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Journal of Thrombosis and Haemostasis :... Jul 2023Current assays that monitor thrombin generation in plasma rely on fluorogenic substrates to follow the kinetics of zymogen activation, which may be complicated by...
BACKGROUND
Current assays that monitor thrombin generation in plasma rely on fluorogenic substrates to follow the kinetics of zymogen activation, which may be complicated by substrate cleavage from other proteases. In addition, these assays depend on activation following cleavage at the prothrombin R320 site and fail to report the cleavage at the alternative R271 site, leading to the shedding of the auxiliary Gla and kringle domains of prothrombin.
OBJECTIVES
To develop a plasma assay that directly monitors prothrombin activation independent of fluorogenic substrate hydrolysis.
METHODS
Cleavage at the R271 site of prothrombin is monitored through loss of Förster resonance energy transfer in plasma coagulated along the extrinsic or intrinsic pathway.
RESULTS
The availability of factor (F)V in plasma strongly influences the rate of prothrombin activation. The rate of thrombin formation is equally perturbed in FV or prothrombin-depleted plasma, implicating that the thrombin-catalyzed feedback reactions that amplify the coagulation response play an important role in generating sufficient amounts of FVa required for the assembly of prothrombinase. Congenital deficiencies in FVIII and FIX significantly slow down cleavage at R271 in plasma coagulated along the extrinsic and intrinsic pathways. Prothrombin activation in FXI-deficient plasma is only perturbed when coagulation is triggered along the intrinsic pathway.
CONCLUSION
The Förster resonance energy transfer assay enables direct monitoring of prothrombin activation through cleavage at R271 without the need for fluorogenic substrates. The assay is sensitive enough to assess how deficiencies in coagulation factors affect thrombin formation.
Topics: Humans; Prothrombin; Thrombin; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Blood Coagulation Factors; Factor Xa
PubMed: 36931601
DOI: 10.1016/j.jtha.2023.03.008 -
Journal of Thrombosis and Thrombolysis Aug 2018The predictive value of factor V Leiden and the G20210A prothrombin mutation regarding recurrent venous thromboembolism (VTE) is limited and does not influence...
The predictive value of factor V Leiden and the G20210A prothrombin mutation regarding recurrent venous thromboembolism (VTE) is limited and does not influence subsequent patient management. Systematic testing for such genetic thrombophilia should be avoided, but to which extent such testing is practiced in a Swiss Hospital is unknown. To examine the current practice of factor V Leiden and/or G20210A prothrombin mutation testing in a University Hospital, and to assess the clinical consequences of testing on patients. 1388 adult patients (48.7% women) with a main diagnosis of VTE hospitalized at the Lausanne university hospital between January 2013 and December 2015. FV Leiden and/or prothrombin G20210A mutation testing was performed in 61 (4.4%) patients with VTE, an average of 20 patients/year. On multivariable analysis, age < 65 years [odds ratio and (95% confidence interval) 5.91 (3.12-11.19)], being admitted in a medical ward [5.71 (2.02-16.16)] and staying in the intensive care unit [0.34 (0.12-0.97)] were associated with thrombophilia testing. No differences were found between patients with and without testing regarding in-hospital mortality [OR and 95% CI for tested vs. non-tested: 0.23 (0.03-1.73), p = 0.153] and length of stay (multivariable adjusted average ± standard error: 16.9 ± 3.3 vs. 20.0 ± 0.7 days for tested and non-tested patients, respectively, p = 0.875). Thrombophilia testing in hospitalized patients with a main diagnosis of VTE is seldom performed. FV Leiden and/or prothrombin G20210A mutation should not be routinely assessed in patients with acute VTE.
Topics: Adult; Factor V; Genetic Predisposition to Disease; Genetic Testing; Hospital Mortality; Hospitals; Humans; Length of Stay; Middle Aged; Mutation; Practice Patterns, Physicians'; Prothrombin; Thrombophilia; Venous Thromboembolism
PubMed: 29922879
DOI: 10.1007/s11239-018-1702-6 -
Analytical Methods : Advancing Methods... Oct 2022Disorders of haemostasis result in both excessive bleeding and clotting and are a major global cause of morbidity and mortality, particularly in the developing world. A...
Disorders of haemostasis result in both excessive bleeding and clotting and are a major global cause of morbidity and mortality, particularly in the developing world. A small number of simple tests can be used to screen and monitor for such dysfunctions, one of which is the prothrombin time (PT) test and associated International Normalisation Ratio (INR). PT/INR is routine in hospital laboratories in developed countries, and can also be performed using point-of-care instruments. However, neither of these approaches is appropriate in low-resource settings. Significant interest has grown in paper-based devices to form the basis of simple and low-cost assays that may have the potential for application in such environments. This study describes the development of a simple, low-cost, paper-based lateral flow prothrombin assay. The assay employed wax printing on chromatography paper to define test channels, with deposition of thromboplastin reagent and calcium chloride onto the resulting strips. These were placed in a test housing and measurement of the flow rates of deposited plasma samples were performed in triplicate. The flow dynamics of the assay was optimised according to the type of paper substrate used, the nature and quantity of the thromboplastin reagent, the amount of calcium chloride required, and the volume of sample employed. An optimised assay configuration demonstrated a dynamic range of 6 mm between normal and factor-deficient plasmas. The assay showed good correlation with laboratory-based PT assay (Yumizen G200) in artificial plasmas in the 9.8 to 36 s range ( = 0.8112). The assay also demonstrated good dynamic range and correlation in patient plasma samples in comparison with hospital PT, with a range of 9.8 to 45 s ( = 0.7209).
Topics: Anticoagulants; Calcium Chloride; Humans; Indicators and Reagents; Prothrombin; Prothrombin Time; Thromboplastin
PubMed: 36048161
DOI: 10.1039/d2ay00965j -
BMC Medical Genetics Oct 2020Thrombophilia is a hypercoagulable state that may have a genetic basis (inherited) or can be acquired. It is a multifactorial condition and only the mutual interactions...
BACKGROUND
Thrombophilia is a hypercoagulable state that may have a genetic basis (inherited) or can be acquired. It is a multifactorial condition and only the mutual interactions between the environment and genes may lead to the development of clinical manifestation. This state is the main factor promoting venous (rarely arterial) thromboembolism (VTE). Inherited thrombophilia is mainly associated with two pathogenic variants in the V coagulation factor (FV) and the prothrombin (FII) genes. The aim of our study was to evaluate the frequency of two pathogenic variants in FII and FV genes as inherited thrombophilia factors in a group within the Polish population in comparison with other described populations.
METHODS
All studied groups consisted of 633 unrelated patients aged between 18 and 70. Individuals in the research group come from the Podlasie region of Poland. Genotyping of FII and FV variants was performed using the 7900HT Fast Real-Time PCR System and were genotyped by TaqMan assay.
RESULTS
The pathogenic allele frequency for A allele was 0.03 (3%) and 0.07 (7%) for FII and FV genes, respectively. The GA/AA genotypes (c.*97G > A variant) were observed in only 33 (5.03%) individuals in the studied group. Additionally, the frequency of GA/AA genotypes was over 17.4% in the coagulation factor V. Co-incidence of heterozygous genotype GA of variants FII and FV genes was observed in only 4 subjects.
CONCLUSION
The FII gene variant shown in our study is less frequent than in other European countries (about 6%). In contrast, the A allele of the FV gene occurs with a frequency similar to that of Northern, Central and South Central Europe (about 5%).
Topics: Adolescent; Adult; Aged; Alleles; Factor V; Female; Gene Frequency; Genetic Predisposition to Disease; Genotype; Humans; Male; Middle Aged; Polymorphism, Single Nucleotide; Prothrombin; Thrombophilia; Young Adult
PubMed: 33036569
DOI: 10.1186/s12881-020-01136-5 -
American Journal of Obstetrics and... Mar 2024More than 150 million women worldwide use oral contraceptives. Women with inherited thrombophilia and carriers of certain thrombophilia gene variants, such as factor V...
BACKGROUND
More than 150 million women worldwide use oral contraceptives. Women with inherited thrombophilia and carriers of certain thrombophilia gene variants, such as factor V Leiden and the prothrombin, are at an increased risk for venous thromboembolism, especially when combined with oral contraceptive use. Venous thromboembolism is a complex disorder involving many genetic risk factors, and recently, polygenic risk scores have been proposed to capture a significant proportion of the genetic risk of venous thromboembolism.
OBJECTIVE
The aim of this study was to estimate the risk for developing venous thromboembolism when initiating oral contraceptive use (first 2 years) and during continued use among women with a high genetic liability.
STUDY DESIGN
We used a prospective study design in which 244,420 participants from the UK Biobank were followed from birth. The effect of oral contraceptive use during the first 2 years and in the remaining years of oral contraceptive use on the risk of developing venous thromboembolism was estimated using a Cox regression with a time-dependent exposure variable. Women were stratified according to their polygenic risk scores and whether they were carriers of factor V Leiden and/or prothrombin variants.
RESULTS
When genetic risk was not considered, an increased risk for venous thromboembolism was observed during the first 2 years of oral contraceptive use (hazard ratio, 3.09; 95% confidence interval, 3.00-3.20) but not during continued use (hazard ratio, 0.92; 95% confidence interval, 0.80-1.05). However, when genetic risk was considered, women in the highest polygenic risk score category had a more pronounced risk of developing a venous thromboembolism during the first 2 years of oral contraceptive use (hazard ratio, 6.35; 95% confidence interval, 4.98-8.09), and a high risk was also observed among factor V Leiden (hazard ratio, 5.73; 95% confidence interval, 5.31-6.17) and prothrombin variant carriers (hazard ratio, 5.23; 95% confidence interval, 4.67 - 5.87). A high polygenic risk score in combination with being a factor V Leiden and prothrombin variant carrier conferred the highest risk for developing a venous thromboembolism during the first 2 years of oral contraceptive use (hazard ratio, 14.8; 95% confidence interval, 9.28-23.6). Women with a high genetic liability also had an increased risk during continued use but it was less pronounced, and the highest risk was conferred to carriers of both factor V Leiden and the prothrombin variant (hazard ratio, 4.93; 95% confidence interval, 3.16-7.7).
CONCLUSION
Evaluating polygenic risk can identify additional venous thromboembolism risk that is not captured in the commonly investigated genes for inherited thrombophilia. Our results indicate that oral contraceptive use is associated with an increased risk for developing a venous thromboembolism, particularly among women with a high genetic predisposition, and that oral contraceptive use dramatically increases the risk thereof short after initiation of use, which decreases with continued use. This suggests that the polygenic risk score could be used to identify women who are at high risk for developing a venous thromboembolism and advise them on alternative methods of contraception.
Topics: Humans; Female; Venous Thromboembolism; Contraceptives, Oral; Prospective Studies; Prothrombin; UK Biobank; Biological Specimen Banks; Thrombophilia; Risk Factors; Contraception; Factor V
PubMed: 37734636
DOI: 10.1016/j.ajog.2023.09.012 -
Advances in Clinical Chemistry 2016Antiphospholipid syndrome (APS) is an autoimmune disorder characterized by thrombosis and/or pregnancy-related morbidity accompanied by persistently positive... (Review)
Review
Antiphospholipid syndrome (APS) is an autoimmune disorder characterized by thrombosis and/or pregnancy-related morbidity accompanied by persistently positive antiphospholipid antibodies (aPL). Current laboratory criteria for APS classification recommend testing for lupus anticoagulant as well as IgG and IgM anticardiolipin, and beta-2 glycoprotein I (anti-β2GPI) antibodies. However, there appears to be a subset of patients with classical APS manifestations who test negative for the recommended criteria aPL tests. While acknowledging that such patients may have clinical features that are not of an autoimmune etiology, experts also speculate that these "seronegative" patients may test negative for relevant autoantibodies as a result of a lack of harmonization and/or standardization. Alternatively, they may have aPL that target other antigens involved in the pathogenesis of APS. In the latter, autoantibodies that recognize a phosphatidylserine/prothrombin (PS/PT) complex have been reported to be associated with APS and may have diagnostic relevance. This review highlights analytical and clinical attributes associated with PS/PT antibodies, taking into consideration the performance characteristics of criteria aPL tests in APS with specific recommendations for harmonization and standardization efforts.
Topics: Antibodies; Antiphospholipid Syndrome; Biomarkers; Humans; Phosphatidylserines; Prothrombin
PubMed: 26975968
DOI: 10.1016/bs.acc.2015.10.003 -
Journal of Thrombosis and Thrombolysis Nov 2021Although a few antiphospholipid syndrome (APS) occurs with acquired thrombotic thrombocytopenic purpura (TTP), the relationship between antiphospholipid antibodies (aPL)...
Although a few antiphospholipid syndrome (APS) occurs with acquired thrombotic thrombocytopenic purpura (TTP), the relationship between antiphospholipid antibodies (aPL) and anti-ADAMTS13 (anti-a disintegrin and metalloprotease with thrombospondin type 1 motif, member 13) antibody remains uncertain. We investigated the relationship between high-risk thrombotic aPL and anti-ADAMTS13 antibody. Two hundred and thirty-seven patients with positive lupus anticoagulant and/or anticardiolipin antibody were included. Anti-βGPI (anti-β-glycoprotein I), anti-βGPIdI (anti-β2-glycoprotein I domain I), anti-PS/PT (anti-phosphatidylserine and prothrombin), ADAMTS13 activity, and anti-ADAMTS13 antibody were measured. Double positivity of anti-βGPI and anti-PS/PT increased thrombotic risk more than three-fold and showed increased positivity of anti-ADAMTS13 antibody in comparison with the double negative group. Double positivity of anti-βGPIdI and anti-PS/PT presented both effects even more. In the linear regression analysis, double positivity of anti-βGPI and anti-PS/PT independently affected the anti-ADAMTS13 antibody level (β = 1.982, P = 0.042). Our results revealed that double positivity of anti-βGPI or anti-βGPIdI and anti-PS/PT increased not only thrombotic risk but also the positivity of anti-ADAMTS13 antibody, especially indicating anti-βGPIdI showed a higher synergistic effect with anti-PS/PT. We suggest a possible association of anti-ADAMTS13 antibody with a high thrombotic risk of APS. Double positivity of anti-βGPI (anti-β-glycoprotein I) and anti-PS/PT (anti-phosphatidylserine and prothrombin) antibodies enhanced not only thrombotic risk but also positivity of anti-ADAMTS13 (anti-a disintegrin and metalloprotease with thrombospondin type 1 motif, member 13) antibody. Furthermore, double positivity of anti-βGPIdI (anti-β2-glycoprotein I domain I) combined with anti-PS/PT even more elevated both thrombosis and positivity of anti-ADAMTS13 antibody. Double positivity of βGPI and anti-PS/PT was found as an independently significant contributing factor to anti-ADAMTS13 antibody level. We suggest the association between anti-ADAMTS13 antibody and the pathophysiology of antiphospholipid syndrome, which should be further evaluated.
Topics: Antibodies, Antiphospholipid; Antiphospholipid Syndrome; Disintegrins; Humans; Phosphatidylserines; Prothrombin; Thrombosis; Thrombospondins; beta 2-Glycoprotein I
PubMed: 33914240
DOI: 10.1007/s11239-021-02406-6 -
The Journal of Biological Chemistry Aug 2021In the penultimate step of the coagulation cascade, the multidomain vitamin-K-dependent zymogen prothrombin is converted to thrombin by the prothrombinase complex...
In the penultimate step of the coagulation cascade, the multidomain vitamin-K-dependent zymogen prothrombin is converted to thrombin by the prothrombinase complex composed of factor Xa, cofactor Va, and phospholipids. Activation of prothrombin requires cleavage at two residues, R271 and R320, along two possible pathways generating either the intermediate prethrombin-2 (following initial cleavage at R271) or meizothrombin (following initial cleavage at R320). The former pathway is preferred in the absence of and the latter in the presence of cofactor Va. Several mechanisms have been proposed to explain this preference, but the role of the sequence and position of the sites of cleavage has not been thoroughly investigated. In this study, we engineered constructs where the sequences DEDSDRAIEGRTATSEYQT and RELLESYIDGRIVEGSDAE were swapped between the R271 and R320 sites. We found that in the absence of cofactor Va, the wild-type sequence at the R271 site is cleaved preferentially regardless of its position at the R271 or R320 site, whereas in the presence of cofactor Va, the R320 site is cleaved preferentially regardless of its sequence. Additional single-molecule FRET measurements revealed that the environment of R271 changes significantly upon cleavage at R320 due to the conformational transition from the closed form of prothrombin to the open form of meizothrombin. Detailed kinetics of cleavage at the R271 site were monitored by a newly developed assay based on loss of FRET. These findings show how sequence and position of the cleavage sites at R271 and R320 dictate the preferred pathway of prothrombin activation.
Topics: Blood Coagulation; Kinetics; Prothrombin
PubMed: 34265300
DOI: 10.1016/j.jbc.2021.100955 -
Hepatology Communications Apr 2022Hepatocellular carcinoma (HCC), the sixth most common cancer worldwide, has an incidence rate equal to mortality. Over 80% of HCC cases occur within a high-risk...
Hepatocellular carcinoma (HCC), the sixth most common cancer worldwide, has an incidence rate equal to mortality. Over 80% of HCC cases occur within a high-risk population, mainly patients with both cirrhosis and chronic hepatitis B or C. With a 5-year survival rate ranging from <16% for advanced HCC to >90% for early stage HCC, there is a high medical need for the early detection of HCC. In this study, we systematically evaluated biomarkers mentioned in international guidelines and peer-reviewed literature for HCC surveillance and diagnosis with the aim of identifying combinations that display high sensitivity and specificity for early stage HCC. Fifty biomarkers were measured in the first sample panel, panel A (n = 110), and subjected to univariate analysis. Of these, 35 biomarkers (38 assays) from panel A and an additional 13 biomarkers from the literature were prioritized for subsequent multivariate evaluation with lasso regression and exhaustive search of two- to four-biomarker combinations (panel B). Panel B included 1,081 samples from patients with HCC (n = 308) or with chronic liver diseases (n = 740). Among all patients, 61.0% had hepatitis B, 32.9% had hepatitis C, and 60.5% had cirrhosis; 40.6% of patients with HCC had early stage cancer. Protein induced by vitamin K absence-II (PIVKA-II; also known as des-gamma-carboxy prothrombin [DCP]) and alpha-fetoprotein (AFP) demonstrated the best clinical performance, both individually and in combination, and the addition of a third biomarker (Lens culinaris agglutinin-reactive fraction of AFP [AFP-L3], cartilage oligomeric matrix protein [COMP], insulin-like growth factor-binding protein 3 [IGFBP3], or matrix metalloproteinase 3 [MMP3]) further increased sensitivity for the detection of both early stage and all-stage HCC. The addition of age and sex to the three-biomarker panel resulted in an improved diagnostic performance. Conclusion: The combination of AFP and PIVKA-II, with either IGFBP3, COMP or MMP3, plus age and sex, demonstrated the best performance for the detection of early- and all-stage HCC. These novel panels performed similar to that of the GALAD score (sex [gender], age, plus serum levels of AFP, AFP-L3 and DCP [PIVKA-II]), a promising screening tool developed for HCC detection.
Topics: Biomarkers; Carcinoma, Hepatocellular; Case-Control Studies; Humans; Liver Cirrhosis; Liver Neoplasms; Matrix Metalloproteinase 3; Prospective Studies; Protein Precursors; Prothrombin; alpha-Fetoproteins
PubMed: 34796691
DOI: 10.1002/hep4.1847 -
Seminars in Thrombosis and Hemostasis Apr 2015A rapid restoration of hemostasis should be regarded as a primary goal for management of critical bleeding, which often represents a life-threatening condition. Among... (Review)
Review
A rapid restoration of hemostasis should be regarded as a primary goal for management of critical bleeding, which often represents a life-threatening condition. Among the various therapeutic strategies available in this clinical setting, we aim to summarize in this narrative review the current status on the use of recombinant-activated factor VII and prothrombin complex concentrates. The safety and effectiveness of these hemostatic agents in reversal of the anticoagulant effects of vitamin K antagonists will be also explored. In addition, their role in the management of acute bleeding associated with the newer direct oral anticoagulants dabigatran, rivaroxaban, and apixaban will be analyzed in a dedicated section.
Topics: Administration, Oral; Animals; Anticoagulants; Blood Coagulation Factors; Factor VIIa; Hemorrhage; Hemostasis; Hemostatics; Humans; Prothrombin; Recombinant Proteins; Vitamin K
PubMed: 24937097
DOI: 10.1055/s-0034-1381234