Activation of Na/H exchange by thrombin in peritoneal mast cells]

Using the fluorescent probe, BCECF, the changes in intracellular pH (pHi) in rat peritoneal mast cells were studied. alpha-Thrombin (0.1 nM) induced biphasic changes in pHi which consisted in a temporary decrease in pH with its subsequent steady increase due to the Na/H exchange activation which was inhibited by EIPA and controlled by extracellular Na+. The biphasic changes in pHi induced by DIP-alpha-thrombin (0.1 pM-1 nM), a catalytically inactive form with an intact recognition site, were similar to those of alpha-thrombin, whereas beta/gamma-thrombin (10-1000 pM), a catalytically active form characterized by structural disturbances in the recognition site, was able to induce only the initial phase of acidification. The thrombin recognition site modulators, alpha 1-thymosin and heparin, blocked the ability of the enzyme to induce the alkalinization of pHi. Nigericin stimulated the Na/H-exchange in mast cells. The rate of the Na/H-exchange activation determined with nigericin, decreased with an increase in the alpha-thrombin dose from 0.1 pM up to 10 nM. Activation of protein kinase C (PKC) in mast cells by PMA used at 1 nM and 10 nM led to the alkalinization of the cytoplasm as a result of the Na/H-exchange activation blocked by EIPA. The PKC inhibitor, H-7, suppressed the pHi increase induced by both PMA and alpha-thrombin. The alpha-thrombin-induced acidification of the cytoplasm was completely blocked by SITS in Ca(2+)-free media, whereas in media with Ca2+ SITS inhibited the pHi decline. Acidification of the cytoplasm by thrombin seems to be due to both Ca2+ influx and activation of Cl- fluxes. It is concluded that the observed activation of the Na/H-exchange by thrombin is induced by a cascade of intracellular reactions involving PKC.     

Alpha-thrombin stimulation of heparin secretion by the peritoneal mast cells in rats

The interaction of alpha-thrombin with connective tissue-type mast cells (CTMC) purified by Ficoll density gradient centrifugation has been examined. It was demonstrated that exposure of CTMC to polymixin (widely used histamine liberator) (3 mg/ml) induced the release of heparin and histamine. Exposure of CTMC to 10(-11) M alpha-thrombin resulted in increase of heparin secretion by 75.5% in relation to basal level. CTMC which were stimulated by very low concentrations of alpha-thrombin (10(-11)-10(-8) M) can release high level of heparin, but not histamine. We have a suggestion that the thrombin specificity is connected with the additional recognition binding site for high molecular substrates (HMS) distinct from the active centre. Unlike alpha-thrombin which has both the active centre and the recognition site for HMS, beta/gamma-thrombin with catalytic activity but with disrupted recognition site induced the heparin release from mast cells only at higher concentrations than alpha-thrombin. It was revealed that DIP-alpha-thrombin without proteolytic activity was unable to activate mast cells in contrast to alpha-thrombin. We consider that alpha-thrombin induced release of heparin by CTMC account for proteolytic and hormone-like activity enzyme by means of both the active centre and the additional recognition site for HMS.     

Binding of FITC-labelled alpha-thrombin by peritoneal mast cells]

The interaction of bovine alpha-thrombin with peritoneal mast cells was studied using FITC-labeled enzyme. Thrombin was modified with FITC in the presence of heparin and was separated from heparin and free FITC by gel-filtration at HPLC yielding FITC-labeled alpha-thrombin with intact additional recognition binding site for high molecular substrates and cell receptors. Equilibrium studies have shown that the binding of thrombin to peritoneal mast cells is active independent, rapid, specific, saturable and reversible. Equilibrium between bound and free thrombin is attained within I min and Scatchard analysis indicates a population of approximately 54 x 10(3) sites/cell with a dissociation constant of 1.3 x 10(-9) M. FITC-labeled alpha-thrombin binds to peritoneal mast cells in a temperature-dependent manner with optimum at 37 degrees C. These results indicate that FITC-labeled alpha-thrombin binds to peritoneal mast cells with high affinity.     

Thymosin beta 4 induced phenotypic changes in Molt-4 leukemic cell line

Thymosin beta 4 was tested for its ability to induce phenotypic changes in the human T-cell line Molt-4. Cells were cultured with nanogram concentrations of thymosin beta 4 for up to 16 days and were analyzed with a panel of monoclonal antibodies, sheep erythrocyte rosetting, peanut agglutinin binding (PNA) and an antibody to the enzyme, terminal deoxynucleotidyl transferase (TdT). Thymosin beta 4 induced Molt-4 cells to reduce the expression of a T-cell lineage specific antigen, with preferential expression on T blast-cells, detected by WT 1 monoclonal antibody. Thymosin beta 4 also induced an increase in sheep erythrocyte rosettes and PNA binding as well as an increased expression of OKT 11 A and OKT 8 in Molt-4 cells. TdT was found to be unchanged, however. Analysis of thymosin beta 4-treated cells with other monoclonal antibodies (OKT 3, OKT 6, OKT 9) showed no change when compared to controls. These results showed that thymosin beta 4 is capable of inducing phenotypic changes in Molt-4 cells. Such changes may represent a differentiation process of these cells through the early stages of the maturation process of thymus-dependent lymphocytes, albeit not to the stage of mature T cells.     

Solid phase synthesis of thymosin beta 9

Thymosin beta 9, a 41 residue thymic polypeptide, has been synthesized by a solid phase method. A modification of the low HF method was used to deprotect and cleave the peptide from the resin. Thymosin beta 9 was then obtained in analytically pure form by a one-step purification procedure in 32% yield. The activity of thymosin beta 9 in the terminal deoxynucleotidyl transferase assay was greater than calf thymus fraction 5, but comparable to thymosin beta 4. In contrast to thymosin alpha 1, neither beta 4 nor beta 9 was active in the rosette inhibition assay.     

The chemistry and biology of thymosin. I. Isolation, characterization, and biological activities of thymosin alpha1 and polypeptide beta1 from calf thymus.

A partially purified extract from thymus tissue termed thymosin Fraction 5 has been shown to reconstitute immunological deficiencies resulting from the lack of thymic function in several animal models, as well as humans with primary and secondary immunodeficiency diseases. Thymosin Fraction 5 consists of a family of polypeptides with molecular weights ranging from 1,000 to 15,000. Several of these polypeptides contribute individually to the biological activity of the parent compound. Two polypeptide components of thymosin Fraction 5, termed thymosin alpha1 and polypeptide beta1, have been characterized chemically and biologically. Thymosin alpha1 is a highly acidic molecule composed of 28 amino acid residues. This polypeptide has potent biological activity and has been found to be 10 to 1,000 times as active as thymosin Fraction 5 in one in vivo and several in vitro bioassay systems designed to measure differentiation and function of thymus-dependent lymphocytes (T cells). Polypeptide beta1, in contrast, is inactive in our bioassay systems, suggesting that it is not involved in thymic hormone action. Sequence analysis and homology studies have indicated that polypeptide beta1, although present in Fraction 5, does not contribute to the biological activity of thymosin Fraction 5.     

Receptors for thymosin alpha 1 on mouse thymocytes

Thymosin alpha 1 is able to act in vitro to stimulate T-cell precursors and to induce surface markers. The initial mechanism by which alpha 1 activates T cells could be the binding of alpha 1 to cell membranes. Using a specific anti-alpha 1 antibody and an indirect immunofluorescence procedure it was found that thymosin alpha 1 binds to the surface of a large portion of murine lymphocytes. Furthermore, thymocytes have been fractionated into immature and mature subpopulations by using the peanut agglutinin (PNA) technique. It was found that PNA+, immature cells showed specific receptors for alpha 1 on the cell membrane.     

Effects of thymosin alpha l on some time-dependent functions in thymectomized mice

The effects of a synthetic thymosin alpha1 on the azathioprine-sensitivity of spleen cells and on the thymic-like activity of serum from adult thymectomized mice, were observed. Thymosin alpha1 is able to restore the lowered levels of these T-dependent functions after in vivo administration.     

Appearance of thymosin alpha 1 in supernatants of monocytes incubated with prothymosin alpha.

Prothymosin alpha, a polypeptide of 109 to 111 amino acid residues, contains the entire thymosin alpha 1 sequence (residues 1-28) at its amino terminal. Human peripheral blood monocytes incubated with prothymosin alpha release thymosin alpha 1 in the culture supernatants. In addition total RNA is found to increase. The production of thymosin alpha 1 involves de novo protein synthesis as shown by the kinetics of this release and its inhibition by actinomycin D and cycloheximide. Thymosin alpha 1 release, possibly in association with HLA-DR, stimulates the proliferation of the T cell population.     

Protective activity of thymosin against opportunistic infections in animal models

Animal models for opportunistic infections were developed by using mice immunosuppressed by 5-FU. These mice were susceptible to various microorganisms, while normal mice had greater tolerance to such microbial infections. In these models, thymosin alpha 1 was found to protect mice against lethal infections with Candida albicans, Listeria monocytogenes, Pseudomonas aeruginosa, and Serratia marcescens when it was administered during 5-FU treatment prior to the infections. Thymosin alpha 1 was effective in some infections at 0.4-400 micrograms/kg/day IP, about 1/100 of the dose required for thymosin fraction 5. Activity was also demonstrated against L-monocytogenes and Ps. aeruginosa by counting the viable bacteria in the liver after infection. The protective activity against Candida, elimination of which macrophages were essential, was abrogated by anti-thymocyte serum and/or carrageenan, indicating that thymosin alpha 1 serves to maintain the functions of macrophages by reducing the damage to T cells by 5-FU. On the other hand, the activity against Pseudomonas infection was not affected by anti-thymocyte serum or carrageenan. It is probable that thymosin alpha 1 also exerts its effect on neutrophils without participation of T cells and macrophages.     

Conformational lability of vitronectin: induction of an antigenic change by alpha-thrombin-serpin complexes and by proteolytically modified thrombin

We previously showed that the alpha-thrombin-antithrombin III complex causes antigenic change in vitronectin as monitored by the monoclonal anti-vitronectin antibody 8E6 (Tomasini & Mosher, 1988). We have extended these studies to other protease-serpin complexes and to gamma-thrombin, a proteolytic derivative of alpha-thrombin. In the presence of heparin, recognition of vitronectin by 8E6 was increased 64- or 52-fold by interaction with the complex of alpha-thrombin and heparin cofactor II or the Pittsburgh mutant (Met358----Arg) of alpha 1-protease inhibitor, respectively. This was comparable to the value obtained with the alpha-thrombin-antithrombin III complex. Factor Xa-serpin complexes were approximately 4-fold less effective than the corresponding thrombin complexes. alpha-Thrombin-serpin complexes but not Xa-serpin complexes formed disulfide-bonded complexes with vitronectin. Antigenic changes and disulfide-bonded complexes were not detected when trypsin- or chymotrypsin-serpin complexes were incubated with vitronectin. gamma-Thrombin caused 7- and 34-fold increases in recognition of vitronectin by MaVN 8E6 in the absence and presence of heparin, respectively. In contrast, alpha-thrombin by itself had no effect. The antigenic change induced by gamma-thrombin was maximal when gamma-thrombin and vitronectin were equimolar, was not dependent on cleavage of vitronectin, and was abolished by inhibition of gamma-thrombin with Phe-Pro-Arg-chloromethyl ketone but not with diisopropyl fluorophosphate. These data indicate that alpha-thrombin is the component in alpha-thrombin-serpin complexes that induces the antigenic change in vitronectin, probably via a region that is preferentially exposed in gamma-thrombin.     

人胸腺素α1在大肠杆菌中的融合表达

利用基因工程表达的方法,在大肠杆菌中通过与GST蛋白融合的方式高效表达了胸腺素α1前体基因,随后经亲和层析和SP强阳离子树脂纯化相结合的方式,得到了胸腺素前体肽段31肽和N端未经乙酰化修饰的28肽。融合蛋白表达量达到菌体总蛋白的35%～40%,样品肽的产量也达到了约200mg/L（肽/发酵液）的产量。经质谱测定,分子量分别为3366和3066。BalB/C小鼠脾脏淋巴细胞体外测活表明,所构建的GSTTα1融合蛋白和纯化后的产物对于淋巴细胞具有比较明显的增殖作用,其中N端未经乙酰化的28肽产物与31肽产物活性相近,均对淋巴细胞具有明显的刺激增殖作用。     

Thymosin alpha 1-like peptides: localization and biochemical characterization in the rat brain and pituitary gland

Using a radioimmunoassay for thymosin alpha 1, endogenous thymosin-like peptides were characterized in the rat brain and pituitary gland. Thymosin alpha 1-like peptides were present in high concentrations in hypothalamus and pituitary extracts. These peptides were characterized using gel filtration techniques and the main peak of immunoreactive thymosin had a molecular weight similar to that of thymosin alpha 1 (3108 daltons). Using HPLC techniques, one main peak of immunoreactivity was present in brain extracts, whereas two peaks were present in pituitary extracts, one of which coeluted with thymosin alpha 1. The discrete regional distribution of thymosin alpha 1-like peptides was investigated and the highest densities of immunoreactive thymosin were present in the median eminence and arcuate nucleus of the hypothalamus, as well as the neurointermediate lobe of the pituitary. Due to the anatomical proximity of immunoreactive thymosin to loci containing known releasing factors and hormones, thymosin alpha 1-like peptides may function as neuroendocrine regulatory agents.     

Role of exosites 1 and 2 in thrombin reaction with plasminogen activator inhibitor-1 in the absence and presence of cofactors.

The cofactors heparin, vitronectin (VN), and thrombomodulin (TM) modulate the reactivity of alpha-thrombin with plasminogen activator inhibitor (PAI-1). While heparin and VN accelerate the reaction by approximately 2 orders of magnitude, TM protects alpha-thrombin from rapid inactivation by PAI-1 in the presence of VN. To understand how these cofactors function, we studied the kinetics of PAI-1 inactivation of alpha-thrombin, the exosite 1 variant gamma-thrombin, the exosite 2 mutant R93,97,101A thrombin, and recombinant meizothrombin in both the absence and presence of these cofactors. Heparin and VN accelerated the second-order association rate constant [k(2) = (7.9 +/- 0.5) x 10(2) M(-)(1) s(-)(1)] of alpha-thrombin with PAI-1 approximately 200- and approximately 240-fold, respectively. The k(2) value for gamma-thrombin [(7.9 +/- 0.7) x 10(1) M(-)(1) s(-)(1)] was impaired 10-fold, but was enhanced by heparin and VN approximately 280- and approximately 75-fold, respectively. Similar to inactivation of gamma-thrombin, PAI-1 inactivation of alpha-thrombin in complex with the epidermal growth factor-like domains 4-6 of TM (TM4-6) was impaired approximately 10-fold. The exosite 2 mutant R93,97,101A thrombin, which was previously shown not to bind heparin, and meizothrombin, in which exosite 2 is masked, reacted with PAI-1 at similar rates in both the absence and presence of heparin [k(2) = (1.3-1.5) x 10(3) M(-)(1) s(-)(1) for R93,97,101A thrombin and k(2) = (3.6-5.1) x 10(2) M(-)(1) s(-)(1) for meizothrombin]. Unlike heparin, however, VN enhanced the k(2) of R93,97,101A thrombin and meizothrombin inactivation approximately 80- and approximately 30-fold, respectively. Continuous kinetic analysis as well as competition kinetic studies in the presence of S195A thrombin suggested that the accelerating effect of VN or heparin occurs primarily by lowering the dissociation constant (K(d)) for formation of a noncovalent, Michaelis-type complex. Analysis of these results suggest that (1) heparin binds to exosite 2 of alpha-thrombin to accelerate the reaction by a template mechanism, (2) VN accelerates PAI-1 inactivation of alpha-thrombin by lowering the K(d) for initial complex formation by an unknown mechanism that does not require binding to either exosite 1 or exosite 2 of alpha-thrombin, (3) alpha-thrombin may have a binding site for PAI-1 within or near exosite 1, and (4) TM occupancy of exosite 1 partially accounts for the protection of thrombin from rapid inactivation by PAI-1 in the presence of vitronectin.     

The COOH-terminal domain of hirudin. An exosite-directed competitive inhibitor of the action of alpha-thrombin on fibrinogen

Hirudin, a potent 65-residue polypeptide inhibitor of alpha-thrombin found in the saliva of the leech Hirudo medicinalis, and fragments thereof are potentially useful as antithrombotic agents. Hirugen, the synthetic N-acetylated COOH-terminal dodecapeptide (Ac-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr(SO3)-Leu) of hirudin was shown in the present study to behave as a pure competitive inhibitor (Ki = 0.54 microM) of human alpha-thrombin-catalyzed release of fibrinopeptide A from human fibrinogen. In contrast to this inhibitory activity, hirugen slightly enhanced (increased kcat/Km 1.6-fold) alpha-thrombin-catalyzed hydrolysis of the fluorogenic tripeptide substrate N-p-Tosyl-Gly-Pro-Arg-7-amino-4-methylcoumarin. These observations indicate that hirugen binds to alpha-thrombin at an exosite distinct from the active site, and that interaction with this exosite is a major determinant of the competence of alpha-thrombin to bind fibrinogen. Consistent with this view, hirugen blocked binding of fibrin II to alpha-thrombin. Studies of the effect of hirugen on the rate of inactivation of alpha-thrombin by antithrombin III (AT), the major plasma inhibitor of alpha-thrombin, indicated that binding of hirugen to alpha-thrombin results in less than a 2.5-fold decrease in the rate of inactivation of alpha-thrombin by AT, both in the absence and presence of heparin. This behavior is distinct from that of active site-directed competitive inhibitors of alpha-thrombin which bind to alpha-thrombin and block both conversion of fibrinogen to fibrin and inactivation of alpha-thrombin by AT. Hirugen, an exosite-directed competitive inhibitor, blocks the interaction of alpha-thrombin with fibrinogen while leaving alpha-thrombin competent to react with AT. Thus, unlike active site-directed competitive inhibitors, hirugen should act in concert with AT and heparin to reduce the amount of fibrinogen that is processed during the lifetime of alpha-thrombin in plasma.     

Developmental changes of serum thymosin alpha 1 and beta 4 in male and male castrated pigs: modulation by testosterone and human chorionic gonadotropin.

Thymic secretory peptides thymosin beta 4 and alpha 1 have possible endocrine roles in both immune and reproductive systems; thus, they should respond to endocrine feedback control mechanisms consistent with gonadal function. In an initial experiment, male pigs (boars; n = 90; 10/time) were bled at 1, 3, 6, 12, 18, 24, 30, 36, and 96 wk of age before and 24 h after hCG stimulation. Thymosin beta 4 concentrations were significantly depressed 24 h after hCG challenge. Testosterone concentrations increased with age up to 36 wk and were further increased with hCG stimulation (p less than 0.01). In a subsequent experiment, boars (n = 12) and barrows (males castrated shortly after birth; n = 12) were blood-sampled, administered hCG, and sampled again 24 h later at 1, 3, 6, 12, 18, and 24 wk of age. Barrows (n = 12) were administered testosterone with the same protocol. Testosterone concentrations increased in boars with maturity and were further increased from the hCG stimulation (p less than 0.01). Thymosin beta 4 concentrations decreased with age in boars and barrows (p less than 0.01), and hCG challenge depressed thymosin alpha 1 and beta 4 concentrations in boars and thymosin beta 4 in barrows (p less than 0.01). Testosterone treatment of barrows also depressed thymosin beta 4 and alpha 1 in barrows (p less than 0.01). The depression of thymosins by hCG treatment points to a role for gonadotropins in altering circulating thymosin concentrations independent of, but in conjunction with, the effect of gonadal steroids.     

Thrombin's enzymatic activity increases permeability of endothelial cell monolayers

Human alpha-thrombin increases the permeability of bovine pulmonary artery endothelial cell (CCL-209) monolayers. To determine if this increase is via an enzymatic or receptor-mediated mechanism, enzymatically active forms of alpha-thrombin and enzymatically inactive forms with cell binding activity were incubated with the monolayers. Enzymatic forms included alpha-thrombin and two digestion products, zeta-thrombin (chymotryptic product with 89% clotting activity) and gamma-thrombin (tryptic product). Enzymatically inactive forms included D-Phe-Pro-Arg-chloromethylketone-(PPACK) alpha-thrombin and diisopropylphosphorofluoridate-(DIP) alpha-thrombin. Cell binding activity of alpha- and PPACK-alpha-thrombin was demonstrated to be similar to each other and comparable to that cited in the literature for DIP-alpha-thrombin. gamma-Thrombin, on the other hand, did not compete for binding of 125I-labeled alpha-thrombin. All enzymatic forms of alpha-thrombin increased endothelial permeability as assessed by the clearance of 125I-albumin across the monolayers. Coincubation of PPACK, an enzymatic site inhibitor, with alpha- or gamma-thrombin prevented the increase in permeability, further indicating that alpha-thrombin increased permeability by its enzymatic activity. Both enzymatically inactive forms of alpha-thrombin with high-affinity binding activity had no effect on permeability. To further examine whether cell binding activity of alpha-thrombin contributed to the increased permeability, a sulfated COOH-terminal fragment of hirudin (hirugen) that binds to the anion-binding site of alpha-thrombin but, unlike hirudin, does not interact with the catalytic site was coincubated with alpha-thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of enzymatic activity of alpha-thrombin by low molecular weight synthetic inhibitor]

The effect of the organophosphoric inhibitor, SA-152, on the fibrinogen-coagulating and TAME-esterase activity of bovine alpha-thrombin was studied. The irreversible inhibition constants (k11 = 1.1 x 10(4) M-1.min-1,Ki = 0.7 x 10(-4) M, k2 = 0.8 min-1 towards the coagulating activity and kII = 0.7 x 10(4) M-1.min-1, Ki = 0.3 x 10(-4) M, k2 = 0.2 min-1 towards the esterase activity) were determined. The SA-152 inactivated alpha-thrombin was dialyzed and incubated with 0.5 M and 2.5 M NaCl and 10 mM TAME. There was no reconstitution of activity of the SA-152 modified alpha-thrombin after dialysis and treatment with high concentrations of NaCl and TAME. Heparin interactions with the anion-binding site of the high molecular weight recognition center in the alpha-thrombin molecule did not significantly influence the values of the kinetic constants for the enzyme inhibition by SA-152. This finding is consistent with the hypothesis on the irreversible binding of SA-152 in the active center of the enzyme.     

Actin-latrunculin A structure and function. Differential modulation of actin-binding protein function by latrunculin A

Latrunculin A is used extensively as an agent to sequester monomeric actin in living cells. We hypothesize that additional activities of latrunculin A may be important for its biological activity. Our data are consistent with the formation of a 1:1 stoichiometric complex with an equilibrium dissociation constant of 0.2 to 0.4 micrometer and provide no evidence that the actin-latrunculin A complex participates in the elongation of actin filaments. Profilin and latrunculin A bind independently to actin, whereas binding of thymosin beta(4) to actin is inhibited by latrunculin A. Potential implications of this differential effect on actin-binding proteins are discussed. From a structural perspective, if latrunculin A binds to actin at a site that sterically influences binding by thymosin beta(4), then the observation that latrunculin A inhibits nucleotide exchange on actin implies an allosteric effect on the nucleotide binding cleft. Alternatively, if, as previously postulated, latrunculin A binds in the nucleotide cleft of actin, then its ability to inhibit binding by thymosin beta(4) is a surprising result that suggests that significant allosteric changes affect the thymosin beta(4) binding site. We show that latrunculin A and actin form a crystalline structure with orthorhombic space group P2(1)2(1)2(1) and diffraction to 3.10 A. A high resolution structure with optimized crystallization conditions should provide insight regarding these remarkable allosteric properties.