Maria Mantur

Study of the mechanisms of aldosterone prothrombotic effect in rats.

Introduction: We investigated the role of primary haemostasis, fibrinolysis, nitric oxide (NO) and oxidative stress as well as mineralocorticoid receptors (MR) in acute aldosterone prothrombotic action. Materials and methods: Venous thrombosis was induced by stasis in Wistar rats. Aldosterone (ALDO; 10, 30, 100 µg/kg/h) was infused for 1 h. Eplerenone (EPL; 100 mg/kg, p.o.), a selective MR antagonist, was administered before ALDO infusion. Bleeding time (BT) and platelet adhesion to collagen were evaluated. The expression of nitric oxide synthase (NOS), NADPH oxidase, superoxide dismutase (SOD) and plasminogen activator inhibitor (PAI-1) was measured. NO, malonyl dialdehyde (MDA) and hydrogen peroxide (H2O2) plasma levels were assayed. Results: Significant enhancement of venous thrombosis was observed after ALDO infusion. ALDO shortened BT and increased platelet adhesion. Marked increases were observed in PAI-1, NADPH oxidase and SOD mRNA levels. MDA and H2O2 levels were augmented in ALDO-treated groups, and NOS expression and NO level were decreased. EPL reduced ALDO effects on thrombus formation, primary haemostasis, PAI-1 expression and MDA level. Conclusion: Short-term ALDO infusion enhances experimental venous thrombosis in the mechanism involving primary haemostasis, fibrinolysis, NO and oxidative stress-dependent pathways. The MR antagonist only partially diminished the ALDO effects, suggesting the involvement of additional mechanisms.

Cerebrospinal fluid leakage—Reliable diagnostic methods

Prompt diagnosis and early treatment of cerebrospinal fluid (CSF) leakage minimizes the risk of severe complications. In patients presenting with clear fluid nasal discharge it is important to identify the nature of the rhinorrhea. The CSF leakage may occur as post-traumatic, iatrogenic, spontaneous or idiopathic rhinorrhea. The differential diagnosis of CSF rhinorrhea often presents a challenging problem. The confirmation of CSF rhinorrhea and localization of the leakage may be diagnosed by CT, MRI cisternography and MRI cisternography in combination with single photon emission tomography or radioisotopic imaging. Although these methods allow estimation of the CSF leakage with high accuracy, they are expensive and invasive procedures. Therefore, biochemical methods are still used in the differentiation. Although the most common diagnostic method for screening CSF leakage is glucose oxidase, its diagnostic sensitivity and specificity is generally unsatisfactory. False negative results may occur with bacterial contamination and false positive results are common in diabetic patients. Glucose detection is not recommended as a confirmatory test. As such, other biomarkers of the CSF leakage, such as beta-2-transferrin (beta-2 trf) and beta-trace protein (betaTP) are necessary to identify and confirm of this condition.

Thrombopoiesis in small for gestational age newborns.

Thrombocytopenia in small for gestational age (SGA) newborns may be due to placental vascular pathology, fetal consumptive coagulopathy and platelet destruction, local imbalance of thromboxane A2 causing placental vasoconstriction and platelet aggregation. Thrombopoiesis in SGA newborns is poorly recognized. In 61 SGA newborns we evaluated thrombocytopoiesis in relation to gender and the rate maturity expressed as <5th percentile and <10th percentile. Female newborns demonstrated higher thrombopoietin (TPO) level at 92.06 pg/ml than male newborns at 79.81 pg/ml. Newborns less developed <5th percentile, showed increased TPO level of 92.0 pg/ml in comparison to <10th percentile of 78.0 pg/ml. This observation is more pronounced in female newborns. Contrary to our expectations we did not find any statistically significant differences in the percentage of reticulated platelets (PLRET) and platelets count in relation to gender and <5th percentile or <10th percentile. We can postulate intrauterine hypoxia is responsible for the increase of erythropoietin and impairment of thrombopoiesis in SGA newborns.

The effect of gestational age on platelet surface expression of CD62P in preterm newborns.

Hemostasis in preterm newborns is characterized by low reserve functional capacity with special reference to the presence of such risk factors as asphyxia or infection. Platelets play vitally important role in hemostasis. Expression of CD62P is a marker of stimulated or activated blood platelets. The study involved a group of 16 preterm newborns, five girls and 11 boys. DAKO QIFIKIT was applied to calculate the number of these antigens. The mean CD 62P expression was found to be 23 792 per platelet. Correlation was found between antigen density and gestational age r = 0.954, p = 0.01. Evident deficit of P-selectin on the surface of platelets in preterm newborns may be at least in part responsible for platelet dysfunction, with special reference to interaction between circulating leukocytes and combating infection.

Changes in PDGF concentration in surgically treated colorectal carcinoma.

PURPOSE The aim of the present study was to asses the effect of tumor advancement, differentiation grade and surgery treatment on PDGF- AB level and platelet (PLT) count depending on the site of blood collection. MATERIAL AND METHODS The study included 38 patients submitted to surgical treatment due to diagnosis of colorectal cancer (CRC) without remote metastasis: G2- 20 patients and group G3- 18 patients. The control group consisted of 24 healthy subjects. In CRC patients the blood samples was collected three times: 1) before surgery, 2) intrasurgically and 3) 90 days after surgery. Serum PDGF- AB concentration was determined by ELISA- Kit reagents. RESULTS PDGF concentration in all the patients was several times higher than in the control group, irrespective of tumor differentiation grade and the site of blood collection. However the level of PDGF- AB in intraoperatively collected arterial blood and venous blood in group G3 (arterial blood- 379.9+/-12.1 ng/ml; venous blood- 398.4+/-13.2 ng/ml) was significantly higher than in group G2 (arterial blood- 169.4+/-88.6 ng/ml; venous blood- 194.2+/-84.0 ng/ml). No significant differences were observed between venous and arterial blood. No correlation was found between the PLT count and PDGF- AB concentration. CONCLUSION High blood PDGF- AB concentration in CRC patients but no significant positive correlation observed between the PLT count and PDGF-AB suggest its neoplastic origin beside PLT. Determination of this factor in blood serum may have an important implication in early diagnosis of CRC, which is the second most common malignant neoplasm with high recurrence rates.

The evaluation of angiogenesis and matrix metalloproteinase-2 secretion in bone marrow of multiple myeloma patients before and after the treatment

PURPOSE Angiogenesis appears to be a prominent feature of many hematological disorders, particularly in multiple myeloma (MM). Progression in MM also involves secretion of the metaloproteinases (MMPs). In this study, the expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and its receptor, in bone marrow trephine biopsy (TB) of thirty six MM patients before and after the treatment or during progression was examined. The MMP-2 secretion was assessed from the same patients. MATERIAL/METHODS Immunohistochemical staining of bone marrow specimens for angiogenic factors and microvessel density (MVD) and bone marrow aspirates for Western blot analysis of MMP-2 expression was performed. RESULTS In active, untreated MM patients, we found statistically significant differences in the expression of angiogenic factors according to the patients after the anti-angiogenic treatment. We found statistical differences of the expression of angiogenic factors between the group of patients with a response after the treatment and the patients who had progression during the treatment. The data showed statistically significant decreased MVD after the treatment. The results showed statistically significant differences between initial secretion of MMP-2 in active, untreated MM patients and patients with a response after the treatment and patients with progression during the treatment. CONCLUSIONS We showed that not only decreased expression of angiogenic cytokines is present after the anti-angiogenic treatment but also activity of MMP-2 in MM patients who responded to the treatment. Combination therapy with the inhibition of the activity of MMPs could represent an interesting therapeutical approach in MM.

Expression of P-selectin (CD62P) on platelets after thrombin and ADP in hypotrophic and healthy, full-term newborns

Abstract Background: Hypotrophic newborns in comparison with eutrophic newborns demonstrate a reduced blood platelet count and therefore may have haemostasis disorders. Expression of P-selectin (CD62P) on the surface of platelets is a marker of stimulated, activated blood platelets. The ability of platelets to react can be determined after the addition of the following activators: strong (thrombin) and weak (ADP). Materials and methods: We studied 48 hypotrophic newborns, 25 females and 23 males, weighing less than the 10th centile and 55 healthy, full-term newborns, 25 females and 30 males. Expression of CD62P on the surface of platelets was examined in the native state, after the addition of thrombin or ADP. Results: Hypotrophic newborns exhibited almost double the percentage of platelets expressing CD62P compared with the control group, 4.21% versus 2.88%. After the addition of thrombin, the percentage was 31.5% versus 12.5%, p < 0.001, whereas after the addition of ADP, the percentage was 9.54% versus 4.5%, p = 0.002. Conclusions: Hypotrophic newborns are capable of greater platelet activation in comparison with healthy term newborns. However, gender does not affect the expression of P-selectin.

Platelet-Derived Microparticles and Platelet Count in Preterm Newborns

Objective: Does formation of platelet-derived microparticles correspond to platelet activation? Methods: The study was performed in 51 preterm newborns, 25 girls and 26 boys. The control group consisted of 55 term newborns, 25 girls and 30 boys. Blood samples were collected from the umbilical artery. The percentage of platelet-derived microparticles and platelet count were determined using flow cytometric analysis based on the CD61-positive antigen. Results: The percentage of platelet-derived microparticles was higher in preterm newborns (5.46) in comparison to term newborns (4.22, p < 0.01). We found 4.61% of platelet-derived microparticles in preterm female newborns and 6.28% in preterm boys (p < 0.0070). The platelet count was 256 × 103 µl in girls and 238 × 103 µl in boys. Female healthy term newborns presented higher values of platelet-derived microparticles (4.4%) than male newborns (4.07%, p = 0.4725, table 1). The platelet count in girls was found to be 308 × 103 µl and in boys 270 × 103 µl. Conclusions: Higher percentage of platelet-derived microparticles in preterm newborns may provide a compensatory mechanism for the hemostatic system.

Bone marrow megakaryocytes, soluble P-selectin and thrombopoietic cytokines in multiple myeloma patients

Abstract The expression of adhesion molecules and other cell-surface molecules is substantial in the communication between plasma cells and bone marrow microenvironment, and may lead to increased proliferation of myeloma cells. Many of the cytokines involved in multiple myeloma (MM) pathogenesis, e.g. thrombopoietin (TPO) and interleukin-6 (IL-6), play a pivotal role in different developmental stages of megakaryocytopoiesis and thrombopoiesis. The principal aim of our study was to explore the relationship between thrombopoietic cytokines, megakaryocytes (MKs) and soluble P-selectin (sP-selectin) levels in MM patients before and after anti-angiogenic treatment. Forty-four patients (20 female and 24 male) with a newly diagnosed MM were examined in three groups, following a division based on the International Staging System, ISS. Plasma levels of TPO, IL-6 and soluble P-selectin (human sP-selectin) were measured by means of ELISA. Bone marrow specimens were studied to determine the number of MKs and the so-called “naked nuclei” (NN), as well as the expression of platelet-derived growth factor (PDGF). The comparison revealed a significantly higher concentration of cytokines and sP-selectin in newly diagnosed MM patients compared to healthy volunteers: for TPO, p = 0.01, IL-6, p = 0.0005 and sP-selectin, p = 0.00008, respectively. Marked differences were observed in the concentration of sP-selectin, expression of PDGF and MKs counts between patients with MM stage I and MM stage III. Statistically meaningful correspondences were also found between MKs versus TPO, NN versus TPO, as well as MKs versus MPV, p = 0.009, p = 0.004 and p = 0.0005, respectively. Furthermore, the analysis exhibited some statistically meaningful divergences between initial concentrations of sP-selectin in subgroups with different response after chemotherapy. The initial concentration of sP-selectin in the group of MM patients with complete or partial remission stood at 31.86 ± 6.13 ng/ml. In the remaining patients (stable disease), the concentration of sP-selectin amounted to 35.15 ± 7.23 ng/ml (p = 0.048). We found a correlation between sP-selectin and IL-6 (ρ = 0.57, p = 0.0004), TPO and IL-6 (ρ = 0.46, p = 0.001) as well as sP-selectin and TPO (ρ = 0.36, p = 0.043), and sP-selectin and PDGF (ρ = 0.36, p = 0.03). Our study has eventually demonstrated that sP-selectin, as a marker of platelet activation, could be a useful marker of maximum response to therapy. Its strong association with another marker like PDGF-AB could further lead to the development of new combinational therapeutic strategies of anti-angiogenic therapy in MM patients.

Does thrombopoiesis in multiple myeloma patients depend on the stage of the disease

PURPOSE Infiltration of the bone marrow by neoplastic plasmocytes in multiple myeloma (MM) patients might impair megakaryocytopoiesis. The aim of the study was to evaluate stage-dependent platelet count (PLT) and thrombopoietin (TPO) concentration in comparison to the control group. We also wanted to establish whether TPO might be recognized as a marker of the stage of the disease. MATERIAL/METHODS The study group consisted of 41 patients (mean age 67.7) with newly diagnosed MM prior to treatment and categorized according to the Durie and Salmon diagnostic classification. The control group consisted of 30 healthy subjects (mean age 65.5). PLT, WBC, RBC and Hb were measured with the use of the haematological analyser. TPO was assayed with the use of ELISA and albumin with the use of the immunonephelometry method. The number of plasma cells in the bone marrow was evaluated in bone marrow smears under light microscopy. RESULTS PLT was not statistically different as compared the control groups, but was stage-dependent. Thrombocytopenia was observed in the III stage of MM. TPO median was significantly higher in study group than in healthy subjects and it was increasing considerably with the stage of the disease. TPO concentration was negatively correlated with albumin and PLT. AUC for TPO was 0.9764. The number of plasma cells in the bone marrow was considerably increasing with the stage of the disease. CONCLUSIONS PLT and TPO in MM patients were stage-dependent. Elevated TPO concentration in MM patients might be an unfavourable marker of the stage of the disease.