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

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.

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

Blood Advances ◽

10.1182/bloodadvances.2020003464 ◽

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.

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

Haemophilia ◽

10.1111/hae.13492 ◽

2018 ◽

Vol 24 (3) ◽

pp. e160-e163 ◽

Cited By ~ 1

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

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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

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.36.suppl_1.43 ◽

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.

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The C536T Transition in the Tissue Factor Pathway Inhibitor (TFPI) Gene Does not Contribute to Risk of Venous Thrombosis among Carriers of Factor V Leiden

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614095 ◽

2000 ◽

Vol 84 (10) ◽

pp. 724-725 ◽

Cited By ~ 6

Author(s):

Robert Luhm ◽

Martin Hessner

Keyword(s):

Venous Thrombosis ◽

Tissue Factor ◽

Tissue Factor Pathway Inhibitor ◽

Factor V Leiden ◽

Factor V ◽

Tissue Factor Pathway

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Interaction between tissue factor pathway inhibitor and factor V levels on the risk of venous thrombosis

Journal of Thrombosis and Haemostasis ◽

10.1111/j.1538-7836.2010.03805.x ◽

2010 ◽

Author(s):

A. E. A. Dahm ◽

I. D. Bezemer ◽

P. M. Sandset ◽

F. R. Rosendaal

Keyword(s):

Venous Thrombosis ◽

Tissue Factor ◽

Tissue Factor Pathway Inhibitor ◽

Factor V ◽

Tissue Factor Pathway

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

Circulation ◽

10.1161/01.cir.0000017361.39256.82 ◽

2002 ◽

Vol 105 (18) ◽

pp. 2139-2142 ◽

Cited By ~ 54

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

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Polymorphisms of the tissue factor pathway inhibitor gene are associated with venous thromboembolism in the antiphospholipid syndrome and carriers of factor V Leiden

Blood Coagulation & Fibrinolysis ◽

10.1097/mbc.0b013e3281eec977 ◽

2007 ◽

Vol 18 (6) ◽

pp. 559-564 ◽

Cited By ~ 21

Author(s):

Lisa F Lincz ◽

Murray J Adams ◽

Fiona E Scorgie ◽

Jim Thom ◽

Ross I Baker ◽

...

Keyword(s):

Venous Thromboembolism ◽

Tissue Factor ◽

Antiphospholipid Syndrome ◽

Tissue Factor Pathway Inhibitor ◽

Factor V Leiden ◽

Factor V ◽

Tissue Factor Pathway ◽

Inhibitor Gene

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Curcumin Prevents Cytokine-Mediated Tissue Factor Pathway Inhibitor Down-Regulation in Human Endothelial Cells

Blood ◽

10.1182/blood.v118.21.1202.1202 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1202-1202

Author(s):

Keiko Maruyama ◽

Eriko Morishita ◽

Yukie Goto ◽

Akiko Sekiya ◽

Hidesaku Asakura ◽

...

Keyword(s):

Endothelial Cells ◽

Tissue Factor ◽

Western Blotting ◽

Tissue Factor Pathway Inhibitor ◽

The Other ◽

Tnf Alpha ◽

Down Regulation ◽

Protein Levels ◽

Other Hand ◽

Tissue Factor Pathway

Abstract Abstract 1202 Objective: Curcumin (diferuloyl methane), an active component of the spice turmeric, has been shown to exhibit anti-inflammatory and antioxidant activities in addition to an anticartinogenic activity in vitro and in vivo. Furthermore, we reported that curcumin inhibited the induction of tissue factor (TF) expression in human umbilical vein endothelial cells (HUVECs) at 52nd ASH 2010. Therefore, curcumin may ameliorate hyper-coagulable state associated with inflammation or oxidative stress. On the other hand, tissue factor pathway inhibitor (TFPI) which is expressed by endothelial cells plays a crucial role in hemostasis by regulating TF-induced initiation of coagulation. This study examined whether curcumin modulates the expression of TFPI in HUVECs. Methods: HUVECs were pretreated with curcumin at the concentration of 20 μM for 3h, washed and stimulated with tumor necrosis factor-alpha (TNF-alpha, 10 ng/ml) for additional 12 or 24h. The mRNA and protein levels of TFPI in the cultured HUVECs were determined by reverse transcriptase polymerase chain reaction (RT-PCR) and western blotting, respectively. To determine whether curcumin affects the MAPK signaling pathways, the phosphorylation of p38 mitogen-activated protein kinase (p38MAPK), extracellular signal-regulated kinase1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK) in the HUVECs were analyzed with western blotting. Additionally, to determine whether curcumin affects nuclear factor-kappa B (NF-kB) pathway, nuclear and cytoplasmic fractions were extracted and protein levels were determined by western blotting for NF-kB (p65), p-IkB and IkB. Results: After TNF-alpha stimulation, TFPI mRNA levels were approximately decreased by 40% compared to the control (p<0.05; Figure 1B). Similarly to the mRNA expression, TFPI protein levels were decreased (Figure 1A). On the other hand, pretreatment of HUVECs with curcumin significantly suppresses TNF-alpha-induced TFPI mRNA and protein down-regulation (p<0.05; Figure 1A, B). Curcumin inhibited TNF-alpha-induced activation of p38MAPK, ERK1/2, and JNK. Moreover, curcumin inhibits TNF-alpha-induced IkB activation in HUVECs. And, translocation of NF-kB from the cytosol into the nucleus by TNF-alpha was inhibited by curcumin. Conclusions: These results indicate that curcumin may suppress the TNF-alpha-induced TFPI down-regulation via NF-kB pathways. Thus, curcumin may offer a novel antithrombotic option for treatment of the hypercoagulable state associated with inflammation. Disclosures: No relevant conflicts of interest to declare.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1705762114 ◽

2017 ◽

Vol 114 (36) ◽

pp. 9659-9664 ◽

Cited By ~ 10

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.

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