A dual-function chymotrypsin-like serine protease with plasminogen activation and fibrinolytic activities from the GRAS fungus, Neurospora sitophila.

In this study, we have isolated and characterized a fibrinolytic enzyme from the GRAS (Generally Recognized as Safe) fungus, Neurospora sitophila. The enzyme was purified by fractional ammonium sulfate precipitation, hydrophobic interaction, ion exchange and gel filtration chromatography to 45.2 fold with a specific activity of 415.6U/mg protein. The native molecular mass of the enzyme was 49kDa, while the denatured molecular mass was 30kDa and 17.5kDa, indicating that the enzyme was a hetero-dimer. It was optimally active at 50°C and pH 7.4 and stable at human physiological temperature and pH. It was found to be a chymotrypsin-like serine protease which cleaved the synthetic chromogenic substrate, N-Succinyl-Ala-Ala-Pro-Phe-pNA for which the apparent Km and Vmax values were 0.24mM and 4.17×10-5mM/s, respectively. The enzyme hydrolyzed all the chains of fibrinogen by cleaving α chain first, followed by ß chain and then γ chain. Moreover, the enzyme possessed dual function of direct fibrinolysis as well as plasminogen activation. Due to its attractive biochemical and fibrinolytic properties and being from a GRAS fungus, the fibrinolytic enzyme has application as a safe and efficient thrombolytic drug.

Anchoring ß-cyclodextrin modified lysine to polymer monolith with biotin: specific capture of plasminogen.

Plasminogen (Plg) is a kind of glycoprotein which plays an important role in cell migration. The determination of Plg content can directly reflect the abnormal manifestation of fibrinolytic system dysfunctions. In the present work, lysine (Lys)-based adsorbents were prepared for the specific capture of Plg through the covalent binding of Lys with a polymer monolithic substrate. Lys was modified with ß-cyclodextrin (ß-CD) via a click reaction and anchored to the substrate with biotin by host-guest interaction. The biotin-Lys-CD based monolithic material was employed for the specific capture of Plg. Combining with mass spectrometry (MS) determinations, the method exhibited a low detection limit of 1.0 fmol with relative standard deviations below 10.0% for Plg. Considering the advantages of simplicity, sensitivity, and high specificity, the developed approach was successfully applied to the determination of Plg in human plasma samples and opened a gallery for testing Plg as a biomarker for the diagnosis of fibrinolytic system dysfunctions.

Isolation and purification of recombinant human plasminogen Kringle 5 by liquid chromatography and ammonium sulfate salting-out.

In this work, a novel method was established to isolate and purify Human plasminogen Kringle 5 (HPK5) as a histidine-tagged fusion protein expressed in Escherichia coli BL21 (DE3). This method consisted of sample extraction using a Ni-chelated Sepharose Fast-Flow affinity column, ammonium sulfate salting-out and Sephadex G-75 size-exclusion column in turn. The purity analysis by SDS-PAGE, high-performance size-exclusion and reversed-phase chromatographies showed that the obtained recombinant fusion HPK5 was homogeneous and its purity was higher than 96%; the activity analysis by chorioallantoic membrane model of chicken embryos revealed that the purified recombinant HPK5 exhibited an obvious anti-angiogenic activity under the effective range of 5.0-25.0 µg/mL. Through this procedure, about 19 mg purified recombinant fusion HPK5 can be obtained from 1 L of original fermentation solution. Approximate 32% of the total recombinant fusion HPK5 can be captured and the total yield was approximately 11%.

A novel antithrombotic coronary stent: lysine-poly(HEMA)-modified cobalt-chromium stent with fibrinolytic activity.

Prevention of coagulation appears not to be possible when a foreign surface is in contact with blood; the alternative concept of a clot lysing surface has therefore been suggested. In this work, a mimic of the fibrinolytic system was constructed on L605 cobalt-chromium coronary stents. Lysine which, immobilized on a surface, has been shown previously to adsorb plasminogen in contact with blood was attached to the stent using poly(2-hydroxyethyl methacrylate) (poly(HEMA)) as a spacer. The lysine-poly(HEMA) modified stent was shown to have low nonspecific protein adsorption and to bind plasminogen in high quantity from plasma. Following exposure to plasma and treatment with tissue plasminogen activator, the lysine-modified stents showed clot-lysing properties in vitro while the unmodified L605 stents did not. It was shown that the modified stents retained their clot lysing properties after 24 h exposure to plasma.

[Isolation and purification of recombinant soluble and non-fusion angiogenesis inhibitor Kringle 5 using chromatography].

The Kringle 5 domain of plasminogen is one of the most potent angiogenesis inhibitors known to date, which can inhibit cell proliferation and migration efficiently. In the study, on the foundation of successful clone and expression of recombinant soluble and non-fusion angiogenesis inhibitor Kringle 5, a two-step chromatographic method, including the use of SP Sepharose Fast Flow cation exchanger and Sephacryl S-100 HR size exclusion chromatography in sequence, was established to separate and purify angiogenesis inhibitor Kringle 5. On the SP Sepharose Fast Flow column, the buffer A consisted of 50.0 mmol/L acetic acid-sodium acetate (pH 5.2), and the buffer B consisted of buffer A with the addition of 0.5 mol/L sodium chloride (pH 5.2); on Sephacryl S-100 HR column, the elution buffer was 5.0 mmol/L phosphate solution (pH 7.0). Through the two-step chromatographic purification process, the purity of the obtained Kringle 5 was more than 98%. In addition, it was found that the obtained Kringle 5 could inhibit the blood vessel growth of chick embryo chorioallantoic membrane effectively. Finally it is concluded that this method can effectively separate active recombinant soluble and non-fusion angiogenesis inhibitor Kringle 5.

Separation and study of the range of plasminogen isoforms in patients with prostate cancer.

Using affinity chromatography, two-dimensional electrophoresis, and MALDI-TOF mass spectrometry, plasminogen isoforms were separated and identified in blood plasma. Healthy donors and patients with prostate cancer in various stages of development were included in the studied sample. With the development of prostate cancer, four additional specific plasminogen isoforms are registered in blood plasma; they are characterized by lower molecular weights and higher pI values compared to isoforms found in the control group.

Characterization of Novel OmpA-Like Protein of Leptospira interrogans That Binds Extracellular Matrix Molecules and Plasminogen

Leptospira interrogans is the etiological agent of leptospirosis, a zoonotic disease of human and veterinary concern. The identification of novel proteins that mediate host-pathogen interactions is important for understanding the bacterial pathogenesis as well as to identify protective antigens that would help fight the disease. We describe in this work the cloning, expression, purification and characterization of three predicted leptospiral membrane proteins, LIC10258, LIC12880 (Lp30) and LIC12238. We have employed Escherichia coli BL21 (SI) strain as a host expression system. Recently, we have identified LIC12238 as a plasminogen (PLG)-binding receptor. We show now that Lp30 and rLIC10258 are also PLG-receptors of Leptospira, both exhibiting dose-dependent and saturating binding (KD, 68.8±25.2 nM and 167.39±60.1 nM, for rLIC10258 and rLIC12880, respectively). In addition, LIC10258, which is a novel OmpA-like protein, binds laminin and plasma fibronectin ECM molecules and hence, it was named Lsa66 (Leptospiral surface adhesin of 66 kDa). Binding of Lsa66 to ECM components was determined to be specific, dose-dependent and saturable, with a KD of 55.4±15.9 nM to laminin and of 290.8±11.8 nM to plasma fibronectin. Binding of the recombinant proteins to PLG or ECM components was assessed by using antibodies against each of the recombinant proteins obtained in mice and confirmed by monoclonal anti-polyhistidine antibodies. Lsa66 caused partial inhibition on leptospiral adherence to immobilized ECM and PLG. Moreover, this adhesin and rLIC12238 are recognized by antibodies in serum samples of confirmed leptospirosis cases. Thus, Lsa66 is a novel OmpA-like protein with dual activity that may promote the attachment of Leptospira to host tissues and may contribute to the leptospiral invasion. To our knowledge, this is the first leptospiral protein with ECM and PLG binding properties reported to date.

Magnetic purification of plasminogen from human plasma by specific lysine affinity.

A novel magnetic adsorbent was prepared by covalently binding lysine onto the surface of nanoparticles via a carbodiimide coupling method. The adsorption of plasminogen onto surface-modified magnetic nanoparticles from human plasma was studied in a batch system. Surface modifications of particles were characterized using Fourier-transformed infrared spectra, transmission electron micrography, and ninhydrin assay. The maximum weight ratio of lysine to the superparamagnetic particles was 30 µmol/mg of particles. Effects of pH and temperature on plasminogen adsorption by the magnetic particles were evaluated. Desorption of plasminogen from the magnetic adsorbent was investigated using aminocaproic acid, a lysine analogue. Using a buffer composed by aminocaproic acid or lysine, plasminogen on the magnetic nanoparticles could be eluted. Overall, the results demonstrated that the lysine-coated magnetic adsorbent increased the efficiency and speed of recovery of plasminogen from human plasma.

Plasmin on adherent cells: from microvesiculation to apoptosis.

Cell activation by stressors is characterized by a sequence of detectable phenotypic cell changes. A given stimulus, depending on its strength, induces modifications in the activity of membrane phospholipid transporters and calpains, which lead to phosphatidylserine exposure, membrane blebbing and the release of microparticles (nanoscale membrane vesicles). This vesiculation could be considered as a warning signal that may be followed, if the stimulus is maintained, by cell detachment-induced apoptosis. In the present study, plasminogen incubated with adherent cells is converted into plasmin by constitutively expressed tPA (tissue-type plasminogen activator) or uPA (urokinase-type plasminogen activator). Plasmin formed on the cell membrane then induces a unique response characterized by membrane blebbing and vesiculation. Hitherto unknown for plasmin, these membrane changes are similar to those induced by thrombin on platelets. If plasmin formation persists, matrix proteins are then degraded, cells lose their attachments and enter the apoptotic process, characterized by DNA fragmentation and specific ultrastructural features. Since other proteolytic or inflammatory stimuli may evoke similar responses in different types of adherent cells, the proposed experimental procedure can be used to distinguish activated adherent cells from cells entering the apoptotic process. Such a distinction is crucial for evaluating the effects of mediators, inhibitors and potential therapeutic agents.

Comparación de la activación y la cinética de laplasmina bufalina con la humana / Ativação e cinética comparativa da plasmina bufalina e a humana / Comparative activation and kinetic of plasmin bufaline with the human one

El plasminógeno es el zimógeno de la plasmina, enzima activada a nivel fisiológico por el activadortisular del plasminógeno y la urokinasa, la plasmina es la enzima encargada de disolver el coágulosanguíneo. En este estudio se compararon la plasmina humana con la bufalina en su forma de activación dezimógeno a enzima y en la afinidad hacia el sustrato cromogénico. Los plasminógenos fueron purificadospor el mismo método de cromatografías de afinidad y cambio iónico. De igual manera las activaciones sehicieron utilizando urokinasa humana en ambos casos. La plasmina bufalina demostró mayor activacióny afinidad (1.35mM) que la plasmina humana (2.16 mM), siendo la bufalina 1.5 veces mas afin al sustratocromogénico que la humana. Este estudio demuestra que el método de purificación de los plasminógenospuede ser el mismo para muchas especies, se demuestra una vez más que las plasminas animales al parecerson más eficientes en la disolución del coágulo o degradación de sustratos, que la plasmina humana.Este estudio indica que la plasmina bufalina puede ser utilizada en los parámetros que se determinanclínicamente en pacientes con problemas cardiovasculares, reduciendo el tiempo de determinación de estosparámetros fibrinolíticos, que pueden dar al médico un margen de tiempo superior para actuar.

The Plasminogen is the zymogene of the Plasmin, enzyme which physiologically is activated by twodifferent enzymes, the tissue plasminogen activator and the urokinase, the plasmin is the enzyme that dissolves blood clots. In this study the human plasmin was compared to the bufaline plasmin, in theactivation from the zymogene to the enzyme form as well as in the affinity to the chromogenic substrate.The two plasminogens were purified by the same chromatographies methods: affinity and ion-exchange.Furthermore, both plasminogens were activated by human urokinase. The bufaline plasmin showed moreactivation and affinity (1.35 mM) that the human plasmin (2.16 mM), in addition, the bufaline plasmindemonstrated a 1.5 times more affinity to the chromogenic substrate that the human plasmin. This studydemonstrated that the plasminogens of several species can be purified by this method. Besides, one moretime the animal’s plasmins probably to be more efficient in the dissolution of blood clots or degradation ofsubstrates than the human plasmin. More over this study indicated that the bufaline plasmin can be usedin clinical determinations of patients with cardiovascular diseases. This also reduces the determinationtime of fibrinolytic parameters that physicians can give, having more time to take appropriate treatment.

O plasminogênio é o zymogen da plasmina, enzima ativada a nivel fisiológico pelo ativador tissulardo plasminogênio e uroquinase, plasmina é a enzima responsável de dissolver o coágulo de sanguíneo.neste estudo foi comparada a plasmina humana com a plasmina búbalina em seu modo de ativaçãode zymogen a enzima e na afinidade substrato cromogênico. Os plasminogênio foram purificados como mesmo método de cromatografia de afinidade e de troca iônica, e as ativações foram feitas usandouroquinase humana nos dois casos. A Búfalo plasmina mostrou maior ativação e afinidade (1.35 mM)que a plasmina humana (2.16 mM), sendo a bufalina 1.5 vezes mais afim ao substrato Cromogênico quea humana. Este estudo mostrou que o método de purificação do plasminogênios pode ser o mesmo paramuitas espécies, alem disso, que as plasminas animais são mais eficientes na dissolução do coáguloo degradação de substratos que a plasmina humana. Este estudo indicou que a plasmina búfalo podeser utilizada nos parâmetros determinados clínicamente em pacientes com problemas cardiovasculares,diminuindo o tempo de determinação destes parâmetros fibrinolíticos, que podem dar ao médico umintervalo de maior tempo para atuar.

Mouse plasminogen has oxidized phosphatidylcholine adducts that are not metabolized by lipoprotein-associated phospholipase A2under basal conditions.

We previously showed that plasminogen (Plg) isolated from the plasma of normal human subjects contains 1-2 moles of oxidized phosphatidylcholine (oxPtdPC) adducts/mole of protein. Moreover, we suggested that these species are generated at the hepatic site and speculated that they may play a role in the reported cardiovascular pathogenicity of Plg. We aimed to determine whether mouse Plg also harbors linked oxPtdPCs and whether these molecules are metabolized by lipoprotein-associated phospholipase A(2)/PAF acetylhydrolase (Lp-PLA(2)/PAF-AH), an enzyme specific for hydrolysis of oxPtdPCs. We determined the total concentration of Plg in plasma samples from control (WT) and Lp-PLA(2)-deficient (KO) mice, we isolated Plg, and assessed its content of oxPtdPCs by immunoblot analyses. We also evaluated whether human recombinant Lp-PLA(2) metabolized Plg-linked oxPtdPCs in vivo and in vitro. WT and KO mice expressed comparable levels (14.4-15.8 mg/dL) of plasma Plg, as determined by ELISA. We observed no differences in the content of oxPtdPC in Plg isolated from the two mouse strains and in parallel no changes in oxPtdPC content in mouse Plg following incubation with pure recombinant Lp-PLA(2). Plg from mouse plasma contains oxPtdPC adducts that are not affected by the action of Lp-PLA(2), suggesting that linkage to Plg protects oxPtdPCs from metabolism during their transport in the plasma. This modification may have important physio-pathological implications related to the function of Plg, oxPtdPCs, or both.

Refolding, purification, and activation of miniplasminogen and microplasminogen isolated from E. coli inclusion bodies.

Two des-kringle derivatives of human plasminogen, microplasminogen and miniplasminogen, have been expressed at high levels as inclusion bodies in Escherichia coli using a T7 expression system. In each case, the isolated inclusion bodies were refolded and purified. A final yield of approximately 10% of total refolded protein was observed in each case. Both refolded molecules were successfully activated to their functional forms, microplasmin and miniplasmin, by the plasminogen activator urokinase. The kinetic properties of the refolded microplasmin and miniplasmin were comparable to full length, native plasmin.

Direct binding of recombinant plasminogen kringle 1-3 to angiogenin inhibits angiogenin-induced angiogenesis in the chick embryo CAM.

Angiogenin is one of the most potent angiogenesis-inducing proteins. Angiostatin is one of the most potent angiogenesis inhibitors, and it contains the first four kringle domains of plasminogen (K1-4). Recombinant human plasminogen kringle 1-3 (rK1-3) was expressed in Escherichia coli and purified to homogeneity. The binding of t-4-aminomethylcyclohexanecarboxylic acid with the purified kringle 1-3 was determined by changes in intrinsic fluorescence. rK1-3 exhibits comparable ligand-binding properties as native human plasminogen kringle 1-3. The purified rK1-3 inhibits neovascularization in the chick embryo chorioallantoic membrane (CAM) assay. Interaction of angiogenin with rK1-3 was examined by immunological binding assay and surface plasmon resonance kinetic analysis, and the equilibrium dissociation constants for the complex, Kd, are 0.89 and 0.18 microM, respectively. rK1-3 inhibits angiogenin-induced angiogenesis in the chick embryo CAM in a concentration-dependent manner. These results indicate that rK1-3 directly binds to angiogenin and thus rK1-3 inhibits the angiogenic activity of angiogenin.

[Two incompatible plasmids coexpress the human endostatin and predigested human plasminogen kringle 5 in E. coli].

OBJECTIVE: To construct the recombinant plasmid pET28a/hES and coexpress the human endostatin (hES) and predigested human plasminogen kringle 5 (predhPK-5) in E. coli. METHODS: The mRNA was extracted from human liver tissue, and the endostatin gene was amplified by RT-PCR, which then was cloned into pET-28a(+). Under screening pressure by ampicillin and kanamycin simultaneously, E. coli BL21 (DE3) was cotransformed with pET28a/hES and pGEX-1lambdaT/predhPK-5 and induced with IPTG to express the recombinant proteins. The stability of cotransformants existing in E. coli was measured through two aspects in the serial culture time and passage number of bacterium. RESULTS: The two incompatible plasmids could be coexisted under the pressure of two antibiotics (ampicillin and kanamycin). After induced with IPTG, both human endostation and predhPK-5 gene were coexpressed, and the recombinant proteins comprised about 20% and 21% of total cell proteins, respectively. The two incompatible plasmids could still be maintained in over 75% E. coli cells for at least 16 hours or after 120 passages under the pressure of two antibiotics. CONCLUSION: The two incompatible plasmids pET28a/hES and pGEX-1lambda T/predhPK-5 may coexpress the recombinant proteins in E. coli. A new method for coexpression of proteins in E. coli containing two incompatible plasmids is proved to be feasible.

Safety profile of the intravitreal streptokinase-plasmin complex as an adjunct to vitrectomy in the rabbit.

BACKGROUND: The generation of an atraumatic posterior vitreous detachment (PVD), a common goal in vitreoretinal surgery, is a challenge, particularly in children and young trauma patients. Plasmin has been proposed as an adjunct to vitrectomy to enzymatically generate a PVD. Low doses of streptokinase-activated plasmin were tested in human pilot studies. This dose-escalation study assesses the safety range of intravitreal human streptokinase-plasmin in rabbits. METHODS: Plasminogen was isolated from human plasma by affinity chromatography, followed by activation with streptokinase (1:1), to generate the streptokinase-plasmin complex. Enzyme doses from 0.1-7 activity units (AU, in 0.1 ml) were injected into the mid-vitreous of 35 eyes; six control eyes were injected with balanced salt solution (BSS, 0.1 ml). Thirty minutes after injection, a two-port vitrectomy was performed. Fundus and slit lamp examinations were performed on days 1 and 7. On days 2 and 7, bright flash electroretinography was performed and compared with preoperative recordings. Some animals receiving higher doses of streptokinase-plasmin (1-7 AU) were followed clinically and with electroretinography for up to 9 months. RESULTS: A mild-to-moderate inflammatory response was seen in both control and plasmin-treated eyes on day 1, but had disappeared completely by day 7 in most eyes. In the 7 AU group, inflammation was stronger and more protracted. Two of three eyes from this group developed wrinkling of the medullary rays; one of them showed discoloration and traction at the medullary rays in the late follow-up. Electroretinograms (ERGs) of vitrectomized control eyes showed the following changes from preoperative values: 48 h, a-wave -11.10% [no significant (n.s.)], b-wave -14.62% (P=0.046); 7 days, a-wave +9.18% (n.s.), b wave +11.69% (n.s.). For the enzyme-treated eyes: 48 h: a-wave -20.43% (P<0.001), b-wave -9.57% (p<0.001); 7 days: a wave -14.21% (P<0.001), b-wave +2.48% (P<0.001). There was no evidence of dose-dependent ERG changes in enzyme-treated eyes at doses up to 5 AU. Groups of up to 3 AU were investigated by light and transmission electron microscopy, without evidence of toxicity. CONCLUSION: Streptokinase-plasmin doses up to 3 AU were found to be safe when injected into rabbit eyes followed by vitrectomy.

Production of recombinant human microplasminogen and pilot study in inducing posterior vitreous detachment.

PURPOSE: To realize the high production of recombinant human microplasminogen (r-mPlg) with Pichia pastoris and demonstrate the efficacy of r-mPlg in inducing posterior vitreous detachment (PVD). METHODS: Recombinant plasmid pAO815-3mPlg was constructed and transformed into SMD1168 cells. Positive recombinant clones were selected with MD plate and cultured in BMG medium, then induced in BMM medium. A protein band corresponding to mPlg with molecular mass of 29 kDa was revealed in SDS-PAGE and confirmed by Western blot. Anion-exchange chromatography and plasminogen activity assay kit were used to obtain purified r-mPlg with biological activity. Twenty eyes of freshly slaughtered pigs were divided into 4 groups, 5 eyes in each group. Group 1 served as normal control. Intravitreal injection of 0.1 ml BSS, 1000 IU/0.1 ml recombinant streptokinase (r-SK) and 1000 IU/0.1 ml r-SK plus 3 U/0.1 ml r-mPlg was done respectively to groups 2, 3, and 4. After incubation at 37 degrees C for 60 min, all eyes were processed for light microscopy, scanning electron microscopy (SEM), and transmission electron microscopy (TEM). RESULTS: r-mPlg, which has potential fibrinolytic activity, was successfully obtained with yield of 30 mg/L and purity of 97%. PVD was demonstrated by SEM in group 4 but not in other three groups. The retina and the inner limiting membrane (ILM) were well preserved in all eyes. CONCLUSION: r-mPlg, which has potential fibrinolytic activity, can be produced through Pichia pastoris expression system. Three U of r-mPlg combined with 1000 IU r-SK was effective in producing PVD without damaging the retina.

Peroxynitrite and fibrinolytic system: the effect of peroxynitrite on plasmin activity.

We have shown that peroxynitrite (ONOO-) inhibits streptokinase-induced conversion of plasminogen to plasmin in a concentration-dependent manner and reduces both amidolytic (IC5o approximately 280 microM at 10 microM concentration of enzyme) and proteolytic activity of plasmin. Spectrophotometric and immunoblot analysis of peroxynitrite-treated plasminogen demonstrates a concentration-dependent increase in its nitrotyrosine residues that correlates with a decreased generation of active plasmin. Peroxynitrite (1 mM) causes the nitration of 2.9 tyrosines per plasminogen molecule. Glutathione, like deferoxamine, partially protects plasminogen from peroxynitrite-induced inactivation and reduces the extent of tyrosine nitration. These data suggest that nitration of plasminogen tyrosine residues by peroxynitrite might play an important role in the inhibition of plasmin catalytic activity.

[Methods and prospects of development of the affinitive sorbents for plasminogen isolation from human blood plasma]. / Puti i perspektivy sozdaniia affinnykh sorbentov dlia vydeleniia plazminogena iz plazmy krovi cheloveka.

The review paper was dedicated to development of the promising photochemical synthesis of affine sorbents for plasminogen isolation from the human blood plasma. Some most interesting, from the viewpoint of practice, types of sorbents and carriers based on high-molecular compounds of natural (organic) or synthetic origin have been considered. The advantages of the use of photochemical synthesis of biospheric sorbent as compared with thermochemical method have been shown. The most promising methods of creation of affine sorbents on the basis of oligomer-polymeric photoinitiators and oligomerpolymeric photosensibilizing (donor-acceptor) systems are presented.

Removal of endotoxin by reverse phase HPLC abolishes anti-endothelial cell activity of bacterially expressed plasminogen kringle 5.

The success of recombinant protein expression/purification in Escherichia coli depends on a high-fidelity system rendering purified proteins free of confounding contaminants such as endotoxin. Here we report on the expression and purification of a cryptic plasminogen-derived domain, kringle 5, which was previously reported to specifically inhibit endothelial cell growth and, therefore, angiogenesis. Using a histidine (HIS)-tag expression and Ni(+)-NTA agarose purification system identical to previous reports, we found that our purified recombinant kringle 5 did inhibit endothelial cell growth, but this activity could not be eradicated by heat denaturing or proteolysis of kringle 5 with various proteases. This led us to suspect the presence of a contaminant in the purified samples. Quantitative endotoxin testing using a limulus amoebocyte lysate assay revealed that all samples purified by Ni(+)-NTA agarose alone harbored high concentrations of endotoxin that could not be removed by additional purification on anion exchange chromatography. Finally, when kringle 5 was rendered endotoxin-free by purification on reverse phase high-performance liquid chromatography (HPLC), there was a complete loss of endothelial cell growth inhibitory activity. These results strongly suggest that endotoxin-free recombinant kringle 5 may not possess anti-angiogenic activity and demonstrates that, especially in angiogenesis type assays, endotoxin contamination can lead to a misinterpretation of results.