scholarly journals Sensitized mutagenesis screen in Factor V Leiden mice identifies thrombosis suppressor loci

2017 ◽  
Vol 114 (36) ◽  
pp. 9659-9664 ◽  
Author(s):  
Randal J. Westrick ◽  
Kärt Tomberg ◽  
Amy E. Siebert ◽  
Guojing Zhu ◽  
Mary E. Winn ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Genetic Risk ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor V Leiden ◽  
Factor V ◽  
Factor Gene ◽  
Mutagenesis Screen ◽  
Function Mechanism ◽  
Whole Exome ◽  
Tissue Factor Pathway

Factor V Leiden (F5L) is a common genetic risk factor for venous thromboembolism in humans. We conducted a sensitized N-ethyl-N-nitrosourea (ENU) mutagenesis screen for dominant thrombosuppressor genes based on perinatal lethal thrombosis in mice homozygous for F5L (F5L/L) and haploinsufficient for tissue factor pathway inhibitor (Tfpi+/−). F8 deficiency enhanced the survival of F5L/LTfpi+/− mice, demonstrating that F5L/LTfpi+/− lethality is genetically suppressible. ENU-mutagenized F5L/L males and F5L/+Tfpi+/− females were crossed to generate 6,729 progeny, with 98 F5L/LTfpi+/− offspring surviving until weaning. Sixteen lines, referred to as “modifier of Factor 5 Leiden (MF5L1–16),” exhibited transmission of a putative thrombosuppressor to subsequent generations. Linkage analysis in MF5L6 identified a chromosome 3 locus containing the tissue factor gene (F3). Although no ENU-induced F3 mutation was identified, haploinsufficiency for F3 (F3+/−) suppressed F5L/LTfpi+/− lethality. Whole-exome sequencing in MF5L12 identified an Actr2 gene point mutation (p.R258G) as the sole candidate. Inheritance of this variant is associated with suppression of F5L/LTfpi+/− lethality (P = 1.7 × 10−6), suggesting that Actr2p.R258G is thrombosuppressive. CRISPR/Cas9 experiments to generate an independent Actr2 knockin/knockout demonstrated that Actr2 haploinsufficiency is lethal, supporting a hypomorphic or gain-of-function mechanism of action for Actr2p.R258G. Our findings identify F8 and the Tfpi/F3 axis as key regulators in determining thrombosis balance in the setting of F5L and also suggest a role for Actr2 in this process.

scholarly journals Whole exome sequencing of ENU-induced thrombosis modifier mutations in the mouse

2017 ◽  
Author(s):  
Kärt Tomberg ◽  
Randal J. Westrick ◽  
Emilee N. Kotnik ◽  
David R Siemieniak ◽  
Guojing Zhu ◽  
...  
Keyword(s):  
Risk Factor ◽  
Venous Thrombosis ◽  
Genetic Risk ◽  
Blood Clotting ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor V Leiden ◽  
Factor V ◽  
Loss Of Function ◽  
Whole Exome ◽  
Tissue Factor Pathway

AbstractAlthough the Factor V Leiden (FVL) gene variant is the most prevalent genetic risk factor for venous thrombosis, only 10% of FVL carriers will experience such an event in their lifetime. To identify potential FVL modifier genes contributing to this incomplete penetrance, we took advantage of a perinatal synthetic lethal thrombosis phenotype in mice homozygous for FVL (F5L/L) and haploinsufficient for tissue factor pathway inhibitor (Tfpi+/-) to perform a sensitized dominant ENU mutagenesis screen. Linkage analysis conducted in the 3 largest pedigrees generated from the survivingF5L/LTfpi+/-mice (‘rescues’) using ENU-induced coding variants as genetic markers was unsuccessful in identifying major suppressor loci. Whole exome sequencing was applied to DNA from 107 rescue mice to identify candidate genes enriched for ENU mutations. A total of 3,481 potentially deleterious candidate ENU variants were identified in 2,984 genes. After correcting for gene size and multiple testing,Arl6ip5was identified as the most enriched gene, though not reaching genome-wide significance. Evaluation of CRISPR/Cas9 induced loss of function in the top 6 genes failed to demonstrate a clear rescue phenotype. However, a maternally inherited (not ENU-induced)de novomutation (Plcb4R335Q) exhibited significant co-segregation with the rescue phenotype (p=0.003) in the corresponding pedigree. Thrombosis suppression by heterozygousPlcb4loss of function was confirmed through analysis of an independent, CRISPR/Cas9-inducedPlcb4mutation (p=0.01).Author summaryAbnormal blood clotting in veins (venous thrombosis) or arteries (arterial thrombosis) are major health problems, with venous thrombosis affecting approximately 1 in every thousand individuals annually in the United States. Susceptibility to venous thrombosis is governed by both genes and environment, with approximately 60% of the risk attributed to genetic influences. Though several genetic risk factors are known, >50% of genetic risk remains unexplained. Approximately 5% of people carry the most common known risk factor, Factor V Leiden. However, only 10% of these individuals will develop a blood clot in their lifetime. Mice carrying two copies of the Factor V Leiden mutation together with a mutation in a second gene called tissue factor pathway inhibitor develop fatal thrombosis shortly after birth. To identify genes that prevent this fatal thrombosis, we studied a large panel of mice carrying inactivating gene changes randomly distributed throughout the genome. We identified several genes as potential candidates to alter blood clotting balance in mice and humans with predisposition to thrombosis, and confirmed this protective function for DNA changes in one of these genes (Plcb4).

scholarly journals A sensitized mutagenesis screen in Factor V Leiden mice identifies novel thrombosis suppressor loci

2016 ◽  
Author(s):  
Randal J. Westrick ◽  
Kärt Tomberg ◽  
Amy E. Siebert ◽  
Guojing Zhu ◽  
Mary E. Winn ◽  
...  
Keyword(s):  
Venous Thromboembolism ◽  
Tissue Factor ◽  
Factor V Leiden ◽  
Coagulation Factor ◽  
Common Disease ◽  
Factor V ◽  
Mutagenesis Screen ◽  
Function Mechanism ◽  
Whole Exome ◽  
Genomic Regions

AbstractFactor V Leiden (F5L) is a common genetic risk factor for venous thromboembolism in humans. We conducted a sensitized ENU mutagenesis screen for dominant thrombosuppressor genes based on perinatal lethal thrombosis in mice homozygous forF5L(F5L/L) and haploinsufficient for tissue factor pathway inhibitor (Tfpi+/−).F8deficiency enhanced survival ofF5L/LTfpi+/−mice, demonstrating thatF5L/LTfpi+/−lethality is genetically suppressible. ENU-mutagenizedF5L/Lmales andF5L/+Tfpi+/−females were crossed to generate 6,729 progeny, with 98F5L/LTfpi+/−offspring surviving until weaning. Sixteen lines exhibited transmission of a putative thrombosuppressor to subsequent generations, with these lines referred to asMF5L(Modifier ofFactor5 Leiden) 1-16. Linkage analysis inMF5L6identified a chromosome 3 locus containing the tissue factor gene (F3). Though no ENU-inducedF3mutation was identified, haploinsufficiency forF3(F3+/−) suppressedF5L/LTfpi+/−lethality. Whole exome sequencing inMF5L12identified anActr2gene point mutation (p.R258G) as the sole candidate. Inheritance of this variant is associated with suppression ofF5L/LTfpi+/−lethality (p=1.7x10−6), suggesting thatActr2p.R258Gis thrombosuppressive. CRISPR/Cas9 experiments to generate an independentActr2knockin/knockout demonstrated thatActr2haploinsufficiency is lethal, supporting a hypomorphic or gain of function mechanism of action forActr2p.R258G. Our findings identifyF8and theTfpi/F3axis as key regulators in determining thrombosis balance in the setting ofF5Land also suggest a novel role forActr2in this process.Significance StatementVenous thromboembolism (VTE) is a common disease characterized by the formation of inappropriate blood clots. Inheritance of specific genetic variants, such as the Factor V Leiden polymorphism, increases VTE susceptibility. However, only ~10% of people inheriting Factor V Leiden develop VTE, suggesting the involvement of other genes that are currently unknown. By inducing random genetic mutations into mice with a genetic predisposition to VTE, we identified two genomic regions that reduce VTE susceptibility. The first includes the gene for blood coagulation Factor 3 and its role was confirmed by analyzing mice with an independent mutation in this gene. The second contains a mutation in the Actr2 gene. These findings identify critical genes for the regulation of blood clotting risk.

Abstract 43: Prothrombinase Assembled with Factor V Leiden is Resistant to Inhibition by Tissue Factor Pathway Inhibitor α Lowering the Procoagulant Threshold for Initiation of Coagulation

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Jeremy P Wood ◽  
Lisa M Baumann Kreuziger ◽  
Susan A Maroney ◽  
Rodney M Camire ◽  
Alan E Mast
Keyword(s):  
Tissue Factor ◽  
Thrombin Generation ◽  
Tissue Factor Pathway Inhibitor ◽  
Platelet Rich Plasma ◽  
Anticoagulant Activity ◽  
Factor V Leiden ◽  
Factor Xa ◽  
Factor V ◽  
Activation Threshold ◽  
Tissue Factor Pathway

Factor V (FV) assembles with factor Xa (FXa) into prothrombinase, the enzymatic complex that converts prothrombin to thrombin. Tissue factor pathway inhibitor α (TFPIα) inhibits prothrombinase by high affinity interactions with FXa-activated FV and the FXa active site, thereby blocking the initiation of coagulation. FV Leiden (FVL) is strongly linked to venous thrombosis through its resistance to degradation by activated protein C (aPC), which enhances the propagation of coagulation. FVL combined with a 50% reduction in TFPI causes severe thrombosis and perinatal lethality in mice, suggesting that FVL also promotes the initiation of coagulation. To examine this possibility, thrombin generation assays initiated with limiting FXa were performed with control or FVL plasma and platelet-rich plasma (PRP). The activation threshold for thrombin generation was 10 to 20 pM FXa in 10 control plasmas, but was 5 pM in 4 of 10 homozygous FVL plasmas. FVL PRP had a similar decrease in the activation threshold. The differences in activation threshold were totally normalized by an anti-TFPI antibody, while exogenous TFPIα and a FV-binding peptide that mimics TFPIα had reduced anticoagulant activity in FVL plasma, revealing that the procoagulant effects of FVL in these assays rely on TFPIα. Next, FVL plasmas were studied in fibrin clot formation assays, as they are sensitive to small amounts of thrombin. In reactions activated with 0.5 pM FXa, 1 of 8 control plasmas, compared to 7 of 8 homozygous FVL plasmas, clotted within 60 minutes, with differences again normalized by the anti-TFPI antibody. In prothrombinase activity assays using purified proteins, TFPIα was a 1.7-fold weaker inhibitor of prothrombinase assembled with FVL compared to FV. Thus, in addition to its aPC-mediated effect on the propagation of coagulation, FVL is resistant to TFPIα inhibition, exerting a procoagulant effect on coagulation initiation. This is evident in responses to small stimuli, where TFPIα blocks clotting in plasmas with FV but not FVL. The TFPIα-mediated modulation of the procoagulant threshold may explain the severe perinatal thrombosis in FVL mice with decreased TFPI and be clinically relevant in the clotting associated with oral contraceptives, which cause acquired TFPI deficiency.

scholarly journals Lethal Perinatal Thrombosis in Mice Resulting From the Interaction of Tissue Factor Pathway Inhibitor Deficiency and Factor V Leiden

Circulation ◽  
2002 ◽  
Vol 105 (18) ◽  
pp. 2139-2142 ◽  
Author(s):  
Daniel T. Eitzman ◽  
Randal J. Westrick ◽  
Xiaoming Bi ◽  
Sara L. Manning ◽  
John E. Wilkinson ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor V Leiden ◽  
Factor V ◽  
Tissue Factor Pathway

Abstract 35: Identification of a Mouse Strain Modifier Locus for Factor V Leiden/Tissue Factor Pathway Inhibitor Dependent Thrombosis

2015 ◽  
Vol 35 (suppl_1) ◽  
Author(s):  
Derrik M Germain ◽  
Amy E Siebert ◽  
Stephanie Verbeek ◽  
Guojing Zhu ◽  
Kärt Tomberg ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Positional Cloning ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor V Leiden ◽  
Modifier Gene ◽  
Significant Advance ◽  
Mouse Strains ◽  
Factor V ◽  
Modifier Locus ◽  
Tissue Factor Pathway

Factor V Leiden (FVL) is the most common genetic risk factor for venous thromboembolism (VTE). FVL is incompletely penetrant as only 10% of FVL heterozygotes develop VTE. Previously, nearly uniform perinatal lethality was observed in C57BL/6J (B6) mice hemizygous for tissue factor pathway inhibitor (Tfpi+/-) on a homozygous FVL background (FVQ/Q). However, inbred mouse strains exhibit significant genetic variation and can exert a profound influence on phenotype. We tested the lethality of FVQ/Q Tfpi+/- on the DBA/2J inbred strain background by crossing FVQ/+ Tfpi+/- to DBA/2J followed by a double heterozygous (FVQ/+ Tfpi+/-) x FVQ/Q cross. Surprisingly, FVQ/Q Tfpi+/- mice on the DBA/2J genetic background are born at expected Mendelian frequencies (N=62, nexp=15, nobs=15, N.S.) and display no overt thrombosis. This demonstrates the existence of dominant antithrombotic DBA/2J strain-specific thrombosis modifier gene(s). Analysis of progeny from a DBA/2J-B6 F1 FVQ/Q Tfpi+/- x B6 FVQ/Q cross yielded equal proportions of FVQ/Q Tfpi+/- and FVQ/Q Tfpi+/+ (N=130, nexp=43, nobs=63, p<0.001) which suggested that multiple modifier genes were contributing to the phenotype. To isolate individual dominant modifier loci, we adopted a serial backcrossing strategy in which F1 FVQ/Q Tfpi+/- mice were serially backcrossed to B6 FVQ/Q mice for 17 generations. A standard mouse positional cloning strategy was used on the 110 surviving FVQ/Q Tfpi+/- mice from these crosses which identified a chromosome 2 modifier locus (148-169 Mb) under genetic selection. Currently there are 16 genes in this locus with known annotated non-synonymous coding variants. Ongoing studies are focused on identifying the responsible genetic DBA/2J variant within this locus. Identification of this modifier gene will represent a significant advance for the genetics of thrombotic disease. Not only will it give insight into the pathways leading to thrombosis, it may also reveal previously unexplored therapeutic targets for the prevention of thrombotic events.

scholarly journals Reduced prothrombinase inhibition by tissue factor pathway inhibitor contributes to the factor V Leiden hypercoagulable state

2017 ◽  
Vol 1 (6) ◽  
pp. 386-395 ◽  
Author(s):  
Jeremy P. Wood ◽  
Lisa M. Baumann Kreuziger ◽  
Paul E. R. Ellery ◽  
Susan A. Maroney ◽  
Alan E. Mast
Keyword(s):  
Tissue Factor ◽  
Blood Coagulation ◽  
Tissue Factor Pathway Inhibitor ◽  
Platelet Rich Plasma ◽  
Factor V Leiden ◽  
Hypercoagulable State ◽  
Factor V ◽  
Key Points ◽  
Tissue Factor Pathway

Key Points FVL platelet-poor and platelet-rich plasma have a reduced threshold for the activation of blood coagulation, which is modulated by TFPIα. Prothrombinase assembled with FVL is less susceptible to inhibition by TFPIα than is prothrombinase assembled with FV.

scholarly journals Total tissue factor pathway inhibitor and venous thrombosis

2010 ◽  
Vol 104 (08) ◽  
pp. 207-212 ◽  
Author(s):  
Pamela Lutsey ◽  
Aaron Folsom ◽  
Susan Heckbert ◽  
Mary Cushman ◽  
Neil Zakai
Keyword(s):  
Risk Factors ◽  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor V Leiden ◽  
Coagulation Factors ◽  
Factor V ◽  
Additive Interaction ◽  
Increased Risk ◽  
Tissue Factor Pathway ◽  
D Dimer

SummaryTissue factor pathway inhibitor (TFPI) inhibits tissue factor, a potent coagulation initiator. Limited evidence suggests that low TFPI levels are associated with increased risk of venous thromboembolism (VTE). We measured total TFPI in a nested case-control study in the Longitudinal Investigation of Thromboembolism Etiology. Control subjects were frequency matched 2:1 to cases on age, sex, race, and cohort. Odds ratios (ORs) for VTE by TFPI levels were computed using logistic regression models adjusting for age, race, sex, coagulation factors (factors VII, VIII, IX, XI, D-dimer), and body mass index (BMI). To evaluate for greater than additive interactions, we calculated the percent relative excess risk due to interaction between TFPI and other VTE risk factors. A total of 534 cases of VTE occurred and matched to 1,091 controls. Mean baseline TFPI in ng/ml (standard deviation) in those who developed VTE and controls was 36.4 (12.8) and 35.0 (11.1), respectively. Higher TFPI was associated with male sex, age, BMI, factors VII, VIII, IX, XI, and D-dimer. TFPI level did not differ by ethnicity, factor V Leiden, or pro-thrombin G20210A. Compared with those in the upper 95%, the bottom 5% of TFPI had an age-, sex-, race-, and study-adjusted OR (95% CI) of 1.35 (0.86, 2.12) for VTE. Adjusting for factors VII, VIII, IX, and XI the OR was 1.93 (1.05, 3.53). Further addition of D-dimer and BMI to this model decreased the OR to 1.70 (0.98, 2.93). Low TFPI did not demonstrate greater than additive interaction with other VTE risk factors.

Total Tissue Factor Pathway Inhibitor and Incident Venous Thrombosis: The Longitudinal Investigation of Thromboembolism Etiology

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3827-3827
Author(s):  
Neil A Zakai ◽  
Pamela Lutsey ◽  
Aaron R Folsom ◽  
Susan Heckbert ◽  
Mary Cushman
Keyword(s):  
Risk Factors ◽  
Tissue Factor ◽  
Odds Ratio ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor V Leiden ◽  
Factor V ◽  
Longitudinal Investigation ◽  
Increased Risk ◽  
Tissue Factor Pathway ◽  
D Dimer

Abstract Introduction: Tissue Factor Pathway Inhibitor (TFPI) inhibits tissue factor, a potent coagulation initiator. Limited evidence suggests that low TFPI levels are associated with increased risk of venous thromboembolism (VT). Methods: VT was ascertained in the Longitudinal Investigation of Thromboembolism Etiology, which combines data from the Atherosclerosis Risk in Communities Study and the Cardiovascular Health Study. Total TFPI was measured in a nested case-control sample. With 12 years of follow-up, 534 cases of new VTE were age-, sex-, and race-frequency matched with 1091 controls. The odds ratio for VT was determined for quartiles of TFPI, and for the bottom 5% versus the top 95% in logistic regression models adjusting for demographics (age, race, gender, study), coagulation factors (Factors VII, VIII, IX, XI, D-dimer), and other VT risk factors. To evaluate for greater than additive interactions we calculated the percent relative excess risk of interaction between TFPI and other VT risk factors. Results: Mean TFPI in ng/mL (standard deviation) in cases and controls was 36.4 (12.8) and 35.0 (11.1), respectively. Increasing quartiles of TFPI were associated with male gender and increasing age, body mass index, factors VII, VIII, IX, XI, and D-dimer (all p<0.05). TFPI quartiles were not related to ethnicity, factor V Leiden, or the prothrombin gene polymorphism. Compared with those in the upper 95%, those in the bottom 5% of TFPI (32 cases, 56 controls) had an age-, sex-, race-, and cohort-adjusted odds ratio of 1.35 (0.86, 2.12) for VT. Adjusting for factors VII, VIII, IX, XI, and D-dimer increased the odds ratio to 1.93 (1.05, 3.53). Further addition of BMI to the coagulation factor adjusted model slightly attenuated the odds ratio to 1.70 (0.98, 2.93). There were no associations of TFPI in quartiles or as a continuous variable with VT. TFPI in the bottom fifth percentile did not demonstrate greater than additive interaction with factor V Leiden or with higher BMI. Conclusions: Associations of high TFPI levels with VT were masked by concurrent higher procoagulant levels. After adjusting for procoagulant factors and D-dimer, TFPI ≤ 5% was associated with a 1.9-fold increased odds of VT. These data suggest high total TFPI may be a response to procoagulant states.

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