Process of fibrin depolymerization and non-enzymatic fibrinolysis in rabbits receiving atherogenic rations with addition of antioxidants

The rabbits were kept on atherogenic ration for 2 months. This diet contained 0.3 mg/kg of cholesterol. In blood plasma of animals the authors observed a sharply reduced non-enzymatic fibrinolysis and depolymerization activity of non-stabilized fibrin. The addition of antioxidants and alpha-tocopherol (10 + 10 mg/kg) for 1 month to the atherogenic ration protected from the disturbance of the system hemostasis and normalized the depolymerization of non-stabilized fibrin.     

Destabilase: an enzyme of medicinal leech salivary gland secretion hydrolyzes the isopeptide bonds in stabilized fibrin

The salivary gland secretion of the leech Hirudo medicinalis contains an enzyme termed by us as destabilase, which hydrolyzes the epsilon-(gamma-glutamyl)-lysine bonds as a result of fibrin stabilization by factor XIIIa in the presence of Ca2+. This hydrolysis, apart from the original lysine and glutamine, is characterized by an appearance of lysine and glutamic acid residues. The accumulation of glutamic acid residues leads to spontaneous depolymerization of the destabilized fibrin. As a result, fluid "spots" of destabilized fibrin depolymerization (DFD) begin to appear at the sites of leech secretion application on the surface of stabilized fibrin plates. The DFD activity of the leech salivary gland secretion manifests itself only in case of stabilized fibrin and increases with an increase in the stabilization degree. Treatment of leech secretion with diisopropylfluorophosphate does not affect the enzyme activity, which is completely blocked by monoiodoacetate. The mechanism of action of leech salivary gland secretion and the enzyme isolated from it, i. e., destabilase, was studied, using a synthetic chromogenic substrate - p-nitroanilide-gamma-glutamic acid. The amidolytic activity of leech salivary gland secretion is 2.2 +/- 0.18 nkat/ml, Km(app) for destabilase is 0.6 X 10(-5) M, V = 5.4 X 10(-3) mol/min.     

Electron microscopic study of the depolymerization process on unstabilized fibrin as affected by urea or monochloracetic acid

With the help of the electronmicroscopic method the non-stabilized fibrin depolymerization has been studied. It has been established that under the action of urea or monochloridacetic acid the gradual transition of fibril clot's structure in globular fibrinolike material takes place. These globules have morphological likeness with the fibrin-monomer molecules and have analogy of the morphological properties to the non-stabilized fibrin dissolution products by complex compounds of heparin. The elimination of urea or monochloridacetic acid from media gives possibility to reconstruct the fibrillar fibrin structure.     

Protective antithrombotic effects of proline-containing peptides under the influence of stress on the animal organism

We discovered that simple proline-containing peptides Gly-Pro, Pro-Gly, Pro-Gly-Pro, and semax had an antistress protective effect on the organism appearing as anticoagulation system activation. Repeated intranasal injection of each of these peptides to rats prior to acute immobilization stress prevented a hypercoagulation response to prolonged stress lasting 60 min. At the same time there was increase of anti-thrombotic, anticoagulant, and fibrin depolymerization activity and recovery of enzymatic fibrinolytic activity. Dipeptides were found to have the greatest antistress effect. Our results showed that semax had a protective effect against enhanced blood coagulability resulting from repeated immobilization stress.     

The effect of tuftsin on fibrin polymerization in vitro and on the hemostatic system when administered intravenously to healthy animals

Taftsine tetrapeptide has antiprocoagulatory properties in vitro in the presence of plasma. Taftsine exerts depolymerization influence on fibrin monomer in concentrations of 10(-1) to 10(-9) mg/ml. At intravenous injection in doses of 1 mg and 300 ug/kg, taftsine causes the increase in plasma clotting time and significant increase in enzymatic and nonenzymatic fibrinolysis. The effect can be observed during 120 min after injection of the peptide.     

An actin-depolymerizing protein (destrin) from porcine kidney. Its action on F-actin containing or lacking tropomyosin

An Mr 19 000 protein (destrin) that has the ability to rapidly depolymerize F-actin in a stoichiometric manner was purified from porcine kidney by sequential chromatography on DNase I-agarose, hydroxyapatite, and Sephadex G-75. Its actin-depolymerizing activity is reversibly controlled by changes in KCl concentration but is insensitive to Ca2+ concentration. The rate of depolymerization of F-actin by destrin is much faster than that of spontaneous depolymerization induced by dilution and is not markedly decreased by the addition of end-blocking reagents such as cytochalasin B. These results suggest that destrin depolymerizes F-actin by interacting directly with F-actin protomers. Binding of muscle tropomyosin to F-actin slows down the rate of destrin-induced depolymerization of F-actin by about 30-fold. The data suggest that the destrin-induced depolymerization occurs from the ends of F-actin when F-actin is complexed with tropomyosin, but it takes place from the entire length of F-actin in the absence of tropomyosin.     

Effects of intact fibrin and partially plasmin-degraded fibrin on kinetic properties of one-chain tissue-type plasminogen activator

A comparative kinetic analysis of the enzymatic activities of one-chain and two-chain tissue-type plasminogen activator (t-PA) demonstrates that two-chain t-PA catalyzes the hydrolysis of the peptide substrate D-Val-Leu-Arg-pNA about 4-fold more effectively than one-chain t-PA. The difference is accounted for almost entirely by a corresponding difference is the kcat values of the enzymes, whereas the Km values are similar. The amidolytic activity of two-chain t-PA is not enhanced by intact or partially plasmin-degraded fibrin. In contrast, the activity of one-chain t-PA is stimulated up to 3.7-fold by intact fibrin and up to 4.7-fold by plasmin-degraded fibrin (fibrin X-fragment). The stimulatory effects are realized via increases in the kcat values. It appears thus that in the presence of fibrin the intrinsically inferior catalytic properties of one-chain t-PA become similar to the properties of two-chain t-PA. The dependency of the activity of one-chain t-PA on the concentration of fibrin monomer is consistent with a single association site of both proteins and an association constant of Kass = 6.25 x 10(6) l/mol. Stimulation of one-chain t-PA by plasmin-degraded fibrin is more complex and appears to involve two different binding sites with association constants of Kass = 0.67 x 10(9) l/mol and Kass = 3.85 x 10(6) l/mol, respectively. The stimulatory effects of fibrin and partially plasmin-degraded fibrin on one-chain t-PA are suppressed by epsilon-aminocaproic acid and by a monoclonal antibody directed against the lysine binding site of t-PA. The latter findings support the notion that fibrin activation of one-chain t-PA is mediated by the lysine binding site on kringel domains of the enzyme.     

Microtubule Depolymerization by the Kinesin-8 Motor Kip3p: A Mathematical Model

Proteins from the kinesin-8 family promote microtubule (MT) depolymerization, a process thought to be important for the control of microtubule length in living cells. In addition to this MT shortening activity, kinesin 8s are motors that show plus-end directed motility on MTs. Here we describe a simple model that incorporates directional motion and destabilization of the MT plus-end by kinesin 8. Our model quantitatively reproduces the key features of length-versus-time traces for stabilized MTs in the presence of purified kinesin 8, including length-dependent depolymerization. Comparison of model predictions with experiments suggests that kinesin 8 depolymerizes processively, i.e., one motor can remove multiple tubulin dimers from a stabilized MT. Fluctuations in MT length as a function of time are related to depolymerization processivity. We have also determined the parameter regime in which the rate of MT depolymerization is length dependent: length-dependent depolymerization occurs only when MTs are sufficiently short; this crossover is sensitive to the bulk motor concentration.     

Determination of depolymerization kinetics of amylose, amylopectin, and soluble starch by Aspergillus oryzae alpha-amylase using a fluorimetric 2-p-toluidinylnaphthalene-6-sulfonate/flow-injection analysis system

This study reports on the determination of the depolymerization kinetics of amylose, amylopectin, and soluble starch by Aspergillus oryzae alpha-amylase using flow-injection analysis with fluorescence detection and 2-p-toluidinylnaphthalene-6-sulfonate as the fluorescent probe. The experimental data points, corresponding to the evolution of the concentration of "detectable" substrate with depolymerization time, were fit to a single exponential decay curve in the case of amylose and to a double exponential decay curve in the cases of amylopectin and soluble starch. For all the assayed substrates, the determined depolymerization rates at time zero correlated well with the initial enzyme and substrate concentrations through the usual Michaelis-Menten hyperbola. Therefore, this methodology allows the determination of alpha-amylase activity using any of these substrates. For amylopectin and soluble starch, the value of the total depolymerization rate at any depolymerization time was the result of the additive contribution of two partial depolymerization rates. In contrast, the total depolymerization rate for amylose was always a single value. These results, in conjunction with the relative time evolution of the two partial depolymerization rates (for amylopectin and soluble starch), are in good agreement with a linear molecular structure for amylose, a "grape-like" cluster molecular structure for amylopectin, and an extensively degraded grape-like cluster structure for soluble starch.     

Gold labeling of thrombin and ultrastructural studies of thrombin-gold conjugate binding by fibrin

Monodispersed thrombin-gold (T-Au) conjugates were prepared by the absorption of a monolayer (3.8 nm thick) of human alpha-thrombin around individual monodispersed colloidal gold particles (16.5 +/- 1.8 nm). Like free molecular thrombin, T-Au conjugates can cause platelet aggregation, plasma clotting, and the release of fibrinopeptides A and B from fibrinogen. At the same thrombin concentration, T-Au conjugates have only one-tenth the fibrinogen-clotting activity of free thrombin and one-third the amidolytic activity of free thrombin. Hirudin can completely inhibit the fibrinogen-clotting activity of both T-Au conjugates and free thrombin, but can inhibit only half of the amidolytic activity of the conjugates. Diisopropyl fluorophosphonate can completely inhibit the fibrinogen-clotting activity and the amidolytic activity of both T-Au conjugates and free thrombin. T-Au conjugates were further characterized by studying the mechanism of their binding to fibrin and the location of the binding site on fibrin. The results of electron microscopic studies showed that T-Au conjugates, but not albumin-Au conjugates, are bound by fibrin. Increasing T-Au conjugate concentrations are associated with an increase in the number of T-Au conjugates binding to fibrin. At 0.1 microM thrombin, 73% of the T-Au conjugates are bound to branch points of the fibrin network with 27% of the T-Au conjugates present in the fibrin strands. At higher thrombin concentration (e.g., 0.5 microM) the percentage of T-Au conjugates bound to locations other than branch points increases to 62%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Construction and characterization of a recombinant chimeric plasminogen activator consisting of a fibrin peptide and a low molecular mass single-chain urokinase

A recombinant chimeric plasminogen activator (f beta/scuPA-32k), with a fibrin beta-chain peptide (comprising Gly15 through Arg 42) linked to the N-terminal of a low molecular mass (32 kDa) single-chain urokinase (scuPA-32k, comprising Leu144 through Leu 411) via a 50 amino acid linker sequence, was produced by expression the corresponding chimeric cDNA in Escherichia coli cells. After refolding in vitro, the chimeric protein was purified to homogeneity by zinc chelate-Sepharose chromatography, Sephacryl S200 chromatography and benzamidine-Sepharose chromatography in sequence. The apparent molecular mass was 36 kDa shown by SDS-PAGE analysis. The special activity was 87,000 IU/mg detected by fibrin plate determination. F beta/scuPA-32k could directly activate plasminogen following Michaelis-Menten kinetics with K(m) = 0.52 microM and k(2) = 0.0024 s(-1). Mediated by plasmin, the single-chain molecule could be converted to the active two-chain molecule. The chimeric protein had 3.3 times higher fibrin affinity than scuPA-32k in the fibrin concentration of 3.2 mg/mL, while the chimeric protein inhibited the fibrin clotting and platelet aggregation. F beta/scuPA-32k showed a higher thrombolytic potency in vitro plasma clot lysis than scuPA-32k and depleted less fibrinogen in plasma. These results showed that the chimeric protein had not only higher fibrinolytic activity but also anti-thrombus activity. Further evaluation of the thrombolytic potential in appropriate animal models is required.     

Activation of gelatinases by fibrin is PA/plasmin system-dependent in human glomerular endothelial cells

Evidence suggests that fibrin deposit is related to severity of glomerulonephropathy. Fibrin is considered to play an active role beyond a haemostatic plug or temporary matrix in response to injury. We have reported that fibrin induced specific morphological changes and up-regulated intercellular adhesion molecule-1 expression of glomerular endothelial cells (GECs). Changes of gelatinases activity have been implicated playing a prominent role in glomerular diseases involving matrix turnover. This study examined whether overlying fibrin influences the expression of gelatinase A and B in cultured human GECs and mechanism underlying the activation. No gelatinase activity was detectable in supernatant of cultured GECs; however, physiological concentration of fibrin (0.5–2.0 mg/ml) induced a dramatic expression of activated MMP-2 and MMP-9 at both mRNA and protein level in a dose and time dependent manner. Increased mRNA level of membrane-type 1 matrix metalloproteinases (MT1-MMPs) was also found. Interestingly, we observed that fibrin also induced the expression of tissue type plasminogen activator (tPA), urokinase type plasminogen activator (uPA) and plasminogen activator inhibitor-1 by casein zymographic and reverse zymographic analysis. Fibrin plate assay revealed the net activity was PA predominant. Serine protease inhibitor aprotinin blocked the conversion of pro-gelatinase A and B to their active forms. The results demonstrate that overlying fibrin increased the secretion of gelatinase A and B from GECs. PA/plasmin proteolytic pathways contributed to the activation of gelatinases.     

Involvement of finger domain and kringle 2 domain of tissue-type plasminogen activator in fibrin binding and stimulation of activity by fibrin.

Human tissue-type plasminogen activator (t-PA) catalyses the conversion of inactive plasminogen into active plasmin, the main fibrinolytic enzyme. This process is confined to the fibrin surface by specific binding of t-PA to fibrin and stimulation of its activity by fibrin. Tissue-type plasminogen activator contains five domains designated finger, growth factor, kringle 1, kringle 2 and protease. The involvement of the domains in fibrin specificity was investigated with a set of variant proteins lacking one or more domains. Variant proteins were produced by expression in Chinese hamster ovary cells of plasmids containing part of the coding sequence for the activator. It was found that kringle 2 domain only is involved in stimulation of activity by fibrin. In the absence of plasminogen and at low concentration of fibrin, binding of t-PA is mainly due to the finger domain, while at high fibrin concentrations also kringle 2 is involved in fibrin binding. In the presence of plasminogen, fibrin binding of the kringle 2 region of t-PA also becomes important at low fibrin concentrations.     

Low molecular weight derivatives of different carrageenan types and their antiviral activity

A number of low molecular weight (LMW) fractions of carrageenans with different structural types were obtained by free radical depolymerization (H₂O₂), mild acid hydrolysis (HCl), and a specific enzyme. Three samples of carrageenans were depolymerized: kappa-carrageenan from Chondrus armatus, kappa-carrageenan from Kappaphycus alvarezii, and kappa/beta-carrageenan from Tichocarpus crinitus with initial molecular weights of 250, 390, and 400 kDa, respectively. The chemical depolymerization by two methods resulted to LMW derivatives of carrageenans with molecular weight from 1.2 to 3.5 kDa. Oligosaccharides of kappa- and kappa/beta-carrageenans with molecular weight of 2.2 and 4.3 kDa, respectively, were obtained after enzymatic depolymerization by recombinant kappa-carrageenase from Pseudoalteromonas carrageenovora. It was shown that the antiviral activity of high molecular weight carrageenans against tobacco mosaic virus was higher than that of their LWM derivatives independently on the depolymerization method. The method of depolymerization had some influence on the antiviral activity of carrageenan. LMW derivatives of kappa- and kappa/beta-carrageenans obtained by mild acid hydrolysis showed higher antiviral activity than the products of free radical depolymerization. The oligosaccharides prepared by enzymatic degradation possessed the lowest activity.     

Species-Specific Functions of Twinfilin in Actin Filament Depolymerization

Twinfilin is a highly conserved member of the actin depolymerization factor homology (ADF-H) protein superfamily, which also includes ADF/Cofilin, Abp1/Drebrin, GMF, and Coactosin. Twinfilin has a unique molecular architecture consisting of two ADF-H domains joined by a linker and followed by a C-terminal tail. Yeast Twinfilin, in conjunction with yeast cyclase-associated protein (Srv2/CAP), increases the rate of depolymerization at both the barbed and pointed ends of actin filaments. However, it has remained unclear whether these activities extend to Twinfilin homologs in other species. To address this, we purified the three mouse Twinfilin isoforms (mTwf1, mTwf2a, mTwf2b) and mouse CAP1, and used total internal reflection fluorescence microscopy assays to study their effects on filament disassembly. Our results show that all three mouse Twinfilin isoforms accelerate barbed end depolymerization similar to yeast Twinfilin, suggesting that this activity is evolutionarily conserved. In striking contrast, mouse Twinfilin isoforms and CAP1 failed to induce rapid pointed end depolymerization. Using chimeras, we show that the yeast-specific pointed end depolymerization activity is specified by the C-terminal ADF-H domain of yeast Twinfilin. In addition, Tropomyosin decoration of filaments failed to impede depolymerization by yeast and mouse Twinfilin and Srv2/CAP, but inhibited Cofilin severing. Together, our results indicate that Twinfilin has conserved functions in regulating barbed end dynamics, although its ability to drive rapid pointed end depolymerization appears to be species-specific. We discuss the implications of this work, including that pointed end depolymerization may be catalyzed by different ADF-H family members in different species.     

Kinetics of actin depolymerization: influence of ions, temperature, age of F-actin, cytochalasin B and phalloidin

Actin, labelled with the fluorescent dye N-(3-pyrenyl)maleimide, was diluted below its critical concentration and depolymerization was followed by measuring the declining fluorescence intensity. The time courses of depolymerization were fitted to a sum of three exponentials. In most cases there was a fast initial phase followed by one or three slower ones. Increasing MgCl2 concentration slowed down depolymerization velocity, as did substitution of Tris-maleate buffer by phosphate buffer. Older F-actin preparations depolymerized more slowly than younger ones. Phalloidin strongly decreased depolymerization velocity even after sonication. In the presence of cytochalasin B depolymerization was more uniformly exponential than in the absence of cytochalasin B; overall depolymerization velocity was decreased by cytochalasin B. The results are discussed on the assumption that depolymerization kinetics reflect the length distribution of actin filaments during depolymerization.     

Anticoagulant low molecular weight heparin does not enhance the activation of plasminogen by tissue plasminogen activator

The activity of tissue plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA) is stimulated by heparin. Heparin binds tightly to t-PA, u-PA, and plasminogen and decreases the usual stimulatory effect of fibrin on t-PA activity. In the present study we have found that low molecular weight heparin (LMW-heparin) preparations obtained by nitrous acid depolymerization or heparinase treatment of standard heparin have different properties with respect to their interaction with the fibrinolytic system. LMW-heparin prepared by either method does not stimulate plasmin formation by t-PA. However, these preparations of heparin still efficiently accelerate the inhibition of thrombin by antithrombin III. Binding data show that LMW-heparin does not bind t-PA and Glu-plasminogen and only binds very weakly to Lys-plasminogen. These results illustrate that it is possible to selectively destroy the fibrinolytic stimulating properties of heparin while leaving the classical anticoagulant characteristics intact.     

Determination of beta-amylase activity by a fluorimetric 2-p-toluidinylnaphthalene-6-sulfonate flow-injection analysis (2, 6-TNS-FIA) method, using amylose and amylopectin as substrates

2-p-Toluidinylnaphthalene-6-sulfonate (2,6-TNS) is a compound which is barely fluorescent in pure water but whose fluorescence can be strongly enhanced if the environment becomes hydrophobic, i.e. by the addition of suitable substrates such as proteins or 1, 4-alpha-D-glucans. The enhancement of fluorescence results from the formation of a 2,6-TNS/substrate complex. For linear and ramified 1, 4-alpha-D-glucans, the fluorescence intensities of the complexes depend linearly on their concentrations but nonlinearly on their average molecular weights (AMW). Thus, the fluorescence detector acts simultaneously as a linear detector concerning the concentration of 1,4-alpha-D-glucan and as a nonlinear mass-selective detector concerning its AMW. These properties have been used for the development of a fluorimetric 2,6-TNS-FIA methodology for the determination of beta-amylase activity, using amylose and amylopectin as substrates. The experimental data points, corresponding to the concentration of "detectable" substrate vs depolymerization time, were fitted using a two-parameter exponential decay curve, and the depolymerization rates at time zero were calculated. The depolymerization rates at time zero vs the corresponding initial substrate concentrations were fitted using the Michaelis-Menten hyperbola and the enzymic constants k(3) and K(m) for amylose (5.93 x 10(-3) g/microKat. min and 1.49 g/L, respectively) and for amylopectin (7.40 x 10(-3) g/microKat+. min and 1.65 g/L, respectively) were determined.     

Characterization of an antibody-urokinase conjugate. A plasminogen activator targeted to fibrin

The plasminogen activator urokinase was linked covalently to a monoclonal antibody specific for the amino terminus of the beta chain of human fibrin by means of the unidirectional cross-linking reagent N-succinimidyl-3-(2-pyridyldithio)propionate. N-Succinimidyl-3-(2-pyridyldithio)propionate allowed the amino groups on urokinase to be coupled to the sulfhydryl groups on iminothiolane (which had been introduced into the antibody before the coupling reaction). The inter-heavy chain sulfhydryl of the Fab' of this antibody was also linked to N-succinimidyl-3-(2-pyridyldithio)propionate-substituted urokinase. The antibody- or Fab'-urokinase complexes were purified by two affinity chromatography steps. In the first, benzamidine was used as ligand for urokinase, in the second, a heptapeptide consisting of the 7 amino-terminal residues of the beta chain of fibrin (beta peptide) was used as ligand for the antibody. The activity of the purified conjugates was compared with that of urokinase alone in an assay measuring lysis of 125I-fibrin monomer covalently linked to Sepharose CL-4B. For any concentration of either urokinase alone or urokinase-antifibrin antibody conjugate, an equivalent amount of lysis (release of labeled peptide from fibrin monomer-Sepharose) was obtained with 1/250 the concentration (with respect to urokinase content) of antifibrin antibody-urokinase conjugate. The antifibrin Fab'-urokinase conjugate exhibited a similar enhancement of activity in comparison with urokinase. Enhanced fibrinolysis was fully inhibited by beta peptide. These results suggest that antibody targeting enhances the concentration of urokinase in the vicinity of immobilized fibrin monomer, thereby also increasing the local conversion of plasminogen to plasmin, which in turn degrades its substrate, fibrin. Univalent antigen-antibody binding is sufficient for optimal efficiency.     

Enhancement of plasminogen activator activity in cultured endothelial cells by granulocyte colony-stimulating factor

A hitherto unknown function of granulocyte colony-stimulating factor (G-CSF) was found using cultured endothelial cells. G-CSF stimulated activity of plasminogen activator (PA) in both extracellular and intracellular milieus of endothelial cells obtained from bovine carotid and aortic artery. This effect was dependent on the concentration of G-CSF added to the culture medium and on the treatment time. The extracellular activity was enhanced approximately 5-fold at a concentration of 5,000 colony-forming unit (CFU)/ml (2.6 nM) and in about a 15-hr treatment period. Analyses by fibrin and reverse fibrin autography revealed that activity of PA was much more increased than that of PA inhibitor in endothelial cells treated with G-CSF.