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)

A defect involving poor anticoagulant response to activated protein C (APC), an anticoagulant serine protease known to inactivate factors Va and VIIIa in plasma, was recently reported and the existence of a novel APC cofactor was suggested. To define the frequency of this defect among 25 venous thrombophilic patients with no identifiable laboratory test abnormality and among 22 patients previously identified with heterozygous protein C or protein S deficiency, the APC-induced prolongation of the activated partial thromboplastin time assay for these patients was compared with results for 35 normal subjects. The results show that this new defect in anticoagulant response to APC is surprisingly present in 52% to 64% of the 25 patients, ie, in the majority of previously undiagnosed thrombophilia cases, but is not present in 20 of 22 heterozygous protein C or protein S deficient patients, suggesting that the new factor is a risk factor independent of protein C or protein S deficiency. The results demonstrate that abnormalities in the anticoagulant protein C pathway are present in the majority of thrombophilic patients.
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PMID:Anticoagulant protein C pathway defective in majority of thrombophilic patients. 814 69

Tripeptide aldehydes such as Boc-D-Phe-Pro-Arg-H (51) exhibit potent direct inhibition of thrombin. This distinction offers important insight for the design of more potent and selective serine protease inhibitors which may be useful pharmacological tools and hold promise for development of clinically useful agents. The structure-activity relationships (SAR) on a series of anticoagulant peptides with high selectivity for the enzyme thrombin are discussed. The SAR is centered on a series of di- and tripeptide arginine aldehydes based on the structure of 51. The structural and conformational role of the amino acid residue in position 1 was investigated by substitution with conformationally restricted aromatic amino acids, aromatic acids, and a dipeptide isostere containing the psi[CH2N] amide bond replacement. Many of these peptides demonstrate potent antithrombotic activity along with selectivity toward thrombin, determined by comparison of in vitro inhibitory effects on trypsin, plasmin, factor Xa, and tissue plasminogen activator. Compound 5f, D-1-Tiq-Pro-Arg-H.sulfate is highly active and the most selective tripeptide aldehyde inhibitor of thrombin reported to date.
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PMID:Highly selective tripeptide thrombin inhibitors. 842 61

A slow, tight-binding inhibitor of thrombin with an apparent molecular mass of about 5 kDa has been isolated from Haemadipsa sylvestris, an Indian leech of the family of Haemadipsidae. The inhibitory activity, called haemadin, is thrombin specific since it does not inhibit other proteases like trypsin, chymotrypsin, factor Xa, or plasmin. NH2-terminal amino acid sequence analysis (residues 1-45) does not reveal any homology to known serine protease inhibitors, including the thrombin-specific inhibitor hirudin. The haemadin cDNA cloned by polymerase chain reaction techniques codes for a polypeptide of 57 amino acid residues preceded by 20 residues of a signal peptide sequence. A synthetic gene coding for the mature haemadin was expressed in Escherichia coli. Recombinant haemadin displays a similar inhibition constant and specific activity as its natural counterpart. Although there is no obvious sequence identity between haemadin and hirudin, both proteins seem to share common mechanisms for thrombin inhibition.
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PMID:Isolation, sequence analysis, and cloning of haemadin. An anticoagulant peptide from the Indian leech. 847 5

In a previous report, we described the molecular cloning, expression, and partial characterization of a second human tissue factor pathway inhibitor (TFPI), which we designated as TFPI-2 [Sprecher, C. A., et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 3353-3357]. Recombinant TFPI-2 inhibited the amidolytic activity of trypsin as well as that of factor VIIa in complex with tissue factor. TFPI-2 recently has been shown to be identical to placental protein 5 (PP5), a glycoprotein originally isolated from placenta that exhibits serine protease inhibitory activity. In the present study, we have examined TFPI-2/PP5 for its ability to inhibit a number of serine proteases involved in blood coagulation and fibrinolysis, inasmuch as TFPI-2/PP5 prolonged the coagulation time of human plasma induced by either tissue factor or contact activation in a dose-dependent manner. In addition to its ability to inhibit the amidolytic and proteolytic activities of the factor VIIa-tissue factor complex, TFPI-2/PP5 strongly inhibited the amidolytic activities of human factor XIa, human plasma kallikrein, and human plasmin with Ki values of 15, 25, and 3 nM, respectively. TFPI-2/PP5 was also a weak inhibitor of the activation of factor X by a complex of human factor IXa and poly(lysine) with an apparent Ki of 410 nM. Heparin markedly enhanced the ability of TFPI-2/PP5 to inhibit factor VIIa-tissue factor both in the solution phase and on cell surfaces. In addition, heparin augmented the inhibition of human factor Xa amidolytic activity at relatively high levels (10-100 nM) of TFPI-2/PP5. No significant inhibition of glandular kallikrein, urinary plasminogen activator, tissue plasminogen activator, human activated protein C, human factor Xa, human thrombin, or leukocyte elastase was observed when these proteases were incubated with TFPI-2 in the absence of heparin.
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PMID:Inhibitory properties of a novel human Kunitz-type protease inhibitor homologous to tissue factor pathway inhibitor. 855 84

We investigated the effect of activated protein C (APC) on lipopolysaccharide (LPS)-induced pulmonary vascular injury in rats to investigate the possible usefulness of APC as a treatment for adult respiratory distress syndrome. Intravenously administered LPS (5 mg/kg) significantly increased pulmonary vascular permeability. APC prevented the LPS-induced increase in pulmonary vascular permeability observed at 6 hours. Heparin plus antithrombin III (ATIII) and active site-blocked factor Xa (DEGR-Xa), a selective inhibitor of thrombin generation, inhibited LPS-induced coagulopathy but did not prevent LPS-induced pulmonary vascular injury. LPS-induced pulmonary vascular injury was significantly attenuated in rats with nitrogen mustard-induced leukocytopenia and in rats treated with ONO-5046, a potent granulocyte elastase inhibitor. Administration of LPS also increased pulmonary accumulation of leukocytes, as evaluated by measurement of myeloperoxidase activity in the lungs. APC significantly reduced LPS-induced increases in pulmonary accumulation of leukocytes at 1 hour. Neither ATIII plus heparin nor DEGR-Xa inhibited leukocyte accumulation. Active site-blocked APC (DIP-APC) prevented neither the LPS-induced pulmonary accumulation of leukocytes nor the LPS-induced increase in pulmonary vascular permeability. These results suggest that the mechanism of APC inhibition of LPS-induced pulmonary vascular injury was independent of its anticoagulant activity and was related to its ability to inhibit accumulation of leukocytes. In addition, these findings suggest that the serine protease activity of APC may be essential to its inhibitory effect on LPS-induced pulmonary accumulation of leukocytes and subsequent pulmonary vascular injury.
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PMID:Activated protein C attenuates endotoxin-induced pulmonary vascular injury by inhibiting activated leukocytes in rats. 855 86

A specific blood coagulation factor X activator was purified from the venom of Ophiophagus hannah by gel filtration and two steps of FPLC Mono-Q column ion-exchange chromatography. It showed a single protein band both in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and alkaline polyacrylamide gel electrophoresis. The mol. wt was estimated to be 62,000 in non-reducing conditions and 64,500 in reducing conditions by SDS-PAGE. The isoelectric point was found to be pH 5.6. The enzyme had weak amidolytic activities toward CBS 65-25, but it showed no activities on S-2266, S-2302, thrombin substrate S-2238, plasmin substrate S-2251 or factor Xa substrate S-2222. It had no arginine esterase activity toward substrate benzoylarginine ethylester (BAEE). The enzyme activated factor X in vitro and the effect was absolutely Ca2+ dependent, with a Hill coefficient of 6.83. It could not activate prothrombin nor had any effect on fibrinogen and thus appeared to act specifically on factor X. The procoagulant activity of the enzyme was almost completely inhibited by serine protease inhibitors like PMSF, TPCK and soybean trypsin inhibitor; partially inhibited by L-cysteine. Metal chelator EDTA did not inhibit its procoagulant activity. These results suggest that the factor X activator from O. hannah venom is a serine protease.
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PMID:Isolation and properties of a blood coagulation factor X activator from the venom of king cobra (Ophiophagus hannah). 859 78

A specific activator of blood coagulation factor X was purified from the venom of Bungarus fasciatus by gel filtration and by ion-exchange chromatography on a Mono-Q column (FPLC). It consisted of a single polypeptide chain, with a mol. wt of 70,000 in reducing and non-reducing conditions. The enzyme had an amidolytic activity towards the chromogenic substrates S-2266 and S-2302 but it did not hydrolyse S-2238, S2251 or S-2222, which are specific substrates for thrombin, plasmin and factor Xa, respectively. The enzyme activated factor X in vitro and the effect was Ca2+ dependent with a Hill coefficient of 7.9. As with physiological activators, the venom activator cleaves the heavy chain of factor X, producing the activated factor Xa alpha. The purified factor X activator from B. fasciatus venom did not activate prothrombin, nor did it cleave or clot purified fibrinogen. The amidolytic activity and the factor X activation activity of the factor X activator from B. fasciatus venom were readily inhibited by serine protease inhibitors such as diisopropyl fluorophosphate (DFP), phenylmethanesulfonyl fluoride (PMSF), benzamidine and by soybean trypsin inhibitor but not by EDTA. These observations suggest that the factor X activator from B. fasciatus venom is a serine protease. It therefore differs from those of activators obtained from Vipera russelli and Bothrops atrox venoms, which are metalloproteinases.
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PMID:An activator of blood coagulation factor X from the venom of Bungarus fasciatus. 859 79

The ability to regulate proteolytic functions is critical to cell biology. We describe events that regulate the initiation of the coagulation cascade on endothelial cell surfaces. The transmembrane protease receptor tissue factor (TF) triggers coagulation by forming an enzymatic complex with the serine protease factor VIIa (VIIa) that activates substrate factor X to the protease factor Xa (Xa). Feedback inhibition of the TF-VIIa enzymatic complex is achieved by the formation of a quaternary complex of TF-VIIa, Xa, and the Kunitz-type inhibitor tissue factor pathway inhibitor (TFPI). Concomitant with the downregulation of TF-VIIa function on endothelial cells, we demonstrate by immunogold EM that TF redistributes to caveolae. Consistently, TF translocates from the Triton X-100-soluble membrane fractions to low-density, detergent-insoluble microdomains that inefficiently support TF-VIIa proteolytic function. Downregulation of TF-VIIa function is dependent on quaternary complex formation with TFPI that is detected predominantly in detergent-insoluble microdomains. Partitioning of TFPI into low-density fractions results from the association of the inhibitor with glycosyl phosphatidylinositol anchored binding sites on external membranes. Free Xa is not efficiently bound by cell-associated TFPI; hence, we propose that the transient ternary complex of TF-VIIa with Xa supports translocation and assembly with TFPI in glycosphingolipid-rich microdomains. The redistribution of TF provides evidence for an assembly-dependent translocation of the inhibited TF initiation complex into caveolae, thus implicating caveolae in the regulation of cell surface proteolytic activity.
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PMID:Ligand-induced protease receptor translocation into caveolae: a mechanism for regulating cell surface proteolysis of the tissue factor-dependent coagulation pathway. 860 63

Coagulation factor Xa is a plasma serine protease that catalyzes prothrombin to thrombin conversion, which, in turn, leads to the generation of the fibrin clot. Of the several parameters that govern the plasma level of factor Xa, control of its catabolism is of crucial importance. However, little is known regarding the mechanisms by which factor Xa is catabolized. In the present study we examine the cellular basis for the uptake and degradation of factor Xa. 125I-Factor Xa was degraded by hepatoma cells and embryonic fibroblasts via a process which required cell surface-bound tissue factor pathway inhibitor (TFPI), a potent inhibitor of factor Xa. Uptake and degradation of cell surface-bound 125I-TFPI was also markedly stimulated in response to factor Xa binding. The intracellular kinetics of 125I-factor Xa and cell surface-bound 125I-TFPI display a strikingly similar pattern, suggesting that factor Xa and cell surface-bound TFPI are taken up as a bimolecular complex. Using cell lines either deficient in low density lipoprotein receptor-related protein, an endocytic receptor that mediates the degradation of uncomplexed TFPI (Warshawsky, I., Broze, G.J., Jr., and Schwartz, A.L. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 6664-6668), or deficient in tissue factor (TF), an integral membrane protein capable of forming quarternary complexes with factor Xa, TFPI, and factor VIIa, we demonstrated that the receptor that mediates the uptake and degradation of factor Xa-TFPI complex was neither low density lipoprotein receptor-related protein nor TF. As the vascular endothelial cell surface retains a substantial pool of TFPI (Sandset, P.M., Alildgaard, U., and Larsen, M.L. (1988) Thromb. Res. 50, 803-813; Novotny, W.F., Brown, S.G., Miletich, J.P., Rader, D.J., and Broze, G.J., Jr. (1991) Blood 78, 387-393), our data suggest that endothelial cell surface TFPI may be actively involved in the clearance of factor Xa from the circulation via mediated uptake and degradation.
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PMID:Receptor-mediated endocytosis of coagulation factor Xa requires cell surface-bound tissue factor pathway inhibitor. 862 21

Acidic phospholipids play an important but incompletely understood role in prothrombin activation. Here we report the effect of short-chain phosphatidylserine (dicaproylphosphatidylserine, C6PS) and the corresponding phosphatidylglycerol (C6PG) and phosphatidylcholine (C6PC) derivatives on the rate of prothrombin activation by factor Xa. The critical micellar concentrations of these short-chained phospholipids have been determined under a variety of conditions that we used for kinetic and structural studies. Under conditions for which these lipids exist in a soluble form, the results demonstrate that: (i) the rate of human prothrombin activation by human factor Xa was enhanced in a calcium-dependent fashion up to 60-fold by addition of C6PS, roughly 20% of the optimal enhancement seen with bovine phosphatidylserine/palmitoyloleoylphosphatidylcholine (25/75 PS/POPC) membranes; (ii) C6PS inhibited the rate of hydrolysis of synthetic factor Xa substrate (S-2765), an effect that was mimicked, but at much lower lipid concentrations, by PS/POPC membranes; (iii) there was no enhancement of prothrombin activation and much less inhibition of hydrolysis of S-2765 by factor Xa in the presence of C6PG or C6PC; and (iv) the thermal denaturation of prothrombin was altered in a calcium-independent but dose-dependent fashion by either C6PS or C6PG. These results have been interpreted in terms of the existence of (a) specific PS binding site(s) on factor Xa (Kd approximately 73 microM) that regulate(s) the activity of this serine protease. Our results do not rule out the possibility that the rate of prothrombin activation is also influenced by a weaker, calcium-independent, and less specific acidic lipid binding site on prothrombin, the occupancy of which results in conformational changes in this protein. The results clearly suggest that PS binding regulates the rate of prothrombin activation.
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PMID:Soluble phospholipids enhance factor Xa-catalyzed prothrombin activation in solution. 865 26


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