Bortezomib (PS-341, or Velcade), reversible inhibitor of 20S proteasome approved for the treatment of multiple myeloma and mantle cell lymphoma, exhibited a cytotoxic effect toward other malignancies including leukaemia. In this study, we have documented that incubation of both HL-60 and K562 leukaemia cells with nanomolar concentrations of bortezomib is associated with the death of HL-60 cells observed within 24 hours of incubation with bortezomib and the death of K562 cells that were observed after 72 hours of incubation with bortezomib. The relative resistance of K562 cells to bortezomib correlated well with significantly higher expression of HSP27, HSP70, HSP90α, HSP90β and GRP75 in these cells. Incubation of both HL-60 and K562 cells with bortezomib induced a cleavage of HSP90β as well as expression of HSP70 and HSP90β but bortezomib did not affect levels of HSP27, HSP90α, GRP75 and GRP78. The death of both types of cells was accompanied with proteolytic activation of caspase 3 that was observed in HL-60 cells and proteolytic degradation of procaspase 3 in K562 cells. Our study has also pointed to essential role of caspase 8 in bortezomib-induced cleavage of HSP90β in both HL-60 and K562 cells. Finally, we have shown that bortezomib induced activation of caspase 9/caspase 3 axis in HL-60 cells, while the mechanism of death of K562 cells remains unknown.

Abstract Background: Rotational thrombelastometry (ROTEM) is a real-time clotting test that provides insight into clotting factors, the fibrinolytic system and platelet function. We obtained the longitudinal values on ROTEM in normal pregnancy and in puerperium. Material and Methods: After ethics committee approval and subject informed consent, citrated blood was sampled from healthy pregnants four times during pregnancy and one time postpartum. As controls we used nonpregnant women undergoing voluntary blood donation. Extem and Intem tests and basic coagulation test were carried out. Results: We included 112 women in our study, 55 non-pregnant women (controls) and 57 healthy pregnants with 5 samplings. The values of maximum clot firmness (MCF - in EXTEM and INTEM) were significantly higher up to 34th-36th week of pregnancy than those in non-pregnant subjects. MCF in 6th-7th week after delivery was significantly higher in both tests. Clotting time (CT) in pregnant women was significantly shorter (EXTEM) compared to non-pregnant subjects. We also found a very strong correlation between MCF and platelet count in all gestational weeks.' Conclusions: Rotation thromboelastometry clearly demonstrates the hypercoagulability in pregnancy and can reflect the higher risk of venous thromboembolism in both pregnancy and puerperium. Strong correlation between MCF and platelet count can suggest role of platelets in hypercoagulability in pregnant women. This study provides a better knowledge about physiological changes in ROTEM measurement during pregnancy and postpartum.

The mechanisms of fibrinolysis have been suggested to be linked to the pathogenesis of peripheral artery disease. The impact of therapeutic angiogenesis on the parameters of fibrinolysis was studied in critical limb ischemia (CLI). CLI patients (N = 29) and blood donors as controls (N = 29) were enrolled. Bone marrow (600 +/- 50 ml) was centrifuged (3200g, 20 min, 22 degrees C), bone marrow-derived mononuclear cells (100-120 ml) were separated by Optipress I and implanted into the ischemic limb using intramuscular injections. ELISA was employed for the assessment of plasma tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) levels. Patients were followed-up prior to the procedure and after 1, 3 and 6 months. All stage-IV patients (N = 22) had ischemic lesions. The lesions resolved in 10 patients. Five patients underwent major amputation; they all were stage-IV. Ischemic lesions persisted in seven patients beyond 6 months. The t-PA levels were higher in patients compared with the healthy controls both at baseline (P < 0.01) and after 6 months (P < 0.05). No significant changes were observed in the t-PA levels during the follow-up. PAI-1 was higher in patients than in the healthy individuals at baseline (P < 0.001) and at month 1 (P < 0.05). However, no difference in PAI-1 levels between the patients and the healthy individuals was found after 3 and 6 months. The PAI-1 levels were significantly downregulated during the follow-up compared with the baseline (P < 0.0001). Therapeutic angiogenesis for the CLI downregulates PAI-1 levels, thus having a systemic effect on fibrinolysis.

The term thrombelastography / thrombelastometry was used to describe the trace produced from measurement of the viscoelastic changes associated with fibrin polymerization. The result of measurement is a compact mapping of the various stages of haemostasis. One of the first real clinical applications of this method was the haemostatic monitoring of liver transplantation and cardiac surgery using extracorporeal circulation. In trauma patients the thrombelastography /thrombelastometry was proved to predict early transfusion requirements. Another authors suggest thrombelastography /thrombelastometry as a possible tool for early identification of pregnant women at increased risk of fetal loss. This article provides overview on the development of thrombelastography / trombelastometry and its possible use in laboratory of haemostasis.

Introduction:Sticky platelet syndrome (SPS) is most likely a hereditary thrombophilia characterized by platelet hyperaggregation after low concentrations of platelet inducersadenosine diphosphate and/or epinephrine. We present 9 kindreds with SPS familial occurrence.Material and Methods:Familial trait of SPS was looked up in the database of the National Center of Hemostasis and Thrombosis. Families with at least 3 SPS-positive members were studied, described, and presented.Results:In the group of 1093 symptomatic patients, SPS was confirmed in 240 cases. Familial occurrence with at least 3 SPS-positive relatives was found in 9 cases.Conclusion:The exact pathogenesis of SPS is not sufficiently explained. Our findings seem to support the idea that SPS might have an autosomal dominant hereditary fashion.

Platelets are required for the recruitment of bone marrow-derived mononuclear cells (BMMNC) into ischemia-induced vasculature, which underlines their key role in angiogenesis. The difference in platelet immunophenotype between healthy controls and patients with critical limb ischemia (CLI) treated with therapeutic angiogenesis (TA) using BMMNC was assessed. The impact of TA on the expression of platelet membrane markers was studied as well. CLI patients (N = 26) and blood donors as controls (N = 21) were enrolled. Bone marrow (600 +/- 50 ml) was centrifuged (3200g, 20 min, 22 degrees C). BMMNC (100-120 ml) were separated by Optipress I and implanted to the ischemic limb using deep intramuscular injections. Flow cytometry was employed for the peripheral blood platelets immunophenotyping. CD41FITC, CD62PE, CD36FITC, CD29FITC antibodies were used. Patients were followed up prior to the procedure and at months 1, 3 and 6. The expression of CD41 was lower in CLI patients than in the controls. P-selectin (CD62P) was higher in CLI patients than in controls at the baseline and at month 6. It was significantly down-regulated at month 3, however not at months 1 and 6 compared to baseline. Platelet GPIV (CD36) was higher at the baseline, but not during the follow-up compared to the controls. beta 1-integrin (CD29) progressively decreased during the follow-up as compared to the baseline value. Platelets in CLI express P-selectin, GPIV and beta 1-integrin more abundantly than platelets of healthy subjects. TA down-regulates the expression of the respective markers. Possible mechanism could be higher clearance of the activated platelets in the ischemic tissues during angiogenesis.

Pregnancy is associated with a tendency to hypercoagulation which minimises the risk of peripartum haemorrhage. Global hemostasis assays, as thromboelastometry (TEM), may be useful for a detection of excessive blood clotting in pregnancy.

Platelet hyperaggregability after low concentrations of platelet agonists adenosinediphoshate (ADP) and/or epinephrine (EPI) referred to as sticky platelet syndrome (SPS) is the second most common thrombophilic state. Clinical symptoms include unexplained arterial and venous thrombotic events, frequently recurrent under oral anticoagulant therapy. The aim was to determine if there is any difference in platelet CD60P (P-selectin) expression between SPS patients and controls.

Aim was to determine the differences in platelet immune-phenotype between patients with critical limb ischemia (CLI) treated with therapeutic angiogenesis (TA) using bone marrow-derived mononuclear cells (BMMNC) and healthy controls and to find out what is the impact of TA on the expression of platelet membrane markers during the 6-months follow up.

The GP6 gene encodes the GPVI, a crucial platelet membrane glycoprotein, for adequate platelet activation, adhesion and aggregation. The objectives of the present study were to assess the genetic variability of the GP6 gene in patients with platelet hyperaggregability phenotype, known as sticky platelet syndrome (SPS) manifesting as deep vein thrombosis (DVT), and/or pulmonary embolism, and in controls; and to evaluate its role in the pathogenesis of venous thromboembolism (VTE) in SPS. Seventy-seven patients with SPS and 77 healthy blood donors as controls were enrolled. Light transmission aggregometry was used to diagnose SPS according to the method of Mammen and Bick. Seven single-nucleotide polymorphisms (SNPs) of the GP6 gene (rs1654410, rs1671153, rs1654419, rs11669150, rs12610286, rs1654431, rs1613662) were assessed using restriction fragment length polymorphism analysis. A significant association between 1613662-G [P < 0.05, odds ratio (OR) 2.087, confidence interval (CI) 1.049-4.148], 1654419-A (P < 0.05, OR 2.161, CI 1.020-4.577) and VTE was found in patients with SPS. The analysis based on SPS type revealed a significantly higher occurrence of 1671153-G (P < 0.05, OR 2.317, CI 1.103-4.865) and 1654419-A (P < 0.05, OR 2.317, CI 1.103-4.865) in the SPS type II compared to the control group. No association between the studied GP6 genotypes and the severity of VTE (pulmonary embolism vs. DVT) was found. In the patients, significant positive relationship between the 1671153-G, 1654419-A, 1613662-G alleles and male sex was observed. GP6 SNPs 1613662-G, 1671153-G and 1654419-A alleles are associated with an increased risk of VTE in SPS. They could contribute to the SPS phenotype.

SPS is most likely a hereditary, autosomal dominant thrombophilia characterized by platelet hyperaggregation after low concentrations of platelet inducers - adenosine diphosphate (ADP) and/or epinephrine (EPI). It manifests with angina pectoris, acute myocardial infarction (MI), transient cerebral ischemic attacks (TIA), stroke, retinal thrombosis, early pregnancy loss syndrome, peripheral arterial thrombosis, and venous thrombosis, sometimes recurrent under oral anticoagulant therapy.

Venous thromboembolism (VTE) is a disease with high prevalence in patients with multiple myeloma (MM). Among haematologic malignancies, multiple myeloma confers a high risk of developing such comlications, with a VTE rate of nearly 10%.

IntroductionType 2 diabetes (DM2) is associated with complex hemostasis disorders characterised with enhanced coagulation, platelet dysfunction and hypofibrinolysis.These pathological conditions predispose type 2 diabetics towards increased morbidity and mortality due to thrombotic complications.In fact, DM2 is considered the most frequent acquired thrombophilic state.Under physiological conditions, fibrinolytic system is responsible for the lysis of fibrin clot, thus maintaining the blood fluidity.It was first described by Astrup in 1956.The most important molecule in fibrinolytic system is plasmin, which cleaves fibrin clot.It is released in its inactive form called plasminogen from the liver to the blood stream and gets activated by tissue plasminogen activator (t-PA), urokinase plasminogen activator (u-PA), clotting factor XII (FXII) and kallikrein.Plasmin deactivation occurs via several molecules.The best known and most important is 2-plasmin inhibitor ( -2PI).Another one, which was described in early nineteen nineties, is called thrombin-activatable fibrinolysis inhibitor (TAFI).Plasminogen activation and thus plasmin formation is inhibited by plasminogen activator inhibitor type 1 (PAI-1), PAI-2 and PAI-3.Fibrinolysis disorder due to disequilibrium between plasminogen activators and its inhibitors is the most characteristic feature of prothrombotic state associated with diabetes.The fibrinolytic activity in DM2 is influenced by numerous factors, the most crucial ones are glycaemic control, insulinaemia, blood lipid profile, blood pressure, genetic factors and different sorts of medication (Brummel-Ziedins et al., 2009, Alzahrani & Ajjan, 2010a). Fibrinolysis Plasminogen and its activationUnder physiological conditions, fibrinolytic system is responsible for the lysis of fibrin clot, thus maintaining the blood fluidity.The central protein in whole fibrinolytic system is plasmin, a serin protease, which cleaves fibrin clot.Plasminogen, the single-chain proenzyme of plasmin, is a protein of 791 amino acids, m.w.92 000 synthesized primarily in the liver (Raum et al., 1980).The lysine-binding domains of plasminogen mediate its specific interactions with fibrin, cell surface receptors and other proteins, including its circulating inhibitor 2-plasmin inhibitor ( 2-PI), which is also known as 2-antiplasmin (Hajjar et al., 1986;Plow et al., 1991).Activation of plasminogen, by cleavage of a single Arg-Val peptide bond at position 560-561 (Holvoet et al., 1985), gives rise to the active serine protease, plasmin.www.intechopen.com

Disturbances of coagulation and fibrinolysis in type 2 diabetes mellitus (DM2) contribute to increased rates of macrovascular complications such as myocardial infarction and ischemic stroke. The aim of the study was to investigate the relationship among plasminogen activator inhibitor 1 (PAI-1), thrombin-activable fibrinolysis inhibitor (TAFI), tissue plasminogen activator (t-PA), prothrombin fragments 1 + 2 (F1 + 2), glycemic control, hypertension, sex and body mass index (BMI) in DM2 patients with normoalbuminuria and microalbuminuria. Forty-two normoalbuminuric (NAU), 42 microalbuminuric (MAU) patients with DM2 and 42 blood donors as control group were enrolled. TAFI, PAI-1, t-PA and F1 + 2 were assessed by enzyme-linked immunosorbent assay (ELISA) in all patients. TAFI was significantly increased in the MAU group, PAI-1 and F1 + 2 were increased in both groups and t-PA was not elevated in either group compared to controls. We found positive correlations in the NAU: TAFI and fibrinogen (r=0.65, P=0.02), PAI-1 and triglycerides (r=0.67, P=0.01), in the MAU: TAFI and F1 + 2 (r=0.48, P=0.02), TAFI and systolic blood pressure (r=0.53, P=0.01), PAI-1 and BMI (r=0.43, P<0.05). We found decreased fibrinolysis in DM2 patients presented with increased PAI-1 in both NAU and MAU. Hypofibrinolysis in MAU is further accented by the elevation of TAFI. TAFI-mediated inhibition of fibrinolysis in DM2 is regulated independently from PAI-1. Patients sex does not affect diabetes-related changes in hemostasis and fibrinolysis. Blood Coagul Fibrinolysis 22:493-498 (C) 2011 Wolters Kluwer Health vertical bar Lippincott Williams & Wilkins.

BACKGROUNDS:Translational medicine is a medical field encompassing basic research and development of new diagnostic and therapeutic strategies for clinical practice. The present scientific paper focuses on our previous experience in the field of chemoresistance testing in patients with oncological diseases.MATERIAL AND METHODS:Since 2005, we sampled 71 patients with a leukaemia (AML, ALL and CML) and 92 patients with a solid tumour (lung and gastrointestinal tract cancer). Malignant cell in vitro drug resistance testing was carried out using cytotoxic methyl-thiazol tetrazolium (MTT) assay.RESULTS:Based on the LC50 (lethal concentration of a drug killing 50% of cell population), we found that patients with acute myeloblastic leukaemia exhibit a greater degree of resistance than patients with acute lymphoblastic leukaemia. In patients with bronchogenic carcinomas, primary resistance to cisplatin was identified in 28% of tested samples, paclitaxel 36%, vincristine 50%, etoposide 56%, vinorelbine 57%, topotecan 62%, gemcitabine 77% and dacarbazine 86%.CONCLUSION:In vitro tests with gastrointestinal tract cancers also suggested high effectiveness of cisplatin (with the exception of gastric carcinoma) that was comparable with 5-fluorouracil. Even though the MTT assay has some limitations (insufficient number of vital cells, possible contamination by non-malignant cells, etc.), this in vitro method proved very effective in testing malignant cell resistance to clinically used cytostatics.