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Microparticles: mediators of cellular and environmental homeostasis
Böing, A.N.
Publication date
2011
Link to publication
Citation for published version (APA):
Böing, A. N. (2011). Microparticles: mediators of cellular and environmental homeostasis.
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Chapter 9
Human alternatively spliced tissue factor is
not secreted and does not trigger coagulation
Anita N. Böing, Chi M. Hau, Auguste Sturk and Rienk Nieuwland
Chapter 9
Human tissue factor (TF), a 45 kDa transmembrane protein, initiates coagulation. In 2003, Bogdanov et al. described an alternatively spliced form of TF (asTF), which lacks a transmembrane domain and is present as a soluble protein1. asTF is produced by various
cell types1-3 and was reported to be present in human plasma, suggesting that asTF is secreted1. At present, the coagulant properties of asTF are disputed. Although recombinant
asTF shortened the clotting time of platelet poor plasma1, asTF was enriched in human
thrombi1 and triggered activation of factor X when secreted by human umbilical vein
endothelial cells (HUVEC)4, asTF expressed by human embryonic kidney (HEK) cells
failed to activate factor X5. To further explore the putative role of asTF in coagulation, we
studied the procoagulant properties of asTF cloned from HUVEC in an expression model of MIA PaCa-2 cells, a human pancreatic TF-deficient cell line2;6-8. Using these cells,
confounding factors such as concurrent expression of TF are excluded.
First, we investigated the production and secretion of asTF by HUVEC. Upon activation with interleukin (IL)-1α (1 hour), both asTF RNA and TF RNA were detectable (data not shown). The asTF protein, however, was not detectable in cell lysates of activated HUVEC (IL-1α, 5 ng/mL; 5 hours), whereas TF was clearly present (Figure 1A). Similarly, the corresponding lysates of microparticles (MP) contained TF but not asTF. In contrast, the MP-free culture supernatant (SN) contained neither asTF nor TF. Thus, expression of asTF RNA in activated HUVEC is not paralleled by translation into the asTF protein, and asTF is neither sorted into MP nor secreted into the culture supernatant.
To investigate production, secretion and procoagulant properties of asTF by MIA PaCa-2 cells, we subcloned cDNA from both asTF and TF into a pDEST N3 gateway vector. The transcript sequences were verified and identical to AF487337 and NM001993, respectively (NCBI). Both asTF cDNA and TF cDNA were transfected into MIA PaCa-2 cells. After transfection, MIA PaCa-2 cells expressed both asTF and TF (Figure 1B), but only TF was detectable in MP and MP-free culture supernatant. Given the facts that (i)
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asTF is secreted. In sum, although MIA PaCa-2 cells translate asTF, asTF is not sorted intoMP or released as a soluble protein into the culture medium.
The procoagulant properties of transfected MIA PaCa-2 cells were studied using cell lysates as described earlier5. A lysate of TF-transfected cells initiated thrombin generation (n=3; Figure 1C), which was inhibited by anti-factor VII(a) but not by anti-factor XII(a). The asTF-transfected cell lysate also initiated a low level of thrombin generation, which was only partially factor VII(a) dependent (Figure 1D). Thrombin generation by lysates from untransfected and EGFP-transfected cells were comparable to the asTF cell lysate (Figures 1E and 1F, respectively), as was the extent of inhibition by anti-factor VII(a), indicating that asTF does not initiate additional thrombin generation in our experimental conditions compared to controls.
To explain thrombin generation by untransfected MIA PaCa-2 cells, we hypothesized that asTF in human plasma binds to membrane fragments, thereby facilitating factor VII(a)-mediated thrombin generation1. However, a comparable level of factor
VII(a)-dependent thrombin generation was observed following reconstitution of a lysate from untransfected cells into normal plasma depleted of asTF and TF (data not shown). Thus, the low level of thrombin generation is not explained by binding of exogenous asTF. Alternatively, we can not exclude that MIA PaCa-2 cells produce very low amounts of procoagulant TF which may be involved in the basal thrombin generation.
Whether asTF is coagulant remains controversial. First, extremely high levels of recombinant asTF, i.e. over 3,000-fold higher than the asTF level in normal plasma, shortened the clotting time of human platelet-poor plasma1, but the relevance of these
results were seriously questioned9.
Thereafter, two studies were published by the same investigators, who investigated the production and procoagulant properties of native asTF from HUVEC4;10. Although in
both studies identical experimental conditions resulted in similar quantities of TF, as detected by Western blot, in the study of Szotowski et al. the amounts of asTF exceeded those of TF, whereas in the study of Eisenrach et al. only small amounts of asTF were found compared to TF. Taken together, even the amount of asTF produced by the only primary cell type studied thus far remains controversial.
Chapter 9
Figure 1. Expression and procoagulant activity of asTF.
A asTF TF th ro m b o p la s ti n un tr a n s fe c te d E G F P ( m o ck) re c o m b inan t as T F TF as T F E G F P ( m o ck) as T F TF unt ra ns fe c te d as T F E G F P ( m o ck) TF un tr a n s fe c te d Cells MP SN MIA PaCa-2 asTF T F th ro m b o p la s ti n Cells re c o m b in a n t a s T F MP SN HUVEC B - + - + - + A asTF TF th ro m b o p la s ti n un tr a n s fe c te d E G F P ( m o ck) re c o m b inan t as T F TF as T F E G F P ( m o ck) as T F TF unt ra ns fe c te d as T F E G F P ( m o ck) TF un tr a n s fe c te d Cells MP SN MIA PaCa-2 asTF T F th ro m b o p la s ti n Cells re c o m b in a n t a s T F MP SN HUVEC B - + - + - + - + - + - + th ro mb in (n mo l/ L ) 0 5 10 15 0 10 20 30 40 0 5 10 15 0 10 20 30 40 th ro m b in (n m o l/L ) 20 30 40 C E 20 30 40 F D th ro mb in (n mo l/ L ) 0 5 10 15 0 10 20 30 40 0 5 10 15 0 10 20 30 40 th ro m b in (n m o l/L ) 20 30 40 C E 20 30 40 F D 0 5 10 15 0 10 20 30 40 0 5 10 15 0 10 20 30 40 th ro m b in (n m o l/L ) 20 30 40 C E 20 30 40 F 20 30 40 F D
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Figure 1 shows the expression and procoagulant activity of asTF. A: HUVEC were non-activated (-) or activated (+) with IL-1α for five hours in serum-free culture medium. Culture supernatant was centrifuged (10 minutes, 180g), and MP were isolated by centrifugation (60 minutes, 18,890g). MP-free culture supernatant (SN) was concentrated using a 3 kDa column. Lysates of HUVEC, MP and SN were subjected to SDS-PAGE under reducing conditions, followed by Western blotting using an antibody recognizing both asTF and TF (4501; American Diagnostica). As secondary antibody, rabbit-anti-goat HRP was used. Subsequently, blots were incubated with lumi-light and exposed for 2.5 hours. B: MIA PaCa-2 cells were left untransfected or transfected (lipofectin; 10 µL) with asTF, TF or EGFP (mock) DNA (1 µg; 48 hours). Samples were prepared and used for Western blot as described for HUVEC. Blots were exposed for 0.5 hour. Lanes entitled “Cells” contained lysates from approximately 40.000 cells each, lanes entitled “MP” contained lysates of MP isolated from serum-free conditioned culture supernatant of 250.000 cells, and lanes entitled “SN” contained lysates from concentrated supernatant from approximately 250.000 cells. As positive controls, thromboplastin (of human origin) and recombinant asTF were used. C-F: Thrombin generation by lysates from untransfected (E) or transfected MIA PaCa-2 cells (C, D, F). Transfected cells (48 hours) were lysed with n-octyl-D-ß-glycopyranoside (15 mmol/L) in HEPES buffer (10 mmol/L) for 15 minutes at 37 °C. Cell lysates were centrifuged to remove debris (5 minutes at 2000g and 4 °C). Lysates were preincubated (30 minutes) without antibodies (filled circles) or with antibodies against human factor VII(a) (open circles) or human factor XII(a) (open triangles). After 30 minutes, the mixtures were added to defibrinated and MP-free normal pool plasma, which also had been preincubated with the indicated antibodies. Thrombin generation was initiated by addition of calcium, and every 30 s a subsample was collected and added to the chromogenic thrombin substrate pefachrome (Pentapharm). After three minutes, thrombin generation was stopped by addition of citric acid and free thrombin formation was monitored by measuring p-nitroaniline (λ=405 nm). Representative thrombin generation curves are shown of thrombin generation by cell-lysates from TF-transfected (C), asTF-transfected (D), untransfected (E) or EGFP (mock)-transfected (F) MIA PaCa-2 cells.
With regard to secretion of asTF by primary cells, the conditioned medium of TNF-α stimulated HUVEC was shown to contain asTF, indicating secretion of asTF. Although this secreted asTF was procoagulant, its activity was less than 0.1% of the corresponding TF activity4.
With regard to asTF-transfected cells, Censarek et al. transfected HEK cells with a FLAG-tagged asTF5. Similar to our findings, asTF was present in cell lysates, absent in MP
and culture supernatant and lacked procoagulant activity. Recently, Hobbs et al. expressed asTF in MIA PaCa-2 cells2. They reported secretion of asTF in the conditioned medium,
but found no shortening of a clotting time, again indicating that asTF lacks procoagulant activity. Taken together, the reported procoagulant activity of asTF was either extremely low compared to TF, or absent.
Despite detectable levels of asTF in MIA PaCa-2 cells, lysates thereof did not initiate additional factor VII-dependent thrombin generation. Various encryption
Chapter 9
membrane environment11-13, dimerization of TF monomers12, and a conformational change
initiated by protein disulfide isomerase (PDI)14. A role for PDI, however, seems unlikely
since asTF lacks both cysteines (Cys186 and Cys209). Whether or not the other mechanisms,
including various post-translational modification mechanisms, affect the ability of asTF to initiate coagulation remains to be determined.
In our present study, we demonstrate that despite expression of asTF RNA in HUVEC, no detectable levels of the corresponding protein were detectable in these cells, their MP or the culture medium. In contrast, when this asTF cDNA was expressed in MIA PaCa-2 cells, the asTF protein became detectable. Also several other pancreatic cells produce and secrete substantial quantities of asTF2. Last year, Hobbs et al. reported a higher
tumor mass in mice injected with asTF-transfected MIA PaCa-2 cells, suggesting a role in tumor growth and angiogenesis. Thus, also based on our present findings, we speculate that the main physiological or pathological role of asTF may not be coagulation but rather angiogenesis or other processes.
References
1. Bogdanov VY, Balasubramanian V, Hathcock J et al. Alternatively spliced human tissue factor: a circulating, soluble, thrombogenic protein. Nat.Med. 2003;9:458-462.
2. Hobbs JE, Zakarija A, Cundiff DL et al. Alternatively spliced human tissue factor promotes tumor growth and angiogenesis in a pancreatic cancer tumor model. Thromb.Res. 2007;120 Suppl 2:S13-S21.
3. Szotowski B, Goldin-Lang P, Antoniak S et al. Alterations in myocardial tissue factor expression and cellular localization in dilated cardiomyopathy. J.Am.Coll.Cardiol. 2005;45:1081-1089.
4. Szotowski B, Antoniak S, Poller W, Schultheiss HP, Rauch U. Procoagulant soluble tissue factor is released from endothelial cells in response to inflammatory cytokines. Circ.Res. 2005;96:1233-1239.
5. Censarek P, Bobbe A, Grandoch M, Schror K, Weber AA. Alternatively spliced human tissue factor (asHTF) is not pro-coagulant. Thromb.Haemost. 2007;97:11-14.
6. Kakkar AK, Chinswangwatanakul V, Tebbutt S, Lemoine NR, Williamson RC. A characterization of the coagulant and fibrinolytic profile of human pancreatic carcinoma cells. Haemostasis 1998;28:1-6.
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10. Eisenreich A, Bogdanov VY, Zakrzewicz A et al. Cdc2-like kinases and DNA topoisomerase I regulate alternative splicing of tissue factor in human endothelial cells. Circ.Res. 2009;104:589-599.
11. Bach R, Rifkin DB. Expression of tissue factor procoagulant activity: regulation by cytosolic calcium. Proc.Natl.Acad.Sci.U.S.A 1990;87:6995-6999.
12. Bach RR, Moldow CF. Mechanism of tissue factor activation on HL-60 cells. Blood 1997;89:3270-3276.
13. Le DT, Rapaport SI, Rao LV. Relations between factor VIIa binding and expression of factor VIIa/tissue factor catalytic activity on cell surfaces. J.Biol.Chem. 1992;267:15447-15454. 14. Ahamed J, Versteeg HH, Kerver M et al. Disulfide isomerization switches tissue factor from
