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Sensors (Basel, Switzerland) Oct 2020Prothrombin-related thrombophilia is a genetic disorder produced by a substitution of a single DNA base pair, replacing guanine with adenine, and is detected mainly by...
Prothrombin-related thrombophilia is a genetic disorder produced by a substitution of a single DNA base pair, replacing guanine with adenine, and is detected mainly by polymerase chain reaction (PCR). A suitable alternative that could detect the single point mutation without requiring sample amplification is the surface plasmon resonance (SPR) technique. SPR biosensors are of great interest: they offer a platform to monitor biomolecular interactions, are highly selective, and enable rapid analysis in real time. Oligonucleotide-based SPR biosensors can be used to differentiate complementary sequences from partially complementary or noncomplementary strands. In this work, a glass chip covered with an ultrathin (50 nm) gold film was modified with oligonucleotide strands complementary to the mutated or normal (nonmutated) DNA responsible for prothrombin-related thrombophilia, forming two detection platforms called mutated thrombophilia (MT) biosensor and normal thrombophilia (NT) biosensor. The results show that the hybridization response is obtained in 30 min, label free and with high reproducibility. The sensitivity obtained in both systems was approximately 4 ΔμRIU/nM. The dissociation constant and limits of detection calculated were 12.2 nM and 20 pM (3 fmol), respectively, for the MT biosensor, and 8.5 nM and 30 pM (4.5 fmol) for the NT biosensor. The two biosensors selectively recognize their complementary strand (mutated or normal) in buffer solution. In addition, each platform can be reused up to 24 times when the surface is regenerated with HCl. This work contributes to the design of the first SPR biosensor for the detection of prothrombin-related thrombophilia based on oligonucleotides with single point mutations, label-free and without the need to apply an amplification method.
Topics: Biosensing Techniques; Humans; Oligonucleotides; Point Mutation; Prothrombin; Reproducibility of Results; Surface Plasmon Resonance; Thrombophilia
PubMed: 33142935
DOI: 10.3390/s20216240 -
Scientific Reports Feb 2018The clotting factor prothrombin exists in equilibrium between closed and open conformations, but the physiological role of these forms remains unclear. As for other...
The clotting factor prothrombin exists in equilibrium between closed and open conformations, but the physiological role of these forms remains unclear. As for other allosteric proteins, elucidation of the linkage between molecular transitions and function is facilitated by reagents stabilized in each of the alternative conformations. The open form of prothrombin has been characterized structurally, but little is known about the architecture of the closed form that predominates in solution under physiological conditions. Using X-ray crystallography and single-molecule FRET, we characterize a prothrombin construct locked in the closed conformation through an engineered disulfide bond. The construct: (i) provides structural validation of the intramolecular collapse of kringle-1 onto the protease domain reported recently; (ii) documents the critical role of the linker connecting kringle-1 to kringle-2 in stabilizing the closed form; and (iii) reveals novel mechanisms to shift the equilibrium toward the open conformation. Together with functional studies, our findings define the role of closed and open conformations in the conversion of prothrombin to thrombin and establish a molecular framework for prothrombin activation that rationalizes existing phenotypes associated with prothrombin mutations and points to new strategies for therapeutic intervention.
Topics: Amino Acid Sequence; Enzyme Activation; Enzyme Precursors; Enzyme Stability; Humans; Kinetics; Models, Molecular; Mutation; Protein Conformation; Protein Engineering; Prothrombin; Structure-Activity Relationship; Thrombin
PubMed: 29440720
DOI: 10.1038/s41598-018-21304-1 -
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 -
Journal of Thrombosis and Haemostasis :... Dec 2006Prothrombin (FII) G20210A mutation and elevated plasma prothrombin activity are known risk factors for venous thrombosis. The risk of venous thrombosis among 19911G...
The association of prothrombin A19911G polymorphism with plasma prothrombin activity and venous thrombosis: results of the MEGA study, a large population-based case-control study.
BACKGROUND
Prothrombin (FII) G20210A mutation and elevated plasma prothrombin activity are known risk factors for venous thrombosis. The risk of venous thrombosis among 19911G carriers of the prothrombin A19911G polymorphism has not been extensively investigated.
OBJECTIVES AND METHODS
We assessed prothrombin activity, FIIG20210A, and FIIA19911G polymorphisms in a large population-based case-control study, the Multiple Environmental and Genetic Assessment (MEGA) study of risk factors for venous thrombosis. Four thousand three hundred and sixty-five consecutive patients with a first episode of deep vein thrombosis of the leg or pulmonary embolism were included. The control group (n = 4779) consisted of partners of patients or persons gathered using a random-digit dialing method. We studied the effect of FIIA19911G polymorphism on prothrombin activity and thrombosis risk, also in combination with factor V Leiden.
RESULTS
Among FII20210-GG control subjects, FII19911-GG carriers had 7.1% [95% confidence interval (CI): 5.7-8.5] higher mean prothrombin activity than FII19911-AA carriers and the risk for GG carriers was 1.43-fold increased compared to AA carriers [odds ratio (OR) 1.43; 95% CI: 1.27-1.61]. Among FII20210-GA control carriers, the mean prothrombin activity in both FII19911-AA and -AG carriers was nearly equivalent [131.7% and 133.4%; mean difference (95% CI) = 1.7% (-7.2-10.7)]. Because of genetic linkage, FII19911-GG carriers were very rare on a FII20210-GA background, as only one FII20210A carrier had FII19911-GG. In FII20210-GA carriers, the OR increased from 3.05 (95% CI: 2.17-4.27) in subjects with FII19911-AA to 3.33 (2.28-4.85) in subjects with FII19911-AG, compared to those with FII20210-GG and FII19911-AA.
CONCLUSIONS
The FIIA19911G polymorphism is associated with mildly elevated prothrombin activity and is a risk factor for venous thrombosis.
Topics: Adenine; Adult; Aged; Case-Control Studies; Factor V; Female; Gene Frequency; Genetic Predisposition to Disease; Guanine; Heterozygote; Humans; Male; Middle Aged; Netherlands; Odds Ratio; Polymorphism, Genetic; Population Surveillance; Prothrombin; Risk Factors; Venous Thrombosis
PubMed: 17059428
DOI: 10.1111/j.1538-7836.2006.02257.x -
Journal of Clinical Pathology Mar 1988
Topics: Adolescent; Biomarkers; Carcinoma, Hepatocellular; Child; Female; Humans; Infant; Liver Neoplasms; Male; Protein Precursors; Prothrombin
PubMed: 2834424
DOI: 10.1136/jcp.41.3.356 -
The Journal of Biological Chemistry Nov 1984A monoclonal antibody JO1 X 1 was prepared against human abnormal prothrombin using the hybridoma technique. The clone secreting this antibody was selected on the basis...
A monoclonal antibody JO1 X 1 was prepared against human abnormal prothrombin using the hybridoma technique. The clone secreting this antibody was selected on the basis of the ability of this antibody to bind to abnormal prothrombin, but not to prothrombin, in the presence of calcium ions. The antibodies were purified by affinity chromatography in EDTA on columns of prothrombin-Sepharose. Bound antibodies were eluted with 15 mM CaCl2. The kinetics of dissociation of antibody from the antibody-prothrombin complex with the addition of calcium ions fit a first-order kinetic model. Increasing CaCl2 concentration increased the rate of antibody-prothrombin dissociation. Ca(II) and Mn(II) inhibited antibody-prothrombin interaction; half-maximal binding was observed at 0.9 and 4 mM, respectively. Mg(II) had little effect on antibody-antigen interaction. The JO1 X 1 antibody bound fragment 1, fragment (1-39), abnormal prothrombin, and prothrombin equivalently in the presence of EDTA, but did not bind to des(1-44)prothrombin in the presence of EDTA or prothrombin in the presence of CaCl2. These results indicate that the monoclonal antibody JO1 X 1 is conformation specific for the calcium-free conformer of prothrombin and directed against an antigenic determinant near the NH2 terminus of prothrombin expressed in the 1-39 region of the protein. This analysis provides confirmation of the presence of a metal-free conformer of prothrombin.
Topics: Animals; Antibodies, Monoclonal; Biomarkers; Calcium Chloride; Enzyme-Linked Immunosorbent Assay; Epitopes; Magnesium; Magnesium Chloride; Manganese; Mice; Mice, Inbred BALB C; Protein Precursors; Prothrombin
PubMed: 6209271
DOI: No ID Found -
The Journal of Biological Chemistry Mar 1975The binding of Ca2+ to prothrombin and the intermediates of prothrombin activation was investigated by equilibrium dialysis using 45Ca2+ as the ligand. Scatchard plots...
The binding of Ca2+ to prothrombin and the intermediates of prothrombin activation was investigated by equilibrium dialysis using 45Ca2+ as the ligand. Scatchard plots of these data indicate that prothrombin (Mr = 70,000) has 10 to 11 Ca2+ binding sites which can be differentiated in terms of their binding affinity. Six of these Ca2+ binding sites have log Kassoc = 3.5 and all are found intact in the NH2-terminal segment (activation intermediate 3, Mr = 23,000) of the prothrombin molecule. Four or five additional weaker binding sites for Cz2+ with log Kassoc = 2.7 present in prothrombin are found intact in the remaining COOH-segment (activation intermediate 1, Mr = 51,000) of the prothrombin molecule. Upon further activation the Ca2+ binding sites residing in intermediate 1 are found intact in activation intermediate 4 (which constitutes the NH2-terminal segment of the intermediate 1 molecule). The remaining COOH-terminal portion (activation intermediate 2, Mr = 41,000) of the intermediate 1 molecule has no affinity for Ca2+. The activation of prothrombin and activation intermediates 1 and 2 was studied using these activators: Factor Xa alone, Factor Xa-Ca+, AND Factor Xa-Ca2+-phospholipid. The rate of thrombin production from prothrombin was progressively increased as Ca2+ and phospholipid were added to the system, whereas no significant increase in the rates of activation of intermediate 1 and 2 was observed. When Factor V was added to the Factor Xa-Ca2+-phospholipid system, the rate of activation of intermediate 1 was greatly enhanced. In the absence of Ca2+, Factor V had no effect on the rate of thrombin formation from intermediate 1. Factor V had no stimulatory effects on the rate of intermediate 2 activation. However, in the presence of an equimolar amount of intermediate 4, Factor V accelerated the conversion of intermediate 2 to thrombin. These studies indicate that the Ca2+ binding sites of the prothrombin molecule are contained in the "pro" fragment (intermediates 3 and 4) of the prothrombin molecule. Intermediate 1 and intermediate 2, both of which lack the strong Ca2+ binding sites of prothrombin, are poor substrates for the Factor Xa-Ca2+-phospholipid complex activation when compared to prothrombin. The addition of Factor V to the catalyst results in acceleration of the activation rate of intermediate 1 and an equimolar mixture if intermediates 2 and 4. These results lead us to conclude that the strong Ca2+ binding sites are the sites of phospholipid binding (intermediate 3), whereas the seak binding sites are the sites of Factor V binding (intermediate 4).
Topics: Binding Sites; Calcium; Enzyme Activation; Factor V; Factor X; Kinetics; Peptide Fragments; Phospholipids; Protein Binding; Prothrombin; Time Factors
PubMed: 1117001
DOI: No ID Found -
Journal of Thrombosis and Haemostasis :... Apr 2011This study was conducted to assess whether newly developed recombinant clotting factor concentrates enable the reversal of dilutional coagulopathy.
BACKGROUND
This study was conducted to assess whether newly developed recombinant clotting factor concentrates enable the reversal of dilutional coagulopathy.
METHODS
In 50 anesthetized pigs, ~60% of the blood volume was withdrawn and replaced with hydroxyethyl starch. Pigs were randomized to receive either 200 mg kg(-1) fibrinogen (n = 10), fibrinogen and 35 IU kg(-1) prothrombin complex concentrate (PCC) (n = 10), fibrinogen and 4 mg kg(-1) recombinant human factor II (rhFII) concentrate (n = 10), fibrinogen and a three-factor combination (3F) of 4 mg kg(-1) rhFII, 0.006 mg kg(-1) recombinant human FVIIa and 0.32 mg kg(-1) recombinant human FX (n = 10), or saline (n = 10). Thereafter, a standardized liver laceration was performed to induce uncontrolled hemorrhage. Survival time and blood loss were determined, and standard coagulation tests and thrombelastometry were performed.
RESULTS
Fibrinogen combined with rhFII or PCC improved survival. Blood loss was significantly decreased in all groups as compared with the animals receiving saline. Clotting time was significantly shortened in the animals treated with fibrinogen and PCC, as well as in those treated with fibrinogen and 3F. One animal died after administration of fibrinogen and PCC.
CONCLUSION
Following hemodilution, a combination of fibrinogen and PCC, rhFII or 3F enhances coagulation and final clot strength. Mortality was reduced statistically significantly only in the animals treated with fibrinogen and rhFII or PCC, whereas administration of the combination of fibrinogen and PCC caused a fatal thromboembolic complication. The combination of fibrinogen and rhFII might be effective in reversing dilutional coagulopathy and may reduce blood loss in cases of dilutional coagulopathy.
Topics: Animals; Blood Coagulation Disorders; Factor X; Prothrombin; Recombinant Proteins; Swine
PubMed: 21255250
DOI: 10.1111/j.1538-7836.2011.04211.x -
PloS One 2015Mannan-binding lectin-associated serine protease-1 (MASP-1), a protein of the complement lectin pathway, resembles thrombin in terms of structural features and substrate...
Mannan-binding lectin-associated serine protease-1 (MASP-1), a protein of the complement lectin pathway, resembles thrombin in terms of structural features and substrate specificity. Due to its interplay with several coagulation factors, it has the ability to induce fibrin clot formation independent of the usual coagulation activation pathways. We have recently shown that MASP-1 activates prothrombin and identified arginine (R) 155, R271, and R393 as potential cleavage sites. FXa cleaves R320 instead of R393, and thrombin cleaves R155 and R284 in prothrombin. Here we have used three arginine-to-glutamine mutants of prothrombin, R271Q, R320Q, R393Q and the serine-to-alanine active site mutant S525A to investigate in detail the mechanism of MASP-1 mediated prothrombin activation. Prothrombin wildtype and mutants were digested with MASP-1 and the cleavage products were analysed by SDS-PAGE and N-terminal sequencing. A functional clotting assay was performed by thrombelastography. We have found that MASP-1 activates prothrombin via two simultaneous pathways, either cleaving at R271 or R393 first. Both pathways result in the formation of several active alternative thrombin species. Functional studies confirmed that both R393 and R320 are required for prothrombin activation by MASP-1, whereas R155 is not considered to be an important cleavage site in this process. In conclusion, we have described for the first time a detailed model of prothrombin activation by MASP-1.
Topics: Blood Coagulation; HEK293 Cells; Humans; Mannose-Binding Protein-Associated Serine Proteases; Models, Biological; Proteolysis; Prothrombin
PubMed: 26645987
DOI: 10.1371/journal.pone.0144633 -
Arteriosclerosis, Thrombosis, and... Aug 2013Individuals with elevated prothrombin, including those with the prothrombin G20210A mutation, have increased risk of venous thrombosis. Although these individuals do not...
OBJECTIVE
Individuals with elevated prothrombin, including those with the prothrombin G20210A mutation, have increased risk of venous thrombosis. Although these individuals do not have increased circulating prothrombotic biomarkers, their plasma demonstrates increased tissue factor-dependent thrombin generation in vitro. The objectives of this study were to determine the pathological role of elevated prothrombin in venous and arterial thrombosis in vivo, and distinguish thrombogenic mechanisms in these vessels.
APPROACH AND RESULTS
Prothrombin was infused into mice to raise circulating levels. Venous thrombosis was induced by electrolytic stimulus to the femoral vein or inferior vena cava ligation. Arterial thrombosis was induced by electrolytic stimulus or ferric chloride application to the carotid artery. Mice infused with prothrombin demonstrated increased tissue factor-triggered thrombin generation measured ex vivo, but did not have increased circulating prothrombotic biomarkers in the absence of vessel injury. After venous injury, elevated prothrombin increased thrombin generation and the fibrin accumulation rate and total amount of fibrin ≈ 3-fold, producing extended thrombi with increased mass. However, elevated prothrombin did not accelerate platelet accumulation, increase the fibrin accumulation rate, or shorten the vessel occlusion time after arterial injury.
CONCLUSIONS
These findings reconcile previously discordant findings on thrombin generation in hyperprothrombinemic individuals measured ex vivo and in vitro, and show elevated prothrombin promotes venous, but not arterial, thrombosis in vivo.
Topics: Animals; Blood Coagulation; Blood Platelets; Carotid Arteries; Chlorides; Disease Models, Animal; Femoral Vein; Ferric Compounds; Fibrin; Humans; Mice; Noxae; Prothrombin; Risk Factors; Thrombophilia; Vena Cava, Inferior; Venous Thrombosis
PubMed: 23723374
DOI: 10.1161/ATVBAHA.113.301607