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Keywords:

  • hemostasis;
  • immunohistochemistry;
  • initiation of coagulation;
  • skin;
  • tissue factor

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

Summary. Background: ‘Idling’ or ongoing low-level activity of the tissue factor (TF) pathway is a postulated mechanism by which the coagulation process can become active without a lag period at sites of injury. Objective: To determine whether TF around cutaneous vessels has bound factor VIIa in the absence of injury, and thus could participate in the idling process. Methods: Immunostaining of mouse skin with antibodies against a 15-residue peptide from the sequence of mouse TF, and against the whole extracellular portion of TF. Results: The whole TF antibody recognized TF in squamous epithelium and around vessels in the dermis. By contrast, the monospecific antibody only recognized TF in the squamous epithelium, but not around vessels. We also found that biotinylated, active site-inhibited FVIIa (FVIIai) bound to tissue sections in the same areas in which TF was recognized by the monospecific antibody (squamous epithelium), but did not bind around vessels. Molecular modeling revealed that FVIIa and FX binding to TF masked a significant part of the surface of the target peptide. Conclusions: In the aggregate, these data are most consistent with the interpretation that TF in perivascular sites has bound FVIIa, even in the absence of any injury. The presence of endogenously bound FVIIa prevents the subsequent binding of the monospecific antibody or exogenous FVIIai to perivascular TF.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

Most descriptions of the coagulation process state that factor VII (FVII) binds to tissue factor (TF) when an injury occurs and blood leaves the vasculature and encounters TF in the extravascular compartment. Assembly of the FVIIa–TF complex then leads to initiation of coagulation. However, there is indirect evidence that this scenario is not accurate. The coagulation factors can leave the vasculature, as do other proteins such as immunoglobulins. They distribute to the extravascular compartment [1] and are found in the lymph in amounts roughly in proportion to their molecular weights [2]. In addition, low levels of antithrombin–FXa complexes are measurable in lymph. Finally, plasma levels of the activation peptide from FX are significantly reduced in FVII-deficient patients but are not different from normal levels in hemophilia patients [3]. These findings suggest that there is a basal level of FVIIa–TF-catalyzed activation of FX in the extravascular space. This ‘idling’ of the TF pathway is prevented from progressing to generation of fibrin by the low availability of the higher molecular weight factors (FVIII and FV) and the absence of platelet surface to support large-scale thrombin generation.

Jesty and Beltrami also proposed that ‘idling’ of the TF pathway occurs [4]. They suggested that the ‘idling’ process limits activation of procoagulants in the baseline state, yet allows a rapid coagulant response when injury does occur. However, they suggested that the major source of ‘idling’ is via intravascular TF.

We now present immunohistochemical evidence that extravascular TF in close proximity to vessels has bound FVII or FVIIa in the absence of a breach in the vessel wall and, thus, is probably involved in the ‘idling’ process.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

Tissue source and processing

Skin tissue from C57B mice was obtained in the process of studies on wound healing under a protocol approved by the Animal Care and Use Committee of the University of North Carolina. Skin from the backs of mice was obtained after sacrifice. The tissue was pinned flat in 10% buffered formalin for 6–12 h before specimens were processed into paraffin. Specimens were bisected and embedded in paraffin blocks for sectioning.

Tissue sections (5 μm) were cut and placed on precleaned Snowcoat X-tra poly-lysine-coated slides for immunohistochemical staining. One slide was also stained with hematoxylin and eosin.

Immunostaining

Slides for immunostaining were placed in a 60 °C oven overnight and allowed to cool before dewaxing. Paraffin was removed from the sections with three 3-min immersions in xylene, and this was followed by rehydration through graded alcohols.

Antigen retrieval was performed by heating to 95 °C in DakoCytomation Target Retrieval Solution for 10 min. Endogenous peroxide activity was blocked by incubation in 3% H2O2 for 20 min. Non-specific binding was blocked with MOM (Mouse on Mouse) Ig blocking reagent (Vector Labs, Burlingame, CA, USA), and primary antibodies were diluted in MOM diluent.

Antibody to ‘whole TF’ was raised in rabbit to the extracellular domain of mouse TF (the kind gift of M. Ezban, Novo Nordisk A/S, Denmark).

Monospecific anti-TF was raised in rabbit in our laboratories to a peptide corresponding to amino acids 155–168 in the mature mouse TF sequence, with a cysteine residue added to the N-terminal end of the peptide: CITYRKGSSTGKKTN-NH2. An antibody preparation raised to this sequence had previously been reported to inhibit mouse TF activity [5].

An antibody preparation to mouse FVII raised in rabbits was the kind gift of K. High (Children’s Hospital of Philadelphia, Philadelphia, PA, USA).

Binding of the primary antibodies was detected with a biotinylated anti-(rabbit IgG) secondary antibody (Vector Labs) at a 1:250 dilution, and visualized with a Dako LASB2 kit (DakoCytomation, Carpinteria, CA, USA), which employs a streptavidin–horseradish peroxidase conjugate and diaminobenzidine (DAB) substrate. The slides were counterstained with Dako Hematoxylin, dehydrated, and coverslipped with Vectamount mounting medium (Vector Labs).

TF in tissue sections was also detected by binding of mouse FVIIa (kind gift of M. Ezban, NovoNordisk A/S, Denmark) that had been reacted with biotinylated Phe-Pro-Arg-chloromethyl ketone (biotin–FPRck; Haematologic Technologies, Essex Junction, VT, USA) [6]. Biotinylated active site-inhibited FVIIa (biotin–FVIIai) binds to TF even more tightly than FVII or FVIIa, and has been used to detect functional TF in a variety of tissues [7]. Mouse FVIIa (8 μm) was incubated overnight at room temperature with biotin–FPRck (100 μm). This process inhibited over 90% of FVIIa activity. The resulting biotin–FVIIai was repurified on a 1-mL HiTrap Q FF column (Amersham Biosciences, Piscataway, NJ, USA). Tissue sections were prepared as for immunohistochemistry, with the exception that they were not subjected to antigen retrieval before blocking. They were incubated with biotin–FVIIai (200 nm) for 1 h, and biotin–FVIIai binding was then visualized with a Dako LASB2 kit (DakoCytomation, Carpinteria, CA, USA) and DAB substrate. The specificity of the staining was verified by the fact that simultaneous addition of an excess of anti-whole TF antibody eliminated staining with biotin–FVIIai.

In one set of experiments, the ability of FVIIa to compete with the monospecific anti-TF antibody was assessed by preincubating tissue sections with mouse FVIIa before immunostaining with the primary antibody.

Molecular modeling

Modeling of the structure of intact TF, the FVIIa–TF complex and the site of binding of the monospecific TF antibody was performed to evaluate the possibility that binding of FVII with or without FX or FIX could block the access of the monospecific antibody to its target sequence. The partial X-ray crystal structure of soluble human tissue factor (sTF)–FVIIa [8] was used as a basis to prepare an all-atom model. This structure is unique among those for coagulation proteins, in that the serine protease cofactor necessary for physiologic function is present. However, certain regions are missing from the structure: sTF – N-terminus residues 1–4, loop residues 85–89, and loop residues 159–162; and FVIIa – residues 143–152. These residues were added and solvent equilibrated by a molecular dynamics procedure that accommodates accurate electrostatics [9]. After 20 ns of simulation, the solvent-accessible surface area (SASA) for sTF residues 155–168 was computed for sTF alone, sTF–FVIIa and sTF–FVIIa–FXa. FXa is activated FX, the product of the activation of zymogen FX, which forms a stable product complex with sTF–FVIIa. We had previously developed a solvent-equilibrated all-atom model [10] for the ternary complex sTF–FVIIa–FXa.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

As shown in Fig. 1, the squamous epithelial cells on the skin surface and in hair follicles stain strongly with the antibody against whole TF, as reported in the literature. The staining tends to be weak in the basal layer, and becomes more intense as the epithelial cells differentiate.

image

Figure 1.  Immunostaining of mouse skin with antibody raised against the whole tissue factor (TF) molecule (left panel) and a monospecific antibody raised against a 15 amino acid sequence from TF (right panel). Tissue was processed and stained as described in Materials and methods. The pattern of staining was consistent in skin samples from 15 different animals. Squamous epithelium, including hair follicles, stained uniformly with the whole TF antibody and more weakly with the monospecific antibody. The antibody against the whole TF revealed staining around all arteries, arterioles, veins and venules in the s.c. tissue, but not around dermal capillaries. Original magnification 40×. The measurement bar applies to both panels.

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The monospecific anti-TF also stained the epithelium and hair follicles, although in a less uniform fashion than did the antiserum to the whole TF molecule. We expected staining to be weaker, as there are fewer epitopes per TF molecule recognized by the monospecific antibody.

As shown in Fig. 2, small arteries and veins are surrounded by a layer of TF when stained with the antibody against whole TF, consistent with what has been reported in the literature. The endothelial cells are negative for TF, but cells surrounding the vessels (pericytes) stain strongly. Strikingly, the monospecific anti-TF did not stain around vessels as did the anti-whole TF, even though the antibody reacted strongly with squamous epithelium in the same sections.

image

Figure 2.  Immunostaining with antibody against the whole tissue factor (TF) molecule, with monospecific antibody against a 15 amino acid sequence from TF, and with antibody against mouse factor VII (FVII). Tissue was processed and stained as described in Materials and methods. These four panels are from the same tissue sample. The pattern of staining was consistent in skin samples from 15 different animals for TF staining and three animals for FVII staining. Pericytes around vessels consistently exhibited TF staining with the antibody against the whole molecule, but not with the monospecific antibody. Blood within the vessel, endothelium and pericytes exhibited staining for FVII. Original magnification 400×.

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Antimouse FVII antibody stained both in and around small vessels, as well as staining the blood within the vessels. This antibody reacts both with zymogen and activated FVII, so we cannot distinguish the activation state of the FVII in the tissue sections. Most notable was the finding that FVII was detected not only within the vasculature, but also at extravascular sites. Endothelial cells as well as surrounding cells stained for FVII. FVII staining was much weaker in epithelium than around vessels. In addition, many mononuclear cells consistent with macrophages stained strongly for FVII. The affinity of FVIIa for TF is quite high, with a Kd in the subnanomolar range. Thus, we felt that the presence of FVII around the vessels made it very likely that the FVII would, in fact, have bound to the TF, to which it was in close proximity.

If perivascular TF had already bound FVIIa in vivo, it would not be able to bind exogenously added FVIIai–biotin in tissue sections. The FVIIai–biotin could not displace the endogenous FVIIa from a complex with TF, because the tissue sections were fixed with formaldehyde, which chemically crosslinks adjacent proteins [11]. Indeed, as shown in Fig. 3, we found that incubation of tissue sections with FVIIai–biotin led to its binding in the squamous epithelium, but not around vessels. Thus, the perivascular TF antigen was not able to bind FVIIa.

image

Figure 3.  Tissue staining with biotinylated FVIIai. Tissue was processed and stained as described in Materials and methods. These four panels are from the same tissue sample. Biotin–FVIIai bound to sites in the squamous epithelium and is seen as the brown staining in (A) and (B). No staining is seen around vessels in the same panels (indicated by arrows) or around the small artery in (C). (D) is the negative control. Original magnification: (A) 40×; (B, D) 100×; (C) 400×.

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We were able to confirm that binding of FVIIa to TF in skin sections blocked subsequent binding of the monospecific antibody as follows. When tissue sections were preincubated with mouse FVIIa before staining with the monospecific anti-TF antibody, the staining in squamous epithelium and hair follicles was abolished, as shown in Fig. 4. By contrast, staining with the polyspecific antibody was not altered by preincubation with mouse FVIIa (data not shown).

image

Figure 4.  Preincubation with mouse FVIIa blocks subsequent immunostaining with monospecific antibody against a 15 amino acid sequence from tissue factor (TF). Tissue was processed and immunostained with monospecific antibody as described in Materials and methods. The bottom panel was from a tissue section preincubated with mouse factor VIIa before immunostaining, and the top panel was incubated at the same time with diluent buffer alone before immunostaining. The original magnification of both panels was 100×.

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Finally, we used a molecular modeling approach to determine whether the epitope detected by the monospecific anti-TF is likely to be directly blocked by prior binding of FVIIa. The modeling is based on data from human TF and FVIIa structures. We computed the SASA in Å2 for residues 155–168 for the complete all-atom models of sTF alone, sTF–FVIIa and sTF–FVIIa–FXa on the basis of our 20-ns all-atom solvent-equilibrated models [9,10]. From viewing the structures in three-dimensional graphics, it is clear that FVIIa contacts sTF in this region, as illustrated in Fig. 5. In the TF–FVIIa–FX complex, FXa contacts this region of TF as well. The SASAs for TF residues 155–168 are: sTF, 1012 Å2; sTF–FVIIa, 704 Å2; and sTF–FVIIa–FXa, 442 Å2. Thus, binding of FVIIa to TF covers about 30% of the 155–168 surface, and binding of FXa covers an additional 25%.

image

Figure 5.  Representation of the molecular model of the human tissue factor (TF)–FVIIa–FX complex. FVIIa is shown in red, TF in blue, and FXa in green. TF residues of interest are shown in yellow, and calcium ions in white. On the left is the model of the complete ternary complex. On the right is a zoom into the region of TF to which the monospecific antibody was raised. It is easy to see how access of the antibody to this site would be blocked by the presence of FVIIa or FVIIa and FX.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

The tissue distribution of TF has previously been assessed by immunohistochemistry [12–14]. Squamous epithelium stains quite strongly. Vascular endothelium does not stain, but there is a ‘coat’ of TF staining in the ‘adventitia’ around vessels. Our immunostaining results with an antibody preparation raised against the entire extracellular domain of mouse TF were entirely consistent with these published results.

By contrast, we found that a monospecific anti-TF antiserum raised to a peptide consisting of residues 155–168 of the mature mouse TF molecule failed to detect TF antigen around vessels in sections of formalin-fixed paraffin-embedded mouse skin. This antiserum reacted very well with TF antigen in squamous epithelium in the same sections. Antiserum raised against the whole mouse TF extracellular domain detected TF antigen both around vessels and in squamous epithelium in adjacent sections from the same tissue specimens. Both antibody preparations were raised in rabbits. Thus, the failure of the monospecific antibody to react with perivascular TF was not due to inability of the antibody to detect TF in paraffin sections, and nor was it due to some peculiarity related to producing the antibody in rabbits or related to the anti-(rabbit IgG) secondary antibody that we used. These results led us to hypothesize that binding of FVIIa to TF at sites around vessels blocked binding of the monospecific antibody to TF at those sites.

As our tissues were fixed in formalin before staining, proteins adjacent to one another in the tissues could be crosslinked together during fixation. Even if the anti-TF antibody had a very high affinity, it could not compete with FVII or FVIIa already bound to TF in vivo, as the FVIIa–TF complex was probably covalently linked together in the tissue sections.

We have also provided direct evidence that FVIIa binding to TF can block binding of the monospecific antibody, as preincubation of tissue sections with mouse FVIIa abolished subsequent staining with the monospecific TF antibody, but did not alter staining with the polyspecific antibody.

We found that FVII was indeed present in and around the vessels. As FVII has a high affinity for TF (Kd in the low nanomolar [15] or subnanomolar range [16]), it is very likely that the two would bind if FVII were available in the extravascular compartment. This interpretation is consistent with the fact that the Gla-containing coagulation factors can leave the vascular space, as they are found in the lymph [2]. In addition, FVII can be synthesized by some stromal cells and mononuclear phagocytes [17], and could reach the perivascular space by this mechanism.

Supporting the interpretation that prior binding of FVIIa blocked binding of the monospecific TF antibody, we found that biotinylated active site-inhibited FVIIa was also unable to bind to TF in a perivascular location, but bound well to TF in squamous epithelium in the same sections. Thus, the presence of FVIIa bound to perivascular TF in vivo most likely blocks subsequent binding of the biotin–FVIIai reagent just as it blocks binding of the monospecific antibody.

Additional pieces of evidence from the literature support the concept that FVIIa is bound to TF outside the vascular space. A much greater proportion of an injected dose of FVII than of another Gla-containing protein, prothrombin, partitions to the extravascular space [18]. Even though the clearance of FVII from the plasma is much faster than that of prothrombin (plasma half-life of 5–6 h for FVII [19] vs. 2–3 days for prothrombin [20]), the whole body fractional catabolic rate of FVII is less than that of prothrombin [18]. These data suggest that FVII has an extended residence time outside the vascular space, although they do not prove that this is a result of its binding to TF in the extravascular space. These data are also consistent with limited clinical observations suggesting that FVII-deficient patients can be maintained on prophylactic therapy with FVIIa two or three times a week [21], an indication that the persistence of FVII in the tissues is considerably longer than its plasma half-life.

On the basis of homology to human TF, some of the residues in the peptide sequence to which the monospecific antibody was raised would be expected to interact with FVIIa in the FVIIa–TF complex or with substrate (FX or FIX), based on mutagenesis studies and molecular modeling [22,23]. However, some of the residues (amino acids 159–162) in the middle of our target peptide (amino acids 155–168) are not visualized in the published FVIIa–TF crystal structures, so we could not be certain of their position. To try to get a more complete assessment of the structure of the epitope recognized by the monospecific antibody and its potential interactions with FVIIa and FX, we used a molecular modeling approach. The missing residues were added and solvent equilibrated by a molecular dynamics procedure. The model was used to calculate the amount of the surface of the target peptide residues that would be solvent exposed in the presence and absence of FVIIa and FX. These calculations suggest that – subject to the assumption of structural equivalence in human and mouse TF – FVIIa binding shields about 30% of the surface of the epitope, and subsequent binding of FX blocks an additional 25% of the surface. Binding of two inhibitory antibodies to TF has been studied in detail, based on the crystal structure of the antibody–TF complexes [24,25]. One of them blocks binding of FX to the FVIIa–TF complex. Conversely, it is plausible that binding of FVIIa to TF could impede binding of an antibody to its target peptide. We have not attempted to address the question of whether the FVIIa–TF substrates, FX or FIX, are bound in a ternary complex at perivascular locations. However, these proteins also leave the vasculature and transit through the tissues into the lymph. Thus, it is likely that FIX and FX bind to FVIIa–TF complexes in the perivascular space in the absence of injury.

The region to which the monospecific TF antibody is directed is fairly close to the site of a disulfide bond that has been reported to modulate TF activity [26,27]. The Cys186–Cys209 disulfide bond of human TF is critical for coagulant activity, and protein disulfide isomerase can inhibit coagulation by targeting this disulfide. A TF mutant (TF C209A) with an unpaired Cys186 has reduced affinity for FVIIa. Thus, it is possible that the state of this disulfide bond might have an effect on the binding of our monospecific antibody. Our preliminary modeling studies suggest that there is little or no change in the structure of the epitope recognized by the monospecific antibody whether or not the disulfide bond is intact or disrupted. Our entire body of evidence is most consistent with the interpretation that the failure of the monospecific antibody to bind at perivascular sites is due to FVIIa having already bound at those sites in vivo. However, we have not addressed the possibility that a conformational change because of a change in the allosteric disulfide bond might also influence antibody binding.

In summary, we cannot directly prove that FVIIa is bound to perivascular TF in the dermis. However, FVIIa antigen is present in this location. Furthermore, exogenously added FVIIa cannot bind to the perivascular TF, and neither can an antibody to a portion of TF that is shielded by prior binding of FVIIa. Therefore, we conclude that perivascular TF is likely to exist in a complex with FVIIa in the absence of any injury.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

Computational resources were provided by The University of North Carolina at Chapel Hill and the Biomedical Unit of the Pittsburgh Supercomputing Center. This work was supported by research grants from Novo Nordisk A/S (D. M. Monroe), The Louise and Gustavus Pfeiffer Foundation, a Beginning Grant-in-Aid from the American Heart Association, and NIH grants U54-HL077878 and HL081395 (G. M. Arepally), NIH grant HL-06350 and NSF grant ITP/APS-0121361 (L. G. Pedersen), and the US Department of Veterans Affairs (M. Hoffman).

Disclosure of Conflict of Interests

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

The authors state that they have no conflict of interest.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References
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