Relationships between Activators and Inhibitors of Plasminogen, and the Progression of Small Abdominal Aortic Aneurysms

OBJECTIVE: plasmin is a common activator of the known proteolytic systems involved in the aneurysmal degradation, and is reported to be associated with the expansion of abdominal aortic aneurysms (AAA). The aim of this study was to study the activating pathways of plasminogen as predictors of the progression of AAA. MATERIALS AND METHODS: one hundred and twelve of 122 male patients with a small AAA (def.: +3cm) were interviewed, examined, had blood samples taken at diagnosis, and scanned annually for 1-5 years (mean 3.5 years), and referred for surgery if the AAA exceeded 5cm in diameter.A random sample of 70 of the 112 cases had plasma levels of urokinase-like-plasminogen activator (uPA), tissue-type-plasminogen activator (tPA), plasminogen-activator-inhibitor-1 (PAI-1), macrophage inhibiting factor (MIF), tumour-growth-factor-beta1 (TGF-beta1), homocysteine, and serum levels of IgA-antibodies against Chlamydia pneumoniae (IgA-CP) and Cotinine (a nicotine metabolite) measured. Spearmans correlation analysis was used for statistics. RESULTS: the annual expansion rate correlated positively with tPA, IgA-CP and S-Cotinine; r =0.37 (p=0.002), 0.29 (p=0.006) and 0.24 (p=0.038), while PAI1, uPA, TGF-beta1, homocysteine, and MIF did not. S-Cotinine did also correlate positively with tPA, r=0.24 (p=0.049). CONCLUSION: the aortic matrix degradation in AAA may be partly caused by an activation of plasminogen by tPA, but apparently not by uPA, which usually dominates matrix degradation. Smoking seems to be a factor for this pathway, while the pathways of IgA-CP and MIF, a new marker of aneurysmal progression, seem different. The latter observations suggest that other proteolytic pathways are involved in the aortic wall degradation in AAA.     

Plasminogen activation in plasma of patients with systemic lupus erythematosus

Summary We measured ₂-plasmin inhibitor-plasmin complexes (PI-PC) in plasma of patients with systemic lupus erythematosus (SLE) to examine the plasminogen activation in SLE. The plasma PI-PC level in 23 patients with SLE was significantly higher than that in 18 normal subjects (P<0.001) and the SLE patients with nephrotic syndrome had higher plasma PI-PC levels than those without nephrotic syndrome (P<0.01). In addition, the plasma PI-PC level was significantly correlated with the level of plasma C3 breakdown products (iC3b/C3dg) in the patients with SLE (r=0.53, P<0.01). These results suggest that plasminogen is activated in plasma of patients with SLE and that the plasminogen activation may be associated with the activation of complement in SLE.     

Cell signalling associated with fibrinolytic ligand binding to human colorectal carcinoma cells

Summary Addition of purified plasmin or plasminogen (0.1 M) to serum-free culture media elevated cellular D-myo-inositol 1,4,5-trisphosphate (InsP ₃) levels in human colorectal carcinoma cells within 1 h to double those of control cells. This was accompanied by decreases in cellular phosphatidylinositol bisphosphate by 40% in cells exposed to fibrinolytic ligands for up to 1 h. The effect was not due to opening of Ca²⁺ channels of the type blocked by 5 M nifedipine, and 100 M EGTA, a Ca²⁺ chelator, did not suppress plasmin's ability to elevate InsP ₃. Binding assays at 4° C with¹²⁵I-labelled plasmin indicated maximum binding within 1 h suggesting that the effects of plasmin may be associated with its cell-binding function. These cells could convert exogenous plasminogen to plasmin with endogenous activation and this was accompanied by a decrease in radioactive phosphatidylinositol well below control levels (13% of control). Our results contribute to evidence for the association of plasmin-binding sites with a signalling system. A cell signalling system indirectly or directly associated with plasmin binding, would permit carcinoma cells to coordinate extracellular fibrinolysis with cell migration and motility through second messengers.Abbreviations InsP ₃ inositol trisphosphate - Ptd phosphatidyl     

纤溶酶-α2抗纤溶酶复合物检测方法的建立和初步临床应用

目的　建立纤溶酶 α2 抗纤溶酶复合物 (PAP)的检测方法。方法　从血浆中纯化PAP作为免疫原制备单克隆抗体 (单抗 ) ,建立双抗体夹心法酶联免疫吸附试验 (ELISA) ,并对该法进行评价。结果　获取了特异针对PAP新抗原的单抗LW 3C10 ,和针对PAP及纤溶酶原 (Plg)的单抗LW 2B9。对PAP的亲和常数分别为 4 6 9× 10 10 mol/L、5 6 2× 10 9mol/L。以它们建立的夹心ELISA检测PAP在 0～ 15 0 μg/L范围内线性良好 ,批内、批间CV分别为 4 0 %～ 5 2 %、11 2 %～ 13 6 % ,回收率为 85%～ 10 5 % ,加入α2 抗纤溶酶 (α2 AP)及纤溶酶 α2 巨球蛋白复合物 (P α2 M)不干扰测定 ,与美国ADI公司PAP试剂盒相关良好 (r=0 96 2 9)。急性髓细胞白血病组、急性心肌梗死组、肝病组、健康老年人组的血浆PAP水平显著高于正常对照组 (P <0 0 0 1)。结论　该法可用于评估纤溶系统激活。     

Breast cancer cell surface annexin II induces cell migration and neoangiogenesis via tPA dependent plasmin generation

Annexin II, an abundant phospholipids binding cell surface protein, binds tPA and functions as a regulator of fibrinolysis. Annexin II also mediates angiogenesis and enhances tumor growth and metastasis. However, the mechanism supporting this role is not known. Using human breast cancer model we show that invasive human breast cancer cells (MDA-MB231) synthesize annexin II and tissue plasminogen activator (tPA). In vitro both annexin II and tPA interacts which in turn convert zymogen plasminogen to reactive enzyme plasmin. Cell surface produced plasmin inhibited the migration of MDA-MB231 cells. Silencing of annexin II gene in MDA-MB231 cells abolished tPA binding therefore inhibited tPA dependent plasmin generation. These annexin II suppressed MDA-MB231 cells showed reduced motility. Immunohistochemical analysis of prediagnosed clinical specimens showed abundant secretion of tPA and expression of annexin II on the surface of invasive human breast cancer cells which correlates with neovascularization of the tumor. Taken together, these data indicate that annexin II may regulate localized plasmin generation in breast cancer. This may be an early event switching breast cancer from the prevascular phase to the vascular phase and thus contributing to aggressive cancer with the possibility of metastasis. The data provide a mechanism explaining the role of annexin II in breast cancer progression and suggest that annexin II may be an attractive target for therapeutic strategies aimed to inhibit angiogenesis and breast cancer.     

Proteomic characterisation of neuronal sphingolipid-cholesterol microdomains: role in plasminogen activation

Sorting of certain membrane proteins requires a mechanism involving rafts, protein-lipid complexes enriched in glycosphingolipids and cholesterol. These microdomains remain at the plasma membrane of different cell types and play a role in signal transduction. Although recent reports have begun to describe molecules associated with rafts, their protein composition remains largely unknown, especially in neuronal cells. To address this question, we have purified detergent-insoluble raft fractions (DRMs) from primary cultures of hippocampal neurons. Bidimensional gel analysis and pharmacological raft lipid manipulation allowed the identification of neuronal raft proteins and their characterisation by MALDI-TOF analysis. Enolases were found among the proteins identified and functional studies demonstrate their participation in plasminogen binding. We also show the specific enrichment in rafts of several other plasminogen binding molecules and the exclusive activation of plasminogen to the protease plasmin in these microdomains. These observations suggest that neuronal rafts may play, in addition to intracellular signaling, a role in extracellular/membrane protein proteolysis.     

Amiloride and urinary trypsin inhibitor inhibit urothelial cancer invasion

OBJECTIVE: To determine the contribution of urokinase-type plasminogen activator (uPA) and plasmin in the invasion of highly invasive urothelial cancer cells. METHODS: We compared expression levels of mRNA and protease activity of uPA and plasmin formation in primary cultures of the noninvasive transitional cell carcinoma, UCT-1, and in the highly invasive type, UCT-2. By using in vitro cell invasion assay system, we evaluated the effects of amiloride and urinary trypsin inhibitor (UTI), which inhibit uPA and plasmin, respectively, on invasion by both cell lines. RESULTS: Expression levels of mRNA, protein, and activities of uPA were significantly higher (p<0.005) and resulted in more plasminogen activation in UCT-2 than in UCT-1. Amiloride and UTI significantly inhibited plasmin formation and the invasion of both cell lines (p<0.001). CONCLUSIONS: High expression levels of mRNA, activities of uPA and high plasmin formation significantly potentiated the invasiveness of urothelial cancer cells. Thus, inhibitors of uPA and plasmin, such as amiloride and UTI, respectively, could be useful therapeutic tools with which to treat urothelial cancer.     

Plasminogen Activator Inhibitor-1 Antisense Oligodeoxynucleotides Abrogate Mesangial Fibronectin Accumulation

Excessive extracellular matrix (ECM) accumulation is the main feature of chronic renal disease including diabetic nephropathy. Plasminogen activator inhibitor (PAI)-1 is known to play an important role in renal ECM accumulation in part through suppression of plasmin generation and matrix metalloproteinase (MMP) activation. The present study examined the effect of PAI-1 antisense oligodeoxynucleotide (ODN) on fibronectin upregulation and plasmin/MMP suppression in primary mesangial cells cultured under high glucose (HG) or transforming growth factor (TGF)-β1, major mediators of diabetic renal ECM accumulation. Growth arrested and synchronized rat primary mesangial cells were transfected with 1 µM phosphorothioate-modified antisense or control mis-match ODN for 24 hours with cationic liposome and then stimulated with 30 mM D-glucose or 2 ng/ml TGF-β1. PAI-1 or fibronectin protein was measured by Western blot analysis. Plasmin activity was determined using a synthetic fluorometric plasmin substrate and MMP-2 activity analyzed using zymography. HG and TGF-β1 significantly increased PAI-1 and fibronectin protein expression as well as decreased plasmin and MMP-2 activity. Transient transfection of mesangial cells with PAI-1 antisense ODN, but not mis-match ODN, effectively reversed basal as well as HG- and TGF-β1-induced suppression of plasmin and MMP-2 activity. Both basal and upregulated fibronectin secretion were also inhibited by PAI-1 antisense ODN. These data confirm that PAI-1 plays an important role in ECM accumulation in diabetic mesangium through suppression of protease activity and suggest that PAI-1 antisense ODN would be an effective therapeutic strategy for prevention of renal fibrosis including diabetic nephropathy.     

Hemostatic Factors in Hypertriglyceridemic Men: Effects of a Fatty Meal before and after Triglyceride-Lowering Treatment with Etofibrate

The aims of this double-blind study were to examine whether in hypertriglyceridemic men the ingestion of a standardized fatty meal alters hemostasis negatively and whether triglyceride-lowering treatment with etofibrate for 6 weeks alters fasting and postprandial hemostasis positively, thus reversing the potential negative effects of a fatty meal on postprandial hemostasis. To answer these questions, we measured markers of hemostasis immediately before a standardized fatty meal, and 4, 6, 8, and 10 hours after the meal in 21 hypertriglyceridemic men both before and after treatment with etofibrate. We found that the concentration of plasmin alpha2antiplasmin complex markedly increased for at least 10 hours after the fatty meal, but that the activation of factor XII and the concentration of prothrombin activation fragment1+2 decreased after the fatty meal. These results on factor XII contradict reported in vitro data. Triglyceride-lowering treatment with etofibrate in 10 of these men for 6 weeks increased fasting and postprandial protein C and plasminogen and also slightly decreased the activation of fXII; however, it did not reverse the postprandial increase of PAP or change the decrease of prothrombin activation fragment1+2. Our findings indicate that postprandial lipoproteins alter markers of hemostasis positively in an antithrombotic and profibrinolytic direction. In addition, triglyceride-lowering treatment with etofibrate only slightly improves markers of fasting and postprandial hemostasis in an antithrombotic and profibrinolytic direction.     

Inactivation of harmful tumour-associated proteolysis by nanoparticulate system

The primary aim in cancer therapy is to deliver anti-cancer drugs to their specific molecular targets in the tumour. Here we present a system composed of poly(d,l-lactide-co-glycolide) nanoparticles, cytokeratin specific monoclonal antibody and cystatin, a potent protease inhibitor, that can neutralize the excessive proteolytic activity associated with the invasive and metastatic potential of breast tumour cells. The antibody provides specific targeting of the delivery system to invasive breast epithelial cells and, additionally, prevents the generation of plasmin, a central extracellular protease involved in malignant progression. Polymeric nanoparticles rapidly enter the targeted cells and release the inhibitor cargo within the endosomes/lysosomes. The inhibitor is capable to inactivate lysosomal cysteine proteases, in particular cathepsin B, which is involved in the degradation of extracellular matrix inside the tumour cells. Our approach, which combines nanoparticulate delivery system with the inhibitory potential against extracellular and intracellular proteases, may improve the efficacy of therapy in patients with breast tumours compared to the application of individual protease inhibitors.