Active Form Plasmin Research Articles

Lysyl oxidase-like 4 promotes the invasiveness of triple-negative breast cancer cells by orchestrating the invasive machinery formed by annexin A2 and S100A11 on the cell surface.

Our earlier research revealed that the secreted lysyl oxidase-like 4 (LOXL4) that is highly elevated in triple-negative breast cancer (TNBC) acts as a catalyst to lock annexin A2 on the cell membrane surface, which accelerates invasive outgrowth of the cancer through the binding of integrin-β1 on the cell surface. However, whether this machinery is subject to the LOXL4-mediated intrusive regulation remains uncertain. Cell invasion was assessed using a transwell-based assay, protein-protein interactions by an immunoprecipitation-Western blotting technique and immunocytochemistry, and plasmin activity in the cell membrane by gelatin zymography. We revealed that cell surface annexin A2 acts as a receptor of plasminogen via interaction with S100A10, a key cell surface annexin A2-binding factor, and S100A11. We found that the cell surface annexin A2/S100A11 complex leads to mature active plasmin from bound plasminogen, which actively stimulates gelatin digestion, followed by increased invasion. We have refined our understanding of the role of LOXL4 in TNBC cell invasion: namely, LOXL4 mediates the upregulation of annexin A2 at the cell surface, the upregulated annexin 2 binds S100A11 and S100A10, and the resulting annexin A2/S100A11 complex acts as a receptor of plasminogen, readily converting it into active-form plasmin and thereby enhancing invasion.

Identification of potent anti-fibrinolytic compounds against plasminogen and tissue-type plasminogen activator employing in silico approaches

The zymogen protease Plasminogen (Plg) and its active form plasmin (Plm) carry out important functions in the blood clot disintegration (breakdown of fibrin fibers) process. Inhibition of plasmin effectively reduces fibrinolysis to circumvent heavy bleeding. Currently, available Plm inhibitor tranexamic acid (TXA) used for treating severe hemorrhages is associated with an increased incidence of seizures which in turn were traced to gamma-aminobutyric acid antagonistic activity (GABAa) in addition to having multiple side effects. Fibrinolysis can be suppressed by targeting the three important protein domains: the kringle-2 domain of tissue plasminogen activator, the kringle-1 domain of plasminogen, and the serine protease domain of plasminogen. In the present study, one million molecules were screened from the ZINC database. These ligands were docked to their respective protein targets using Autodock Vina, Schrödinger Glide, and ParDOCK/BAPPL+. Thereafter, the drug-likeness properties of the ligands were evaluated using Discovery Studio 3.5. Subsequently, we subjected the protein-ligand complexes to molecular dynamics simulation of 200 ns in GROMACS. The identified ligands P76(ZINC09970930), C97(ZINC14888376), and U97(ZINC11839443) for each protein target are found to impart higher stability and greater compactness to the protein-ligand complexes. Principal component analysis (PCA) implicates, that the identified ligands occupy smaller phase space, form stable clusters, and provide greater rigidity to the protein-ligand complexes. Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) analysis reveals that P76, C97, and U97 exhibit better binding free energy (ΔG) when compared to that of the standard ligands. Thus, our findings can be useful for the development of promising anti-fibrinolytic agents. Communicated by Ramaswamy H. Sarma

Association between the -844 G>A, HindIII C>G, and 4G/5G PAI-1 Polymorphisms and Susceptibility to Multiple Sclerosis in Western Mexican Population.

Introduction Multiple sclerosis is an inflammatory disease, where fibrin deposition and the impairment in its degradation have been shown to play an important role in the demyelination process. Tissue plasminogen activator (tPA) is a serine protease that enhances the conversion of plasminogen into its active form plasmin, the principal tPA inhibitor is the PAI-1. Several PAI-1 polymorphisms impact its gene expression and protein activity. Furthermore, the aim of this study was to investigate the association between the - 844 G>A, HindIII C>G, and 4G/5G PAI-1 polymorphisms and susceptibility to MS. Material and Methods The study group included 400 Mexican mestizo subjects: 200 unrelated patients and 200 unrelated individuals identified as control subjects. The analysis of PAI-1 polymorphisms was performed by polymerase chain reaction-restriction fragment length polymorphism. Results A significant association was found between the CG genotype of the HindIII C>G PAI-1 polymorphism and susceptibility to MS (OR = 1.58, p = 0.03); moreover, the frequency of 5G allele and 5G/5G genotype of the 4G/5G PAI-1 polymorphism was statistically significant (OR = 1.36 and p = 0.04 and OR = 2.43 and p = 0.02, respectively). With respect to the relation between the scores of progression (EDSS) and severity (MSSS), no association was found between EDSS and genotypes of the PAI-1 polymorphisms analyzed. Regarding MSSS, male that carries genotype GA of the -844 G>A and genotype 4G/5G of the 4G/5G PAI-1 polymorphisms showed a significant association with an increase of media of MSSS in comparison with females (p = 0.01 in both cases).

Photonic activation of plasminogen induced by low dose UVB.

Activation of plasminogen to its active form plasmin is essential for several key mechanisms, including the dissolution of blood clots. Activation occurs naturally via enzymatic proteolysis. We report that activation can be achieved with 280 nm light. A 2.6 fold increase in proteolytic activity was observed after 10 min illumination of human plasminogen. Irradiance levels used are in the same order of magnitude of the UVB solar irradiance. Activation is correlated with light induced disruption of disulphide bridges upon UVB excitation of the aromatic residues and with the formation of photochemical products, e.g. dityrosine and N-formylkynurenine. Most of the protein fold is maintained after 10 min illumination since no major changes are observed in the near-UV CD spectrum. Far-UV CD shows loss of secondary structure after illumination (33.4% signal loss at 206 nm). Thermal unfolding CD studies show that plasminogen retains a native like cooperative transition at ~70 ºC after UV-illumination. We propose that UVB activation of plasminogen occurs upon photo-cleavage of a functional allosteric disulphide bond, Cys737-Cys765, located in the catalytic domain and in van der Waals contact with Trp761 (4.3 Å). Such proximity makes its disruption very likely, which may occur upon electron transfer from excited Trp761. Reduction of Cys737-Cys765 will result in likely conformational changes in the catalytic site. Molecular dynamics simulations reveal that reduction of Cys737-Cys765 in plasminogen leads to an increase of the fluctuations of loop 760–765, the S1-entrance frame located close to the active site. These fluctuations affect the range of solvent exposure of the catalytic triad, particularly of Asp646 and Ser74, which acquire an exposure profile similar to the values in plasmin. The presented photonic mechanism of plasminogen activation has the potential to be used in clinical applications, possibly together with other enzymatic treatments for the elimination of blood clots.

Abstract 547: Excess Glucose Dowregulates Factors for Invasion and Induces an Anti-angiogenic Milieu in First Trimester Cytotrophoblast Cell

Objective: Diabetes mellitus is a known risk factor for the development of preeclampsia (preE). First trimester cytotrophoblastic (CTB) cells invade the decidua using the serine protease plasmin. The enzymatic conversion from the inactive plasminogen to the active form plasmin, is performed by urokinase plasminogen activator (uPA), which is regulated by plasminogen activator inhibitor 1 (PAI-1) in CTBs. Disruption of placental development, setting the stage for preE development is believed, in part, to be caused by a dysfunction of the invasive nature of CTBs. The aim of this study is to determine whether excess glucose induces anti-invasive and anti-angiogenic effects in CTBs in vitro . Methods: Human CTB cells (Sw. 71) were derived from first trimester placenta. The CTB cells were treated with 45, 135, 225, 495 or 945 mg/dL glucose for 72h. Thereafter, cell lysates were utilized to measure uPA and PAI-1 protein expression and phosphorylation of p38 MAPK by western blot. The mRNA expression of uPA and PAI-1 in glucose-induced CTB cell lysates was measure by qPCR. Levels of angiogenic and anti-angiogenic factors (sFLT-1, sENG, VEGF165, PlGF) and the level of inflammatory cytokine IL-6 were measured in the culture media by the commercially available kits. Statistical comparisons were performed using analysis of variance with Dunnett's post hoc tests. Results: Both uPA and PAI-1 protein and mRNA expression were downregulated (p<0.05) in CTB cells treated with >135 mg/dL glucose as compared to those in basal (45 mg/dL). The anti-angiogenic factors (sENG and sFLT-1) were upregulated, while the angiogenic factors (VEGF165 and PlGF) were downregulated in the presence of >135 mg/dL glucose. The level of IL-6 was higher (p<0.05) in the presence of glucose >135 mg/dL compared to basal (45 mg/dL) conditions. The p38 MAPK phosphorylation was upregulated (p<0.05) in excess glucose-treated CTBs. Conclusions: Exposure of CTB cells to glucose at levels associated with diabetes causes a disruption in the invasive profile of CTB cells by decreasing the synthesis of uPA and PAI-1 (both protein and mRNA expression); by downregulating expression of VEGF165 and PlGF; and by upregulating expression of sENG, sFLT-1, and IL-6. Moreover, glucose induced a stress signaling in CTBs.

Longistatin is an unconventional serine protease and induces protective immunity against tick infestation

Classical serine proteases use the conserved Ser/His/Asp catalytic triad to hydrolyze substrates. Here, we show that longistatin, a salivary gland protein with two EF-hand domains from the vector tick Haemaphysalis longicornis, does not have the conserved catalytic triad, but still functions as a serine protease. Longistatin was synthesized in and secreted from the salivary glands of ticks, and is injected into host tissues during the acquisition of blood-meals. Longistatin hydrolyzed fibrinogen, an essential plasma protein in the coagulation cascade, and activated plasminogen, into its active form plasmin, a serine protease that dissolves fibrin clots. Longistatin efficiently hydrolyzed several serine protease-specific substrates showing its specificity to the amide bond of Arg. Longistatin did not hydrolyze synthetic substrates specific for other groups of proteases. The enzyme was active at a wide range of temperatures and pHs, with the optimum at 37°C and pH 7. Its activity was efficiently inhibited by various serine protease inhibitors such as phenylmethanesulfonyl fluoride (PMSF), aprotinin, antipain, and leupeptin with the estimated IC50 of 278.57μM, 0.35μM, 41.56μM and 198.86μM, respectively. In addition, longistatin was also potently inhibited by Zinc (Zn2+) in a concentration-dependent manner with an IC50 value of 275μM, and the inhibitory effect of Zn2+ was revived by ethylenediaminetetra acetic acid (EDTA). Immunization studies revealed that longistatin sharply induced high levels of protective IgG antibodies against ticks. Immunization with longistatin reduced repletion of ticks by about 54%, post engorgement body weight by >11% and molting of nymphs by ∼34%; thus, the vaccination trial was ∼73% effective against tick infestation. Taken together, our results suggest that longistatin is a new potent atypical serine protease, and may be an interesting candidate for the development of anti-tick vaccines.

Longistatin, a Plasminogen Activator, Is Key to the Availability of Blood-Meals for Ixodid Ticks

Ixodid ticks are notorious blood-sucking ectoparasites and are completely dependent on blood-meals from hosts. In addition to the direct severe effects on health and productivity, ixodid ticks transmit various deadly diseases to humans and animals. Unlike rapidly feeding vessel-feeder hematophagous insects, the hard ticks feed on hosts for a long time (5−10 days or more), making a large blood pool beneath the skin. Tick's salivary glands produce a vast array of bio-molecules that modulate their complex and persistent feeding processes. However, the specific molecule that functions in the development and maintenance of a blood pool is yet to be identified. Recently, we have reported on longistatin, a 17.8-kDa protein with two functional EF-hand Ca++-binding domains, from the salivary glands of the disease vector, Haemaphysalis longicornis, that has been shown to be linked to blood-feeding processes. Here, we show that longistatin plays vital roles in the formation of a blood pool and in the acquisition of blood-meals. Data clearly revealed that post-transcriptional silencing of the longistatin-specific gene disrupted ticks' unique ability to create a blood pool, and they consequently failed to feed and replete on blood-meals from hosts. Longistatin completely hydrolyzed α, β and γ chains of fibrinogen and delayed fibrin clot formation. Longistatin was able to bind with fibrin meshwork, and activated fibrin clot-bound plasminogen into its active form plasmin, as comparable to that of tissue-type plasminogen activator (t-PA), and induced lysis of fibrin clot and platelet-rich thrombi. Plasminogen activation potentiality of longistatin was increased up to 4 times by soluble fibrin. Taken together, our results suggest that longistatin may exert potent functions both as a plasminogen activator and as an anticoagulant in the complex scenario of blood pool formation; the latter is critical to the feeding success and survival of ixodid ticks.

The role of streptococcal plasmin(ogen) binding in infective endocarditis

The ability of viridans group streptococci (VGS) to bind human plasminogen and its subsequent activation into plasmin may contribute to the pathogenesis of streptococcal endocarditis. The increased proteolytic activity acquired through cell-bound plasmin may lead to a decreased stability of the streptococcal vegetation and possible embolisation. Twenty-two infective endocarditis isolates and 16 non-infective endocarditis isolates were screened for their ability to bind plasminogen through the quantification of its active form plasmin, using the colorimetric substrate D-Val-Leu-Lys p-nitroanilide. The species of the VGS assessed expressed a universal capability to bind human plasminogen, although they did so with differing affinities and independently of the site of isolation.

Pre-clinical studies of plasmin: Superior benefit-to-risk ratio of plasmin compared to tissue plasminogen activator

Currently-used thrombolytic agents are plasminogen activators (PA). While effective for treating thrombotic disease, PA use is associated with unavoidable hemorrhagic complications in susceptible individuals. Thus, there is an urgent need for new agents or approaches that provide greater hemostatic safety without sacrificing thrombolytic efficacy. Evidence now strongly indicates that ‘direct-acting’ thrombolytics, which do not require conversion of the precursor plasminogen to the active form plasmin, offer such a potential. The biochemical properties of plasmin provide a theoretical foundation for safe and effective therapy, based on thrombolytic efficacy upon local catheter delivery of agent and neutralization of circulating agent by its inhibitor (α-2 antiplasmin) to prevent bleeding complications. In vitro and animal studies support these predictions. In comparison with tissue-plasminogen activator (t-PA), plasmin shows a distinct benefit-to-risk advantage. First, plasmin is equally effective as t-PA in thrombolysis and may be superior for treating thrombi in totally-occluded vessels. Second, whereas t-PA causes bleeding from vascular trauma sites in animals when infused at dosages used for thrombolysis (0.5–1 mg/kg), plasmin exhibits safety at therapeutic dosages. In fact, plasmin can be used at several fold higher concentrations than is needed for thrombolysis, thereby providing a significant safety margin that is not attainable for t-PA or other PAs. Phase I trials with plasmin in hemodialysis graft occlusion and peripheral arterial occlusion have thus far confirmed the hemostatic safety of plasmin.

Use of Concomitant Glycoprotein IIb/IIIa Inhibitors with Catheter-directed Peripheral Arterial Thrombolysis

THROMBOSIS of a peripheral artery or bypass graft can result in severely compromised nutrient blood flow to the limb. If left untreated, gangrene can develop and amputation may be necessary. When peripheral arterial thrombosis is diagnosed, percutaneous delivery of thrombolytic agents offers the opportunity to restore circulation and expose any underlying culprit lesion responsible for the occlusion. All clinically available thrombolytic agents are plasminogen activators; they convert plasminogen to its active form plasmin. Fibrin-bound plasmin then cleaves the insoluble fibrin polymer, forming smaller, soluble fibrin degradation products. If all goes as planned, antegrade arterial blood flow is restored and limb perfusion is returned to normal. Thrombolytic agents were first discovered in the early 1930s, when Tillett and Garner (1) noted fibrinolytic properties of a product of the bacterium streptococcus. Of necessity, however, clinical use of this “streptococcal fibrinolysin” awaited the formulation of a suitably pure form of the agent. After investigation in animal models of vascular thrombosis (2) and after testing intrapleural administration of the newly renamed agent streptokinase in patients with loculated hemothoraces (3), Tillett and colleagues (4) infused the agent into the veins of a small number of volunteers; this seminal work was published in 1955. Just 1 year later, Cliffton and Grunnet (5) published a report on the administration of a streptokinase and plasminogen mixture to dissolve pathologic occlusive thrombi in peripheral vessels. Interestingly, some of Cliffton’s patients were treated through a primitive catheter-directed intraarterial approach (5,6). Unfortunately, innovation in the development of thrombolytic strategies slowed dramatically thereafter. Novel approaches to intraarterial thrombolytic therapy awaited the development of adequate guide wire and catheter technical armamentaria and the emergence of a cadre of interventionalists skillful in the use of percutaneous techniques. Although quantum leaps would not be realized for years to come, a group led by Dotter (7) popularized catheter-directed thrombolytic infusion in the 1970s with use of low-dose streptokinase in an effort to limit distant bleeding complications. A decade later, McNamara and Fischer (8) published excellent results with catheter-directed urokinase (UK) administered in very high doses for just a few hours, then in decreasing doses in a stepwise fashion over a period as long as 2 days. By the decade of the 1990s, UK was the most commonly employed peripheral thrombolytic agent. The Rochester (9), STILE (10), and TOPAS (11,12) randomized clinical trials established the utility of catheter-directed thrombolysis in comparison with primary operative therapy. Virtually all thrombolytic cases in the United States were performed with use of UK, an agent that became the workhorse of the peripheral interventionalist. Then, with little warning, the US Food and Drug Administration raised concerns about the manufacture of UK, and its manufacturer, Abbott Laboratories, voluntarily withdrew UK from the marketplace in early 1999 (13). Although the controversy over UK was at first greeted with gloom by interventional radiologists and vascular surgeons, the absence of this familiar agent prompted investigation into alternative thrombolytic agents and treatment strategies. Alteplase, reteplase, and tenecteplase were studied (14). Many practitioners became relatively comfortable with the alternate agents, although anecdotal impressions surfaced suggesting that these alternative agents may be associated with an increased risk of hemorrhage. So while clinicians were comfortable adding full systemic heparinization to a regimen of intraarterial UK, heparin was kept to minimal, almost homeopathic doses with the other agents (15). In this renewed era of investigation stimulated by the loss of UK from the marketplace, clinicians sought alternate treatment strategies to enhance the efficacy of thrombolytic therapy. These investigations arose after realFrom the Department of Vascular Surgery, Desk S40, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, Ohio 44195. Received March 10, 2004; accepted March 10. Address correspondence to K.O.; E-mail: ourielk@ccf.org

Involvement of Urokinase-Type Plasminogen Activator in Acantholysis Induced by Pemphigus IgG

Pemphigus IgG induces acantholysis in skin organ culture without the involvement of complement. Urokinase-type plasminogen activator, a proteolytic enzyme, has been implicated in the development of acantholysis. To test this hypothesis, we prepared a rabbit anti-urokinase antibody, which inhibited the plasminogen activator activity in normal human epidermis and in cultured keratinocytes. When added to skin organ cultures along with pemphigus IgG, anti-urokinase IgG completely prevented the development of acantholysis. Normal or preimmune rabbit IgG had no effect on pemphigus IgG-induced acantholysis. Plasminogen activator converts the zymogen plasminogen to its active form plasmin, a broad specificity serine proteinase. When high concentrations of plasminogen alone were added to skin organ culture, acantholysis of the pemphigus foliaceous type was induced. Anti-urokinase antibody also inhibited plasminogen-induced acantholysis. These results strongly support a pivotal role for plasminogen activator in the development of acantholysis.