Tissue-type Plasminogen Research Articles

Polyphosphate nanoparticles enhance the fibrin stabilization by histones more efficiently than linear polyphosphates.

Beyond the three-dimensional fibrin network, the mechanical and lytic stability of thrombi is supported by the matrix of neutrophil extracellular traps (NETs) composed of polyanionic DNA meshwork with attached proteins including polycationic histones. Polyphosphates represent another type of polyanions, which in their linear form are known to enhance the fibrin stabilizing effects of DNA and histones. However, in vivo polyphosphates are also present in the form of nanoparticles (PolyP-NP), the interference of which with the fibrin/NET matrix is poorly characterized. To compare the effects of linear and nanoparticulate polyphosphates, and their combinations with relevant NET components (DNA, histone H3) on fibrin formation, structure, and lysis in in vitro assays focusing on histone-polyphosphate interactions. Transmission electron microscopy and dynamic light scattering for stability of the PolyP-NP preparations. Turbidimetry for kinetics of fibrinogen clotting by thrombin and fibrin dissolution by tissue-type plasminogen activator/plasminogen. Scanning electron microscopy for fibrin structure. Surface plasmon resonance for strength of histone-PolyP interactions. Both linear PolyP and PolyP-NP accelerated the fibrin formation and slowed down its dissolution and these effects were strongly dependent on the number of individual PolyP particles and not on their size. Addition of DNA did not modify significantly the PolyP-NP effects on fibrin formation and lysis. Both linear and nanoparticulate PolyP counteracted the effect of histone in the acceleration of fibrinogen clotting by thrombin. PolyP-NP, but not linear PolyP enhanced the prolongation of lysis time in fibrin containing histone and caused more pronounced thickening of the fibrin fibers than the linear form. Finally, PolyP-NP bound weaker to histone than the linear form. The interaction of PolyP with histone was a stronger modulator of fibrin formation and lysis than its interaction with DNA. In addition, the PolyP nanoparticles enhanced the thrombus stabilizing effects of histone more effectively than linear PolyP.

Changes in Biomarkers of Coagulation, Fibrinolytic, and Endothelial Functions for Evaluating the Predisposition to Venous Thromboembolism in Patients With Hereditary Thrombophilia.

The changes in the coagulation, fibrinolytic, and endothelial functions are correlated with the pathophysiology of the thromboembolic diseases during acute illness. However, these changes in patients with hereditary thrombophilia who were not in the acute stage of venous thromboembolism (VTE) are unclear. A panel of 4 biomarkers, including thrombin–antithrombin complex (TAT), plasmin-α2-plasmin inhibitor complex (PIC), tissue-type plasminogen activator/plasminogen activator inhibitor-1 complex (t-PAIC), and soluble thrombomodulin (sTM), were assayed in 100 healthy controls and 100 patients with thrombophilia. Although significantly higher concentrations of TAT, PIC, t-PAIC, and sTM were observed in patients with thrombophilia than in healthy controls, 70 patients showed absolutely normal levels of the above 4 biomarkers. Among the other 30 patients who had at least 1 biomarker out of the corresponding reference interval, 26 of them presented elevated PIC with or without increased TAT. Except for sTM, other 3 biomarkers did not show significant differences in patients with previous VTE compared to those without. Patients with single episode of VTE had obviously lower t-PAIC than those with multiple episodes of VTE, whereas the levels of TAT, PIC, and sTM were unassociated with the number of thrombosis episodes. Most thrombophilia patients who were not in the acute stage of VTE showed normal coagulation, fibrinolytic, and endothelial functions. Thus, we were unable to show that the one-time response of this panel was clinically helpful in determining thrombosis risk in thrombophilia individuals. Future studies should focus on the dynamic monitoring during the chronic phase of VTE to offer special advantages for patients with thrombophilia.

Circulating Plasminogen Activator Inhibitor-1 (PAI-1) Is Reduced By In Vivo Thrombin Generation and Subsequent Formation of Activated Protein C (APC)

Background: APC has been suggested to contribute to a hyperfibrinolytic state in various acquired coagulation disorders, including coagulopathies induced by trauma and sepsis. However, in vivo evidence for the proposed underlying mechanism of proteolytic degradation of PAI-1 by APC remains inconclusive. Recently, we have shown increased APC generation in response to in vivo thrombin formation in carriers of the factor V Leiden mutation (FVL). In this approach of stimulated hemostasis activity pattern evaluation (SHAPE), in vivo thrombin formation was triggered by low-dose administration of recombinant activated factor VII (rFVIIa). Aim of the present study was to investigate the resulting effects on the fibrinolytic system. Methods: The study population consisted of 30 FVL carriers (thereof 13 with a history of venous thromboembolism) and 15 healthy controls. Blood samples were collected immediately before and during a period of 8 hours following injection of 15 µg/kg rFVIIa. Plasma levels of APC were quantified using an oligonucleotide-based enzyme capture assay (OECA). Other monitored parameters included plasma levels of prothrombin activation fragment 1+2 (F1+2), tissue-type plasminogen (t-PA), PAI-1, plasminogen, α2-antiplasmin, plasmin-α2-antiplasmin complexes (PAP), soluble fibrin monomers, d-dimer, and thrombin-activatable fibrinolysis inhibitor (TAFI). Results: At baseline, FVL carriers showed higher median levels of APC in comparison to the controls (1.39 vs. 0.86 pmol/L, P=.001), higher PAI-1 levels (30.1 vs. 15.3 ng/mL, P=.002) and lower plasminogen levels (94.8 vs. 110.4%, P=6·10-4). The other baseline parameters did not differ significantly. In both cohorts a comparable increase of F1+2 was observed after administration of rFVIIa, whereas APC increased more (P=.004) in FVL carriers (by 6.40 pmol/L) than in healthy controls (by 2.14 pmol/L), Concurrently, median PAI-1 levels decreased more (P=.007) in FVL carriers (by 19.8 pmol/L) than in healthy controls (by 8.0 pmol/L) (Figure 1). TAFI levels decreased temporarily, from 104.8 to 94.4% (P=.007) in FVL carriers and from 105.0 to 92.1% (P=2·10-4) in healthy controls. PAP levels increased from 164 to 209 ng/mL (P<10-4) in FVL carriers and from 154 to 189 ng/mL (P=.023) in the control group. The extent of these changes did not differ between the two cohorts. D-dimer level increased only in FVL carriers, from 0.34 to 0.41 mg/L (P=.008). t-PA and the other parameters did not show significant changes after rFVIIa administration. Conclusion: Increased APC formation rates in FVL carriers were associated with a greater decline of PAI-1 levels in the absence of interfering changes in t-PA levels. These data provide further in vivo evidence that APC down-regulates PAI-1. Overall, the SHAPE approach utilized here does not induce a significant profibrinolytic response, even in patients with thrombophilia. Disclosures Reda: Grifols: Honoraria; Shire: Honoraria. Winterhagen:SOBI: Honoraria. Berens:Pfizer: Honoraria; Shire: Honoraria; Biotest: Honoraria; CSL Behring: Honoraria; Novo Nordisk: Honoraria; Baxter: Honoraria. Müller:NovoNordisk: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; Siemens: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees. Oldenburg:CSL Behring: Consultancy, Research Funding, Speakers Bureau; NovoNordisk: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Research Funding, Speakers Bureau; Swedish Orphan Biovitrum: Consultancy, Speakers Bureau; Grifols: Consultancy, Speakers Bureau; Takeda (Shire): Consultancy, Research Funding, Speakers Bureau; Chugai: Consultancy, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Biotest: Consultancy, Research Funding, Speakers Bureau; Octapharma: Consultancy, Research Funding, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau. Rühl:CSL Behring: Honoraria; Bayer: Honoraria; Shire: Honoraria; SOBI: Honoraria; Sanofi Genzyme: Honoraria; Grifols: Honoraria.

Plasmin‐dependent proteolysis of tissue factor pathway inhibitor in a mouse model of endotoxemia

The development of a procoagulant state in sepsis, owing to aberrant expression of tissue factor (TF) and a sharp decrease in the level of its major inhibitor, TF pathway inhibitor (TFPI), could lead to microthrombotic organ failure. The mechanism for the decline in TFPI activity in the lung could involve plasmin-mediated cleavage of the inhibitor. To investigate the effect of plasmin generation on lung-associated TFPI activity, in normal conditions and during infusion of endotoxin (lipopolysaccharide [LPS]) in mice. Plasmin generation and TFPI activity were assayed in the lungs of mice deficient in tissue-type plasminogen (Plg) activator (t-PA) or Plg, at 2 h after LPS or saline injection. The sharp loss of lung-associated TFPI activity at 2 h after LPS challenge paralleled the abrupt increase in plasmin generation. TFPI activity was significantly retained in both t-PA(-/-) and Plg(-/-) mice, which are unable to generate plasmin. The increased plasmin generation during the early stages of sepsis could cleave/inactivate TFPI and thus lead to thrombotic complications.

Association of plasma t-PA and PAI-1 with carotid atherosclerosis in patients with type 2 diabetes

The association of plasma tissue type plasmingogen activator (t-PA) and plasminogen activator inhibitor (PAI-1) with carotid atherosclerosis in patients with type 2 diabetes was explored.The subjects with type 2 diabetes (n =91) and control subjects (n =30) were enrolled for this study.According to carotid artery intima media thickness in Color Doppler Ultrasonography,the subjects with type 2 diabetes were divided into four groups:normal carotid group(group 1,n =25),lining thickening group (group 2,n =30),stable plaques group (group 3,n =23),carotid stenosis group(group 4,n =13).Plasma t-PA and PAI-1 levels were measured by ELISA.Compared with the control group,there was no significant change in the levels of plasma t-PA and PAI-1 in group 1 (P＞0.05),plasma t-PA activity was decreased significantly(P＜0.05) and PAI-1 activity increased obviously (P＜0.05) in group 2,group 3,and group 4.Carotid atherosclerosis degree was negatively correlated with t-PA(r=-0.723,P＜0.01) and positively correlated with PAI-1 (r=0.851,P＜0.01).The results suggested that the abnormal fibrinolysis function may take part in the development of atherosclerosis of carotid arteries in patients with type 2 diabetes. Key words: Tissue type plasmingogen activator; Plasminogen activator inhibitor-1 ; Carotid atherosclerosis; Diabetes mellitus, type 2

Promotor polymorphisms of plasminogen activator inhibitor-1 and other thrombophilic genotypes in cerebral venous thrombosis: a case-control study in adults

Plasminogen activator inhibitor 1 (PAI-1) is the main inhibitor of tissue-type and urokinase-type plasminogen activator. A 4G/5G polymorphism in the promoter region of the PAI-1 gene has been reported to enhance the plasma levels of PAI-1. In particular, the 4G allele (guanosine deletion) has been linked with increased plasma PAI-1 levels, which may lead to impaired activity of the fibrinolytic system, thus increasing the incidence of thrombotic events. The aim of this case-control study was to analyze whether variants of the PAI-1 promotor genotype 4G/4G, 4G/5G and 5G/5G, in particular the 4G/5G-variant, constitute an independent risk factor of cerebral venous thrombosis (CVT). A total of 136 consecutive patients with proven CVT were compared to 1,054 DNA specimens of healthy controls from a population-based cohort. PAI-1 promotor polymorphisms were evaluated using polymerase chain reaction. No significant association of CVT with PAI-1 4G/5G was found in either the additive (OR 1.04; 95 % CI 0.78-1.38) or in the dominant model (OR 1.24; 95 % CI 0.72-2.13). Also, the prevalence of the other genotypes (4G/4G and 5G/5G) in patients was not significantly different from controls. When considering the variants of the PAI-1 promoter genotype in combination with known genetical thrombophilias, no differences were found either. As was expected, the prothrombin (G20210A) genotype was confirmed as an independent risk factor for CVT. We conclude that the 4G allele of the PAI-1 polymorphism does not increase the risk of CVT in adults.

Proteinase-activated receptors in the endometrium and endometriosis

Proteinase-activated receptors (PARs) are G protein-coupled receptors activated by various proteinases. PARs play important roles in haemostasis, thrombosis, and inflammation. PAR₁ and PAR₂ are expressed in endometrial cells from the eutopic endometrium and endometriotic cells derived from endometriotic lesions. A typical activator of PAR₁, thrombin, and a typical activator of PAR₂, tryptase, are produced in the endometrium as well as endometriotic lesions. PAR₁ activation in endometrial stromal cells induces production of vascular endothelial growth factor and matrix metalloproteinases, and increases activities of tissue-type and urokinase-type plasminogen activator. PAR₂ activation in endometrial stromal cells stimulates interleukin (IL)-8 and stem cell factor production and proliferation of the cells. PAR₁ activation in endometriotic stromal cells induces production of IL-8, monocyte chemotactic protein-1, and cyclooxygenase-2, and proliferation of the cells. PAR₂ activation in endometriotic stromal cells increases secretion of IL-6 and IL-8, and the number of the cells. These findings indicate a wide range of function of PAR₁ and PAR₂ in the endometrium and endometriosis, and suggest PAR₁ and PAR₂ as possible therapeutic targets for endometriosis.

TGF-β1 Target Genes in Human Fibrotic Disease: From Molecular Mechanisms to Clinically-Relevant Therapies

Interstitial fibrosis is an end-stage pathophysiological feature of several clinically-important diseases involving the pulmonary, hepatorenal and cardiovascular systems and a common response to chronic injury, non-resolving inflammation or long-standing metabolic disease. Increased deposition of extracellular matrix (e.g., fibronectin, collagen), produced by persistently activated myofibroblasts, and attenuated matrix degradation results in disease progression. Disruption of normal tissue architecture eventually culminates in organ failure. Matrix turnover, in the context of both normal and abnormal wound repair, is regulated by several factors among which the most prominent ones are members of the transforming growth factor-β (TGF-β) superfamily. TGF-β1 stimulates fibronectin and collagen synthesis/accumulation at the wound site while impacting the extent and locale of plasmin/matrix metalloproteinaseinitiated stromal remodeling. This pericellular proteolytic cascade is highly-dependent on the generation of plasmin by urokinase (uPA) and tissue-type (tPA) plasminogen activators that, in turn, are subject to regulation by plasminogen activator inhibitor-1. In the normal response to injury, PAI-1 titrates the uPA/tPA/plasmin/MMP system to facilitate repair. Under pathologic conditions, excessive levels of PAI-1 at the site of injury suppress proteolytic activity promoting ECM accumulation and development of fibrosis. The continued clarification of mechanisms that control expression of disease-causative factors, such as PAI-1, will likely lead to the development of gene-specific therapies to inhibit the initiation and progression of the fibrotic process as well as to address the more difficult clinical challenge of reversal of established disease.

Neuronal Nitric Oxide Synthase (nNOS)/NO, An Accelerator of Melanoma Progression, is a Potential Target for Chemoprevention

Neuronal Nitric Oxide Synthase (nNOS)/NO, An Accelerator of Melanoma Progression, is a Potential Target for Chemoprevention

Anticoagulant and antithrombotic activity of a new peptide pENW (pGlu-Asn-Trp)

The aim was to test a newly discovered oligopeptide, pENW (pGlu-Asn-Trp), for its anticoagulant and antithrombotic activity in vivo, and try to investigate its underlying mechanisms. We measured coagulation time by the glass slide method and bleeding time by cutting of mice tails. The thrombosis models employed here included an arterio-venous shunt model and inferior vena ligation model. An ELISA (enzyme-linked immunosorbent assay) was used to analyse t-PA/PAI (tissue-type plasminogen activator/plasminogen activator inhibitor) in the blood drawn from the rats with thrombosis. The ultrastructural changes of the endothelium in the vessels developing thrombosis were observed under a transmission electron microscope. We found that pENW-treated mice exhibited a prolonged coagulation time in a dose-dependent manner, but not an extended haemorrhage time. On the other hand, pENW significantly inhibited thrombus formation in both arterio-venous shunt models and inferior vena ligation models. Plasma t-PA/PAI was significantly higher as measured by ELISA. Transmission electron microscope photos of pENW-treated groups also displayed a better condition than model controls, with less erythrocytes in the vascular lumens. In addition, pENW concentration-dependently inhibited aggregation of platelets induced by ADP (adenosine 5'-diphosphate sodium salt) in rabbit platelet-rich plasma. These findings support the suggestion that pENW possesses antithrombotic activity and could be a promising drug in the prevention and treatment of unwanted clot formation.

Anticoagulant and antithrombotic activity of a new peptide pENW (pGlu-Asn-Trp)

Abstract Objectives The aim was to test a newly discovered oligopeptide, pENW (pGlu-Asn-Trp), for its anticoagulant and antithrombotic activity in vivo, and try to investigate its underlying mechanisms. Methods We measured coagulation time by the glass slide method and bleeding time by cutting of mice tails. The thrombosis models employed here included an arterio-venous shunt model and inferior vena ligation model. An ELISA (enzyme-linked immunosorbent assay) was used to analyse t-PA/PAI (tissue-type plasminogen activator/plasminogen activator inhibitor) in the blood drawn from the rats with thrombosis. The ultrastructural changes of the endothelium in the vessels developing thrombosis were observed under a transmission electron microscope. Key findings We found that pENW-treated mice exhibited a prolonged coagulation time in a dose-dependent manner, but not an extended haemorrhage time. On the other hand, pENW significantly inhibited thrombus formation in both arterio-venous shunt models and inferior vena ligation models. Plasma t-PA/PAI was significantly higher as measured by ELISA. Transmission electron microscope photos of pENW-treated groups also displayed a better condition than model controls, with less erythrocytes in the vascular lumens. In addition, pENW concentration-dependently inhibited aggregation of platelets induced by ADP (adenosine 5′-diphosphate sodium salt) in rabbit platelet-rich plasma. Conclusions These findings support the suggestion that pENW possesses antithrombotic activity and could be a promising drug in the prevention and treatment of unwanted clot formation.

Increased soluble urokinase plasminogen activator receptor (suPAR) is associated with thrombosis and inhibition of plasmin generation in paroxysmal nocturnal hemoglobinuria (PNH) patients

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired genetic disorder of the bone marrow that produces intravascular hemolysis, proclivity to venous thrombosis, and hematopoietic failure. Mutation in the PIG-A gene of a hematopoietic stem cell abrogates synthesis of glycosylphosphoinositol (GPI) anchors and expression of all GPI-anchored proteins on the surface of progeny erythrocytes, leukocytes, and platelets. Urokinase plasminogen activator receptor (uPAR), a GPI-linked protein expressed on neutrophils, mediates endogenous thrombolysis through a urokinase-dependent mechanism. Here we show that membrane GPI-anchored uPAR is decreased or absent on granulocytes and platelets of patients with PNH, while soluble uPAR (suPAR) levels are increased in patients' plasma. Serum suPAR concentrations correlated with the number of GPI-negative neutrophils and were highest in patients who later develop thrombosis. In vitro, suPAR is released from PNH hematopoietic cells and from platelets upon activation, suggesting that these cells are the probable source of plasma suPAR in the absence of GPI anchor synthesis and trafficking of uPAR to the cell membrane. In vitro, the addition of recombinant suPAR results in a dose-dependent decrease in the activity of single-chain urokinase. We hypothesized that suPAR, prevents the interaction of urokinase with membrane-anchored uPAR on residual normal cells.

Antithrombin inhibits bronchoalveolar activation of coagulation and limits lung injury during Streptococcus pneumoniae pneumonia in rats*

Alveolar fibrin deposition is a hallmark of pneumonia. It has been proposed that natural inhibitors of coagulation, including activated protein C, antithrombin, and tissue factor pathway inhibitor, exert lung-protective effects via anticoagulant and possibly anti-inflammatory pathways. We investigated the role of these natural anticoagulants in Streptococcus pneumoniae pneumonia. A controlled in vivo laboratory study. Research laboratory of a university hospital. Total of 98 male Sprague-Dawley rats. Rats were challenged intratracheally with S. pneumoniae (serotype 3, 10(6) colony forming units), inducing pneumonia. Rats were randomized to intravenous treatment with normal saline, activated protein C, antithrombin, tissue factor pathway inhibitor, heparin, or tissue-type plasminogen activator. Rats infected with S. pneumoniae had increased thrombin-antithrombin complexes in bronchoalveolar lavage fluid, with decreased levels of antithrombin activity and fibrin degradation products. Administration of activated protein C, antithrombin, and tissue factor pathway inhibitor significantly limited these procoagulant changes. Furthermore, antithrombin treatment resulted in less bacterial outgrowth of S. pneumoniae and less histopathologic damage in lungs. Anticoagulant treatment attenuates pulmonary coagulopathy during S. pneumoniae pneumonia. Antithrombin seems to exert significant lung-protective effects in pneumococcal pneumonia in rats.

Biology of the peritoneum in normal homeostasis and after surgical trauma

The peritoneum is a serous membrane, which has a protective function for the contents of the abdominal cavity. It maintains homeostasis by allowing exchange of molecules and production of peritoneal fluid, thus providing an environment in which intra-abdominal organs can function properly. When traumatized, whether by surgery or due to inflammatory processes, a series of responses come into action to regenerate the injured part of the peritoneum. The inflammatory reaction causes influx of inflammatory cells but also activates resident mesothelial cells, ultimately leading to a fibrinous exudate. Depending on the severity of the trauma this exudate is transient due to fibrinolysis, or becomes more dense as a result of fibroblasts persisting, leading to fibrinous adhesions. A pivotal role is taken by the enzyme plasmin and its promotors and inhibitors; it is mainly the tissue-type plasminogen activator/plasminogen activator inhibitor ratio which determines the rate of fibrinolysis and therefore the rate of adhesion formation. The rate of injury determines the rate and extent of the inflammatory response to that injury; in its turn the inflammatory reaction determines the extent of adhesion formation. One should realize this when performing intra-abdominal surgery, which is in fact operating inside the peritoneal organ.

Autologous fibrin-coated small-caliber vascular prostheses improve antithrombogenicity by reducing immunologic response

We have recently developed a thrombin-free fibrin-coated vascular prosthesis that has a high performance rate in producing graft antithrombogenicity. We hypothesized that autologous, compared with xenologous, fibrin coatings could improve the antithrombogenicity of grafts by reducing immunologic response. Autologous fibrin-coated vascular prostheses and/or xenologous fibrin-coated vascular prostheses (internal diameter, 2 mm; length, 2.5 cm) were implanted in the bilateral carotid arteries of 50 Japanese white rabbits. They were classified into 2 groups by the selection of grafts in the individual: group I (autologous fibrin-coated vascular prosthesis and xenologous fibrin-coated vascular prosthesis); and group II (group IIa: both autologous fibrin-coated vascular prostheses, or group IIx: both xenologous fibrin-coated vascular prostheses). During a maximum of 180 days after implantation, we evaluated the thrombotic, inflammatory, and immunologic responses associated with both types of graft. All grafts were patent at each end point. In group I, both platelet deposition and anti-graft antibodies in autologous fibrin-coated vascular prostheses were significantly less than those in xenologous fibrin-coated vascular prostheses until postoperative day 30. At postoperative day 10, there were significantly fewer CD45-positive infiltrating cells in autologous fibrin-coated vascular prostheses, and intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and nuclear factor-kappa B expression in autologous fibrin-coated vascular prostheses were less than those in xenologous fibrin-coated vascular prostheses. The neointimal hyperplasia in autologous fibrin-coated vascular prostheses was significantly decreased at postoperative day 180. In group II, serial changes of serum levels of immunoglobulin M, immunoglobulin G, interleukin-1beta, and tissue-type plasminogen activator/plasminogen activator inhibitor-1 ratio in autologous fibrin-coated vascular prostheses were significantly less than those in xenologous fibrin-coated vascular prostheses. In both grafts, platelet deposition significantly correlated with serum immunoglobulin G level and tissue-type plasminogen activator/plasminogen activator inhibitor-1 ratio. These findings suggest that autologous fibrin coating in thrombin-free fibrin-coated vascular prostheses improve antithrombogenicity by reducing immunologic response and have a potential for clinical use in hybrid small-caliber vascular grafts.

Regulation of plasminogen activators in human thyroid follicular cells and their relationship to differentiated function

Human thyroid cells in culture take up and organify (125)I when cultured in TSH (acting through cAMP) and insulin. They also secrete urokinase (uPA) and tissue-type (tPA) plasminogen activators (5-100 IU/10(6)cells/day). TSH and insulin both decreased secreted PA activity (PAA), uPA and tPA protein and their mRNAs. Autocrine fibroblast growth factor increased secreted PAA and inhibited thyroid cell (125)I uptake. Epidermal growth factor (EGF) and the protein kinase C (PKC) activator, TPA significantly increased PAA and inhibited thyroid differentiated function, (TPA > EGF). For TPA, effects were rapid, increased PAA secretion and decreased (125)I uptake being seen at 4 h whereas for EGF, a 24 h incubation was required. qRT-PCR showed significantly increased mRNA expression of uPA with lesser effects on tPA. Aprotinin, which inhibits PAA, increased (125)I uptake but did not abrogate the effects of TPA and EGF. The MEKK inhibitor, PD98059 partially reversed the effects of EGF and TPA on PAA, and largely reversed the effects of EGF but not TPA on differentiated function. PKC inhibitors bisindoylmaleimide 1, and the specific PKCbeta inhibitor, LY379196 completely reversed the effects of TPA on (125)I uptake and PAA whereas EGF effects were unaffected. TPA inhibited follicle formation and this effect was blocked by LY379196 but not PD98059. We conclude that in thyroid cells, MAPK activation inversely correlates with (125)I uptake and directly correlates with PA expression, in contrast to the effects of cAMP. TPA effects on iodide metabolism, dissolution of follicles and uPA synthesis are mediated predominantly through PKCbeta whereas EGF exerts its effects through MAPK but not PKCbeta.

Proteolytic Processing of the Laminin α3 G Domain Mediates Assembly of Hemidesmosomes but Has No Role on Keratinocyte Migration

Laminin-5 (Lm5), the major adhesion ligand of basal epithelial cells, undergoes complex extracellular proteolytic processing that influences cell adhesion and migration. In tumor cell lines, the proteolytic truncation of the C-terminal G domain of the Lm alpha3 chain induces assembly of hemidesmosomes and downregulates cell migration. To define the biological functions of the alpha3 G domain processing in physiological conditions, we have expressed a series of mutant alpha3 complementary DNA in human primary alpha3-null keratinocytes immortalized by human papillomavirus E6E7 (HKalpha3 cells). Using monolayer and organotypic cell cultures we show that: (1) the hinge region between subdomains G3 and G4 carries the proteolytic cleavage sites; (2) nucleation of the hemidesmosomal proteins is independent of the proteolytic maturation of the alpha3 G domain, whereas formation of mature hemidesmosomes relies on proteolytic cleavage of alpha3; and (3) the proteolytic processing plays no role in cell migration, which suggests that nucleation of hemidesmosomal structures in culture does not reflect the migratory potential of the epithelial cells. Our results also demonstrate that HKalpha3 cells are a unique model system, which will be useful to dissect the functions and molecular interactions of Lm5.

In uremia, plasma levels of anti-protein C and anti-protein S antibodies are associated with thrombosis

Vascular access thrombosis is an important cause of morbidity in patients with end-stage renal failure on maintenance hemodialysis (MHD). However, little is known about its risk factors. The present study was undertaken to evaluate the role of coagulation factors, fibrinolytic factors, and anti-phospholipid antibodies (aPL). In particular, we have evaluated the role of anti-protein C and anti-protein S antibodies in patients on MHD with and without thrombosis because no data are available in the literature. The study group comprised 30 patients with thrombotic complications (TC), 40 patients matched for age, sex, and dialytic age with no thrombotic complications (NTC) and 400 controls. We have measured: anti-protein C antibodies, anti-protein S antibodies, anticardiolipin antibodies (ACA), anti-beta2-glycoprotein antibodies (beta2-GPI), and anti-prothrombin antibodies (aPT), along with prothrombin time, fibrinogen, plasminogen, protein C, protein S, anti-thrombin III, APC-resistance test, D-dimer, tissue-type plasminogen's activator, plasminogen activator inhibitor-1 (PAI-1), prothrombin fragment 1+2, factors of the intrinsic and extrinsic pathway, C-reactive protein, and homocysteine. There were no significant differences between groups for prothrombin time, fibrinogen, plasminogen, protein C, protein S, anti-thrombin III, activated protein C (APC) resistance, D-dimer, tPA, C-reactive protein, Factors II, X, and VII. The anti-beta2-GP1 and aPT were elevated in both TC and NTC patients, compared to the control group. Significant differences between TC and NTC groups were found for anti-protein C and anti-protein S antibodies, ACA-IgM, PAI-1, Factor VIII, prothrombin fragments 1+2, and homocysteine. The most novel finding was a significant elevation of anti-protein C antibodies and anti-protein S antibodies in the TC group (i.e., in patients on MHD with thrombosis of vascular access). It indicates that other pathogenetic mechanisms in addition to endothelial damage may cause hypercoagulability in uremia.

Plasminogen interaction with Trypanosoma cruzi

The ability of Trypanosoma cruzi to interact with plasminogen, the zimogenic form of the blood serin protease plasmin, was examined. Immunohistochemistry studies revealed that both forms, epimastigotes and metacyclic trypomastigotes, were able to fix plasminogen in a lysine dependant manner. This interaction was corroborated by plasminogen activation studies. Both forms of the parasite enhanced the plasminogen activation by tissue-type plasminogen activator. The maximal enhancements obtained were 15-fold and 3.4-fold with epimastigotes and metacyclic trypomastigotes, respectively, as compared to plasminogen activation in absence of cells. Ligand-blotting analysis of proteins extracted with Triton X-114 from a microsomal fraction of epimastigotes revealed at least five soluble proteins and one hydrophobic protein able to bind plasminogen.

Effect of blood passage through the pulmonary circulation on fibrinolytic parameters.

The lung vasculature bed has a unique fibrinolytic potential, which has not yet been completely elucidated. We investigated the effect of blood passage through the pulmonary circulation on the values of fibrinolytic parameters in plasma. Forty-seven patients (16 women, 31 men, mean age 54 years, range 21-67 years) who had undergone elective cardiac catheterization were included in the study. The blood samples were taken simultaneously from the right atrium and the left ventricle. The following fibrinolytic parameters were measured: tissue-type plasminogen (t-PA) antigen and activity, plasminogen activator inhibitor-1 (PAI-1) antigen and activity, and euglobulin clot lysis time (ECLT). No difference was found between the samples obtained from the right atrium and the left ventricle with respect to t-PA antigen: 8.1 (6.7-11.3) vs 8.4 (5.9-11.0) ng/ml; t-PA activity: 92 (5-680) vs 62 (32-696) IU/ml; PAI-1 antigen: 8.4 (5.5-14.3) vs 8.7 (6.2-13.1) ng/ml; and ECLT: 5.5 (4.1-9.1) vs 5.6 (4.1-8.5) 1 000/min. In contrast, PAI activity decreased significantly: 7.9 (5.8-10.3) vs 7.4 (6.0-10.4) IU/ml, P < 0.01. Patients with and without pulmonary hypertension did not differ in any of measured parameters, either in the right atrium or in the left ventricle. These results show that under basal conditions fibrinolytic activity which is not attributed to t-PA is elevated in lung vasculature. Further, basal fibrinolytic activity in the lungs is not influenced by pulmonary hypertension.