Regulation of the Incorporation of Tissue Factor into Microparticles by Serine Phosphorylation of the Cytoplasmic Domain of Tissue Factor

Abstract

The mechanisms that regulate the incorporation and release of tissue factors (TFs) into cell-derived microparticles are as yet unidentified. In this study, we have explored the regulation of TF release into microparticles by the phosphorylation of serine residues within the cytoplasmic domain of TF. Wild-type and mutant forms of TF, containing alanine and aspartate substitutions at Ser 253 and Ser 258 , were overexpressed in coronary artery and dermal microvascular endothelial cells and microparticle release stimulated with PAR2 agonist peptide (PAR2-AP). The release of TF antigen and activity was then monitored. In addition, the phosphorylation state of the two serine residues within the released microparticles and the cells was monitored for 150 min. Increased levels of circulating endothelial cell-derived microparticles are recognized as a cause and prognostic marker for vascular disease and injury (1-3). Moreover, these microparticles have been shown to be able to support the induction of thrombosis through a TF-dependent mechanism (3, 4). The mechanisms that regulate the incorporation and release of TF 2 into cell-derived microparticles are as yet unidentified. Furthermore, it appears that cellular activation (5) for example through activation of PKC (6) can induce microparticle release although this is not completely understood. The proteolytic activation of PAR2 by enzymes such as factor Xa and trypsin is known to result in the translocation of PKC␣ to the membrane (7). It has previously been shown that the incubation of cells with trypsin can induce the release of TF into membrane-derived vesicles (8). Furthermore, PKC␣ is known to phosphorylate the cytoplasmic domain of TF at Ser 253 (7, 9), whereas Ser 258 is part of a proline-directed kinase consensus sequence (10), which becomes phosphorylated separately (11) initiated through the phosphorylation of Ser 253 (9). The cytoplasmic domain of TF is not required for the procoagulant activity of TF or de-encryption of this protein (12, 13). Moreover, it has been reported that substitution of the cytoplasmic domain of TF with the cytoplasmic domain of decayaccelerating factor does not alter the release of TF in cells that are capable of releasing TF-containing microparticles without activation (14). However, it is known that cells spontaneously release decay-accelerating factor under standard culture conditions (15). In this study, by aspartate substitution (to mimic phosphoserine) (16) or alanine substitution (to prevent phosphorylation) of two of the serine residues within the cytoplasmic domain of TF, we investigated the contribution of these residues to the regulation of TF incorporation and release into cell-derived microparticles. EXPERIMENTAL PROCEDURESPlasmid Vectors-The pCMV-XL5-TF plasmid for the expression of full-length human TF was obtained from OriGene. Aspartate and alanine substitutions at Ser 253 and Ser 258were carried out to produce mutated constructs (pCMV-XL5-TF Asp253 , pCMV-XL5-TF Ala253 , pCMV-XL5-TF Asp258 , and pCMV-XL5-TF Ala258 ) ...

Accumulation of tissue factor in endothelial cells promotes cellular apoptosis through over-activation of Src1 and involves β1-integrin signalling

“…The pCMV6-Ac-TF-tGFP plasmid DNA (OriGene/Insight Biotechnology, Wembley, UK) was used to express wildtype or a variant form of TF, as previously described [46][47][48]. Preparations of pCMV6-Ac-tGFP (control) and pCMV6-Ac-TF Ala253 -tGFP were described previously [46]. Human coronary artery endothelial cells (HCAEC), devoid of endogenous TF were cultured in MV media containing 5% (v/v) foetal calf serum (FCS) and growth supplements (PromoCell, Heidelberg, Germany).…”

“…Transfection of the cells was carried out using TransIT-2020 (Geneflow, Lichfield, UK) according to the manufacturer's instructions. Cells were permitted to express the proteins for 48 h and the expression of the TF variants was confirmed by flow cytometry [46,49]. Prior to experiments, cells were pre-adapted to respective serum-free media for 1 h and then activated by incubation with PAR2-AP peptide (SLIGKV; 20 µM).…”

Factor VIIa‐induced interaction with integrin controls the release of tissue factor on extracellular vesicles from endothelial cells

“…PAR2 activation with the direct agonist SLIGRL promotes the release of TF + EVs from tumor and endothelial cells, and this process is regulated by intracellular interactions of the TF cytoplasmic domain . Differential centrifugation confirmed that TF is completely recovered in the 16 000 × g EV fraction (Fig.…”

“…TF released on EVs by PAR2 activation is associated with integrin b 1 and is derived from distinct cellular pools PAR2 activation with the direct agonist SLIGRL promotes the release of TF + EVs from tumor and endothelial cells, and this process is regulated by intracellular interactions of the TF cytoplasmic domain [17][18][19]26,27]. Differential centrifugation confirmed that TF is Tissue factor (TF) released on extracellular vesicles (EVs) upon protease-activated receptor (PAR) 2 activation is derived from two distinct cellular pools.…”

Regulation of tissue factor coagulant activity on cell surfaces

“…Studies demonstrating that various PKC activators and inhibitors influence TF activity (see rev [6,114]) reflect the importance of PKC‐mediated phosphorylation for TF gene transcription and protein expression rather than TF phosphorylation per se influencing the coagulant activity of TF. However, it has been reported recently that TF phosphorylation regulates TF incorporation into microparticles and its release [115].…”