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)

To study the requirements for factor-IXa binding to platelets and factor-X activation, we examined the consequences of chemical modification (factor IXMOD) or enzymatic removal (factor IXDES) of gamma-carboxyglutamic acid (Gla) residues. In the presence of factor VIIIa and factor X, there were 344 (+/- 52) binding sites/platelet for factor IXaMOD (apparent dissociation constant [kdapp] = 4.5 +/- 0.9 nmol/L) and 275 (+/- 35) sites/platelet for factor IXaDES (kdapp = 5.0 +/- 0.8 nmol/L) compared with 580 (+/-65) sites/platelet for normal factor IXa (factor IXaN) (kdapp = 0.61 +/- 0.1 nmol/L) and 300 (+/-62) sites/platelet for factor IX (kdapp = 2.9 +/- 0.29 nmol/L). The concentrations of factor IXaN, factor IXaMOD and factor IXaDES required for half-maximal rates of factor-Xa formation were 0.67 nmol/L, 3.5 nmol/L, and 6.7 nmol/L. Whereas maximal velocities (Vmax) of factor Xa formation by factor IXaMOD (approximately 0.8 nmol/L.min-1) and factor IXaN (approximately 10.5 nmol/L.min-1), turnover numbers (kcat expressed as moles of factor Xa formed per minute per mole of factor IXa bound), and values of catalytic efficiency (kcat/Km) were normal, indicating that the decreased rates of factor X activation observed with factor IXaMOD and factor IXaDES are solely a consequence of the abnormal binding of these proteins to thrombin-activated platelets in the presence of factor VIIIa and factor X. Thus, factor IXa binding to platelets is mediated in part, but not exclusively, by high-affinity Ca2+ binding sites in the Gla domain of factor IX.
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PMID:Role of gamma-carboxyglutamic acid residues in the binding of factor IXa to platelets and in factor-X activation. 173 85

Fibrinogen Ledyard was discovered in a 10-year-old boy with a mild bleeding history. His father had the same defect and a bleeding history after surgery. Both patients were heterozygous. The plasma fibrinogen concentration was normal immunologically (335 mg/dL) and very low functionally (52 mg/dL). Purified fibrinogen Ledyard had a prolonged polymerization, which was somewhat corrected by addition of Ca2+ ions. High performance liquid chromatography (HPLC) analyses of the fibrinopeptides released by thrombin showed 1 mol of fibrinopeptide A (FPA) and 2 mol of fibrinopeptide B (FPB) released per mole of fibrinogen Ledyard. Steady-state kinetic parameters were evaluated for release of FPA by thrombin. When the concentration of fibrinogen Ledyard was corrected to 50% of total protein, because only 50% of fibrinogen Ledyard can release FPA, the kinetic constants were similar to those of control fibrinogen (Km = 7.5 mumol/L for A alpha chain, kcat = 54 s-1). This finding indicates that the cleavage site of the A alpha chain in these abnormal molecules may not interact with the catalytic site of thrombin. The three chains of fibrinogen Ledyard were isolated on reverse-phase C4-HPLC. The sequence of the amino terminus of A alpha chain showed that Arg in position 16 was replaced by Cys in the abnormal molecules. Approximately half of fibrinogen Ledyard (52%) was clotted by reptilase, suggesting that fibrinogen Ledyard may consist of 50% normal homodimers (A alpha Arg16 . A alpha Arg16) and 50% abnormal homodimers (A alpha Cys16 . A alpha Cys16). Abnormal molecules could form disulfide bond between the A alpha Cys16 residues. Thus, the abnormal molecules have a different structure that does not bind to thrombin. Probably the abnormality of polymerization of fibrinogen Ledyard results from the interaction of the abnormal molecules with normal fibrin monomers, so that the growth of fibrin protofibrils is inhibited. This abnormal fibrinogen supports adenosine diphosphate-induced platelet aggregation in a normal manner.
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PMID:Fibrinogen Ledyard (A alpha Arg16----Cys): biochemical and physiologic characterization. 191 64

The ability of the COA-SET Fibrin monomer (COA-SET FM) test to detect soluble fibrin was evaluated by comparing the results of the COA-SET FM test with fibrinopeptide A (FPA) determinations following thrombin incubation of plasma or whole blood. In addition, two semiquantitative tests (erythrocytes-agglutination test (FM-test) and ethanol gelation test (EGT] were included in the study. Under the experimental conditions used, the COA-SET FM test proved less sensitive than the FPA-assay. There was a strong correlation between the results obtained by the two tests (r = 0.86, p = 0.0001). When solely regarding low levels of soluble fibrin, however, the correlation was weaker (r = 0.59, p = 0.0003). The FM-test was less sensitive than the COA-SET FM test, but more sensitive than EGT at normal and low fibrinogen concentrations. At high fibrinogen concentrations, however, EGT proved more sensitive than the FM-test. Knowing that 1-2 moles of FPA are released per mole of fibrin monomers formed, a discrepancy was observed between the FPA concentrations and the fibrin monomer concentrations as determined by the COA-SET FM test, the FPA levels being 2-25 times higher than the fibrin monomer levels. The discrepancy was greatest at incipient fibrinogen-fibrin transformation and at high plasma fibrinogen levels. This may suggest that fibrinogen in some way interfered with the stimulating effect of fibrin on the t-PA catalyzed activation of plasminogen, the principle upon which the COA-SET FM test is based.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Comparison of methods for detecting soluble fibrin in plasma. An in vitro study. 232 70

The interaction of thrombin with alpha 2-macroglobulin (alpha 2M) was characterized by monitoring conformational changes and measuring the increase of free sulfhydryl groups during the reaction. Under experimental conditions where [thrombin] greater than [alpha 2M], the conformational change, measured by increases in the fluorescence of 6-(p-toluidino)-2-naphthalenesulfonate, and thiol group appearance displayed biphasic kinetics. The initial rapid phase results in the formation of a stable complex, the appearance of two sulfhydryl groups, the cleavage of approximately half of the Mr 180 000 subunits, and a conformational change that is not as extensive as that which occurs with trypsin. The slower phase is associated with the appearance of two additional sulfhydryl groups, increased cleavage of the Mr 180 000 subunit, and additional conformational changes. The available evidence suggests that the slow phase results from hydrolysis of the Mr 180 000 subunit(s) due to proteolysis of the alpha 2M-thrombin complex by free thrombin. Experiments with 125I-thrombin document the binding of 1 mol of thrombin/mol of alpha 2M that is not dissociated upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the complex. At higher ratios of thrombin to alpha 2M, a second mole of thrombin will reversibly associate with the 1:1 alpha 2M-thrombin complex. Under conditions where [thrombin] less than [alpha 2M], biphasic kinetics were not observed, and the conformational change, sulfhydryl appearance, and hydrolysis of the Mr 180 000 subunit were found to follow second-order kinetics.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Thrombin-induced conformational changes of human alpha 2-macroglobulin: evidence for two functional domains. 241 17

This study characterizes the structural and functional significance of sulfhydryl residues in human plasma heparin cofactor II (HCII). For quantification of sulfhydryl groups, the extinction coefficient of HCII was redetermined and found to be 0.593 ml mg-1 cm-1 using second-derivative spectroscopy and multicomponent analysis assuming 4, 10, and 2 residues of tryptophan, tyrosine, and tyrosine-O-sulfate per mole of protein, respectively. The results show that tyrosine-O-sulfate residues in HCII and in cholecystokinin peptide fragments (as model compounds) do not significantly contribute to the absorbance spectrum from 280 to 300 nm. A total of three sulfhydryl groups per mole of HCII was detected by Ellman's reagent titration, with or without treatment with dithioerythritol, indicating the absence of intramolecular disulfide bonds. Incubation of HCII with 0.1-10 mM dithioerythritol did not diminish its heparin-enhanced thrombin inhibition activity. Treatment with various sulfhydryl-specific reagents, including p-mercuribenzoate, HgCl2, and N-substituted maleimide derivatives, inactivated HCII. Titration with Ellman's reagent after these reactions identified the modification site as a cysteinyl residue(s). However, complete methanethio derivatization of the sulfhydryl groups of HCII using methyl methanethiosulfonate did not alter heparin-catalyzed thrombin inhibition. These results indicate that the sulfhydryl groups of HCII are not essential for thrombin inhibition. HCII differs from antithrombin III, which contains an essential disulfide bond for heparin-dependent thrombin inhibition (Longas, M. O., et al. (1980) J. Biol. Chem. 255, 3436). Furthermore, within the "serpin" (serine proteinase inhibitor) superfamily, HCII resembles chicken ovalbumin in occurrence of sulfhydryl residues and reactivity with various sulfhydryl group-directed compounds.
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PMID:Structure-function relationships in heparin cofactor II: spectral analysis of aromatic residues and absence of a role for sulfhydryl groups in thrombin inhibition. 342 30

Sphingosine inhibited protein kinase C activity and phorbol dibutyrate binding. When the mechanism of inhibition of activity and phorbol dibutyrate binding was investigated in vitro using Triton X-100 mixed micellar methods, sphingosine inhibition was subject to surface dilution; 50% inhibition occurred when sphingosine was equimolar with sn-1,2-dioleoylglycerol (diC18:1) or 40% of the phosphatidylserine (PS) present. Sphingosine inhibition was modulated by Ca2+ and by the mole percent of diC18:1 and PS present. Sphingosine was a competitive inhibitor with respect to diC18:1, phorbol dibutyrate, and Ca2+. Increasing levels of PS markedly reduced inhibition by sphingosine. Since protein kinase C activity shows a cooperative dependence on PS, the kinetic analysis of competitive inhibition was only suggestive. Sphingosine inhibited phorbol dibutyrate binding to protein kinase C but did not cause protein kinase C to dissociate from the mixed micelle surface. Sphingosine addition to human platelets blocked thrombin and sn-1,2-dioctanoylglycerol-dependent phosphorylation of the 40-kDa (47 kDa) dalton protein. Moreover, sphingosine was subject to surface dilution in platelets. The mechanism of sphingosine inhibition is discussed in relation to a previously proposed model of protein kinase C activation. The possible physiological role of sphingosine as a negative effector of protein kinase C is suggested and a plausible cycle for its generation is presented. The potential physiological significance of sphingosine inhibition of protein kinase C is further established in accompanying papers on HL-60 cells (Merrill, A. H., Jr., Sereni, A. M., Stevens, V. L., Hannun, Y. A., Bell, R. M., Kinkade, J. M., Jr. (1986) J. Biol. Chem. 261, 12010-12615) and human neutrophils (Wilson, E., Olcott, M. C., Bell, R. M., Merrill, A. H., Jr., and Lambeth, J. D. (1986) J. Biol. Chem. 261, 12616-12623). These results also suggest that sphingosine will be a useful inhibitor for investigating the function of protein kinase C in vitro and in living cells.
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PMID:Sphingosine inhibition of protein kinase C activity and of phorbol dibutyrate binding in vitro and in human platelets. 346 88

The prothrombin activator from the venom of Oxyuranus scutellatus (Taipan snake) was purified by gel filtration on Sephadex G-200 and ion-exchange chromatography on QAE-Sephadex. The activator is a large protein with a molecular weight of approximately 300,000, which is composed of subunits of Mr 110,000 and 80,000 and two disulfide-linked polypeptides of Mr 30,000. One or both of these Mr 30,000 subunits contain the active site. The venom activator readily converts Factor Xa-specific chromogenic substrates and is also able to activate prothrombin (Km = 166 microM, Vmax = 2.5 mumol of prothrombin activated per min/mg of venom). Gel electrophoretic analysis of prothrombin activation indicates that the venom activator randomly cleaves the Arg274-Thr275 and Arg323-Ile324 bonds of prothrombin since both thrombin and meizothrombin are formed as reaction products. Venom-catalyzed prothrombin activation is not affected by bovine Factor Va but is greatly stimulated by phospholipids plus Ca2+ ions. This stimulatory effect is explained by a decrease of the Km for prothrombin. In the presence of 50 microM phospholipid vesicles (25% phosphatidylserine/75% phosphatidylcholine; mole/mole), the Km is 0.34 microM and the Vmax is 7.1 mumol of prothrombin activated per min/mg of venom. The purified venom activator contains gamma-carboxyglutamic acid residues which presumably function in the interaction between the venom activator and phospholipids. Treatment of the activator with 0.8 M NaSCN strongly reduces its ability to activate prothrombin but has no effect on its amidolytic activity. The prothrombin-converting activity of the NaSCN-treated activator can be restored with bovine Factor Va. During prolonged gradient gel electrophoresis, the Mr 300,000 activator dissociates into smaller subunits. This causes a loss of the prothrombin-converting activity, while the amidolytic activity is recovered in a protein with an apparent molecular weight of 57,000. This protein can, however, rapidly activate prothrombin in the presence of Factor Va or in the presence of a protein component of Mr 220,000 that also migrates on the gel. These results suggest that the prothrombin activator from the O. scutellatus venom is a multimeric protein complex consisting of a Factor Xa-like enzyme and a Factor Va-like cofactor.
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PMID:Prothrombin activation by an activator from the venom of Oxyuranus scutellatus (Taipan snake). 353 Nov 98

Two thrombin independent reactions involving polymerization and gelation of fibrinogen (FBG) and of FBG and fibronectin (FN) are described. In the first reaction FXIII, in the presence of calcium ions, induces oligomerization and eventually complete gelation of FBG, i.e. formation of fibrinogenin. FBG dimers and probably also higher oligomers are formed by the crosslinking of gamma-chains prior to gelation. During gelation the A alpha-chains also become completely crosslinked. These reactions are enhanced by a variety of thiol compounds. With DTT, reduction of specific disulfides in the A alpha-chain of FBG appear to be responsible for the enhancement. In the second reaction, FXII catalyzes the formation of heteropolymers of FBG-FN. These complexes eventually form visible particulate matter called heteronectin. Dimers consisting of 1 mole FBG and 1 mole FN form first, followed by the appearance of higher order heteronectin intermediates. In heteronectin the A alpha-chain of FBG provides the linkage to FN. Thiols also enhance the heteronectin reaction. Formation of fibrinogen and/or heteronectin depends upon the initial relative concentrations of FBG and FN. At equimolar concentrations mainly heteronectin is formed. During clotting of normal whole blood, thrombin induced fibrin formation is the initial event followed by rapid fibrinogen formation. Addition of iodoacetamide (an inhibitor of FXIII) to whole blood prevents the formation of fibrinogenin. These findings suggest that the fibrinogen pathway is important in vivo.
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PMID:Alternative pathways in blood coagulation. 360 76

In order to ascertain the possibility that platelet serotonin uptake may occur during storage of platelet concentrates (PC) at 22 degrees C with agitation, the high-performance liquid chromatographic procedure was applied to determine serotonin uptake by platelets. Studies at 22 degrees C showed that platelets stored for 4 days exhibited a significant serotonin uptake with a Vmax value of 2.4 X 10(-19) mole/platelet/min and a Km value of 0.62 X 10(-6) M. Incubation of PC with 5 X 10(-6) M serotonin for 1 day at 22 degrees C increased their serotonin contents from 2.2 to 4.2 X 10(-7) mole/10(11) platelets. Thrombin stimulation caused about 80% release of intracellular serotonin from fresh as well as stored platelets, which contained standard serotonin in the same amount as the original amount. These results suggest that a significant serotonin uptake of platelets might occur during in vitro storage at 22 degrees C and stored platelets have retained abilities to sequester extracellular serotonin into dense granules.
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PMID:Serotonin uptake at 22 degrees C of stored platelets. 381 Jun 39

We have used both proteolysis and reconstitution experiments to characterize the determinants for LDL receptor binding of HTG-VLDL. In these studies, we showed that the removal of approximately one mole of apo E per mole of HTG-VLDL (Sf 100-400) and HTG-VLDL (Sf 60-100) by thrombin-specific cleavage results in loss of receptor binding and concomitant loss of suppression of HMG-CoA reductase. This is in direct contrast to the lack of effect thrombin cleavage has on the receptor-mediated uptake of LDL, an apo B-mediated process. We were able to reconstitute receptor binding in thrombin-treated HTG-VLDL (Sf 100-400) by the specific reincorporation of one mole of apo E into the VLDL. The incorporation of one mole of apo E into normal non-suppressive VLDL (Sf 60-400) also enables this lipoprotein to bind to the receptor as effectively as LDL. Trypsin, which destroys apo E-mediated, but not apo B-mediated binding to the LDL receptor, abolishes binding of HTG-VLDL (Sf 100-400) and HTG-VLDL (Sf 60-100), but not that of HTG-VLDL (Sf 20-60), IDL, or LDL to the LDL receptor. Therefore, we conclude that apo E of the appropriate conformation is required for receptor-mediated uptake by the LDL receptor of large TG-rich lipoproteins (Sf greater than 60). This conformation of apo E is probably related to the surface on which it is found (i.e., size of the particle) and the mode of incorporation into the phospholipid surface (i.e., transferred from plasma HDL). In large TG-rich particles, it appears that the intact apo E is necessary for the proper orientation of the molecule on the surface, with the carboxy-terminal one-third needed to anchor the apoprotein to the phospholipid surface. We believe that the binding of apo E to the LDL receptor is a redundant system and is used as a backup system in abnormal pathological states such as hypertriglyceridemia, abetalipoproteinemia, and hypobetalipoproteinemia. In the case of hypertriglyceridemia, where the lipolysis mechanism is overloaded, the abnormal binding of HTG-VLDL (Sf greater than 60) provides an alternate catabolic route for their removal from plasma. In the cases of a beta- and hypobetalipoproteinemia, where the normal particles for cholesterol delivery are either absent or at low levels, apo E-containing particles can serve to deliver cholesterol to cells as has been recently observed in vitro.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Expression of LDL receptor binding determinants in very low density lipoproteins. 390 64


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