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)

Human tissue factor pathway inhibitor (TFPI) is a plasma protease inhibitor that consists of three tandem Kunitz-type inhibitor domains flanked by a negatively charged NH2 terminus and a positively charged COOH-terminal tail. Previous studies have shown that the first and second Kunitz-type domains in TFPI are involved in the inhibition of factor VIIa and factor Xa activity, respectively. In the present study, we have compared the inhibitory properties of full-length recombinant TFPI and a truncated form of TFPI lacking the third Kunitz-type domain and COOH-terminal tail (TFPI1-161) with respect to inhibition of factor VIIa-tissue factor complexes on the surface of a human bladder carcinoma cell line J82. Full-length TFPI and TFPI1-161 were kinetically indistinguishable with respect to neutralization of the proteolytic activity of preformed complexes of factor VIIa-tissue factor on the J82 cell surface in the absence of factor Xa. Equimolar amounts of factor Xa augmented the anticoagulant activity of both preparations of TFPI to the same extent, and both preparations of TFPI were equally effective in inhibiting factor VIIa-tissue factor amidolytic activity in solution phase. In addition, plasma concentrations of both forms of TFPI, in stoichiometric complex with factor Xa, inhibited cell surface factor VIIa-tissue factor proteolytic activity markedly faster than plasma levels of antithrombin III, even in the presence of 1 unit/ml heparin. The results of displacement studies suggested slight differences in the affinity of the two TFPI molecules for the cell surface in that approximately 5% of a VIIa.TF.Xa.TFPI1-161 quaternary complex on J82 cells was displaceable from the cell surface by high concentrations of factor VIIa (10-100 nM), whereas only 1-2% of a VIIa.TF.Xa.TFPI complex was displaceable under comparable conditions. Pretreatment of the cells with TFPI/Xa alone or together with R152E factor VII, followed by factor VIIa treatment, revealed significant differences in the two TFPI forms with respect to the degree with which offered factor VIIa could restore factor X activation on the cell surface. These differences notwithstanding, our collective findings indicate that the third Kunitz-type domain and/or COOH-terminal tail of TFPI is not essential for the inhibition of cell surface factor VIIa-tissue factor complexes and suggests that TFPI1-161 may be a useful therapeutic agent in the treatment of thromboembolic episodes.
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PMID:Inhibitory properties of full-length and truncated recombinant tissue factor pathway inhibitor (TFPI). Evidence that the third Kunitz-type domain of TFPI is not essential for the inhibition of factor VIIa-tissue factor complexes on cell surfaces. 847 15

Prekallikrein, the precursor to the serine proteinase kallikrein, circulates in plasma in an equimolar complex with H-kininogen. The binding to H-kininogen is mediated by the kallikrein heavy chain consisting of four "apple" domains, A1-A4, which attaches to H-kininogen with high specificity and affinity (KD = 83 nM). At least two distinct portions of the kallikrein heavy chain form this H-kininogen binding site: a proximal segment located in the NH2-terminal fragment of the heavy chain encompassing A1, and distal segment(s) located in COOH-terminal fragment spanning domains A2-A4. The proximal binding segment has been located to amino acid positions 56-86 of A1. To precisely map the distal binding segment, we have identified monoclonal antibodies directed to the COOH-terminal fragment which interfere with the H-kininogen-prekallikrein complex formation. Monoclonal antibody 13G11 binds to recombinant apple domain A4 but not to domain A3 of the prekallikrein heavy chain. Deletion mutagenesis of domain A4 narrowed down the target epitope of 13G11 to the center portion of domain A4, positions 284-331. Direct binding studies of H-kininogen to various domain A4 constructs revealed that the distal H-kininogen binding portion is located on a segment of 48 residues, which overlaps the 13G11 epitope. Hence the tight interaction of H-kininogen and prekallikrein is mediated by at least two separate sequence segments located in domains A1 and A4, respectively, of the prekallikrein heavy chain. The isolated distal binding segment significantly prolongs the partial thromboplastin time of reconstituted Williams plasma thus stressing the critical role of the prekallikrein-H-kininogen complex formation in the initiation of the endogenous blood coagulation cascade.
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PMID:Mapping of the discontinuous kininogen binding site of prekallikrein. A distal binding segment is located in the heavy chain domain A4. 866 5

Psoriasin is a novel chemotactic inflammatory protein that possesses weak similarity to the S100 family members of Ca(2+)-binding proteins, and that is highly up-regulated in hyperproliferative psoriatic keratinocytes. Here we have used the psoriasin cDNA to express recombinant human (rh) psoriasin in Escherichia coli as a fusion protein containing a hexa His tag and a factor Xa cleavage site in the NH2-terminus. The protein was purified by affinity chromatography on Ni(2+)-nitrilotriacetic acid agarose, digested with factor Xa, further purified by ion-exchange chromatography and characterized by two-dimensional (2-D) gel electrophoresis and NH2-terminal sequencing. The ability of rh psoriasin to bind Ca2+, Zn2+, and Mg2+ was determined by dialysis experiments. We found that rh psoriasin may bind at least seven molecules of Ca2+ in KCl and several molecules in NaCl, with an affinity for the first bound molecule of 1.3-1.6 x 10(4) M-1. This indicates that psoriasin may cooperatively bind several molecules of Ca2+ when present in the extracellular space, or putatively, if localized in subcellular compartments where the concentration of Ca2+ is relatively high. At least eight molecules of Zn2+ were bound in KCl and four in NaCl, with an affinity just below 1 x 10(4) M-1 for the first molecule. Thus psoriasin does not bind significant amounts of Zn2+ at physiological concentrations. Mg2+ and Ca2+ are bound anti-cooperatively and binding of each of the ions (Ca2+, Zn2+, or Mg2+), is accompanied by conformational changes that move tyrosine residues to more hydrophobic areas.
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PMID:Expression and divalent cation binding properties of the novel chemotactic inflammatory protein psoriasin. 898 13

Factor Xa and thrombin (factor IIa) activate factor VIII (fVIII) by different proteolytic pathways. Thrombin cleaves fVIII at Arg372 between the A1 and A2 domains, at Arg740 between the A2 and B domains, and at Arg1689 between the B and A3 domains to form an A1/A2/A3-C1-C2 heterotrimer. We now report a stable porcine fVIIIaXa preparation obtained by Mono S HPLC at pH 6. NH2-terminal sequence analysis of purified subunits of fVIIIaXa revealed that factor Xa cleaves fVIII at Arg219 within the A1 domain and at Arg490 within the A2 domain, as well as at Arg372, Arg740, and Arg1689. Analytical ultracentrifugation of the fVIIIaXa preparation yielded results consistent with a single, 148 kDa species, similar to previous results with fVIIIaIIa [Lollar, P., & Parker, C. G. (1989) Biochemistry 28, 666-674]. Thus, the major species in the fVIIIaXa preparation contains five subunits, including fragments of the A1 and A2 domains that remain noncovalently bound. Fluorescence anisotropy measurements indicated there was no difference in the affinity of fVIIIaXa and fVIIIaIIa for a fluorescent dye-labeled, active-site-blocked derivative of porcine factor IXa. Additionally, the fVIIIaXa preparation bound dye-labeled factor IXa with 1:1 stoichiometry, indicating that all fVIIIaXa molecules in the preparation can bind factor IXa. However, fVIIIaXa had 4-fold less procoagulant activity than fVIIIaIIa. Kinetic analysis of fVIIIa cofactor activity using purified factor IXa and factor X suggested this difference is due to greater activity of fVIIIaIIa relative to fVIIIaXa within the intrinsic fXase complex, rather than a difference in their stabilities.
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PMID:Subunit structure and function of porcine factor Xa-activated factor VIII. 923 79

The effect of human neutrophil elastase (HNE) on human factor V (F.V) or alpha-thrombin-activated human factor V (F.Va) was studied in vitro by prothrombinase assays, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and NH2-terminal sequence analysis. Incubation of F.V (600 nmol/L) with HNE (2 nmol/L) in the presence of Ca2+ resulted in a time-dependent increase in its cofactor activity. In contrast, treatment of F.Va (600 nmol/L) with HNE (60 nmol/L) in the presence of Ca2+ resulted only in a time-dependent decrease in its cofactor activity. Under the conditions of these experiments, the maximum extent of F.V activation accomplished by incubation with HNE was approximately 65% to 70% of that observed with alpha-thrombin in presence of Ca2+. The extent of both the HNE-dependent enhancement in F.V cofactor activity and the HNE-dependent decrease in F.Va cofactor activity was not influenced by the addition of phosphatidylcholine/phosphatidylserine (PCPS) vesicles (50 micromol/L). The HNE-derived cleavage products of F.V, which correlated with increased cofactor activity, as demonstrated by SDS-PAGE under reducing conditions, were different from those generated using alpha-thrombin. Treatment of F.V (600 nmol/L) with HNE (2 nmol/L) in the presence of Ca2+ resulted in the production of three closely spaced doublets of: 99/97, 89/87, and 76/74 kD whose appearance over time correlated well with the increased cofactor activity as judged by densitometry. Treatment of F.Va (600 nmol/L) with HNE (60 nmol/L) in the presence of Ca2+ resulted in the cleavage of both the 96 kD heavy chain and the 74/72 kD light chain into products of: 56, 53, 35, 28, 22, and 12 kD. Although densitometry indicated that both the heavy and light chains of F.Va were hydrolyzed by HNE, cleavage of the 96 kD heavy chain was more extensive during the time period (10 to 30 minutes) of the greatest loss of F.Va cofactor activity. NH2-terminal sequence analysis of F.V treated with HNE indicated cleavage at Ile819 and Ile1484 under conditions during which the procofactor expressed enhanced cofactor activity in the prothrombinase complex. NH2-terminal sequence analysis of F.Va treated with HNE indicated cleavage at Ala341, Ile508, and Thr1767 under conditions, which the cofactor became inactivated, as measured by prothrombinase activity. The activation and inactivation cleavage sites are close to those cleaved by the physiological activator and inactivator of F.V and F.Va, namely alpha-thrombin (Arg709 and Arg1545) and Activated Protein C (APC) (Arg306 and Arg506), respectively. These results indicate that HNE can generate proteolytic products of F.V, which initially express significantly enhanced procoagulant cofactor activity similar to that observed following activation with alpha-thrombin. In contrast, HNE treatment of F.Va resulted only in the loss of its cofactor activity, but again, this is similar to that observed following inactivation by APC.
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PMID:Human neutrophil elastase activates human factor V but inactivates thrombin-activated human factor V. 924 37

Blood compatibilities of functional group-grafted and heparin-immobilized polyurethanes (PUs) were investigated using in vitro thrombus formation, plasma recalcification time (PRT), activated partial thromboplastin time (APTT), platelet adhesion and activation, and peripheral blood mononuclear cell (PBMC) activation. In the experiment with plasma proteins, PRT was shortened on amine group-grafted PU (PU-NH2) but prolonged on heparin-immobilized polyurethane (PU-Hep) when compared to PU control. APTT was significantly prolonged on PU-Hep, suggesting the binding of immobilized heparin to antithrombin III. The percentage of platelet adhesion was slightly increased by the introduction of functional groups such as carboxylic acid and primary amine on PU surfaces, but significantly decreased by the immobilization of heparin on the same substrate. The percentage of serotonin released from platelets adhered on surface-modified PUs was increased with increase of platelet adhesion. In the PBMC experiment, cells adhered less on heparin-immobilized PUs than on functional group-grafted PUs, and the production levels of tumour necrosis factor mRNAs from the cells stimulated by heparin-immobilized PU (PU-N-Hep) were smaller than those by the other substrates.
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PMID:In vitro blood compatibility of functional group-grafted and heparin-immobilized polyurethanes prepared by plasma glow discharge. 924 47

Human factor X is a two-chain, 58-kDa, vitamin K-dependent blood coagulation zymogen. The light chain of factor X consists of an NH2-terminal gamma-carboxyglutamic acid (Gla) domain, followed by a few helical hydrophobic residues and the two epidermal growth factor-like domains, whereas the heavy chain contains the serine protease domain. In this study, native factor X was found to contain three classes of Ca2+-binding sites: two high affinity (Kd 100 +/- 30 microM), four intermediate affinity (Kd 450 +/- 70 microM), and five to six low affinity (Kd 2 +/- 0.2 mM). Decarboxylated factor X in which the Gla residues were converted to Glu retained the two high affinity sites (Kd 140 +/- 20 microM). In contrast, factor X lacking the Gla domain as well as a part of the helical hydrophobic residues (des-44-X) retained only one high affinity Ca2+-binding site (Kd 130 +/- 20 microM). Moreover, a synthetic peptide composed of residues 238-277 (58-97 in chymotrypsinogen numbering) from the protease domain of factor X bound one Ca2+ with high affinity (Kd 150 +/- 20 microM). From competitive inhibition assays for binding of active site-blocked factor Xa to factor Va in the prothrombinase complex, the Kd for peptide-Va interaction was calculated to be approximately 10 microM as compared with 30 pM for factor Xa and approximately 1.5 microM for decarboxylated factor Xa. A peptide containing residues 238-262(58-82) bound Ca2+ with reduced affinity (Kd approximately 600 microM) and did not inhibit Xa:Va interaction. In contrast, a peptide containing residues 253-277(73-97) inhibited Xa:Va interaction (Kd approximately 10 microM) but did not bind Ca2+. In additional studies, Ca2+ increased the amidolytic activity of native and des-44-Xa toward a tetrapeptide substrate (benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide) by approximately 1.6-fold. The half-maximal increase was observed at approximately 150 microM Ca2+ and the effect was primarily on the kcat. Ca2+ also significantly protected cleavage at Arg-332-Gln-333(150-151) in the protease domain autolysis loop. Des-44-Xa in which the autolysis loop was cleaved possessed </=5% of the amidolytic activity of the noncleaved form; however, the S1 binding site was not affected, as determined by the p-aminobenzamidine binding. Additionally, autolysis loop-cleaved, active site-blocked native factor Xa was calculated to have approximately 10-fold reduced affinity for factor Va as compared with that of the noncleaved form.
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PMID:Interaction of calcium with native and decarboxylated human factor X. Effect of proteolysis in the autolysis loop on catalytic efficiency and factor Va binding. 926 43

A modified human beta2 receptor, designated 0K-beta2, was developed for site-specific labeling at the amino terminus with amine reactive fluorescent probes. 0K-beta2 has the following modifications: (1) all 16 lysines in the wild-type beta2 receptor were mutated to arginines, (2) a FLAG epitope preceded by a cleaved hemagglutinin signal sequence was fused to the amino terminus, and (3) a hexahistidine tail was added to the carboxyl terminus. The FLAG epitope and hexahistidine tail were added to facilitate purification while lysine to arginine mutations eliminate potential labeling sites for amine-reactive fluorescent probes. The remaining primary amines in the 0K-beta2 receptor, the amino terminal amine and the epsilon-amine of Lys3, both reside in the amino-terminal FLAG epitope. The 0K-beta2 receptor expressed in Sf9 insect cells exhibited ligand binding and G-protein coupling characteristics similar to the wild-type beta2 receptor. The modified receptor was labeled with fluorescamine, an amine-reactive fluorescent probe. Proteolysis with factor Xa showed that labeling was confined to the amino terminus of the 0K-beta2 receptor. Our results demonstrate site-specific fluorescamine labeling at the amino terminus of the 0K-beta2 receptor, a lysine-depleted beta2 receptor that retains functional characteristics of the wild-type receptor.
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PMID:Site-specific fluorescence labeling of the beta2 adrenergic receptor amino terminus. 939 50

The inactivation of factor Va was examined on primary cultures of human umbilical vein endothelial cells (HUVECs), either after addition of activated protein C (APC) or after addition of alpha-thrombin and protein C (PC) zymogen. Factor Va proteolysis was visualized by Western blot analysis using a monoclonal antibody (alpha HVaHC No. 17) to the factor Va heavy chain (HC), and cofactor activity was followed both in a clotting assay using factor V-deficient plasma and by quantitation of prothrombinase function. APC generation was monitored using the substrate 6-(D-VPR)amino-1-naphthalenebutylsulfonamide (D-VPR-ANSNHC4H9), which permits quantitation of APC at 10 pmol/L. Addition of APC (5 nmol/L) to an adherent HUVEC monolayer (3.5 x 10(5) cells per well) resulted in a 75% inactivation of factor Va (20 nmol/L) within 10 minutes, with complete loss of cofactor activity within 2 hours. Measurements of the rate of cleavage at Arg506 and Arg306 in the presence and absence of the HUVEC monolayer indicated that the APC-dependent cleavage of the factor Va HC at Arg506 was accelerated in the presence of HUVECs, while cleavage at Arg306 was dependent on the presence of the HUVEC surface. Factor Va inactivation proceeded with initial cleavage of the factor Va HC at Arg506, generating an M(r) 75,000 species. Further proteolysis at Arg306 generated an M(r) 30,000 product. When protein C (0.5 mumol/L), alpha-thrombin (1 nmol/L), and factor Va (20 nmol/L) were added to HUVECs an APC generation rate of 1.56 +/- 0.11 x 10(-14) mol/min per cell was observed. With APC generated in situ, cleavage at Arg506 on the HUVEC surface is followed by cleavage at Arg306, generating M(r) 75,000 and M(r) 30,000 fragments, respectively. In addition, the appearance of two novel products derived from the factor Va HC are observed when thrombin is present on the HUVEC surface: the HC is processed through limited thrombin proteolysis to generate an M(r) 97,000 fragment, which is further processed by APC to generate an M(r) 43,000 fragment. NH2-terminal sequence analysis of the M(r) 97,000 fragment revealed that the thrombin cleavage occurs in the COOH-terminus of the intact factor Va HC since both the intact HC as well as the M(r) 97,000 fragment have the same sequence. Our data demonstrate that the inactivation of factor Va on the HUVEC surface, initiated either by APC addition or PC activation, follows a mechanism whereby cleavage is observed first at Arg506 followed by a second cleavage at Arg306. The latter cleavage is dependent on the availability of the HUVEC surface. This mechanism of inactivation of factor Va is similar to that observed on synthetic phospholipid vesicles.
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PMID:Protein C activation and factor Va inactivation on human umbilical vein endothelial cells. 940 54

The structure of two selective inhibitors, Ac-Tyr-Ile-Arg-Ile-Pro-NH2 and Ac-(4-Amino-Phe)-(Cyclohexyl-Gly)-Arg-NH2, in the active site of the blood clotting enzyme factor Xa was determined by using transferred nuclear Overhauser effect nuclear magnetic resonance (NMR) spectroscopy. They represent a family of peptidic inhibitors obtained by the screening of a vast combinatorial library. Each structure was first calculated by using standard computational procedures (distance geometry, simulated annealing, energy minimization) and then further refined by systematic search of the conformation of the inhibitor docked in the active site and repeating the simulated annealing and energy minimization. The final structure was optimized by molecular dynamics simulations of the inhibitor-complex in water. The NMR restraints were kept throughout the refinement. The inhibitors assume a compact, very well defined conformation, embedded into the substrate binding site not in the same way as a substrate, blocking thus the catalysis. The model allows to explain the mode of action, affinity, and specificity of the peptides and to map the active site.
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PMID:Mapping the active site of factor Xa by selective inhibitors: an NMR and MD study. 951 42


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