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

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

Immunotherapy with Interleukin-2 (IL-2) and LAK cells has shown antitumoral activity in metastatic cancer patients. So far, thrombocytopenia is the major side effect reported in hemostasis. We have studied coagulation parameters in 6 patients treated with r-Met Hu IL-2 [ala-125]. In each case, we have observed a significant fall in prothrombin time, fibrinogen, protein C, anti-thrombin III, plasminogen, alpha 2-antiplasmin and all of the clotting factors except factor VIII. There was a significant increase in the activated thromboplastin time. No significant modifications of the D-Dimer test, fibrin-fibrinogen degradation products (FDP) and thrombin time were observed. Our data suggest that r-Met Hu IL-2 [ala-125] could interfere with the hepatic synthesis of the clotting factors and their inhibitors.
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PMID:Blood coagulation abnormalities during adoptive immunotherapy with interleukin-2 (r-Met Hu IL-2 [ala 125]). 200 36

Exhaustive extraction of human platelets with 6 M guanidine-HCl, and 5% beta-mercaptoethanol, followed by 5% SDS resulted in a sedimentable material which showed fibrous structure by transmission electron microscopy. When platelets treated with 8 M urea, 50 mM DTT and 2% SDS were applied on a 3% solubilizable acrylamide gel a high molecular weight material could be also isolated which was highly crosslinked by epsilon(gamma-glutamyl)lysine bonds. Its amino acid composition was Asp 110, Glu 119, Ser 55, Gly 70, Arg 33, Thr 41, Ala 112, Pro 93, Tyr 35, Val 18, Met 55, Cys 46, IIe 47, Leu 71, Phe 27, Lys 76 expressed as residue per 1000. The quantity of platelet polymer material as well as the amount of epsilon(gamma-glutamyl)lysine bond was slightly higher in thrombin activated platelets. The insoluble matrix of resting platelets reacts with antibodies against spectrin, alpha-actinin, actin, myosin, tropomyosin. The matrix from activated platelets has shown reaction with additional antibodies including ones against blood coagulation factor XIIIa, fibrinogen, von Willebrand factor, thrombospondin, tubulin and filamin. The presence of an epsilon(gamma-glutamyl)lysine cross-linked cell matrix in platelets is consistent with the observation of a similar structure in other cells.
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PMID:The presence of a covalently cross-linked matrix in human platelets. 200 80

There has been major interest in the potential interaction between blood coagulation and inflammation. Most of the effort has focused on cellular interactions involving platelets and polymorphonuclear leukocytes (PMNS). The recent discovery of tissue kallikrein(TK) activity in PMNs prompted the study of the possible role of thrombin(IIa) in this process. Human PMNs were isolated by density gradient centrifugation. Human IIa was compared with fMLP with respect to chemotaxis and enzyme release. Results from the challenges by IIa and fMLP were compared to a NaCl control using Student's paired t-test. IIa was a potent chemotactic agent for PMNs (p less than or equal to 0.0121) and stimulated the release of TK (p less than or equal to 0.0001) as determined by hydrolysis of S-2266. FMLP significantly stimulated PMN chemotaxis (p less than or equal to 0.0028) but had no effect on TK release. Release of TK was confirmed by Western Blot analysis and 35S-methionine incorporation into a 35 KD protein after IIa challenge. These results demonstrate that IIa is chemotactic for PMNs and can cause release of tissue kallikrein demonstrating a direct role for blood coagulation in the regulation of the inflammatory response.
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PMID:Linkage between blood coagulation and inflammation: stimulation of neutrophil tissue kallikrein by thrombin. 201 25

A study was made of the blood and tissue oxygen regime in patients with vibratory disease (VD) induced by local vibration and of the importance of lipid peroxidation (LPO) in oxygenation disorders. Venous hyperoxia, a decrease of the arteriovenous difference according to oxygen, the percentage of oxygen utilization by tissues, shift of the acid-base balance towards metabolic acidosis were established, attesting to tissue hypoxia that increased with the gravity of VD. The importance of a steady activation of LPO and depression of the antioxidant system in the pathogenesis of hypoxia associated with VD was supported by the correlation analysis data on oxygen balance and LPO, the functional and metabolic characteristics of red blood cells (according to the viscosity of red blood cell suspension and the content in the cells of SH-groups, lipoproteins and histidine) and platelets (according to aggregation in response to ADP and thrombin) as well as by the level of blood serum fluorescence. The authors provide evidence for the use of antioxidants (a complex of alpha-tocopherol with ascorbic acid and methionine and calcium antagonists of the nifedipine group), giving a membranostabilizing effect, in multimodality treatment of patients afflicted with VD.
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PMID:[Cell-membrane aspects of the pathogenesis of hypoxia in vibration disease induced by local vibration]. 204 32

Inhibition of thrombin by heparin cofactor (HCII) is accelerated approximately 1000-fold by heparin or dermatan sulfate. We found recently that the mutation Arg189----His decreases the affinity of HCII for dermatan sulfate but not for heparin (Blinder, M. A., Andersson, T. R., Abildgaard, U., and Tollefsen, D. M. (1989) J. Biol. Chem. 264, 5128-5133). Other investigators have implicated Arg47 and Lys125 of anti-thrombin (homologous to Arg103 and Lys185 of HCII) in heparin binding. To investigate the corresponding residues in HCII, we have constructed amino acid substitutions (Arg103----Leu, Gln, or Trp; Lys185----Met, Asn, or Thr) by oligonucleotide-directed mutagenesis of the cDNA and expressed the products in Escherichia coli. The recombinant HCII variants were assayed for binding to heparin-Sepharose and for inhibition of thrombin in the presence of various concentrations of heparin or dermatan sulfate. All of the Arg103 variants bound to heparin with normal affinity. Furthermore, inhibition of thrombin by the Arg103----Leu variant occurred at a normal rate in the absence of a glycosaminoglycan and was accelerated by normal concentrations of heparin and dermatan sulfate. These results indicate that HCII, unlike anti-thrombin, does not require a positive charge at this position for the interaction with heparin or dermatan sulfate. The Arg103----Gln and Arg103----Trp variants inhibited thrombin at about one-third of the normal rate in the absence of a glycosaminoglycan, suggesting that these mutations exert an effect on the reactive site (Leu444-Ser445) of HCII. All of the Lys185 variants bound to heparin with decreased affinity but inhibited thrombin at approximately the normal rate in the absence of a glycosaminoglycan. These variants required greater than 10-fold higher concentrations of heparin to accelerate inhibition of thrombin and were not stimulated significantly by dermatan sulfate, suggesting that heparin and dermatan sulfate interact with Lys185 of HCII. These results provide evidence that the glycosaminoglycan-binding site in HCII includes Lys185 but not Arg103, both of which were predicted to be involved by homology to anti-thrombin.
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PMID:Site-directed mutagenesis of arginine 103 and lysine 185 in the proposed glycosaminoglycan-binding site of heparin cofactor II. 210 20

Guanine nucleotide-binding regulatory proteins, or G proteins, mediate the interaction of agonist receptors on the platelet surface with phospholipase C and adenylyl cyclase. To better understand this process, we have used several approaches to identify which G proteins are present in platelets, normal human megakaryocytes, and human erythroleukemia (HEL) cells, a leukemic cell line with megakaryocytic features. Because platelet and HEL cell responses to thrombin are inhibited by pertussis toxin, we have focused upon the members of the Gi family, whose alpha subunits can be ADP-ribosylated by that toxin. Western blots with antisera specific for Gi alpha demonstrated the presence in both platelets and HEL cells of the three best-described forms of this protein: Gi alpha 1, Gi alpha 2, and Gi alpha 3. Based upon immunoprecipitation studies with [35S]-methionine-labeled HEL cells, their relative abundance appears to be Gi alpha 2 much greater than Gi alpha 3 greater than Gi alpha 1. A HEL cell cDNA library screened with the Gi alpha antisera produced clones encoding Gi alpha 2 and Gi alpha 3 that had sequences similar to those reported from other sources. Gi alpha-specific probes created from these cDNA clones confirmed the presence of mRNA encoding Gi alpha 2 and Gi alpha 3 in both platelets (by Northern blotting) and megakaryocytes (by in situ hybridization). Thus the pertussis toxin substrates that have previously been detected in platelets and HEL cells are shown to be members of the Gi alpha family, all of which are candidates for interaction with receptors for thrombin and other agonists.
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PMID:Identification of the pertussis toxin-sensitive G proteins in platelets, megakaryocytes, and human erythroleukemia cells. 211 27

We have undertaken a structural and functional analysis of recombinant plasminogen activator inhibitor type 1 (PAI-1) produced in Escherichia coli using site-directed mutagenesis and immunochemistry. Expression of recombinant PAI-1 yielded an inhibitor that was functionally indistinguishable from PAI-1 made in human endothelial cells. Mutations in both the reactive center P1 and P1' residues (Arg-Met) and a putative secondary binding site for plasminogen activators on PAI-1 have been engineered to assess their functional effects. The inhibition of a panel of serine proteases, including plasminogen activators, trypsin, elastase, and thrombin, has been studied. Substitution of the P1 arginine residue with lysine or the P1' residue with either valine or serine had no detectable effect on the rate of inhibition of plasminogen activators. However, replacement of both P1 and P1' by Met-Ser produced a variant with no detectable plasminogen activator inhibitor activity. Mutations introduced into either Asp102 or Lys104 in the second site did not affect the rate of inhibition of plasminogen activators. Complementary immunochemical experiments using antibodies directed against the same two regions of the PAI-1 protein confirm that the reactive center is the primary determinant of inhibitory activity and that the putative second site is not a necessary functional region.
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PMID:Mutational and immunochemical analysis of plasminogen activator inhibitor 1. 212 Feb 33

Active-site-blocked, fluorescent derivatives of tPA (Activase) and a variant (delta FEIX) which lacks the finger and epidermal growth factor-like domains and possesses Asn to Gln and Val to Met mutations at residues 117 and 245, respectively, were prepared. The binding of these to fibrin was studied by adding them at systematically varying concentrations to fibrinogen, at a fixed concentration, inducing clotting with thrombin, separating free and bound tPA or delta FEIX by centrifugation, and measuring the concentration of unbound material by extrinsic fluorescence. Similar studies were performed with Glu and Lys-plasminogen, using intrinsic fluorescence. epsilon-amino caproic acid (EACA) was utilized to distinguish kringle-dependent from finger-dependent binding. In the absence of EACA, delta FEIX-bound fibrin through a single class of sites with Kd = 0.69 microM and n = 1.34 delta FEIX/fibrin. The binding of delta FEIX was completely inhibited by EACA and 50% displacement occurred at [EACA] = 300 microM. Fibrin-bound tPA was only partially displaced with EACA. In the presence of 30 mM EACA, tPA binding reflected a single class of sites with Kd = 0.26 microM and n = 0.60 tPA/fibrin. In the absence of EACA, tPA binding was complex, typified by downwardly curved Scatchard plots, and was consistent with interactions of the two classes of sites, characterized by Kd = 0.13 microM, n = 0.60 and Kd = 0.61 microM, n = 1.23. These were attributed to finger and kringle-dependent interactions, respectively. Under the experimental conditions employed, Glu-plasminogen exhibited no binding to fibrin, whereas Lys-plasminogen bound to a single class of sites with Kd = 0.25 microM and n = 1.02 plasminogen/fibrin. This binding was completely inhibited by EACA and 50% displacement occurred at [EACA] = 28 microM. Competition experiments indicated that Lys-plasminogen does not displace either tPA or delta FEIX from fibrin. From these results the conclusions are drawn that tPA can interact with intact fibrin by two different and independent modes, involving, respectively, the finger and kringle 2 domains, and neither of these modes are competitive with the kringle-dependent binding of Lys-plasminogen.
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PMID:The dissociation constants and stoichiometries of the interactions of Lys-plasminogen and chloromethyl ketone derivatives of tissue plasminogen activator and the variant delta FEIX with intact fibrin. 212 71

Site-directed mutagenesis was used to create hirudin in which Asn52 was replaced by methionine. Cyanogen bromide cleavage at this unique methionine resulted in two fragments. These fragments have been used to study the kinetic mechanism of the inhibition of thrombin by hirudin and to identify areas of the two molecules which interact with each other. The binding of the C-terminal fragment (residues 53-65) to thrombin resulted in a decrease in the Michaelis constant for the substrate D-phenylalanylpipecolylarginyl-p-nitroanilide (DPhe-Pip-Arg-NH-Ph). The N-terminal fragment (residues 1-52) was a competitive inhibitor of thrombin. There was a small amount of cooperativity in the binding of the two fragments. Whereas hirudin and its C-terminal fragment protected alpha-thrombin against cleavage by trypsin, the N-terminal fragment did not. Hirudin and the N-terminal fragment completely prevented the cleavage of alpha-thrombin by pancreatic elastase while the C-terminal fragment afforded a lesser degree of protection. The results of these experiments with trypsin and elastase are discussed in terms of interaction areas on thrombin and hirudin.
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PMID:Use of fragments of hirudin to investigate thrombin-hirudin interaction. 218 Jun 97

The physiological function of the serpin (serine proteinase inhibitor) heparin cofactor II (HCII) is not well understood. A role for HCII as an inhibitor of thrombin in the presence of dermatan sulfate and heparin has been proposed. Neutrophils (PMN) are the major cellular component of acute inflammation. HCII can be proteolytically inactivated by cathepsin G (CG) and elastase (LE), which are released by stimulated PMN. We have recently shown that reaction products of HCII with CG and LE are potent chemotaxins for PMN. Monocytes (monos) appear later in the course of inflammation than do PMN. They differentiate into macrophages in the tissues and participate in healing of damaged tissue and initiating immune responses. We found that the proteolysis products of HCII were chemotactic for monocytes in a fashion similar to their effects on PMN. At 10(-8) to 10(-9) M, the chemotactic activity of HCII proteolysis products was comparable to that of 10(-8) M N-formyl-Met-Leu-Phe (fMLP). The chemotactic activity of HCII-proteinase reaction products is mediated by a different mechanism than that of alpha 1 proteinase inhibitor (alpha 1 PI)-LE complexes or fMLP. Our data suggest that chemotactic activity generated by proteolysis of HCII is not due to the conformational change induced by cleavage of the exposed loop near the reactive site nor by release of the reactive site peptide. We also compared the effects of HCII reaction products and fMLP on expression of Mac-1 and p150,95 adhesive proteins. Mac-1 has been implicated in mono adhesion and chemotaxis and as a potential initiator of coagulation. The surface expression of Mac-1 was not increased above control levels by incubation of leukocytes with HCII digests, even though fMLP did increase surface Mac-1. Proteolysis products of HCII could play a role in the initial influx of PMN into a thrombus, and in the transition from acute to chronic inflammation, or to granulation and healing.
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PMID:Characteristics of the chemotactic activity of heparin cofactor II proteolysis products. 219 22


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