lysine-Sepharose Affinity Chromatography Research Articles

Bacillolysin MA, a Novel Bacterial Metalloproteinase That Produces Angiostatin-like Fragments from Plasminogen and Activates Protease Zymogens in the Coagulation and Fibrinolysis Systems

We isolated a novel protease that converts plasminogen to angiostatin-like fragments (BL-angiostatins) from a culture of Bacillus megaterium A9542 through a single-step chromatography on CM-cellulose. The protease, designated bacillolysin MA (BL-MA), belongs to a family of neutral metalloproteinases based on the nucleotide sequence of its gene. At an enzyme:substrate ratio of 1:540, BL-MA cleaved human plasminogen mainly at Ser441-Val442 to form BL-angiostatin and miniplasminogen with a K(m) of 3.0 +/- 0.8 microM and a k(cat) of 0.70 +/- 0.09 s(-1). The resulting BL-angiostatins inhibited the proliferation, migration, and tube formation of vascular endothelial cells at concentrations of 1-10 microg/ml. Although BL-MA failed to activate plasminogen, it increased urokinase-catalyzed activation of plasminogen caused by production of miniplasminogen, which is highly susceptible to activation. In addition, BL-MA was active in converting prourokinase, prothrombin, coagulation factor X, and protein C to their active forms. BL-MA enhanced both the clotting of human plasma and clot dissolution in the presence of prourokinase. Thus, BL-MA affects blood coagulation and fibrinolysis systems and can be used to produce angiostatin-like plasminogen fragments and active serine proteases of human plasma.

Stimulation of Vascular Smooth Muscle Cell Proliferation and Migration by Apolipoprotein(a) Is Dependent on Inhibition of Transforming Growth Factor-β Activation and on the Presence of Kringle IV Type 9

Elevated plasma concentrations of lipoprotein(a) are a risk factor for the development of a variety of atherosclerotic disorders. Despite intensive study, the mechanisms by which lipoprotein(a) promotes these disorders remain to be unequivocally defined. It has been demonstrated that lipoprotein(a), through its unique constituent apolipoprotein(a) (apo(a)), stimulates vascular smooth muscle cell (SMC) migration and proliferation. These effects arise from the ability of apo(a) to inhibit the formation of active transforming growth factor beta (TGF-beta) from its latent precursor, which in turn is caused by the ability of apo(a) to decrease the formation of plasmin from its precursor plasminogen. We utilized a battery of recombinant apo(a) variants that represent systematic deletions of the various domains in the molecule to further probe the mechanism underlying the effect of apo(a) on SMC responses. All recombinant apo(a) variants that contained kringle IV type 9 were able to stimulate SMC proliferation and migration and to decrease the formation of active TGF-beta; conversely all recombinant apo(a) variants lacking kringle IV type 9 had no effect on these parameters. The kringle IV type 9-dependent effects of apo(a) on SMC proliferation required the presence of plasminogen, suggesting for the first time that this kringle mediates the ability of apo(a) to inhibit pericellular plasmin formation.

Generation and role of angiostatin in human platelets

AbstractPlatelets regulate new blood vessel growth, because they contain a number of angiogenesis promoters and inhibitors. Additionally, platelets contain matrix metalloproteinases (MMPs), which when released mediate platelet adhesion and aggregation, and plasminogen, a fibrinolytic system enzyme that serves to limit blood clot formation. Enzymatic cleavage of plasminogen by MMPs generates angiostatin, an angiogenesis inhibitor. Therefore, we examined whether platelets generate angiostatin during aggregation in vitro. Platelets were isolated from healthy human donors and then aggregated with collagen, thrombin, or HT-1080 fibrosarcoma cells. Angiostatin was detected by Western blot analysis in the platelet releasates of all blood donors irrespective of the aggregating agent used. Platelet pellet homogenates showed the presence of angiostatin in all donors, which was released upon aggregation. Furthermore, platelet-derived angiostatin was isolated and purified by lysine-Sepharose affinity chromatography from collagen-aggregated platelet releasates. Bioassay of platelet-derived angiostatin showed that it inhibited the formation of capillary structures by human umbilical vein endothelial cells (HUV-EC-Cs) in an in vitro angiogenesis model. Inhibition of angiostatin in platelet releasates promoted the formation of capillary structures by HUV-EC-Cs. We conclude that healthy human platelets contain angiostatin, which is released in active form during platelet aggregation, and platelet-derived angiostatin has the capacity to inhibit angiogenesis.

A New Method of Purifying Fibrinogen with Both Biological and Immunological Activity from Human Plasma

Using a combination of Cohn ethanol fractionation, virus inactivation, glycine and sodium chloride precipitation, and lysine-Sepharose affinity chromatography, a unique and rapid simplified method was developed to obtain highly purified fibrinogen for diagnostic use with both biological (Clauss method) and immunological (Jacobsson method) activity. Yield was 0.66 g of fibrinogen per liter of starting pooled plasma, and the purified product showed good agreement in activity with the starting material. The purified fibrinogen solution contained over 95% clottable protein and had a clear appearance. No degradation was observed after urokinase treatment and the preparation provided good precision in fibrinogen measurement compared to pooled plasma. The simplified method was, thus, shown to result in a high-purity fibrinogen preparation, suitable for in vitro diagnostic use, as well as for use to prepare a fibrinogen reference material and to perform fibrinogen quality control using an automated coagulation analyzer.

Characterization of the Interactions of Plasminogen and Tissue and Vampire Bat Plasminogen Activators with Fibrinogen, Fibrin, and the Complex of d-Dimer Noncovalently Linked to Fragment E

Vampire bat plasminogen activator (b-PA) causes less fibrinogen (Fg) consumption than tissue-type plasminogen activator (t-PA). Herein, we demonstrate that this occurs because the complex of D-dimer noncovalently linked to fragment E ((DD)E), the most abundant degradation product of cross-linked fibrin, as well as Fg, stimulate plasminogen (Pg) activation by t-PA more than b-PA. To explain these findings, we characterized the interactions of t-PA, b-PA, Lys-Pg, and Glu-Pg with Fg and (DD)E using right angle light scattering spectroscopy. In addition, interactions with fibrin were determined by clotting Fg in the presence of various amounts of t-PA, b-PA, Lys-Pg, or Glu-Pg and quantifying unbound material in the supernatant after centrifugation. Glu-Pg and Lys-Pg bind fibrin with Kd values of 13 and 0.13 microM, respectively. t-PA binds fibrin through two classes of sites with Kd values of 0.05 and 2.6 microM, respectively. The second kringle (K2) of t-PA mediates the low affinity binding that is eliminated with epsilon-amino-n-caproic acid. In contrast, b-PA binds fibrin through a single kringle-independent site with a Kd of 0.15 microM. t-PA competes with b-PA for fibrin binding, indicating that both activators share the same finger-dependent site on fibrin. Glu-Pg binds (DD)E with a Kd of 5.4 microM. Lys-Pg binds to (DD)E and Fg with Kd values of 0.03 and 0.23 microM, respectively. t-PA binds to (DD)E and Fg with Kd values of 0.02 and 0.76 microM, respectively; interactions were eliminated with epsilon-amino-n-caproic acid, consistent with K2-dependent binding. Because it lacks a K2-domain, b-PA does not bind to either (DD)E or Fg, thereby explaining why b-PA is more fibrin-specific than t-PA.

High-level secretion in Pichia pastoris and biochemical characterization of the recombinant kringle 2 domain of tissue-type plasminogen activator.

The kringle 2 (K2) domain of tissue-type plasminogen activator (tPA) has been expressed in Pichia pastoris cell lines GSI 15 and KM71. This construct contained a hexahistidine sequence at the C-terminus of the kringle to aid in purification by immobilized metalion-affinity chromatography. The exact amino acid sequence of the isolated kringle was EAEAYV-[K2tPA]SR(H)6, where [K2tPA] represents amino acid sequence residues C1-C82 of the kringle domain (residues 180-261 of tPA). The clones of the yeast transformants provided large amounts of the recombinant (r)-[K2tPA]-containing polypeptide at levels that allowed ready purification of several hundred mg from shake flasks and near-gram levels from a high-biomass fermenter. Purification of the kringle domain directly from cell-conditioned media was accomplished in a single step by either immobilized Ni(+)-affinity chromatography or lysine-Sepharose affinity chromatography. N-linked glycans were present on approx. 30% of this yeast-expressed material, at N5 of the kringle (corresponds to N11 of the particular construct, N184 of full-length tPA). The expressed recombinant kringle recognized a conformation-specific monoclonal antibody generated against tPA that is directed to the K2 domain of the protein, interacted properly with various omega-amino acid ligands, and showed signature conformational properties when studied by differential scanning calorimetry and high-resolution 1H-NMR. The results demonstrate that the P. pastoris system can be employed to obtain large amounts of secreted and properly folded kringle domains.

Simple and rapid procedure for the purification of lipoprotein(a)

Lipoprotein(a) [Lp(a)] is a low-density lipoprotein-like particle displaying strong athero-thrombotic properties. Highly purified Lp(a) is increasingly requested for standardization of Lp(a) measurements and for biological studies. Several procedures have been described for Lp(a) separation and purification but none of them appear completely suitable. We present here a procedure for Lp(a) purification based on sequential elutions after lysine-Sepharose affinity chromatography. We were able to identify four distinct subspecies of Lp(a) showing different affinity to ϵ-amino groups of lysine-Sepharose, simply by modifying molarity and pH of the eluents; the fraction obtained in highly purified state represented the major form and could be eluted with 0.5 M sodium phosphate buffer (pH 4.4). Advantages of this procedure are represented by simplicity, rapidity and final yield.

Identification of Two Functionally Distinct Lysine-binding Sites in Kringle 37 and in Kringles 32−36 of Human Apolipoprotein(a)

The well documented association between high plasma levels of lipoprotein(a) (Lp(a)) and cardiovascular disease might be mediated by the lysine binding of apolipoprotein(a) (apo(a)), the plasminogen-like, multikringle glycoprotein in Lp(a). We employed a mutational analysis to localize the lysine-binding domains within human apo(a). Recombinant apo(a) (r-apo(a)) with 17 plasminogen kringle IV-like domains, one plasminogen kringle V-like domain, and a protease domain or mutants thereof were expressed in the human hepatocarcinoma cell line HepG2. The lysine binding of plasma Lp(a) and r-apo(a) in the culture supernatants of transfected HepG2 cells was analyzed by lysine-Sepharose affinity chromatography. Wild type recombinant Lp(a) (r-Lp(a)) revealed lysine binding in the range observed for human plasma Lp(a). A single accessible lysine binding site in Lp(a) is indicated by a complete loss of lysine binding observed for r-Lp(a) species that contain either a truncated r-apo(a) lacking kringle IV-37, kringle V, and the protease or a point-mutated r-apo(a) with a Trp-4174-->Arg substitution in the putative lysine-binding pocket of kringle IV-37. Evidence is also presented for additional lysine-binding sites within kringles 32-36 of apo(a) that are masked in Lp(a) as indicated by an increased lysine binding for the point mutant (Cys-4057-->Ser), which is unable to assemble into particles. An important role of these lysine-binding site(s) for Lp(a) assembly is suggested by a decreased assembly efficiency for deletion mutants lacking either kringle 32 or kringles 32-35.

The Activation of Type 1 and Type 2 Plasminogen by Type I and Type II Tissue Plasminogen Activator

Tissue plasminogen activator (tPA) was fractionated using lysine-Sepharose affinity chromatography. Type I, type II, and a minor peak with high affinity for lysine (designated type D) tPA were recovered. In an indirect amidolytic assay involving native human Glu-plasminogen and fibrin, type II tPA showed a 2-fold higher activity than type I. To explore the combinatorial effect of the variable glycosylation status of both tPA and plasminogen, kinetic constants for fibrin-dependent plasminogen activation were determined for combinations of type I, II, and D tPA with type 1 and 2 plasminogen. Within a 4-fold range, the fastest rate was achieved from the combination of type D (type II + D) tPA and type 2 plasminogen. N-Glycosylation of plasminogen increased the Km value for activation by all tPA variants; N-glycosylation of type I tPA at Asn184 decreased the kcat (turnover) values for the fibrin-dependent activation of plasminogen over type II tPA, while type D tPA showed the highest turnover rate. In the presence of fibrinogen fragments, N-glycosylation of plasminogen at site 289 modulates the kinetics of association of enzyme and substrate, while N-glycosylation at site 184 on tPA modulates the turnover rate of the enzyme.

Cloning, expression, and characterization of human apolipoprotein(a) kringle IV37.

A portion of kringle IV37 (KIV37) of apolipoprotein (a), (apo(a)), was polymerase chain reaction-cloned from human liver cDNA. The protein product of this clone was expressed in Escherichia coli as a poly histidine fusion protein. Based on recovery of purified fusion apo(a) KIV37 protein expression levels were estimated to be 10 mg/g of E. coli cell paste. Mass spectral analysis showed the molecular mass of fusion apo(a) KIV37 to be 12,260 +/- 1 daltons. Almost all fusion apo(a) KIV37 was expressed as inclusion bodies and had to be refolded. Fusion apo(a) KIV37 was isolated from the inclusion bodies and purified by lysine-Sepharose affinity chromatography by eluting with 0.2 M epsilon-aminocaproic acid. The fusion protein was treated with thrombin to yield a homogeneous, functional apo(a) KIV37 domain composed of 92 amino acids having a molecular mass of 10,510 +/- 1 daltons. N-terminal protein sequencing and amino acid analysis have confirmed the sequence and composition of apo(a) KIV37. The molar extinction coefficient, epsilon, for apo(a) KIV37 was determined to be 3.1 x 10(4) M-1 cm-1, and the pI was measured to be 6.7 +/- 0.1. In addition, the dissociation constants, Kd, for a series of 11 lysine analogs have been determined by measuring the change in intrinsic fluorescence of apo(a) KIV37 upon saturable binding with these compounds. Kd values ranged from 4.2 +/- 0.9 microM for trans-4-(aminomethyl)cyclohexanecarboxylic acid to 4.6 +/- 0.4 mM for L-arginine. Apo(a) KIV37 binds to plasmin-treated fibrinogen with an EC50 value of 14 +/- 1.2 microM and prevents the binding of Lp(a) to plasmin-treated fibrinogen with an IC50 value of 16 +/- 6 microM. Lp(a) binds to the plasmin-treated fibrinogen surface with an EC50 value of approximately 1.0 +/- 0.3 nM. These studies demonstrate that apo(a) KIV37 can be expressed at high levels, refolded properly, and used as a fully functional lysine-binding domain. In addition, these results also demonstrate that apo(a) KIV37 provides the major interaction of Lp(a) with fibrinogen. One additional weak binding site in Lp(a) is adequate to describe overall Lp(a) binding to fibrinogen.

Lysine-Binding Heterogeneity of Lp(a): Consequences for Fibrin Binding and Inhibition of Plasminogen Activation

Lipoprotein(a) [Lp(a)] is recognized as an independent risk factor for atherosclerosis. Lp(a) consists of a LDL-like moiety with an additional glycoprotein, apo(a), linked to apolipoprotein B-100. Apo(a) has a high homology with plasminogen (Pg). In vivo, Pg is activated on a fibrin surface by tissue Pg activator (tPA). We prepared Lp(a) from plasma by sequential ultracentrifugation followed by lysine-sepharose affinity chromatography. We found that a changing (donor dependent) fraction of the Lp(a) did not bind to lysine-sepharose. This fraction, designated Lp(a)lys-, was further purified using gel filtration. Bound Lp(a) [Lp(a)lys+] was eluted with 0.2 M EACA. Apo(a) isoforms in both fractions were identical. In contrast Lp(a)lys+ inhibited Pg activation by tPA in vitro (IC50% 20 mg/l), whereas Lp(a)lys- did not. In addition Lp(a)lys- did not bind to CNBr-digested fibrinogen whereas Lp(a)lys+ did (Kd, app = 0.2 nM). Therefore we conclude that a changing donor dependent fraction of human plasma Lp(a) does not inhibit Pg activation in vitro and does not bind to CNBr-digested fibrinogen.

Purification and characterization of the D2 cell adhesion protein: analysis of the postnatally regulated polymorphic forms and their cellular distribution.

The developmentally regulated, D2 cell adhesion protein has been purified from 10-12 day old rat synaptosomes by sequential hydroxyapatite chromatography, wheat germ lectin affinity chromatography and gel filtration. The purified protein was found to be composed of two polypeptide components of 200 and 140 kd molecular weight which comprised 0.5-1.0% of total synaptosomal membrane protein. Lysine-Sepharose affinity chromatography could further separate the purified protein into sialic acid-rich and sialic acid-poor forms. Immunoblot analysis of whole brain homogenates and synaptosomes with an antiserum raised against the purified protein (anti-D2) revealed the presence of three immunologically related polypeptides of 200, 140, and 115 kd molecular weight. These polypeptides, which appeared as a diffuse zone (greater than 200 kd) in fetal material, were found to developmentally regulate by altering their relative expression. This was particularly marked in the 200 kd component. Furthermore, the 200 kd polypeptide appeared to be neuron-specific as both the 140 and 115 kd components were common to synaptosomes and primary cultures of astrocytes.

Isolation of the fourth component of guinea pig complement and its single polypeptide chain precursor from plasma

Guinea pig C4 (C4 gp)§ was isolated from plasma by polyethyleneglycol-4000 (PEG-4000) precipitation, lysine-Sepharose affinity chromatography removal of plasminogen and plasmin, DEAE-Sephacel, SP-Sephadex chromatography, and Sepharose CL-6B gel filtration. Both phenylmethylsulfonyl fluoride (PMSF) and ethylenediamine tetraacetate (EDTA) were present in all Chromatographie buffers, and each C4 pooled concentrate was made 0.5 m M in DFP. The overall yield for three consecutive preparations averaged 3.1% of the initial plasma hemolytic C4 activity and the average specific activity rose to 139,223 U/mg, representing an average purification factor of 157-fold. The preparation of C4 gave a single band on Ouchterlony analysis with antiserum to guinea pig serum or to purified C4. Analytical alkaline discontinuous gel electrophoresis revealed a dominant protein band and functional C4 eluted from replicate gels in this region. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of unreduced C4 gave a mol. wt of 200,000, whereas SDS-PAGE of reduced and alkylated C4 demonstrated subunits of 93,000, 75,000 and 33,000 mol. wt as well as a small amount of non-reducible 200,000 mol. wt material. Elution of the 200,000 mol. wt material recognized by SDS-PAGE under reducing conditions revealed antigenic relatedness to either the α- or β-chain of C4, suggesting that it represents a pro-C4 single chain polypeptide.

Plasminogen activator in the ascitic fluid Associated with peritonitis carcinomatosa

Larger amounts of FDP were found in the ascitic fluid associated with peritonitis carcinomatosa than in the non-malignant fluid. By Todd's fibrin slide method, the dissolution of fibrin was observed around free cancer cells in the ascites, but not around the granulocytes. Therefore, it was suggested that the origin of FDP in malignant ascitic fluid was the plasminogen activator dependent on the cancer cells.By Lysine-Sepharose affinity chromatography, plasminogen activator was partially purified 4, 500 folds from ascitic fluid with peritonitis carcinomatosa of breast cancer (FDP 1, 280μg/ml). Euglobulin fraction of the malignant ascites was applied to the column on Lysine-Sepharose 4B, and eluted with 0.5M NaCl, 1M NaCl and 0.1M acetic acid. The activator was observed in the fraction of 1M NaCl elution, and plasmin in the fraction of 0.1M acetic acid.On Ouchterlony's method, a precipitin line was not observed between the activator and anti-urokinase rabbit serum.This result indicates that this plasminogen activator might be different from urokinase.

Coagulation and fibrinolysis in hepatic cirrhosis

In order to clarify coagulation and fibrinolysis in hepatic cirrhosis following items were examined.1) Counts of megakaryocyte and its classification. 2) Lifespan of 5Cr-labeled platelet. 3) Platelet volume and its distribution (Coulter Counter ZB). 4) Platelet spreading test (Breddin's method). 5) Platelet aggregation (Evans Aggregometer). 6) Platelet Factor-3 (Kaolin). 7) Serum phospholipids and its fractionation. 8) Hepaplastin test (Eisai pharmaceutical Co, test for II, Vll, X factors) 9) Plasma fibrinogen (Tyrosine). 10) FDP (HIT). 11) Protamine sulfate test (Lipinski's). 12) Total plasmin (fibrin plate). 13) Plasminogen and antiplasmin (lysine sepharose affinity chromatography) 14) Factor XIII (Urayama's). from these examinations following facts were clarified.1) Reduction of platelet counts is due to disturbance of platelet formation and enhanced breakdown of platelets and may either due to pooling in the spleen.The remarkable reduction and its characteristic asthenic pattern of platelet aggregation may suggest hypofunction of the platelet cell. It may be supported with the facts of the large spreading tendency of the platelet and reduction of PF-3 availability.2) Hepaplastin test clearly shows reduction of extrinsic factor in cirrhosis of the liver.3) In regard to f ibrinolysis, in case of the hepatic cirrhosis, plasma fibrinogen tends to decrease, on the other hand protamind sulfate test tend to show higher value.Total plasma plasmin increased remarkably, on the other hand, plasminogen and antiplasmin slightly decreased. Factor XIII decreased either in case of cirrhosis.Whether the enhanced fibrinolysis may occur primary or secondary must be examined more in detail.

Mechanisms involved in enhancement of plasma fibrinolytic activity by chloroform

The effects of a single 1-min extraction with chloroform (CHCl3) on plasma fibrinolytic activity has been examined by 125I-fibrin solid phase assay, using normal plasma and plasma depleted of plasminogen (PLG) by lysine-Sepharose affinity chromatography. Fibrinolytic activity of normal plasma is increased (40%-175%), and more than 95% of antiplasmin activity is removed. The increase is demonstrable in PLG- depleted plasma, and is not inhibited by tranexamic acid (0.01 M). Purified PLG is not activated to plasmin by ChCl3 treatment. Bio-Gel A 0.5 m fractionation of CHCl3-extracted, PLG-depleted plasma reveals fractions with the following activities: (1) streptokinase-activatable, PLG-independent fibrinolytic activities; (2) PLG activator activities; and (3) plasmin-stimulated but PLG-independent fibrinolytic activities, which include activities inhibited by hexadimethrine bromide and which cofractionate in part with plasmin-stimulated procoagulant activities. In addition, similar fractionation of nonextracted plasma reveals two non-plasmin fibrinolytic activities (approximately 30,000 and 13,000 daltons) activated by streptokinase and plasmin, respectively. The findings indicate that the enhanced fibrinolytic activity resulting from CHCl3 treatment is independent of plasmin as the ultimate fibrinolytic enzyme, although activities stimulated by plasmin may contribute, and that such treatment is a useful maneuver for study of PLG-dependent and PLG-independent fibrinolytic mechanisms, and their interactions.

Fibrinolytic enzyme system in blood of rats with urinary bladder tumor induced by oral administration of N-butyl-N-(4-hydroxybutyl)-nitrosamine (author's transl)

There are many reports on the relationship among malignant tumors, blood clotting and fibrinolytic enzyme system. The present report describes the fibrinolytic enzyme system in plasma of rats with experimental urinary bladder tumor. The plasma was separated into three fractions with lysinesepharose affinity chromatography. The materials were Wistar-Imamichi strain male rats which had been administered a dose of 0.02mg/head/day of N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) from 8 weeks old to 16 weeks old and then sacrified at 28 weeks old. Fraction-I did not contain either plasmin (PL) or plasminogen activator (PLg-act). Though the normal rat plasma sometimes has a slight antiplasmic action, the plasma of rats which had been administered BBN had a marked antiplasmic action. However, the action did not correspond with bladder tumor and hyperplasia. The rat plasma had an antiurokinase activity irrespective of BBN-administration. Sometimes fraction-II had also PLg-act irrespective of BBN-administration. On the rats which had been administered BBN, the activity of PLg-act in the bladder tumor-group and in the hyperplasia-group had an increasing tendency which was more marked than that in the unchanged group. Fraction-III revealed mainly the PL-activity. Normal rats had no activated PL, but the animals administered BBN revealed PL-activity. The PL-activity in the unchanged group had a more marked increasing tendency that in the bladder tumor group and hyperplasia group.