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The Journal of Biological Chemistry Aug 1985To study the specific role of gamma-carboxyglutamic acid (Gla) residues in prothrombin, we have isolated a series of partially carboxylated prothrombin variants from a...
To study the specific role of gamma-carboxyglutamic acid (Gla) residues in prothrombin, we have isolated a series of partially carboxylated prothrombin variants from a patient with a hereditary defect in vitamin K-dependent carboxylation (Goldsmith, G. H., Pence, R. E., Ratnoff, O. D., Adelstein, D. A., and Furie, B. (1982) J. Clin. Invest. 69, 1253-1260). The three variant prothrombins, purified by DEAE-Sephacel, immunoaffinity chromatography, and preparative gel electrophoresis, were indistinguishable from prothrombin in molecular weight, amino acid composition, and NH2-terminal amino acid sequence, with the exception of Gla residues. Variant prothrombin 1, with 8 Gla residues, had 66% of the coagulant activity of prothrombin, one high affinity metal-binding site (Kd = 15 nM), and three lower affinity sites (Kd = 2.7 microM); prothrombin contained two high affinity (36 nM) and four lower affinity sites (Kd = 1 microM). Ca(II) induced a 23% decrease in the intrinsic fluorescence of variant prothrombin 1 fragment 1, compared to a 35% decrease in that of prothrombin fragment 1. The phospholipid binding activity of variant prothrombin 1 was 44% that of prothrombin. Variant prothrombin 2 and variant prothrombin 3, with 4 and 6 Gla residues, respectively, had about 5% of prothrombin coagulant activity and a single high affinity and two lower affinity metal-binding sites and exhibited no phospholipid binding activity. Variant prothrombin 3 fragment 1 and variant prothrombin 2 fragment 1 demonstrated 18 and 13% of Ca(II)-induced fluorescence quenching, respectively. Abnormal prothrombin, with 1 Gla residue, had 8% of prothrombin coagulant activity, a single lower affinity (1 microM) metal-binding site, and 13% Ca(II)-induced fluorescence quenching of the fragment 1 species and did not bind to phospholipid. These results indicate that Gla residues define the metal binding properties of prothrombin. Most, if not all, of the Gla residues are required for complete prothrombin function, and the prothrombin coagulant activity correlates to the phospholipid binding activity of the prothrombin species.
Topics: Amino Acid Sequence; Amino Acids; Calcium; Chromatography, Affinity; Gadolinium; Genetic Variation; Humans; Phospholipids; Protein Binding; Prothrombin; Spectrometry, Fluorescence
PubMed: 4019472
DOI: No ID Found -
Thrombosis Et Diathesis Haemorrhagica Jul 1968
Topics: Animals; Cattle; Chromatography; Hydrogen-Ion Concentration; Prothrombin; Resins, Plant
PubMed: 5751240
DOI: No ID Found -
JAMA Jun 1969
Topics: Absorption; Blood Coagulation Tests; Calcium Phosphates; Chromatography; Coumarins; Factor IX; Factor VII Deficiency; Hemophilia B; Hemostasis; Humans; Hypoprothrombinemias; Liver Diseases; Prothrombin
PubMed: 5818828
DOI: No ID Found -
The Journal of Biological Chemistry Feb 2003Prothrombinase cleaves prothrombin at Arg(271) and Arg(320) to produce thrombin. The kinetics of cleavage of five recombinant prothrombins were measured: wild-type...
Prothrombinase cleaves prothrombin at Arg(271) and Arg(320) to produce thrombin. The kinetics of cleavage of five recombinant prothrombins were measured: wild-type prothrombin (WT-II), R155A/R284A/R271A prothrombin (rMZ-II), R155A/R284A/R320A prothrombin (rP2-II), S525C prothrombin labeled with fluorescein (WT-II-F*), and R155A/R284A/R271A/S525C prothrombin labeled with fluorescein (rMZ-II-F*). rMZ-II and rP2-II are cleaved only at Arg(320) and Arg(271), respectively, to yield the intermediates meizothrombin and prethrombin-2, respectively. WT-II-F* and rMZ-II-F* were labeled at Cys(525) with fluorescein; cleavage was monitored by enhanced fluorescence. Activation kinetics of WT-II, rMZ-II, and rP2-II indicated that the catalytic efficiency of cleavage at Arg(320) was increased by 30,000-fold by the cofactor factor Va, as was the conversion of prothrombin to thrombin. However, factor Va increased cleavage at Arg(271) only by 34-fold. Although WT-II competitively inhibited cleavage of WT-II-F*, rMZ-II or rP2-II did not inhibit completely even at saturating concentrations. However, rMZ-II and rP2-II together inhibited WT-II-F* cleavage competitively. Both WT-II and rMZ-II competitively inhibited rMZ-II-F* cleavage, whereas rP2-II did not. A model of prothrombin activation that includes two equilibrating forms of prothrombinase, each recognizing one of the cleavage sites, is quantitatively consistent with all of the experimental observations. Therefore, we conclude that the kinetics of prothrombin activation can be described by a "ping-pong"-like mechanism.
Topics: Arginine; Binding Sites; Binding, Competitive; DNA; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Enzyme Precursors; Factor Va; Fluorescein; Humans; Kinetics; Models, Biological; Models, Theoretical; Mutagenesis, Site-Directed; Prothrombin; Recombinant Proteins; Thrombin; Thromboplastin; Time Factors
PubMed: 12496269
DOI: 10.1074/jbc.M206413200 -
Zhonghua Xue Ye Xue Za Zhi = Zhonghua... Sep 1998
Review
Topics: DNA, Antisense; Hemorrhage; Humans; Point Mutation; Prothrombin; Venous Thrombosis
PubMed: 11189491
DOI: No ID Found -
Journal of Thrombosis and Haemostasis :... May 2013Activated prothrombin complex concentrates (APCCs), utilized in bypassing therapy for hemophiliacs with inhibitor, contain factors (Fs) VII, FII, FIX and FX, and their...
BACKGROUND AND OBJECTIVES
Activated prothrombin complex concentrates (APCCs), utilized in bypassing therapy for hemophiliacs with inhibitor, contain factors (Fs) VII, FII, FIX and FX, and their active forms. A recent report has demonstrated that mixtures of APCC and FVIII potentiated thrombin generation, in vitro, in plasma from patients with severe hemophilia A, but the mechanism(s) involved remains unknown.
RESULTS
APCC (0.05 U mL(-1) ) increased FVIII activity ~ 4-fold within 1 min in one-stage clotting assays, followed by a return to initial levels within 10 min. This reaction was dependent on the presence of tissue factor and phospholipid. Thrombin generation produced from APCC was ~ 3.5-fold greater in the presence of FVIII than that in its absence. SDS-PAGE analysis revealed that APCC sequentially proteolyzed the heavy chain of FVIII at Arg(372) and Arg(740) , followed by cleavage at Arg(336) . Proteolysis was prevented by FVIIa inhibitor, but not by hirudin, supporting the concept that APCC itself possessed the potential to activate FVIII in early coagulation phases, and that FVIIa in APCC contributed mainly to this reaction. APCC-mediated FVIII activation was unaffected by the addition of anti-FVIII inhibitor antibodies, irrespective of epitope specificity. Anti-C2 type 1 inhibitors, however, diminished the inactivation phase of the APCC reaction by inhibiting cleavage at Arg(336) .
CONCLUSION
Small amounts of APCC, relative to the standard concentration used for clinical purposes, could activate FVIII directly, even in the presence of anti-FVIII antibodies. Combination therapy based on mixtures of APCC and FVIII could have significant beneficial implications for the treatment of hemophilia A patients with inhibitors.
Topics: Autoantibodies; Complex Mixtures; Electrophoresis, Polyacrylamide Gel; Factor VIII; Hemostasis; Humans; Proteolysis; Prothrombin
PubMed: 23517528
DOI: 10.1111/jth.12197 -
Clinical Chemistry Nov 1987PIVKA-II (Protein Induced by Vitamin K Absence) is abnormal des-carboxylated prothrombin, which is present in vitamin K deficiency or in patients using warfarin. With a... (Comparative Study)
Comparative Study
PIVKA-II (Protein Induced by Vitamin K Absence) is abnormal des-carboxylated prothrombin, which is present in vitamin K deficiency or in patients using warfarin. With a sensitive method for PIVKA-II, biochemical vitamin K deficiency can be established before clinical symptoms occur. We give an overview of methods used to detect PIVKA-II, and four selected methods are inter-compared: (a) measuring total factor II including PIVKA-II by using Echis carinatus snake venom as an activator of prothrombin; (b) measuring PIVKA-II by using snake venom as an activator of factor II after adsorption of functional factor II onto barium sulfate; (c) electrophoresis-immunofixation method; and (d) enzyme immunoassay. We found d to be the most sensitive and reliable method for PIVKA-II.
Topics: Adsorption; Barium Sulfate; Biomarkers; Electrophoresis; Endopeptidases; Enzyme Activation; Humans; Immunoenzyme Techniques; Immunologic Techniques; Protein Precursors; Prothrombin; Viper Venoms; Vitamin K Deficiency
PubMed: 3315304
DOI: No ID Found -
Blood May 2024Antiprothrombin antibodies are found in antiphospholipid patients, but how they interact with prothrombin remains elusive. Prothrombin adopts closed and open forms. We...
Antiprothrombin antibodies are found in antiphospholipid patients, but how they interact with prothrombin remains elusive. Prothrombin adopts closed and open forms. We recently discovered type I and type II antibodies and proposed that type I recognizes the open form. In this study, we report the discovery and structural and functional characterization in human plasma of a type I antibody, POmAb (prothrombin open monoclonal antibody). Using surface plasmon resonance and single-molecule spectroscopy, we show that POmAb interacts with kringle-1 of prothrombin, shifting the equilibrium toward the open form. Using single-particle cryogenic electron microscopy (cryo-EM), we establish that the epitope targeted by POmAb is in kringle-1, comprising an extended binding interface centered at residues R90-Y93. The 3.2-Å cryo-EM structure of the complex reveals that the epitope overlaps with the position occupied by the protease domain of prothrombin in the closed state, explaining the exclusive binding of POmAb to the open form. In human plasma, POmAb prolongs phospholipid-initiated and diluted Russell's viper venom clotting time, which could be partly rescued by excess phospholipids, indicating POmAb is an anticoagulant but exerts a weak lupus anticoagulant effect. These studies reveal the structural basis of prothrombin recognition by a type I antiphospholipid antibody and uncover an exciting new strategy to achieve anticoagulation in human plasma.
Topics: Humans; Antibodies, Antiphospholipid; Antibodies, Monoclonal; Blood Coagulation; Cryoelectron Microscopy; Epitopes; Kringles; Protein Binding; Prothrombin
PubMed: 38437497
DOI: 10.1182/blood.2023022942 -
Polski Tygodnik Lekarski May 1956
Topics: Hematologic Tests; Hemostatics; Humans; Postoperative Period; Prothrombin; Surgical Procedures, Operative
PubMed: 13349765
DOI: No ID Found -
Blood Jan 1975Data are presented to support the concept that the vitamin K-dependent coagulation factors are adsorbed onto the platelet membrane and that these make up part of the...
Data are presented to support the concept that the vitamin K-dependent coagulation factors are adsorbed onto the platelet membrane and that these make up part of the "plasma atmosphere" necessary for the aggregation of platelets by various agents. Together with evidence that other coagulation factors also are part of the plasma atmosphere, it is suggested that the aggregation reaction is part of the coagulation sequence. The immunologic approach to demonstrating constituents of the platelet membrane promises to be a highly specific technique for studying further the constituents of the platelet membrane and their reactions in hemostasis.
Topics: Blood Coagulation Factors; Humans; Immune Sera; Platelet Aggregation; Platelet Aggregation Inhibitors; Prothrombin
PubMed: 803116
DOI: No ID Found