-
Thrombosis and Haemostasis Jul 2022The antiphospholipid syndrome is characterized by antibodies directed against phospholipid-binding proteins and phospholipids attached to cell membrane receptors,...
The antiphospholipid syndrome is characterized by antibodies directed against phospholipid-binding proteins and phospholipids attached to cell membrane receptors, mitochondria, oxidized lipoproteins, and activated complement components. When antibodies bind to these complex antigens, cells are activated and the coagulation and complement cascades are triggered, culminating in thrombotic events and pregnancy morbidity that further define the syndrome. The phospholipid-binding proteins most often involved are annexins II and V, β-glycoprotein I, prothrombin, and cardiolipin. A distinguishing feature of the antiphospholipid syndrome is the "lupus anticoagulant." This is not a single entity but rather a family of antibodies directed against complex antigens consisting of β-glycoprotein I and/or prothrombin bound to an anionic phospholipid. Although these antibodies prolong in vitro clotting times by competing with clotting factors for phospholipid binding sites, they are not associated with clinical bleeding. Rather, they are thrombogenic because they augment thrombin production in vivo by concentrating prothrombin on phospholipid surfaces. Other antiphospholipid antibodies decrease the clot-inhibitory properties of the endothelium and enhance platelet adherence and aggregation. Some are atherogenic because they increase lipid peroxidation by reducing paraoxonase activity, and others impair fetal nutrition by diminishing placental antithrombotic and fibrinolytic activity. This plethora of destructive autoantibodies is currently managed with immunomodulatory agents, but new approaches to treatment might include vaccines against specific autoantigens, blocking the antibodies generated by exposure to cytoplasmic DNA, and selective targeting of aberrant B-cells to reduce or eliminate autoantibody production.
Topics: Antiphospholipid Syndrome; Female; Humans; Lupus Coagulation Inhibitor; Phospholipids; Placenta; Pregnancy; Prothrombin; Thrombosis; beta 2-Glycoprotein I
PubMed: 34794200
DOI: 10.1055/a-1701-2809 -
Blood Jun 2022The intrinsic and extrinsic pathways of the coagulation cascade converge to a common step where the prothrombinase complex, comprising the enzyme factor Xa (fXa), the...
The intrinsic and extrinsic pathways of the coagulation cascade converge to a common step where the prothrombinase complex, comprising the enzyme factor Xa (fXa), the cofactor fVa, Ca2+ and phospholipids, activates the zymogen prothrombin to the protease thrombin. The reaction entails cleavage at 2 sites, R271 and R320, generating the intermediates prethrombin 2 and meizothrombin, respectively. The molecular basis of these interactions that are central to hemostasis remains elusive. We solved 2 cryogenic electron microscopy (cryo-EM) structures of the fVa-fXa complex, 1 free on nanodiscs at 5.3-Å resolution and the other bound to prothrombin at near atomic 4.1-Å resolution. In the prothrombin-fVa-fXa complex, the Gla domains of fXa and prothrombin align on a plane with the C1 and C2 domains of fVa for interaction with membranes. Prothrombin and fXa emerge from this plane in curved conformations that bring their protease domains in contact with each other against the A2 domain of fVa. The 672ESTVMATRKMHDRLEPEDEE691 segment of the A2 domain closes on the protease domain of fXa like a lid to fix orientation of the active site. The 696YDYQNRL702 segment binds to prothrombin and establishes the pathway of activation by sequestering R271 against D697 and directing R320 toward the active site of fXa. The cryo-EM structure provides a molecular view of prothrombin activation along the meizothrombin pathway and suggests a mechanism for cleavage at the alternative R271 site. The findings advance our basic knowledge of a key step of coagulation and bear broad relevance to other interactions in the blood.
Topics: Cryoelectron Microscopy; Factor V; Factor Va; Factor Xa; Prothrombin; Thromboplastin
PubMed: 35427420
DOI: 10.1182/blood.2022015807 -
Expert Review of Proteomics Dec 2014The structure of prothrombin has eluded investigators for decades but recent efforts have succeeded in revealing the architecture of this important clotting factor....
The structure of prothrombin has eluded investigators for decades but recent efforts have succeeded in revealing the architecture of this important clotting factor. Unanticipated features have emerged outlining the significant flexibility of the zymogen due to linker regions connecting the γ carboxyglutamic domain, kringles and protease domain. A new, structure-based framework helps in defining a molecular mechanism of prothrombin activation, rationalizes the severe bleeding phenotypes of several naturally occurring mutations and identifies targets for drug design.
Topics: Humans; Mutation; Protein Structure, Tertiary; Prothrombin
PubMed: 25327788
DOI: 10.1586/14789450.2014.971763 -
The Journal of Biological Chemistry Dec 1975Human prothrombin has been purified from American Red Cross Factor IX concentrates. Studies of the activation of the human prothrombin with the use of sodium dodecyl... (Comparative Study)
Comparative Study
Human prothrombin has been purified from American Red Cross Factor IX concentrates. Studies of the activation of the human prothrombin with the use of sodium dodecyl sulfate electrophoretic analysis of activation products indicated that human prothrombin activation is similar to bovine prothrombin activation. Molecular weight analysis of human prothrombin and intermediated by sodium dodecyl sulfate co-electrophoresis with bovine prothrombin and its intermediates resulted in molecular weights of 70,000 for prothrombin, 51,000 for intermediate 1, 41,000 for intermediate 2, 23,000 for intermediate 3, and 13,000 for intermediate 4. Amino acid compositions of human prothrombin and intermediates are similar to those for bovine prothrombin and intermediates. NH2-terminal sequence studies of human prothrombin, intermediates, and alpha-thrombin A and B chains placed the intermediates in the parent human prothrombin molecule as described for the bovine system. Intermediate 3 is the NH2-terminal of prothrombin, and intermediate 1 is the COOH-terminal segment of the zymogen. Intermediate 4 is the NH2-terminal of intermediate 1. Intermediate 2', the immediate precursor of alpha-thrombin, is the COOH-terminal segment of intermediate 1. In general, a high degree of homology in the primary structure of prothrombin and intermediates was observed between the human and bovine system. The NH2-terminal sequences of human intermediate 2' and alpha-thrombin A chain are identical. However, human intermediate 2' isolated in a manner identical with that used for the isolation of bovine intermediate 2 is homologous with bovine intermediate 2, beginning with residue 14.
Topics: Amino Acid Sequence; Amino Acids; Animals; Cattle; Chromatography, Affinity; Enzyme Activation; Factor IX; Humans; Macromolecular Substances; Molecular Weight; Prothrombin
PubMed: 1238394
DOI: No ID Found -
The Journal of Applied Laboratory... Nov 2020Protein induced by vitamin K absence-II (PIVKA-II) is produced by the liver during hepatoma and upon warfarin administration. Those patients have disturbed protein...
BACKGROUND
Protein induced by vitamin K absence-II (PIVKA-II) is produced by the liver during hepatoma and upon warfarin administration. Those patients have disturbed protein synthesis and glycosylation in the liver. This decreases the number of γ-carboxyglutamyl (Gla) residues on prothrombin, converting prothrombin into PIVKA-II. The mechanism of this conversion, however, is not clearly understood.
METHODS
Prothrombin was isolated from healthy and warfarin-treated individuals whose liver function of protein production was quantitatively normal. Glycan structures in the purified prothrombin containing PIVKA-II were qualitatively analyzed by high performance liquid chromatography after labeling the glycan with fluorophore 2-aminobenzamide.
RESULTS
The concentration of PIVKA-II was significantly higher in the warfarin-treated individuals than in the healthy individuals (P< 0.001). Although protein production in the liver was normal in both groups, the concentration of prothrombin was lower in the warfarin-treated individuals than in the healthy individuals (P < 0.001). The main glycan was A2 in the healthy and warfarin-treated individuals (86.6 ± 4.4% and 85.6 ± 3.4%, respectively). Eight types of glycan were characterized in both groups, although generation of PIVKA-II in the warfarin-treated individuals did not lead to variation in glycosylation of prothrombin.
CONCLUSIONS
Warfarin therapy leads to lower amounts of prothrombin and Gla residues within prothrombin without exerting qualitative and quantitative change in glycan profile and protein synthetic function in the liver.
Topics: Biomarkers; Humans; Protein Precursors; Protein Processing, Post-Translational; Prothrombin; Warfarin
PubMed: 32594109
DOI: 10.1093/jalm/jfaa069 -
Biosensors Sep 2022Thrombin is a serine protease with an essential role in homeostasis and blood coagulation. During vascular injuries, thrombin is generated from prothrombin, a plasma... (Review)
Review
Thrombin is a serine protease with an essential role in homeostasis and blood coagulation. During vascular injuries, thrombin is generated from prothrombin, a plasma protein, to polymerize fibrinogen molecules into fibrin filaments. Moreover, thrombin is a potent stimulant for platelet activation, which causes blood clots to prevent bleeding. The rapid and sensitive detection of thrombin is important in biological analysis and clinical diagnosis. Hence, various biosensors for thrombin measurement have been developed. Biosensors are devices that produce a quantifiable signal from biological interactions in proportion to the concentration of a target analyte. An aptasensor is a biosensor in which a DNA or RNA aptamer has been used as a biological recognition element and can identify target molecules with a high degree of sensitivity and affinity. Designed biosensors could provide effective methods for the highly selective and specific detection of thrombin. This review has attempted to provide an update of the various biosensors proposed in the literature, which have been designed for thrombin detection. According to their various transducers, the constructions and compositions, the performance, benefits, and restrictions of each are summarized and compared.
Topics: Aptamers, Nucleotide; Biosensing Techniques; DNA; Fibrin; Fibrinogen; Prothrombin; Thrombin
PubMed: 36140153
DOI: 10.3390/bios12090767 -
Clinical and Applied... Sep 2018Clotting factor defects are usually associated with bleeding. About 2 decades ago, 2 polymorphisms, one of FII (G20210A) and another of FV (Arg506Gln), have been shown... (Review)
Review
Clotting factor defects are usually associated with bleeding. About 2 decades ago, 2 polymorphisms, one of FII (G20210A) and another of FV (Arg506Gln), have been shown to be associated with prothrombotic state and venous thrombosis. As a consequence, FII and FV could be considered both as prohemorrhagic factors and prothrombotic conditions. Recently, it has been shown that missense mutations in the prothrombin gene of amino acid Arg596 of exon 14 to Leu596, Gln596, or Trp596 caused the appearance of a thrombophilic state and venous thrombosis. These mutated FII are not associated with bleeding, but only with venous thrombosis. Furthermore, they are all heterozygotes for the mutations. No missense mutation associated with thrombosis has been discovered so far for FV. As a consequence, the prothrombotic activity of FII is the result of a polymorphism and of a missense mutation, whereas that of FV derives only from a polymorphism. The observation that a clotting factor defect may be associated with both bleeding or venous thrombosis depending on the site of the mutation has caused an extensive reevaluation of the blood clotting mechanism.
Topics: Amino Acid Substitution; Animals; Factor V; Hemorrhage; Humans; Mutation, Missense; Polymorphism, Genetic; Prothrombin; Venous Thrombosis
PubMed: 29690772
DOI: 10.1177/1076029618770741 -
Journal of Thrombosis and Haemostasis :... Jun 2013The proteolytic conversion of prothrombin to thrombin catalyzed by prothrombinase is one of the more extensively studied reactions of blood coagulation. Sophisticated... (Review)
Review
The proteolytic conversion of prothrombin to thrombin catalyzed by prothrombinase is one of the more extensively studied reactions of blood coagulation. Sophisticated biophysical and biochemical insights into the players of this reaction were developed in the early days of the field. Yet, many basic enzymological questions remained unanswered. I summarize new developments that uncover mechanisms by which high substrate specificity is achieved, and the impact of these strategies on enzymic function. Two principles emerge that deviate from conventional wisdom that has otherwise dominated thinking in the field. (i) Enzymic specificity is dominated by the contribution of exosite binding interactions between substrate and enzyme rather than by specific recognition of sequences flanking the scissile bond. Coupled with the regulation of substrate conformation as a result of the zymogen to proteinase transition, novel mechanistic insights result for numerous aspects of enzyme function. (ii) The transition of zymogen to proteinase following cleavage is not absolute and instead, thrombin can reversibly interconvert between zymogen-like and proteinase-like forms depending on the complement of ligands bound to it. This establishes new paradigms for considering proteinase allostery and how enzyme function may be modulated by ligand binding. These insights into the action of prothrombinase on prothrombin have wide-ranging implications for the understanding of function in blood coagulation.
Topics: Prothrombin; Substrate Specificity; Thrombin
PubMed: 23809130
DOI: 10.1111/jth.12217 -
Journal of Thrombosis and Haemostasis :... Aug 2019Blood coagulation factor Va serves an indispensable role in hemostasis as cofactor for the serine protease factor Xa. In the presence of an anionic phospholipid membrane... (Review)
Review
Blood coagulation factor Va serves an indispensable role in hemostasis as cofactor for the serine protease factor Xa. In the presence of an anionic phospholipid membrane and calcium ions, factors Va and Xa assemble into the prothrombinase complex. Following formation of the ternary complex with the macromolecular zymogen substrate prothrombin, the latter is rapidly converted into thrombin, the key regulatory enzyme of coagulation. Over the years, multiple binding sites have been identified in factor Va that play a role in the interaction of the cofactor with factor Xa, prothrombin, or the anionic phospholipid membrane surface. In this review, an overview of the currently available information on these interactive sites in factor Va is provided, and data from biochemical approaches and 3D structural protein complex models are discussed. The structural models have been generated in recent years and provide novel insights into the molecular requirements for assembly of both the prothrombinase and the ternary prothrombinase-prothrombin complexes. Integrated knowledge of functionally important regions in factor Va will allow for a better understanding of factor Va cofactor activity.
Topics: Binding Sites; Blood Coagulation; Cell Membrane; Factor Va; Factor Xa; Humans; Models, Molecular; Phospholipids; Protein Binding; Protein Interaction Domains and Motifs; Prothrombin; Structure-Activity Relationship; Thromboplastin
PubMed: 31102425
DOI: 10.1111/jth.14487 -
Arteriosclerosis, Thrombosis, and... Oct 2015As a novel class of therapeutics, aptamers, or nucleic acid ligands, have garnered clinical interest because of the ease of isolating a highly specific aptamer against a... (Review)
Review
As a novel class of therapeutics, aptamers, or nucleic acid ligands, have garnered clinical interest because of the ease of isolating a highly specific aptamer against a wide range of targets, their chemical flexibility and synthesis, and their inherent ability to have their function reversed. The following review details the development and molecular mechanisms of aptamers targeting specific proteases in the coagulation cascade. The ability of these anticoagulant aptamers to bind to and inhibit exosite function rather than binding within the active site highlights the importance of exosites in blocking protein function. As both exosite inhibitors and reversible agents, the use of aptamers is a promising strategy for future therapeutics.
Topics: Aptamers, Nucleotide; Blood Coagulation; Clinical Trials, Phase I as Topic; Clinical Trials, Phase II as Topic; Female; Humans; Male; Molecular Targeted Therapy; Prothrombin; Sensitivity and Specificity; Serine Endopeptidases; Thrombin
PubMed: 26315404
DOI: 10.1161/ATVBAHA.115.300131