Shedding of tissue factor (TF)-containing microparticles rather than alternatively spliced TF is the main source of TF activity released from human cancer cells


J. W. Rak, Henderson Research Center, 711 Concession Street, Rm. 216, Hamilton, Ontario, Canada L8V 1C3.
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Cancer is commonly associated with an increased risk of thrombosis, which contributes significantly to patient morbidity and mortality. Tissue factor (TF), the primary cellular initiator of coagulation, has been implicated in this process as it is highly expressed on the surface of cancer cells in many tumor types [1]. Furthermore, elevated levels of circulating TF have been detected in cancer patients [2] and may contribute to this systemic hypercoagulability. TF procoagulant activity in the circulation may originate from various sources, e.g. cell membrane-derived microparticles [3,4] and leukocytes [5]. A recent report describes a soluble form of human TF (designated asHTF) that can be generated by alternative splicing of the gene [6]. The asHTF isoform, which contains the extracellular domain of TF, but lacks the transmembrane and cytoplasmic domains, could potentially represent an important source of circulating procoagulant activity. We therefore investigated whether cancer cells express alternatively spliced TF (asHTF), as this could be relevant for dissemination of their procoagulant activity.

RNA extracted from tumor cell lines was reverse-transcribed in a reaction mixture containing 100 pmol oligo-dT primer, 0.5 mm dNTP, 100 U Moloney Murine Leukemia Virus (MMLV)-reverse transcriptase, and RNase Block (Stratagene, La Jolla, CA, USA) in the supplied reaction buffer. The reaction product was used in subsequent polymerase chain reactions containing 800 nm primers, 0.2 mm dNTP, and 5 U Platinum Taq polymerase (Invitrogen, Burlington, Canada) in a PTC-100 Programmable Thermal Controller (MJ Research, Watertown, MA, USA). TF and asHTF were amplified by 30 cycles of 94 °C, 50 °C and 72 °C, each for 40 s, using forward (5′-CAGGCACTACAAATACTGTGGCAG-3′) and reverse (5′-TGCAGTAGCTCCAACAGTGCTTCC-3′) primers corresponding to TF nts 221–244 and 1013–1036, respectively. TF and asHTF were discriminated on a 2% agarose gel as bands of 815 and 656 bp, respectively. As a control, the GAPDH gene was amplified using the following primers: 5′-TCGGAGTCAACGGATTTGGTCGTA-3′ (forward) and 5′-AGCCTTCTCCATGGTGGTGAAGA-3′ (reverse).

All tumor cells in a panel comprised of glioblastoma, melanoma, gastric, colorectal and squamous cell carcinoma cell lines were found to express the asHTF transcript; however, this species constituted only a small fraction of the total TF mRNA produced by these cells (Fig. 1a). Alternative splicing of TF by colorectal cancer cells does not appear to be related to cell transformation, as normal colonic epithelial cells (FHC) also express asHTF, albeit at extremely low levels (Fig. 1b). Human umbilical vein endothelial cells (HUVEC) express neither TF nor asHTF under basal conditions (Fig. 1b).

Figure 1.

Cancer cells release tissue factor (TF) procoagulant activity in two forms. (a) Expression of alternatively spliced TF (asHTF) in human tumor cell lines, as determined by reverse transcriptase-polymerase chain reaction. U343, Human glioma; KATO III, SNU-5 and MKN-74, gastric carcinoma; HCT116, colorectal carcinoma; A431, squamous cell carcinoma; WM1341B and WM983A, melanoma cells. (b) Normal colonic epithelial cells (FHC) express both TF and asHTF; human umbilical vein endothelial cells (HUVEC) express neither transcript. (c) TF activity in conditioned medium of tumor cells is associated with shed membrane vesicles pelleted by ultracentrifugation. Inset: electron micrograph of negatively stained A431 microvesicles. One unit of TF activity is defined as the activity of the 1/104 dilution of rabbit brain thromboplastin standard. (d) Shedding of TF-containing microvesicles is the predominant source of TF activity released from human cancer cells.

Two of the tumor cell lines, HCT116 colorectal carcinoma and A431 squamous cell carcinoma, were studied in further detail. TF activity was detected in conditioned medium of both HCT116 and A431 cells using a chromogenic assay for factor Xa generation [7], performed in both the presence and absence of phospholipid vesicles (50 µm PC:PS, 7 : 3; Avanti Polar Lipids, Inc., Alabaster, AL, USA), prepared as described [8]. Moreover, the level of cell-free TF activity released corresponded to the amount of TF mRNA and protein produced by these cells (data not shown). Since viable cells may actively shed plasma membrane-derived vesicles (known as microvesicles or microparticles) containing procoagulant activity [4], we examined whether TF is released from tumor cells in this form. After clearing conditioned medium of cell debris (500 × g, 15 min then 800 × g, 20 min) and ultracentrifugation (100 000 × g), the bulk of TF activity was found to be associated with pelleted tumor microvesicles, rather than with the supernatant fraction, containing asHTF (Fig. 1c). These TF-containing vesicles could be visualized by flow cytometry with a monoclonal anti-TF antibody (American Diagnostica, Greenwich, CT, USA), as well as by electron microscopy (Fig. 1c, data not shown).

Thus, TF activity is secreted from cancer cells predominantly in association with shed membrane vesicles rather than as soluble asHTF. Tumor microvesicles have been studied in the context of tumor cell invasiveness [9], escape from immune surveillance [10], and more recently, participation in angiogenesis [11]. Our findings suggest that shed microvesicles constitute the main source of TF activity released by cancer cells, and in this fashion could contribute to the prothrombotic effects associated with cancer.