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International Journal of Biological... Apr 2018A dysfunctional prothrombin gene characterized by novel point mutation at Arg553 to Gln residue in Deep vein thrombosis (DVT) patient which we designated as "Prothrombin...
A dysfunctional prothrombin gene characterized by novel point mutation at Arg553 to Gln residue in Deep vein thrombosis (DVT) patient which we designated as "Prothrombin Amrita" was previously reported from our lab. The mutation occurred at nucleotide 20030 in exon 14 and was confirmed by restriction enzyme digestion. Arg553 has been reported as one of the key residues for the binding of cofactor Na ion in the thrombin protein. Structural analysis revealed the molecular mechanism behind the coagulant form of thrombin due to point Arg553Gln mutation near the cofactor Na ion region. Molecular electrostatic potential maps and molecular dynamics (MD) simulation of the wild type and mutated thrombin showed the key role played by the Na ion for its coagulant mechanism by analysing the charge distribution and nature of the hydrogen bonding at the mutated region of interest. We observed maintenance of the fast or procoagulant form of dysfunctional prothrombin due to changes in the charge distribution by this mutation and thereby also keeping strong hydrogen bonding network revealed by MD simulation between prothrombin and Na ion. This molecular mechanism might be the main cause for DVT in patients with this dysfunctional prothrombin gene.
Topics: Amino Acid Substitution; Humans; Molecular Dynamics Simulation; Point Mutation; Protein Domains; Prothrombin; Structure-Activity Relationship; Venous Thrombosis
PubMed: 29382582
DOI: 10.1016/j.ijbiomac.2018.01.084 -
The Journal of Biological Chemistry Oct 1988The present study investigates the unique contribution of the NH2-terminal 33 residues of prothrombin, the gamma-carboxyglutamic acid (Gla) domain, to the Ca(II) and...
The present study investigates the unique contribution of the NH2-terminal 33 residues of prothrombin, the gamma-carboxyglutamic acid (Gla) domain, to the Ca(II) and phospholipid-binding properties of prothrombin. Two Gla domain peptides, 1-42 and 1-45, produced by chymotryptic cleavage of prothrombin fragment 1 (residues 1-156 of the amino terminus of bovine prothrombin) and isolated by anion-exchange chromatography were utilized to characterize the Gla domain of prothrombin. This investigation utilized several experimental approaches to examine the properties of the Gla domain peptides. These studies were somewhat hampered by the metal ion-induced insolubility of the peptides. However, the 1-45 peptide was specifically radioiodinated, which facilitated the study of this peptide at low concentrations. In contrast to prothrombin fragment 1, the intrinsic fluorescence of both 1-42 and 1-45 was not quenched upon the addition of 1 mM Ca(II) or any concentration of Mg(II). Equilibrium dialysis studies revealed that the 1-42 peptide bound three Ca(II) ions noncooperatively, whereas fragment 1 binds seven Ca(II) ions in a positive cooperative manner. Ca(II)-promoted conformational changes are observed by comparison of electrophoretic mobility changes in the presence of increasing Ca(II) concentrations. Prothrombin, fragment 1, and the Gla domain peptides 1-42 and 1-45 exhibited similar electrophoretic mobility behavior in the presence of Ca(II) ions. The radiolabeled 1-45 peptide was found to comigrate with phospholipid vesicles on gel permeation chromatography in the presence of Ca(II). Fragment 1 was shown to inhibit this Ca(II)-dependent phospholipid binding of 1-45, demonstrating that the 1-45 peptide does possess the necessary phospholipid-binding structure. Furthermore, a metal ion-dependent conformational monoclonal antibody, F9.29, was inhibited from binding fragment 1 by the 1-42 peptide.
Topics: Amino Acid Sequence; Animals; Calcium; Cattle; Chymotrypsin; Kinetics; Magnesium; Peptide Fragments; Phospholipids; Protein Binding; Prothrombin
PubMed: 3170544
DOI: No ID Found -
European Journal of Biochemistry Jan 2000There is considerable interest in determining the role of prothrombin fragments, especially urinary prothrombin fragment 1 (UPTF1), in the pathogenesis of calcium...
There is considerable interest in determining the role of prothrombin fragments, especially urinary prothrombin fragment 1 (UPTF1), in the pathogenesis of calcium oxalate (CaOx) urinary calculi. This fragment is present in abundance in the matrix of CaOx crystals generated in human urine in vitro and has also been detected in human urinary stones containing calcium. More recently, prothrombin gene expression has been reported in the human kidney. However, studies examining the renal biosynthesis of prothrombin or perhaps only its fragments during experimental lithogenesis, and in consequence, the role of UPTF1 in stone formation, cannot be carried out in humans. The aim of this investigation therefore was to determine whether prothrombin gene expression is present in the rat kidney. Total RNA was isolated from the kidneys and livers of 12 rats. Using reverse transcriptase PCR, mRNAs corresponding to the thrombin and fragment 1 + 2 (F1+2) regions of prothrombin were analysed by agarose gel electrophoresis. The expression of glyceraldehyde 3-phosphate dehydrogenase was also examined to determine whether the quality of the tissue mRNAs was adequate for analyses. The amplified products were identified by sequence analysis. All kidneys displayed evidence of expression of the thrombin and F1+2 domains of the prothrombin gene. Furthermore, the sequences of these PCR-derived products from kidney were identical to those from liver. This suggests that the prothrombins secreted by these two organs are identical. The fact that prothrombin biosynthesis occurs in both the human and rat kidney presents an opportunity for using established rat models of stone disease to evaluate the influence of lithogenic conditions on prothrombin gene expression, and the potential role of UPTF1 in vivo.
Topics: Animals; Base Sequence; Disease Models, Animal; Gene Expression; Gene Expression Profiling; Kidney; Liver; Male; Molecular Sequence Data; Peptide Fragments; Prothrombin; RNA, Messenger; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; Sequence Analysis, DNA; Thrombin; Urinary Calculi
PubMed: 10601851
DOI: 10.1046/j.1432-1327.2000.00954.x -
Thrombosis and Haemostasis Sep 1998Several studies indicated that activation of the clotting system may promote the growth and the invasive behavior of tumor cells. In the present study, we evaluated the...
Several studies indicated that activation of the clotting system may promote the growth and the invasive behavior of tumor cells. In the present study, we evaluated the migratory response of various melanoma cell lines to several clotting factors and prothrombin derivatives (thrombin, fragment 1, fragment 2 and kringle 1 fragment). Prothrombin, thrombin and fragment 1 stimulated chemotaxis of the murine (K-1735 M2, X21) and human A375 (SM) melanoma cell lines. Prothrombin and prothrombin fragment 1 showed their maximal chemotactic activity at 0.5 approximately 1 microM. Chemotaxis induced by thrombin was inhibited by hirudin, but not that induced by prothrombin or fragment 1. Other clotting proteins and the fragment 2 and kringle 1 fragment of prothrombin did not elicit chemotactic activity. Checkerboard analysis indicated that motility was directional with a significant chemokinetic component. The K-1735 M2 cells also migrated in a concentration-dependent manner to substratum-bound insoluble prothrombin, thrombin or fragment 1. Ligand binding assays showed that both prothrombin and fragment 1 bound to K-1735 M2 cells with apparent Kds of 0.5 microM. This binding was inhibited by an excess concentration of unlabeled prothrombin and fragment 1 but not by similar concentrations of other prothrombin fragments. These findings suggest that prothrombin and its fragment 1 exert chemotactic activity on melanoma cells by different mechanisms and different binding sites from that induced by thrombin.
Topics: Animals; Cell Movement; Humans; Melanoma; Mice; Neoplasm Invasiveness; Peptide Fragments; Prothrombin; Skin Neoplasms; Tumor Cells, Cultured
PubMed: 9759619
DOI: No ID Found -
Thrombosis Research Aug 1991The hepatic asialoglycoprotein receptor (AGPR) system can efficiently internalize and degrade circulating glycoproteins which lack terminal sialic acids on their...
The hepatic asialoglycoprotein receptor (AGPR) system can efficiently internalize and degrade circulating glycoproteins which lack terminal sialic acids on their carbohydrate chains. Since pro-thrombin is a glycosylated plasma protein, possible involvement of AGPR system in its clearance from circulation was evaluated. The half lives of bovine 125I-prothrombin and 125I-asialoprothrombin, injected intravenously into rats, were 192 and 1.8 minutes, respectively. Asialoprothrombin appeared to be cleared by the hepatic AGPRs since 33% of it accumulated in the liver at 30 minutes and its clearance was competitively blocked by simultaneous administration of increasing amounts of asialofetuin. Only 5% of prothrombin accumulated in the liver at 3 hours and injections asialofetuin in amounts capable of saturating the AGPR for the duration of four asialoprothrombin half lives had no effect on the disappearance of prothrombin. Our observations indicate that, although asialoprothrombin is readily cleared from plasma by the AGPR system, prothrombin is not. Thus these receptors do not appear to be involved in physiological processes that control prothrombin half life.
Topics: Animals; Asialoglycoprotein Receptor; Cattle; Half-Life; Humans; Liver; Male; Metabolic Clearance Rate; Prothrombin; Rats; Rats, Inbred Strains; Receptors, Immunologic
PubMed: 1957274
DOI: 10.1016/0049-3848(91)90133-h -
The Journal of Biological Chemistry Dec 1979
Topics: Amino Acids; Animals; Antibodies; Antigen-Antibody Reactions; Cattle; Epitopes; Immunoassay; Immunodiffusion; Prothrombin; Radioimmunoassay; Warfarin
PubMed: 91611
DOI: No ID Found -
Biochimie 1976A procedure for the preparation of highly purified pig prothrombin is described. Compared to the initial clotting activity of the starting plasma, this protein was...
A procedure for the preparation of highly purified pig prothrombin is described. Compared to the initial clotting activity of the starting plasma, this protein was purified 776 times with a final yield of 8 per cent. The purified zymogen showed a specific activity of 1,460 NH units/mg of protein , a molecular weight of 65,000 as determined by SDS-polyacrylamide disc gel electroesis, E 1.0 mg/ml 1.0 cm, 280 nm = 1.45 at pH 7.0 and the following amino acid composition: Asx 51, Thr 38, Glx 62, Pro 23, Gly44, Ala 25, Half-Cys 30, Val 35, Met 3, Ile 30, Leu 32, Tyr 19, Phe 22, Lys 36, His 8, Arg 28, and Trp 13, which accounts for a minimum molecular weight of 59,370 (carbohydrates not computed). Alanine was found as the only N-terminal residue. Carboxypeptidases A and B failed to release any C-terminal residue. By hydrazinolysis however 0.4 mole of serine was released per mole of prothrombin. The activation of crude and chromatographed pig prothrombin was investigated.
Topics: Amino Acids; Animals; Electrophoresis, Disc; Humans; Molecular Weight; Prothrombin; Species Specificity; Swine
PubMed: 953054
DOI: 10.1016/s0300-9084(76)80219-2 -
The Journal of Urology Jan 2005We investigated the effects of urinary prothrombin fragment 1 in the formation of calcium oxalate urolithiasis.
PURPOSE
We investigated the effects of urinary prothrombin fragment 1 in the formation of calcium oxalate urolithiasis.
MATERIALS AND METHODS
Fresh urine and renal parenchyma from patients with calcium oxalate calculus and normal controls were collected. Urinary prothrombin fragment 1 was isolated and purified from urine. It was identified by sodium dodecyl sulfide-polyacrylamide gel electrophoresis and analysis of its first 13 N-amino acids. The inhibitory activity of urinary prothrombin fragment 1 on calcium oxalate crystal growth was tested by the seeded crystallization technique. Meanwhile, the gamma-carboxyglutamic acid composition of urinary prothrombin fragment 1 was analyzed by a previously described method and genetic mutation of the gamma-carboxyglutamic acid domain of urinary prothrombin fragment 1 from renal parenchyma was detected by polymerase chain reaction-single strand conformational polymorphism sequencing.
RESULTS
The gamma-carboxyglutamic acid composition of urinary prothrombin fragment 1 was significantly decreased from normal (24.4 to 1.7 mol/1,000 amino acids) in patients with calcium oxalate calculus. The mean growth index +/- SD of urinary prothrombin fragment 1 to calcium oxalate crystals was 42.3 +/- 4.2 compared with the normal index of 19.2 +/- 2.8 (p <0.01). The polymerase chain reaction-single strand conformational polymorphism sequencing technique revealed no genetic mutation of the gamma-carboxyglutamic acid domain of urinary prothrombin fragment 1 in patients with calcium oxalate calculus.
CONCLUSIONS
The gamma-carboxyglutamic acid composition of urinary prothrombin fragment 1 as well as its ability to inhibit calcium oxalate crystal growth was significantly decreased in patients with calcium oxalate calculus. This was not caused by genetic mutation of the gamma-carboxyglutamic acid domain of urinary prothrombin fragment 1. It is important to elucidate the mechanisms of calcium oxalate stones in view of urinary prothrombin fragment 1.
Topics: 1-Carboxyglutamic Acid; Adult; Amino Acids; Calcium Oxalate; Electrophoresis, Polyacrylamide Gel; Humans; Middle Aged; Peptide Fragments; Polymerase Chain Reaction; Polymorphism, Single-Stranded Conformational; Protein Precursors; Prothrombin; Urinary Calculi
PubMed: 15592049
DOI: 10.1097/01.ju.0000146847.24571.c8 -
Purification and properties of a prothrombin activator from the venom of Notechis scutatus scutatus.The Journal of Biological Chemistry Aug 1985The prothrombin activator present in the venom of the mainland tiger snake (Notechis scutatus scutatus) was purified to homogeneity by gel chromatography on Sephadex...
The prothrombin activator present in the venom of the mainland tiger snake (Notechis scutatus scutatus) was purified to homogeneity by gel chromatography on Sephadex G-200 followed by ion-exchange chromatography on SP-Sephadex. The venom activator has an apparent molecular weight of 54,000. It consists of a heavy chain (Mr = 32,000) and a light chain (Mr = 23,000) held together by one or more disulfide bridges. The active site is located at the heavy chain region of the molecule. The venom activator contains 8 gamma-carboxyglutamic acid residues/molecule. Gel electrophoretic analysis of prothrombin activation indicates that the venom activator is capable of cleaving both the Arg 274-Thr 275 and Arg 323-Ile 324 bonds of bovine prothrombin. The order of bond cleavage appears to be random since prethrombin-2 and meizothrombin occur as intermediates during prothrombin activation. Prothrombin activation by the venom activator alone is very slow. This is explained by the unfavorable kinetic parameters for the reaction (Km for prothrombin = 105 microM, Vmax = 0.0025 nmol of prothrombin activated per min/microgram of venom activator). Phospholipids plus Ca2+ and Factor Va greatly stimulate venom-catalyzed prothrombin activation. In the presence of 50 microM phospholipid vesicles composed of 20 mol % phosphatidylserine and 80 mol % phosphatidylcholine, the Km drops to 0.2 microM, whereas there is hardly any effect on the Vmax. Factor Va causes a 3,500-fold increase of the Vmax (8.35 nmol of prothrombin activated per min/microgram of venom activator) and a 10-fold decrease of the Km (9.5 microM). The most favorable kinetic parameters are observed in the presence of both 50 microM phospholipid and Factor Va (Km = 0.16 microM, Vmax = 27.9 nmol of prothrombin activated per min/microgram of venom activator). These changes of the kinetic parameters explain the stimulatory effects of Factor Va and phospholipid on venom-catalyzed prothrombin activation. The venom activator slowly converts the Factor Xa-specific chromogenic substrates CH3SO2-D-leucyl-glycyl-L-arginine-p-nitroanilide and N-benzoyl-L-isoleucyl-L-glutamyl-(piperidyl)-glycyl-L-arginyl-p-nitroani lide hydrochloride. Factor Va causes a 7-fold stimulation of chromogenic substrate conversion by the venom activator. This stimulation appears to be the result of the formation of a tight 1:1 complex between the venom activator and Factor Va.
Topics: 1-Carboxyglutamic Acid; Animals; Calcium; Elapid Venoms; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Kinetics; Molecular Weight; Peptide Hydrolases; Phospholipids; Prothrombin; Snakes
PubMed: 3894355
DOI: No ID Found -
Journal of Chromatography. A Jan 2014Prothrombin (coagulation Factor II) is a complex multidomain glycoprotein that plays a central role in blood coagulation. It is the zymogen precursor to the protease...
Prothrombin (coagulation Factor II) is a complex multidomain glycoprotein that plays a central role in blood coagulation. It is the zymogen precursor to the protease thrombin that catalyzes the formation of the fibrin clot and regulates a multitude of other cellular responses related to coagulation and hemostasis. For the biological activity of prothrombin, the vitamin K dependent posttranslational modification of glutamic acid residues to gamma-carboxylglutamic acid is of crucial importance. Prothrombin can be recombinantly expressed using mammalian cell culture. However, the product is a heterogeneous mixture of variants with different degrees of carboxylation, requiring separation of closely related charge isoforms. A second challenge for purification is the need to remove traces of the product-related impurity thrombin, a protease, to extremely low levels. In this work, we describe a purification strategy that provides solutions to both challenges and results in an efficient and robust process for active recombinant prothrombin. We also describe the analytical characterization of recombinant prothrombin by HPLC, LC-MS/MS, and complementary biochemical assays.
Topics: Amino Acid Sequence; Chromatography, High Pressure Liquid; Humans; Molecular Sequence Data; Protein Processing, Post-Translational; Prothrombin; Recombinant Proteins; Tandem Mass Spectrometry
PubMed: 24369997
DOI: 10.1016/j.chroma.2013.12.024