Identification of the integrin-binding site on coagulation factor VIIa required for proangiogenic PAR2 signaling

Key Points The FVIIa integrin-binding motif is required for TF-FVIIa complex formation with integrin β1 and proangiogenic signaling. The arf6 integrin recycling pathway controls TF-FVIIa signaling and cell surface availability for procoagulant activity.

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Modulating the Antithrombin-Mediated in Vivo Clearance of Coagulation Factor VIIa

Blood ◽

10.1182/blood.v124.21.4233.4233 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4233-4233 ◽

Cited By ~ 1

Author(s):

Henrik Østergaard ◽

Lene Hansen ◽

Hermann Pelzer ◽

Henrik Agersø ◽

Anette A. Pedersen ◽

...

Keyword(s):

Complex Formation ◽

Factor Viia ◽

Coagulation Factor ◽

Site Directed Mutagenesis ◽

Wild Type ◽

Sprague Dawley ◽

Resistant Variant ◽

Opposite Behavior

Abstract The short half-life of coagulation factor VIIa (FVIIa) in circulation is the result of elimination through multiple pathways of which inactivation by the plasma inhibitor antithrombin (AT) accounts for as much as 65% of the total clearance in humans. Remarkably, the rate of inhibition in vivo is about 30 times greater than the uncatalyzed rate of inhibition in vitro suggesting the presence of rate enhancing components in vivo (Agersø et al. (2011) J Thromb Haemost, 9:333-338). Prime candidates include endogenous heparin-like glycosaminoglycans (GAGs) potentiating the reactivity of antithrombin, or tissue factor (TF) which upon binding to FVIIa increases its susceptibility to inhibition. In the present study site-directed mutagenesis of FVIIa was undertaken to identify variants with altered AT reactivity in order to explore the relationship between the reactivity of FVIIa with AT in vitro and in vivo as well as the nature of endogenous rate enhancing components. The pharmacokinetic properties of FVIIa variants were determined in Sprague Dawley rats as this model recapitulates the aspects of AT-mediated FVIIa clearance observed in humans and allows for interaction of human FVIIa with endogenous rat TF. Similar to the human situation, inactivation of wild-type FVIIa in rat is evident as an accumulation of circulating FVIIa-AT complexes and a progressive divergence of the pharmacokinetic profiles representing FVIIa clot activity and total FVIIa antigen. Initially, the ability to modulate the in vivo complex formation with AT was investigated using two FVIIa variants exhibiting enhanced (>200%) or reduced (<10%) in vitro reactivity with AT, respectively, regardless of the type of cofactor present. Reflecting the in vitro reactivity, clot activity and antigen PK profiles in rats were found to coincide for the AT resistant variant along with essentially no detectable AT complex formation, whereas exacerbated AT complex formation and clot activity:antigen discrepancy was observed for the variant exhibiting enhanced in vitro reactivity. Interestingly, among the generated FVIIa variants with altered AT reactivity, two subsets were identified that displayed differential in vitro reactivity with AT depending on the type cofactor present. Accordingly, one group exhibited a greater susceptibility to inhibition relative to wild-type FVIIa in the presence of heparin but not in the presence of TF, while the other group demonstrated the opposite behavior. Endowed with the ability to report on the cofactor identity from the rate of inhibition relative to wild-type FVIIa, variants from each group were tested for their tendency to accumulate as complexes with AT following intravenous administration to rats. Supporting a contribution from endogenous GAGs to the in vivo inactivation of FVIIa, the measured in vivo peak levels of accumulated FVIIa-AT complexes were found to directly correlate with the in vitro rate constants determined for the variants in the presence of heparin, but not when the cofactor was TF or the combination of TF and heparin. Altogether, these results 1) demonstrate a direct relationship between the in vitro reactivity of FVIIa with AT in the presence of heparin and the clearance of FVIIa through this pathway in vivo, and 2) identify heparin-like GAGs as the likely rate enhancing component of FVIIa inhibition in vivo. Disclosures Østergaard: Novo Nordisk A/S: Employment. Hansen:Novo Nordisk A/S: Employment. Pelzer:Novo Nordisk A/S: Employment. Agersø:Novo Nordisk A/S: Employment. Pedersen:Novo Nordisk A/S: Employment. Glue:Novo Nordisk A/S: Employment. Johnsen:Novo Nordisk A/S: Employment. Andersen:Novo Nordisk A/S: Employment. Bjelke:Novo Nordisk A/S: Employment. Breinholt:Novo Nordisk A/S: Employment. Stennicke:Novo Nordisk A/S: Employment. Gandhi:Novo Nordisk A/S: Employment. Olsen:Novo Nordisk A/S: Employment. Hermit:Novo Nordisk A/S: Employment.

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Identification Of The Coagulation Factor VIIa Integrin Binding Site Essential For Pro-Angiogenic Tissue Factor Signaling

Blood ◽

10.1182/blood.v122.21.3566.3566 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3566-3566

Author(s):

Andrea S Rothmeier ◽

Barbara M Mueller ◽

Henrik Ostergaard ◽

Wolfram Ruf

Keyword(s):

Complex Formation ◽

Tissue Factor ◽

Wound Repair ◽

Coagulation Factor ◽

Cytoplasmic Domain ◽

Melanoma Cells ◽

Coagulation Cascade ◽

Wild Type ◽

Integrin Β1 ◽

Erk Phosphorylation

Abstract The cellular initiator of the coagulation cascade, tissue factor (TF), plays pivotal roles in primary hemostasis and wound repair. In vitro evidence point to specific functions of the TF-FVIIa complex in inducing a pro-angiogenic and migratory program in keratinocytes dependent on activation of G protein-coupled signaling of protease activated receptor 2 (PAR2). Accordingly, mice with low levels of FVIIa have delayed cutaneous wound healing and pathological angiogenesis in genetic mouse models of cancer progression is impaired in mice that either lack PAR2 or the TF cytoplasmic domain. Our previous data demonstrated that TF cytoplasmic domain signaling regulates α3β1-dependent migration and that FVIIa promotes the association of TF with β1 integrin heterodimers, but the functional contributions of this interaction to TF-dependent signaling remained unclear. Here, we identified a KGE motif in the FVIIa protease domain that is crucial for FVIIa-dependent complex formation of TF with integrins. Mutation of the negatively charged E26 to A affected neither the catalytic activity of FVIIa, nor significantly FXa generation in a purified system and on cell surfaces. Wild-type FVIIa induced co-immunoprecipitation of TF with keratinocyte-expressed α3β1 integrin. In contrast, FVIIa E26A was severely impaired in promoting association of TF with integrins in a pull-down assay with an antibody that recognized the active conformational state of the integrin β1. Previous transcriptome analysis has indicated that TF-FVIIa produces signaling distinct from activation of PAR2 with agonist peptide. In keratinocytes, wild-type FVIIa induced PI-3 kinase-dependent AKT and ERK phosphorylation, whereas ERK was phosphorylated independent of PI-3 kinase in cells stimulated with the PAR2 agonist SLGIRL. Mutation of FVIIa E26 required for integrin ligation abolished AKT phosphorylation and attenuated ERK phosphorylation, indicating that activation of the PI-3 kinase pathway specifically depends on complex formation of TF with integrins in the context of PAR2 cleavage. Consistently, FVIIa E26A inefficiently supported the PI-3 kinase-, ERK- and PAR2-dependent induction of the pro-angiogenic cytokine IL-8. Mutation of FVIIa E26 similarly reduced complex formation with integrin β1 and attenuated PI-3 kinase- and ERK-dependent IL-8 induction in melanoma cells. Furthermore, the mutation impaired stimulation of migration in melanoma and breast cancer cells. In addition, deletion of the cytoplasmic domain-deleted TF transfected into TF-negative melanoma cells resulted in significantly reduced IL-8 induction by FVIIa in comparison to controls. However, the TF cytoplasmic domain was not required for FVIIa-induced complex formation with integrins, demonstrating direct contributions of the TF cytoplasmic domain to pro-angiogenic signaling. Taken together, these data elucidate the molecular details by which ligand binding promotes complex formation between TF and integrins and provide a novel mechanism of protease selective signaling of a promiscuous PAR in angiogenesis and wound repair. Disclosures: Ostergaard: Novo Nordisk A/S: Employment.

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Tissue factor–mediated endocytosis, recycling, and degradation of factor VIIa by a clathrin-independent mechanism not requiring the cytoplasmic domain of tissue factor

Blood ◽

10.1182/blood.v97.6.1712 ◽

2001 ◽

Vol 97 (6) ◽

pp. 1712-1720 ◽

Cited By ~ 23

Author(s):

Carsten B. Hansen ◽

Charles Pyke ◽

Lars C. Petersen ◽

L. Vijaya Mohan Rao

Keyword(s):

Cell Surface ◽

Tissue Factor ◽

Factor Viia ◽

Coagulation Factor ◽

Cytoplasmic Domain ◽

Cell Surface Expression ◽

Functional Protein ◽

Coated Pits ◽

Bhk Cells ◽

Independent Mechanism

Endocytosis and recycling of coagulation factor VIIa (VIIa) bound to tissue factor (TF) was investigated in baby hamster kidney (BHK) cells stably transfected with TF or TF derivatives. Cell surface expression of TF on BHK cells was required for VIIa internalization and degradation. Approximately 50% of cell surface–bound VIIa was internalized in one hour, and a majority of the internalized VIIa was degraded soon thereafter. Similar rates of VIIa internalization and degradation were obtained with BHK cells transfected with a cytoplasmic domain-deleted TF variant or with a substitution of serine for cysteine at amino acid residue 245 (C245S). Endocytosis of VIIa bound to TF was an active process. Acidification of the cytosol, known to inhibit the internalization via clathrin-coated pits, did not affect the internalization of VIIa. Furthermore, receptor-associated protein, known to block binding of all established ligands to members of the low-density lipoprotein receptor family, was without an effect on the internalization of VIIa. Addition of tissue factor pathway inhibitor/factor Xa complex did not affect the internalization rate significantly. A substantial portion (20% to 25%) of internalized VIIa was recycled back to the cell surface as an intact and functional protein. Although the recycled VIIa constitutes to only approximately 10% of available cell surface TF/VIIa sites, it accounts for 65% of the maximal activation of factor X by the cell surface TF/VIIa. In summary, the present data provide evidence that TF-dependent internalization of VIIa in kidney cells occurs through a clathrin-independent mechanism and does not require the cytoplasmic domain of TF.

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VP7 Mediates the Interaction of Rotaviruses with Integrin αvβ3 through a Novel Integrin-Binding Site

Journal of Virology ◽

10.1128/jvi.78.20.10839-10847.2004 ◽

2004 ◽

Vol 78 (20) ◽

pp. 10839-10847 ◽

Cited By ~ 41

Author(s):

Selene Zárate ◽

Pedro Romero ◽

Rafaela Espinosa ◽

Carlos F. Arias ◽

Susana López

Keyword(s):

Sialic Acid ◽

Cell Surface ◽

Surface Protein ◽

Integrin Αvβ3 ◽

Surface Proteins ◽

Binding Motif ◽

Independent Manner ◽

Multistep Process ◽

Integrin Binding Site ◽

Viral Surface Protein

ABSTRACT Rotavirus entry is a complex multistep process that depends on the trypsin cleavage of the virus spike protein VP4 into polypeptides VP5 and VP8 and on the interaction of these polypeptides and of VP7, the second viral surface protein, with several cell surface molecules, including integrin αvβ3. We characterized the effect of the trypsin cleavage of VP4 on the binding to MA104 cells of the sialic acid-dependent virus strain RRV and its sialic acid-independent variant, nar3. We found that, although the trypsin treatment did not affect the attachment of these viruses to the cell surface, their binding was qualitatively different. In contrast to the trypsin-treated viruses, which initially bound to the cell surface through VP4, the non-trypsin-treated variant nar3 bound to the cell through VP7. Amino acid sequence comparison of the surface proteins of rotavirus and hantavirus, both of which interact with integrin αvβ3 in an RGD-independent manner, identified a region shared by rotavirus VP7 and hantavirus G1G2 protein in which six of nine amino acids are identical. This region, which is highly conserved among the VP7 proteins of different rotavirus strains, mediates the binding of rotaviruses to integrin αvβ3 and probably represents a novel binding motif for this integrin.

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Abstract 70: P2X7-Induced Thiol-Disulfide Exchange is Critical for the Coupling of Coagulation and Inflammation.

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.33.suppl_1.a70 ◽

2013 ◽

Vol 33 (suppl_1) ◽

Author(s):

Andrea S Rothmeier ◽

Christian Furlan-Freguia ◽

Patrizia Marchese ◽

Brian Petrich ◽

Zaverio M Ruggeri ◽

...

Keyword(s):

Cell Surface ◽

Thioredoxin Reductase ◽

Integrin Β1 ◽

Disulfide Exchange ◽

Upstream Regulator ◽

Molecular Events ◽

Pathway Activation ◽

Purinergic P2x7 Receptor ◽

Recycling Pathway ◽

Key Steps

Macrophages are important players in the maintenance of tissue homeostasis, but promote inflammation through the release of IL1β triggered by danger signals in form of extracellular ATP that activates the purinergic P2X7 receptor. We found that P2X7 signaling also contributes to thrombosis by inducing thiol-dependent tissue factor (TF) activation coupled to procoagulant microparticles (MP) release. In the present study, we identified thiol-regulated proteins released on MP and based on this information delineated key steps in the P2X7-induced generation of prothrombotic TF + MP. We find that TF procoagulant activity of LPS/IFNγ primed macrophages is controlled by internalization through the arf6/integrin-recycling pathway. Activation of P2X7 inactivates arf6 and prevents TF internalization, but additional steps are required to generate highly procoagulant MP carrying TF and integrin β1. Imaging of cell surface TF by confocal microscopy shows translocation of TF onto filopodia that form in response to P2X7 activation. Blocking raft mobility does not inhibit filopodia formation, but rather specifically prevents TF and integrin β1 trafficking and release on MP. We show that filopodia formation is dependent on thioredoxin reductase (TRXR). Remarkably, thioredoxin (TRX), the direct substrate of TRXR, is entirely released from the cytosol. Pharmacological inhibition of TRXR blocks both TRX release and reductive changes on the cell surface and MP, identifying the molecular events that change the extracellular redox environment. TRXR-mediated externalization of TRX was also required for activation of the inflammasome and caspase1 leading to IL1β processing and release. These data elucidate the molecular events required for the generation of highly procoagulant TF + MP and identifies TRXR-TRX dependent thiol-disulfide exchange as common upstream regulator responsible for the induction of inflammation and coagulation in innate immune cells.

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Studies of the Mechanism for Enhanced Cell Surface Factor VIIa/Tissue Factor Activation of Factor X on Fibroblast Monolayers after Their Exposure to N-Ethylmaleimide

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648973 ◽

1994 ◽

Vol 72 (06) ◽

pp. 848-855 ◽

Cited By ~ 34

Author(s):

Dzung The Le ◽

Samuel I Rapaport ◽

L Vijaya Mohan Rao

Keyword(s):

Cell Surface ◽

Tissue Factor ◽

Factor Viia ◽

Cell Damage ◽

Specific Binding ◽

Surface Membrane ◽

Annexin V ◽

Factor X ◽

Binding Studies ◽

Anionic Phospholipid

SummaryFibroblast monolayers constitutively expressing surface membrane tissue factor (TF) were treated with 0.1 mM N-ethylmaleimide (NEM) for 1 min to inhibit aminophospholipid translocase activity without inducing general cell damage. This resulted in increased anionic phospholipid in the outer leaflet of the cell surface membrane as measured by the binding of 125I-annexin V and by the ability of the monolayers to support the generation of prothrombinase. Specific binding of 125I-rVIIa to TF on NEM-treated monolayers was increased 3- to 4-fold over control monolayers after only brief exposure to 125I-rVIIa, but this difference progressively diminished with longer exposure times. A brief exposure of NEM-treated monolayers to rVIIa led to a maximum 3- to 4-fold enhancement of VIIa/TF catalytic activity towards factor X over control monolayers, but, in contrast to the binding studies, this 3- to 4-fold difference persisted despite increasing time of exposure to rVIIa. Adding prothrombin fragment 1 failed to diminish the enhanced VIIa/TF activation of factor X of NEM-treated monolayers. Moreover, adding annexin V, which was shown to abolish the ability of NEM to enhance factor X binding to the fibroblast monolayers, also failed to diminish the enhanced VIIa/TF activation of factor X. These data provide new evidence for a possible mechanism by which availability of anionic phospholipid in the outer layer of the cell membrane limits formation of functional VIIa/TF complexes on cell surfaces.

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