Human Plasminogen Activator Research Articles

Substrate kringle-mediated catalysis by the streptokinase-plasmin activator complex: Critical contribution of kringle-4 revealed by the mutagenesis approaches

Streptokinase (SK) is a protein co-factor with a potent capability for human plasminogen (HPG) activation. Our previous studies [1] have indicated a major role of long-range protein–protein contacts between the three domains (alpha, beta, and gamma) of SK and the multi-domain HPG substrate (K1–K5CD). To further explore this phenomenon, we prepared truncated derivatives of HPG with progressive removal of kringle domains, like K5CD, K4K5CD, K3–K5CD (K3K4K5CD), K2–K5CD (K2K3K4K5CD) and K1–K5CD (K1K2K3K4K5CD). While urokinase (uPA) cleaved the scissile peptide in the isolated catalytic domain (μPG) with nearly the same rate as with full-length HPG, SK-plasmin showed only 1–2% activity, revealing mutually distinct mechanisms of HPG catalysis between the eukaryotic and prokaryotic activators. Remarkably, with SK.HPN (plasmin), the ‘addition’ of both kringles 4 and 5 onto the catalytic domain showed catalytic rates comparable to full length HPG, thus identifying the dependency of the “long-range” enzyme–substrate interactions onto these two CD-proximal domains. Further, chimeric variants of K5CD were generated by swapping the kringle domains of HPG with those of uPA and TPA (tissue plasminogen activator), separately. Surprisingly, although native-like catalytic turnover rates were retained when either K1, K2 or K4 of HPG was substituted at the K5 position in K5CD, these were invariably lost once substituted with the evolutionarily more distant TPA- and uPA-derived kringles. The present results unveil a novel mechanism of SK.HPN action in which augmented catalysis occurs through enzyme–substrate interactions centered on regions in substrate HPG (kringles 4 and 5) that are spatially distant from the scissile peptide bond.

Investigation of Pharmacodynamics, Pharmacology, and Toxicology of Domestic Human Prourokinase

The comparison of the effect of fibrinolytic activity of recombinant human prourokinase (rhpro-UK) and urokinase(UK) with rabbits indicated rhpro-UK and UK all decreased resolution time of globular proteins, and the affect of rhpro-UK on parameter of fibrinolysis of rabbits was less than UK. The solution of thrombin of rhpro-UK in vitro was increased with dosage of rhpro-UK, and when rhpro-UK was compared with isodose UK, the difference is significant. Both rhpro-UK and UK all was marked solution of thrombin from Chinese piglets, and it were not evident effect on fibrinogen, plasminogen, and a2-antiplasmin of the piglets. The affect of rhpro-UK on bleeding time, clotting time, bleeding amount of unit time was less than UK. It indicated that the side effect of rhpro-UK was distinguished less than UK. The investigation of pharmacology of ileums of mice, rats, dogs, and guinea pigs indicated that rhpro-UK was not marked affect on behavior, state, central nervous system, cardiovascular system, respiratory system, and digestive system of the tested animals. But tested dogs were bleeding at location of operation, and the blood of the animal was heparinized. The studis of toxicology of rhpro-UK indicated that rhpro-UK was median lethal dose of 97.5 mg/kg in mice. When The Beagle dogs was administed rhpro-UK of 2, 8, 28 mg/kg, the toxic reaction was not observed, except for temporal hyperemia of gum, extend of clotting time, increasing of total cholerythrin, total proteins, albumin of the dogs, the direct damage of organs for the medicine was not observed in examination of pathology in the dosages of 8 and 28 mg/kg, any toxic reaction was not observed in dosage of 2 mg/kg. The special toxic experiments proved that rhpro-UK was not of mutagenesis and teratogenesis.

HIF-1α in Epidermis: Oxygen Sensing, Cutaneous Angiogenesis, Cancer, and Non-Cancer Disorders

Besides lung, postnatal human epidermis is the only epithelium in direct contact with atmospheric oxygen. Skin epidermal oxygenation occurs mostly through atmospheric oxygen rather than tissue vasculature, resulting in a mildly hypoxic microenvironment that favors increased expression of hypoxia-inducible factor-1α (HIF-1α). Considering the wide spectrum of biological processes, such as angiogenesis, inflammation, bioenergetics, proliferation, motility, and apoptosis, that are regulated by this transcription factor, its high expression level in the epidermis might be important to HIF-1α in skin physiology and pathophysiology. Here, we review the role of HIF-1α in cutaneous angiogenesis, skin tumorigenesis, and several skin disorders.

The Binding Mechanism of a Peptidic Cyclic Serine Protease Inhibitor

Serine proteases are classical objects for studies of catalytic and inhibitory mechanisms as well as interesting as therapeutic targets. Since small-molecule serine protease inhibitors generally suffer from specificity problems, peptidic inhibitors, isolated from phage-displayed peptide libraries, have attracted considerable attention. Here, we have investigated the mechanism of binding of peptidic inhibitors to serine protease targets. Our model is upain-1 (CSWRGLENHRMC), a disulfide-bond-constrained competitive inhibitor of human urokinase-type plasminogen activator with a noncanonical inhibitory mechanism and an unusually high specificity. Using a number of modified variants of upain-1, we characterised the upain-1–urokinase-type plasminogen activator complex using X-ray crystal structure analysis, determined a model of the peptide in solution by NMR spectroscopy, and analysed binding kinetics and thermodynamics by surface plasmon resonance and isothermal titration calorimetry. We found that upain-1 changes both main-chain conformation and side-chain orientations as it binds to the protease, in particular its Trp3 residue and the surrounding backbone. The properties of upain-1 are strongly influenced by the addition of three to four amino acids long N-terminal and C-terminal extensions to the core, disulfide-bond-constrained sequence: The C-terminal extension stabilises the solution structure compared to the core peptide alone, and the protease-bound structure of the peptide is stabilised by intrapeptide contacts between the N-terminal extension and the core peptide around Trp3. These results provide a uniquely detailed description of the binding of a peptidic protease inhibitor to its target and are of general importance in the development of peptidic inhibitors with high specificity and new inhibitory mechanisms.

SecretedBacillus anthracisproteases target the host fibrinolytic system

The fibrinolytic system is often the target for pathogenic bacteria, resulting in increased fibrinolysis, bacterial dissemination, and inflammation. The purpose of this study was to explore whether proteases NprB and InhA secreted by Bacillus anthracis could activate the host's fibrinolytic system. NprB efficiently activated human pro-urokinase plasminogen activator (pro-uPA), a key protein in the fibrinolytic cascade. Conversely, InhA had little effect on pro-uPA. Plasminogen activator inhibitors (PAI)-1, 2 and the uPA receptor were also targets for NprB in vitro. InhA efficiently degraded the thrombin-activatable fibrinolysis inhibitor (TAFI) in vitro. Mice infected with B. anthracis showed a significant decrease in blood TAFI levels. In another mouse experiment, animals infected with isogenic inhA deletion mutants restored TAFI levels, while the levels in the parent strain decreased. We propose that NprB and InhA may contribute to the activation of the fibrinolytic system in anthrax infection.

Embolization with Onyx in Iatrogenic Bleeding of the Gluteal Region

We report a unique method using transcatheter Onyx embolization in a bleeding due to morphine injection in the gluteal region. A 47-year-old man with a rare blood type presented a painful gluteal hematoma due to iatrogenic injury. A computed tomographic angiography verified bleeding from a suspected branch of the deep femoral artery. Because of the unbearable pain, the hematoma was evacuated by means of computed tomography (CT)-guided puncture and the insertion of a pigtail catheter combined with the injection of a human plasminogen activation agent (t-PA). The initial result was positive. To stop the bleeding, angiographic embolization with Onyx was successfully used. Onyx can be used in small vessel bleedings and might offer the advantage of selective embolization in cases where the access to the bleeding vessel is challenging or time-consuming.

Elucidation of the Contribution of Active Site and Exosite Interactions to Affinity and Specificity of Peptidylic Serine Protease Inhibitors Using Non-Natural Arginine Analogs

There is increasing interest in developing peptides for pharmacological intervention with pathophysiological functions of serine proteases. From phage-displayed peptide libraries, we previously isolated peptidylic inhibitors of urokinase-type plasminogen activator, a potential target for intervention with cancer invasion. The two peptides, upain-1 (CSWRGLENHRMC) and mupain-1 (CPAYSRYLDC), are competitive inhibitors of human and murine urokinase-type plasminogen activator, respectively. Both have an Arg as the P1 residue, inserting into the S1 pocket in the active site of the enzymes, but their specificity depends to a large extent on interactions outside the enzymes' active sites, so-called exosite interactions. Here we describe upain-2 (CSWRGLENHAAC) and the synthesis of a number of upain-2 and mupain-1 variants in which the P1 Arg was substituted with novel non-natural Arg analogs and achieved considerable improvement in the affinity of the peptides to their targets. Using chimeras of human and murine urokinase-type plasminogen activator as well as X-ray crystallography, we delineated the relative contribution of the P1 residue and exosite interactions to the affinity and specificity of the inhibitors for their target enzyme. The effect of inserting a particular non-natural amino acid into the P1 position is determined by the fact that changes in interactions of the P1 residue in the S1 pocket lead to changed exosite interactions and vice versa. These findings are of general interest when the affinities and specificities of serine protease inhibitors to be used for pharmacological intervention are considered and could pave the way for potential drug candidates for the treatment of cancer.

Rock bream ( Oplegnathus fasciatus) serpin, protease nexin-1: Transcriptional analysis and characterization of its antiprotease and anticoagulant activities

Protease nexin-1 (PN-1) is a serine protease inhibitor (SERPIN) protein with functional roles in growth, development, patho-physiology and injury. Here, we report our work to clone, analyze the expression profile and characterize the properties of the PN-1 gene in rock bream (Rb), Oplegnathus fasciatus. RbPN-1 encodes a peptide of 397 amino acids (AA) with a predicted molecular mass of 44 kDa and a 23 AA signal peptide. RbPN-1 protein was found to harbor a characteristic SERPIN domain comprised of a SERPIN signature and having sequence homology to vertebrate PN-1s. The greatest identity (85%) was observed with PN-1 from the three-spined stickleback fish, Gasterosteus aculeatus. The functional domains, including a heparin binding site and reactive centre loop were conserved between RbPN-1 and other fish PN-1s; in particular, they were found to correspond to components of the human plasminogen activator inhibitor 1, PAI-1. Phylogenetic analysis indicated that RbPN-1 was closer to homologues of green spotted pufferfish and Japanese pufferfish. Recombinant RbPN-1 demonstrated antiprotease activity against trypsin (48%) and thrombin (89%) in a dose-dependent manner, and its antithrombotic activity was potentiated by heparin. The anticoagulant function prolonged clotting time by 3.7-fold, as compared to the control in an activated partial thromboplastin time assay. Quantitative real-time PCR results indicated that RbPN-1 is transcribed in many endogenous tissues at different levels. Lipopolysaccharide (LPS) stimulated a prolonged transcriptional response in hematic cells, and Rb iridovirus up-regulated the RbPN-1 mRNA level in hematic cells to a maximum of 3.4-fold at 12 h post-infection. Interestingly, LPS and Edwardsiella tarda significantly induced the RbPN-1 transcription at the late phase of infection. In vivo studies indicated that injury response caused a temporal suppression in RbPN-1 transcription, in conjunction with that of another SERPIN, rock bream heparin cofactor II, RbHCII. Taken together, our findings suggest that PN-1 functions as an antiprotease and anticoagulant and that SERPINs (PN-1 and HCII) are likely to contribute to immunity and post-injury responses.

Identification through Combinatorial Random and Rational Mutagenesis of a Substrate-interacting Exosite in the γ Domain of Streptokinase

To identify new structure-function correlations in the γ domain of streptokinase, mutants were generated by error-prone random mutagenesis of the γ domain and its adjoining region in the β domain followed by functional screening specifically for substrate plasminogen activation. Single-site mutants derived from various multipoint mutation clusters identified the importance of discrete residues in the γ domain that are important for substrate processing. Among the various residues, aspartate at position 328 was identified as critical for substrate human plasminogen activation through extensive mutagenesis of its side chain, namely D328R, D328H, D328N, and D328A. Other mutants found to be important in substrate plasminogen activation were, namely, R319H, N339S, K334A, K334E, and L335Q. When examined for their 1:1 interaction with human plasmin, these mutants were found to retain the native-like high affinity for plasmin and also to generate amidolytic activity with partner plasminogen in a manner similar to wild type streptokinase. Moreover, cofactor activities of the mutants precomplexed with plasmin against microplasminogen as the substrate as well as in silico modeling studies suggested that the region 315-340 of the γ domain interacts with the serine protease domain of the macromolecular substrate. Overall, our results identify the presence of a substrate specific exosite in the γ domain of streptokinase.

Metals affect the structure and activity of human plasminogen activator inhibitor‐1. II. Binding affinity and conformational changes

Human plasminogen activator inhibitor type 1 (PAI-1) is a serine protease inhibitor with a metastable active conformation. The lifespan of the active form of PAI-1 is modulated via interaction with the plasma protein, vitronectin, and various metal ions. These metal ions fall into two categories: Type I metals, including calcium, magnesium, and manganese, stabilize PAI-1 in the absence of vitronectin, whereas Type II metals, including cobalt, copper, and nickel, destabilize PAI-1 in the absence of vitronectin, but stabilize PAI-1 in its presence. To provide a mechanistic basis for understanding the unusual modulation of PAI-1 structure and activity, the binding characteristics and conformational effects of these two types of metals were further evaluated. Steady-state binding measurements using surface plasmon resonance indicated that both active and latent PAI-1 exhibit a dissociation constant in the low micromolar range for binding to immobilized nickel. Stopped-flow measurements of approach-to-equilibrium changes in intrinsic protein fluorescence indicated that the Type I and Type II metals bind in different modes that induce distinct conformational effects on PAI-1. Changes in the observed rate constants with varying concentrations of metal allowed accurate determination of binding affinities for cobalt, nickel, and copper, yielding dissociation constants of ∼40, 30, and 0.09 μM, respectively. Competition experiments that tested effects on PAI-1 stability were consistent with these measurements of affinity and indicate that copper binds tightly to PAI-1.

Metals affect the structure and activity of human plasminogen activator inhibitor‐1. I. Modulation of stability and protease inhibition

Human plasminogen activator inhibitor type 1 (PAI-1) is a serine protease inhibitor with a metastable active conformation. Under physiological conditions, half of the inhibitor transitions to a latent state within 1-2 h. The interaction between PAI-1 and the plasma protein vitronectin prolongs this active lifespan by ∼50%. Previously, our group demonstrated that PAI-1 binds to resins using immobilized metal affinity chromatography (Day, U.S. Pat. 7,015,021 B2, March 21, 2006). In this study, the effect of these metals on function and stability was investigated by measuring the rate of the transition from the active to latent conformation. All metals tested showed effects on stability, with the majority falling into one of two types depending on their effects. The first type of metal, which includes magnesium, calcium and manganese, invoked a slight stabilization of the active conformation of PAI-1. A second category of metals, including cobalt, nickel and copper, showed the opposite effects and a unique vitronectin-dependent modulation of PAI-1 stability. This second group of metals significantly destabilized PAI-1, although the addition of vitronectin in conjunction with these metals resulted in a marked stabilization and slower conversion to the latent conformation. In the presence of copper and vitronectin, the half-life of active PAI-1 was extended to 3 h, compared to a half-life of only ∼30 min with copper alone. Nickel had the largest effect, reducing the half-life to ∼5 min. Together, these data demonstrate a heretofore-unknown role for metals in modulating PAI-1 stability.

Generation of transgenic chicks using an oviduct-specific expression system

We successfully replaced the ovalbumin gene of a magnum region in chickens with a human plasminogen activator. We constructed pL-eGFP, pL-tPAGFP and pL-2.8OVtPAGFP vectors and cultured 293FT chicken embryo fibroblasts, chicken primordial germ cells, Hela C127 cells, and oviduct epithelial cells. All vectors were expressed in the transfected cells, except pL-2.8OVtPAGFP vector, which was only expressed in oviduct epithelial cells. A lentivirus with pL-2.8OVtPAGFP was injected in fertilized eggs; 11 chicks hatched in the G₀ generation, four of them carried the tPAGFP. Two cockerels from the G₀ generation were crossed with four wild-type hens. Three chicks in G₁ carried the tPAGFP. We concluded that by using an oviduct-specific vector for transfection, human recombinant plasminogen activator protein can be expressed in the oviducts of laying hens. This character is inherited and can be reproduced with a need for repeated transfection.

Pharmacokinetics and safety of a new bioengineered thrombolytic agent, human tissue urokinase type plasminogen activator in Chinese healthy volunteers

The aim of our study was to investigate the pharmacokinetics and safety of human tissue urokinase type plasminogen activator (HTUPA) in healthy Chinese subjects after intravenous administration. Thirty-two subjects were given intravenous injection doses of 5-35 mg of HTUPA for safety evaluation. Twenty-four subjects were given 10, 20 or 30 mg HTUPA for pharmacokinetic assessment. Safety and tolerance were evaluated by monitoring adverse events, laboratory parameters, electrocardiography and vital signs. HTUPA concentration in human serum samples was determined by an enzyme-linked immunosorbent assay (ELISA) method. The main pharmacokinetic parameters were calculated by DAS software. HTUPA was generally well tolerated and in the whole study course no serious adverse events occurred. The main pharmacokinetic parameters were as follows: geometric mean [95% confidence interval, CI] for t1/2 were 1.5 (1.4, 1.6), 1.3 (1.2, 1.4), and 1.2 (1.2, 1.3) h, AUC0-t were 1.0 (0.7, 1.3), 2.1 (1.5, 2.7), and 5.6 (4.7, 6.6) mg h L(-1), AUC0-∞ were 1.1 (0.8, 1.3), 2.1 (1.5, 2.7), and 5.8 (4.7, 6.7) mg h L(-1) for 10, 20, and 30 mg group, respectively. The main pharmacokinetic parameters were not significantly different between males and females (P>0.05). No serious adverse events were reported by the subjects or revealed by clinical or laboratory examinations, suggesting the given doses were safe and well tolerated.

Contribution of Plasminogen Activation towards the Pathogenic Potential of Oral Streptococci

Oral streptococci are a heterogeneous group of human commensals, with a potential to cause serious infections. Activation of plasminogen has been shown to increase the virulence of typical human pathogenic streptococci such as S. pneumoniae. One important factor for plasminogen activation is the streptococcal α-enolase. Here we report that plasminogen activation is also common in oral streptococci species involved in clinical infection and that it depends on the action of human plasminogen activators. The ability to activate plasminogen did not require full conservation of the internal plasminogen binding sequence motif FYDKERKVY of α-enolase that was previously described as crucial for increased plasminogen binding, activation and virulence. Instead, experiments with recombinant α-enolase variants indicate that the naturally occurring variations do not impair plasminogen binding. In spite of these variations in the internal plasminogen binding motif oral streptococci showed similar activation of plasminogen. We conclude that the pathomechanism of plasminogen activation is conserved in oral streptococci that cause infections in human. This may contribute to their opportunistic pathogenic character that is unfurled in certain niches.

The omptins of Yersinia pestis and Salmonella enterica cleave the reactive center loop of plasminogen activator inhibitor 1.

Plasminogen activator inhibitor 1 (PAI-1) is a serine protease inhibitor (serpin) and a key molecule that regulates fibrinolysis by inactivating human plasminogen activators. Here we show that two important human pathogens, the plague bacterium Yersinia pestis and the enteropathogen Salmonella enterica serovar Typhimurium, inactivate PAI-1 by cleaving the R346-M347 bait peptide bond in the reactive center loop. No cleavage of PAI-1 was detected with Yersinia pseudotuberculosis, an oral/fecal pathogen from which Y. pestis has evolved, or with Escherichia coli. The cleavage and inactivation of PAI-1 were mediated by the outer membrane proteases plasminogen activator Pla of Y. pestis and PgtE protease of S. enterica, which belong to the omptin family of transmembrane endopeptidases identified in Gram-negative bacteria. Cleavage of PAI-1 was also detected with the omptins Epo of Erwinia pyrifoliae and Kop of Klebsiella pneumoniae, which both belong to the same omptin subfamily as Pla and PgtE, whereas no cleavage of PAI-1 was detected with omptins of Shigella flexneri or E. coli or the Yersinia chromosomal omptins, which belong to other omptin subfamilies. The results reveal a novel serpinolytic mechanism by which enterobacterial species expressing omptins of the Pla subfamily bypass normal control of host proteolysis.

Sustained thromboprophylaxis mediated by an RBC-targeted pro-urokinase zymogen activated at the site of clot formation

AbstractPlasminogen activators (PAs) are used to treat life-threatening thrombosis, but not for thromboprophylaxis because of rapid clearance, risk of bleeding, and central nervous system (CNS) toxicity. We describe a novel strategy that may help to overcome these limitations by targeting a thrombin-activated PA pro-drug to circulating red blood cells (RBCs). We fused a single chain antibody (scFv Ter-119) that binds to mouse glycophorin A (GPA) with a variant human single-chain low molecular weight urokinase construct that can be activated selectively by thrombin (scFv/uPA-T). scFv/uPA-T bound specifically to mouse RBCs without altering their biocompatibility and retained its zymogenic properties until converted by thrombin into an active 2-chain molecule. As a result, RBC-bound scFv/uPA-T caused thrombin-induced fibrinolysis. One hour and 48 hours after intravenous (IV) injection in mice, approximately 70% and approximately 35% of scFv/uPA-T was retained in the blood, respectively, and approximately 95% of the circulating scFv/uPA-T remained bound to RBCs. A single IV injection of scFv/uPA-T provided effective prophylaxis against arterial and venous thrombosis for up to 24 hours. Thus, prophylactic delivery of RBC-targeted PA pro-drugs activated selectively at the site of clot formation represents a new approach to prevent thrombosis in clinical settings where the risk of clotting is high.

Combinatorial gene therapy renders increased survival in cirrhotic rats

BackgroundLiver fibrosis ranks as the second cause of death in México's productive-age population. This pathology is characterized by acummulation of fibrillar proteins in hepatic parenchyma causing synthetic and metabolic disfunction. Remotion of excessive fibrous proteins might result in benefit for subjects increasing survival index. The goal of this work was to find whether the already known therapeutical effect of human urokinase Plasminogen Activator and human Matrix Metalloprotease 8 extends survival index in cirrhotic animals.MethodsWistar rats (80 g) underwent chronic intoxication with CCl4: mineral oil for 8 weeks. Cirrhotic animals were injected with a combined dose of Ad-delta-huPA plus Ad-MMP8 (3 × 1011 and 1.5 × 1011 vp/Kg, respectively) or with Ad-beta-Gal (4.5 × 1011) and were killed after 2, 4, 6, 8 and 10 days. Then, liver and serum were collected. An additional set of cirrhotic animals injected with combined gene therapy was also monitored for their probability of survival.ResultsOnly the cirrhotic animals treated with therapeutical genes (Ad-delta-huPA+Ad-MMP-8) showed improvement in liver fibrosis. These results correlated with hydroxyproline determinations. A significant decrement in alpha-SMA and TGF-beta1 gene expression was also observed. Cirrhotic rats treated with Ad-delta-huPA plus Ad-MMP8 had a higher probability of survival at 60 days with respect to Ad-beta-Gal-injected animals.ConclusionA single administration of Ad-delta-huPA plus Ad-MMP-8 is efficient to induce fibrosis regression and increase survival in experimental liver fibrosis.

Effects on human plasminogen conformation and activation rate caused by interaction with VEK-30, a peptide derived from the group A streptococcal M-like protein (PAM)

In vertebrates, fibrinolysis is primarily carried out by the serine protease plasmin (Pm), which is derived from activation of the zymogen precursor, plasminogen (Pg). One of the most distinctive features of Pg/Pm is the presence of five homologous kringle (K) domains. These structural elements possess conserved Lys-binding sites (LBS) that facilitate interactions with substrates, activators, inhibitors and receptors. In human Pg (hPg), K2 displays weak Lys affinity, however the LBS of this domain has been implicated in an atypical interaction with the N-terminal region of a bacterial surface protein known as PAM ( Pg-binding group A streptococcal M-like protein). A direct correlation has been established between invasiveness of group A streptococci and their ability to bind Pg. It has been previously demonstrated that a 30-residue internal peptide (VEK-30) from the N-terminal region of PAM competitively inhibits binding of the full-length parent protein to Pg. We have attempted to determine the effects of this ligand–protein interaction on the regulation of Pg zymogen activation and conformation. Our results show minimal effects on the sedimentation velocity coefficients ( S° 20,w) of Pg when associated to VEK-30 and a direct relationship between the concentration of VEK-30 or PAM and the activation rate of Pg. These results are in contrast with the major conformational changes elicited by small-molecule activators of Pg, and point towards a novel mechanism of Pg activation that may underlie group A streptococcal (GAS) virulence.

Abstract 5580: Nanolytic Approach to Prompt Revascularization in Acute Ischemic Stroke

Background: Vascular-constrained fibrin-specific nanolytic agents could permit early revascularization in the setting of acute ischemic stroke. Development of fibrin-targeted nanolytics requires optimized surface co-presentation of homing ligands and fibrinolytic enzymes on nanoparticles without compromising bioactivity. Objective : The study optimized human urokinase coupling and anti-fibrin homing ligand density for targeted thrombolytic efficacy and demonstrated the concept in vivo. Methods and Results: Minimal thiolation (N-succinimidyl-S-acetylthioacetate, SATA) of urokinase (i.e., 1 to 3 SATA/molecule) diminished human clot dissolution time relative to the native molecule; greater degrees of thiolation (3 to 6 SATA/molecule) severely decreased activity. For dog clots, which digested slower than human clots, 1 to 3 SATA/urokinase did not further impair fibrinolysis rate. Concomitant coupling of thiolated urokinase and anti-fibrin antibody (50:5, 100:10, 200:20, and 400:40 ratio, respectively) to perfluorocarbon nanoparticles resulted in optimal clot dissolution for the 100:10 and 200:20 formulations. Increasing the surface urokinase-to-antibody ratio from 0:10 to 50:10, 100:10, 200:10, and 400:10 increased particle size from 273 nm (0:10) to 340 nm at the 50:10 and 100:10 enzyme levels, and to more than 400 nm with 200 urokinase molecules per particle. Acoustic microscopy (25MHz) was used to quantify binding of the fibrin-targeted PFC nanoparticles to acoustically transparent human clots. Fibrin targeting with 10 antibodies per particle provided minimal acoustic enhancement in comparison with the relatively increased (p<0.05) but equivalent signals obtained with 20 or 40 antibodies per particle. Dissolution of canine femoral clots, created by electrical injury, was monitored with low MI ultrasound under open circulation conditions following in situ targeting with the nanolytic system. Substantial subjective clot dissolution and fragmentation was observed in three animals over 60 minutes, demonstrating the nanolytic concept. Conclusion: Nanolytic agents can target fibrinolytic enzymes to lyse nascent thrombus, which may permit early revascularization in acute ischemic stroke with minimal hemorrhagic risk.

Plasmin plays a key role in the regulation of profibrogenic molecules in hepatic stellate cells

Plasmin role in transforming growth factor-beta (TGF-beta)-responsive gene regulation remains to be elucidated. Also, plasmin action on co-repressor Ski-related novel protein N (SnoN) and differential activation of matrix metalloproteinases (MMPs) are unknown. Thus, the role of plasmin on profibrogenic molecule expression, SnoN transcriptional kinetics and gelatinase activation was investigated. Hepatic stellate cells (HSC) were transduced with adenovirus-mediated human urokinase plasminogen activator (Ad-huPA) (4 x 10(9) viral particles/ml). Overexpression of urokinase plasminogen activator and therefore of plasmin, was blocked by tranexamic acid (TA) in transduced HSC. Gene expression was monitored by reverse transcriptase polymerase chain reaction. HSC-free supernatants were used to evaluate MMP-2 and MMP-9 by zymography. SnoN, TGF-beta and tissue inhibitor of metalloproteinase (TIMP)-1 were analysed by Western blot. Plasmin and SnoN expression kinetics were evaluated in bile duct-ligated (BDL) rats. Plasmin overexpression in Ad-huPA-transduced HSC significantly decreased gene expression of profibrogenic molecules [alpha1(I)collagen 66%, TIMP-1 59%, alpha-smooth muscle actin 90% and TGF-beta 55%]. Interestingly, both SnoN gene and protein expression increased prominently. Plasmin inhibition by TA upregulated the profibrogenic genes, which respond to TGF-beta-intracellular signalling. In contrast, SnoN mRNA and protein dropped importantly. Plasmin-activated MMP-9 and MMP-2 in HSC supernatants. Taken together, these findings indicate that MMP-9 activation is totally plasmin dependent. SnoN levels significantly decreased in cholestatic-BDL rats (82%) as compared with control animals. Interestingly, hepatic plasmin levels dropped 46% in BDL rats as compared with control. Plasmin plays a key role in regulating TGF-beta-responding genes. In particular, regulation of TGF-beta-co-repressor (SnoN) is greatly affected, which suggests SnoN as a cardinal player in cholestasis-induced fibrogenesis.