"Soluble Tissue Factor" in the 21st Century: Definitions, Biochemistry, and Pathophysiological Role in Thrombus Formation.

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Semin Thromb Hemost. Author manuscript; available in PMC 2016 April 11. Published in final edited form as: Semin Thromb Hemost. 2015 October ; 41(7): 700–707. doi:10.1055/s-0035-1556049.

“Soluble Tissue Factor” in the 21st Century: Definitions, Biochemistry, and Pathophysiological Role in Thrombus Formation Vladimir Y. Bogdanov, PhD1 and Henri H. Versteeg, PhD2

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1Division

of Hematology/Oncology, Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio 2Department of Internal Medicine, Section of Thrombosis and Hemostasis, Leiden University Medical Center, Leiden, The Netherlands

Abstract

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Tissue factor (TF), the main trigger of blood coagulation, is essential for normal hemostasis. Over the past 20 years, heightened intravascular levels and activity of TF have been increasingly perceived as an entity that significantly contributes to venous as well as arterial thrombosis. Various forms of the TF protein in the circulation have been described and proposed to be thrombogenic. Aside from cell and vessel wall-associated TF, several forms of non–cell-associated TF circulate in plasma and may serve as a causative factor in thrombosis. At the present time, no firm consensus exists regarding the extent, the vascular setting(s), and/or the mechanisms by which such TF forms contribute to thrombus initiation and propagation. Here, we summarize the existing paradigms and recent, sometimes paradigm-shifting findings elucidating the structural, mechanistic, and pathophysiological characteristics of plasma-borne TF.

Keywords tissue factor; thrombosis; microparticle; alternative splicing; atherosclerosis

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Two decades have now passed since, as perhaps best exemplified by stellar articles from Eisenberg and colleagues on clot surface-bound factor X (FXa), whole blood thrombi were customarily referred to as structures that “presumably lack tissue factor.”1 As the 20th century was coming to a close, this concept began to unravel rapidly and the experimental evidence to the contrary started to accumulate exponentially, beginning with the landmark studies by Toschi et al and Marmur et al pointing to tissue factor (TF) being present in thrombogenic atherosclerotic plaques and plaque-associated organized thrombi.2,3 Key et al and Giesen et al demonstrated the presence of biologically active TF in the blood of healthy individuals4,5 and, perhaps most pointedly for the purpose of this overview, Mallat et al demonstrated that > 95% of the TF activity in the plaque material is associated with micro-

Address for correspondence Vladimir Y. Bogdanov, PhD, Division of Hematology/Oncology, Department of Internal Medicine, University of Cincinnati College of Medicine, 3125 Eden Avenue, Suite 1316, Cincinnati, OH 45267 ([email protected]);. Henri H. Versteeg, PhD, Section of Thrombosis and Hemostasis, Department of Internal Medicine, Leiden University Medical Center, Albinusdreef 2, Room C7-14, 2333 ZA Leiden, The Netherlands ([email protected]).

Bogdanov and Versteeg

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particles (MPs) of white cell origin.6 Schecter et al expanded the set of cell types likely serving as the source of releasable TF in the plaque to vascular smooth muscle cells (SMCs) that, upon stimulation with various agonists, deposited TF in the medium as a protein incorporated in MPs ≤ 200 nm as assessed by flow cytometry.7 Shortly thereafter, the Sturk group concluded that MPs that circulate in the blood of healthy subjects not only lack TF antigen and/or activity, but may rather have anticoagulant properties,8 while patients with multiple organ dysfunction syndrome and sepsis in fact do have circulating MPs bearing active TF.9 Almost immediately after that, Diamant et al reported TF+ MPs in the plasma of type 2 diabetics, long known to be at risk for thrombosis.10 Shet et al convincingly demonstrated that TF+ MPs present in sickle blood shortens clotting times in a TFdependent manner, thus solidifying the concept that active TF protein does circulate in human plasma and thus likely contributes to blood thrombogenicity, at least in certain patently prothrom-botic states.11 As the 21st century was taking hold, the concept of “non– cell-associated TF,” “blood-borne TF,” “plasma TF,” and so on, was expanded even further to include a novel entity: an alternatively spliced form of TF that lacks a transmembrane domain and can thus be secreted by cells as a free protein not inserted into and/or associated with the plasma membrane.12

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Throughout this confiagration of evolving paradigms, paradigm shifts, and crosscutting sets of findings pertaining toTF protein in the soluble compartment of blood, the term “soluble TF” was used continuously, if not all too consistently, in much of the literature on the subject (see, e.g., the study carried out by Khan et al13). Today, the term “soluble TF (sTF)” remains poorly defined and continues to cause considerable confusion, especially among newcomers in the field. Here, we first cover the definition(s) of “sTF” and problems associated with the use of this term, and then discuss more recent developments in the field pointing to non– cell-associated circulating TF as a possible contributor to venous thrombosis, arterial thrombosis, and whether, in 2015, at least some forms of “sTF” should still be perceived as viable and attractive antithrombotic targets.

“Soluble TF”: Definitions, Molecules, Mechanisms

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As described in landmark studies by Osterud, Niemetz, and Key and colleagues, circulating monocytes most likely account for well over 90% of blood-borne TF activity, especially when they are activated by a variety of means.4,14,15 However, much of blood-monocyte TF is functionally inert and several biochemical mechanisms are able to elicit TF’s “decryption,” that is, the process by which TF gains ability to serve as a catalytic cofactor for FVIIa—the topic comprehensively covered elsewhere.16 In this light, the presence of non–cell-associated TF in the circulation continues to be of much interest because TF+ MPs in blood, even if readily procoagulant, are nonetheless grossly diluted and only their gradual accretion on the surface of a growing thrombus may thus provide a means to periodically generate a burst of thrombin and fibrin deposition, thus driving thrombus growth.17 Cloning of TF cDNA in the late 1980s18 enabled not only the generation of highly specific antibodies recognizing the TF protein, but also manipulation of the TF protein via recombinant DNA methodology to create structurally and functionally diverse TF molecules. One of the earliest examples of the latter is a form of TF truncated at the juncture

Semin Thromb Hemost. Author manuscript; available in PMC 2016 April 11.


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of its extracellular and transmembrane domains, created by Morrissey and colleagues for the purpose of measuring the level of FVIIa subfractions in pools of total FVII.19 This recombinant form of TF is, inherently, biochemically soluble inasmuch as it can exist in solution without detergents, and not in association with lipid particles. This version of “sTF,” was ultimately used to crystallize the TF/FVIIa complex.20 A naturally occurring form of biochemically soluble TF was subsequently described in human and mouse, termed “alternatively spliced TF” (asTF).12,21 Like sTF, asTF lacks a transmembrane domain yet unlike sTF, it lacks a juxtamembrane region of the extracellular domain encoded by exon 5 and contains a unique C-terminus not present in the full-length TF (flTF).22 Recently, studies in the Mackman laboratory led to the identification of truncated, presumably degraded form(s) of flTF that circulate in mice bearing flTF-expressing tumors23; it is not known whether such form(s) of the flTF protein exist in the human circulation.

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The mechanisms underlying the functions of TF and asTF are substantially distinct inasmuch as flTF relies on its role of the enzymatic cofactor for FVIIa to trigger either FXa/ FIXa production, or engagement of protease-activated receptors (PARs), primarily PAR2, while asTF’s mode of action is predominantly nonproteolytic and exerted via asTF interacting with a noncanonical binding site of integrin subsets, which elicits an array of signaling cascade-mediated effects resulting in angiogenesis, cell proliferation and/or migration, and monocyte recruitment.24 Both human and mouse asTF exhibit very low procoagulant activity in the presence of phosphatidylserine (PS)-containing vesicles, and both are found in spontaneously formed mural thrombi12,21; however, (patho)physiological significance of this hypomorphic asTF cofactor activity is a subject of debate.

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From a purely biochemical point of view, the only forms of “soluble TF” described to date comprise sTF (recombinant, structure known), asTF (naturally occurring, structure known), and “truncated” flTF in tumor-bearing mice (occurring in a mouse model, structure unknown, Fig. 1). That notwithstanding, any and all forms of TF that are (1) not cellassociated, and (2) circulate in plasma continue to be referred to as “sTF” in the literature.25 Over the past decade, TF+ MPs have received particular attention and it is thus warranted to discuss some recent advances on plasma MP composition in general, as well as the flTF/ asTF axis as it pertains to MPs formed and released by TF-expressing cells (Fig. 2). The study by Arraud et al tackled the issue of morphology, size, phenotype, and concentrations of MPs (or, as the authors designated them, extracellular vesicles [EVs]) present in normal human plasma,26 using a novel experimental approach based on sedimentation of labeled EVs on electron microscopy grids; the results were compared with those obtained by conventional flow cytometry.

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Several potentially paradigm-shifting observations were reported: first, less than 1% of all plasma EVs contained externalized PS—required for flTF and asTF cofactor activity — which is at odds with the generally accepted view of EV/MP formation27; second, human plasma was shown to contain, in addition to spherical EVs of various sizes, tubular-shaped EVs with the average length of approximately 2 μm that, while numerically accounting for about 5% of all EVs, amounted to a far more substantial portion of total membrane surface exposed by all plasma EV subpopulations; third, Arraud et al determined that large, intacterythrocyte size EVs (6–8 μm in diameter, the remnants of the red blood cells void of



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hemoglobin [so-called “Eryghosts”]), when detected by flow cytometry, exhibited forward scatter/side scatter properties indistinguishable from those of 0.5 μm microbe-ads, underscoring the inadequacy of conventional flow cytometry as the method of choice when assessing the size of plasma particulates.

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Another recent study by Ashcroft et al28 determined the size distribution of blood MPs using atomic force microscopy and microfluidics, and found the majority of MPs to be in the 30 to 90 nm range, adding to the evidence that flow cytometry is highly unsuitable to measure characteristics of these circulating vesicles. Further studies using innovative approaches such as those employed by Arraud et al and Ashcroft et al are needed to ascertain whether any of their observations may impact our understanding of plasma TF+ MPs’ structure, cellular source, and/or their abundance in health and disease. In a fortuitously timely manner, related issues were also addressed by Davila et al,29 who utilized an orthotopic model of pancreatic cancer to examine the nature and coagulant properties of the TF protein released from a tumor into the circulation. It has been postulated and subsequently confirmed by several groups that tumor vessels are “leaky” compared with normal vessels, which allows ample systemic release of proteins and particles by cancer tissue, exemplified by pancreatic ductal adenocarcinoma (PDAC).30 The human PDAC cell line used by Davila et al, L3.6pl, expresses both flTF and asTF; nanoparticle tracking analysis (NTA) and dynamic light scattering approaches were employed to assess plasma MPs. Two interesting points warrant highlighting: first, the TF protein released into the circulation of tumor-bearing mice was mainly nonsedimentable even at 100,000g (which is in agreement with another NTA-based study recently published by Ettelaie et al that examined MPs in human plasma31), and it exhibited procoagulant activity only when combined with PS+ phospholipids; second, asTF protein was released by L3.6pl cells as a free protein and in association with MPs in cell culture, yet, in the plasma of tumor-bearing mice, it was found to be exclusively associated with MPs.29 These observations, while reemphasizing the previously postulated tenet that “functionally inert” TF present in plasma may readily exert its FVIIa cofactor function when/if deposited on a PS-positive surface, for example, activated endothelium/monocyte and/or growing thrombus,32 call into question whether asTF should be thought of as a protein that exists exclusively in a biochemically soluble state in vivo: asTF requires PS+ phospholipids to serve as a hypomorphic cofactor for FVIIa,33 and asTF’s contribution to slow, long-term thrombus propagation, especially under low shear/venous flow conditions, may thus be appreciable. Moreover, Davila et al demonstrated that flTF that circulates on MPs with a very small diameter (~30 nm, most likely exosomes as evidenced by positivity for such markers as CD63, Hsp70, and flotillin-1), cannot be effectively sedimented from plasma by ultracentrifugation, introducing yet another reason to avoid the term “sTF”: clearly, more structurally precise and, ideally, isoform- specific terminology should be used when describing non–cell-associated plasma TF. In the remainder of our overview, we will use the term “plasma TF” and point to whether the “plasma TF” being discussed comprises flTF, asTF, their combination, or “total TF,” that is, TF protein species produced by F3 (Cf3 in the mouse) whose “splicing identity” is uncertain (Table 1).



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Plasma Tissue Factor in Venous Thrombosis The concept of plasma TF promoting venous thrombosis is a generally accepted one, and largely rests on experimental evidence obtained in mouse models as exemplified by the work performed over 10 years ago by Biró et al.34 That being said, direct evidence of TF protein’s presence in human venous thrombi has not been rigorously studied, which is understandable because even trace amounts of active TF are sufficient to induce thrombus initiation, as well as propagation, when low-shear flow rates and gradual accretion of active TF on the outer edge of a clot are taken into account.35

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Recently, several studies were published whose primary aim was to tackle plasma TF’s correlative and/or causative properties in benign as well as malignant prothrombotic states. As we are writing this review, no firm consensus exists as to whether TF+ MP levels and/or activity correlate with an increased risk of venous thromboembolism (VTE) in benign disease: just to name a few recent examples, Willemze et al reported that in patients with the antiphospholipid syndrome (APS), TF activity of plasma MPs was significantly higher compared with subjects with asymptomatic antiphospholipid antibodies, yet it did not differ among APS subjects with pregnancy-related morbidity (PM), thrombosis, and/or PM + thrombosis.36 Echoing these observations, Thaler et al found that in patients with acute unilateral symptomatic and unprovoked deep vein thrombosis (DVT) of the lower limb, TF activity of plasma MPs was the same as that present in the plasma of healthy control subjects, and it did not differ significantly between patients with proximal or distal DVT and those with or without residual DVT after 6 months.37


On the other hand, Walenga et al reported that in patients with minor lower limb trauma, VTE was significantly and positively associated with higher TF levels in platelet poor plasma that persisted for 5 weeks.38 One possible explanation of the divergent results reported by Thaler et al and Walenga et al is of course the difference in the underlying pathology (absence of trauma in the former and presence of trauma = tissue damage in the latter); another possible factor is that Thaler et al measured MP-associated TF activity, whereas Walenga et al assessed total TF antigen detectable in plasma using a commercial enzyme-linked immunosorbent assay. Even in the settings featuring less divergent clinical scenarios and tools, there are contradicting reports: Ye et al found a positive association between TF+ MPs and recurrent VTE, but not a first VTE39; however, a recent study by our group did not find such a link.40 In cancer, as exemplified by PDAC—a solid malignancy that carries one of the highest levels of risk for venous thrombosis30—there is also no firm consensus as to whether plasma TF is a contributing factor to this comorbidity, with earlier articles from Khorana et al suggesting that it is,41 whereas a more recent report from Thaler et al, while corroborating that TF+ MPs are in fact elevated in the plasma of patients with metastatic PDAC, suggesting that TF+ MPs do not significantly contribute to clot formation.42 As in studies of benign disease, the reasons for such discrepancies likely have to do with differences in/limitations of the techniques used to isolate and analyze TF+MPs, and the properties of the MP species themselves in various patient populations. With regard to the latter, in 2012 Wang et al provided several new and important insights into the mechanisms



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that likely contribute to a prothrombotic state in PDAC.23 Using murine models and human PDAC cell lines, Wang et al demonstrated that (1) orthotopic tumors are far more procoagulant than subcutaneously grown tumors and release higher levels of TF in circulation; (2) the half-life of MPs released by a tumor into circulation is much shorter if the MP surfaces are rich in mucin-1; and (3) the TF protein expressed by the tumor itself, aside from TF+ MPs, plays a critical role in systemic activation of coagulation. These findings point to the extreme complexity of a prothrombotic state in PDAC; we note that the cell lines used by Wang et al expressed flTF, yet not asTF.

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Over the past decade, the pancreas-asTF axis also received considerable attention in several laboratories: we were the first to document coexpression of flTF and asTF messenger RNA (mRNA) in the human pancreas,12 and Johansson et al documented the presence of asTF protein in human pancreas tissue soon thereafter43; in 2006, asTF expression was reported in a variety of human PDAC cell lines44 and the following year, Hobbs et al described increased angiogenesis and tumor mass produced by a subcutaneously grown PDAC cell line MiaPaCa2 that is void of endogenous TF expression, and was genetically modified to express asTF.45 Hobbs et al were unable to explain the mechanisms responsible, the integrinmediated nature of which was concomitantly and independently discovered and described by our laboratories.46

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As it pertains to this overview, we recently reported that a highly aggressive, flTF and asTFpositive PDAC cell line termed Pt45.P1, when engineered to overexpress asTF yet not flTF, releases MPs that are enriched in surface flTF as well as asTF and exhibit higher levels of TF-dependent procoagulant activity47; we note that orthotopically grown Pt45.P1 tumors overexpressing asTF release asTF in the circulation at the levels found in the plasma of patients suffering from aggressive PDAC.48 Further studies are planned to address the relative contributions of tumor TF, plasma TF, and that of the two splice variants of the TF protein to a prothrombotic state in PDAC and/or other malignancies characterized by heightened TF expression in cancer lesions.

Plasma TF in Arterial Thrombosis

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There is solid epidemiological evidence that venous thrombosis likely contributes to arterial thrombosis,49 and the concept of an unstable atherosclerotic plaque rupture being the trigger of arterial thrombus initiation is well accepted; as we mentioned in the introduction, plaque material is abundant in TF+ MPs that very procoagulant. However, TF-driven arterial thrombus propagation is a bit more controversial: unlike venous circulation, arterial circulation features high-wall shear rates and the thrombus-propagating TF must not only originate from the circulating blood, but also contribute to the growth of a platelet-rich “white clot.”35,50 The existence of “extracellular TF” in the organized plaque-associated thrombus reported by Marmur et al3 was followed by studies aimed to investigate this non–cell-associated TF in more detail. In 2002, Himber et al reported that arterial thrombectomy specimens contain, in addition to the TF-positive leukocytes, extracellular TF in the form of vesicles associated with platelets and fibrin51; at the same time, perfusion chamber studies performed by



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Balasubramanian et al revealed that, under arterial wall shear rates, circulating TF protein only colocalizes with platelets when they are activated, for example, via exposure to collagen surfaces.52

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The origin and/or physiological relevance of platelet TF is another controversial area that is covered in detail elsewhere53; that said, there is little doubt that circulating TF—plasma TF and/or cell-associated TF (e.g., monocyte TF)—can be transferred to platelets under shear conditions and, by inference, contribute to arterial clot formation.54,55 Recently, Palmerini et al reported that in coronary thrombi obtained by aspiration in subjects with acute STsegment elevation myocardial infarction (STEMI), TF staining was invariably positive in monocytes yet only occasionally observed in neutrophils and/or platelets, and the levels of detectable TF antigen in the thrombi did not correlate with the levels of TF activity56; because the aspirated material can comprise (1) acute thrombus (the immediate cause of STEMI); (2) organized plaque-associated thrombus, and (3) dislodged plaque/vessel wall material, it is hard to conclude whether the study by Palmerini et al revealed new causal cues to the effect that circulating TF (either cell-associated or plasma TF) is a contributor to coronary occlusion. That being said, we do note that blood components evidently comprise a far more substantial contributor to individual propensity for arterial thrombosis when compared, side-by-side, to the factors present in the vessel wall.57


Aside from untreated atherosclerotic plaques, which become more unstable and thus prone to rupture/thrombosis as they progress, invasively treated plaques, for example, via percutaneous transluminal angioplasty commonly performed on coronary arteries, are also prone to thrombotic occlusion that occurs under extremely high shear rates due to neointimal narrowing largely driven by SMC proliferation.58 Our initial studies of flTF and asTF in spontaneously formed arterial thrombi revealed that these two TF forms are readily detectable and colocalize in autopsy specimens (human), and occlusive thrombi formed spontaneously as the result of a PCA-like experimental procedure (mouse).12,21 As it pertains to asTF’s plaque promoting properties, in 2011 we showed that asTF is abundant in lipid-rich plaques and, when compared with flTF, is a more potent inducer of monocyte recruitment to and through endothelial monolayers59; in 2013, we posited that asTF may thus promote vasa vasorum remodeling leading to plaque destabilization.60 Recently, Giannarelli et al reported that flTF and asTF mRNA and protein levels are lower in stable type IV carotid plaques compared with macrophage-rich, instable type VI plaques.61 The in vitro studies did not break new ground, neither in terms of the major sources of asTF in the plaque, that is, CD68+ cells as we described before,59 nor in terms of the mechanisms engaged by asTF to promote plaque progression, that is, integrin-mediated activation of MAPK/Akt pathways yielding neovascularization partially via vascular endothelial growth factor induction under hypoxic conditions as shown by our groups and that of Rauch and colleagues.46,47,62,63 Of note is a rather peculiar outcome of the in vivo studies performed by Giannarelli et al,61 that is, the increased neo-intimal formation in response to asTF that was transduced via lentivirus into carotid lesions on the ApoE −/− background: even though the authors did not perform SMC-specific tissue staining, it is well known that SMC comprise the bulk of neointima that develops in rodent models64 and the obtained phenotype thus appears at odds



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with their findings in human lesions inasmuch as asTF-high type VI plaques had less SMC compared with asTF-low type IV plaques.61 Unfortunately, no flTF transduction was performed by Giannarelli et al to compare the effects of flTF versus asTF in this setting. One possible explanation may be that human asTF protein was expressed in a murine vessel: our groups recently reported that murine asTF, while very similar to the human asTF in terms of its integrin-mediated proangiogenic/monocyte recruitment potential, utilizes a slightly distinct repertoire of integrins to exert its biological functions65 and no studies were performed to date that compared human versus murine asTF’s effects on human and/or murine SMC. We note that we initially identified murine asTF in cultured murine SMC, and showed that it elicits minimal procoagulant activity in the presence of PS-containing phospholipids.21

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As to the pathophysiological significance of the total coagulant potential in the plaque, recent studies performed by Borissoff et al put forward a tenet that compared with advancedstage plaques, early-stage plaques have elevated de novo biosynthesis of TF and other clotting factors and thus carry more procoagulant potential yet they are less pro-thrombotic because—in part due to their very potent ability to produce fibrin—they are less prone to rupture66; unfortunately, Borissoff et al did not examine the flTF/asTF axis in the set of plaques they analyzed. Clearly, better-designed studies are needed to dissect the relative contribution of plaque asTF to lesion stability, as well as its possible yet still unproven role in the propagation of arterial thrombus.

Remaining Questions and Emerging Concepts Targeting Plasma Tissue Factor to Reduce Thrombosis: Yes/No? How? Where?


As much as one would hope that TF—the molecule at the top of the coagulation cascade whose inhibition, by inference, would most profoundly impact thrombus formation—may comprise a viable therapeutic target, the risk of bleeding associated with targeting flTF (the form that, to date, continues to be of most interest in the field of thrombosis) is quite considerable. On the other hand, targeting asTF, the molecule that is most likely dispensable for normal hemostasis, certainly remains a viable option. As discussed above, asTF’s role in elevating the risk of thrombosis may be indirect, for example, by increasing the release of procoagulant flTF into circulation by cancer lesions and promoting atherosclerosis47,59; thus, it is possible that targeting asTF in solid tissue, rather than in plasma, may be more attractive: our monoclonal anti-asTF antibody has shown efficacy in stemming breast cancer growth in vivo,62 and it reduces metastatic spread and release of asTF into the circulation of mice bearing PDAC tumors.67 However, antibodies are relatively large and tissue penetration may thus be an obstacle; with that in focus, our groups have initiated studies to map the regions of asTF that interact with integrins, which we hope will enable generation of small inhibitory peptides (e.g., nanobodies) and/or single-molecule compounds with tissue penetrance superior to that of an antibody. Further studies will also most certainly be performed to better delineate the potential predictive and/or prognostic biomarker value of plasma TF—be that either TF+ MPs or free/ MP-associated asTF—in various disease states.



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An Emerging Paradigm: Targeting Vascular Tissue Factor via Intrinsic Pathway


As it pertains to arterial thrombosis, the reduction of its triggering factors, for example, vessel wall TF—also remains of much interest, as does the inhibition of arterial thrombus propagation, the process that evidently engages the elements of the intrinsic/contact pathway to a rather substantial extent68: inasmuch as, such as asTF, the inhibition of the “upstream” intrinsic pathway factors is not expected to produce serious bleeding complications, this avenue of antithrombotic intervention seems attractive. Very recently, two independent studies revealed that, rather surprisingly, there seems to be an “added benefit” to targeting the intrinsic pathway to prevent arterial thrombosis per se: Puy et al reported that FXIa upregulates the extrinsic pathway by inactivating tissue factor pathway inhibitor,69 while Stavrou et al convincingly demonstrated that in prekallikrein null mice, the reduction of arterial thrombosis is in part due to a reduction of total TF mRNA, antigen, and activity in the aortic wall.70 Thus, a heretofore unappreciated feedback loop may exist comprising TFinitiated arterial thrombus formation that, when propagated via intrinsic pathway, further potentiates TF procoagulant activity in the vasculature. We greatly look forward to future studies divining this interpathway relationship which may very well lead to the identification of the contact pathway elements comprising the most attractive candidates for therapeutic intervention.

Conclusion

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Every year, more studies—experimental and epidemiological —are published that aim to add new insights into the contribution of plasma TF to thrombus formation; cumulatively, evidence continues to amass to the effect that plasma TF can and does contribute to thrombogenesis, at least in some settings. That being said, the great variety of techniques and approaches currently employed across the globe to study the relative contribution of plasma TF versus blood cell-associated TF versus vessel wall TF continue to render the interpretation of the obtained data very challenging. As we mentioned, direct targeting of the TF coagulant function to reduce or prevent thrombosis may be risky due to bleeding complications, not dissimilar to and perhaps even more severe than those observed with antiplatelet drugs.71 However, targeting TF expression at the mRNA level may be in principle, not only safer, but also splice form-specific: for instance, distinct splicing factors promote flTF and asTF mRNA biosynthesis.72,73 We hope that new technological advances will soon emerge that enable highly specific and efficacious in vivo targeting of the splicing machinery, which would allow better dissection of flTF and asTF’s roles in various disease states, most critically cardiovascular disease and cancer.

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Acknowledgments We are grateful to Dusten Unruh (University of Cincinnati, Ohio) for his help with the drawings, and to Prof. JohnBjarne Hansen (University of Tromso, Norway) for his insightful suggestions. This work was supported in part by NIH/NCI grants R21CA160293 and R01CA190717 to V.Y.B. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.



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References


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Author Manuscript Fig. 1.

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Different forms of TF protein that have been referred to as “soluble TF” in the literature. TF ectodomain: in green—the first (nonregulatory) disulfide bond; in yellow—the second (allosteric, regulatory) disulfide bond. Degraded flTF was observed in plasma in a mouse model; its structure and existence in human circulation await verification. flTF, full-length TF; TF, tissue factor.

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Author Manuscript Author Manuscript Author Manuscript Fig. 2.

Modes of TF protein release into the circulation from a cancer lesion (top), and a ruptured atherosclerotic plaque (bottom). asTF can be released as a free protein, and in association with microparticles. asTF, alternatively spliced TF; TF, tissue factor.

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Table 1

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Summary of concepts from the current report “Soluble tissue factor (TF)”: why the term “plasma TF” is preferred “Soluble TF” can mean either biochemically soluble, or simply non–cell-associated forms of circulating TF Full-length TF (flTF) and alternatively spliced TF (asTF) are both able to circulate in association with microparticles (MPs) Plasma MPs exhibit vast structural diversity, as does plasma TF Plasma TF and venous thrombosis No consensus as to whether elevated TF+ MP heighten the risk of thrombosis in benign disease and/or cancer Divergent methods of studying TF antigen and activity are likely responsible for the lack of such a consensus asTF may potentiate plasma thrombogenicity indirectly in pancreatic cancer Plasma TF and arterial thrombosis flTF and asTF antigen and TF pro-coagulant activity are detectable in thrombi associated with arterial plaques Human and murine asTF promote neovascularization and monocyte recruitment non-proteolytically, via β-integrins More “pro-coagulant” plaques are likely to be less “thrombogenic” because they are more stable Emerging concepts

Author Manuscript

Targeting asTF, rather than flTF, in cancer and possibly atherosclerosis is attractive due to low risk of bleeding Targeting the intrinsic pathway is a promising way to stem arterial thrombosis Positive feedback loop in arterial thrombosis: the intrinsic pathway potentiates TF activity in the vasculature

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