Inhibition of tissue factor:factor VIIa–catalyzed factor IX and factor X activation by TFPI and TFPI constructs

Peraramelli, Sameera; Thomassen, M. Christella L. G. D.; Heinzmann, Alexandra C.A.; Rosing, Jan; Hackeng, Tilman M.; Hartmann, Rudolf; Scheiflinger, F.; Dockal, Michael

doi:10.1111/jth.12713

Cited by 25 publications

(19 citation statements)

References 52 publications

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“…Ndonwi and Broze reported that protein S does not directly stimulate TFPIα inhibition of TF/FVIIa whereas Peraramelli et al showed that protein S does directly stimulate inhibition of TF/FVIIa and thereby also inhibits the activation of FIX by TF/FVIIa. [36][37][38] Whether FV-Short affects these reactions remains to be elucidated. Protein S may also function as a carrier for TFPIα because protein S deficient individuals have low TFPIα and furthermore immune depletion of protein S is reported to results in decreased TFPIα.…”

Section: Fv-short Binds Tfpiα With High Affinity and Through Mechanismsmentioning

confidence: 99%

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

Dahlbäck

Guo

Livaja-Koshiar

et al. 2018

Research and Practice in Thrombosis and Haemostasis

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Essentials FV‐Short, a normal splice isoform of Factor V, binds tissue factor pathway inhibitor (TFPIα) with high affinity.FV‐Short functions as a synergistic TFPIα cofactor with protein S in inhibition of Factor Xa.FV‐Short is much more efficient as TFPIα cofactor than full length FV.TFPIα‐cofactor activity of FV‐Short is lost upon activation of coagulation by thrombin‐mediated cleavage. Background FV‐Short is a normal splice variant of Factor V (FV) having a short B domain, which exposes a high affinity‐binding site for tissue factor pathway inhibitor α (TFPIα). FV‐Short and TFPIα circulate in complex in plasma.ObjectivesThe aim was to elucidate whether FV‐Short affects TFPIα as inhibitor of coagulation FXa and to test whether the TFPIα‐cofactor activity of protein S is influenced by FV‐Short.MethodsRecombinant FV, wild‐type FV‐Short and a FV‐Short thrombin‐cleavage resistant variant were expressed and purified. The influence of FV and FV‐Short variants and/or protein S on the FXa inhibitory activity of TFPIα was monitored both in a purified system and in a plasma‐based thrombin generation assay.Results FV‐Short had intrinsically weak TFPIα‐cofactor activity but with protein S present, FV‐Short yielded efficient inactivation of FXa. Protein S alone did not promote full TFPIα‐activity. Intact FV was inefficient at low protein S concentrations and had 10‐fold lower activity compared to FV‐Short at physiological protein S levels. Activation of FV‐Short by thrombin resulted in the loss of the TFPIα‐cofactor activity. The synergistic TFPIα‐cofactor activity of FV‐Short and protein S was also demonstrated in plasma using a thrombin generation assay.Conclusions FV‐Short and protein S are highly efficient, synergistic cofactors to TFPIα in the regulation of FXa activity, whereas full length FV has lower activity. Our results suggest the formation of an efficient FXa‐inhibitory complex between FV‐Short, TFPIα and protein S on the surface of negatively charged phospholipids.

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Section: Fv-short Binds Tfpiα With High Affinity and Through Mechanismsmentioning

confidence: 99%

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

Dahlbäck

Guo

Livaja-Koshiar

et al. 2018

Research and Practice in Thrombosis and Haemostasis

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“…Because the TFPI concentration necessary for efficient inhibition of FXa (5) is higher than the free full-length TFPI concentration in plasma (0.25–0.5 n m ) (6, 7), TFPI is on its own a poor inhibitor of FXa. Protein S functions as a cofactor for TFPI, efficiently enhancing TFPI inhibition of FXa (8, 9) and the inhibition of FXa activation by TF-VIIa (10). The protein S enhancement of TFPI is dependent on a direct interaction involving Glu-226 (9) and Arg-199 (11) within the TFPI K3 domain and laminin G-like domain 1 of the protein S sex hormone-binding globulin (SHBG)-like region (12, 13).…”

Section: Introductionmentioning

confidence: 99%

Factor V has an anticoagulant cofactor activity that targets the early phase of coagulation

Santamaria

Reglińska-Matveyev

Gierula

et al. 2017

Journal of Biological Chemistry

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Tissue factor pathway inhibitor (TFPI), the main inhibitor of initiation of coagulation, exerts an important anticoagulant role through the factor Xa (FXa)-dependent inhibition of tissue factor/factor VIIa. Protein S is a TFPI cofactor, enhancing the efficiency of FXa inhibition. TFPI can also inhibit prothrombinase assembly by directly interacting with coagulation factor V (FV), which has been activated by FXa. Because full-length TFPI associates with FV in plasma, we hypothesized that FV may influence TFPI inhibitory function. Using pure component FXa inhibition assays, we found that although FV alone did not influence TFPI-mediated FXa inhibition, it further enhanced TFPI in the presence of protein S, resulting in an ∼8-fold reduction in Ki compared with TFPI alone. A FV variant (R709Q/R1018Q/R1545Q, FVΔIIa) that cannot be cleaved/activated by thrombin or FXa also enhanced TFPI-mediated inhibition of FXa ∼12-fold in the presence of protein S. In contrast, neither activated FV nor recombinant B-domain-deleted FV could enhance TFPI-mediated inhibition of FXa in the presence of protein S, suggesting a functional contribution of the B domain. Using TFPI and protein S variants, we show further that the enhancement of TFPI-mediated FXa inhibition by protein S and FV depends on a direct protein S/TFPI interaction and that the TFPI C-terminal tail is not essential for this enhancement. In FXa-catalyzed prothrombin activation assays, both FV and FVΔIIa (but not activated FV) enhanced TFPI function in the presence of protein S. These results demonstrate a new anticoagulant (cofactor) function of FV that targets the early phase of coagulation before prothrombinase assembly.

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“…High nanomolar TFPI concentrations were required for inhibition of TF/FVIIa‐mediated FIXa generation (Fig. C; K i = 33.6 ± 11.1 n m ) . Partial abrogation of inhibition at 200 n m TFPI was obtained with 100 n m mAb2F22 (Fig.…”

Section: Resultsmentioning

confidence: 90%

“…From the progress curves of FIX activation, rates of FIX activation (first derivative of the progress curves) were calculated. From the rates of FIX activation, the percentage inhibition of FVIIa/TF was calculated and plotted as function of TFPI concentration and fitted to the Langmuir binding isotherm as previously described . Chromogenic substrate conversion was measured at 405 nm in a Spectramax reader.…”

Section: Methodsmentioning

confidence: 99%

Factor Xa and VIIa inhibition by tissue factor pathway inhibitor is prevented by a monoclonal antibody to its Kunitz‐1 domain

Augustsson

Svensson

Kjær

et al. 2018

Journal of Thrombosis and Haemostasis

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Background Initiation of coagulation is induced by binding of activated factor VII (FVIIa) to tissue factor (TF) and activation of factor X (FX) in a process regulated by tissue factor pathway inhibitor (TFPI). TFPI contains three Kunitz-type protease inhibitor domains (K1-K3), of which K1 and K2 block the active sites of FVIIa and FXa, respectively. Objective To produce a monoclonal antibody (mAb) directed towards K1, to characterize the binding epitope, and to study its effect on TFPI inhibition. Methods A monoclonal antibody, mAb2F22, was raised against the N-terminal TFPI(1-79) fragment. Binding data were obtained by surface plasmon resonance analysis. The Fab-fragment of mAb2F22, Fab2F22, was expressed and the structure of its complex with TFPI(1-79) determined by X-ray crystallography. Effects of mAb2F22 on TFPI inhibition were measured in buffer- and plasma-based systems. Results mAb2F22 bound exclusively to K1 of TFPI (K ~1 nm) and not to K2. The crystal structure of Fab2F22/TFPI (1-79) mapped an epitope on K1 including seven residues upstream of the domain. TFPI inhibition of TF/FVIIa amidolytic activity was neutralized by mAb2F22, although the binding epitope on K1 did not include the P1 residue. Binding of mAb2F22 to K1 blocked TFPI inhibition of the FXa amidolytic activity and normalized hemostasis in hemophilia human A-like plasma and whole blood. Conclusion mAb2F22 blocked TFPI inhibition of both FVIIa and FXa activities and mapped a FXa exosite for binding to K1. It reversed TFPI feedback inhibition of TF/FVIIa-induced coagulation and restored clot formation in FVIII-neutralized human plasma and blood.

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Inhibition of tissue factor:factor VIIa–catalyzed factor IX and factor X activation by TFPI and TFPI constructs

Abstract: To cite this article: Peraramelli S, Thomassen S, Heinzmann A, Rosing J, Hackeng TM, Hartmann R, Scheiflinger F, Dockal M. Inhibition of tissue factor:factor VIIa-catalyzed factor IX and factor X activation by TFPI and TFPI constructs. J Thromb Haemost 2014; 12: 1826-37.

Cited by 25 publications

References 52 publications

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

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

Factor V has an anticoagulant cofactor activity that targets the early phase of coagulation

Factor Xa and VIIa inhibition by tissue factor pathway inhibitor is prevented by a monoclonal antibody to its Kunitz‐1 domain

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