In prothrombin G20210A carriers the genetic mutation and the history of Venous Thrombosis contribute both to thrombin generation independently of factor II plasma levels

SummarySeveral hereditary and acquired risk factors for venous thromboembolism (VTE) are associated with impaired down-regulation of thrombin formation via the protein C pathway. To identify individuals at risk, functional tests are needed that estimate the risk to develop venous thrombosis.We determined the effects of hereditary and acquired risk factors of venous thrombosis on an APC resistance test that quantifies the influence of APC on the time integral of thrombin formation (the endogenous thrombin potential, ETP) initiated in plasma via the extrinsic coagulation pathway. APC sensitivity ratios (APCsr) were determined in plasma from carriers of factor VLeiden (n = 56) or prothrombin G20210A (n = 18), of individuals deficient in antithrombin (n = 9), protein C (n = 7) or protein S (n = 14) and of women exposed to acquired risk factors such as hormone replacement therapy (n = 49), oral contraceptive use (n = 126) or pregnancy (n = 35). We also analysed combinations of risk factors (n = 60).The thrombin generation-based APC resistance test was sensitive for the factor VLeiden and prothrombin G20210A mutation, to protein S deficiency, hormone replacement therapy, oral contraceptive use and pregnancy. The assay was not influenced by antithrombin-or protein C deficiency. The presence of more than one risk factor of venous thrombosis resulted in more pronounced APC resistance. The APCsr of individuals with a single or combined risk factors of VTE correlated well with reported risk increases.The thrombin generation-based APC resistance test identifies individuals at risk for venous thrombosis due to acquired risk factors and/or hereditary thrombophilic disorders that affect the protein C pathway.

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Correlation of Genetic Polymorphisms Detected by Geneohm EPLEX™ Electrochemical Platform with Functional Study for Venous Thrombosis.

Blood ◽

10.1182/blood.v104.11.4054.4054 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4054-4054

Author(s):

Bo Xu ◽

Steven Thompson ◽

Carol Koenigberger ◽

James Pettay ◽

Arkadiy Silbergleit ◽

...

Keyword(s):

Risk Factors ◽

Venous Thrombosis ◽

Genetic Polymorphisms ◽

Factor V Leiden ◽

Mthfr C677t ◽

Plasma Homocysteine ◽

Functional Study ◽

Prothrombin G20210a ◽

Factor V ◽

History Of

Abstract Venous thrombosis (VT) is a multi-factorial disorder with both congenital and acquired risk factors. Mutations in several genes, such as factor V, prothrombin and methylene tetrahydrofolate reductase (MTHFR), are considered risk factors for thrombophilia. Since multiple mutations compound the risk for (VT), simultaneous discovery of mutations could directly alter patient management. In this study, we employed the GeneOhm ePlex™ platform to simultaneously detect genetic polymorphisms for six markers: factor V Leiden (FVL) and HR2A45374G, prothrombin G20210A, MTHFR C677T and A1298C, and plasminogen activator inhibitor 1 (4G/5G). Fifty-one patient samples were selected. Each sample was genotyped for all six markers on the GeneOhm ePlex™ electrochemical array and data from functional studies were analyzed and compared to the genotyping results. Among the 51 patients, 16 were tested for activated protein C resistance and the average values were 1.22, 1.76 and 2.64 for FVL homozygous, heterozygous and wild type normal patients, respectively. In addition, the average plasma homocysteine levels measured in 17 patients were 15.40, 6.42 and 11.93, 12.63 mmol/L for MTHFR C677T homozygous, heterozygous and MTHFR A1298C heterozygous and C677T/A1298C double heterozygous, respectively. Furthermore, 10 out of 11 patients with history of deep venous thrombosis (DVT) and/or pulmonary embolism (PE) displayed genetic abnormalities in FVL or prothrombin G20210A. The other patient with history of both DVT and PE showed homozygous in MTHFR C677T with high plasma homocysteine level (22.3 mmol/L) and heterozygous mutation in PAI-1. This study demonstrates the principle of multiplexed molecular diagnostics for the polymorphisms associated with thrombophilia and the utility of the GeneOhm ePlex platform. The study is being expanded to test a larger set of samples to establish the relationship between genetic polymorphism and corresponding clinical outcome for all six markers.

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C0372 Enhanced thrombin generation in women with history of oral contraception related venous thrombosis

Thrombosis Research ◽

10.1016/j.thromres.2012.08.049 ◽

2012 ◽

Vol 130 ◽

pp. S118-S119

Author(s):

Saša Anžej Doma ◽

Maja Vučnik ◽

Mojca Božič Mijovski ◽

Polona Peternel ◽

Mojca Stegnar

Keyword(s):

Venous Thrombosis ◽

Thrombin Generation ◽

Oral Contraception ◽

History Of

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Venous thrombosis and multiple prothrombotic gene defects

Hämostaseologie ◽

10.1055/s-0037-1619580 ◽

2003 ◽

Vol 23 (03) ◽

pp. 117-120

Author(s):

C. Mengis ◽

F. Demarmels Biasiutti

Keyword(s):

Risk Factors ◽

Venous Thrombosis ◽

Thrombotic Event ◽

Prothrombin G20210a ◽

Thrombotic Risk ◽

History Of ◽

Fv Leiden ◽

Gene Defects

SummaryWe describe the case of a 30-year-old woman with homozygous prothrombin G20210A transition and heterozygous FV Leiden mutation and a history of postpartum venous thrombosis. Despite the high thrombotic risk to be assumed in the presence of this combined thrombophilia our patient suffered her first venous thrombotic event only at the age of 29 years during the puerperium of her first pregnancy. This fact supports the concept of venous thrombosis as a multicausal disease, with interaction of genetic and acquired risk factors. The case presented also stresses the importance of performing complete thrombophilia investigation.

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P1251THROMBIN GENERATION ASSAY: A POTENTIAL TOOL TO STRATIFY THROMBOTIC RISK IN HEMODIALYSIS PATIENTS

Nephrology Dialysis Transplantation ◽

10.1093/ndt/gfaa142.p1251 ◽

2020 ◽

Vol 35 (Supplement_3) ◽

Author(s):

Ileana Kalikatzaros ◽

Massimo Radin ◽

Irene Cecchi ◽

Savino Sciascia ◽

Giacomo Forneris ◽

...

Keyword(s):

Ischemic Stroke ◽

Venous Thrombosis ◽

Thrombin Generation ◽

Previous History ◽

International Normalized Ratio ◽

Vascular Events ◽

Thrombotic Risk ◽

Thrombin Generation Assay ◽

Autoimmune Conditions ◽

History Of

Abstract Background and Aims Patients with Chronic Kidney Disease (CKD) in hemodialysis (HD) show both high thrombotic and hemorrhagic risks. However, routine laboratory techniques aimed to evaluate haemostasis, i.e. activated prothrombin time (PT) and activated partial thromboplastin time (aPTT), are not sensitive enough to detect mild hypocoagulable or hypercoagulable states in this population. Indeed, these methods evaluate the start-up phase of the coagulation, but omit the amplification stage in which an exponential increase of thrombin generation occurs. Thrombin generation assay (TGA) is a second-level global coagulative test able to evaluate thrombin generation and decay. So far the TGA has never been used for assessing thrombotic risk in HD patients. Method This is a monocentric observational retrospective study conducted at San Giovanni Bosco Hospital and University of Turin, Italy. After chart-reviewing of all patients with CKD in HD, we enrolled: Group A) 100 Patients with CKD in HD, treated or not treated with warfarin Group B) 60 Patients treated with Warfarin with normal kidney function Group C) 60 Healthy Controls Results Compared to healthy donor patients on hemodialysis that were not treated with warfarin had significantly lower tLag (mean tLag 8.2±3.4 vs. 9.7±2.9, p < 0.05), lower tPeak (mean tPeak 14.3±6 vs. 16.2±4.7, p < 0.05), lower Peak (mean Peak 151.8±77.4 vs. 209.2±103.8, p < 0.001) and lower AUC (mean AUC 1624.5±564.4 vs. 2023±489.2, p < 0.001) (Figure 1). Compared to controls with normal renal function treated with warfarin, HD patients treated with warfarin had higher tLag (mean tLag 10.5±3.3 vs. 8.3±2.1, p < 0.05), higher tPeak (mean tPeak 16.5±4.9 vs. 13±2.9, p < 0.05). Among HD patients who were not treated with warfarin, those with autoimmune conditions showed a pro-thrombotic TGA profile when compared to HD patients without autoimmune diseases, with significantly higher Peak (mean Peak 188.4±30 vs. 149.9±78.7, p < 0.05) and higher AUC (mean AUC 2066.9±138.2 vs. 1601.5±569, p < 0.001). Similarly, compared to patients without previous history of vascular events (59), patients with previous ischemic stroke or venous thrombosis (41), had significantly lower tLag (mean tLag 8±2.9 vs. 14.2±8.5, p < 0.001), lower tPeak (mean tPeak 14±5.6 vs. 21.7±12.3, p <0.05), higher Peak (mean Peak 154.9±76.8 vs. 71.83±49.2, p<0.05) and higher AUC (mean AUC 1653.7±548.7 vs. 863.4±501.4, p < 0.05). Of note, a significant positive relationship was detected between the International Normalized Ratio (INR) and both tLag (Pearson 0.46, p <0.001) and tPeak (Pearson 0.35, p <0.001). INR was inversely correlated to Peak (Pearson -0.47, p <0.001) and AUC (Pearson -0.61, p <0.001) (Figure 2). Conclusion Identifying patients at high risk for cardiovascular diseases and thrombosis has an important impact on the management of patients with CKD in HD. In this study, we observed a prothrombotic TGA profile in patients with CKD in HD, especially those with autoimmune conditions or previous history of arterial events (especially ischemic stroke) or venous thrombosis. Prospective studies are needed to evaluate the possible clinical use of TGA as thrombotic risk stratification tool in HD patients.

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Plasma Constitutive Serum Amyloid A4 Is Procoagulant and Is Elevated in Venous Thrombosis Patients

Blood ◽

10.1182/blood.v126.23.3486.3486 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3486-3486

Author(s):

John H. Griffin ◽

Jose A. Fernandez ◽

Ranjeet Kumar Sinha ◽

Darlene J. Elias ◽

Hiroshi Deguchi

Keyword(s):

Venous Thrombosis ◽

Plasma Levels ◽

Thrombin Generation ◽

Conflicts Of Interest ◽

Oxidized Ldl ◽

Serum Amyloid ◽

Procoagulant Activity ◽

Clinical Event ◽

Coagulation Assays ◽

Sds Page

Abstract Regulation of thrombin generation by the factors (F) Xa:Va complex is key for achieving hemostasis and avoiding thrombosis, i.e., for the hemostatic balance. Plasma lipoproteins and minor abundance lipids can influence the hemostatic balance. For example, oxidized LDL is procoagulant, HDL is anticoagulant as an APC:protein S cofactor, certain glycosphingolipids are anticoagulant cofactors, and acyl carnitines and lysosulfatides inhibit the FXa;Va complex. To identify novel lipoprotein factors that affect FXa:Va activity (prothrombinase (IIase) activity), lipoprotein fractions from ten adult varied subjects were made by ultracentrifugation and, following dialysis into Hepes-buffered saline, screened for their procoagulant activity in purified IIase assays. Each lipoprotein fraction, except that from one subject (#939) with only 22% activity, similarly enhanced thrombin generation by IIase. SDS page analysis of these lipoprotein fractions showed a very low level of one protein band (14 kDa) for subject #939. Proteomics analysis of that band from normal lipoprotein fractions implied this 14 kDa band was constitutive serum amyloid A4 (SAA4). The biologic functions of SAA4 are unclear. To assess its procoagulant activity, recombinant 14 kDa SAA4 was studied in various coagulation assays. When SAA4 was added to IIase assays, it caused a robust procoagulant activity in the presence of FVa but not in the absence of FVa. Plasma-based coagulation assays, done without phospholipids added, were also used, including FXIa-induced thrombin generation, and FXa-1-stage clotting assays. Recombinant SAA4 was mildly or strongly procoagulant in these plasma-based assays. SAA4 dose-dependently shortened the FXa-induced clotting time from 320 sec to 55 sec; and SAA4 increased thrombin generation by 15 % in normal plasma and by 85 % in plasma from subject #939. Direct interactions between recombinant SAA4 and purified FXa were studied using SPR (Octet Red™) with immobilized-FXa subjected to titrations of SAA4. Kinetic constants gave a value for Kd apparent of 25 nM for SAA4 binding to FXa. Thus, SAA4 binds FXa and is procoagulant by enhancing IIase activity. We then assessed potential clinical relevance of SAA4's procoagulant activity. SAA4 is a plasma apolipoprotein, mainly made in the liver, with a constant plasma level of 4 µM. It is not an acute phase protein, unlike SAA1 and SAA2. SAA4 contains 112 residues and plasma contains two monomeric isoforms (14 kDa and 19 kDa) due to the absence or presence of N-glycosylation at Asn-76. The plasma levels of monomeric SAA4 (combined 14 and 19 kDa isoforms) were determined by quantitative IR immunoblotting (non-reduced SDS PAGE) using the Odyssey IR imaging system (Li-Cor Biosciences) for 110 venous thrombosis (VTE) subjects and 110 matched controls from the Scripps Venous Thrombosis Registry. The Registry samples were from male and female adults without cancer or lipid altering drugs who were < 55 yrs old and gave fasting blood samples > 3 months after the clinical event (see H. Deguchi et al, BLOOD 2015). When the relative fluorescence unit (RFU) values for SAA4 14 and 19 kDa monomer bands for 110 normal subjects were normalized to a median of 100 %, patients with VTE had significantly higher plasma levels of monomeric SAA4 (median 116.4 RFU vs median 100 RFU, p=0.0005) (see median values in Figure). The odds ratio (OR) for the 75%-ile cut-off was 2.1 (95% CI = 1.2 - 3.8, p=0.01) (see Figure). Thus, SAA4 monomers are elevated in VTE patients compared to matched controls for the Scripps Venous Thrombosis Registry. Further studies are needed to replicate this finding and to gather more data towards deciphering whether the link between elevated SAA4 and VTE is causal, consequential or coincidental. However, the fact that recombinant 14 kDa SAA4 binds FXa and is procoagulant in purified IIase assays and in plasma-based clotting assays provides biological plausibility for a causal prothrombotic role for SAA4. These findings also raise the possibility that SAA4 procoagulant activity might contribute to normal hemostasis. Further studies are needed to clarify the details for the procoagulant effects of SAA4 on coagulation pathways. In summary, these results show that elevated monomeric plasma levels of SAA4 are strongly linked to VTE in adults (< 55 yrs old) and that SAA4 itself is a potential enhancer of thrombin generation in plasma. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

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Circulating Activated Protein C in Thrombophilia Carriers

Blood ◽

10.1182/blood.v128.22.4948.4948 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4948-4948

Author(s):

Thijs E van Mens ◽

Joost C.M. Meijers ◽

Saskia Middeldorp

Keyword(s):

Venous Thrombosis ◽

Lower Limit ◽

Protein C ◽

Thrombin Generation ◽

Activated Protein C ◽

Factor V Leiden ◽

Prothrombin G20210a ◽

Factor V ◽

Limit Of Quantification ◽

Inherited Thrombophilia

Abstract Background: Inherited thrombophilias are genetic disorders in which mutation carriers have an elevated risk of venous thromboembolism through abnormalities in the coagulation cascade. These abnormalities all lead to increased thrombin generation. The mutations, of which factor V Leiden and prothrombin G20210A are the most common, therefore likely increase thrombin mediated protein C activation in plasma. Previous findings have however been inconsistent. Increased activation of protein C in inherited thrombophilia would be interesting in light of various unexplained phenotypes described in thrombophilia carriers. Examples of such phenotypes include improved fertility, increased risk of miscarriage, protection from diabetic nephropathy, decreased susceptibility to and mortality from sepsis and decreased mortality in acute respiratory distress syndrome. These do not appear directly related to increased coagulation in carriers. Activated protein C (APC) possesses a wide range of signaling functions and interactions with multiple pathways. These result in anti-apoptotic, anti-inflammatory, gene-expression, regenerative and endothelial stabilizing effects. Such properties can easily be thought to play a role in the above described phenotypes. APC has indeed been shown to possess beneficial properties in numerous animal injury models. Due to its pleiotropic nature, APC might be a promising candidate for further research into the unexplained phenotypes observed in inherited thrombophilia. Aim: To investigate if plasma APC concentrations are higher in thrombophilia carriers as compared to non-carriers. Methods: We performed a cross-sectional observational study comparing the APC plasma levels of factor V Leiden and prothrombin G20210A mutation hetero- and homozygotes with non-carriers. Exclusion criteria comprised use of anticoagulant medication and recent venous thrombosis or risk factors for venous thrombosis. We measured APC using a recently developed highly sensitive oligonucleotide-based capture assay, with a limit of detection of 0.022 ng/ml and the ability to quantify APC upward of 0.116 ng/ml (lower limit of quantification) (Müller et al., 2012). In addition we determined APC-protein C inhibitor complex (APC-PCI) as a secondary measure of protein C activation, and prothrombin fragment 1+2 (F1+2) concentration as a measure of thrombin generation using immunoassays. Parametric and non-parametric descriptive and inferential statistics were applied as appropriate. Results: We included 19 thrombophilia carriers and 18 non-carriers (Table 1). APC was detectable in 47% of carriers and in 39% of non-carriers (p = 0.74). APC was above the lower limit of quantification in only 19% of all subjects, with no difference between the groups (Figure 1). The median APC-PCI concentration in carriers and non-carriers were 5 AU (IQR 3.5-10.5) vs. 5 AU (IQR 3.0-8.0) (p = 0.338); and mean F1+2 concentrations were 266 pmol/L and 194 pmol/L in carriers and non-carriers respectively (p = 0.075). Discussion: We did not find increased circulating APC concentrations in thrombophilia carriers. Given the low number of subjects with quantifiable APC in the study, elevated APC levels in carriers versus non-carriers cannot be fully excluded. Local elevation at the site of thrombin formation still seems plausible, and our data do show a trend towards increased thrombin generation in thrombophilia carriers. However, we also show that systemic concentrations are generally below 0.116 ng/ml, which is an order of magnitude lower than concentrations previously reported as physiological levels. A prominent role for APC in non-coagulation related thrombophilia phenotypes might therefore be questioned. References Müller, J. et al. (2012). Journal of Thrombosis and Haemostasis : JTH, 10(3), 390-8. Measured APC concentrations above the limit of quantification, according to thrombophilia carriership status. Measured APC concentrations above the limit of quantification, according to thrombophilia carriership status. Figure 1 Figure 1. Disclosures Middeldorp: Boehringer Ingelheim: Consultancy; GSK: Consultancy, Honoraria; Aspen: Consultancy, Honoraria; BMS/Pfizer: Consultancy, Honoraria; Bayer: Consultancy; Daiichi Sankyo: Consultancy, Honoraria; Sanquin: Consultancy.

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Thrombin generation and D-dimer concentrations in a patient cohort investigated for venous thromboembolism. Relations to venous thrombosis, factor V Leiden and prothrombin G20210A. The LIST study

Thrombosis Research ◽

10.1016/j.thromres.2008.12.033 ◽

2009 ◽

Vol 124 (2) ◽

pp. 178-184 ◽

Cited By ~ 17

Author(s):

Roza Chaireti ◽

Cecilia Jennersjö ◽

Tomas L. Lindahl

Keyword(s):

Venous Thromboembolism ◽

Venous Thrombosis ◽

Thrombin Generation ◽

Factor V Leiden ◽

Prothrombin G20210a ◽

Factor V ◽

Patient Cohort ◽

D Dimer

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Idiopathic venous thrombosis

Hämostaseologie ◽

10.1055/s-0037-1616877 ◽

2006 ◽

Vol 26 (01) ◽

pp. 52-54 ◽

Cited By ~ 3

Author(s):

P. A. Kyrle

Keyword(s):

Chronic Disease ◽

Venous Thrombosis ◽

Vitamin K ◽

Clinical Benefit ◽

Plasma Levels ◽

Vitamin K Antagonists ◽

Antithrombin Deficiency ◽

Optimal Duration ◽

Risk Of Treatment

SummaryVenous thrombosis is a chronic disease with a recurrence rate of approximately 30% within 5-8 years. The optimal duration of secondary thromboprophylaxis in these patients entails balancing the risk of recurrence against the risk of treatment-associated bleeding. There is agreement that patients with a first idiopathic venous thrombosis should receive vitamin K antagonists for at least 3-6 months. Convincing trials showing a clinical benefit in terms of morbidity or mortality with respect to expansion of anticoagulation beyond 6 months are lacking. Nevertheless, some subgroups of patients with venous thrombosis may benefit from indefinite anticoagulation. Thus, patients with antithrombin deficiency, combined or homozygous defects, more than one unprovoked episode of thrombosis, the lupus anticoagulant or high factor VIII plasma levels are good candidates for long-term prevention.

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