Ex vivo platelet morphology assessment of chronic myeloid leukemia patients before and after Imatinib treatment.

Chronic myeloid leukemia (CML) is a myeloproliferative disease and the first line treatment is through the administration of Imatinib, a first generation tyrosine kinase inhibitor. Thrombocytosis and bleeding irregularities are common in CML, however, the morphological variations in CML patients' platelets are not well documented. In this study, ex vivo platelet morphology of control participants, as well as CML patients were assessed before and after Imatinib treatment. The topographical and structural morphology of platelets were determined via scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Qualitative data of SEM and TEM revealed that CML patient's platelets were prone to aggregation and coagulation at time of diagnosis; the samples that were not aggregated at time of diagnosis showed typical discoid shaped platelets, which was comparable to control participants' platelets. TEM results of CML patients' platelets at diagnosis showed that internal granular constituents including dense bodies were decreased in comparison to control participants. In all CML patients, platelets appeared activated after 6 months of treatment with Imatinib with membrane structure abnormalities and constituent variations. Research to date has primarily focused on the effects of CML on leukocyte populations, however, the results of the current study implicate the impact of CML pathogenesis on platelets, seemingly as a result of alterations in normal hematopoiesis. In addition, the impact of Imatinib treatment on platelet morphology was also established, indicating an increase in platelet activation. Recognizing and understanding the impact of CML disease progression on platelets is of importance to aid improved patient treatment. RESEARCH HIGHLIGHTS: In the study, results from SEM and TEM indicated that CML patient's platelets were prone to aggregation at time of diagnosis, and activation after Imatnib treatment. Platelet samples that did not aggregate had decreased internal granular constituents.

Platelet Function, Role in Thrombosis, Inflammation, and Consequences in Chronic Myeloproliferative Disorders.

Platelets are conventionally defined as playing a vital role in homeostasis and thrombosis. This role has over the years transformed as knowledge regarding platelets has expanded to include inflammation, cancer progression, and metastasis. Upon platelet activation and subsequent aggregation, platelets release a host of various factors, including numerous pro-inflammatory factors. These pro-inflammatory factors are recruiters and activators of leukocytes, aiding in platelets' immune regulating function and inflammatory function. These various platelet functions are interrelated; activation of the inflammatory function results in thrombosis and, moreover, in various disease conditions, can result in worsened or chronic pathogenesis, including cancer. The role and contribution of platelets in a multitude of pathophysiological events during hemostasis, thrombosis, inflammation, cancer progression, and metastasis is an important focus for ongoing research. Platelet activation as discussed here is present in all platelet functionalities and can result in a multitude of factors and signaling pathways being activated. The cross-talk between inflammation, cancer, and platelets is therefore an ideal target for research and treatment strategies through antiplatelet therapy. Despite the knowledge implicating platelets in these mentioned processes, there is, nevertheless, limited literature available on the involvement and impact of platelets in many diseases, including myeloproliferative neoplasms. The extensive role platelets play in the processes discussed here is irrefutable, yet we do not fully understand the complete interrelation and extent of these processes.

Apoptotic profiling of chronic myeloid leukaemia patients' platelets ex vivo before and after treatment with Imatinib.

Chronic myeloid leukaemia (CML) is a malignancy of the haematopoietic stem cells. The first line of treatment for CML, especially in developing countries, remains the first-generation tyrosine kinase inhibitor, Imatinib. Patients with CML are frequently diagnosed with platelet abnormalities. However, the specific mechanism of platelet abnormalities in CML remains unclear and poorly understood. The aim of this study was therefore to determine the apoptotic profiles of CML patients ex vivo on platelets before and after treatment with Imatinib. Blood samples of healthy volunteers and CML patients at diagnosis and after 6 months treatment with Imatinib were collected. Platelet counts, viability and activation were determined. Results showed that CML patients' platelet counts were elevated upon diagnosis and these levels statistically significantly decreased after 6 months of treatment. Platelet activation was significantly increased after 6 months of treatment compared to levels at diagnosis (P-value < .05). Similarly, platelet apoptosis was also increased after 6 months of treatment. Abnormalities in platelet functioning found in this study may partly be due to clonal proliferation of haematopoietic cells in CML patients, specifically of megakaryocyte precursors as well as the inhibition of platelet tyrosine kinase's and the inhibition of platelet-derived growth factor.

Critical evaluation of equations for serum osmolality: Proposals for effective clinical utility.

BACKGROUND: Many studies have assessed the predictive accuracy of serum osmolality equations. Different approaches for selecting a usable equation were compared using thirty published equations and patient data from a regional hospital laboratory. METHODS: Laboratory records were extracted with same-sample results for measured serum osmolality, sodium, potassium, urea and glucose analysed in a regional hospital laboratory between 1/1/2017-31/12/2018. Differences were analysed using Passing-Bablok and difference (Bland-Altman) analysis. Three approaches were compared: the shotgun approach, adjusting for bias, and deriving a novel equation using multivariate analysis. The criteria for success included bias ≤0.7%, a 230 - 400 mOsm/kg range, and osmolal gap (OG) 95% reference limits within ±10 mOsm/kg. RESULTS: The majority of equations produced proportionally negative-biased results. The shotgun approach identified two equations (EQ19, EQ6) with bias ≤0.7% but unworkable OG reference limits. The bias adjustment approach produced several equations with bias ≤ 0.7% and OG reference limits within or equivalent to ±10 mOsm/kg. A novel equation generated by us (1.89Na+ + 1.71 K+ + 1.08 Urea + 1.08 Glucose + 13.7) improved with the adjustment of bias and was not superior to the adjusted published equations. CONCLUSION: Few published equations are immediately usable. Adjustment of bias derives several usable equations of which the best had OG ranges <20 mOsm/kg. We conclude that adjustment of bias can generate equations of equal or superior performance to that of novel equations.

Eryptosis: An Erythrocyte's Suicidal Type of Cell Death.

Erythrocytes play an important role in oxygen and carbon dioxide transport. Although erythrocytes possess no nucleus or mitochondria, they fulfil several metabolic activities namely, the Embden-Meyerhof pathway, as well as the hexose monophosphate shunt. Metabolic processes within the erythrocyte contribute to the morphology/shape of the cell and important constituents are being kept in an active, reduced form. Erythrocytes undergo a form of suicidal cell death called eryptosis. Eryptosis results from a wide variety of contributors including hyperosmolarity, oxidative stress, and exposure to xenobiotics. Eryptosis occurs before the erythrocyte has had a chance to be naturally removed from the circulation after its 120-day lifespan and is characterised by the presence of membrane blebbing, cell shrinkage, and phosphatidylserine exposure that correspond to nucleated cell apoptotic characteristics. After eryptosis is triggered there is an increase in cytosolic calcium (Ca2+) ion levels. This increase causes activation of Ca2+-sensitive potassium (K+) channels which leads to a decrease in intracellular potassium chloride (KCl) and shrinkage of the erythrocyte. Ceramide, produced by sphingomyelinase from the cell membrane's sphingomyelin, contributes to the occurrence of eryptosis. Eryptosis ensures healthy erythrocyte quantity in circulation whereas excessive eryptosis may set an environment for the clinical presence of pathophysiological conditions including anaemia.

An overview of the role of platelets in angiogenesis, apoptosis and autophagy in chronic myeloid leukaemia.

Amongst males, leukaemia is the most common cause of cancer-related death in individuals younger than 40 years of age whereas in female children and adolescents, leukaemia is the most common cause of cancer-related death. Chronic myeloid leukaemia (CML) is a chronic leukaemia of the haematopoietic stem cells affecting mostly adults. The disease results from a translocation of the Philadelphia chromosome in stem cells of the bone marrow. CML patients usually present with mild to moderate anaemia and with decreased, normal, or increased platelet counts. CML represents 0.5% of all new cancer cases in the United States (2016). In 2016, an estimated 1070 people would die of this disease in the United States. Platelets serve as a means for tumours to increase growth and to provide physical- and mechanical support to elude the immune system and to metastasize. Currently there is no literature available on the role that platelets play in CML progression, despite literature reporting the fact that platelet count and size are affected. Resistance to CML treatment with tyrosine kinase inhibitors can be as a result of acquired resistance ensuing from mutations in the tyrosine kinase domains, loss of response or poor tolerance. In CML this resistance has recently become linked to bone marrow (BM) angiogenesis which aids in the growth and survival of leukaemia cells. The discovery of the lungs as a site of haematopoietic progenitors, suggests that CML resistance is not localized to the bone marrow and that the mutations leading to the disease and resistance to treatment may also occur in the haematopoietic progenitors in the lungs. In conclusion, platelets are significantly affected during CML progression and treatment. Investigation into the role that platelets play in CML progression is vital including how treatment affects the cell death mechanisms of platelets.

Ex vivo Determination of an Estradiol Analogue-Induced Changes on Platelet Morphology and Angiogenic Biomarkers.

Angiogenesis is a closely controlled biological process that takes place during fetal development of blood vessels and wound healing, and includes the development of new blood vessels from preexisting blood vessels. Tumor angiogenesis is a means by which tumors obtain oxygen, nutrition and promote tumor growth. Angiogenesis-regulating proteins are therefore ideal biomarkers in the study of tumor pathophysiology. In our laboratory, a new in silico-designed analogue of 2-methoxyestradiol has been synthesized with angiogenic properties, namely 2-ethyl-3-O-sulfamoyl-estra-1,3,5(10)16-tetraene (ESE-16). The ex vivo influence of ESE-16 on angiogenesis and morphology in platelets of healthy participants was investigated. Scanning electron microscopy revealed no morphological changes in ESE-16-treated platelets. The possible antiangiogenic effect of ESE-16-exposed platelets was determined by means of flow cytometry measurement of angiogenic protein levels, which were significantly increased after platelets were added to tumorigenic breast epithelial cells. This indicates that binding of platelets to cancer cells causes differential release of platelet constituents. Vascular endothelial growth factor levels were decreased in platelets, whereas platelet-derived growth factor and matrix metallopeptidase-9 levels were not significantly affected in platelets. In light of the above-mentioned data, further investigation of ESE-16's influence on morphology and angiogenic markers in platelets of cancer patients is warranted.

Ex vivo apoptotic and autophagic influence of an estradiol analogue on platelets.

BACKGROUND: Platelets are known contributors to the vascularization, metastasis and growth of tumors. Upon their interaction with cancer cells they are activated resulting in degranulation and release of constituents. Since the apoptotic- and autophagic effects of 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10)16-tetraene (ESE-16) has been shown to occur in vitro and this compound was designed to bind to carbonic anhydrase II (CAII), the possible occurrence of these cell death mechanisms in platelets as circulatory components, is of importance. METHODS: Scanning electron microscopy was used to assess morphological changes in platelets after exposure to ESE-16. The possible apoptotic- and autophagic effect of ESE-16 in platelets was also determined by means of flow cytometry through measurement of Annexin V-FITC, caspase 3 activity, autophagy related protein 5 levels and light chain 3-I to light chain 3-II conversion. RESULTS: Scanning electron microscopy revealed no changes in ESE-16-treated platelets when compared to vehicle-treated samples. Apoptosis detection by Annexin V-FITC and measurement of caspase 3 activity indicated that there was no increase in apoptosis when platelets were exposed to ESE-16. The incidence of autophagy by measurement of autophagy related protein 5 levels and light chain 3-I to light chain 3-II conversion showed that exposure to ESE-16 did not cause the incidence of autophagy in platelets. CONCLUSION: This is the first ex vivo study reporting on involvement of apoptosis- and autophagy-related targets in platelets after exposure to ESE-16, warranting further investigation in platelets of cancer patients.

Ultrastructural changes of erythrocytes in whole blood after exposure to prospective in silico-designed anticancer agents: a qualitative case study.

BACKGROUND: Novel, in silico-designed anticancer compounds were synthesized in our laboratory namely, 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10),15-tetraen-17-ol (ESE-15-ol) and 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10)16-tetraene (ESE-16). These compounds were designed to have improved bioavailability when compared to their source compound, 2-methoxyestradiol. This theoretically would be due to their increased binding affinity to carbonic anhydrase II, present in erythrocytes. Since the novel compounds under investigation are proposed to be transported within erythrocytes bound to carbonic anhydrase II, the morphological effect which they may exert on whole blood and erythrocytes is of great significance. A secondary outcome included revision of previously reported procedures for the handling of the whole blood sample. The purpose of this study was twofold. Firstly, the ultrastructural morphology of a healthy female's erythrocytes was examined via scanning electron microscopy (SEM) after exposure to the newly in silico-designed compounds. Morphology of erythrocytes following exposure to ESE-15-ol and ESE-16 for 3 minutes and 24 hours at 22°C were described with the use of SEM. The haemolytic activity of the compounds after 24 hours exposure were also determined with the ex vivo haemolysis assay. Secondly, storage conditions of the whole blood sample were investigated by determining morphological changes after a 24 hour storage period at 22°C and 37°C. RESULTS: No significant morphological changes were observed in the erythrocyte morphology after exposure to the novel anticancer compounds. Storage of the whole blood samples at 37°C for 24 hours resulted in visible morphological stress in the erythrocytes. Erythrocytes incubated at 22°C for 24 hours showed no structural deformity or distress. CONCLUSIONS: From this research the optimal temperature for ex vivo exposure of whole blood samples to ESE-15-ol and ESE-16 for 24 hours was determined to be 22°C. Data from this study revealed the potential of these compounds to be applied to ex vivo study techniques, since no damage occurred to erythrocytes ultrastructure under these conditions. As no structural changes were observed in erythrocytes exposed to ESE-15-ol and ESE-16, further ex vivo experiments will be conducted into the potential effects of these compounds on whole blood. Optimal incubation conditions up to 24 hours for whole blood were established as a secondary outcome.

An estrogen analogue and promising anticancer agent refrains from inducing morphological damage and reactive oxygen species generation in erythrocytes, fibrin and platelets: a pilot study.

BACKGROUND: 2-Methoxyestradiol is known to have antitumour and antiproliferative action in vitro and in vivo. However, when 2-methoxyestradiol is orally administered, it is rapidly oxidized by the enzyme 17β-hydroxysteriod dehydrogenase in the gastrointestinal tract. Therefore, 2-methoxyestradiol never reaches high enough concentrations in the tissue to be able to exert these antitumour properties. This resulted in the in silico-design of 2-methoxyestradiol analogues in collaboration with the Bioinformatics and Computational Biology Unit (UP) and subsequent synthesis by iThemba Pharmaceuticals (Pty) Ltd (Modderfontein, Midrand, South Africa). One such a novelty-designed analogue is 2-ethyl-3-O-sulphamoyl-estra-1, 3, 5(10)16-tetraene (ESE-16). METHODS: This pilot study aimed to determine the morphological effect and possible generation of reactive oxygen species by ESE-16 on erythrocytes and platelet samples (with and without added thrombin) by means of scanning electron microscopy, transmission electron microscopy and flow cytometry. RESULTS: Erythrocytes and platelets were exposed to ESE-16 at a concentration of 180nM for 24 hours. Scanning- and transmission electron microscopy indicated that ESE-16 did not cause changes to erythrocytes, platelets or fibrin networks. Flow cytometry measurements of hydrogen peroxide and superoxide indicated that ESE-16 does not cause an increase in the generation of reactive oxygen species in these blood samples. CONCLUSION: Further in vivo research is warranted to determine whether this novel in silico-designed analogue may impact on development of future chemotherapeutic agents and whether it could be considered as an antitumour agent.

Ultrastructural changes of erythrocytes in whole blood after exposure to prospective in silico-designed anticancer agents: a qualitative case study

BACKGROUND: Novel, in silico-designed anticancer compounds were synthesized in our laboratory namely, 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10),15-tetraen-17-ol (ESE-15-ol) and 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10)16-tetraene (ESE-16). These compounds were designed to have improved bioavailability when compared to their source compound, 2-methoxyestradiol. This theoretically would be due to their increased binding affinity to carbonic anhydrase II, present in erythrocytes. Since the novel compounds under investigation are proposed to be transported within erythrocytes bound to carbonic anhydrase II, the morphological effect which they may exert on whole blood and erythrocytes is of great significance. A secondary outcome included revision of previously reported procedures for the handling of the whole blood sample. The purpose of this study was twofold. Firstly, the ultrastructural morphology of a healthy female's erythrocytes was examined via scanning electron microscopy (SEM) after exposure to the newly in silico-designed compounds. Morphology of erythrocytes following exposure to ESE-15-ol and ESE-16 for 3 minutes and 24 hours at 22°C were described with the use of SEM. The haemolytic activity of the compounds after 24 hours exposure were also determined with the ex vivo haemolysis assay. Secondly, storage conditions of the whole blood sample were investigated by determining morphological changes after a 24 hour storage period at 22°C and 37°C. RESULTS: No significant morphological changes were observed in the erythrocyte morphology after exposure to the novel anticancer compounds. Storage of the whole blood samples at 37°C for 24 hours resulted in visible morphological stress in the erythrocytes. Erythrocytes incubated at 22°C for 24 hours showed no structural deformity or distress. CONCLUSIONS: From this research the optimal temperature for ex vivo exposure of whole blood samples to ESE-15-ol and ESE-16 for 24 hours was determined to be 22°C. Data from this study revealed the potential of these compounds to be applied to ex vivo study techniques, since no damage occurred to erythrocytes ultrastructure under these conditions. As no structural changes were observed in erythrocytes exposed to ESE-15-ol and ESE-16, further ex vivo experiments will be conducted into the potential effects of these compounds on whole blood. Optimal incubation conditions up to 24 hours for whole blood were established as a secondary outcome.