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Annals of Internal Medicine Oct 1976
Topics: Blood Coagulation; Disseminated Intravascular Coagulation; Dose-Response Relationship, Drug; Hemophilia A; Humans; Prothrombin
PubMed: 970783
DOI: 10.7326/0003-4819-85-4-531 -
The Journal of Biological Chemistry Aug 2013The zymogen prothrombin is composed of fragment 1 containing a Gla domain and kringle-1, fragment 2 containing kringle-2, and a protease domain containing A and B...
The zymogen prothrombin is composed of fragment 1 containing a Gla domain and kringle-1, fragment 2 containing kringle-2, and a protease domain containing A and B chains. The prothrombinase complex assembled on the surface of platelets converts prothrombin to thrombin by cleaving at Arg-271 and Arg-320. The three-dimensional architecture of prothrombin and the molecular basis of its activation remain elusive. Here we report the first x-ray crystal structure of prothrombin as a Gla-domainless construct carrying an Ala replacement of the catalytic Ser-525. Prothrombin features a conformation 80 Å long, with fragment 1 positioned at a 36° angle relative to the main axis of fragment 2 coaxial to the protease domain. High flexibility of the linker connecting the two kringles suggests multiple arrangements for kringle-1 relative to the rest of the prothrombin molecule. Luminescence resonance energy transfer measurements detect two distinct conformations of prothrombin in solution, in a 3:2 ratio, with the distance between the two kringles either fully extended (54 ± 2 Å) or partially collapsed (≤34 Å) as seen in the crystal structure. A molecular mechanism of prothrombin activation emerges from the structure. Of the two sites of cleavage, Arg-271 is located in a disordered region connecting kringle-2 to the A chain, but Arg-320 is well defined within the activation domain and is not accessible to proteolysis in solution. Burial of Arg-320 prevents prothrombin autoactivation and directs prothrombinase to cleave at Arg-271 first. Reversal of the local electrostatic potential then redirects prothrombinase toward Arg-320, leading to thrombin generation via the prethrombin-2 intermediate.
Topics: Crystallography, X-Ray; Energy Transfer; Models, Molecular; Protein Conformation; Prothrombin; Static Electricity
PubMed: 23775088
DOI: 10.1074/jbc.M113.466946 -
Biochemistry Dec 2002Antiphospholipid antibodies interact with phospholipid membranes via lipid binding plasma proteins, mostly, prothrombin and beta(2)-glycoprotein I. Using ellipsometry,...
Antiphospholipid antibodies interact with phospholipid membranes via lipid binding plasma proteins, mostly, prothrombin and beta(2)-glycoprotein I. Using ellipsometry, we characterized prothrombin-mediated binding of lupus anticoagulant (LA) positive IgG, isolated from patients with antiphospholipid syndrome, to phosphatidylserine (PS)-containing membranes. LA IgG did not bind to membranes in the absence of prothrombin, but addition of prothrombin resulted in high-affinity binding of prothrombin-LA IgG complexes; half-maximal binding was attained at IgG and prothrombin concentrations of 10 microg/mL and 4 nM, respectively. Adsorption to membranes containing 10-40 mol % PS revealed that membrane-bound rather than solution-phase prothrombin determines the adsorption kinetics. Depletion of prothrombin and LA IgG from the solution results in rapid desorption which is strongly inhibited by addition of prothrombin but not of LA IgG. Prothrombin-mediated adsorption of monovalent Fab1 fragments prepared from patient LA IgG was negligible, indicating that monovalent interaction between prothrombin and LA IgG is weak. The kinetics of adsorption and desorption indicate that divalent binding of LA IgG to prothrombin at the lipid membrane occurs.
Topics: Antibodies, Antiphospholipid; Binding Sites, Antibody; Biosensing Techniques; Humans; Immunoassay; Immunoglobulin G; Immunosorbents; Kinetics; Lipid Bilayers; Lupus Coagulation Inhibitor; Phosphatidylserines; Protein Binding; Prothrombin; Silicon
PubMed: 12450402
DOI: 10.1021/bi026408l -
Scientific Reports Feb 2019The fragment 2 domain (F2) of prothrombin and its interaction with factor (F) Va is known to contribute significantly to prothrombinase-catalyzed activation of...
The fragment 2 domain (F2) of prothrombin and its interaction with factor (F) Va is known to contribute significantly to prothrombinase-catalyzed activation of prothrombin. The extent to which the F2-FVa interaction affects the overall thrombin generation, however, is uncertain. To study this interaction, nuclear magnetic resonance spectroscopy of recombinant F2 was used to identify seven residues within F2 that are significantly responsive to FVa binding. The functional role of this region in interacting with FVa during prothrombin activation was verified by the FVa-dependent inhibition of thrombin generation using peptides that mimic the same region of F2. Because six of the seven residues were within a 9-residue span, these were mutated to generate a prothrombin derivative (PT6). These mutations led to a decreased affinity for FVa as determined by surface plasmon resonance. When thrombin generation by an array of FXa containing prothrombinase components was monitored, a 54% decrease in thrombin generation was observed with PT6 compared with the wild-type, only when FVa was present. The functional significance of the specific low-affinity binding between F2 and FVa is discussed within the context of a dynamic model of molecular interactions between prothrombin and FVa engaging multiple contact sites.
Topics: Amino Acid Sequence; Binding Sites; Factor Va; Humans; Kinetics; Peptide Fragments; Protein Binding; Protein Interaction Domains and Motifs; Protein Interaction Maps; Protein Structure, Secondary; Prothrombin; Sequence Analysis, Protein
PubMed: 30792421
DOI: 10.1038/s41598-019-38857-4 -
The Journal of Laboratory and Clinical... Feb 1983Specific immunoassays have been developed for forms of human prothrombin that vary in their degree of carboxylation. Human abnormal (des-gamma-carboxyl) prothrombin was...
Specific immunoassays have been developed for forms of human prothrombin that vary in their degree of carboxylation. Human abnormal (des-gamma-carboxyl) prothrombin was isolated in 18% yield from the plasma of a patient treated with warfarin. The purified protein migrated as a single band in electrophoresis and contained an average of three gamma-carboxyglutamic acid residues per molecule. A specific antibody subpopulation was isolated from rabbit anti-abnormal prothrombin antiserum by using affinity chromatography. These antibodies, which bound to abnormal prothrombin but which cross-reacted minimally with prothrombin, were used to establish an immunoassay specific for abnormal prothrombin. In parallel, a specific antibody subpopulation, anti-prothrombin: Ca(II), was isolated from rabbit anti-prothrombin antiserum by conformation perturbation affinity chromatography. This antibody, which bound prothrombin but minimally cross-reacted with abnormal prothrombin, was used to establish a specific immunoassay for native prothrombin. An anti-prethrombin 1 subpopulation bound abnormal prothrombin and prothrombin equivalently and was used for an immunoassay that measured total prothrombin. These assays permit the quantitation of abnormal prothrombin and prothrombin in plasma and serum. The level of native prothrombin antigen correlates precisely with the functional prothrombin activity. These assays provide an example of the use of specific antibodies against functionally important antigenic surfaces to monitor properties of coagulation proteins with the precision and reliability of immunoassy.
Topics: Electrophoresis, Polyacrylamide Gel; Humans; Immunoassay; Methods; Prothrombin; Warfarin
PubMed: 6822761
DOI: No ID Found -
PLoS Pathogens Sep 2022The majority of adenovirus (Ad) vectors are based on human Ad type 5, which is a member of Ad species C. Species C also includes the closely-related types 1, 2, 6, 57...
The majority of adenovirus (Ad) vectors are based on human Ad type 5, which is a member of Ad species C. Species C also includes the closely-related types 1, 2, 6, 57 and 89. It is known that coagulation factors bind to Ad5 hexon and play a key role in the liver tropism of Ad5 vectors, but it is unclear how coagulation factors affect vectors derived from other species C Ads. We evaluated species C Ad vectors both in vitro and following intravenous injection in mice. To assess the impact of hexon differences, we constructed chimeric Ad5 vectors that contain the hexon hypervariable regions from other species C types, including vectors with hexon mutations that decreased coagulation factor binding. After intravenous injection into mice, vectors with Ad5 or Ad6 hexon had strong liver tropism, while vectors with chimeric hexon from other Ad types had weaker liver tropism due to inhibition by natural antibodies and complement. In addition, we discovered a novel ability of hexon to bind prothrombin, which is the most abundant coagulation factor in blood, and we found striking differences in the affinity of Ads for human, mouse and bovine coagulation factors. When compared to Ad5, vectors with non-Ad5 species C hexons had considerably higher affinity for both human and mouse prothrombin. Most of the vectors tested were strongly dependent on coagulation factors for liver transduction, but vectors with chimeric Ad6 hexon showed much less dependence on coagulation factors than other vectors. We found that in vitro neutralization experiments with mouse serum predicted in vivo behavior of Ad5 vectors, but in vitro experiments did not predict the in vivo behavior of vectors based on other Ad types. In sum, hexons from different human Ad species C viruses confer diverse properties on vectors, including differing abilities to target the liver.
Topics: Adenoviridae; Adenoviruses, Human; Animals; Capsid Proteins; Cattle; Genetic Vectors; Humans; Mice; Prothrombin; Transduction, Genetic
PubMed: 36156097
DOI: 10.1371/journal.ppat.1010859 -
Thrombosis Research Jun 1991An abnormal prothrombin has been detected in a 26-year-old female, who had no history of excessive bleeding. Prothrombin activity was approximately 10% when measured...
An abnormal prothrombin has been detected in a 26-year-old female, who had no history of excessive bleeding. Prothrombin activity was approximately 10% when measured using either the classical one-stage assay or the assay with Echis carinatus venom, whereas prothrombin antigen level was normal. In keeping with current nomenclature practices, the abnormal prothrombin was designated "Prothrombin Himi". The electrophoretic behavior and calcium binding properties of Prothrombin Himi did not differ significantly from normal. Prothrombin Himi was isolated by chromatography on Q-Sepharose. Electrophoretic migration of the purified abnormal prothrombin on SDS-PAGE was normal. Upon prothrombin activation by Echis carinatus venom, the clotting activity produced from Prothrombin Himi was only 37% of the normal level after 90 minutes of the activation time, where as the amidolytic activity was almost the same as normal. The cleavage patterns of Prothrombin Himi by factor Xa or Echis carinatus venom investigated by SDS-PAGE, were found to be normal. These results indicate that Prothrombin Himi was characterized by a defective thrombin enzymatic activity.
Topics: Adult; Blood Coagulation Tests; Endopeptidases; Enzyme Activation; Factor Xa; Female; Humans; Pedigree; Peptide Fragments; Prothrombin; Thrombin
PubMed: 1926060
DOI: 10.1016/0049-3848(91)90373-5 -
The American Journal of Gastroenterology Sep 1990Des-gamma-carboxy prothrombin is an abnormal prothrombin which increases in the plasma of patients with hepatocellular carcinoma. To clarify the process of...
Measurement of immunoreactive prothrombin, des-gamma-carboxy prothrombin, and vitamin K in human liver tissues: overproduction of immunoreactive prothrombin in hepatocellular carcinoma.
Des-gamma-carboxy prothrombin is an abnormal prothrombin which increases in the plasma of patients with hepatocellular carcinoma. To clarify the process of des-gamma-carboxy prothrombin synthesis, immunoreactive prothrombin, des-gamma-carboxy prothrombin, and vitamin K (phylloquinone and menaquinone) concentrations were determined in human liver tissue, including hepatocellular carcinoma. In the patients with elevated plasma des-gamma-carboxy prothrombin levels, both immunoreactive prothrombin and des-gamma-carboxy prothrombin significantly increased in hepatoma tissues compared with non-cancerous liver tissue. On the other hand, no significant difference was observed in the endogenous vitamin K (K1, MK-4, MK7) concentrations between hepatoma and noncancerous portions, in either the cases with or without increase of plasma des-gamma-carboxy prothrombin. These data strongly suggested that in the patients with an increase of plasma des-gamma-carboxy prothrombin, overproduction of prothrombin in hepatoma plays in important role in the synthesis of des-gamma-carboxy prothrombin.
Topics: Analysis of Variance; Biomarkers; Carcinoma, Hepatocellular; Enzyme-Linked Immunosorbent Assay; Humans; Liver Diseases; Liver Neoplasms; Protein Precursors; Prothrombin; Vitamin K; alpha-Fetoproteins
PubMed: 1697141
DOI: No ID Found -
Proceedings of the National Academy of... May 1977The amino acid sequence of the nonthrombin half of human prothrombin is presented. Prothrombin fragment 1 has 155 amino acid residues as compared with 156 for the bovine...
The amino acid sequence of the nonthrombin half of human prothrombin is presented. Prothrombin fragment 1 has 155 amino acid residues as compared with 156 for the bovine equivalent. Ten gamma-carboxyglutamic acid residues are at the same location in each species. Human prothrombin fragment 2 contains 118 amino acid residues, as does the similar bovine fragment. Comparing bovine and human prothrombin fragment 1 we found 131 residues to be identical (84%). In prothrombin fragment 2, 84 residues were identical (71%). Assuming a time span of 90 million years since the radiation of several orders of placental mammals, prothrombin fragments 1 and 2 incorporated one substitution site per 100 amino acid sites every 11.2 and 6.3 million years, respectively. Internal homology is acribed to partial gene duplication, with the most likely crossover point located between residues 60-61 and residues 165-166.
Topics: Amino Acid Sequence; Humans; Peptides; Prothrombin
PubMed: 266717
DOI: 10.1073/pnas.74.5.1969 -
Biochemistry Feb 1987Structural studies on a hereditarily abnormal prothrombin, prothrombin Tokushima, have been performed to identify the difference responsible for its reduced fibrinogen...
Structural studies on a hereditarily abnormal prothrombin, prothrombin Tokushima, have been performed to identify the difference responsible for its reduced fibrinogen clotting activity upon conversion to thrombin. The prothrombin sample used was from a heterozygote but contained exclusively a defective prothrombin molecule, since the patient was heterozygous for both dysprothrombinemia and hypoprothrombinemia. Amino acid sequence analysis of a peptide isolated from a lysyl endopeptidase digest of the abnormal thrombin indicated that Arg-418 (equivalent to Asn-101 in the chymotrypsin numbering system) had been replaced by Trp. This amino acid substitution can result from a single nucleotide change in the codon for Arg-418 (CGG----TGG). The Arg----Trp replacement found in the thrombin portion of prothrombin Tokushima appears to reduce its interaction with various substrates including fibrinogen and platelet receptors and accounts for the recurrent bleeding episode observed in the propositus.
Topics: Amino Acid Sequence; Arginine; Chromatography, High Pressure Liquid; Fibrinogen; Heterozygote; Humans; Peptide Fragments; Peptide Mapping; Prothrombin; Thrombin; Tryptophan
PubMed: 3567158
DOI: 10.1021/bi00378a020