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  • br A hypercoagulable state is characteristic of

    2020-08-18


    A hypercoagulable state is characteristic of cancer patients, shown
    Corresponding authors.
    E-mail addresses: [email protected] (K. Pather), [email protected] (T.N. Augustine).
    by thrombocytosis, heightened Cyclophosphamide Cyclophosphamide of platelet activation mar-kers and platelet-derived microparticles [6,17]. Breast cancer itself is a risk factor for cancer-associated thrombosis [18,19,20,21]. Treatment strategies for hormone-responsive breast cancer have improved mor-tality rates; however, they are associated with increased risk for thromboembolic events [19,20,22]. Tamoxifen, a selective oestrogen receptor modulator (SERM) and the predominant treatment prescribed for oestrogen-receptor positive breast tumours in pre- and post-meno-pausal women [22], increases survival and reduces the risk of recur-rence of invasive and non-invasive breast cancer when used for a minimum of 5 years [23,24] but is associated with significant risk of thromboembolic complications [20,22,25–27]. Anastrozole, a third generation non-steroidal aromatase inhibitor (AI) that reduces oestra-diol bioavailability is clinically more effective in reducing thrombotic complications in postmenopausal women [27]; however, risk remains elevated compared to that of tumour-free individuals, with combination AI and chemotherapy use heightening the risk of venous thromboem-bolism (VTE) [20].
    Recent studies highlight that platelets may play a greater part in venous thrombosis than previously described [28,29]. Clinically, ele-vated platelet count, platelet hyperaggregability, and heightened levels of CD62P+ (P-selectin) microparticles are suggested as risk factors for VTE in cancer patients [17,28]. In vitro studies have confirmed the ability of mammary carcinoma cell lines to induce platelet activation demonstrated by P-selectin expression (exposure) [27], ultrastructural alterations [30], and aggregation via GPIb-IX and GPIIb/IIIa activation [31,32]; and platelet-dependent induction of metastasis [2,27]. Ta-moxifen has been extensively investigated; however, contradictory la-boratory results have been produced, some of which do not reflect the prothrombotic clinical situation [20,22,25–27]. Tamoxifen-induced platelet activation is linked to its ability to facilitate Ca2+ entry [33–35]. Nevertheless, recent studies show that despite this Tamoxifen inhibits washed platelet aggregation, particularly under collagen sti-mulation [26]. At high concentrations Tamoxifen pre-treatment of platelets decreases MCF7 cell metastatic potential and inhibits induced platelet activation as identified by P-selectin expression (exposure) [27]. These discordant results may reflect differences in hormone-therapy dosage and experimental method in platelet isolation, with the loss of plasma in the preparation of washed platelets linked to studies refuting Tamoxifen-induced platelet activation and aggregation [13].
    To clarify contradictory results regarding Tamoxifen-induced pla-telet activation; and determine the effect of Anastrozole-induced pla-telet activation, we present the effects of physiological doses of Anastrozole and Tamoxifen on luminal phenotype MCF7- and T47D-induction of platelet activation. Pre-treatment of breast cancer cells allows for mimicking the cumulative effect of the drugs, as would occur in vivo, followed by exposure to whole blood. While aggregometry re-mains the gold standard for investigating platelet aggregation in disease states, we describe an early stage in the coagulation process that may impact tumour processes, by assessing platelet activation. Briefly, as previously described by our laboratory, we use an index that describes P-selectin expression in relation to the number of platelets expressing the marker [10,36]. However, as noted previously, the lack of P-selectin expression could erroneously be interpreted as inactivation, whereas it could reflect progression to late stage activation [10]. Thus, we couple our analysis with ultrastructural assessment of platelet morphology to better understand the phenomenon of platelet activation in light of hormone-therapy.
    2. Methods
    MCF7 and T47D breast cancer cells, obtained from ATCC (Virginia, USA) were cultured in 75 cm2 Nunc culture flasks to passage numbers of 36 and 29 respectively. MCF7 cells were propagated in DMEM  Thrombosis Research 177 (2019) 51–58
    (Dulbecco's Modified Eagles Medium, Lonza, Walkersville, MD, USA), with 10% FBS (Foetal Bovine Serum, Gibco, Life Technologies, CA, USA), 0.1% P/S (Penicillin/Streptomycin, Sigma-Aldrich, St. Louis, MO, USA). T47D cells were propagated in RPMI (Roswell Park Memorial Institute medium) with 0.2 units/ml bovine insulin, 10% FBS and 0.1% P/S. Cells were cryopreserved in 10% DMSO (Dimethyl sulfoxide, Saarchem, Johannesburg, South Africa), with 60% FBS and 30% DMEM at −80 °C for subsequent experimentation.