Filamin-A is required for the incorporation of tissue factor into cell-derived microvesicles

We previously reported that the incorporation of tissue factor (TF) into cell-derived microvesicles (MVs) is regulated by the phosphorylation of the cytoplasmic domain of TF. Since the cytoskeletal protein filamin-A is known to bind to the cytoplasmic domain of TF in a phosphorylation-dependent manner, the involvement of filamin-A in the incorporation of TF into MVs was examined. Endothelial cells were transfected to express TF, whereas MDA-MB-231 cells were used to examine endogenously expressed TF. MV release was induced by activating protease-activated receptor-2 (PAR2). Partial suppression of filamin-A expression using two different filamin-A siRNA sequences resulted in significant reductions in the incorporation of TF antigen into MVs as determined by TF-ELISA and western blot analysis, and was reflected in reduced thrombin-generation and FXa-generation capacities of these MVs. Deletion of the cytoplasmic domain of TF also resulted in reduced incorporation of TF into MVs, whereas the suppression of filamin-A expression had no additional effect on the incorporation of truncated TF into MVs. Partial suppression of filamin-A expression had no effect on the number and size distribution of the released MVs. However, >90 % suppression of filamin-A expression resulted in increased MV release, possibly as a result of increased instability of the plasma membrane and underlying cytoskeleton. In conclusion, the presence of filamin-A appears to be essential for the incorporation of TF into MVs following PAR2 activation, but is not required for the process of MV formation and release following PAR2 activation

Duramycin-induced calcium release in cancer cells

Introduction: Duramycin through binding with phosphatidylethanolamine (PE) has shown potential to be an effective anti-tumour agent. However its mode of action in relation to tumour cells is not fully understood. Methods: PE expression on the surface of a panel of cancer cell lines was analysed using duramycin and subsequent antibody labelling then analysed by flow cytometry. Cell viability was also assessed via flow cytometry using annexin V and propidium iodide (PI). Calcium ion (Ca²⁺) release by tumour cells in response to duramycin was determined by spectrofluorometry following incubation with Fluo-3, AM. Confocal microscopy was performed on the cancer cell line AsPC-1 to assess real time cell response to duramycin treatment. Results: Duramycin was able to detect cell surface PE expression on all 15 cancer cell lines screened, which was shown to be duramycin concentration dependent. However higher concentrations induced necrotic cell death. Duramycin induced calcium ion (Ca²⁺) release from the cancer cell lines also in a concentration and time dependent manner. Confocal microscopy showed an influx of PI into the cells over time and induced morphological changes. Conclusion: Duramycin induces Ca²⁺ release from cancer cell lines in a time and concentration dependent relationship

Accumulation of tissue factor in endothelial cells induces cell apoptosis, mediated through p38 and p53 activation

We previously reported that high levels of tissue factor (TF) can induce cellular apoptosis in endothelial. In this study, TF-mediated mechanisms of induction of apoptosis were explored. Endothelial cells were transfected to express wild-type TF. Additionally, cells were transfected to express Asp253-substituted, or Ala253-substitued TF to enhance or prevent TF release respectively. Alternatively, cells were pre-incubated with TF-rich and TF-poor microvesicles. Cell proliferation, apoptosis and the expression of cyclin D1, p53, bax and p21 were measured following activation of cells with PAR2-agonist peptide. Greatest levels of cell proliferation and cyclin D1 expression were observed in cells expressing wild-type or Asp253-substituted TF. In contrast, increased cellular apoptosis was observed in cells expressing Ala253-substituted TF, or cells pre-incubated with TF-rich microvesicles. The level of p53 protein, p53-phosphorylation at ser33, p53 nuclear localisation and transcriptional activity, but not p53 mRNA, were increased in cells expressing wild-type and Ala253-substituted TF, or in cells pre-incubated with TF-rich microvesicles. However, the expression of bax and p21 mRNA, and Bax protein were only increased in cells pre-incubated with TF-rich microvesicle and in cells expressing Ala253-substituted TF. Inhibition of the transcriptional activity of p53 using pifithrin-α suppressed the expression of Bax. Finally, siRNA–mediated suppression of p38α, or inhibition using SB202190 significantly reduced the p53 protein levels, p53 nuclear localisation and transcriptional activity, suppressed Bax expression and prevented cellular apoptosis. In conclusion, accumulation of TF within endothelial cell, or sequestered from the surrounding can induce cellular apoptosis through mechanisms mediated by p38, and involves the stabilisation of p53

Chemotherapy treatment of multiple myeloma patients increases circulating levels of endothelial microvesicles

Correspondenc

Antithrombotic therapy in palliative care

Management of venous thromboembolism (VTE) in patients in advanced cancer can be difficult due to the increased risk of recurrent and extending VTE despite therapeutic anticoagulation, and of bleeding due to or exacerbated by anticoagulation. Currently, best practice is long term administration of low molecular weight heparin (LMWH), but a recurrent VTE and bleeding rate remains, and some patients have contra-indications to anticoagulation. Newer anticoagulants such as oral anti-thrombin agents and biotinylated idrapurinux may have a role in the future.Management of venous thromboembolism (VTE) in patients in advanced cancer can be difficult due to the increased risk of recurrent and extending VTE despite therapeutic anticoagulation, and of bleeding due to or exacerbated by anticoagulation. Currently, best practice is long term administration of low molecular weight heparin (LMWH), but a recurrent VTE and bleeding rate remains, and some patients have contra-indications to anticoagulation. Newer anticoagulants such as oral anti-thrombin agents and biotinylated idrapurinux may have a role in the future

Apixaban suppresses the release of TF-positive microvesicles and restrains cancer cell proliferation through directly inhibiting TF-fVIIa activity

The activation of protease-activated receptor (PAR)-2 by factor Xa (fXa) promotes the release of tissue factor-positive microvesicles (TF + MV), and contributes to proliferation in cancer cells. This study examined the ability of direct oral anticoagulants (DOACs), apixaban and rivaroxaban, to inhibit the release of TF + MV from two cell lines (MDA-MB-231 and AsPC-1) as well as cell proliferation. Activation of the cells with fXa (10 nM) enhanced the release of TF + MV but was suppressed in the presence of either DOAC. These MVs were found to contain fVIIa, but not fXa. Incubation of cell lines with apixaban (1.8 μM) but not rivaroxaban (1.8 μM), in the absence of fXa decreased the release of TF + MV below that of resting cells, in a PAR2-dependent manner. Furthermore, incubation with apixaban reduced the proliferation rate in both cells lines. Incubation of purified fVIIa with apixaban but not rivaroxaban resulted in complete inhibition of fVIIa proteolytic activity as measured using two fVIIa chromogenic substrates. Pre-incubation of the cells with an inhibitory anti-fVIIa antibody, with apixaban or the blocking of PAR2 suppressed the release of TF + MV to a comparable level, and reduced cell proliferation but the effect was not cumulative. This study has established that the activation of PAR2 by TF-fVIIa complex is the principal mediator in augmenting the release of TF + MV as well as cancer cell proliferation. Importantly, for the first time we have shown that apixaban selectively inhibits the proteolytic activity of fVIIa as well as the signalling arising from the TF-fVIIa complex

Cancer patients’ experiences of the diagnosis and treatment of incidental pulmonary embolism (a qualitative study)

Background: The diagnosis of symptomatic cancer-associated thrombosis often causes distress and alarm for patients, especially for those unaware of the risk, or the signs and symptoms to look out for. There are few data about cancer patients’ experiences of incidentally diagnosed pulmonary embolism (IPE), where lack of warning (recognised signs, symptoms) may cause delayed diagnosis and aggravate distress. Objectives: To explore cancer patients’ experience of the diagnosis of and living with incidental pulmonary embolism treated with anticoagulation. Methods: A qualitative study using modified grounded theory approach. Semi-structured interviews were conducted as part of a mixed- methods prospective observational survey study of consenting patients with IPE. Data were subjected to thematic analysis. The qualitative findings are presented. Findings: Eleven participants were interviewed (mean age 68.3 years, range 38–82 years; various forms of cancer and stages). Three major themes and one cross-cutting theme were generated. Theme (1): IPE is experienced in the context of cancer and concomitant comorbidities. Issues are understood in the shadow of–and often overshadowed by—current serious illness. Theme (2): Being diagnosed with IPE. Misattribution to cancer or other comorbidities caused delay in help-seeking and diagnosis. Theme (3): Coping with anticoagulation. Participants’ incorporated anticoagulation treatment and its effects into their daily routine with acceptance and stoicism. Finally, the cross-cutting theme relates to a lack of information and uncertainty, contributing to distress throughout the experience. Conclusion: The diagnosis of IPE was upsetting and unexpected. Expert and timely information was valued by those with IPE. Education called for about the increased risk of cancer-associated thrombosis and the signs and symptoms to be aware of

Activation of PAR2 by tissue factor induces the release of the PTEN from MAGI proteins and regulates PTEN and Akt activities

Tissue factor (TF) signalling has been associated with alterations in Akt activity influencing cellular survival and proliferation. TF is also shown to induce signalling through activation of the protease activated receptor (PAR)2. Seven cell lines were exposed to recombinant-TF (rec-TF), or activated using a PAR2-agonist peptide and the phosphorylation state of PTEN, and the activities of PTEN and Akt measured. Furthermore, by measuring the association of PTEN with MAGI proteins a mechanism for the induction of signalling by TF was proposed. Short term treatment of cells resulted in de-phosphorylation of PTEN, increased lipid-phosphatase activity and reduced Akt kinase activity in most of the cell lines examined. In contrast, continuous exposure to rec-TF up to 14 days, resulted in lower PTEN antigen levels, enhanced Akt activity and increased rate of cell proliferation. To explore the mechanism of activation of PTEN by TF, the association of "membrane-associated guanylate kinase-with inverted configuration" (MAGI)1–3 proteins with PTEN was assessed using the proximity ligation assay and by co-immunoprecipitation. The interaction of PTEN with all three MAGI proteins was transiently reduced following PAR2 activation and explains the changes in PTEN activity. Our data is first to show that PAR2 activation directly, or through exposure of cells to TF releases PTEN from MAGI proteins and is concurrent with increases in PTEN phosphatase activity. However, prolonged exposure to TF results in the reduction in PTEN antigen with concurrent increase in Akt activity which may explain the aberrant cell survival, proliferation and invasion associated with TF during chronic diseases

Cancer patients’ experiences of living with venous thromboembolism: A systematic review and qualitative thematic synthesis

Background: Cancer-Associated thrombosis is common. Recommended treatment is daily injected low-molecular-weight heparin for 6months. Most studies focus on prophylaxis and treatment; few have explored patients’ experience. Aims To identify and synthesise the available literature concerning patients’ experience of cancer associated thrombosis. Design Systematic literature review and qualitative thematic synthesis. MethodsMEDLINE, Embase, CINAHL, PsychINFO (until 10/2016; limited to English) were searched. Eligible papers were qualitative studies of adult patients’ experience of cancer-associated thrombosis. Two researchers screened titles/abstracts/papers against inclusion criteria with recourse to a third for disagreements. Critical Appraisal Skills Programme qualitative checklist tool was used for quality appraisal. Results1397 articles were identified. Five qualitative studies (total n=92; age range 32 to 84 years) met the inclusion criteria. Participants had various cancer types. Most had advanced disease and were receiving palliative care. Four major themes emerged from the data: knowledge deficit (patients and clinicians); effects of cancer associated thrombosis (physical and psychological); effects of anticoagulation; coping strategies. ConclusionThe cancer journey is difficult in itself, but thrombosis was an additional, frightening and unexpected burden. Although the association between cancer and thromboembolism is well known, cancer patients are not educated routinely about the risk or warning symptoms/signs of thromboembolism which may otherwise be misattributed to the cancer by patient and clinician alike. This systematic review highlights the impact of cancer-associated thrombosis on the lives of cancer patients, and calls for education for patients and clinicians to be part of routine care, and further work to address this patient priority

Mesenteric desmoid tumor developing on the site of an excised gastrointestinal stromal tumor

We present a case of a rare and unusual occurrence of a desmoid tumor at the site of a resected gastrointestinal stromal tumor and mimicking a recurrence, with a brief discussion of the management of desmoid tumors