The interaction of factor V and tissue factor pathway inhibitor in a myeloma patient with acquired factor V deficiency

Haemophilia ◽  
2018 ◽  
Vol 24 (3) ◽  
pp. e160-e163 ◽  
Author(s):  
E. De Maertelaere ◽  
E. Castoldi ◽  
I. Van haute ◽  
D. Deeren ◽  
K. M. Devreese
Keyword(s):  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor V ◽  
Myeloma Patient ◽  
Factor V Deficiency ◽  
Tissue Factor Pathway

scholarly journals Elevated levels of tissue factor pathway inhibitor in patients with mild to moderate bleeding tendency

2021 ◽  
Vol 5 (2) ◽  
pp. 391-398
Author(s):  
Dino Mehic ◽  
Alexander Tolios ◽  
Stefanie Hofer ◽  
Cihan Ay ◽  
Helmuth Haslacher ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Genetic Alterations ◽  
Factor V ◽  
Bleeding Tendency ◽  
Healthy Controls ◽  
Single Nucleotide ◽  
Time To Peak ◽  
Coagulation Inhibitors ◽  
Tissue Factor Pathway

Abstract High levels of tissue factor pathway inhibitor (TFPI), caused by a longer TFPIα half-life after binding to a factor V splice variant and variants in the F5 gene, were recently identified in 2 families with an as-yet-unexplained bleeding tendency. This study aimed to investigate free TFPIα in a well-characterized cohort of 620 patients with mild to moderate bleeding tendencies and its association to genetic alterations in the F5 gene. TFPIα levels were higher in patients with bleeding compared with healthy controls (median [interquartile range], 8.2 [5.5-11.7] vs 7.8 [4.3-11.1]; P = .026). A higher proportion of patients had free TFPIα levels more than or equal to the 95th percentile compared with healthy controls (odds ratio [OR] [95% confidence interval (CI)], 2.82 [0.98-8.13]). This was pronounced in the subgroup of patients in whom no bleeding disorder could be identified (bleeding of unknown cause [BUC; n = 420]; OR [95% CI], 3.03 [1.02-8.98]) and in platelet function defects (PFDs) (n = 121; OR [95% CI], 3.47 [1.09-11.08]). An increase in free TFPIα was associated with a mild delay in thrombin generation (prolonged lag time and time to peak), but not with alterations in routinely used global clotting tests. We could neither identify new or known genetic variations in the F5 gene that are associated with free TFPIα levels, nor an influence of the single-nucleotide variant rs10800453 on free TFPIα levels in our patient cohort. An imbalance of natural coagulation inhibitors such as TFPIα could be an underlying cause or contributor for unexplained bleeding, which is most probably multifactorial in a majority of patients.

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

scholarly journals Characterization of the Interaction between Tissue Factor Pathway Inhibitor α (TFPIα) and Factor V Species

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 485-485
Author(s):  
Teodolinda Petrillo ◽  
Cornelis Van 't Veer ◽  
Rodney M. Camire
Keyword(s):  
Tissue Factor ◽  
Western Blotting ◽  
Tissue Factor Pathway Inhibitor ◽  
Specific Binding ◽  
Single Point ◽  
Single Point Mutation ◽  
Factor V ◽  
High Affinity ◽  
Thrombin Cleavage ◽  
Tissue Factor Pathway

Activation of factor V (FV) involves removal of its central B-domain following proteolysis at R709, R1018 and R1545. Two evolutionary conserved regions (basic region; BR; residues 964-1008 and acidic region 2; AR2; residues 1493-1538) of the B-domain play an essential role in keeping FV inactive. FV derivatives lacking the BR but retaining AR2 (FVAR) have cofactor-like properties while the BR fragment added in trans blocks their procoagulant function. Physiological important forms of FVAR include: platelet FV, FXa activated FV and FV-short. The latter is a splice variant lacking most of the B-domain, including the BR, yet retains AR2. In normal plasma, FV-short represents <2% of the total FV but is overexpressed in patients affected by the East-Texas bleeding disorder due to a single point mutation or deletion in exon 13. In plasma, FV-short forms a complex with tissue factor pathway inhibitor α (TFPIα) through a high affinity interaction between AR2 and the basic C-terminal region of TFPIα (TFPIα-BR; residues 249-264) which is homologous to FV-BR. It has also been found that FV interacts with TFPIα via its BR, albeit with reduced affinity compared to FVAR. Furthermore, TFPIα and FV levels in plasma appear linked suggesting FV may act as carrier for TFPIα. Collectively these results are puzzling considering the mechanism by which these proteins are thought to interact. How can FV, with its endogenous BR engaged in interactions with AR2, simultaneously interact with TFPIα? To gain more insight into this question, we characterized the binding of TFPIα to different physiologic FV species including full-length (fl) FV, FVa, FV-short and other FVAR species. In direct binding measurements, we found that fluorescently labelled TFPIα-BR (OG488-TFPIα-BR) bound FV-short with high affinity (Kd = 0.66 nM). Unlabeled TFPIα and TFPIα-BR displaced OG488-TFPIα-BR from FVshort equivalently indicating specific binding of the BR region of TFPIα to FV-short. No detectable binding was observed to FVa and the OG488-TFPIα-BR also failed to bind fl-FV. These data indicated that AR2 is required for binding to TFPIα-BR and that the endogenous BR in fl-FV is associated with AR2 and precludes binding to TFPIα-BR. In support of this, thrombin cleavage of FV-short over time during binding measurements showed a gradual and marked decreased in fluorescence which correlated with cleavage at R1545 and release of AR2 as observed by western blotting. Cleavage of fl-FV by thrombin during the binding assay transiently increased fluorescence, indicating that TFPIα-BR binds to cleaved FV which correlated with removal of the endogenous BR (cleavage at R709 and R1018) as shown by western blotting. Subsequent cleavage at R1545 resulted in a decrease in fluorescence and hence binding. Using a FV-derivative that cannot be cleaved at R1018 (R1018Q), no binding of TFPIα-BR could be detected upon thrombin incubation, despite cleavage at R709. Together these data indicate that 1) cleavage of FV at R709 has little, if any influence on disrupting the BR-AR2 interaction; 2) cleavage at R1018 releases endogenous FV BR allowing TFPIα to engage via AR2; and 3) cleavage at R1545 removes AR2 eliminating TFPIα binding. Our data suggests that intramolecular binding of FV BR to AR2 has high affinity. To further assess the difference in apparent affinity of the intramolecular BR for AR2 compared to TFPIα-BR, we compared rates of FV-short activation (± TFPIα-BR) by thrombin to fl-FV and monitored cleavage at R1545. Based on the data, we estimate that intramolecular FV BR binds at least 25-50-fold tighter compared to TFPIα-BR binding to FV-short. Overall, we conclude that TFPIα via its BR binds to FV-short and cleaved forms of FV which retain AR2 but have its BR removed. TFPIa binding to these FV species not only blocks procoagulant function but also delays further cleavage at R1545. FVa and fl-FV do not bind TFPIα and are not regulated by this anticoagulant. Fl-FV must first be cleaved at R709 and R1018 prior to any possible TFPIα binding/regulation. Our data support the findings that TFPIα regulates the procoagulant function of FV-short and dampens thrombin generation by delaying the generation of FVa by tuning the activity of FVAR during the initiation of coagulation. This is especially evident when the coagulation stimulus is weak (e.g. low tissue factor), and much less important with a strong stimulus (e.g. high tissue factor) where other anticoagulant mechanisms dominate. Disclosures Camire: Pfizer: Research Funding.

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 Factor V-short and protein S as synergistic tissue factor pathway inhibitor (TFPIα) cofactors

2017 ◽  
Vol 2 (1) ◽  
pp. 114-124 ◽  
Author(s):  
Björn Dahlbäck ◽  
Li Jun Guo ◽  
Ruzica Livaja-Koshiar ◽  
Sinh Tran
Keyword(s):  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Protein S ◽  
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.

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