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Effects of HC and IFN-α on PV RBC adhesion
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by thrombo-hemorrhagic complications and a propensity to transform into myelofibrosis and acute leukemia.5
Figure 1. Calreticulin expression is decreased in polycythemia vera (PV) patients under hydroxycarbamide (HC) and interferon-α (IFN) treatments. Quantification of Calr expression normalized by actin from (A) 4 PV patients before (UT) and after (HC) HC treat- ment and (B) 11 control (CT), 19 UT, 11 HC, and 7 IFN patients. Horizontal lines repre- sent medians: 0.9883, 2.023, 0.8760 and 0.8760, respectively.
In this study, we investigated the effects of HC and IFN treatments on the expression of membrane proteins in cir- culating RBCs of PV patients. We show that HC and IFN each have a different impact on the expression and func- tion of erythroid membrane proteins. While both drugs seem to normalize Calr expression, HC (but not IFN) enhances the expression of several proteins, including Lu/BCAM and CD147 adhesion proteins, and further exacerbates RBC adhesion to laminin.
Methods
Patients and blood samples
The study obtained ethical approval from Comité de Protection des Personnes Ile de France VII (PP 14-035); blood samples from 53 PV patients, followed at Saint Louis Hospital, Paris, were obtained with informed consent. Patients are divided in 3 groups: 25 patients (16 men and 9 women; mean age: 63±14 years) were treated with phlebotomy in addition to low-dose aspirin; 20 patients (11 men and 9 women; mean age: 64±16 years) were treated with HC [mean dose 0.8 g/day (0.3-1.5g/day)]; 12 patients (9 men and 3 women; mean age: 50±13 years) were treated with PEG-IFN-α according to international and local guidelines20 [mean dose 115 mg/week (45-180 mg/week)]. The patient groups are small, and not necessarily matched for age or sex, partially due to the treatment guidelines of IFN and HC. Four patients from the first category were treated with HC during the study; blood sam- ples from these 4 patients were collected before and during HC- treatment, at least six months after starting the treatment. All HC- treated patients were compliant to the treatment as they had com- plete hematologic response and increased mean corpuscular vol- ume (MCV). JAK2V617F mutation was observed in all patients. Blood samples from 16 regular healthy donors at the Etablissement Français du Sang were also analyzed in this study (9 women and 7 men; mean age: 40±12 years, range 25-67 years). The 2 control (CT) blood samples used for the proteomic analysis, CT1 and CT2, were chosen to match as well as possible with the PV patient group of the study; they were from 67 and 57 year-old donors, respectively.
Blood samples and ghost preparation
Blood samples were collected on sodium heparin tubes. Buffy coat was removed and RBCs were cryopreserved at Centre National de Référence pour les Groupes Sanguins, Paris. RBC membranes were prepared by hypotonic lysis and washed with 5 mM sodium phosphate, pH 8.0 containing 0.2 mM phenylmethyl- sulfonylfluoride.
Reducing the vascular risk and preventing transformation are the main purpose of PV treatment. According to current guidelines, low-risk PV patients are managed with phle- botomy and aspirin, whereas high-risk patients receive cytoreduction with hydroxycarbamide (HC) or interferon- α (IFN).6 New therapeutic approaches aim at developing small molecules as specific inhibitors of JAK2 mutated forms.7 HC, also known as hydroxyurea, is commonly used to treat PV patients requiring cytoreductive therapy. HC is a cytostatic non-alkylating hydroxylated urea analog that inactivates ribonucleotide reductase, the enzyme that catal- yses the conversion of ribonucleotides to deoxyribonu- cleotides during de novo DNA synthesis,8 leading to the interruption of DNA synthesis and cell death at the S- phase.9 HC does not target the malignant clone and there- fore appears unable to eradicate it. The efficacy of HC in preventing thrombosis was suggested in several random- ized clinical trials,10 but is still not proven. HC alone or in combination with other therapies may increase the risk of acute leukemia in MPN.11
Interferon-α is a non-leukemogenic drug that, in some cases, induces cytogenetic remissions or reversion from monoclonal to polyclonal patterns of hematopoiesis, and may specifically target the malignant clone.12 Its wide- spread use was hampered by its parenteral administration, cost and toxicity. The development of pegylated forms (PEG-IFN-α) increased tolerance and efficacy in IFN-treat- ed patients.13
Increased thrombotic risk in PV patients is associated with high levels of hemoglobin, impaired rheology, and increased viscosity resulting from erythrocytosis.14-16 Targeting a hematocrit below 45% seems to decrease the thrombotic risk of these patients.10 An additional parame- ter that might contribute to this risk was brought to light by our work showing abnormal activation of adhesion proteins in PV red blood cells (RBC).17,18 More recently, using a proteomic approach, we also found that mem- branes of PV RBCs had abnormal expression of several proteins including the adhesion protein Lu/BCAM (Lutheran/Basal Cell Adhesion Molecule) and proteins from the endoplasmic reticulum, such as calreticulin (Calr) and calnexin.19 These findings indicate that JAK2V617F not only impacts cell proliferation, but also induces changes in the repertoire of erythroid proteins expressed in circulat- ing RBCs, which might contribute to the circulatory com- plications described in PV.
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