Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

---

Query: EC:3.4.21.5 (thrombin)
33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

This report describes a French Canadian family whose members exhibit a high incidence of allo- and autoantibodies to antigens present on both platelets and endothelial cells. This is correlated with various HLA specificities known to be associated with autoimmunity, such as A1, B8, DR3, and, in some cases, with clinical disorders, including nephritis, hypertension, and thrombocytopenia. Immunoblot analysis using platelet and endothelial cell lysates showed serum antibodies to a 75 kDa endothelial cell surface polypeptide and to polypeptides with apparent mass of 115 kDa and 26 kDa found on both platelets and endothelial cells. This 115 kDa internal platelet protein was also found in a variety of other cell types, such as mononuclear cells, and increased following cell activation. Monoclonal antibody immunobilization assays were used to characterize the 26 kDa polypeptide; in three of the four patients tested, an antibody to leukocyte differentiation antigen CD9 was identified. The asymptomatic child of the propositus also exhibited an autoantibody against an 80 kDa platelet protein which was sensitive to thrombin digestion, suggesting that this polypeptide may be platelet glycoprotein V. In addition, P1A1 alloantibody was identified in one sister who had given birth to a severely thrombocytopenic boy and who herself had a severe vascular rejection to a cadaver kidney 2 years prior to this study. The propositus also developed hypertensive renal disease following a pregnancy and became dialysis dependent. Thus, members of this family have developed a variety of antibodies, particularly to platelet and endothelial cell antigens. Some subjects have remained asymptomatic in spite of having autoantibodies. However, others have been seriously ill, and their immune response to these antigens is believed to have played a role in the pathogenesis of their neonatal alloimmune thrombocytopenic purpura, hypertensive renal disease, renal graft rejection, and thrombocytopenia.
...
PMID:Characterization of novel platelet and endothelial cell target antigens in a family with genetic susceptibility to autoimmunity. 174 38

Human zeta-thrombin, a catalytically competent serine proteinase, arises from a single chymotryptic cleavage at Trp-148 in alpha-thrombin to generate two nonconvalently associated polypeptide segments designated zeta 1-thrombin (the 36-residue A-chain disulfide linked to B-chain residues B1-148) and zeta 2-thrombin (B149-259). We report here the expression of recombinant zeta 2-thrombin in Escherichia coli and the reconstitution of catalytically competent zeta-thrombin by combination of zeta 1-thrombin with recombinant zeta 2-thrombin. A DNA fragment encoding zeta 2-thrombin was cloned into a pATH2 expression vector as a trpE-zeta 2 fusion gene, in which a factor Xa cleavage site was inserted between the trpE and the zeta 2-thrombin gene. High-level expression of this fusion protein was achieved under the control of the E. coli trp promoter. The expressed zeta 2-thrombin was liberated from the fusion protein by factor Xa cleavage, reduced with DTT, and purified to homogeneity by reverse-phase HPLC. Oxidation of the reduced zeta 2-thrombin in the presence of 80 microM CuSO4 and 6 M urea at pH 8.15 yielded material that was indistinguishable on HPLC from zeta 2-thrombin isolated by resolution of human zeta-thrombin. Catalytically active zeta-thrombin was generated by combination of recombinant zeta 2-thrombin with zeta 1-thrombin that was isolated by resolution of human zeta-thrombin. Recombinant zeta-thrombin displayed catalytic activities, toward a small chromogenic substrate and fibrinogen, that were similar to those of alpha-thrombin prepared from human blood plasma and zeta-thrombin obtained by treatment of alpha-thrombin with chymotrypsin.(ABSTRACT TRUNCATED AT 250 WORDS)
...
PMID:Reconstitution of catalytically competent human zeta-thrombin by combination of zeta-thrombin residues A1-36 and B1-148 and an Escherichia coli expressed polypeptide corresponding to zeta-thrombin residues B149-259. 175 88

The growth of capillary endothelial cells (BCE) is an important regulatory step in the formation of capillary blood vessels. In vivo, the proliferation of these cells is stringently controlled. In vitro they can be stimulated by polypeptide growth factors, such as acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF). Since bFGF is synthesized and stored by vascular endothelial cells, this mitogen may play an important role in an autocrine growth regulation during angiogenesis. Here, evidence is presented for induction of the mRNA of bFGF by bFGF itself. A similar increase of bFGF mRNA was observed in response to thrombin and after treatment with phorbol ester. These results suggest that an autocrine loop may exist that may serve to modulate the mitogenic response in BCE under various physiological conditions, (e.g., wound healing and new capillary formation).
...
PMID:Transcriptional regulation of basic fibroblast growth factor gene expression in capillary endothelial cells. 175 80

Recombinant DNA technologies now allow the preparation of virtually any polypeptide sequence. Very efficient expression systems for prokaryotic and eukaryotic cells have been developed which may yield large quantities of the desired protein. Bacterial systems are still the most widely used while alternative organisms are often considered when post-translational modifications could influence the biological behaviour of the product. For hirudin or its analogues, two important molecular characteristics should be taken into account. First, it is necessary that no extra amino acid residue, such as the initial methionine, is present on the NH2 end of the recombinant polypeptide. It is known that a free N-terminal sequence is crucial for the thrombin inhibitory activity. Second, a sulphate group on tyrosine at position 63 is found in natural hirudin extracted from leeches. Such post-translational modification has never been observed for all the recombinant hirudin preparations reported to date even though the importance of the sulphate group on the in vitro and in vivo activity of hirudin has not yet been clarified. Finally, the recombinant DNA methodology of choice for the commercial development of hirudin must also take into consideration yield and cost factors which ultimately will affect the widespread use of this product particularly if it has to compete with heparin. We will review our work on the preparation of recombinant hirudin describing bacterial and insect cell expression systems and addressing some of the questions mentioned above.
...
PMID:Production of the HV1 variant of hirudin by recombinant DNA methodology. 177 78

Human parathyroid hormone, hPTH, an 84 amino acid polypeptide, was produced intracellularly in Escherichia coli as a fusion protein, linked to the C-terminus of a 15 kD IgG-binding protein. Approximately 100 mg fusion protein was obtained per liter fermentation medium. To test the efficiency of two alternative enzymatic cleavage methods, two fusion proteins differing only in the linker region were constructed. Cleavage of a Phe-Phe-Pro-Arg linker was obtained with bovine thrombin and cleavage of a Phe-Ala-His-Tyr linker with recombinant H64A subtilisin. Both enzymes yielded the correct N-terminus and cleaved their respective linkers quantitatively, although additional internal cleavage sites in hPTH were detected and characterized. The linker cleavage conditions were optimized and hPTH was purified to homogeneity. Thrombin cleavage resulted in a final yield of 5 mg hPTH/L, while H64A subtilisin cleavage was more specific and gave 8 mg/L. The purified recombinant product was identical to native hPTH and exhibited full biological activity in an adenylate cyclase assay.
...
PMID:Thrombin and H64A subtilisin cleavage of fusion proteins for preparation of human recombinant parathyroid hormone. 179 10

The interaction of fibrinogen with its receptors on platelet surfaces leads to platelet aggregation. A snake-venom peptide, trigramin, has previously been demonstrated to inhibit platelet aggregation by acting as a fibrinogen-receptor antagonist. By means of gel filtration, ionic-exchange chromatography and reverse-phase h.p.l.c., a potent platelet-aggregation inhibitor, triflavin, has now been purified from the venom of Trimeresurus flavoviridis. The purified triflavin is a single-chain polypeptide, consisting of about 71 amino acid residues with a molecular mass of 7600 Da, and its N-terminal sequence is Gly-Glu-Glu-Cys-Asp. Triflavin dose-dependently inhibited human platelet aggregation stimulated by ADP, adrenaline, collagen, thrombin or prostaglandin endoperoxide analogue U46619 in preparations of platelet-rich plasma, platelet suspension and whole blood. Its IC50 ranged from 38 to 84 nM, depending on the aggregation inducer used and the platelet preparation. However, triflavin apparently did not affect the platelet shape change and ATP-release reactions caused by these agonists. Triflavin inhibited fibrinogen-induced aggregation of human elastase-treated platelets in a dose-dependent manner, indicating that it directly interferes with the binding of fibrinogen to its receptors on platelet membranes exposed by elastase treatment. Additionally, triflavin dose-dependently blocked 125I-labelled fibrinogen binding to ADP-activated platelets. In conclusion, triflavin inhibits platelet aggregation through the blockade of fibrinogen binding to fibrinogen receptors on platelet membranes.
...
PMID:A potent antiplatelet peptide, triflavin, from Trimeresurus flavoviridis snake venom. 185 63

Triflavin, an antiplatelet peptide containing Arg-Gly-Asp, purified from Trimeresurus flavoviridis venom, inhibits aggregation of human platelets stimulated by a variety of agonists. It blocks aggregation through interference with fibrinogen binding to its specific receptor on the platelet surface membrane in a competitive manner, but it has no apparent effect on intracellular events, such as thromboxane B2 formation, phosphoinositides breakdown and intracellular Ca2+ mobilization of thrombin-activated platelets. In this study, we determined the complete sequence of triflavin, which is composed of a single polypeptide chain of 70 amino acids. Its sequence is rich in cysteine and contains Arg-Gly-Asp at residues 49-51 in the carboxy-terminal domain. Triflavin shows about 68% identity of amino acid sequence with trigramin, which is a specific antagonist of the fibrinogen receptor associated with glycoprotein IIb/IIIa complex. [125I]Triflavin binds to unstimulated and ADP-stimulated platelets in a saturable manner and its Kd values are estimated to be 76 and 74 nM, respectively; the corresponding numbers of binding sites are 31,029 and 34,863 per platelet, respectively. [125I]Triflavin binding is blocked by Gly-Arg-Gly-Asp-Ser in a competitive manner. EDTA, the Arg-Gly-Asp-containing peptides (including naturally occurring polypeptides, trigramin and rhodostomin), and monoclonal antibody, 7E3, raised against GP IIb/IIIa complex, inhibit [125I]triflavin binding to unstimulated and ADP-stimulated human platelets. In conclusion, triflavin specifically binds to fibrinogen receptor associated with GP IIb/IIIa complex and its binding site is located at or near GP IIb/IIIa complex, overlapping with those of 7E3 and another Arg-Gly-Asp-containing polypeptide, rhodostomin.(ABSTRACT TRUNCATED AT 250 WORDS)
...
PMID:Triflavin, an antiplatelet Arg-Gly-Asp-containing peptide, is a specific antagonist of platelet membrane glycoprotein IIb-IIIa complex. 186 44

A heterozygous hereditary fibrinogen variant, fibrinogen Bern III, has been characterized. The proposita and her daughter showed prolonged thrombin time and reptilase time, as well as a markedly reduced fibrinogen concentration as determined by functional clotting assay. Fibrinogen was purified from the proposita's plasma and subjected to biochemical characterization. The delayed fibrin formation was shown to result from impaired release of fibrinopeptide A. Thrombin was found to cleave an extended fibrinopeptide A (A alpha 1-19) from the reduced polypeptide chains of the abnormal fibrinogen. Amino acid analysis of this fragment indicated that the arginine residue, located at the physiological thrombin cleavage site, was replaced by cysteine. The functional defect of the fibrinogen variant Bern III is due to the amino acid substitution A alpha 16 Arg----Cys.
...
PMID:[Fibrinogen Bern III: a further case of hereditary fibrinogen variants with substitution A alpha 16 Arg----Cys]. 189 1

Factor VIII delta II is a genetically engineered deletion variant of factor VIII expressed by recombinant Chinese hamster ovary cells, in which a major portion of the central (B) domain and a part of the light chain (Pro771-Asp1666) are missing. After immunoaffinity purification, the kinetics of thrombin cleavage of the novel molecule was analysed by SDS/PAGE, Western blotting and N-terminal amino acid sequencing. Thrombin first cleaves factor VIII delta II at Arg740-Ser741 to generate the 90-kDa heavy chain and an 80-kDa fusion polypeptide consisting of the remaining portion of the B domain and the 73-kDa light chain. The 90-kDa fragment is further cleaved, giving rise to 50-kDa and 40-kDa fragments while the 80-kDa fragment generates a 71/73-kDa doublet. The 71/73-kDa doublet, 50-kDa and 40-kDa fragments were further analysed by N-terminal amino acid sequencing and found to correspond to the predicted amino acid sequences. Our study shows that, in spite of the 900 amino acid deletion present in factor VIII delta II, the essential structural elements required for thrombin activation are conserved.
...
PMID:Thrombin cleavage analysis of a novel antihaemophilic factor variant, factor VIII delta II. 190 Feb 36

A recombinant Factor VIII (Factor VIII-delta II) consists of a unique polypeptide chain of 165 kDa deleted from the major part of the B-domain and from the cleavage site at Arg-1648-Glu-1649 found in plasma-derived Factor VIII. It was expressed in mammalian cells in serum-free medium containing von Willebrand factor and purified by a one-step immunopurification. The recombinant Factor VIII was characterized as a single active peak when subjected to f.p.l.c., in contrast with the plasma-derived molecule. Its coagulant activity was decreased in the presence of EDTA, suggesting that a bivalent ion is required, as for plasma-derived Factor VIII. The activation by thrombin and the inactivation by activated protein C were studied and the resulting molecular forms were analysed by f.p.l.c. and SDS/PAGE. The results clearly demonstrate that, despite the structural differences between plasma-derived and recombinant Factor VIII, activation and inactivation of Factor VIII-delta II generate proteolysed complexes similar to that described for plasma-derived Factor VIII. Thus this deleted recombinant Factor VIII, which is processed similarly to plasma-derived Factor VIII, should be normally integrated in the regulation system of Factor X activation in the blood-coagulation cascade.
...
PMID:Structural and functional characterization of Factor VIII-delta II, a new recombinant Factor VIII lacking most of the B-domain. 190 11


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---