Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0027960 (mole)
 21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

When 125I-labeled thrombin was incubated with washed human platelets or with the supernatant solution of activated platelets, it formed a NaDodSO4-stable complex of apparent mass greater than 450 000 daltons. Formation of the complex was temperature dependent; with 20 nM thrombin incubated with the supernatant solution of ionophore-activated platelets, the initial rate of formation of the stable complex was 1 nM thrombin/min at 37 degrees C, 50 times the rate at 22 degrees C. Thrombin with all free amino groups methylated was still reactive. Active-site-blocked thrombin formed the complex only slowly. The complex that formed with active thrombin was not dissociated by hydroxylamine in urea. Reduction with 2-mercaptoethanol dissociated the complex, and its formation was blocked by the sulfhydryl-blocking agents iodoacetamide and 4,4'-dithiodipyridine. The complex was thus unlike those of thrombin and alpha 2-macroglobulin or antithrombin III, but it had characteristics of a disulfide-linked complex. Of the secreted proteins, albumin and glycoprotein G adhered to an activated thiol-Sepharose column, indicating that they contained free thiol groups. Purified glycoprotein G and thrombin formed a complex similar to the complex formed when thrombin was incubated with the supernatant solution of activated platelets. The purified glycoprotein bound 2.6 mol of radioactive N-ethylmaleimide/mol of protein, indicating three sulfhydryl groups per mole. After reacting with purified glycoprotein G, thrombin developed a new sulfhydryl group. It is concluded that glycoprotein G (thrombin-sensitive protein, thrombospondin) and thrombin form a dissociable complex that leads to a covalent complex by thiol-disulfide exchange of a thiol group on glycoprotein G and a disulfide on thrombin.
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PMID:Formation of a stable complex of thrombin and the secreted platelet protein glycoprotein G (thrombin-sensitive protein, thrombospondin) by thiol-disulfide exchange. 643 45

At pH 4.1, bovine thrombin reacts rapidly with N-bromo-succinimide to yield modified enzyme containing oxidized tryptophan residue. Both fibrinogen clotting activity and esterase activity are reduced considerably when three moles of tryptophan residues per mole of thrombin are oxidized, but the Michaelis constants for synthetic substrates are not appreciably altered. Reaction of NBS also results in a decrease in the affinity of thrombin for heparin. The dissociation constant for heparin-thrombin complex is increased by 2.6-fold due to the modification of one tryptophan residue. However, the magnitude of the increase in the dissociation constant remains the same for modified enzymes containing approximately two or three oxidized tryptophan residues. The rate constant for the inactivation of thrombin by antithrombin III is increased by 2.5-fold due to the modification of a single tryptophan residue. This increase in rate constant is not further amplified when more than one tryptophan residue is oxidized. In contrast, in the presence of heparin the rate of inactivation of modified and unmodified thrombins by antithrombin III are not significantly different. Thus, the heparin-sensitized inactivation of thrombin by antithrombin III is affected by the modification of one tryptophan residue. Spectrophotometric titrations of the phenolic hydroxyl groups suggest that the structural environments of tyrosyl groups for both unmodified and modified thrombin containing one oxidized tryptophan residue, are similar. The temperature for half loss of catalytic activity of control and NBS-modified thrombin, containing one oxidized tryptophan, are 52 and 51.5 degrees C respectively. It appears that the one tryptophan residue of thrombin is situated at or close to the binding site of heparin.
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PMID:Catalytic and regulatory functions of N-bromosuccinimide-modified bovine thrombin. 652 42

A dysprothrombin designated prothrombin Quick, is isolated from the plasma of an individual with < 2% of normal functional prothrombin activity and 34% of the normal prothrombin level by immunologic assay. With Factor Xa or taipan snake venom as activators, a fragmentation pattern identical to that of normal prothrombin is observed on gel electrophoresis in dodecylsulfate. This evidence combined with the observed barium citrate adsorption of prothrombin Quick and the low activity suggests that the defect in prothrombin Quick is in the thrombin portion of the molecule. Thrombin Quick is isolated and comigrates with thrombin on dodecyl sulfate gel electrophoresis, either reduced or nonreduced. The activity of thrombin Quick on several biological substrates of thrombin is investigated. Relative to normal thrombin, thrombin Quick is 1/200 as active on fibrinogen and 1/20-1/50 as effective in activating Factors V and VIII and aggregating platelets. A complex with antithrombin III is detected by dodecyl sulfate gel electrophoresis. Further investigation with the active site titrant, dansylanginine-N-(3-ethyl-1,5-pentanediyl)amide showed that the thrombin Quick preparation has the same affinity for the titrant as thrombin, but apparently only 40% active sites per mole protein are titrable.
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PMID:Identification of a congenital dysthrombin, thrombin Quick. 677 45

The cholesterol to phospholipid mole ratio (C/PL) of human platelets was increased 1.3-fold or maintained at a normal value by incubating platelets with sonicated dispersions of cholesterol and phosphatidylcholine (PC) (C/PL = 3 or 1, respectively). Thrombin-induced mobilization of [3H]arachidonic acid from prelabeled phospholipids and subsequent formation of labeled cyclo-oxygenase and lipoxygenase products were increased in cholesterol-enriched platelets as a function of thrombin concentration. Elevated platelet cholesterol content affected thrombin-induced changes in platelet phospholipids: (a) hydrolysis of PC was more sensitive to thrombin and was markedly enhanced over a wide range of thrombin concentrations (0.1-2 units/ml); (b) hydrolysis of phosphatidylinositol (PI) was increased at thrombin concentrations greater than or equal to 0.2 unit/ml. Increased metabolism of [3H]arachidonic acid in stimulated cholesterol-enriched platelets was due to loss of [3H]arachidonate from PC at 0.1 unit/ml of thrombin. At higher thrombin concentrations (0.2-2 units/ml) it reflected enhanced hydrolysis of predominantly PC, but also PI. We conclude that cholesterol, possibly through its effect on platelet lipid organization, influences arachidonic acid metabolism in stimulated plates by promoting enhanced activity of platelet phospholipase(s) for liberation of arachidonic acid.
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PMID:Effect of membrane cholesterol on phospholipid metabolism in thrombin-stimulated platelets. Enhanced activation of platelet phospholipase(s) for liberation of arachidonic acid. 708 8

Affinity-purified polyclonal antibodies prepared against a synthetic peptide corresponding to sequence 18-29 from the N-terminus of rabbit alpha-skeletal actin reacted with G- and F-actin. Epitope mapping experiments with thrombin and hydroxylamine cleaved actin, and immunochemical assays verified the specificity of antibodies for the 18-29 sequence on actin. The binding of up to 0.5 mol of IgG per mole of actin did not affect the rigor binding of myosin subfragment 1 (S-1) to actin. Similarly, the binding of IgG to actin was not changed by a complete saturation of actin by S-1. In contrast to this, the weak acto-S-1 interactions in the presence of ATP were strongly inhibited by the 18-29 antibodies. At 25 degrees C, the acto-S-1 ATPase activity was inhibited by IgG stronger than the binding of S-1.ATP gamma S to actin. Thus, at this temperature, a catalytic inhibition of the acto-S-1 system appears to account at least in part for the antibody effect. Acto-S-1 ATPase activities at 25 degrees C were inhibited also by F(ab)(18-29). At 5 degrees C, the acto-S-1 ATPase activity and the binding of S-1.ATP to actin were inhibited approximately to the same extent by IgG(18-29). These results are discussed in terms of S-1 binding sites on actin and the possible role of sequence 18-29 in actomyosin interactions.
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PMID:Role of sequence 18-29 on actin in actomyosin interactions. 768 58

Recombinant p56lck tyrosine kinase was purified to near homogeneity from a baculovirus/insect cell expression system. Treatment with thrombin proteolytically removed the C-terminal 54 amino acids from p56lck. Processed enzyme migrated on sodium dodecyl sulfate (SDS) gels with a M(r) approximately 6,000 lower than intact enzyme. Analytical ultracentrifugation of intact and processed p56lck gave M(r)'s of 62,600 and 56,200, respectively, confirming that the thrombin treated enzyme existed in solution as a processed polypeptide and that there was no anomalous migration in SDS gels due to thrombin treatment. Simultaneous multispeed analysis of sedimentation equilibrium data demonstrated that both intact and processed enzyme can dimerize with a weak binding constant in the range of 200-300 microM. Purified intact p56lck incorporated 2 mol of [32P]P(i) per mole of enzyme. Purified processed p56lck incorporated only 1 mol of [32P]P(i) per mole of enzyme. The loss of 1 mol of [32P]P(i) per mole of enzyme after thrombin deletion of the C-terminus demonstrates that p56lck undergoes autophosphorylation at the C-terminus. The data are consistent with autophosphorylation at tyrosine 505, which has previously been thought to be a regulatory phosphorylation site, but which now must also be considered as an autophosphorylation site.
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PMID:Dimerization of native and C-terminally proteolyzed p56lck tyrosine kinase. 787 93

Snake venoms, especially from the Crotalidae family, contain a variety of enzymes that prevent blood coagulation by virtue of their fibrinolytic enzymes. Nineteen snake venoms were screened for fibrinolytic activity and the highest activity was found in the venom of Crotalus basiliscus basiliscus venom. The active principle, basilase, was isolated, purified, and found to have fibrinolytic and fibrinogenolytic activity. It had a molecular weight of 22,000 and 1 mol of zinc per mole of protein associated with it. The proteolytic activity of the enzyme against dimethyl casein was inhibited by ethylenediaminetetraacetic acid and alpha 2-macroglobulin. It did not inactivate alpha 2-macroglobulin. Basilase did not have any of the following activities: thrombin-like, factor X-like, protein C activating, or urokinase-like. It caused neither hemorrhage nor platelet aggregation. In spite of its proteolytic activity, basilase did not hydrolyze the membranes of platelets. Basilase had 24% alpha-helix, 31% beta-sheet, 25% turns, and 20% unordered structure, as determined by Fourier Transform Infrared spectroscopy. Basilase is an enzyme that hydrolyzes fibrin directly without activation of plasminogen.
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PMID:Biochemical characterization of basilase, a fibrinolytic enzyme from Crotalus basiliscus basiliscus. 789 51

BACKGROUND. Hypofibrinogenemia and increased fibrin(ogen) degradation products in acute leukemia have been attributed to intravascular thrombin generation triggered by leukemic cells. However, the strict relationship between fibrinogen catabolism and turnover of fibrinopeptide A (FPA), which is a sensitive and specific marker of thrombin activity, has not been evaluated in acute leukemia (AL) with or without disseminated intravascular coagulation (DIC) to see whether mechanisms other than thrombin activity could be responsible for fibrinogen consumption. We report here the 125I-fibrinogen kinetics and FPA measurements in 19 patients with AL, 6 of them with DIC. METHODS AND RESULTS. Radiolabelled fibrinogen kinetics were studied in all the patients concomitantly with the start of chemotherapy. Fibrinopeptide A was measured by a radioimmunoassay at time of diagnosis and during chemotherapy. The kinetics of disappearance of radiolabelled fibrinogen where biphasic, with a rapid phase in the first 1-3 days of chemotherapy and a subsequent slow phase associated with the reduction or disappearance of blast cells. Patients with DIC had a significantly shorter half-life and turnover than patients without DIC. The latter group had significant shortening of these parameters in comparison to normal subjects. The thrombin-dependent catabolic rate of fibrinogen, calculated from the mean level of FPA during the first phase of disappearance curve and by assuming 2 moles of FPA generated per mole of fibrinogen, was similar in patients without DIC and in normal subjects, whereas patients with DIC had a significantly higher catabolic rate, even though the increase was not sufficient to account for all the turnover of fibrinogen. No relationship was observed between fibrinogen turnover and FPA turnover.
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PMID:Fibrinogen survival and fibrinopeptide A in acute leukemia. 803 60

Changes in platelet cytoplasmic free calcium levels were investigated in contact with cast film surfaces of a block copolymer containing 2-hydroxyethyl methacrylate (HEMA) and styrene (St) (0.5 mole fraction HEMA). These copolymer surfaces demonstrate microdomain alternating lamellae structures composed of hydrophilic HEMA domains (5 nm width) and hydrophobic St domains (20 nm width). The results were compared with those obtained from platelets contacting a random copolymer of HEMA-St (0.5 mole fraction HEMA) and from homopolymers of polystyrene (PSt) and poly(2-hydroxyethyl methacrylate) (PHEMA). Cytoplasmic free calcium levels in platelets contacting the microdomain structured surfaces of the HEMA-St block copolymer remained relatively constant in contrast to the significant increases observed for the radically prepared HEMA-St copolymer, PSt, and PHEMA surfaces. Adhering platelets were stimulated by exogenously introduced thrombin and calcium ionophore A23187 20 min after platelet adherence to the polymer surfaces. Only platelets on the block copolymer surfaces showed active metabolic responses. These results suggest that adhering platelets on the microdomain structured surfaces maintain high sensitivities to external stimulation due to an intrinsic strong inhibition of platelet functional changes induced by surface contact.
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PMID:Prevention of changes in platelet cytoplasmic free calcium levels by interaction with 2-hydroxyethyl methacrylate/styrene block copolymer surfaces. 811 39

Phosphatidylserine (PS) is normally restricted to the inner leaflet of the plasma membrane of cells (including blood platelets). Upon cell activation PS may become exposed to the outer surface of the cell. Cell membranes with surface exposed PS at the outside form a catalytic surface for coagulation reactions. When platelets are activated with ionophore or with thrombin in combination with thapsigargin, calcium induced scrambling of phospholipids takes place, resulting in PS exposure. Concomitant with PS exposition structural changes take place. On resting and activated platelets we combined the immunocytochemical detection of surface exposed PS with (ultra)structural information. Blood platelets were activated in the presence of annexin V, a protein which binds to PS in the presence of Ca2+. Annexin V was found to bind to lipid bilayers containing more than 5 mole % PS as estimated by binding of fluorescent-labelled annexin V to liposomes with varying PS concentrations. After vitrification, freeze-substitution and embedding of the platelets, annexin V was located on ultra thin sections, as detected by an anti-annexin V antibody and gold labelled protein A. Upon activation, the platelets show two different forms; irregular platelets with unchanged cytoplasm and round cells with apparently diluted cytoplasm. Activation with ionophore initially resulted in both forms, but after ten minutes only round platelets with diluted cytoplasm were observed. Both forms of these platelets as well as the microvesicles were found to be annexin V positive. However upon activation with thrombin in combination with thapsigargin, only the round cells with diluted cytoplasm and microvesicles were annexin V positive, whereas platelets with unchanged cytoplasm, even when microvesicles are present, are negative for annexin V.
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PMID:Ultrastructural detection of surface exposed phosphatidylserine on activated blood platelets. 856 Apr 27


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