relation to menopause, hormone receptor or nodal status. CONCLUSION: Tissue PAI-1 antigen levels and tumor severity seem to be associated with the PAI-1 4G/5G polymorphism. Further studies with a larger number of patients are needed to clarify the influence of this polymorphism in breast cancer.     

Inhibition of plasminogen activation by polymerized ampicillin

The polymerized β-lactam antibiotic ampicillin inhibits the proteolytic activity of human plasmin upon ¹²⁵I-labeled fibrin clots. The inhibition is dose-dependent, with half-maximal inhibition occurring at 1.25 mM of the polymerized antibiotic. Polymerized ampicillin also inhibits binding of plasmin to fibrin, and 38% inhibition of binding occurs at 10 mM of the antibiotic. Furthermore, polymerized ampicillin inhibits the activation of plasminogen by either urokinase-like plasminogen activator (uPA) or tissue type-plasminogen activator (tPA). At 7.5 mM of polymerized ampicillin, the uPA-mediated plasminogen activation is suppressed by 94%, and half-maximal inhibition is obtained at 0.66 mM. The direct activity of uPA on the chromogenic substrate L-pyroglutamyl-glycyl-L-arginine p-nitroanilide hydrochloride (S-2444) is unaffected by polymerized ampicillin levels of up to 10 mM. The inhibitory effects of the polymerized antibiotic on the activation of plasminogen by both uPA and tPA is totally abolished in presence of fibrin. These interactions may serve as a novel model for ligands that enhance the clot-specificity of thrombolytic agents.     

Maximal PAI-1 inhibition in vivo requires neutralizing antibodies that recognize and inhibit glycosylated PAI-1

Plasminogen activator inhibitor-1 (PAI-1) regulates the activity of t-PA and u-PA and is an important inhibitor of the plasminogen activator system. Elevated PAI-1 levels have been implicated in the pathogenesis of several diseases. Prior to the evaluation of PAI-1 inhibitors in humans, there is a strong need to study the effect of PAI-1 inhibition in mouse models.In the current study, four monoclonal antibodies previously reported to inhibit recombinant PAI-1 in vitro, were evaluated in an LPS-induced endotoxemia model in mice. Both MA-33H1F7 and MA-MP2D2 exerted a strong PAI-1 inhibitory effect, whereas for MA-H4B3 and MA-124K1 no reduced PAI-1 activity was observed in vivo. Importantly, the lack of PAI-1 inhibition observed for MA-124K1 and MA-H4B3 in vivo corresponded with the absence of inhibition toward glycosylated mouse PAI-1 in vitro.Three potential N-glycosylation sites were predicted for mouse PAI-1 (i.e. N209, N265 and N329). Electrophoretic mobility analysis of glycosylation knock-out mutants before and after deglycosylation indicates the presence of glycan chains at position N265.These data demonstrate that an inhibitory effect toward glycosylated PAI-1 is a prerequisite for efficient PAI-1 inhibition in mice. Our data also suggest that PAI-1 inhibitors for use in humans must preferably be screened on glycosylated PAI-1 and not on recombinant non-glycosylated PAI-1.     

Heterogeneous glycosylation patterns of human PAI-1 may reveal its cellular origin

The main inhibitor of intravascular fibrinolysis is plasminogen activator inhibitor 1 (PAI-1) which binds to and irreversibly inhibits tissue plasminogen activator (tPA). PAI-1 is present in blood, both in platelets and in plasma, and PAI-1 levels are associated with risk of atherothrombosis. Several tissues express PAI-1 but the source of plasma PAI-1 is not known. We recently found that platelets can de novo synthesize PAI-1 and the amount synthesized in vitro in 24 hours is 35-fold higher than required to maintain normal plasma levels. Recombinant human PAI-1 expressed in different cell types or secreted naturally by human cell lines, exhibit heterogeneous glycosylation patterns. The aim of this study was to investigate the hypothesis that platelets might be the source of plasma PAI-1 and that the cellular source of PAI-1 can be determined by its tissue-specific glycosylation pattern. PAI-1 was isolated from platelets, macrophages, endothelial cells, adipose tissue, as well as plasma from lean and obese subjects. The glycosylation was analyzed by nanoLC-MS/MS. PAI-1 isolated from cell lysates and conditioned media from macrophages, endothelial cells, and adipose tissue expressed heterogeneous glycosylation patterns. By contrast, no glycans were detected on PAI-1 isolated from plasma or platelets from healthy lean individuals. Hence, our data suggest that platelets may be the main source of plasma PAI-1 in lean individuals. Interestingly, plasma PAI-1 from obese subjects had a glycan composition similar to that of adipose tissue suggesting that obese subjects with elevated PAI-1 levels may have a major contribution from other tissues.     

The impact of cardiac ischemia and reperfusion on markers of activated haemostasis and fibrinolysis during cardiopulmonary bypass: comparison of plasma levels in arterial and coronary venous blood

INTRODUCTION: Activation of coagulation and fibrinolysis is common among patients undergoing cardiopulmonary bypass (CPB) surgery. Little is known, however, about the impact of myocardial ischemia and reperfusion on coagulation activation and fibrinolysis in this clinical setting. STUDY DESIGN AND METHODS: We determined the levels of coagulation activation and fibrinolysis markers (CAFM) in 19 patients with severe coronary heart disease (CHD) during CPB surgery. FXIIa, tissue factor (TF), FVIIa, tissue plasminogen activator/plasminogen activator inhibitor-1 complexes (tPA/PAI-1), prothrombin fragments 1+2 (F1+2), D-dimers (DD) and plasmin-plasmin inhibitor complexes (PPI) were measured at baseline, prior to and after cardioplegic myocardial ischemia. Simultaneous blood samples were drawn from the aorta and the coronary sinus to evaluate arteriovenous CAFM plasma level gradients. RESULTS: Myocardial ischemia induced significant increases in gradients of FXIIa and F1+2 levels across the coronary circulation without influencing systemic levels of these markers significantly. Systemic levels of FXIIa, tPA/PAI-1, F1+2, DD and PPI increased significantly during CPB operation. There was a significant linear correlation between FXIIa, FVIIa, F1+2, DD and PPI. CONCLUSIONS: Myocardial ischemia induces contact activation and thrombin generation rather than release of tPA and might thus contribute to postoperative thromboembolic complications. Surgery itself and CPB cause activation of coagulation and fibrinolysis as already described. A significant association between FXIIa, FVIIa, F1+2, DD and PPI suggests a relationship between contact activation, thrombin generation, fibrin formation and fibrinolysis.     

Chronic relapsing experimental allergic encephalomyelitis (CREAE) in plasminogen activator inhibitor-1 knockout mice: the effect of fibrinolysis during neuroinflammation

During neuroinflammation in multiple sclerosis (MS) fibrinogen, not normally present in the brain or spinal cord, enters the central nervous system through a compromised blood–brain barrier. Fibrin deposited on axons is ineffectively removed by tissue plasminogen activator (tPA), a key contributory factor being the upregulation of plasminogen activator inhibitor-1 (PAI-1). Aims: This study investigated the role of PAI-1 during experimental neuroinflammatory disease. Methods: Chronic relapsing experimental allergic encephalomyelitis (CREAE), a model of MS, was induced with spinal cord homogenate in PAI-1 knockout (PAI-1^−/−) and wild type (WT) mice, backcrossed onto the Biozzi background. Results: Disease incidence and clinical severity were reduced in PAI-1^−/− mice, with animals developing clinical signs significantly later than WTs. Clinical relapses were absent in PAI-1^−/− mice and the subsequent reduction in neuroinflammation was coupled with a higher capacity for fibrinolysis in spinal cord samples from PAI-1^−/− mice, in association with increased tPA activity. Axonal damage was less apparent in PAI-1^−/− mice than in WTs, implicating fibrin in both inflammatory and degenerative events during CREAE. Conclusions: PAI-1 is a potential target for therapy in neuroinflammatory degenerative diseases, allowing effective fibrin removal and potentially reducing relapse rate and axonal damage.     

Localization of urokinase-type plasminogen activator, its receptor, and inhibitors in mouse forebrain during postnatal development

Proteolytic enzymes are postulated to play a role in cell migration and synapse organization during brain development. Among these, urokinase-type plasminogen activator (uPA) has been studied in neoplastic and cultured brain cells extensively. We hypothesized that uPA, its receptor, and its inhibitors would be expressed in immature glial and neuronal cells in postnatal mouse forebrain. Immature cortical neurons were immunoreactive for uPA, its receptor, and its substrate plasminogen peaking at the end of postnatal week two, consistent with the postulated role in synaptogenesis. Immunoreactivity for uPA receptor was also observed on astroglial cells in vitro. Neither it nor uPA were convincingly detected in subventricular zone precursor cells, immature white matter or pre-labeled immature cells that had been transplanted into brain. Plasminogen activator inhibitor type 1 immunoreactivity was observed on endothelia up to 12 days age, and type 2 was observed to surround immature cells. We conclude, based on the spatial and temporal distribution of immunoreactivity, that uPA and its receptor may be relatively more important for synaptogenesis, remodeling, and reactive processes than for cell migration in developing mouse brain.