Authors: Nicola Pozzi, Enrico Di Cera

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

Authors: David Green

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

Authors: Eliza A Ruben, Brock Summers, Michael J Rau...

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

Review

Authors: Antonio Girolami, Elisabetta Cosi, Silvia Ferrari...

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

Comparative Study

Authors: M R Downing, R J Butkowski, M M Clark...

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

Authors: Kathleen Freson

Topics: Protein C; Protein S; Prothrombin; Blood Coagulation

PubMed: 37199157
DOI: 10.1161/ATVBAHA.123.319442

Review

Authors: Rebecca S Woodruff, Bruce A Sullenger

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

Review

Authors: S Krishnaswamy

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

Review

Authors: Mark Schreuder, Pieter H Reitsma, Mettine H A Bos...

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

Authors: Daria Korolova, Viktoriya Gryshchenko, Tamara Chernyshenko...

BACKGROUND

Knowing the variability of blood coagulation responses to liver damage of different origins can provide a key to curing liver tissues or to mitigating treatment side effects. The aim of the present work was to compare the changes in the main components of hemostasis under experimental drug-induced hepatosis and hepatitis in rats.

METHODS

We modeled diclofenac-induced hepatitis and tetracycline-induced hepatosis. Hemostasis response was gauged by measuring fibrinogen, factor X, protein C (PC), and prothrombin in plasma. The decarboxylated form of prothrombin was detected by measuring prothrombin index and ecamulin index. Platelet reactivity was studied using aggregometry.

RESULTS

Both hepatitis and hepatosis decreased the synthesis of fibrinogen, factor X, and prothrombin. However, protein carboxylation was not disrupted in hepatosis but was much impaired in hepatitis. PC decreased in both models as a consequence of its consumption possibly during inflammatory response. Platelet aggregation rate was lower in hepatosis but higher in hepatitis.

CONCLUSIONS

Our findings imply the need for a thorough monitoring of the hemostasis system in liver diseases to avoid possible thrombotic complications. Its state indicates the disorder's rate and character.

Topics: Rats; Animals; Prothrombin; Factor X; Blood Coagulation Factors; Fibrinogen; Liver Diseases; Hemostatics; Chemical and Drug Induced Liver Injury

PubMed: 36574273
DOI: 10.1002/ame2.12301