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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 -
Clinical Immunology (Orlando, Fla.) Apr 2024Our study aimed to evaluate the presence, clinical associations, and potential mechanistic roles of non-criteria antiphospholipid antibodies (aPL) and circulating...
Our study aimed to evaluate the presence, clinical associations, and potential mechanistic roles of non-criteria antiphospholipid antibodies (aPL) and circulating calprotectin, a highly stable marker of neutrophil extracellular trap release (NETosis), in pediatric APS patients. We found that 79% of pediatric APS patients had at least one non-criteria aPL at moderate-to-high titer. Univariate logistic regression demonstrated that positive anti-beta-2 glycoprotein I domain 1 (anti-D1) IgG (p = 0.008), anti-phosphatidylserine/prothrombin (aPS/PT) IgG (p < 0.001), and aPS/PT IgM (p < 0.001) were significantly associated with venous thrombosis. Positive anti-D1 IgG (p < 0.001), aPS/PT IgG (p < 0.001), and aPS/PT IgM (p = 0.001) were also associated with non-thrombotic manifestations of APS, such as thrombocytopenia. Increased levels of calprotectin were detected in children with APS. Calprotectin correlated positively with absolute neutrophil count (r = 0.63, p = 0.008) and negatively with platelet count (r = -0.59, p = 0.015). Mechanistically, plasma from pediatric APS patients with high calprotectin levels impaired platelet viability in a dose-dependent manner.
Topics: Humans; Child; Antibodies, Antiphospholipid; Antiphospholipid Syndrome; Biomarkers; beta 2-Glycoprotein I; Immunoglobulin G; Immunoglobulin M; Prothrombin; Leukocyte L1 Antigen Complex
PubMed: 38355030
DOI: 10.1016/j.clim.2024.109926 -
Beijing Da Xue Xue Bao. Yi Xue Ban =... Dec 2023To investigate whether anti-phosphatidylserine/prothrombin antibodies and its IgG or IgM subtypes were correlated with unexplained recurrent miscarriages.
OBJECTIVE
To investigate whether anti-phosphatidylserine/prothrombin antibodies and its IgG or IgM subtypes were correlated with unexplained recurrent miscarriages.
METHODS
In our a single-center retrospective study, 283 patients with at least one unexplained miscarriage who visited the Third Hospital of Peking University between January 2021 and August 2023, aged between 18-40 years, and tested for anti-phosphatidylserine/prothrombin antibodies IgG or IgM subtypes, were included. The patients with either positive IgG or IgM anti-phosphatidylserine/prothrombin antibody were regarded as positive for anti-phosphatidylserine/prothrombin antibody. SPSS 26.0 software was used for statistical analysis. Chi-square test and Logistic regression analysis were used to study the correlation of anti-phosphatidylserine/prothrombin antibodies and its IgG or IgM subtypes with unexplained recurrent miscarriages. And the diagnostic sensitivity, specificity, the positive predictive value, the negative predictive value of anti-phosphatidylserine/prothrombin antibodies and its IgG or IgM subtypes in unexplained miscarriages was calculated with four-fold table.
RESULTS
Chi-square analysis showed that anti-phosphatidylserine/prothrombin antibodies and its IgM subtypes were correlated with recurrent miscarriages (both < 0.05), while the IgG subtype was not correlated with recurrent miscarriages (>0.05). After adjusting with anticardiolipin antibodies, anti-β glycoprotein antibodies, lupus anticoagulants, antinuclear antibodies, and age by Logistic regression analysis, anti-phosphatidylserine/prothrombin antibodies were correlated with unexplained recurrent miscarriages (=2.084, 95% 1.045-4.155, < 0.05), and anti-phosphatidylserine/prothrombin antibody IgM subtypes were correlated with unexplained recurrent miscarriages (=2.368, 95% 1.187-4.722, < 0.05).The sensitivity of anti-phosphatidylserine/prothrombin antibody in recurrent miscarriage was 65.43%, the specificity was 48.51%, the positive predictive value was 33.76%, and the negative predictive value was 77.78%. In the patients with recurrent miscarriages with negative classical antiphospholipid antibodies, the sensitivity of anti-phosphatidylserine/prothrombin antibody was 59.09%, the specificity was 63.23%, the positive predictive value was 40.63%, and the negative predictive value was 78.40%. The sensitivity of the anti-phosphatidylserine/prothrombin antibody IgM subtype for the diagnosis of recurrent miscarriage was 65.43%, the specificity was 50.99%, the positive predictive value was 34.87%, and the negative predictive value was 78.63%.
CONCLUSION
Anti-phosphatidylserine/prothrombin antibody and IgM subtype antibody are correlated with unexplained recurrent miscarriages in patients with at least one unexplained miscarriage. Whether positive anti-phosphatidylserine/prothrombin antibody or IgM subtype could predict future unexplained recurrent miscarriages warrants a prospective study.
Topics: Pregnancy; Female; Humans; Adolescent; Young Adult; Adult; Prothrombin; Retrospective Studies; Phosphatidylserines; Prospective Studies; beta 2-Glycoprotein I; Antibodies, Antiphospholipid; Antiphospholipid Syndrome; Antibodies, Anticardiolipin; Abortion, Habitual; Immunoglobulin G; Immunoglobulin M
PubMed: 38101789
DOI: 10.19723/j.issn.1671-167X.2023.06.016 -
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 -
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 -
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 -
Blood Jun 2024The factor V Leiden (FVL; rs6025) and prothrombin G20210A (PTGM; rs1799963) polymorphisms are 2 of the most well-studied genetic risk factors for venous thromboembolism...
The factor V Leiden (FVL; rs6025) and prothrombin G20210A (PTGM; rs1799963) polymorphisms are 2 of the most well-studied genetic risk factors for venous thromboembolism (VTE). However, double heterozygosity (DH) for FVL and PTGM remains poorly understood, with previous studies showing marked disagreement regarding thrombosis risk conferred by the DH genotype. Using multidimensional data from the UK Biobank (UKB) and FinnGen biorepositories, we evaluated the clinical impact of DH carrier status across 937 939 individuals. We found that 662 participants (0.07%) were DH carriers. After adjustment for age, sex, and ancestry, DH individuals experienced a markedly elevated risk of VTE compared with wild-type individuals (odds ratio [OR] = 5.24; 95% confidence interval [CI], 4.01-6.84; P = 4.8 × 10-34), which approximated the risk conferred by FVL homozygosity. A secondary analysis restricted to UKB participants (N = 445 144) found that effect size estimates for the DH genotype remained largely unchanged (OR = 4.53; 95% CI, 3.42-5.90; P < 1 × 10-16) after adjustment for commonly cited VTE risk factors, such as body mass index, blood type, and markers of inflammation. In contrast, the DH genotype was not associated with a significantly higher risk of any arterial thrombosis phenotype, including stroke, myocardial infarction, and peripheral artery disease. In summary, we leveraged population-scale genomic data sets to conduct, to our knowledge, the largest study to date on the DH genotype and were able to establish far more precise effect size estimates than previously possible. Our findings indicate that the DH genotype may occur as frequently as FVL homozygosity and may confer a similarly increased risk of VTE.
Topics: Humans; Prothrombin; Factor V; Female; Male; Heterozygote; Middle Aged; United Kingdom; Biological Specimen Banks; Aged; Risk Factors; Venous Thromboembolism; Adult; Thrombosis; Genetic Predisposition to Disease; Genotype; Polymorphism, Single Nucleotide; UK Biobank
PubMed: 38498041
DOI: 10.1182/blood.2023023326 -
Scientific Reports Jan 2020The equilibrium between active E and inactive E* forms of thrombin is assumed to be governed by the allosteric binding of a Na ion. Here we use molecular dynamics...
The equilibrium between active E and inactive E* forms of thrombin is assumed to be governed by the allosteric binding of a Na ion. Here we use molecular dynamics simulations and Markov state models to sample transitions between active and inactive states. With these calculations we are able to compare thermodynamic and kinetic properties depending on the presence of Na. For the first time, we directly observe sodium-induced conformational changes in long-timescale computer simulations. Thereby, we are able to explain the resulting change in activity. We observe a stabilization of the active form in presence of Na and a shift towards the inactive form in Na-free simulations. We identify key structural features to quantify and monitor this conformational shift. These include the accessibility of the S1 pocket and the reorientation of W215, of R221a and of the Na loop. The structural characteristics exhibit dynamics at various timescales: Conformational changes in the Na binding loop constitute the slowest observed movement. Depending on its orientation, it induces conformational shifts in the nearby substrate binding site. Only after this shift, residue W215 is able to move freely, allowing thrombin to adopt a binding-competent conformation.
Topics: Amino Acid Motifs; Humans; Kinetics; Molecular Dynamics Simulation; Protein Binding; Protein Conformation; Prothrombin; Sodium; Thrombin
PubMed: 31974511
DOI: 10.1038/s41598-020-57822-0