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1
Davis-Harrison, Rebecca L., Narjes Tavoosi, Mary Clay, John M. Boettcher, Chad M. Rienstra, and James H. Morrissey. "Structural Insights Into How Clotting Proteins with GLA Domains Bind to Membrane Surfaces." Blood 116, no. 21 (November 19, 2010): 1141. http://dx.doi.org/10.1182/blood.v116.21.1141.1141.
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Abstract Abstract 1141 Most steps in the blood coagulation cascade obligatorily take place on membrane surfaces and are dependent on the exposure of phosphatidylserine (PS). Many coagulation proteins bind to PS-containing membrane bilayers in a calcium-dependent manner via gamma-carboxyglutamate-rich (GLA) domains. In spite of their importance, a clear picture of how GLA domains bind to the membrane interface has yet to emerge. A further intriguing aspect of the membrane's role in blood coagulation is that certain phospholipids, most notably phosphatidylethanolamine (PE), strongly synergize with PS to promote clotting reactions. The mechanisms of this synergy, and of PE's contribution to GLA domain binding, are poorly understood – although a number of hypotheses have been put forward. We now propose a new hypothesis to explain GLA domain binding to membranes, which we term the ABC (Anything But Choline) hypothesis; it invokes two main types of protein-phospholipid interactions: a single L-serine-specific binding site in each GLA domain; and multiple “phosphate-specific” interactions in which the phosphate groups of non-phosphatidylcholine phospholipids form coordination complexes with the tightly bound calcium ions in GLA domains. We have utilized liposomes and nanoscale phospholipid bilayers (Nanodiscs) in studies employing a series of techniques including solid-state NMR (SSNMR) and surface plasmon resonance (SPR) to address the mechanism of GLA domain-membrane interactions. We provide direct evidence in favor of the ABC hypothesis for GLA domain binding to membrane surfaces. Using SSNMR, we demonstrate that two distinct PS headgroup conformations are induced by binding of calcium ions, and that a third, novel PS headgroup conformation is induced when the prothrombin GLA domain engages the membrane. SPR studies have allowed for the determination of thermodynamic profiles of GLA domains interacting with phospholipid bilayers containing PS and/or PE, providing further insights to the mechanisms of GLA domain-membrane interactions. Disclosures: No relevant conflicts of interest to declare.
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Majumder, Rinku, Judson Craft, Alireza Rezaie, R. Barry, and Dougald Monroe. "Role of Gamma-Carboxyglutamic Acid (GLA) Domain in Phosphatidylserine (PS)-Regulated Activity of Factor IXa." Blood 110, no. 11 (November 16, 2007): 2696. http://dx.doi.org/10.1182/blood.v110.11.2696.2696.
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Abstract Both factors Xa and IXa are vitamin-K-dependent serine proteases that consist of disulfide-linked heavy and light chains. The heavy chain contains the serine protease active site, while the light chain contains the GLA (gamma-carboxyglutamic acid) domain, EGF-I (epidermal growth factor-like region) and EGF-II domains. Effect of PS on FXa: We have extensively studied the interaction of factor Xa with 1, 2-dicaproyl-sn-glycero-3-phospho-L-serine (C6PS). C6PS is a soluble analogue of phosphatidylserine, which is present in platelet membranes; it serves as a model for the effect of membrane-bound PS on factor Xa activity and structure. We located three C6PS binding sites to different domains of factor Xa using a combination of activity, circular dichroism, fluorescence, and equilibrium dialysis measurements, showing that : the Gla domain binds C6PS only in the absence of calcium (kd ∼ 1 mM); a calcium-dependent, regulatory, PS-binding site exists in the EGFN domain when linked by calcium to the Gla domain; and a second calcium-requiring site exists in the EGFC-catalytic domains and shares residues with the substrate recognition site. We have shown that full functional response to C6PS requires linkage of the Gla, EGFNC, and catalytic domains in the presence of calcium. Recently we have observed that the proteolytic activity of des EGFN factor Xa is not affected in the presence of C6PS, locating more precisely a significant component of the PS-triggered regulatory site to the EGFN domain. Efect of PS on FIXa: We have previously reported that C6PS induces a calcium-dependent conformational change in factor IXa that regulates the amidolytic and proteolytic activities. We have also shown that factor IXa binds 2 molecules of C6PS. Here we have examined the role of the GLA domain in this C6PS regulation of factor IXa using a GLA-domainless variant of factor IXa (GD-IXa). In the absence of the GLA domain, binding to C6PS has no effect on the rate of proteolytic or amidolytic activity of GD-IXa toward both factor X and synthetic substrates. The binding of C6PS to GD-IXa studied using intrinsic tryptophan fluorescence (Kd = ∼50μM) was different from that for native factor IXa (Kd = ∼2μM). The critical micelle concentration (CMC) of C6PS under the conditions of these experiments (∼300μM) was much greater than the C6PS concentrations used in our experiments. We conclude that: C6PS does bind to GD-IXa, but at a reduced affinity compared to factor IXa; C6PS does not regulate the amidolytic or proteolytic activities of factor IXa in the absence of GLA domain. This could be due to two C6PS sites existing in FIXa, with the regulatory site requiring the Gla domain (as observed for FXa), or to a requirement for the Gla domain for the regulatory activity of a single site. Future studies looking at the stoichiometry of binding of C6PS with GD-IXa will help distinguish between these possibilities. Supported by Supported by NHLBI (HL 072827).
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Geng, Jie-Ping, and Francis J. Castellino. "The Propeptides of Human Protein C, Factor VII, and Factor IX Are Exchangeable with Regard to Directing Gamma-Carboxylation of these Proteins." Thrombosis and Haemostasis 76, no. 02 (1996): 205–7. http://dx.doi.org/10.1055/s-0038-1650555.
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SummaryThe specificity of the propeptide sequence in directing vitamin Independent post-translational γ-carboxylation has been assessed by examination of the extent of processing of chimeric constructs of blood coagulation factor VII (fVII), factor IX (fIX) and protein C (PC). One chimera consisted of a protein in which the γ-carboxyglutamic acid (Gla)/helical stack domain of PC (amino acid residues 1 to 46) was replaced by that of fIX (residues 1 to 47) in an otherwise intact PC. Another consisted of the same construction of a fVII/PC Gla domain-based mutant protein. The final chimera contained the leader/propeptide sequence of PC (residues -42 to -1) replaced by that of fIX (residues -46 to -1). In each case, all Glu-precursor Gla residues in the Gla domains of the proteins were fully processed to Gla. These results demonstrate that the propeptides of fIX and PC are capable of directing γ-carboxylation of the Gla regions of either protein, that the propeptide of PC can fully function in γ-carboxylation of the Gla region of fVII, and further suggest that, with regard to γ-carboxylation, communications between the propeptides and Gla domains in intact proteins are general in nature.
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Allan, J. A., A. J. P. Docherty, P. J. Barker, N. S. Huskisson, J. J. Reynolds, and G. Murphy. "Binding of gelatinases A and B to type-I collagen and other matrix components." Biochemical Journal 309, no. 1 (July 1, 1995): 299–306. http://dx.doi.org/10.1042/bj3090299.
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Matrix sequestration of matrix metalloproteinases may be important for the facilitation of remodelling events and the migration of cells through the extracellular matrix. Using an ELISA technique we studied the ability of pro and active forms of gelatinases A and B (GLA and GLB) to bind to matrix components and the contribution made by the different enzyme domains. Pro and active forms of GLA and GLB bound to type-I and type-IV collagens, gelatin and laminin films. Binding to collagens occurred exclusively via the N-terminal portion of the molecule in both of the gelatinases; deletion of the fibronectin-like domain in GLA abolished binding. Fibronectin was shown to compete with GLA, confirming that binding occurs through this domain. GLA and GLB competed for binding to collagen type I, whereas collagenase and stromelysin bound to different sites and could be co-localized with the gelatinases. We conclude that gelatinases have different binding specificities from those previously documented for stromelysin and collagenase, which bind through their C-terminal domains to collagen fibrils.
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Gopinath, Subash C. B., Yasuo Shikamoto, Hiroshi Mizuno, and Penmetcha K. R. Kumar. "Snake-venom-derived Factor IX-binding protein specifically blocks the γ-carboxyglutamic acid-rich-domain-mediated membrane binding of human Factors IX and X." Biochemical Journal 405, no. 2 (June 27, 2007): 351–57. http://dx.doi.org/10.1042/bj20061737.
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A potent anticoagulant protein, IX-bp (Factor IX binding protein), has been isolated from the venom of Trimeresurus flavoviridis (habu snake) and is known to bind specifically to the Gla (γ-carboxyglutamic acid-rich) domain of Factor IX. To evaluate the molecular basis for its anticoagulation activity, we assessed its interactions with various clotting factors. We found that the anticoagulation activity is primarily due to binding to the Gla domains of Factors IX and X, thus preventing these factors from recognizing phosphatidylserine on the plasma membrane. The present study suggests that ligands that bind to the Gla domains of Factors IX and X may have the potential to become novel anticoagulants.
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Szymanski, D. B., R. A. Jilk, S. M. Pollock, and M. D. Marks. "Control of GL2 expression in Arabidopsis leaves and trichomes." Development 125, no. 7 (April 1, 1998): 1161–71. http://dx.doi.org/10.1242/dev.125.7.1161.
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More than twenty genes are required for the correct initiation, spacing, and morphogenesis of trichomes in Arabidopsis. The initial selection of trichome precursors requires the activity of both the GLABROUS1 (GL1) and TRANSPARENT TESTA GLABROUS (TTG) genes. The GLABRA2 (GL2) gene is required for subsequent phases of trichome morphogenesis such as cell expansion, branching, and maturation of the trichome cell wall. Previous studies have shown that GL2 is a member of the homeodomain class of transcription factors. Here we report a detailed analysis of GL2 expression in the shoot using anti-GL2 antibodies and the GUS reporter gene fused to the GL2 promoter. The GL2 expression profile in the shoot is complex, and involves spatial and temporal variation in developing leaves and trichomes. Two separate promoter domains that are expressed in trichomes were identified. GL2, like GL1, is expressed in developing trichomes and in cells surrounding trichomes during early stages of trichome development. Unlike GL1, GL2 expression persists in mature trichomes. It was found that while GL1 and TTG were not required for the initiation of GL2 expression in the non-trichome cells, the presence of a functional GL1 or TTG gene was able to increase GL2 expression in these cells compared to ttg gl1 plants. The hypothesis that GL1 regulates aspects of GL2 expression is consistent with epistatic analysis of gl1 and gl2 and the expression patterns of GL1 and GL2. In support of this hypothesis, it was found that ectopic expression of GL1 in the presence of ectopic expression of the maize R gene, which can bypass the requirement for TTG, can ectopically activate GL2 transcription.
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Im, U., and M. Kanakidou. "Impacts of East Mediterranean megacity emissions on air quality." Atmospheric Chemistry and Physics 12, no. 14 (July 23, 2012): 6335–55. http://dx.doi.org/10.5194/acp-12-6335-2012.
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Abstract. Megacities are large urban agglomerations with intensive anthropogenic emissions that have significant impacts on local and regional air quality. In the present mesoscale modeling study, the impacts of anthropogenic emissions from the Greater Istanbul Area (GIA) and the Greater Athens Area (GAA) on the air quality in GIA, GAA and the entire East Mediterranean are quantified for typical wintertime (December 2008) and summertime (July 2008) conditions. They are compared to those of the regional anthropogenic and biogenic emissions that are also calculated. Finally, the efficiency of potential country-based emissions mitigation in improving air quality is investigated. The results show that relative contributions from both cities to surface ozone (O3) and aerosol levels in the cities' extended areas are generally higher in winter than in summer. Anthropogenic emissions from GIA depress surface O3 in the GIA by ~ 60% in winter and ~ 20% in summer while those from GAA reduce the surface O3 in the GAA by 30% in winter and by 8% in summer. GIA and GAA anthropogenic emissions contribute to the fine particulate matter (PM2.5) levels inside the cities themselves by up to 75% in winter and by 50% (GIA) and ~ 40% (GAA), in summer. GIA anthropogenic emissions have larger impacts on the domain-mean surface O3 (up to 1%) and PM2.5 (4%) levels compared to GAA anthropogenic emissions (<1% for O3 and ≤2% for PM2.5) in both seasons. Impacts of regional anthropogenic emissions on the domain-mean surface pollutant levels (up to 17% for summertime O3 and 52% for wintertime fine particulate matter, PM2.5) are much higher than those from Istanbul and Athens together (~ 1% for O3 and ~ 6% for PM2.5, respectively). Regional biogenic emissions are found to limit the production of secondary inorganic aerosol species in summer up to 13% (non-sea-salt sulfate (nss-SO42−) in rural Athens) due to their impact on oxidant levels while they have negligible impact in winter. Finally, the responses to country-based anthropogenic emission mitigation scenarios inside the studied region show increases in O3 mixing ratios in the urban areas of GIA and GAA, higher in winter (~ 13% for GIA and 2% for GAA) than in summer (~ 7% for GIA and <1% for GAA). On the opposite PM2.5 concentrations decrease by up to 30% in GIA and by 20% in GAA with the highest improvements computed for winter. The emission reduction strategy also leads to domain-wide decreases in most primary pollutants like carbon monoxide (CO) or nitrogen oxides (NOx) for both seasons. The results show the importance of long range transport of pollutants for the air quality in the East Mediterranean. Thus, improvements of air quality in the East Mediterranean require coordinated efforts inside the region and beyond.
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Sun, Yong-Hui, Lei Shen, and Björn Dahlbäck. "Gla domain–mutated human protein C exhibiting enhanced anticoagulant activity and increased phospholipid binding." Blood 101, no. 6 (March 15, 2003): 2277–84. http://dx.doi.org/10.1182/blood-2002-06-1691.
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Protein C is a member of the vitamin K– dependent protein family. Proteins in this family have similar γ-carboxyglutamic acid (Gla)–rich domains, but their affinities for negatively charged phospholipid membranes vary more than 1000-fold. We have shown that it is possible to enhance anticoagulant activity and membrane affinity of protein C by selective mutagenesis of the Gla domain. In this study, 3 new mutants, Q10G11N12 (QGN), S23E32D33Y44 (SEDY), and Q10G11N12S23E32D33Y44 (QGNSEDY), were created. In plasma-based coagulation assays, the activated form of QGNSEDY (QGNSEDY-APC) demonstrated approximately 20-fold higher anticoagulant activity than wild-type activated protein C (WT APC), while QGN-APC and SEDY-APC did not. Both normal activated factor V (FVa) and FVa Leiden (Arg506Gln) were degraded much more efficiently by QGNSEDY-APC than by WT APC in the presence as well as in the absence of protein S. Binding of protein C variants to negatively charged phospholipid membranes was investigated using light scattering and the BIAcore technique. QGNSEDY demonstrated 3- to 7-fold enhanced binding as compared with WT protein C, suggesting the membrane affinity to be influenced by several residues located at different parts of the Gla domain. The anticoagulant activity as well as phospholipid binding ability was only enhanced when multiple regions of the Gla domain were modified. The results provide insights into the molecular mechanisms that are involved in determining the binding affinity of the interaction between Gla domains and phospholipid membranes. The unique properties of QGNSEDY-APC suggest this APC variant possibly to have greater therapeutic potential than WT APC.
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Chen, Shu-wen, Jean-François Schved, Jean-Luc Pellequer, and Muriel Giansily-Blaizot. "Model of a Ternary Complex between Activated Factor VII, Tissue Factor and Factor IX." Thrombosis and Haemostasis 88, no. 07 (2002): 74–82. http://dx.doi.org/10.1055/s-0037-1613157.
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SummaryUpon binding to tissue factor, FVIIa triggers coagulation by activating vitamin K-dependent zymogens, factor IX (FIX) and factor X (FX). To understand recognition mechanisms in the initiation step of the coagulation cascade, we present a three-dimensional model of the ternary complex between FVIIa:TF:FIX. This model was built using a full-space search algorithm in combination with computational graphics. With the known crystallographic complex FVIIa:TF kept fixed, the FIX docking was performed first with FIX Gla-EGF1 domains, followed by the FIX protease/EGF2 domains. Because the FIXa crystal structure lacks electron density for the Gla domain, we constructed a chimeric FIX molecule that contains the Gla-EGF1 domains of FVIIa and the EGF2-protease domains of FIXa. The FVIIa:TF:FIX complex has been extensively challenged against experimental data including site-directed mutagenesis, inhibitory peptide data, haemophilia B database mutations, inhibitor antibodies and a novel exosite binding inhibitor peptide. This FVIIa:TF:FIX complex provides a powerful tool to study the regulation of FVIIa production and presents new avenues for developing therapeutic inhibitory compounds of FVIIa:TF:substrate complex.
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Davis-Harrison, Rebecca L., Narjes Tavoosi, Vincent S. Pureza, and James H. Morrissey. "Phospholipid Synergy in Prothrombinase Activity." Blood 118, no. 21 (November 18, 2011): 1175. http://dx.doi.org/10.1182/blood.v118.21.1175.1175.
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Abstract Abstract 1175 Most steps in the blood coagulation cascade obligatorily take place on membrane surfaces and are dependent on the exposure of phosphatidylserine (PS). Previous studies from our lab and others have shown that phosphatidylethanolamine (PE) poorly supports clotting reactions by itself, but strongly synergizes with PS to promote several membrane-dependent steps in the blood clotting cascade, although the mechanism for PE-PS synergy has been unclear. We recently put forward a new mechanistic explanation – which we termed the ABC or Anything But Choline hypothesis – for how PS and PE synergize to enhance factor X (fX) activation by the factor VIIa-tissue factor complex (Tavoosi et al., J. Biol. Chem. 286:23247–53, 2011). The membrane contribution to this reaction is dominated by the affinity of fX for the membrane surface; since fX binds to membranes via its gamma-carboxyglutamate-rich (GLA) domain, the ABC hypothesis therefore focuses on the mechanisms by which GLA domains engage the phospholipid bilayer. We identified two main types of GLA domain-phospholipid interactions: a single phospho-L-serine-specific binding site in each GLA domain; and multiple ”phosphate-specific” interactions in which the phosphate groups of non-phosphatidylcholine phospholipids form coordination complexes with the tightly bound calcium ions in GLA domains. In the current study, we test the ABC hypothesis in the context of the prothrombinase complex – i.e., activation of prothrombin by the membrane-bound complex of fXa and factor Va (fVa). Using a variety of approaches including surface plasmon resonance analyses, we measured the contributions of varying phospholipid compositions to the membrane binding affinities of fXa, fVa and prothrombin, as well as to the enzymatic activity of prothrombinase. Our results suggest that phospholipid synergy in prothrombinase activity differs in certain respects from that observed for the factor VIIa-tissue factor complex. Not only did PS synergize with PE for enhancing the activity of prothrombinase, but phosphatidylglycerol (PG) and phosphatidylacid (PA) also synergized with PE, albeit more weakly than with PS (i.e., significantly higher levels of PG or PA in the presence of PE were required to achieve prothrombinase activities comparable to mixtures of PS and PE). In contrast, PE failed to synergize with either PG or PA to support fX activation by the factor VIIa-tissue factor complex. These differences primarily arise from differential membrane binding of the substrates for these two complexes (fX for factor VIIa-tissue factor and prothrombin for prothrombinase). The data suggest that the phospho-L-serine-specific binding site in the GLA domain of prothrombin may not be as stringent as that of fX, as high levels of PG or PA can substitute for PS in membrane binding of prothrombin but not for fX. This study provides further insights into the membrane's role in regulating blood clotting reactions, specifically the binding interactions between GLA domains and membrane surfaces. Disclosures: No relevant conflicts of interest to declare.
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Gilbert, Gary E., Anu Bhimavarapu, Patricia Price, and Marc Jacquemin. "Antibody to C1 Domain of Factor VIII Alters Interaction of Factor Xase Complex with Factor X." Blood 104, no. 11 (November 16, 2004): 1738. http://dx.doi.org/10.1182/blood.v104.11.1738.1738.
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Abstract The role of the C1 domain in function of factor VIII has not been clearly defined. In contrast, functional interactions have been identified for the three A domains and the C2 domain. We hypothesized that the C1 domain of factor VIII participates in both phospholipid binding and interaction with factor X and/or factor IXa. We evaluated inhibition of the factor Xase complex by LE2E9, a human inhibitor IgG4k mAb against C1. We utilized altered catalytic activity of the factor Xase complex in a defined assay to report the inhibition by LE2E9. Inhibition by LE2E9 was also evaluated when soluble phosphatidylserine replaced vesicles to support the factor Xase complex and when Gla-domainless factor X was the substrate. The deglycosylated form of LE2E9 was also evaluated to better define the mechanism through which LE2E9 exerts its effect. We found that LE2E9 bound to factor VIIIa with an apparent KD of 0.5 nM. The apparent affinity of factor VIIIa for sonicated phospholipid vesicles of phosphatidylserine:phosphatidylethanolamine:phosphatidylcholine 4:20:76 increased 3-fold in the presence of LE2E9. The apparent affinity of factor VIIIa for factor IXa was not significantly changed. The KM of the factor VIIIa-factor IXa complex was 20 ± 2 nM with LE2E9 vs. 40 ± 2 nM without. LE2E9 decreased the Vmax by 77 ± 6% indicating that the affinity of factor X for the factor Xase complex is increased while the rate of cleavage is decreased. When Gla-domainless factor X was used as the substrate for the factor Xase complex, LE2E9 did not inhibit activity indicating that inhibition occurs via an interaction that involves the factor X Gla domain. When the factor VIIIa-factor IX complex was supported by dihexanoyl phosphatidylserine rather than phospholipid vesicles the inhibition of Vmax was 47% indicating that the inhibitory effect does not require a phospholipid bilayer. Deglycosylated LE2E9 did not significantly change the KM but decreased the Vmax by 22% while both antibodies bound to factor VIII with the same affinity. These results suggest that LE2E9 inhibition relates largely to interaction of a carbohydrate moiety with factor VIII or factor X rather than binding the core C1 epitope. We conclude that LE2E9 decreases the KM, and the Vmax for the factor VIIIa-factor IXa complex, but only when the factor X Gla domain is present. These results suggest that in the factor Xase complex the C1 domain of factor VIII is intimately associated with the Gla domain of factor X and that interaction between these domains enhances the kcat for the factor VIIIa-factor IXa complex.
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Dayer, Mohammad Reza, Omid Ghayour, and Mohammad Saaid Dayer. "Mechanism of Protein-Z-Mediated Inhibition of Coagulation Factor Xa by Z-Protein-Dependent Inhibitor: A Molecular Dynamic Approach." ISRN Hematology 2012 (March 20, 2012): 1–8. http://dx.doi.org/10.5402/2012/762728.
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Protein Z is a plasma protein functioning as a carrier for ZPI. Protein Z also accelerates inhibitory effect of ZPI on factor Xa by 1000-fold. Inhibition of coagulation cascade via FXa by ZPI and other serpins is very important safety factor for normal homeostasis protecting human life against unwanted thrombosis. In the present work using native structure of PZ, ZPI, FXa and in a dynamic simulation, using NAMD software, the ternary complex was studied in an up to 10 nanoseconds protocol. Rely on trajectory analyses, we postulated that PZ binds ZPI by using its SP-like domain and through noncovalent forces. PZ then transfers ZPI through-out the blood, and by using its GLA domain and a bivalent cation of calcium, PZ binds to phospholipid bilayers (e.g., platelet) where the FXa is preallocated. In case of PZ-ZPI binding to plasma membrane, a series of complementary interactions take place between FXa, and PZ-ZPI complex including interactions between RCL loop of ZPI and catalytic site of FXa and some take place between long arm of PZ (composed of GLA, EGF1, and EGF2 domains) and GLA domain of FXa. In our claim these complementary interactions lead PZ to bind correctly to prelocated FXa.
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Vadivel, Kanagasabai, Sayeh Agah, Amanda S. Messer, Duilio Cascio, Madhu S. Bajaj, Sriram Krishnaswamy, Charles T. Esmon, Kaillathe Padmanabhan, and S. Paul Bajaj. "Mg2+ Is Required for Optimal Folding of the γ-Carboxyglutamic Acid (Gla) Domains of Vitamin K-Dependent Clotting Factors At Physiological Ca2+." Blood 118, no. 21 (November 18, 2011): 1172. http://dx.doi.org/10.1182/blood.v118.21.1172.1172.
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Abstract Abstract 1172 Equilibrium dialysis experiments indicate that the Gla domain of coagulant protein factor (F) VIIa and of anticoagulant protein C (PC) each bind seven Ca2+ in the absence and four in the presence of physiologic Mg2+ (0.6 mM). The previous x-ray structure of FVIIa/soluble (s) tissue factor (TF) in the presence of 5 mM Ca2+/45 mM Mg2+ (5Ca/45Mg) (Bajaj et al., J Biol Chem, 281, 24873–24888, 2006) revealed that sites 2,3,5 and 6 were occupied by Ca2+ while sites 1, 4 and 7 contained Mg2+ (Tulinsky numbering, Biochemistry 31, 2554–2566, 1992). Since these concentrations of metal ions were supraphysiologic, we sought to obtain crystals under concentrations of Ca2+ and Mg2+ approaching those found in blood. We have solved the structures of FVIIa/sTF under three new conditions: 2.5 mM Ca2+/1.25 mM Mg2+ at 2.8 Å (2.5Ca/1.25Mg); 5 mM Ca2+/2.5 mM Mg2+ at 1.8 Å (5Ca/2.5Mg); and 45 mM Ca2+/5 mM Mg2+ at 1.7 Å (45Ca/5Mg). For 2.5Ca/1.25Mg, the Gla domain of FVIIa was disordered indicating insufficient concentrations of Ca2+/Mg2+ to fold the Gla domain. Interestingly, folding of the Ω-loop for 5Ca/2.5Mg was similar to that reported under 5Ca/45Mg, with identical positions for four Ca2+ and three Mg2+. In contrast, while the folding of the Ω-loop at 45Ca/5Mg was similar to that reported only in the presence of Ca2+ (Banner et al., Nature, 380, 41–46,1996); however, positions 1 and 7 contained Mg2+ (Fig. 1A). Thus, it would appear that four Ca2+ and three Mg2+ ions are bound to the circulating FVII/FVIIa. Moreover, circulating FVII/FVIIa will need high Ca2+ concentrations to have its position four switched from Mg2+ to Ca2+.Fig. 1.A) The superimposed structures of FVIIa Gla domains from 5Ca/2.5Mg (1.8 Å) and 45Ca/5Mg (1.7 Å) showing differences in the Ω-loops. B) The superimposed structures of Gla domain in FVIIa (45Ca/5Mg) and in 5Ca/5Mg PC-EPCR (1.6 Å) showing similar Ω-loop conformations.Fig. 1. A) The superimposed structures of FVIIa Gla domains from 5Ca/2.5Mg (1.8 Å) and 45Ca/5Mg (1.7 Å) showing differences in the Ω-loops. B) The superimposed structures of Gla domain in FVIIa (45Ca/5Mg) and in 5Ca/5Mg PC-EPCR (1.6 Å) showing similar Ω-loop conformations. All seven metal sites in the structure of PC-Gla complexed to endothelial protein C receptor (EPCR) had been refined with Ca2+ despite the presence of 5 mM Ca2+ and 5 mM Mg2+ in the crystallization buffer (Oganesyan et al., J Biol Chem, 277, 24851–24854, 2002). Based on our findings with VIIa/sTF, we revisited the PC-Gla/EPCR structure. We determined that positions 1 and 7 are Mg2+ sites while 2, 3, 4, 5 and 6 are Ca2+ sites in the PC-Gla/EPCR structure (Fig. 1B). This contrasts to four Ca2+ and three Mg2+ bound to VIIa/TF at 5Ca/2.5Mg. Since five Ca2+ and two Mg2+ were seen in VIIa/sTF at 45Ca/5Mg, it would appear that position 4 can accommodate either Ca2+ or Mg2+ depending upon conditions. We propose that at near physiologic Ca2+ and Mg2+, ligation of the Gla domain with EPCR and possibly phospholipid (PL) membranes facilitates substitution of the Mg2+ bound at the position four with Ca2+. This metal switch may be essential to achieve a favorable conformation of the Gla domain Ω-loop for optimal ligand of PL binding. Mg2+ also enhanced the Ca2+-dependent interaction of FVIIa or activated protein C (APC) to PL assessed by surface plasmon resonance. Binding of FVIIa and APC each was saturated by the physiological concentration of Ca2+ (1.1 mM) in the presence of physiological Mg2+ (0.6 mM). In contrast, only half-saturable binding was observed at the physiological concentration of Ca2+ in the absence of Mg2+. Further, 0.6 mM Mg2+ potentiated (∼2.5-fold) the PL-dependent activation of FX by FVIIa/TF at 1.1 mM Ca2+. Similarly, Mg2+ potentiated (∼3-fold) the activation of FX by FVIIa/TF assembled on the endotoxin-stimulated monocyte surface. PL-dependent inactivation of FVa by APC was also enhanced ∼3-fold by 0.6 mM Mg2+ at 1.1 mM Ca2+. At saturating Ca2+ (5 mM), the activation of FX by FVIIa/TF or the inactivation of FVa by APC was similar to that with 1.1 mM Ca2+/0.6 mM Mg2+. Thus Mg2+ at physiologic concentrations augments PL- or natural membrane-dependent coagulation and anticoagulation at the plasma concentration of Ca2+. We propose that vitamin K-dependent clotting and anti-clotting proteins circulate in blood with four Ca2+ ions bound. The remaining three (or more in FIX and FX) divalent metal binding sites in each Gla domain are occupied by Mg2+. The conformation of the Ω-loop in circulating vitamin K- dependent proteins is not favorable for binding to PL but it is achieved by switching Mg2+ at position four to Ca2+. Thus, the metal ion at position four regulates PL-dependent coagulation and anticoagulation reactions. Disclosures: No relevant conflicts of interest to declare.
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Wilkerson, Emily M., Barbara Bates, Kraig T. Kumfer, Nicholas M. Riley, Brad S. Schwartz, and Joshua J. Coon. "Novel Sites of Gamma Carboxylation in Human Factor VII." Blood 128, no. 22 (December 2, 2016): 3759. http://dx.doi.org/10.1182/blood.v128.22.3759.3759.
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Abstract Introduction Gamma- (γ-) carboxylation of glutamate residues is a vitamin K-dependent post-translational modification critical to the function of several plasma proteins. Found in the N-terminal domains of specific proteins, most of which are involved in hemostasis, these γ-carboxyglutamate residues (Gla) help mediate binding of divalent cations and are essential to protein function. Coagulation factor VII(a) bears 10 known Gla residues as characterized by N-terminal sequencing, yet Thim et al.( Biochemistry 27:7785 1988) used amino acid analysis to quantify 11.0 mol of Gla/mol of protein for plasma-derived factor VII(a). We used mass spectrometry to map and validate Gla residues of coagulation factor VII(a) to potentially identify Gla residues outside of the Gla domain. Methods Four sources of factor VII(a) (2 plasma-derived and 2 recombinant) were extracted, digested, and analyzed by tandem mass spectrometry (LC-MS/MS). Each sample was digested with trypsin and chymotrypsin to provide orthogonal coverage. Peptides derived from factor VII(a) proteolysis were analyzed on a nanoLC coupled to a quadrupole-Orbitrap-quadrupole linear ion trap mass spectrometer (Orbitrap Fusion Lumos Thermo Scientific). Multiple fragmentation methods were used to map and validate the sites including collisional based dissociation (CAD), higher energy collisional activated dissociation (HCD), electron transfer dissociation (ETD), and electron-transfer/higher-energy electron transfer activation (EThcD). Tandem MS spectra were collected at resolution 30K at 200 m/z, and data were processed using MaxQuant, COMPASS, and Proteome Discoverer. All identified sites were validated through manual annotation of spectra. We estimate that any site with >1% occupancy will be identified as Gla with this method. To validate select sites of novel gamma carboxylation, synthetic peptides were made for 4 different sites that were consistently identified in factor VII(a) from all 4 sources. Synthetic peptides were analyzed using MS methods described above, generating "true positives" to match with peptides identified from the factor VII(a) sources. Spectra from the synthetic peptides and factor VII from each source were compared using manual spectral annotation. Results In addition to identifying known Gla residues at positions 6, 7, 19, 20, 29, and 35 of factor VII(a), we detected and validated 9 novel Gla residues outside of the N-terminal Gla domain. Novel sites include residues 94, 116, 132, 219, 215, 229, 265, 196 and 385. Four of these residues (210, 220, 296 and 385) were identified as Gla in all 4 sources of factor VII(a) and were validated with synthetic peptides using a combination of fragmentation methods, providing high confidence in their characterization. Published crystallographic data suggest that residues 210 and 220 of factor VIIa-tissue factor are closely approximated to a Ca2+ ion complexed to the C-terminal protease domain; this is not the case for residues 296 and 385. We continue to refine the technique to map the Gla residues (novel and known), and to quantify the fraction of factor VII(a) molecules from each source that contain the modification at each site, in order to better incorporate our data with established studies showing >90% occupancy at each of the 10 Gla domain sites. These data suggest there is room to expand our understanding of how carboxylation contributes to specific protein function, in order to provide more comprehensive understanding of this post-translational modification, and refine our understanding of hemostatic mechanisms. Disclosures No relevant conflicts of interest to declare.
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Vadivel, Kanagasabai, Amy E. Schmidt, Duilio Cascio, Kaillathe Padmanabhan, Sriram Krishnaswamy, Hans Brandstetter, and S. Paul Bajaj. "Structure of human factor VIIa–soluble tissue factor with calcium, magnesium and rubidium." Acta Crystallographica Section D Structural Biology 77, no. 6 (May 14, 2021): 809–19. http://dx.doi.org/10.1107/s2059798321003922.
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Coagulation factor VIIa (FVIIa) consists of a γ-carboxyglutamic acid (GLA) domain, two epidermal growth factor-like (EGF) domains and a protease domain. FVIIa binds three Mg2+ ions and four Ca2+ ions in the GLA domain, one Ca2+ ion in the EGF1 domain and one Ca2+ ion in the protease domain. Further, FVIIa contains an Na+ site in the protease domain. Since Na+ and water share the same number of electrons, Na+ sites in proteins are difficult to distinguish from waters in X-ray structures. Here, to verify the Na+ site in FVIIa, the structure of the FVIIa–soluble tissue factor (TF) complex was solved at 1.8 Å resolution containing Mg2+, Ca2+ and Rb+ ions. In this structure, Rb+ replaced two Ca2+ sites in the GLA domain and occupied three non-metal sites in the protease domain. However, Rb+ was not detected at the expected Na+ site. In kinetic experiments, Na+ increased the amidolytic activity of FVIIa towards the synthetic substrate S-2288 (H-D-Ile-Pro-Arg-p-nitroanilide) by ∼20-fold; however, in the presence of Ca2+, Na+ had a negligible effect. Ca2+ increased the hydrolytic activity of FVIIa towards S-2288 by ∼60-fold in the absence of Na+ and by ∼82-fold in the presence of Na+. In molecular-dynamics simulations, Na+ stabilized the two Na+-binding loops (the 184-loop and 220-loop) and the TF-binding region spanning residues 163–180. Ca2+ stabilized the Ca2+-binding loop (the 70-loop) and Na+-binding loops but not the TF-binding region. Na+ and Ca2+ together stabilized both the Na+-binding and Ca2+-binding loops and the TF-binding region. Previously, Rb+ has been used to define the Na+ site in thrombin; however, it was unsuccessful in detecting the Na+ site in FVIIa. A conceivable explanation for this observation is provided.
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Ahnström, Josefin, Helena M. Andersson, Kevin Canis, Eva Norstrøm, Yao Yu, Björn Dahlbäck, Maria Panico, Howard R. Morris, James T. B. Crawley, and David A. Lane. "Activated protein C cofactor function of protein S: a novel role for a γ-carboxyglutamic acid residue." Blood 117, no. 24 (June 16, 2011): 6685–93. http://dx.doi.org/10.1182/blood-2010-11-317099.
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Abstract Protein S has an important anticoagulant function by acting as a cofactor for activated protein C (APC). We recently reported that the EGF1 domain residue Asp95 is critical for APC cofactor function. In the present study, we examined whether additional interaction sites within the Gla domain of protein S might contribute to its APC cofactor function. We examined 4 residues, composing the previously reported “Face1” (N33S/P35T/E36A/Y39V) variant, as single point substitutions. Of these protein S variants, protein S E36A was found to be almost completely inactive using calibrated automated thrombography. In factor Va inactivation assays, protein S E36A had 89% reduced cofactor activity compared with wild-type protein S and was almost completely inactive in factor VIIIa inactivation; phospholipid binding was, however, normal. Glu36 lies outside the ω-loop that mediates Ca2+-dependent phospholipid binding. Using mass spectrometry, it was nevertheless confirmed that Glu36 is γ-carboxylated. Our finding that Gla36 is important for APC cofactor function, but not for phospholipid binding, defines a novel function (other than Ca2+ coordination/phospholipid binding) for a Gla residue in vitamin K–dependent proteins. It also suggests that residues within the Gla and EGF1 domains of protein S act cooperatively for its APC cofactor function.
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Ruf, Wolfram, Justin Shobe, S. Mohan Rao, Craig D. Dickinson, Arthur Olson, and Thomas S. Edgington. "Importance of Factor VIIa Gla-Domain Residue Arg-36 for Recognition of the Macromolecular Substrate Factor X Gla-Domain†." Biochemistry 38, no. 7 (February 1999): 1957–66. http://dx.doi.org/10.1021/bi982254r.
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Yegneswaran, Subramanian, Phuong Nguyen, John Griffin, and Andrew Gale. "Prothrombin amino terminal region helps protect coagulation factor Va from proteolytic inactivation by activated protein C." Thrombosis and Haemostasis 101, no. 01 (2009): 55–61. http://dx.doi.org/10.1160/th08-07-0491.
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SummaryThe hypothesis that prothrombin (FII) protects coagulation factor Va (FVa) from proteolytic inactivation by activated protein C (APC) was tested using purified proteins. FII dose-dependently protected FVa from APC proteolysis under conditions where competition of proteins for binding to negatively-charged phospholipid surface was not relevant (i.e. either at high phospholipid vesicle concentrations or using soluble dicaproylphosphatidylserine at levels below its critical micellar concentration). Cleavages in FVa at both Arg506 and Arg306 by APC were inhibited by FII. FII did not alter the amidolytic activity of APC towards chromogenic oligopeptide substrates or inhibit FVIIIa inactivation by APC, implying that the FII-mediated protection of FVa from APC proteolysis was due to the ability of FII to inhibit protein-protein interactions between FVa and APC. FII also protected FVa from inactivation by Gla-domainless APC, ruling out a role for the APC Gla domain for these observations. To identify domains of FII responsible for the observed phenomenon, various forms or fragments of FII were employed. Biotin-PheProArg-CMK-inhibited meizothrombin and fII-fragment 12 protected FVa from proteolysis by APC. In contrast, no significant protection of FVa from APC cleavage was observed for Gladomainless-FII, prethrombin-1, prethrombin-2, FII fragment 1 or active site inhibited-thrombin (DEGR-thrombin). Overall, these data demonstrate that the Gla domain of FII linked to kringle 1 and 2 is necessary for the ability of FII to protect FVa from APC cleavage and support the general concept that assembly of the FII activation complex (FXaFVaFIIlipid surface) protects FVa from APC inactivation so that the procoagulant, thrombin generating pathway can act unhindered by APC. Only following FII activation and dissociation of the FII Gla domain fragments from the FII-ase complex, can APC inactivate FVa and down-regulate thrombin generation.
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Nakabayashi, Toru, Kazuhiro Mizukami, Sumiyoshi Naitoh, Mika Takeda, Yasuo Shikamoto, Takafumi Nakagawa, Hiroki Kaneko, et al. "Protein C Sapporo (protein C Glu 25 → Lys): A heterozygous missense mutation in the Gla domain provides new insight into the interaction between protein C and endothelial protein C receptor." Thrombosis and Haemostasis 94, no. 11 (2005): 942–50. http://dx.doi.org/10.1160/th05-05-0326.
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SummaryInteraction of the γ-carboxyglutamic acid (Gla) domain of protein C with endothelial protein C receptor (EPCR) is a critical step for efficient activation of protein C, though interactions by mutants in the Gla domain of protein C with EPCR have been rarely evaluated. We identified a 44-year-old Japanese woman with a history of recurrent thromboembolism as an inherited missense mutation, the first such case reported in Japan, which involved a protein C Gla 25 mutation. Total protein C antigen and Gla protein C antigen levels in the proband were normal. Protein C activity measured with an anticoagulant assay was reduced, whereas that measured with an amidolytic assay was normal. She was therefore phenotypically diagnosed as type IIb protein C deficiency. Direct sequencing of the PCR fragments revealed a heterozygous G toA transition at nucleotide position 1462 in exon 3, which predicted an amino acid substitution of Glu 25 by Lys. Her mother and one son were also heterozygous for this mutation. A molecular dynamics simulation of Gla 25→Lys/EPCR complex in water suggested that the affinity between the molecules was decreased compared to the wild type Gla domain/EPCR complex. Since Gla 25 has been shown to play an important role in protein C function, not only in membrane phospholipid binding but also in binding to EPCR, our findings provide new insight into the mechanism by which the Glu 25→Lys mutation induces type IIb protein C deficiency in individuals.
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Gaussem, Pascale, Sophie Gandrille, Jérôme Duchemin, Joseph Emmerich, Martine Alhenc-Gelas, Marie-Françoise Aillaud, and Martine Aiach. "Influence of Six Mutations of the Protein C Gene on the Gla Domain Conformation and Calcium Affinity." Thrombosis and Haemostasis 71, no. 06 (1994): 748–54. http://dx.doi.org/10.1055/s-0038-1642517.
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SummaryThe protein C Gla domain was studied in six families presenting a type II hereditary deficiency characterized by low activity in a coagulation assay and normal activity in an amidolytic assay. Five of these mutations, previously described by our group, affected Arg-5, Arg-1, Arg 229 and Ser 252. We report here the first natural Glu 7 to Asp mutation in a sixth family.We evaluated the binding of the mutated protein C to Hn, a monoclonal antibody (mAb) known to recognize the sequence Phe 4 to Arg 9 of the Gla domain; the presence of calcium ions suppresses the recognition of this epitope by Hn. Mutation of Arg 229 to Gin and Ser 252 to Asn did not modify the inhibition of protein C binding, whereas the Arg-1 to His mutation resulted in a loss of inhibition in the presence of CaCl2. This suggests that the protein C of this patient shows impaired carboxylation.The protein C from patients bearing the mutations Arg-5 to Trp, Arg-1 to Cys and Glu 7 to Asp bound poorly to Hn mAb, even in the absence of calcium ions. The calcium affinity of the Gla domain was studied by pseudo-affinity chromatography, in which protein C was successively eluted from a Mono Q column by CaCl210 mM and NaCl 0.6 M. Protein C from the patient bearing the Arg-5 to Asp mutation had a normal elution profile, suggesting that a modification of the propeptide cleavage site impairs the conformation of the Gla domain but not carboxylation. Conversely, the patient bearing the mutation Arg-1 to Cys had an abnormal elution profile, showing that calcium binding affinity was impaired probably due to a defective carboxylation (the behaviour of the Glu 7 to Asp mutant lacking one Gla residue validated our experimental approach).These results confirm that mutations in the serine protease domain do not modify the conformation of the N-terminal part of the Gla domain. Arg-1 to His, Arg-1 to Cys and Glu 7 to Asp mutations strongly modified affinity for calcium ions, whereas the Arg-5 to Trp mutation probably modifies the conformation of the Gla domain.
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Flores-Ibarra, Andrea, Federico M. Ruiz, Sabine Vértesy, Sabine André, Hans-Joachim Gabius, and Antonio Romero. "Preliminary X-ray crystallographic analysis of an engineered variant of human chimera-type galectin-3 with a shortened N-terminal domain." Acta Crystallographica Section F Structural Biology Communications 71, no. 2 (January 28, 2015): 184–88. http://dx.doi.org/10.1107/s2053230x15000023.
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How lectins translate sugar-encoded information into cellular effects not only depends on glycan recognition. Other domains of the protein can contribute to the functional profile of a lectin. Human galectin-3 (Gal-3), an adhesion/growth-regulatory galectin, is composed of three different domains and is thus called a chimera-type protein. In addition to the carbohydrate-recognition domain, this lectin encompasses an N-terminal domain consisting of a peptide harbouring two phosphorylation sites and nine non-triple-helical collagen-like repeats. This region plays an as yet structurally undefined role in Gal-3 aggregation and ligand recognition. To date, crystallization of full-length Gal-3 has not been achieved. With the aim of providing structural insights into this modular organization, a Gal-3 variant was crystallized maintaining the terminal peptide and three of the nine collagen-like repeats. The crystals belonged to the orthorhombic space groupP212121, with unit-cell parametersa= 94.04,b= 97.96,c= 236.20 Å, and diffracted to a resolution of 3.3 Å.
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Niwa, K., M. Takebe, T. Sugo, Y. Kawata, J. Mimuro, S. Asakura, Y. Sakata, et al. "A gamma Gly-268 to Glu substitution is responsible for impaired fibrin assembly in a homozygous dysfibrinogen Kurashiki I." Blood 87, no. 11 (June 1, 1996): 4686–94. http://dx.doi.org/10.1182/blood.v87.11.4686.bloodjournal87114686.
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A new type of gamma Gly-268 (GGA) to Glu (GAA) substitution has been identified in a homozygous dysfibrinogen by analyses of the affected polypeptide and its encoding gene derived from a 58 year-old man manifesting no major bleeding or thrombosis. The functional abnormality was characterized by impaired fibrin assembly most likely due to failure to construct properly aligned double-stranded fibrin protofibrils. This presumption was deduced from the following findings: (1) Factor XIIIa-catalyzed cross-linking of the fibrin gamma-chains progressed in a normal fashion, indicating that the contact between the central E domain of one fibrin monomer and the D domain of another took place normally; (2) Nevertheless, factor XIIIa-catalyzed cross-linking of the fibrinogen gamma-chains was obviously delayed, suggesting that longitudinal association of D domains of different fibrin monomers, ie, D:D association was perturbed; (3) Plasminogen activation catalyzed by tissue-type plasminogen activator was not as efficiently facilitated by polymerizing fibrin monomer derived from the patient as by the normal counterpart. Therefore, gamma Gly-268 would not be involved in the 'a' site residing in the D domain, which functions as a complementary binding site with the thrombin-activated 'A' site in the central E domain, but would be rather involved in the D:D self association sites recently proposed for human fibrinogen. Thus, the gamma Glu-268 substitution newly identified in this homozygous dysfibrinogen seems to impair proper alignment of adjacent D domains of neighboring fibrin molecules in the double-stranded fibrin protofibril, resulting in delayed fibrin gel formation.
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Mason, A. W. "Free quotients of infinite rank of GL2 over Dedekind domains." Proceedings of the Royal Society of Edinburgh: Section A Mathematics 129, no. 1 (1999): 77–84. http://dx.doi.org/10.1017/s0308210500027475.
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This paper is concerned with integral domains R, for which the factor group SL2(R)/U2(R) has a non-trivial, free quotient, where U2(R) is the subgroup of GL2(R) generated by the unipotent matrices. Recently, Krstić and McCool have proved that SL2(P[x])/U2(P[x]) has a free quotient of infinite rank, where P is a domain which is not a field. This extends earlier results of Grunewald, Mennicke and Vaserstein.Any ring of the type P[x] has Krull dimension at least 2. The purpose of this paper is to show that result of Krstić and McCool extends to some domains of Krull dimension 1, in particular to certain Dedekind domains. This result, which represents a two-dimensional anomaly is the best possible in the following sense. It is well known that SL2(R) = U2(R), when R is a domain of Krull dimension zero, i.e. when R is a field. It is already known that for some arithmetic Dedekind domains A, the factor group SL2(A)/U2(A) has a free quotient of finite (and not infinite) rank.
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HOUBEN, Roger J. T. J., Dirk T. S. RIJKERS, Thomas B. STANLEY, Francine ACHER, Robert AZERAD, Sanna-Maria KÄKÖNEN, Cees VERMEER, and Berry A. M. SOUTE. "Characteristics and composition of the vitamin K-dependent γ-glutamyl carboxylase-binding domain on osteocalcin." Biochemical Journal 364, no. 1 (May 8, 2002): 323–28. http://dx.doi.org/10.1042/bj3640323.
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Two different sites on vitamin K-dependent γ-glutamyl carboxylase (VKC) are involved in enzyme—substrate interaction: the propeptide-binding site required for high-affinity substrate binding and the active site for glutamate carboxylation. Synthetic descarboxy osteocalcin (d-OC) is a low-Km substrate for the VKC, but unique since it possesses a high-affinity recognition site for the VKC, distinct from the propeptide which is essential as a binding site for VKC. However, the exact location and composition of this VKC-recognition domain on d-OC has remained unclear until now. Using a stereospecific substrate analogue [t-butyloxycarbonyl-(2S,4S)-4-methylglutamic acid-Glu-Val (S-MeTPT)] we demonstrate in this paper that the high affinity of d-OC for VKC cannot be explained by a direct interaction with either the active site or with the propeptide-binding site on VKC. It is shown using various synthetic peptides derived from d-OC that there are two domains on d-OC necessary for recognition: one located between residues 1 and 12 and a second between residues 26 and 39, i.e. at the C-terminal side of the γ-carboxyglutamate (Gla) domain. Both internal sequences contribute substantially to the efficiency of carboxylation. On the basis of these data we postulate the presence of a second high-affinity substrate-binding site on VKC capable of specifically binding d-OC, which is the first vitamin K-dependent substrate of which the VKC binding domain is interrupted by the Gla domain.
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Saller, François, Bruno O. Villoutreix, Aymeric Amelot, Tahar Kaabache, Bernard F. Le Bonniec, Martine Aiach, Sophie Gandrille, and Delphine Borgel. "The γ-carboxyglutamic acid domain of anticoagulant protein S is involved in activated protein C cofactor activity, independently of phospholipid binding." Blood 105, no. 1 (January 1, 2005): 122–30. http://dx.doi.org/10.1182/blood-2004-06-2176.
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Abstract We expressed 2 chimeras between human protein S (PS) and human prothrombin (FII) in which the prothrombin γ-carboxyglutamic acid (Gla) domain replaced the PS Gla domain in native PS (GlaFII-PS) or in PS deleted of the thrombin-sensitive region (TSR) (GlaFII-ΔTSR-PS). Neither PS/FII chimera had activated protein C (APC) cofactor activity in plasma clotting assays or purified systems, but both bound efficiently to phospholipids. This pointed to a direct involvement of the PS Gla domain in APC cofactor activity through molecular interaction with APC. Using computational methods, we identified 2 opposite faces of solvent-exposed residues on the PS Gla domain (designated faces 1 and 2) as potentially involved in this interaction. Their importance was supported by functional characterization of a PS mutant in which the face 1 and face 2 PS residues were reintroduced into GlaFII-PS, leading to significant APC cofactor activity, likely through restored interaction with APC. Furthermore, by characterizing PS mutants in which PS face 1 and PS face 2 were individually replaced by the corresponding prothrombin faces, we found that face 1 was necessary for efficient phospholipid binding but that face 2 residues were not strictly required for phospholipid binding and were involved in the interaction with APC.
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Boman, Annette L., Paul D. Salo, Melissa J. Hauglund, Nicole L. Strand, Shelly J. Rensink, and Olga Zhdankina. "ADP-Ribosylation Factor (ARF) Interaction Is Not Sufficient for Yeast GGA Protein Function or Localization." Molecular Biology of the Cell 13, no. 9 (September 2002): 3078–95. http://dx.doi.org/10.1091/mbc.e02-02-0078.
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Golgi-localized γ-ear homology domain, ADP-ribosylation factor (ARF)-binding proteins (GGAs) facilitate distinct steps of post-Golgi traffic. Human and yeast GGA proteins are only ∼25% identical, but all GGA proteins have four similar domains based on function and sequence homology. GGA proteins are most conserved in the region that interacts with ARF proteins. To analyze the role of ARF in GGA protein localization and function, we performed mutational analyses of both human and yeast GGAs. To our surprise, yeast and human GGAs differ in their requirement for ARF interaction. We describe a point mutation in both yeast and mammalian GGA proteins that eliminates binding to ARFs. In mammalian cells, this mutation disrupts the localization of human GGA proteins. Yeast Gga function was studied using an assay for carboxypeptidase Y missorting and synthetic temperature-sensitive lethality between GGAs andVPS27. Based on these assays, we conclude that non-Arf-binding yeast Gga mutants can function normally in membrane trafficking. Using green fluorescent protein-tagged Gga1p, we show that Arf interaction is not required for Gga localization to the Golgi. Truncation analysis of Gga1p and Gga2p suggests that the N-terminal VHS domain and C-terminal hinge and ear domains play significant roles in yeast Gga protein localization and function. Together, our data suggest that yeast Gga proteins function to assemble a protein complex at the late Golgi to initiate proper sorting and transport of specific cargo. Whereas mammalian GGAs must interact with ARF to localize to and function at the Golgi, interaction between yeast Ggas and Arf plays a minor role in Gga localization and function.
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Wildgoose, Peter, Tony Jørgensen, Yutaka Komiyama, Tomohiro Nakagaki, Anders Pedersen, and Walter Kisiel. "The Role of Phospholipids and the Factor VII Gla-Domain in the Interaction of Factor VII with Tissue Factor." Thrombosis and Haemostasis 67, no. 06 (1992): 679–85. http://dx.doi.org/10.1055/s-0038-1648522.
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SummaryWhether or not the factor VII Gla-domain is involved in the high-affinity interaction of factor VII and tissue factor via calcium-dependent interactions with surrounding phospholipids is unknown. To investigate this, we have purified the factor VII Gla-peptide (FVII-GP) from digested recombinant human factor VII a and assessed its effect on factor VII: tissue factor interactions. FVII-GP inhibited the activation of factor X by factor Vila in the presence of either soluble or cell surface tissue factor halfmaximally at 0.5 μM and 2.7 μM, respectively. However, FVII-GP failed to inhibit the specific binding of factor Vila to cell-surface tissue factor, and did not inhibit the ability of tissue factor to stimulate the amidolytic activity of factor Vila. Unrelipidated tissue factor apoprotein stimulated the amidolytic activity of factor Vila to the same extent as relipidated tissue factor apoprotein. These findings suggest that the factor VII Gla-domain does not directly interact with tissue factor, but rather is important for calcium binding and concomitant expression of other factor VII epitopes necessary for tissue factor recognition and binding. To test this hypothesis, we have prepared a monoclonal antibody against a putative factor VII epitope that participates in the interaction of factor VII with cell-surface tissue factor (peptide 195-206) and assessed its ability to bind to factor VII in the presence and absence of calcium. Binding of this monoclonal antibody (PW-4) to intact factor VII a was calcium-dependent and could be inhibited in a dose-dependent manner by peptide 195-206. The antibody reacted with Gla-domainless factor Vila, but only 37% as compared to intact factor Vila. In addition, PW4 as well as its Fab’ fragment, inhibited factor Vila binding to cell-surface tissue factor. These studies indicate that the factor VII Gla-domain does not provide structural elements that contribute to the formation of a stable factor VII/VII a-tissue factor binary complex. The factor VII Gla-domain appears to be necessary, however, in binding calcium ions and inducing a calcium-dependent conformational change in factor VII/VII a that expresses one or more neoepitopes that participates in the interaction of factor VII/VII a with the extracellular domain of tissue factor apoprotein.
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Soeda, Tetsuhiro, Keiji Nogami, Masahiro Takeyama, Kenichi Ogiwara, Kazuhiko Tomokiyo, Yoichi Sakata, Akira Yoshioka, and Midori Shima. "Role of the Direct Interaction between Factor VIII C2 and Factor IXa Gla Domain in the Factor Xase Complex." Blood 110, no. 11 (November 16, 2007): 2687. http://dx.doi.org/10.1182/blood.v110.11.2687.2687.
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Abstract Factor VIII functions as a cofactor for factor IXa in the anionic phospholipid surface-dependent conversion of factor X to Xa. It is well-known that the A2 and A3 domains of factor VIII interact with the catalytic domain and EGF2 domain of factor IXa, respectively. Recently, Furie et al. have reported that the Gla domain of factor IXa (factor IXa-GD) interacts with the light chain of factor VIII. However, the factor IXa-GD-interactive site on the light chain remained to be investigated. In the current study, the recombinant C2 (rC2) domain of factor VIII was prepared using a yeast secretion system. ELISA-based assay in the absence of phospholipid showed the Glu-Gly-Arg-active site modified factor IXa (EGR-factor IXa) bound to the immobilized rC2 domain dose-dependently, and the binding ability was maximum under the condition of 150 mM NaCl/1 mM CaCl2. This binding was competitively inhibited by the addition of excess of factor VIII or rC2 domain, supporting the specificity of this interaction. Furthermore, the presence of high ionic strength and the metal-ion chelator EDTA blocked this binding by ∼95 and ∼75%, respectively. Surface plasmon resonance-based assay showed that the binding affinity (Kd) of rC2 domain for EGR-factor IXa was 108 ± 15.5 nM. GD less-factor IXa, deleting the GD completely, failed to bind to rC2 domain. A monoclonal antibody against factor IXa-GD specific for calcium-dependent conformation (mAbIXa-GD) also inhibited (∼ 95%) the rC2 domain binding to EGR-factor IXa in a dose-dependent manner (IC50; 758 nM), suggesting the authentic of the C2 domain and factor IXa-GD interaction. The addition of rC2 domain or mAbIXa-GD inhibited the factor IXa-catalyzed factor X activation with factor VIIIa in the absence of phospholipid (IC50; 15.7 μM or 43.2 nM, respectively), whilst both any little affected in the absence of factor VIIIa. In addition, the ∼8-kDa C2 fragment obtained by V8 protease digestion (residues 2182–2259) bound directly to EGR-factor IXa. Taken together, these results indicate that factor VIII C2 domain directly interacts with factor IXa-GD via both the electrostatic- and calcium-dependent interactions. Furthermore, our results provide the first evidence for an essential role of the C2 domain in the association between factor VIII and factor IXa in the factor Xase complex.
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Hirst, Jennifer, Margaret R. Lindsay, and Margaret S. Robinson. "Golgi-localized, γ-Ear-containing, ADP-Ribosylation Factor-binding Proteins: Roles of the Different Domains and Comparison with AP-1 and Clathrin." Molecular Biology of the Cell 12, no. 11 (November 2001): 3573–88. http://dx.doi.org/10.1091/mbc.12.11.3573.
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We have previously identified a novel family of proteins called the GGAs (Golgi-localized, γ-ear-containing, ADP-ribosylation factor-binding proteins). These proteins consist of an NH2-terminal VHS domain, followed by a GAT domain, a variable domain, and a γ-adaptin ear homology domain. Studies from our own laboratory and others, making use of both yeast and mammals cells, indicate that the GGAs facilitate trafficking from the trans-Golgi network to endosomes. Here we have further investigated the function of the GGAs. We find that GGA-deficient yeast are not only defective in vacuolar protein sorting but they are also impaired in their ability to process α-factor. Using deletion mutants and chimeras, we show that the VHS domain is required for GGA function and that the VHS domain from Vps27p will not substitute for the GGA VHS domain. In contrast, the γ-adaptin ear homology domain contributes to GGA function but is not absolutely required, and full function can be restored by replacing the GGA ear domain with the γ-adaptin ear domain. Deleting the γ-adaptin gene together with the twoGGA genes exacerbates the phenotype in yeast, suggesting that they function on parallel pathways. In mammalian cells, the association of GGAs with the membrane is extremely unstable, which may account for their absence from purified clathrin-coated vesicles. Double- and triple-labeling immunofluorescence experiments indicate that the GGAs and AP-1 are associated with distinct populations of clathrin-coated vesicles budding from the trans-Golgi network. Together with results from other studies, our findings suggest that the GGAs act as monomeric adaptors, with the four domains involved in cargo selection, membrane localization, clathrin binding, and accessory protein recruitment.
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Hirst, Jennifer, Winnie W. Y. Lui, Nicholas A. Bright, Nicholas Totty, Matthew N. J. Seaman, and Margaret S. Robinson. "A Family of Proteins with γ-Adaptin and Vhs Domains That Facilitate Trafficking between the Trans-Golgi Network and the Vacuole/Lysosome." Journal of Cell Biology 149, no. 1 (April 3, 2000): 67–80. http://dx.doi.org/10.1083/jcb.149.1.67.
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We have cloned and characterized members of a novel family of proteins, the GGAs. These proteins contain an NH2-terminal VHS domain, one or two coiled-coil domains, and a COOH-terminal domain homologous to the COOH-terminal “ear” domain of γ-adaptin. However, unlike γ-adaptin, the GGAs are not associated with clathrin-coated vesicles or with any of the components of the AP-1 complex. GGA1 and GGA2 are also not associated with each other, although they colocalize on perinuclear membranes. Immunogold EM shows that these membranes correspond to trans elements of the Golgi stack and the TGN. GST pulldown experiments indicate that the GGA COOH-terminal domains bind to a subset of the proteins that bind to the γ-adaptin COOH-terminal domain. In yeast there are two GGA genes. Deleting both of these genes results in missorting of the vacuolar enzyme carboxypeptidase Y, and the cells also have a defective vacuolar morphology phenotype. These results indicate that the function of the GGAs is to facilitate the trafficking of proteins between the TGN and the vacuole, or its mammalian equivalent, the lysosome.
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Degani, Genny, Alessandra Altomare, Stefania Digiovanni, Beatrice Arosio, Guenter Fritz, Angela Raucci, Giancarlo Aldini, and Laura Popolo. "Prothrombin is a binding partner of the human receptor of advanced glycation end products." Journal of Biological Chemistry 295, no. 35 (July 14, 2020): 12498–511. http://dx.doi.org/10.1074/jbc.ra120.013692.
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The receptor for advanced glycation end products (RAGE) plays a key role in mammal physiology and in the etiology and progression of inflammatory and oxidative stress-based diseases. In adults, RAGE expression is normally high only in the lung where the protein concentrates in the basal membrane of alveolar Type I epithelial cells. In diseases, RAGE levels increase in the affected tissues and sustain chronic inflammation. RAGE exists as a membrane glycoprotein with an ectodomain, a transmembrane helix, and a short carboxyl-terminal tail, or as a soluble ectodomain that acts as a decoy receptor (sRAGE). VC1 domain is responsible for binding to the majority of RAGE ligands including advanced glycation end products (AGEs), S100 proteins, and HMGB1. To ascertain whether other ligands exist, we analyzed by MS the material pulled down by VC1 from human plasma. Twenty of 295 identified proteins were selected and associated to coagulation and complement processes and to extracellular matrix. Four of them contained a γ-carboxyl glutamic acid (Gla) domain, a calcium-binding module, and prothrombin (PT) was the most abundant. Using MicroScale thermophoresis, we quantified the interaction of PT with VC1 and sRAGE in the absence or presence of calcium that acted as a competitor. PT devoid of the Gla domain (PT des-Gla) did not bind to sRAGE, providing further evidence that the Gla domain is critical for the interaction. Finally, the presence of VC1 delayed plasma clotting in a dose-dependent manner. We propose that RAGE is involved in modulating blood coagulation presumably in conditions of lung injury.
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Coutu, Daniel L., Jian Wui Wu, Georges-Etienne Rivard, Mark D. Blostein, and Jacques Galipeau. "Periostin Is a Previously Uncharacterised Vitamin K Dependent γ-Carboxyglutamic Acid (Gla) Containing Protein Expressed by Marrow-Derived Mesenchymal Stromal Cells." Blood 110, no. 11 (November 16, 2007): 1927. http://dx.doi.org/10.1182/blood.v110.11.1927.1927.
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Abstract The modification of glutamic acid residues to g-carboxyglutamic acid (Gla) is a post-translational modification catalyzed by the vitamin K-dependent γ-glutamylcarboxylase enzyme. Despite ubiquitous expression of the γ-carboxylation machinery in mammalian tissues, only 12 Gla-containing proteins have so far been identified in humans. Because bone tissue is the second most abundant source of Gla-proteins after the liver, we sought to identify Gla proteins secreted by bone-marrow derived mesenchymal stromal cells (MSCs), a precursor to all non-hematopoietic cells in bones. We used a proteomics approach to screen the secretome of MSCs with a combination of 2D gel electrophoresis and tandem mass spectrometry. The most abundant Gla-protein secreted by MSCs was identified as periostin, a previously unrecognized γ-carboxylated protein. In silico aminoacid sequence analysis of periostin demonstrated the presence of four consensus γ-carboxylase recognition sites embedded within fasciclin-like protein domains. The carboxylation of periostin was confirmed by immunoprecipitation using anti-Gla antibodies and could be inhibited by warfarin in MSCs. In conclusion, periostin is a novel vitamin K-dependent γ-carboxylated protein distinguished from other Gla-proteins by the presence of multiple γ-carboxylase recognition sites and MSCs are an abundant source of periostin including its γ-carboxylated variant.
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Qureshi, Shabir H., Likui Yang, Chandrashekhara Manithody, Jong-Sup Bae, and Alireza R. Rezaie. "Functional properties and active-site topographies of factor X Gla- and prothrombin Gla-domain chimeras of activated protein C." Biochimica et Biophysica Acta (BBA) - General Subjects 1780, no. 9 (September 2008): 1080–86. http://dx.doi.org/10.1016/j.bbagen.2008.05.004.
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Cattaneo, Valentina, María V. Tribulatti, and Oscar Campetella. "Galectin-8 tandem-repeat structure is essential for T-cell proliferation but not for co-stimulation." Biochemical Journal 434, no. 1 (January 27, 2011): 153–60. http://dx.doi.org/10.1042/bj20101691.
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Gal (galectin)-8 is a tandem-repeat Gal containing N-CRDs (Nterminal carbohydrate-recognition domains) and C-CRDs (C-terminal carbohydrate-recognition domains) with differential glycan-binding specificity fused by a linker peptide. Gal-8 has two distinct effects on CD4 T-cells: at high concentrations it induces antigen-independent proliferation, whereas at low concentrations it co-stimulates antigen-specific responses. Associated Gal-8 structural requirements were dissected in the present study. Recombinant homodimers N–N (two N-terminal CRD chimaera) and C–C (two C-terminal CRD chimaera), but not single C-CRDs or N-CRDs, induced proliferation; however, single domains induced co-stimulation. These results indicate that the tandem-repeat structure was essential only for the proliferative effect, suggesting the involvement of lattice formation, whereas co-stimulation could be mediated by agonistic interactions. In both cases, C–C chimaeras displayed higher activity than Gal-8, indicating that the C-CRD was mainly involved, as was further supported by the strong inhibition of proliferation and co-stimulation in the presence of blood group B antigen, specifically recognized by this domain. Classic Gal inhibitors (lactose and thiodigalactoside) prevented proliferation but not co-stimulatory activity, which was inhibited by 3-O-β-D-galactopyranosyl-D-arabinose. Interestingly, Gal-8 induced proliferation of naïve human CD4 T-cells, varying from non- to high-responder individuals, whereas it promoted cell death of phytohaemagglutinin or CD3/CD28 pre-activated cells. The findings of the present study delineate the differential molecular requirements for Gal-8 activities on T-cells, and suggest a dual activity relying on activation state.
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Blostein, Mark D., Isabelle Rajotte, and Ines Hasanbasic. "γ-Carboxylation Is Not Required for gas6-Axl Binding but Is Necessary for gas6-Dependent Endothelial Cell Survival." Blood 106, no. 11 (November 16, 2005): 3702. http://dx.doi.org/10.1182/blood.v106.11.3702.3702.
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Abstract Gas6 is a novel member of the vitamin K-dependent family of γ-carboxylated proteins and is a ligand for the receptor tyrosine kinase Axl. Gas6-Axl interactions have been shown to mediate cell survival in vascular endothelium. Although the receptor-binding portion of gas6 lies in the C-terminus, the significance of the N-terminal γ-carboxylated residues (Gla domain) is not clear. To address this question, we recombinantly produced both carboxylated and decarboxylated gas6 and confirmed that the latter is not γ-carboxylated by both Western blotting and fluorescence spectroscopy. Using DAPI staining and flow cytometry, we show that carboxylated gas6 mediates protection of endothelial cells from serum starvation-induced apoptosis whereas decarboxylated gas6 does not. Furthermore, carboxylated gas6, but not decarboxylated gas6, activates Axl and phosphorylates Akt during gas6-Axl-mediated protection of endothelial cells. Interestingly, although decarboxylated gas6 cannot activate Axl and rescue endothelium from serum starvation-induced apoptosis, it can inhibit the survival effect of carboxylated gas6. To further explore the properties of Gla domain of gas6, the binding each form of gas6 to endothelial cells was determined. Both carboxylated and decarboxylated gas6 bind to endothelial cells with an equal affinity of 50 nM. The binding of both forms of gas6 to endothelium is inhibited by an antibody to the extracellular domain of Axl thereby demonstrating that the Gla domain is not required for the direct binding of the C-terminus of gas6 to Axl. These findings support the conclusion that inhibition of gas6-mediated survival by decarboxylated gas6 is not mediated through inhibition of binding of gas6 to its receptor Axl. Rather, the Gla domain of gas6 has a key role by impacting Axl activation via heretofore unknown mechanism(s). Taken together, the results of this study suggest a novel role for γ-carboxylation in gas6 function.
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Ahmad, Nisar, Liaqat Iqbal, and Irfan Ullah. "Language Domains: The Sociolinguistic Significance of Pashto-English Hybridization." Global Language Review V, no. III (September 30, 2020): 108–16. http://dx.doi.org/10.31703/glr.2020(v-iii).12.
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Domains divide language according to the particular context of its use. Concerning different contexts of research, the domain varies. The purpose of the present research is to explore the sociolinguistic significance of Pashto-English hybridization in the language domains. To explore the area, TV programmes from a Pashto channel Khyber News were selected through purposive sampling. As two episodes from each programme were selected, thus a total of ten programmes were taken for analysis that makes about 10 hours of recording. For data analysis, Kachru’s (1978) framework was used. The findings show that according to different domains of language use, the frequency of hybridized words varied. In the domain of Government and Administration and School and Education, mostly words were hybridized, followed by the domain of Economy, Playground and Street, Military and Courts. The findings reveal that domains have sociolinguistic significance that determines the extent to which language is hybridized.
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Mannucci, Pier, Manijeh Lak, Giacomo Mancuso, Maria Mazzucconi, Angiola Rocino, P. Jenkins, Stephen Perkins, and Sepideh Akhavan. "Identification and Three-dimensional Structural Analysis of Nine Novel Mutations in Patients with Prothrombin Deficiency." Thrombosis and Haemostasis 84, no. 12 (2000): 989–97. http://dx.doi.org/10.1055/s-0037-1614161.
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SummaryProthrombin deficiency is an autosomal recessive disorder associated with a moderately severe bleeding tendency. In this study, 13 patients with prothrombin deficiency were screened for the presence of alterations in the prothrombin gene, and nine novel candidate mutations were identified. Of 11 patients with hypoprothrombinemia, ten are homozygous for five mutations and one patient is a compound heterozygote. The two patients with dysprothrombinemia are homozygous for two mutations. Eight of nine mutations are missense ones associated with single amino acid substitutions in the propeptide (Arg-1Gln, Arg-2Trp), the kringle-1 (Asp118Try) and kringle-2 (Arg220Cys) domains and the catalytic serine protease domain (Gly330Ser, Ser354Arg, Arg382His and Arg538Cys). The ninth mutation is an in-frame deletion of 3 bp that results in the omission of one amino acid (del Lys 301/302). The combination of these missense mutations with crystal structures for α-thrombin and the prothrombin fragments 1 and 2 resulted in new insight into the function of α-thrombin. The hypoprothrombinemia mutations were inferred to affect either the cleavage of the propeptide from the Gla domain, the stability of the kringle-1 and −2 domains, or the close association of the A and B chains of the serine protease domain. The dysprothrombinemia mutations were inferred to directly affect catalytic function through their location at the active site crevice or exosite 1 within the serine protease domain.
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Chen, Yu. "Homomorphisms of two-dimensional linear groups over Laurent polynomial rings and Gaussian domains." Mathematical Proceedings of the Cambridge Philosophical Society 109, no. 2 (March 1991): 287–97. http://dx.doi.org/10.1017/s0305004100069759.
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Let GL2(R) be the general linear group of 2 × 2 invertible matrices in M2(R) over a commutative ring R with 1 and SL2(R) be the special linear group consisting of 2 × 2 matrices over R with determinant 1. In this paper we determine the homomorphisms from GL2 and SL2, as well as their projective groups, over Laurent polynomial rings to those groups over Gaussian domains, i.e. unique factorization domains (cf. Theorems 1, 2, 3 below). We also consider more generally the homomorphisms of non-projective groups over commutative rings containing a field which are generated by their units (cf. Theorems 4 and 5). So far the homomorphisms of two-dimensional linear groups over commutative rings have only been studied in some specific cases. Landin and Reiner[7] obtained the automorphisms of GL2(R), where R is a Euclidean domain generated by its units. When R is a type of generalized Euclidean domain with a degree function and with units of R and 0 forming a field, Cohn[3] described the automorphisms of GL2(R). Later, Cohn[4] applied his methods to the case of certain rings of quadratic integers. Dull[6] has considered the automorphisms of GL2(R) and SL2(R), along with their projective groups, provided that R is a GE-ring and 2 is a unit in R. McDonald [9] examined the automorphisms of GL2(R) when R has a large unit group. The most recent work of which we are aware is that of Li and Ren[8] where the automorphisms of E2(R) and GE2(R) were determined for any commutative ring R in which 2, 3 and 5 are units.
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Preston, Roger J. S., Eva Ajzner, Cristina Razzari, Stalo Karageorgi, Sonia Dua, Björn Dahlbäck, and David A. Lane. "Multifunctional Specificity of the Protein C/Activated Protein C Gla Domain." Journal of Biological Chemistry 281, no. 39 (July 25, 2006): 28850–57. http://dx.doi.org/10.1074/jbc.m604966200.
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Wang, Stephanie X., Eugene Hur, Carolyn A. Sousa, Linda Brinen, Eric J. Slivka, and Robert J. Fletterick. "The Extended Interactions and Gla Domain of Blood Coagulation Factor Xa†." Biochemistry 42, no. 26 (July 2003): 7959–66. http://dx.doi.org/10.1021/bi027320a.
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Lind, Bent, Anders H. Johnsen, and Sixtus Thorsen. "Naturally Occurring Arg−1 to His Mutation in Human Protein C Leads to Aberrant Propeptide Processing and Secretion of Dysfunctional Protein C." Blood 89, no. 8 (April 15, 1997): 2807–16. http://dx.doi.org/10.1182/blood.v89.8.2807.
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Abstract The dysfunctional protein C from a thrombophilic patient heterozygote for a G1388 to A converting the codon for Arg−1 to His was purified from plasma and characterized. N-terminal amino acid sequence analysis of the light chain of the protein C demonstrated that the dysfunctional protein C is elongated with one amino acid, namely the mutated His. This finding is compatible with disruption by the mutated His of the original basic propeptidase recognition sequence (Arg−5-Ile-Arg-Lys-Arg−1), resulting in a shift of the cleavage site to a new position, Lys−2-His−1, which follows an alternative basic amino acid propeptidase recognition sequence (Arg−5-Ile-Arg-Lys−2). Because the mutation affects the propeptide that directs the γ-carboxylation converting Glu to Gla residues in the Gla domain, it was investigated whether the mutation impaired this reaction. Gla fragment obtained by cleavage of the dysfunctional protein C light chain with endoproteinase Asp-N was isolated by reverse-phase high-performance liquid chromatography, methylated, and subjected to N-terminal sequence analysis. The methylation step enabled the positive identification of Gla residues as well as the determination of the relative amount of Gla and Glu residues at each of the nine γ-carboxylation sites of the Gla domain. The analysis showed that all nine potential γ-carboxylation sites of the dysfunctional protein C were normally carboxylated. This result is compatible with the notion that position −1 is not a part of the recognition element for the γ-carboxylase. In conclusion, evidence is provided showing that the mutation leads to aberrant propeptide processing and secretion of dysfunctional normally carboxylated protein C extended with the mutated His.
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Francischetti, Ivo M. B., Jesus G. Valenzuela, John F. Andersen, Thomas N. Mather, and José M. C. Ribeiro. "Ixolaris, a novel recombinant tissue factor pathway inhibitor (TFPI) from the salivary gland of the tick, Ixodes scapularis: identification of factor X and factor Xa as scaffolds for the inhibition of factor VIIa/tissue factor complex." Blood 99, no. 10 (May 15, 2002): 3602–12. http://dx.doi.org/10.1182/blood-2001-12-0237.
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Saliva of the hard tick and Lyme disease vector, Ixodes scapularis, has a repertoire of compounds that counteract host defenses. Following sequencing of an I scapularis salivary gland complementary DNA (cDNA) library, a clone with sequence homology to tissue factor pathway inhibitor (TFPI) was identified. This cDNA codes for a mature protein, herein called Ixolaris, with 140 amino acids containing 10 cysteines and 2 Kunitz-like domains. Recombinant Ixolaris was expressed in insect cells and shown to inhibit factor VIIa (FVIIa)/tissue factor (TF)–induced factor X (FX) activation with an inhibitory concentration of 50% (IC50) in the picomolar range. In nondenaturing gel, Ixolaris interacted stoichiometrically with FX and FXa but not FVIIa. Ixolaris behaves as a fast-and-tight ligand of the exosites of FXa and γ-carboxyglutamic acid domainless FXa (des-Gla-FXa), increasing its amidolytic activity. At high concentration, Ixolaris attenuates the amidolytic activity of FVIIa/TF; however, in the presence of DEGR-FX or DEGR-FXa (but not des-Gla-DEGR-FXa), Ixolaris becomes a tight inhibitor of FVIIa/TF as assessed by recombinant factor IX (BeneFIX) activation assays. This indicates that FX and FXa are scaffolds for Ixolaris in the inhibition of FVIIa/TF and implies that the Gla domain is necessary for FVIIa/TF/Ixolaris/FX(a) complex formation. Additionally, we show that Ixolaris blocks FXa generation by endothelial cells expressing TF. Ixolaris may be a useful tool to study the structural features of FVIIa, FX, and FXa, and an alternative anticoagulant in cardiovascular diseases.
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Pavani, Giulia, Lacramioara Ivanciu, Katherine A. Stafford, and Paris Margaritis. "A Single Amino Acid in the Gla Domain Mouse FVIIa Allows Its Binding to the Endothelial Protein C Receptor and Enhances Its Coagulant Activity In Vivo." Blood 126, no. 23 (December 3, 2015): 3489. http://dx.doi.org/10.1182/blood.v126.23.3489.3489.
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Abstract The human protein C (PC) interaction with the endothelial PC receptor (EPCR) is mediated through the PC Gla domain, via key amino acids Phe4 and Leu8. Specifically, substitution of Leu8 with Val from human prothrombin abolishes the PC-EPCR interaction. The Gla domain of human Factor VII (FVII) shares these positions with PC and, consequently, the EPCR binding capacity. In the mouse, a commonly used in vivo model, the sequence determinants of the Gla domain of mouse PC (mPC) interaction with mouse EPCR (mEPCR) are not known. Remarkably, mouse FVII (mFVII) and its activated form (mFVIIa) have poor affinity for mouse EPCR. We previously described a variant of mFVIIa (mFVIIa-FMR) that contained the Leu4->Phe, Leu8->Met and Trp9->Arg from the mPC Gla domain. We found that this molecule was functionally similar to mFVIIa and could bind mEPCR. Using mFVIIa-FMR as surrogate to study the mPC-mEPCR interaction, we highlighted the importance of the Phe4/Met8/Arg9 in the mPC-mEPCR interaction. We also found that mFVIIa-FMR had enhanced hemostatic properties when infused at 3 mg/kg after FeCl3 carotid artery injury in hemophilic mice (vessel occlusion was 2.5 times faster than mFVIIa). In order to further refine whether the mEPCR binding capacity of mPC is coordinated by any/all of Phe4/Met8/Arg9 positions, we previously generated single variants of mPC at these positions using the corresponding amino acids of mFVIIa (that has poor interaction with mEPCR). We found that Phe4 is the sole determinant of specificity of the mPC-mEPCR interaction. Moreover, when Phe4 was placed in mFVIIa, we found that mFVIIa-Phe4 had activity similar to mFVIIa and bound mEPCR on cells (or in solution to soluble mEPCR) with a Kd of ~350nM. This was of similar magnitude to the mEPCR affinity of a mFVIIa variant with the entire mPC Gla domain (~200 nM), indirectly suggesting that Phe4 determines both the specificity and affinity of mPC to mEPCR. Since mFVIIa-FMR showed improved hemostatic properties in vivo as a result of mEPCR binding, enhancing the EPCR-FVIIa binding may generate improved human FVIIa molecules for the treatment of bleeding. Here we wanted to provide proof-of-concept using limited Gla domain modifications. For this, we utilized mFVIIa-Phe4, a minimally modified mFVIIa molecule, described above. Specifically, hemophilia B animals were subjected to a 7.5% FeCl3 injury of their carotid artery for 2 minutes; after 10 minutes mice were infused with 3 mg/kg of mFVIIa or mFVIIa-Phe4. Time to vessel occlusion was determined by monitoring blood flow. Hemostatically normal mice occluded in 13.3 ± 3.0 min. We found that infusion of mFVIIa resulted in vessel occlusion at 8.9 ± 1.7 min. However, mice that received mFVIIa-Phe4 reached vessel occlusion within 4.5 ± 2.4 min, ~2.5 times faster than mFVIIa-infused mice (P<0.01). This was similar to that we previously observed with mFVIIa-FMR infusion after injury in hemophilia B mice. Our results suggest the following: (1) Phe4 in the mPC Gla domain confers the specificity and affinity to mEPCR; (2) a single Phe4 substitution in mFVIIa is the only requirement for enhancing its clotting function in vivo. These data reveal another difference between human and mouse systems that may affect EPCR-dependent functions of other vitamin K-dependent proteins. Moreover, our results suggest the possibility that minimally modified variants of FVIIa with respect to EPCR binding may have more favorable hemostatic properties for clinical use. Disclosures Ivanciu: Bayer Hemophilia Awards Program: Research Funding. Margaritis:Novo Nordisk A/S: Research Funding; Bayer Hemophilia Awards Program: Research Funding.
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Souri, Masayoshi, Hiroki Iwata, Wei Guang Zhang, and Akitada Ichinose. "Unique secretion mode of human protein Z: its Gla domain is responsible for inefficient, vitamin K–dependent and warfarin-sensitive secretion." Blood 113, no. 16 (April 16, 2009): 3857–64. http://dx.doi.org/10.1182/blood-2008-07-171884.
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Abstract Protein Z is a vitamin K–dependent plasma glycoprotein that is involved in the regulation of blood coagulation. Plasma concentrations of protein Z vary widely between subjects and are greatly reduced during warfarin therapy. We developed a sensitive and quantitative assay for protein secretion using a secretory luciferase to explore the mode of secretion of protein Z compared with that of factor X. Protein Z secretion was much less efficient than factor X and was totally dependent upon added vitamin K, while factor X secretion was not. Protein Z secretion was highly sensitive to warfarin treatment of the synthesizing cells. In contrast, although factor X secretion was not precluded by warfarin, its γ-carboxylation was completely blocked. An exchange of the propeptide and/or γ-carboxyglutamic acid domain between protein Z and factor X reproduced the inefficient and warfarin-sensitive secretion pattern of protein Z, and vice versa. Joining of the propeptide and γ-carboxyglutamic acid domain to luciferase also demonstrated that the γ-carboxyglutamic acid domain of protein Z was responsible for its warfarin-sensitive secretion. Thus, it was concluded that the difference observed in secretion patterns of protein Z and factor X was mainly based on the structure of their γ-carboxyglutamic acid domains.
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Davis-Harrison, Rebecca L., Mary Clay, Chad M. Rienstra, and James H. Morrissey. "Molecular Basis of Phospholipid Synergy in Promoting Blood Coagulation Reactions." Blood 120, no. 21 (November 16, 2012): 1109. http://dx.doi.org/10.1182/blood.v120.21.1109.1109.
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Abstract Abstract 1109 Most steps in the blood coagulation cascade obligatorily take place on membrane surfaces and are dependent on the exposure of phosphatidylserine (PS). Many coagulation proteins bind to PS-containing membrane bilayers in a calcium-dependent manner via gamma-carboxyglutamate-rich (GLA) domains. Furthermore, certain phospholipids, most notably phosphatidylethanolamine (PE), strongly synergize with PS to promote clotting reactions. We have recently been investigating the molecular basis of lipid-Ca2+ and lipid-GLA interactions in blood clotting using solid-state NMR (SSNMR) studies that employ isotopically labeled PS. Our studies have revealed that Ca2+ induces two predominant conformations of the PS headgroup (Boettcher et al., Biochem. 50:2264–73, 2011). We now report the quantification of the intermolecular and intramolecular atomic distances within Ca2+-induced PS nanoclusters using SSNMR. We have also expanded SSNMR studies to phospholipid bilayers composed of trinary mixtures of PS, PC, and PE as a means to further investigate the impact of PE on PS headgroup conformations in an intact lipid bilayer (PE-PS synergy). Initial results suggest that incorporation of PE into the bilayer alters the Ca2+: PS ratio required to fully saturate the L-serine headgroups of PS. Additionally, PE appears to affect the predominant PS headgroup conformations in the presence of Ca2+, and may induce a third L-serine conformation. SSNMR studies utilizing isotopically labeled PE headgroups are currently underway which should provide novel information concerning PE's interactions with PS, Ca2+, and/or GLA domains. Previous SSNMR studies from our lab have also indicated that a fraction of L-serine headgroups in PS adopt a new conformation when bovine prothrombin fragment 1 is bound to PS/PC bilayers (Tavoosi et al., J. Biol. Chem. 286:23247–53, 2011). These data support the idea that GLA domains contain an L-serine-specific binding site. We have now bound human prothrombin fragment 1 to membranes containing labeled PS headgroups for use in SSNMR studies. In addition, we have also employed surface plasmon resonance (SPR) experiments to investigate the interactions of both human and bovine prothrombin with bilayers containing PS and PE. These data demonstrate that binding affinities and binding footprints are similar for both prothrombin proteins, and that PE enhances binding affinities and increases the number of GLA binding sites for both proteins. Together, results from SSNMR and SPR studies suggest that different GLA domains use similar molecular mechanisms to interact with the membrane surface. Disclosures: No relevant conflicts of interest to declare.
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Mille-Baker, Blandine, Suely M. Rezende, Rachel E. Simmonds, Philip J. Mason, David A. Lane, and Michael A. Laffan. "Deletion or replacement of the second EGF-like domain of protein S results in loss of APC cofactor activity." Blood 101, no. 4 (February 15, 2003): 1416–18. http://dx.doi.org/10.1182/blood-2002-08-2353.
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Human protein S (PS), a cofactor of anticoagulant-activated protein C (APC), is a modular protein containing 4 epidermal growth factor (EGF)–like domains. EGF1 appears to mediate PS interaction with APC, but the roles of EGFs 2, 3, and 4 are less clear. We synthesized PS variants lacking single EGF domains (EGF2, 3, or 4) and assessed their APC cofactor activity in a factor Va inactivation assay. The variant lacking EGF2 (variant 134) showed the most dramatic loss of activity (∼10% of recombinant wild-type PS activity). Replacement of EGF2 by an additional EGF3 (variant 1334) resulted in a comparable loss of activity, suggesting that the loss of a specific rather than “spacer” function of EGF2 was responsible. We confirmed that the variant 134 had a functional γ-carboxyglutamic acid (Gla) domain and that EGF1 was correctly folded. This is the first clear evidence that EGF2 is required for the expression of PS activity.
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Mason, A. W. "Groups generated by elements with rational fixed points." Proceedings of the Edinburgh Mathematical Society 40, no. 1 (February 1997): 19–30. http://dx.doi.org/10.1017/s0013091500023403.
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Let R be a commutative integral domain and let S be its quotient field. The group GL2(R) acts on Ŝ = S ∪ {∞} as a group of linear fractional transformations in the usual way. Let F2(R, z) be the stabilizer of z ∈ Ŝ in GL2(R) and let F2(R) be the subgroup generated by all F2(R, z). Among the subgroups contained in F2(R) are U2(R), the subgroup generated by all unipotent matrices, and NE2(R), the normal subgroup generated by all elementary matrices.We prove a structure theorem for F2(R, z), when R is a Krull domain. A more precise version holds when R is a Dedekind domain. For a large class of arithmetic Dedekind domains it is known that the groups NE2(R),U2(R) and SL2(R) coincide. An example is given for which all these subgroups are distinct.
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Arcario, Mark J., and Emad Tajkhorshid. "Characterizing Membrane-Bound Forms of Coagulation Factors at Atomic Resolution: Exploring Differences In Membrane Affinity." Blood 116, no. 21 (November 19, 2010): 1139. http://dx.doi.org/10.1182/blood.v116.21.1139.1139.
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Abstract Abstract 1139 Membrane binding constitutes a key step in the activation of coagulation factors. This process largely hinges on specialized “floating” domains, most prominently the GLA and the C1/C2 domains found in almost all coagulation proteins. Currently, the details of how these domains interact with the membrane are largely unknown, due primarily to the lack of atomic models describing the proteins' membrane-bound forms. In sharp contrast to integral membrane proteins, studying insertion of membrane floating proteins has proven extremely challenging, mainly due to the unknown depth of membrane penetration. Although several computational studies have attempted to study the process using conventional models of the membrane, these methods are often prohibitively costly and inefficient due, in part, to the slow diffusion of the lipid molecules, which is required to accommodate the insertion of the floating domain. Here, we present a study on the membrane-bound forms of several important hemostasis factors, namely, human factor IX (hfIX), human factor × (hfX), human protein C (hPrC), human protein S (hPrS), and human protein Z (hPrZ), which utilize the GLA domain for membrane binding, as well as human factor V (hfV) and human factor VIII (hfVIII) in which the C1/C2 domain plays this role. To make the binding event accessible to molecular dynamics (MD) simulations, we have developed a novel membrane mimetic system, composed of a highly mobile hydrophobic core and explicit head groups that allow one to study specific interactions between the floating domains and lipid molecules. These interactions determine not only the specificity of binding to certain biological membranes, but also the molecular basis of the variable binding affinity of coagulation factors. These two factors are of utmost importance in physiology of blood coagulation, in designing mutations of altered affinity, and in developing novel pharmacological agents for thrombotic disorders. The floating domains were placed in the membrane-mimetic system and simulated under physiological conditions. Given the efficiency of the method, we have been able to capture spontaneous binding of these coagulation factors to the membrane (Figure 1) multiple times, despite starting from various initial orientations ensuring that each coagulation factor converged to the same final, membrane-bound structure over multiple trials. From these simulations, we have been able to elucidate the residues important for association with the membrane, as well as several unique phosphatidylserine (PS) specific binding sites in hfIX, hfX, hPrC, hPrS, and hPrZ. Using the membrane-bound structures of these coagulation factors together with free energy calculations, we have been able to elucidate important differences between the binding of high-affinity and low-affinity coagulation factors to the membrane. Disclosures: No relevant conflicts of interest to declare.
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Agah, Sayeh, Amanda Sutton, William H. Velander, and S. Paul Bajaj. "Role of Mg2+ in Extrinsic and Intrinsic Coagulation Under Physiologic Conditions." Blood 112, no. 11 (November 16, 2008): 2024. http://dx.doi.org/10.1182/blood.v112.11.2024.2024.
Full textAbstract:
Abstract Ca2+ is an obligatory factor for both the extrinsic and intrinsic pathways of coagulation. In majority of in vitro studies, investigators use saturating concentrations of Ca2+ (5 to 10 mM) for FVIIa/tissue factor (TF) activation of factor IX (FIX), and factor X (FX) (extrinsic coagulation), as well as for the activation of FIX by FXIa, FX by FIXa/FVIIIa, and prothrombin by FXa/FVa (intrinsic coagulation). However, the concentration of Ca2+ in plasma is only 1.1 mM, which is considerably below the saturating concentration needed for optimal coagulation. Importantly, plasma also contains 0.6 mM Mg2+ that could compensate for subsaturating concentrations of Ca2+ in promoting coagulation. Previous studies have attempted to clarify this concept in FIX, FX and prothrombin activation. However, these studies are sparse and in virtually all cases not detailed. We have systematically examined the role of plasma concentration of Mg2+ (in addition to the plasma concentration of Ca2+) in promoting all Ca2+ dependent steps of extrinsic and intrinsic coagulation and compared it with the saturating concentration of Ca2+. The Km (~ 100 nM) for activation of FIX by FXIa was similar in the presence of plasma concentrations of Ca2+/Mg2+ or 5 mM Ca2+. Furthermore, the Km and Vmax for the activation of FX and FIX by FVIIa/TF were essentially similar for both conditions. The Km and Vmax for the activation of FX by FVIIIa/FIXa ± phospholipid, and prothrombin by FXa/FVa ± phospholipid were also indistinguishable in these two different metal ion conditions. Notably, when only plasma concentrations of Ca2+ (1.1mM or 1.7mM) were used in all reactions mentioned above, coagulation proceeded at suboptimal rates. In further studies, we used Biacore to investigate the binding of FXIa and FVIII to FIXa, soluble TF to FVIIa, and FVa to FXa. Soluble TF, dansyl-Glu-Gly-Arg (dEGR)-IXa and dEGR-Xa were coupled to CM5 chips in the presence of 10 mM Ca2+. At 5 mM Ca2+, the binding of FXIa to dEGR-IXa was characterized by a Kd of ~40 nM, binding of FVIII to dEGR-IXa by a Kd of ~100 nM, and FVa to dEGR-Xa by a Kd of ~120 nM. In the presence of plasma concentrations of Ca2+ and Mg2+, binding constants were similar to those obtained in the presence of 5 mM saturating Ca2+ concentration. Additional 45Ca2+ binding studies using equilibrium dialysis and prothrombin fragment 1, dEGR-VIIa and decarboxylated dEGR-VIIa, FIX and decarboxylated FIX, and FX and decarboxylated FX, indicated that in the g-carboxyglutamic acid (Gla) domain, 2-3 Ca2+ binding sites (Shikimoto, et al., J. Biol. Chem. 278, 24090-24094, 2003; Wang, et al., Biochemistry42, 7959-7966, 2003; Bajaj, et al., J. Biol. Chem.281, 24873-24888, 2006) out of seven core divalent ion binding sites (Soriano-Garcia et al., Biochemistry31, 2554-2566, 1992) could be replaced by Mg2+. Conversely, Mg2+ could not displace the Ca2+ binding sites in the epidermal growth factor-like domain 1 (EGF1) and protease domains of FIX or FX. Overall these studies indicate that (1) saturating concentrations of Ca2+ used in in vitro investigations are valid representations of coagulation studies, except for that Mg2+ compensates for suboptimal concentrations of Ca2+ under physiological conditions; (2) two of the Ca2+-binding sites in the Gla domain (numbers 1 and 7, per Tulinsky numbering (Soriano-Garcia et al., Biochemistry31, 2554-2566, 1992)), and possibly a third site (number 4) are specific for Mg2+ under physiologic conditions; and (3) the Ca2+-binding sites in the EGF1 and protease domains are specific for Ca2+ and can not be occupied by Mg2+ under physiologic conditions. In conclusion, Ca2+ and Mg2+ act in concert to promote optimal coagulation under physiologic conditions. Mg2+ alone does not promote coagulation since it cannot bind to the Ca2+ specific sites in the Gla domain necessary for folding of the Gla domain omega loop.
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Yang, Likui, and Alireza Rezaie. "Calcium-binding sites of the thrombin-thrombomodulin-protein C complex: Possible implications for the effect of platelet factor 4 on the activation of vitamin K-dependent coagulation factors." Thrombosis and Haemostasis 97, no. 06 (2007): 899–906. http://dx.doi.org/10.1160/th06-12-0697.
Full textAbstract:
SummaryThe Ca2+-dependence of protein C activation by thrombin in complex with thrombomodulin (TM) containing chondroitin sulfate (CS) exhibits saturation at ~0.5–1 mM Ca2+, but withTM lacking CS, it has a distinct optimum at ~0.1 mM Ca2+. Since the substrate protein C has multiple Ca2+-binding sites, and the cofactor TM also interacts with Ca2+, the basis for differences in Ca2+ effect on protein C activation by thrombin in complex with TM containing or lacking CS is not known. In this study, by using full-length and Gla-domainless mutants of protein C whose activation by thrombin is independent of either Ca2+ or both Ca2+ and TM, we demonstrate that i) the Ca2+ occupancy of a high-affinity binding site in TM is essential for the high-affinity interaction of the cofactor with thrombin, ii) the Ca2+ occupancy of a binding site (KD ~50 μM) in the catalytic domain of protein C is required for the substrate recognition by the thrombin-TM complex, however, at this concentration of Ca2+ the Gla domain of protein C is not folded properly and thus interacts with exosite-2 of thrombin in complex with TM that lacks CS but not withTM that contains CS, and finally iii) platelet factor 4 can nonspecifically interact with the Gla domain of protein C and other coagulation factors to influence their activation only at subphysiological concentrations of Ca2+.