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:3.4.21.6 (thromboplastin)
13,278 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The purpose of this study was to purify and characterize the agent responsible for the antimetastatic activity of an extract of the salivary glands (SGE) of the Mexican leech Haementeria officinalis. When administered intravenously in mice on the same day as the intravenous inoculation of T241 sarcoma cells, SGE markedly reduces the number and size of lung tumor colonies. In designing a purification protocol for the antimetastatic agent, we postulated that the antimetastatic agent would also display anticoagulant activity. Thus, we discovered that heparin affinity chromatography followed by anion-exchange chromatography results in a fraction highly enriched in both potent anticoagulant activity and potent antimetastatic activity. Approximately, 200-300 micrograms of purified protein is isolated from 150 mg of SGE. As little as 15 micrograms of this material inhibits tumor cell metastasis to the same extent as 1.0 mg of the unfractionated SGE. When analyzed on sodium dodecyl sulfate gels the active fraction consists mainly of one polypeptide band having an apparent molecular weight of approximately 17,000 under either reducing or nonreducing conditions. The protein has a pI of approximately 9.5 and a molecular weight of approximately 17,000 under nondenaturing conditions. A specific antiserum prepared against the 17,000-dalton protein indicated that this protein is the major anticoagulant and antimetastatic agent of leech salivary gland extract. We have termed this anticoagulant, antimetastatic agent "antistasin." We hypothesize that antistatin inhibits coagulation via factor Xa, and not thrombin, since factor Xa, but not thrombin, is rapidly inactivated upon addition of antistasin. The mechanism of antistasin's antimetastatic activity is currently under investigation.
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PMID:Isolation and characterization of antistasin. An inhibitor of metastasis and coagulation. 368 95

The conversion of the blood coagulation zymogen prothrombin to thrombin is associated with the production of several cleavage intermediates and products. In contrast to earlier studies of prothrombin cleavage in chemically defined systems, the current investigation examines the fragmentation of human prothrombin in normal plasma. Radiolabeled prothrombin was added to platelet-poor relipidated normal human plasma, and clotting was initiated with the addition of Ca(II) and kaolin. Analysis of the radiolabeled prothrombin cleavage products by polyacrylamide gel electrophoresis in the presence of dodecyl sulfate and beta-mercaptoethanol identified a heretofore unobserved product of prothrombin activation with an apparent molecular weight of 45,000. This product was identified as fragment 1 X 2 X 3, the NH2-terminal 286 amino acids of prothrombin. The product was isolated from a prothrombin digest by immunoaffinity chromatography using anti-prothrombin:Ca(II) antibodies and by preparative gel electrophoresis. Its amino-terminal sequence is identical to that of prothrombin. Digestion of this product with either Factor Xa or thrombin yields, at a minimum, fragment 1 X 2 and fragment 1. Amino-terminal sequence analysis of the products obtained by digestion with Factor Xa of the unknown activation product indicated 3 amino acid residues at each cycle consistent with the presence of fragment 1, fragment 2, and fragment 3. To unambiguously identify the COOH-terminal amino acid sequence of the product, its factor Xa digestion products were separated by reverse-phase high performance liquid chromatography. Edman degradation of one peptide revealed the complete sequence of fragment 3. On this basis, we identify the Mr 45,000 polypeptide as fragment 1 X 2 X 3 and indicate that it is a prominent product of prothrombin conversion to thrombin when activation occurs in plasma.
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PMID:Prothrombin fragment 1 X 2 X 3, a major product of prothrombin activation in human plasma. 375 58

Two different lipophilic photoreagents, [3H]adamantane diazirine and 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine (TID), have been utilized to examine the interactions of blood coagulation factor Va with calcium, prothrombin, factor Xa, and, in particular, phospholipid vesicles. With each of these structurally dissimilar reagents, the extent of photolabeling of factor Va was greater when the protein was bound to a membrane surface than when it was free in solution. Specifically, the covalent photoreaction with Vl, the smaller subunit of factor Va, was 2-fold higher in the presence of phosphatidylcholine/phosphatidylserine (PC/PS, 3:1) vesicles, to which factor Va binds, than in the presence of 100% PC vesicles, to which the protein does not bind. However, the magnitude of the PC/PS-dependent photolabeling was much less than has been observed previously with integral membrane proteins. It therefore appears that the binding of factor Va to the membrane surface exposes Vl to the lipid core of the bilayer, but that only a small portion of the Vl polypeptide is exposed to, or embedded in, the bilayer core. Addition of either prothrombin or active-site-blocked factor Xa to PC/PS-bound factor Va had little effect on the photolabeling of Vl with TID, but reduced substantially the covalent labeling of Vh, the larger subunit of factor Va. This indicates that prothrombin and factor Xa each cover nonpolar surfaces on Vh when the macromolecules associate on the PC/PS surface. It therefore seems likely that the formation of the prothrombinase complex involves a direct interaction between Vh and factor Xa and between Vh and prothrombin.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Interaction of blood coagulation factor Va with phospholipid vesicles examined by using lipophilic photoreagents. 382 92

The amino acid sequence of protein Z has been determined from sequence analysis performed on fragments obtained by chemical and enzymatic degradations. The polypeptide consists of a single chain containing 396 amino acid residues (Mr 43 677). Comparison with the vitamin K-dependent plasma proteins reveals an extensive homology. The N-terminal part, containing 13 gamma-carboxyglutamic acid and one beta-hydroxyaspartic acid residue, is extensively homologous to and of similar length to the light chain of factor X. The remainder of protein Z is homologous to the serine proteases and of similar size to the heavy chain of factor Xa, but of the active site residues only aspartic acid-102 is present. Histidine-57 and serine-195 are replaced in protein Z by threonine and alanine, respectively. The physiological function of protein Z is still uncertain.
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PMID:Amino acid sequence of bovine protein Z: a vitamin K-dependent serine protease homolog. 388 70

An anticoagulant fraction was isolated from the homogenate of human umbilical cord arteries, using Sephadex gel filtration and DEAE-Sephacel chromatography. Analysis with dodecyl sulfate/polyacrylamide gel electrophoresis and inactivation studies using proteolytic enzymes indicate that the anticoagulant activity is associated with a polypeptide with an apparent Mr of 32 000. The anticoagulant inhibits thromboplastin as well as factor Xa induced clotting but does not affect thrombin initiated fibrin formation. The anticoagulant inhibits the activation of prothrombin by the complete prothrombinase complex, by phospholipid bound factor Xa but not by free factor Xa. The inhibition is instantaneous and independent of the incubation time over the whole range of concentrations tested. Therefore, the anticoagulant is unlikely to be a phospholipase or a protease. Its action does not resemble that of the plasma protease inhibitors, but it probably interferes with the phospholipid--clotting factor interactions.
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PMID:Isolation and partial purification of a novel anticoagulant from arteries of human umbilical cord. 389 92

The blood-clotting protein prothrombin can be converted to thrombin in free solution by the proteolytic enzyme, activated factor X. When prothrombin is bound to the surface of phospholipid vesicles, the rate of thrombin generation is increased more than 30-fold over the rate of unbound prothrombin. If the prothrombin activation process is terminated after a time interval in which less than 10% of the expected thrombin has been produced, two major products are found in the activation mixture. These products have been termed intermediate 1, a precursor of thrombin, and fragment 1. The conversion of intermediate 1 to thrombin is not accelerated by phospholipid nor can binding of this intermediate to phospholipid particles be demonstrated. In contrast, fragment 1, the other activation product derived from prothrombin, binds to phospholipid particles under the same conditions as prothrombin. On the basis of these observations, we propose that the prothrombin molecule contains a specific region of polypeptide chain for binding to phospholipid particles. This specific polypeptide region or lipid interaction site is part of the nonthrombin-forming activation fragment.
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PMID:A polypeptide region of bovine prothrombin specific for binding to phospholipids. 451 4

A 38-year-old male patient with a life-long history of easy bruising and mild bleeding had a prolonged activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT). Reptilase (Bathrops atrox) clotting time was normal. His undiluted and diluted plasma prolonged the APTT, PT, and TT of normal plasma. Fibrin produced from patient plasma was insoluble in 5 M urea. Plasma fibrinogen level was increased when measured as clottable protein and by Laurell electroimmunoassay. Specific assays of plasma factors II, V, VII, X, VIII, IX, XI, and XII were normal. A circulating antithrombin in patient plasma was excluded by demonstrating normal thrombin-induced platelet aggregation of gel-separated platelets in the presence of patient plasma. Purified patient fibrinogen reproduced the anticoagulant effect of patient plasma. Patient fibrinogen antigen was similar to normal fibrinogen antigen by immunodiffusion, immunoelectrophoresis (pH 5.2 and 8.6), and crossed immunoelectrophoresis. His unreduced purified fibrinogen had normal migration on polyacrylamide slab gels. Also, the migration in gel slabs of A alpha, B beta and gamma-polypeptide chains, produced by mercaptoethanol reduction of purified patient fibrinogen, was similar to reduced normal fibrinogen. Thrombin-induced total fibrinopeptide release was normal. However, fibrin monomers produced from patient fibrinogen by thrombin (devoid of fibrinopeptides A and B) reaggregated abnormally; fibrin monomers produced by reptilase (devoid of only fibrinopeptides A) reaggregated normally. Fibrin generated from patient plasma in the presence of factor XIII and calcium, was defective in the formation of covalently bonded alpha-alpha polymers and demonstrated an increased susceptibility to the lytic effects of plasmin (generated in vitro by the addition of streptokinase).
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PMID:Fibrinogen Houston: a dysfibrinogen exhibiting defective fibrin monomer aggregation and alpha-chain cross-linkages. 616 39

Protein C inhibitor was isolated from human plasma using conventional chromatographic technique consisting of barium citrate adsorption, polyethylene glycol fractionation, DEAE-Sepharose CL-6B treatment, ammonium sulfate fractionation, dextran sulfate-agarose chromatography, gel filtration on ACA-44, and DEAE-Sephacel chromatography. The purified protein C inhibitor is a single polypeptide chain with an apparent Mr = 57,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The inhibitor is heterogeneous in pI: six pIs exist between pH 7.4 and 8.6. The inhibitor was shown to be different from the already known plasma protease inhibitors by chemical and immunological analyses. It migrates to the late alpha 1-globulin region on agarose gel electrophoresis. The inhibitor reduced the amidolytic activity of activated protein C noncompetitively by forming a 1:1 molar complex with the enzyme, determined by the use of a fluorogenic substrate toward activated protein C (Boc-Leu-Ser-Thr-Arg-4-methylcoumaryl-7-amide). The inhibition constant (Ki) of the inhibitor against activated protein C was 5.8 x 10(-8) M. The inhibitor also blocked the prolongation of activated partial thromboplastin time by activated protein C. The immunoglobulin which was produced by the inhibitor completely removed the inhibitory activity present in normal human plasma against activated protein C. This suggests that the inhibitor which we have isolated is the only inhibitor in plasma against activated protein C.
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PMID:Protein C inhibitor. Purification from human plasma and characterization. 629 98

Factor VII has been purified to homogeneity from bovine plasma by a procedure that includes affinity purification on an immunoadsorbent column. Recovery was determined by both coagulant assay and liquid scintillation counting, using 3H-factor VII as an internal standard. The purification factor calculated by both methods was approximately 120,000-fold, with a final yield of approximately 18%. Homogeneity was assessed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The material migrated as a single polypeptide chain of 53,000 daltons, and following activation by factor Xa, the one-chain zymogen was quantitatively converted to two-chain factor VIIa. Conversion of affinity-purified factor VII to factor VIIa resulted in up to a 119-fold activation of the coagulant activity, which is 2.7-4 times greater than the activatability reported for factor VII prepared by other methods. Zur et al. calculated that pure factor VII, uncontaminated by traces of factor VIIa, would be activated 123-fold upon conversion to factor VIIa. The close agreement between observed activatability of affinity-purified factor VII and the theoretical prediction suggests that we have isolated factor VII essentially free of factor VIIa. The purification data from three lots of bovine plasma yield an estimate for the plasma concentration of factor VII from 10.1 nM to 18.5 nM.
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PMID:Immunoaffinity purification of bovine factor VII. 636 51

Human factor IX circulates as a single-chain glycoprotein. Upon activation in vitro, it is cleaved into disulfide-linked light and heavy chains and an activation peptide. After reduction of activated 125I-factor IX, the heavy and light chains are readily identified by gel electrophoresis. A direct, immunoradiometric assay for factor IXa was developed to assess activation of factor IX for proteases that cleaved it. The assay utilized radiolabeled antithrombin III with heparin to identify the active site and antibodies to distinguish factor IX. After cleavage of factor IX by factor XIa, factor VIIa-tissue thromboplastin complex, or the factor X-activating enzyme from Russell's viper venom, antithrombin III bound readily to factor IXa. Cleavage of 125I-factor IX by trypsin, chymotrypsin, and granulocyte elastase in the presence of calcium yielded major polypeptide fragments of the sizes of the factor XIa-generated light and heavy chains. Kallikrein did not cleave the zymogen. Nonactivation cleavage was noted by thrombin, but only in the absence of calcium. When the immunoradiometric assay was used to assess trypsin-cleaved factor IX, the product bound antithrombin III, but not maximally. After digesting with insolubilized trypsin, clotting activity confirmed activation. In contrast, incubation of factor IX with elastase (Takaki A et al, J Clin Invest 71:1706, 1983) or chymotrypsin did not lead to generation of an antithrombin III-binding site, despite their digestion of 125I-factor IX into heavy and light chain-sized fragments. In evaluating activation of factor IX, physical evidence of activation cleavages does not necessarily correlate with generation of an active site.
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PMID:Cleavage and activation of human factor IX by serine proteases. 638 97


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