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:3.4.21.5 (thrombin)
33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Resistance to the anticoagulant action of activated protein C, APC resistance, is a highly prevalent risk factor for venous thrombosis among individuals of Caucasian origin. In most cases, APC resistance is associated with a single missense mutation in the gene for coagulation factor V (FV (Leiden)), which predicts the replacement of Arg (506) with a Gln at one of the cleavage sites for APC in factor V. Factor V is a Janus-faced protein with dual functions, serving as an essential nonenzymatic cofactor in both pro- and anticoagulant pathways. Procoagulant factor Va, generated after proteolysis by thrombin or factor Xa, is a cofactor to factor Xa in the activation of prothrombin, whereas anticoagulant factor V, generated after proteolysis by APC, functions as a cofactor in the APC-mediated degradation of FVIIIa. The FV (Leiden) mutation affects the anticoagulant response to APC at two distinct levels of the coagulation pathway, as it impairs degradation of both activated factor V and activated factor VIII, the latter effect inasmuch as FVLeiden is a poor APC cofactor. Several other genetic traits, some of them quite common, are known to affect the anticoagulant response to APC, but none of them cause the same severe APC-resistance phenotype as FV (Leiden) and their importance as risk factors for thrombosis is unclear. A poor APC response may also result from acquired conditions, some of which are clearly involved in the pathogenesis of venous thrombosis. Venous thrombosis is a typical multifactorial disease, the pathogenesis of which involves multiple gene-gene and gene-environment interactions. In many patients with severe thrombophilia, APC resistance is found as a contributing risk factor.
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PMID:Congenital and acquired activated protein C resistance. 1519 91

Thrombin derived from bovine sources commonly is used to arrest bleeding during surgical procedures. However, complications such as postoperative hemorrhage can occur because of the development of cross-reactive anti-bovine antibodies that inhibit human coagulation factor V. It would thus be advantageous to develop techniques to generate human thrombin. This study evaluated thrombin produced from human plasma using a new Thrombin-Processing Device (TPD). Plasma was introduced into the TPD, mixed with an ethanol/ CaCl2 reagent, incubated for 1 h, and the harvested thrombin was assayed for activity and the ability to activate platelets by in vitro assays. TPD-produced thrombin activity was found to be 51.8 +/- 12.4 IU/mL (n = 145). TPD-produced thrombin also stimulated P-selectin (CD62) expression (83 +/- 13% of the platelet population) and Annexin V binding (10.3 +/- 2% of the platelet population) on platelets in a similar fashion to commercial thrombin (P-selectin expression: 88 +/- 3%; Annexin-V binding: 11.4 +/- 3%). Compared with CaCl2 and batroxobin, TPD-produced thrombin had a significantly greater ability to activate platelets. TPD-produced thrombin from human plasma has consistent activity and significantly activates platelets and, thus, may have attractive applications such as the production of autologous thrombin for surgical patients.
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PMID:Quality of thrombin produced from the patient's own plasma using the TPD, a new Thrombin-processing Device. 1611 59

Activated coagulation factor V (FVa) functions as a cofactor to factor Xa (FXa) in the conversion of prothrombin (PT) to thrombin. This essential procoagulant reaction, despite being the subject of extensive investigation, is not fully understood structurally and functionally. To elucidate the structure of the FXa-FVa complex, we have performed protein:protein (Pr:Pr) docking simulation with the pseudo-Brownian Pr:Pr docking ICM package and with the shape-complementarity Pr:Pr docking program PPD. The docking runs were carried out using a new model of full-length human FVa and the X-ray structure of human FXa. Five representative models of the FXa-FVa complex were in overall agreement with some of the available experimental data, but only one model was found to be consistent with almost all of the reported experimental results. The use of hybrid docking approach (theoretical plus experimental) is definitively important to study such large macromolecular complexes. The FXa-FVa model we have created will be instrumental for further investigation of this macromolecular system and will guide future site directed mutagenesis experiments.
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PMID:Proposed structural models of the prothrombinase (FXa-FVa) complex. 1643 49

Blood coagulation factor V (FV) is a multifunctional protein that circulates in human plasma as a precursor molecule which can be activated by thrombin or activated factor X (FXa) in order to express its cofactor activity in prothrombin activation. FV activation is achieved by limited proteolysis after Arg709, Arg1018, and Arg1545 in the FV molecule. The venoms of Daboia russelli and Daboia lebetina contain a serine protease that specifically activates FV by a single cleavage at Arg1545. We have predicted the three-dimensional structure of these enzymes using comparative protein modeling techniques. The plasminogen activator from Agkistrodon acutus, which shows a high degree of homology with the venom FV activators and for which a high-quality crystallographic structure is available, was used as the molecular template. The RVV-V and LVV-V models provide for the first time a detailed and accurate structure of a snake venom FV activator and explain the observed sensitivity or resistance toward a number of serine protease inhibitors. Finally, electrostatic potential calculations show that two positively charged surface patches are present on opposite sides of the active site. We propose that both FV activators achieve their exquisite substrate specificity for the Arg1545 site via interactions between these exosites and FV.
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PMID:Structural models of the snake venom factor V activators from Daboia russelli and Daboia lebetina. 1680 18

Fish oil, containing (n-3) PUFA, is associated with a moderate reduction in cardiovascular disease through a multifactorial mechanism involving a decrease in plasma lipids and anticoagulant activity. Two intervention studies on subjects at risk were performed to determine the relation of these 2 fish-oil effects. In study 1, 54 overweight subjects consumed 3.1 g (n-3) PUFA daily. In study 2, which involved 42 overweight patients with type 2 diabetes, 20 subjects consumed (n-3) PUFA, whereas 22 others ingested a preparation rich in (n-6) PUFA. Tissue factor-induced thrombin generation (thrombin potential) was determined as an integrated measure of plasma coagulant activity. In both studies, multivariate analysis indicated a strong clustering of fasting concentrations of triacylglycerols, prothrombin, factor V, factor VII, and factor X with one another at baseline. This cluster of factors determined partly the interindividual variation in thrombin generation, of which prothrombin and triacylglycerol concentrations were the main determinants. In both healthy subjects and diabetes patients, high triacylglycerol concentrations (>1.69 mmol/L) at baseline were closely linked to a strong fish oil-induced lowering of triacylglycerol and coagulation factor V, VII, and X concentrations, and thrombin generation. We conclude that high fasting triacylglycerol concentrations predict high procoagulant activity and a lowering of thrombin potential with dietary fish oil.
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PMID:Plasma triacylglycerol and coagulation factor concentrations predict the anticoagulant effect of dietary fish oil in overweight subjects. 1718 93

Blood coagulation factor V circulates as a procofactor with little or no procoagulant activity. It is activated to factor Va by thrombin following proteolytic removal of a large central B-domain. Although this reaction is well studied, the mechanism by which bond cleavage and B-domain release facilitate the transition to the active cofactor state has not been defined. Here we show that deletion or substitution of specific B-domain sequences drives the expression of procoagulant function without the need for proteolytic processing. Conversion to the constitutively active cofactor state is related, at least in part, to a cluster of amino acids that is highly basic and well conserved across the vertebrate lineage. Our findings demonstrate that discrete sequences in the B-domain serve to stabilize the inactive procofactor state, with proteolysis primarily functioning to remove these inhibitory constraints. These unexpected results provide new insight into the mechanism of factor V activation.
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PMID:Inhibitory sequences within the B-domain stabilize circulating factor V in an inactive state. 1738 73

Neutrophils and monocytes express cathepsin G and can also bind to activated platelets, thus they can be localized to the site of active coagulation. Previous studies have suggested that cathepsin G inactivated coagulation factor VIII (FVIII) and was thus anticoagulant. But other studies have indicated procoagulant functions for cathepsin G in activation of coagulation factor V or activation of platelets among other possible mechanisms. Therefore, it remains unclear if cathepsin G is anticoagulant or procoagulant. We investigated the effects of human neutrophil cathepsin G on FVIII/VIIIa. Cathepsin G activates FVIII to a partially active form while having only a minor inactivating effect on thrombin-activated FVIIIa. This inactivation is mostly due to decreased stability of FVIIIa since a disulfide bond that prevents A2 subunit dissociation from FVIIIa prevents any loss of activity due to cathepsin G proteolysis. FVIII that has been cleaved by cathepsin G can still be activated by thrombin if A2 subunit dissociation is prevented. Cathepsin G cleavages of FVIII are limited to a few specific sites that are mostly located near known activating and inactivating cleavage sites. Cathepsin G cleavage sites near to thrombin cleavage sites likely contribute to the partial activation of FVIII. Therefore, it is possible that cathepsin G from neutrophils and monocytes may provide some pro-coagulant effect by activating FVIII.
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PMID:Cathepsin G, a leukocyte protease, activates coagulation factor VIII. 1821 33

Activated coagulation factor V (FVa) is an important cofactor that accelerates thrombin production. In human blood, 25% of the factor V (FV) is stored in platelets, complexed to the polymeric, FV binding protein multimerin 1 (MMRN1). The light chain of FV is required for MMRN1 binding, and its C2 domain contains a MMRN1 binding site that overlaps phospholipid binding residues essential for FVa procoagulant function. The homologous structures and roles of the FVa light chain C1 and C2 domains led us to investigate if the C1 domain also contains a MMRN1 binding site. The MMRN1 binding properties of FV constructs were tested by modified enzyme-linked immunoassays, before and after thrombin activation. The constructs tested included the combined C1 and C2 domain deleted FV, and B-domain deleted forms of FV containing C1 domain point mutations or combined C1 and C2 domain phospholipid binding site mutations. The MMRN1 binding site in FV/FVa was mapped to a large region that included the C1 domain phospholipid binding residues Y1956 and L1957. The FV construct with combined C1 and C2 domain phospholipid binding site mutations had no MMRN1 binding, highlighting the critical role of the FV C1 and C2 domain phospholipid binding residues in MMRN1 binding. Our data update the information on the structural features of FV and FVa important for MMRN1 binding, and suggest that the extended MMRN1 binding site in the C1 and C2 domains is important for the storage of FV-MMRN1 complexes in platelets.
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PMID:Location of the multimerin 1 binding site in coagulation factor V: an update. 1845 76

Activated coagulation factor V functions as a cofactor to factor Xa in the conversion of prothrombin to thrombin. Based on the introduction of extra carbohydrate side chains in recombinant factor V, we recently proposed several regions in factor Va to be important for factor Xa binding. To further define which residues are important for factor Xa binding, we prepared fifteen recombinant factor V variants in which clusters of charged amino acid residues were mutated, mainly to alanines. The factor V variants were expressed in COS-1 cells, and their functional properties evaluated in a prothrombinase-based assay, as well as in a direct binding test. Four of the factor V variants, 501A/510A/511D, 501A/510A/511D/513A, 513A/577A/578A, and 501A/510A/511D/513A/577A/578A exhibited markedly reduced factor Xa-cofactor activity tested in the prothrombinase assay, and reduced binding affinity as judged by the direct binding assay. These factor Va variants were normally cleaved at Arg-506 by activated protein C, and the interaction between the factor Xa-factor Va complex and prothrombin was unaffected by the introduced mutations. Based on the integration of all available data, we propose a key factor Xa binding surface to be centered on Arg-501, Arg-510, Ala-511, Asp-513, Asp-577, and Asp-578 in the factor Va A2 domain. These residues form an elongated charged factor Xa binding cluster on the factor Va surface.
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PMID:Mapping of the factor Xa binding site on factor Va by site-directed mutagenesis. 1850 57

Human coagulation factor V (FV), a non-enzymatic cofactor of the prothrombinase complex, is required for the rapid generation of thrombin. FV deficiency is a rare autosomal recessive bleeding disorder. We describe two novel mutations, Tyr91Asn and Asp2098Tyr, found in two probands with a residual FV activity of 51% and 4%, respectively. Modelling and structural analysis of these mutations were performed following short-duration molecular dynamics (MD) simulation. Asp2098Tyr lead to abolishment of the highly conserved salt bridge Asp2098-Arg2171 presumably required for structural integrity of the C2 domain. MD studies suggest that additional conformational changes resulting from this mutation involve local rearrangements at Tyr2063 and Tyr2064 and so affect the phospholipid-membrane binding. MD modelling of the Try91Asn mutant revealed a conformational change nearby the Cu(2+) binding site that could affect overall stabilization of the heavy and light chains. These findings suggest that both mutations influence the structural integrity of FV protein. Transient expression data of wild-type and mutant FV variants in 293T human embryonic kidney cells showed FV-specific activity reduced to 26% for Asp2098Tyr and 56% for Tyr91Asn compared to that of wild-type. Thus, both the data from the short duration molecular dynamic simulation and from expression analysis indicate alterations of the FV protein variants that explain the clinical phenotype.
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PMID:Modelling and expression studies of two novel mutations causing factor V deficiency. 1898 19


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