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Tissue factor expression in blood cells
Thrombosis Research 125 (2010) S31–S34
Contents lists available at ScienceDirect
Thrombosis Research j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / t h r o m r e s
Review Article
Tissue factor expression in blood cells Bjarne Østerud ⁎ Department of Medical Biology, Faculty of Health Sciences, University of Tromsø, 9037 Tromsø, Norway
a r t i c l e
i n f o
a b s t r a c t The popular concept of TF serving predominantly as a hemostatic envelope encapsulating the vascular bed, has recently been challenged by the observation that blood of healthy individuals may form TF-induced thrombus under conditions entailing shear stress and activated platelets, corroborating the notion of blood borne TF. Accordingly, small amounts of TF activity is detected in calcium ionophore-stimulated monocytes, whereas it is questionable whether neutrophils and eosinophils express TF. Still there are contradicting reports on TF synthesis and expression in activated platelets, but when using a very sensitive and specific assay for TF activity measurements, we fail to detect TF activity associated with platelets activated with various agonists. However, activated platelets may play a role in decrypting monocyte TF activity in a process entailing transfer of TF to activated platelets in a P-seelctin –PSGL-1 reaction whereby inactive TF (encrypted) becomes active through the availability of clusters of phosphatidylserine. Microparticles from plasma of healthy subjects possess weak TF-like activity which is not inactivated by anti-TF antibody. Endothelial cells are well documented to synthesize TF by several agonists in vitro. In contrast, there is little evidence that these cells are capable of synthesizing TF in vivo, and a recent report fails to show that TF on the endothelium may play any role in thrombin generation in a murine endotoxemia model. It may be concluded that monocytes are the only blood cells that synthesize and express TF and which may be the only source for TF-induced thrombosis when the endothelium is intact. © 2010 Elsevier Ltd. All rights reserved.
Available online 10 February 2010 Keywords: Tissue factor monocytes granulocytes platelets endothelial cells microparticles
Contents Tissue factor (TF) in monocytes . . . . Lack of tissue factor in granulocytes . . TF in microparticles. . . . . . . . . . TF expression in platelets? . . . . . . TF expression in endothelial cells (ECs) Conflict of interest . . . . . . . . . . Acknowledgement . . . . . . . . . . References . . . . . . . . . . . . . .
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Tissue factor (TF) in monocytes The classical concept of TF distribution has until quite recently been that TF is entirely localized in the extravascular system, i.e. the vessel wall prevents blood from contact with TF. Within the human blood, monocytes have been recognized as the only cell type that can be induced to synthesize TF de novo. Recently this has been challenged, first by the reports of TF associated with the neutrophils, and then by the reports of the presence of TF in platelets. Furthermore,
⁎ Fax: + 47 776 45350. E-mail address: [email protected]. 0049-3848/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2010.01.032
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S31 S32 S32 S32 S33 S33 S33 S33
circulating monocytes have been suggested to possess encrypted TF [For reviews see 1, 2]. Our own experience suggests that the only active TF in blood may be associated with circulating CD14 positive monocytes at a rate of 1-2% [3]. This TF, in small concentrations, is constitutively expressed in an encrypted form and is decrypted by calcium ionophore in vitro and probably through activation of the monocytes in vivo [4]. Although several theories have been suggested for the decryption phenomenon of TF in cells, this may just be a question of available phosphatidylserine (PS) in larger quantities (clusters) in the fluid microenviroment of the TF antigen in the cell membrane, as has recently been suggested [5]. Several years ago we showed that platelets in a granulocyte dependent reaction amplified TF activity expression in LPS and PMA stimulated monocytes of whole
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B. Østerud / Thrombosis Research 125 (2010) S31–S34
blood [6]. This may be part of the decryption of TF in the cell membrane of monocytes by generating more available PS from the activated platelet membrane. Lack of tissue factor in granulocytes In contrast to the reports of TF activity expression in human neutrophils and eosinophils [7–10], we showed that human granulocytes, isolated from stimulated whole blood, contain very low levels of TF activity, whereas plated resting or LPS/PMA-stimulated granulocytes did not possess any TF activity or antigen [11]. This may suggest that granulocytes acquire TF but do not synthesize it themselves. Our data demonstrated that in experiments, when whole blood was reconstituted with TF-silenced monocytes and stimulated with LPS, the minute TF activity in granulocytes was even further reduced to nearly non detectable levels. Acquisition of monocyteexpressed TF-yellow fluorescent protein (TF-YFP) fusion protein by granulocytes in whole blood further confirmed the validity of the transfer hypothesis of TF from monocytes to granulocytes in the blood. Furthermore, using the membrane-targeted myr-YFP, which was not transferred from monocytes to granulocytes under conditions of LPS-stimulated whole blood, we suggest that TF-YFP transfer is specific to TF and is not a random shedding/fusion of monocyte membranes to/with other cells [11]. Similarly, we showed by flow cytometric [fluorescence-activated cell sorting (FACS)] analysis of isolated human eosinophils revealed no surface expression of TF antigen in resting or stimulated eosinophils [12]. Immunoblotting of eosinophil lysates did not show any TF protein under resting or stimulated conditions. The lysates of resting or stimulated eosinophils contained no detectable levels of TF procoagulant activity. In contrast, monocytes, stimulated in plasma or medium, possessed readily detectable TF levels on the cell surface and in cell lysates as detected by FACS and immunoblotting. We found no detectable TF mRNA levels in resting or stimulated eosinophils by real-time polymerase chain reaction (PCR), whereas in monocytes TF mRNA levels were significantly increased after stimulation [12]. TF in microparticles There have also been conflicting reports on TF associated with microparticles. Obviously TF-rich microparticles may be found in the circulation of patients with various diseases, e.g. severe septicaemia, unstable angina, etc [for review see 13) whereas the presence of TF associated with microparticles in the blood of healthy individuals may be questioned. We found that the TF activity of microparticles from non-stimulated blood was not detecable [4,14]. Although microparticles from LPS-stimulated blood had some TF-activity, it was less than 0.5% of that in monocytes from similarly stimulated blood. Correspondingly, the TF activity of microparticles from LPS+PMA stimulated blood was only 0.7% of that of monocytes from similarly stimulated blood. These findings are in accordance with the notion that in healthy individuals TF is hardly present in circulating microparticles, suggesting that what is measured as TF antigen in plasma must either be associated with even smaller particles (exosomes) or represent some soluble form(s), e.g. alternatively spliced TF (asTF) [15]. Parhami-Seren et al [16], using their highly sensitive and specific fluorescence-based double monoclonal antibody immunoassay for TF antigen, reported plasma TF levels below the nominal quantitative limit of their assay (2 pM; approx. 60-70 pg/ml) in almost 80% of the tested healthy individuals, which is far below what has been claimed by others [16]. Furthermore, they found no evidence of active TF in plasma, since an inhibitory anti-TF antibody had no effect on the clotting time. This accords well with our own observations on the lack of effect when adding anti-TF antibody to freshly drawn non anticoagulated blood, on subsequent measurement of clotting time using
free oscillation rheometry (ReoRox 4 instrument)(Østerud and Olsen, unpublished data). In contrast to our results Bogdanov et al [17], using immunocapture and relipidation technique, found that more than 40% of the TF activity in plasma of normal people was sedimented with the microparticles whereas the rest was soluble TF. Considering that our own TF assay, in spite of being a lot more sensitive than the one used for assaying the relipidated TF, fails to detect active TF associated with the microparticles, it seems pertinent to question whether circulating inactive soluble TF may become active in blood by the interaction with the membrane of activated platelets through a relipidation. We believe that microparticles exert procoagulant activity mainly through their exposure of PS, serving as the mandatory template for tenase and prothrombinase. Apparently many studies utilizing the commercial Actichrome assay have erroneously reported high levels of TF activity, probably derived from PS in their test material in combination with intrinsic activation of FX, as documented by Bogdanov et al [17]. In agreement with this view are the results of a very recent study by Ollivier et al [18], where endogenous TF activity levels in plasma were measured using the calibrated automated thrombogram assay (CAT). Their results imply that plasma of unstimulated blood contains negligible TF activity, since the major part of the procoagulant activity is associated with phospholipids, probably PS exposed on microparticles. Even platelet free plasma from blood subjected to 5 hrs LPS treatment contained less than 0.2 pM (about 6-7 pg/ml) TF antigen associated with the microparticles. TF expression in platelets? The association of TF with platelets was first suggested in a study in which platelets were required for the rapid appearance of TF belonging to the blood, and platelet conjugates were identified as major sites of TF presentation in the blood [19]. An extension of this study revealed TF stored within α-granula and the open canalicular system of the platelets [20]. Following activation with either collagen or thrombin, TF activity was exposed on the platelet membrane. Recently, other reports have corroborated the notion of the presence of TF in platelets. Thus Siddiqui et al [21] claimed that collagenstimulated platelets expressed TF activity, although they did not test whether this TF activity could be blocked by anti-TF antibodies, whereas Camera et al [22]claimed the presence of functionally active, membrane-associated, immunoreactive TF in activated platelets of healthy individuals and detectable TF mRNA in unstimulated platelets. Recently, Butenas et al [23] challenged the many reports of bloodborne TF. In this study they failed to detect any TF antigen on blood mononuclear cells in the absence of LPS stimulation, nor was the presence of any TF antigen detected on platelets, whether in unstimulated or LPS-stimulated blood or on washed and activated platelets. They concluded that there is an absence of measurable amounts of active TF in blood and plasma from healthy individuals (<20 fM), in apparent contradiction to other studies indicating the presence of up to 37 pM amounts of TF in plasma [24]. However, the controversy of the presence, synthesis and functional activity of TF in platelets still persists. Thus, Panes et al [25] claimed also that TF was present in quiescent platelets and was enhanced by the activation. Furthermore, neo-synthesis of TF by platelets was reported in resting platelets as well as activated. Recently they proposed that only von Willebrand Factor (vWF) plus Ristocetin might induce TF activity in platelets in short time and that cell membranes needed to be intact (not lysed) in order to detect TF activity [26]. The TF activity seemed to be independent of adding FVII and was not affected by anti-TFPI. Using a highly sensitive and specific assay for TF activity, we detected only trace amounts of TF activity and no TF antigen in platelets from blood incubated with and without LPS stimuli. [14]. This minor amount of TF like activity associated with the non
B. Østerud / Thrombosis Research 125 (2010) S31–S34
activated platelets could not be neutralized by anti-TF antibody, whereas the TF activity in platelets from LPS-stimulated blood was partially inactivated by the same antibody. We believe that relatively weak TF activity associated with platelets in stimulated blood is derived from TF-expressing monocytes, as was also shown previously by two other groups. [27,28]. In a follow up study, TRAP activated platelet rich plasma, was tested for expression of TF activity using anti-TF antibody and anti-TFPI antibody. The low TF like activity measured in our TF assay (0.6-1.1 mU/ml blood) was enhanced by adding anti-TFPI (1.3-1.8 mU/ml blood) and this enhanced activity was not altered by adding anti-TF antibody. In control experiments, the two antibodies together neutralized more than 97 % of the very high TF activity in LPS-stimulated monocytes of whole blood. Using inactive FVIIa (rhFVIIai) in our TF activity assay, had no effect on the TF activity measured in the platelets whereas it almost totally blocked the TF activity in stimulated monocytes. We have therefore concluded that neither resting nor activated platelets fail to express TF activity in our system. However, splicing of TF pre-mRNA, which is present in platelets, has been claimed to generate TF protein as well as activity [29], but we have failed to detect TF activity in activated platelets even after several hours incubation with various agonists (unpublished data). TF expression in endothelial cells (ECs) A number of groups found TF in cultured human endothelial cells (HUVECs) upon perturbation [for review see 30]. It was demonstrated first that thrombin was an agonist of TF induction in HUVECs and later LPS, IL-1ß and TNFα were shown to induce TF in ECs. [for review see 31]. Until 1987 it had not been possible to identify TF localization in various tissues as generation of monospecific antibodies by using purified TF was not available. The new technique of affinity purifying TF as well as the new knowledge of making epitope-defined monoclonal antibodies to human TF, made it possible for immunohistochemical localization of TF. In this way, Drake et al [32] using the latter technique reported of undetectable TF in endothelium as well as peripheral blood cells. The histochemical localization of TF was confirmed and extended a year later by Fleck et al [33]. Although some reports claim TF expression in ECs in vivo, direct evidence is lacking. The fact that even in severe endotoxemia (lethal doses of E. coli) TF was only found in granular structure on the cell surface of the ECs which also contained PSGL-1, is a strong indication that leukocyte-derived microvesicles might deliver TF to the EC surface [34]. In agreement with this Pawlinsky et al [35] by using mice with cell-type specific deletion of gene in an endotoxemia model, showed that when only the ECs had reduced TF expression, there was no reduction in thrombin generation, indicating that TF may not be expressed by the ECs. In conclusion, we strongly believe that the only blood cells capable of synthesizing TF are the monocytes where TF is constitutively expressed in a few circulating cells in healthy individuals. Under several pathophysiologocal conditions where the monocytes are activated, large amounts of TF may be expressed. This TF can be transferred from the monocytes to activated platelets as well as granulocytes in the form of microparticles/microvesicles whereby particularly the activated platelets associated with TF become extremely thromogenic as they also expose phosphatidylserine (PS) on their surface. Conflict of interest None. Acknowledgement This work was supported by grants from the Norwegian Research Council.
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References [1] Mackman N, Tilley RE, Key NS. Role of the extrinsic pathway of blood coagulation in hemostasis and thrombosis. Arterioscler Thromb Vasc Biol 2007;27(8): 1687–93. [2] Østerud B, Bjørklid E. Sources of tissue factor. Semin Thromb Hemost 2006;32(1): 11–23. [3] Egorina EM, Sovershaev MA, Bjorkøy G, Gruber FX, Olsen JO, Parhami-Seren B, et al. Intracellular and surface distribution of monocyte tissue factor: application to intersubject variability. Arterioscler Thromb Vasc Biol 2005;25(7):1493–8. [4] Østerud B, Breimo ES, Olsen JO. Blood borne tissue factor revisited. Thromb Res 2008;122:432–4. [5] Morrissey JH, Pureza V, Davis-Harrison RL, Sligar SG, Rienstra CM, Kijac AZ, et al. Protein–membrane interactions: blood clotting on nanoscale bilayers. J Thromb Haemost 2009;7(2) Abstract SA-TH-018. [6] Halvorsen H, Olsen JO, Østerud B. Granulocytes enhance LPS-induced tissue factor activity in monocytes via an interaction with platelets. J Leukoc Biol 1993;54(4): 275–82. [7] Todoroki H, Nakamura S, Higure A, Okamoto K, Takeda S, Nagata N, et al. Neutrophils express tissue factor in a monkey model of sepsis. Surgery Feb 2000;127(2): 209–16. [8] Maugeri N, Brambilla M, Camera M, Carbone A, Tremoli T, Donati MB, et al. Human polymorphonuclear leukocytes produce and express functional tissue factor upon stimulation. J Thromb Haemost 2006;4:1323–30. [9] Ritis K, Doumas M, Mastellos D, Micheli A, Giaglis S, Magotti P, et al. A novel C5a receptor-tissue factor cross-talk in neutrophils links innate immunity to coagulation pathways. J Immunol 2006;177:4794–802. [10] Moosbauer C, Morgenstern E, Cuvelier SL, Manukyan D, KBidzhekov K, Albrecht S, et al. Eosinophils are a major intravascular location for tissue factor storage and exposure. Blood 2007;109:995–1002. [11] Egorina EM, Sovershaev MA, Olsen JO, Østerud B. Granulocytes do not express but acquire monocyte-derived tissue factor in whole blood: evidence for a direct transfer. Blood 2008;111:1208–16. [12] Sovershaev MA, Lind KF, Devold H, Jørgensen TØ, Hansen JB, Østerud B. and Egorina EM.No evidence for the presence of tissue factor in high-purity preparations of immunologically isolated eosinophils. J Thromb Haemost 2008;6 (10):1742–9. [13] Butenas S, Orfeo T. Mann KG.Tissue factor in coagulation: Which? Where? When? Arterioscler Thromb Vasc Biol 2009;29(12):1989–96. [14] Østerud B, Olsen JO. and Bjørklid E What is blood borne tissue factor? Thromb Res 2009;124(5):640–1. [15] Bogdanov VY, Balasubramanian V, Hathcock J, Vele O, Lieb M, Nemerson Y. Alternatively spliced human tissue factor: a circulating, soluble, thrombogenic protein. Nat Med 2003;9:458–62. [16] Parhami-Seren B, Butenas S, Krudysz-Amblo J, Mann KG. Immunologic quantitation of tissue factors. J Thromb Haemost 2006;4(8):1747–55. [17] Bogdanov VY, Cimmino G, Tardos JG, Tunstead JR, Badimon JJ. Assessment of plasma tissue factor activity in patients presenting with coronary artery disease: limitations of a commercial assay. J Thromb Haemost 2009;7(5): 894–7. [18] Ollivier V, Wang J, Manly D, Machlus KR, Wolberg AS, Jandrot-Perrus M, et al. Detection of endogenous tissue factor levels in plasma using the calibrated automated thrombogram assay. Thromb Res 2010;125(1):90–6. [19] Zillmann A, Luther T, Muller M, Kotzsch M, Spannagl M, Kauke T, et al. Plateletassociated tissue factor contributes to the collagen-triggered activation of blood coagulation. Biochem Biophys Res Commun 2001;281:603–9. [20] Muller I, Klocke A, Alex M, Kotzsch M, Luther T, Morgenstern E, et al. Intravascular tissue factor initiates coagulation via circulating microvesicles and platelets. FASEB J 2003;17:476–8. [21] Siddiqui FA, Desai H, Amirkhosravi A, Amaya M, Francis JL. The presence and release of tissue factor from human platelets. Platelets 2002;13:247–53. [22] Camera M, Frigerio M, Toschi V, Brambilla M, Rossi F, Cottell DC, et al. Platelet activation induces cell-surface immunoreactive tissue factor expression, which is modulated differently by antiplatelet drugs. Arterioscler Thromb Vasc Biol 2003;23:1690–6. [23] Butenas S, Bouchard BA, Brummel-Ziedins KE, Parhami-Seren B, Mann KG. Tissue factor activity in whole blood. Blood 2005;105:2764–70. [24] So AK, Varisco PA, Kemkes-Matthes B, Herkenne-Morard C, Chobaz-Péclat V, Gerster JC, et al. Arthritis is linked to local and systemic activation of coagulation and fibrinolysis pathways. J Thromb Haemost 2003;1(12):2510–5. [25] Panes O, Matus V, Sáez CG, Quiroga T, Pereira J, Mezzano D. Human platelets synthesize and express functional tissue factor. Blood 2007;109(12):5242–50. [26] Mezzano D, Panes O. Regulation of platelet-expressed tissue factor. J Thromb Haemost 2009;7(Supplement 2) Abstract AS-MO-041. [27] Rauch U, Bonderman D, Bohrmann B, Badimon JJ, Himber J, Riederer MA, et al. Transfer of tissue factor from leukocytes to platelets is mediated by CD15 and tissue factor. Blood 2000;96:170–5. [28] Del Conde I, Shrimpton CN, Thiagarajan P. and López JA.Tissue-factor-bearing microvesicles arise from lipid rafts and fuse with activated platelets to initiate coagulation. Blood 2005;106(5):1604–11. [29] Todoroki H, Nakamura S, Higure A, Okamoto K, Takeda S, Nagata N, et al. Neutrophils express tissue factor in a monkey model of sepsis. Surgery Feb 2000;127(2): 209–16. [30] Østerud B, Bjørklid E. Sources of tissue factor. Semin Thromb Hemost Feb 2006;32: 11–23 Review. [31] Bevilacqua MP, Gimbrone Jr MA. Inducible endothelial functions in inflammation and coagulation. Semin Thromb Hemost 1987 Oct;13(4):425–33.
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[32] Drake TA, Ruf W, Morrissey JH, Edgington TS. Functional tissue factor is entirely cell surface expressed on lipopolysaccharide-stimulated human blood monocytes and a constitutively tissue factor-producing neoplastic cell line. J Cell Biol 1989 Jul;109(1):389–95. [33] Fleck RA, Rao LV, Rapaport SI, Varki N. Localization of human tissue factor antigen by immunostaining with monospecific, polyclonal anti-human tissue factor antibody. Thromb Res Jul 15 1990;59(2):421–37.
[34] Lupu C, Westmuckett AD, Peer G, Ivanciu L, Zhu H, Taylor Jr FB, et al. Tissue factordependent coagulation is preferentially up-regulated within arterial branching areas in a baboon model of Escherichia coli sepsis. Am J Pathol 2005 Oct;167(4): 1161–72. [35] Pawlinski R, Mackman N. Cellular sources of tissue factor that contribute to activation of coagulation in endotoxemic mice. J Thromb Haemost 2009;7 (Supplement 2) Abstract AS-MO-042.
Contents lists available at ScienceDirect
Thrombosis Research j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / t h r o m r e s
Review Article
Tissue factor expression in blood cells Bjarne Østerud ⁎ Department of Medical Biology, Faculty of Health Sciences, University of Tromsø, 9037 Tromsø, Norway
a r t i c l e
i n f o
a b s t r a c t The popular concept of TF serving predominantly as a hemostatic envelope encapsulating the vascular bed, has recently been challenged by the observation that blood of healthy individuals may form TF-induced thrombus under conditions entailing shear stress and activated platelets, corroborating the notion of blood borne TF. Accordingly, small amounts of TF activity is detected in calcium ionophore-stimulated monocytes, whereas it is questionable whether neutrophils and eosinophils express TF. Still there are contradicting reports on TF synthesis and expression in activated platelets, but when using a very sensitive and specific assay for TF activity measurements, we fail to detect TF activity associated with platelets activated with various agonists. However, activated platelets may play a role in decrypting monocyte TF activity in a process entailing transfer of TF to activated platelets in a P-seelctin –PSGL-1 reaction whereby inactive TF (encrypted) becomes active through the availability of clusters of phosphatidylserine. Microparticles from plasma of healthy subjects possess weak TF-like activity which is not inactivated by anti-TF antibody. Endothelial cells are well documented to synthesize TF by several agonists in vitro. In contrast, there is little evidence that these cells are capable of synthesizing TF in vivo, and a recent report fails to show that TF on the endothelium may play any role in thrombin generation in a murine endotoxemia model. It may be concluded that monocytes are the only blood cells that synthesize and express TF and which may be the only source for TF-induced thrombosis when the endothelium is intact. © 2010 Elsevier Ltd. All rights reserved.
Available online 10 February 2010 Keywords: Tissue factor monocytes granulocytes platelets endothelial cells microparticles
Contents Tissue factor (TF) in monocytes . . . . Lack of tissue factor in granulocytes . . TF in microparticles. . . . . . . . . . TF expression in platelets? . . . . . . TF expression in endothelial cells (ECs) Conflict of interest . . . . . . . . . . Acknowledgement . . . . . . . . . . References . . . . . . . . . . . . . .
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Tissue factor (TF) in monocytes The classical concept of TF distribution has until quite recently been that TF is entirely localized in the extravascular system, i.e. the vessel wall prevents blood from contact with TF. Within the human blood, monocytes have been recognized as the only cell type that can be induced to synthesize TF de novo. Recently this has been challenged, first by the reports of TF associated with the neutrophils, and then by the reports of the presence of TF in platelets. Furthermore,
⁎ Fax: + 47 776 45350. E-mail address: [email protected]. 0049-3848/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2010.01.032
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circulating monocytes have been suggested to possess encrypted TF [For reviews see 1, 2]. Our own experience suggests that the only active TF in blood may be associated with circulating CD14 positive monocytes at a rate of 1-2% [3]. This TF, in small concentrations, is constitutively expressed in an encrypted form and is decrypted by calcium ionophore in vitro and probably through activation of the monocytes in vivo [4]. Although several theories have been suggested for the decryption phenomenon of TF in cells, this may just be a question of available phosphatidylserine (PS) in larger quantities (clusters) in the fluid microenviroment of the TF antigen in the cell membrane, as has recently been suggested [5]. Several years ago we showed that platelets in a granulocyte dependent reaction amplified TF activity expression in LPS and PMA stimulated monocytes of whole
S32
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blood [6]. This may be part of the decryption of TF in the cell membrane of monocytes by generating more available PS from the activated platelet membrane. Lack of tissue factor in granulocytes In contrast to the reports of TF activity expression in human neutrophils and eosinophils [7–10], we showed that human granulocytes, isolated from stimulated whole blood, contain very low levels of TF activity, whereas plated resting or LPS/PMA-stimulated granulocytes did not possess any TF activity or antigen [11]. This may suggest that granulocytes acquire TF but do not synthesize it themselves. Our data demonstrated that in experiments, when whole blood was reconstituted with TF-silenced monocytes and stimulated with LPS, the minute TF activity in granulocytes was even further reduced to nearly non detectable levels. Acquisition of monocyteexpressed TF-yellow fluorescent protein (TF-YFP) fusion protein by granulocytes in whole blood further confirmed the validity of the transfer hypothesis of TF from monocytes to granulocytes in the blood. Furthermore, using the membrane-targeted myr-YFP, which was not transferred from monocytes to granulocytes under conditions of LPS-stimulated whole blood, we suggest that TF-YFP transfer is specific to TF and is not a random shedding/fusion of monocyte membranes to/with other cells [11]. Similarly, we showed by flow cytometric [fluorescence-activated cell sorting (FACS)] analysis of isolated human eosinophils revealed no surface expression of TF antigen in resting or stimulated eosinophils [12]. Immunoblotting of eosinophil lysates did not show any TF protein under resting or stimulated conditions. The lysates of resting or stimulated eosinophils contained no detectable levels of TF procoagulant activity. In contrast, monocytes, stimulated in plasma or medium, possessed readily detectable TF levels on the cell surface and in cell lysates as detected by FACS and immunoblotting. We found no detectable TF mRNA levels in resting or stimulated eosinophils by real-time polymerase chain reaction (PCR), whereas in monocytes TF mRNA levels were significantly increased after stimulation [12]. TF in microparticles There have also been conflicting reports on TF associated with microparticles. Obviously TF-rich microparticles may be found in the circulation of patients with various diseases, e.g. severe septicaemia, unstable angina, etc [for review see 13) whereas the presence of TF associated with microparticles in the blood of healthy individuals may be questioned. We found that the TF activity of microparticles from non-stimulated blood was not detecable [4,14]. Although microparticles from LPS-stimulated blood had some TF-activity, it was less than 0.5% of that in monocytes from similarly stimulated blood. Correspondingly, the TF activity of microparticles from LPS+PMA stimulated blood was only 0.7% of that of monocytes from similarly stimulated blood. These findings are in accordance with the notion that in healthy individuals TF is hardly present in circulating microparticles, suggesting that what is measured as TF antigen in plasma must either be associated with even smaller particles (exosomes) or represent some soluble form(s), e.g. alternatively spliced TF (asTF) [15]. Parhami-Seren et al [16], using their highly sensitive and specific fluorescence-based double monoclonal antibody immunoassay for TF antigen, reported plasma TF levels below the nominal quantitative limit of their assay (2 pM; approx. 60-70 pg/ml) in almost 80% of the tested healthy individuals, which is far below what has been claimed by others [16]. Furthermore, they found no evidence of active TF in plasma, since an inhibitory anti-TF antibody had no effect on the clotting time. This accords well with our own observations on the lack of effect when adding anti-TF antibody to freshly drawn non anticoagulated blood, on subsequent measurement of clotting time using
free oscillation rheometry (ReoRox 4 instrument)(Østerud and Olsen, unpublished data). In contrast to our results Bogdanov et al [17], using immunocapture and relipidation technique, found that more than 40% of the TF activity in plasma of normal people was sedimented with the microparticles whereas the rest was soluble TF. Considering that our own TF assay, in spite of being a lot more sensitive than the one used for assaying the relipidated TF, fails to detect active TF associated with the microparticles, it seems pertinent to question whether circulating inactive soluble TF may become active in blood by the interaction with the membrane of activated platelets through a relipidation. We believe that microparticles exert procoagulant activity mainly through their exposure of PS, serving as the mandatory template for tenase and prothrombinase. Apparently many studies utilizing the commercial Actichrome assay have erroneously reported high levels of TF activity, probably derived from PS in their test material in combination with intrinsic activation of FX, as documented by Bogdanov et al [17]. In agreement with this view are the results of a very recent study by Ollivier et al [18], where endogenous TF activity levels in plasma were measured using the calibrated automated thrombogram assay (CAT). Their results imply that plasma of unstimulated blood contains negligible TF activity, since the major part of the procoagulant activity is associated with phospholipids, probably PS exposed on microparticles. Even platelet free plasma from blood subjected to 5 hrs LPS treatment contained less than 0.2 pM (about 6-7 pg/ml) TF antigen associated with the microparticles. TF expression in platelets? The association of TF with platelets was first suggested in a study in which platelets were required for the rapid appearance of TF belonging to the blood, and platelet conjugates were identified as major sites of TF presentation in the blood [19]. An extension of this study revealed TF stored within α-granula and the open canalicular system of the platelets [20]. Following activation with either collagen or thrombin, TF activity was exposed on the platelet membrane. Recently, other reports have corroborated the notion of the presence of TF in platelets. Thus Siddiqui et al [21] claimed that collagenstimulated platelets expressed TF activity, although they did not test whether this TF activity could be blocked by anti-TF antibodies, whereas Camera et al [22]claimed the presence of functionally active, membrane-associated, immunoreactive TF in activated platelets of healthy individuals and detectable TF mRNA in unstimulated platelets. Recently, Butenas et al [23] challenged the many reports of bloodborne TF. In this study they failed to detect any TF antigen on blood mononuclear cells in the absence of LPS stimulation, nor was the presence of any TF antigen detected on platelets, whether in unstimulated or LPS-stimulated blood or on washed and activated platelets. They concluded that there is an absence of measurable amounts of active TF in blood and plasma from healthy individuals (<20 fM), in apparent contradiction to other studies indicating the presence of up to 37 pM amounts of TF in plasma [24]. However, the controversy of the presence, synthesis and functional activity of TF in platelets still persists. Thus, Panes et al [25] claimed also that TF was present in quiescent platelets and was enhanced by the activation. Furthermore, neo-synthesis of TF by platelets was reported in resting platelets as well as activated. Recently they proposed that only von Willebrand Factor (vWF) plus Ristocetin might induce TF activity in platelets in short time and that cell membranes needed to be intact (not lysed) in order to detect TF activity [26]. The TF activity seemed to be independent of adding FVII and was not affected by anti-TFPI. Using a highly sensitive and specific assay for TF activity, we detected only trace amounts of TF activity and no TF antigen in platelets from blood incubated with and without LPS stimuli. [14]. This minor amount of TF like activity associated with the non
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activated platelets could not be neutralized by anti-TF antibody, whereas the TF activity in platelets from LPS-stimulated blood was partially inactivated by the same antibody. We believe that relatively weak TF activity associated with platelets in stimulated blood is derived from TF-expressing monocytes, as was also shown previously by two other groups. [27,28]. In a follow up study, TRAP activated platelet rich plasma, was tested for expression of TF activity using anti-TF antibody and anti-TFPI antibody. The low TF like activity measured in our TF assay (0.6-1.1 mU/ml blood) was enhanced by adding anti-TFPI (1.3-1.8 mU/ml blood) and this enhanced activity was not altered by adding anti-TF antibody. In control experiments, the two antibodies together neutralized more than 97 % of the very high TF activity in LPS-stimulated monocytes of whole blood. Using inactive FVIIa (rhFVIIai) in our TF activity assay, had no effect on the TF activity measured in the platelets whereas it almost totally blocked the TF activity in stimulated monocytes. We have therefore concluded that neither resting nor activated platelets fail to express TF activity in our system. However, splicing of TF pre-mRNA, which is present in platelets, has been claimed to generate TF protein as well as activity [29], but we have failed to detect TF activity in activated platelets even after several hours incubation with various agonists (unpublished data). TF expression in endothelial cells (ECs) A number of groups found TF in cultured human endothelial cells (HUVECs) upon perturbation [for review see 30]. It was demonstrated first that thrombin was an agonist of TF induction in HUVECs and later LPS, IL-1ß and TNFα were shown to induce TF in ECs. [for review see 31]. Until 1987 it had not been possible to identify TF localization in various tissues as generation of monospecific antibodies by using purified TF was not available. The new technique of affinity purifying TF as well as the new knowledge of making epitope-defined monoclonal antibodies to human TF, made it possible for immunohistochemical localization of TF. In this way, Drake et al [32] using the latter technique reported of undetectable TF in endothelium as well as peripheral blood cells. The histochemical localization of TF was confirmed and extended a year later by Fleck et al [33]. Although some reports claim TF expression in ECs in vivo, direct evidence is lacking. The fact that even in severe endotoxemia (lethal doses of E. coli) TF was only found in granular structure on the cell surface of the ECs which also contained PSGL-1, is a strong indication that leukocyte-derived microvesicles might deliver TF to the EC surface [34]. In agreement with this Pawlinsky et al [35] by using mice with cell-type specific deletion of gene in an endotoxemia model, showed that when only the ECs had reduced TF expression, there was no reduction in thrombin generation, indicating that TF may not be expressed by the ECs. In conclusion, we strongly believe that the only blood cells capable of synthesizing TF are the monocytes where TF is constitutively expressed in a few circulating cells in healthy individuals. Under several pathophysiologocal conditions where the monocytes are activated, large amounts of TF may be expressed. This TF can be transferred from the monocytes to activated platelets as well as granulocytes in the form of microparticles/microvesicles whereby particularly the activated platelets associated with TF become extremely thromogenic as they also expose phosphatidylserine (PS) on their surface. Conflict of interest None. Acknowledgement This work was supported by grants from the Norwegian Research Council.
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