[Coagulation activity of circulating membrane microparticles in patients with cardiovascular diseases]. / Koaguliatsionnaia aktivnost' tsirkuliruiushchikh membrannykh mikrochastits u patsientov s serdechno-sosudistymi zabolevaniiami.

Membrane microparticles (MP) are released by activated or damaged cells and are able to accelerate blood clotting (coagulation). MP possess coagulation activity since all of them contain on their surface phosphatidylserine (PS), a substrate for the assembly of coagulation complexes, and some of them tissue factor (TF), the primary initiator of coagulation cascade reactions. We compared the coagulation activity and amount of MP in the blood of healthy donors (n=34) and patients with myocardial infarction (MI) (n=32), advanced atherosclerosis (AA) (n=32) and idiopathic pulmonary arterial hypertension (IPAH) (n=19). Total MP fraction was obtained from blood plasma by sedimentation at 20000 g, 30 min. The coagulation activity of PM isolated from 100 µl of donor and patient plasma was determined using a modified recalcification test. MP were added to substrate plasma devoid of endogenous MF, plasma was recalcified, and clotting was recorded by changes in optical density (A450), determining lag phase (min) and maximum rate (Vmax, %A450/min). MP were counted by flow cytometry as PS+ particles (lactadgerin-FITC staining) smaller than 1 µm and their concentration was expressed as 105 MP/µl plasma. MP in all patient groups accelerated plasma clotting more effectively than donor MP. Lag phase compared with donors (11.8 [11.0-13.1] median and interquartile range) was shorter in patients with AA (8.8 [7.0-10.3], p.

Use of Thrombodynamics for revealing the participation of platelet, erythrocyte, endothelial, and monocyte microparticles in coagulation activation and propagation.

BACKGROUND AND OBJECTIVE: For many pathological states, microparticles are supposed to be one of the causes of hypercoagulation. Although there are some indirect data about microparticles participation in coagulation activation and propagation, the integral hemostasis test Thrombodynamics allows to measure micropaticles participation in these two coagulation phases directly. Demonstrates microparticles participation in coagulation activation by influence on the appearance of coagulation centres in the plasma volume and the rate of clot growth from the surface with immobilized tissue factor.Methods: Microparticles were obtained from platelets and erythrocytes by stimulation with thrombin receptor-activating peptide (SFLLRN) and calcium ionophore (A23187), respectively, from monocytes, endothelial HUVEC culture and monocytic THP cell culture by stimulation with lipopolysaccharides. Microparticles were counted by flow cytometry and titrated in microparticle-depleted normal plasma in the Thrombodynamics test. RESULTS: Monocyte microparticles induced the appearance of clotting centres through the TF pathway at concentrations approximately 100-fold lower than platelet and erythrocyte microparticles, which activated plasma by the contact pathway. For endothelial microparticles, both activation pathways were essential, and their activity was intermediate. Monocyte microparticles induced plasma clotting by the appearance of hundreds of clots with an extremely slow growth rate, while erythrocyte microparticles induced the appearance of a few clots with a growth rate similar to that from surface covered with high-density tissue factor. Patterns of clotting induced by platelet and endothelial microparticles were intermediate. Platelet, erythrocyte and endothelial microparticles impacts on the rate of clot growth from the surface with tissue factor did not differ significantly within the 0-200·103/ul range of microparticles concentrations. However, at concentrations greater than 500·103/ul, erythrocyte microparticles increased the stationary clot growth rate to significantly higher levels than do platelet microparticles or artificial phospholipid vesicles consisting of phosphatidylcholine and phosphatidylserine. CONCLUSION: Microparticles of different origins demonstrated qualitatively different characteristics related to coagulation activation and propagation.

[Coagulation properties of erythrocyte derived membrane microparticles]. / Koaguliatsionnye svoistva membrannykh mikrochastits éritrotsitov.

Membrane microparticles (MP) produced upon cell activation and/or damage possess coagulation activity, i.e. ability to accelerate blood clotting. They contain on their surface phosphatidylserine (PS), a substrate for assembling coagulation enzymatic complexes, and some of them tissue factor (TF), the initiator of clotting cascade reactions. In this study coagulation properties of MP derived from erythrocytes have been investigated. These MP were obtained from donor's erythrocytes activated with ionophore A23187 as well as from outdated erythrocyte concentrates for transfusion. MP were counted by flow cytometry. Coagulation activity of MP was examined by modified plasma recalcification assay. Involvement of PS and TF in this reaction was assessed using PS blocker lactadherin and anti-TF antibodies. TF activity in MP was measured by its ability to activate factor X in a chromogenic assay. Size of MP was evaluated by dynamic light scattering. Properties of erythrocyte MP were compared with previously characterized (using the same methodological approaches) MP derived from platelets and monocytic THP-1 cells, lacking and containing TF, respectively. Erythrocyte MP accelerated plasma clotting, but less actively than MP from platelets and MP from THP-1 cells, which demonstrated maximal activity. Lactadherin completely inhibited coagulation activity of all MP. Anti-TF antibodies did not affect clotting parameters in the presence of platelet and erythrocyte MP, but slowed clotting in the presence of MP from THP-1 cells. TF activity was not detected in erythrocyte and platelet MP, unlike MP from THP-1 cells expressing active TF. MP derived from erythrocytes were smaller than MP from platelets and THP-1 cells, with average diameter about 200 nm and 400 nm respectively. Thus, MP from erythrocyte possess less ability to accelerate plasma clotting in comparison with MP from platelet and THP-1 cells. The data obtained suggest that lesser coagulation activity of erythrocyte MP in comparison with MP from THP-1 cells is due to the absence of TF in erythrocyte MP (in contrast to MP from THP-1 cells) and to their smaller size, and in comparison with MP from platelets (which as erythrocyte MP do not express TF) is due to their smaller size only.

Comparison of the size of membrane microparticles of different cellular origin by dynamic light scattering.

Size of membrane microparticles (MPs) from blood plasma and MPs produced in vitro by activated endothelial cells (ECs), monocytes, THP-1 monocytic cells, granulocytes, and platelets was evaluated by dynamic light scattering. MPs were sedimented from the culture media, cell supernatants, and plasma at 20 000 g for 30 min. Average diameters of all types of MPs ranged from 300 to 600 nm. Plasma MPs had the smallest size. Close sizes were registered for MPs from platelets and THP-1 cells. MPs from monocytes were larger, and MPs from granulocytes and ECs were the largest ones. The data obtained indicate that the size of membrane MPs depends on the type of their cell-producers.

Activity of Tissue Factor in Microparticles Produced in vitro by Endothelial Cells, Monocytes, Granulocytes, and Platelets.

Activity of tissue factor (TF) in membrane microparticles (MPs) produced in vitro by endothelial cells (ECs), monocytes, THP-1 monocytic cells, granulocytes, and platelets was investigated. ECs were isolated from human umbilical vein, and monocytes, granulocytes, and platelets - from the blood of healthy donors. ECs, monocytes, and THP-1 cells were activated by bacterial lipopolysaccharide, granulocytes - by lipopolysaccharide or phorbol myristate acetate, and platelets - by SFLLRN, thrombin receptor-activating peptide. MPs were sedimented from the culture medium or supernatant of activated cells at 20,000g for 30 min. Coagulation activity of MPs was analyzed in a modified recalcification assay by assessing their effects on coagulation of donor plasma depleted of endogenous MPs (by centrifuging at 20,000g for 90 min). MPs from all cell types accelerated plasma coagulation. Antibodies blocking TF activity prolonged coagulation lag-phase in the presence of MPs from ECs, monocytes, and THP-1 cells (by 2.7-, 2.0-, and 1.8-fold, respectively), but did not influence coagulation in the presence of MPs from granulocytes and platelets. In accordance with these data, TF activity measured by its ability to activate factor X was found in MPs from ECs, monocytes, and THP-1 cells, but not in MPs from granulocytes and platelets. The data obtained indicate that active TF is present in MPs produced in vitro by ECs, monocytes, and THP-1 cells, but not in MPs derived from granulocytes and platelets.

Enzymatic antioxidant system of endotheliocytes.

It is shown that endothelial cells from human umbilical vein have a reduced activity and gene expression of the "classic" antioxidant enzymes (Cu,Zn-superoxide dismutase, catalase, and Se-containing glutathione peroxidase). At the same time, a high expression level of peroxiredoxin genes was identified in the same endothelial cells, which obviously indicates the predominant involvement of these enzymes in protecting the endothelium from the damaging effect of free radical peroxidation.

Platelet subpopulation bearing leukocyte specific antigen and tissue factor.

Platelets bearing leukocyte antigen CD45 were identified in the blood of patients with myocardial infarction (MI) and healthy donors by flow cytofluorimetry. Part of these platelets contained tissue factor (TF)-primary initiator of blood clotting. The number of CD45+ and CD45+/TF+ platelets in MI patients at the first day was comparable with their level in healthy donors, but was increased at 8-12 days after MI onset. At that time in some patients the amount of CD45+ and CD45+/TF+ platelets reached 5-6 and 2-3% of their total number. It is assumed that CD45+/TF+ platelets could be formed as a result of platelet interaction with leukocytes or leukocyte produced membrane microparticles.

[Effect of malondialdehyde on deformability of human umbilical vein endothelial cells].

In experiments on cultured endothelial cells of human umbilical vein, we found that incubation of these cells during 24 hours with malondiaidehyde (cone. 200 microM) led to over two-fold increase in the cell stiffness. These data suggest that manifold augmentation of malondialdehyde concentration during oxidative stress may be accompanied by a considerable weakening of endothelium-dependent shear stress-induced arterial dilation.

Activation and damage of endothelial cells upon hypoxia/reoxygenation. Effect of extracellular pH.

Disturbances of blood flow upon vascular occlusions and spasms result in hypoxia and acidosis, while its subsequent restoration leads to reoxygenation and pH normalization (re-alkalization) in ischemic sites of the vascular bed. The effect of hypoxia/reoxygenation on activation and stimulation of apoptosis in cultured human endothelial cells was studied. The cells were subjected to hypoxia (2% O2, 5% CO2, 93% N(2)) for 24 h followed by reoxygenation (21% O2, 5% CO2, 74% N(2)) for 5 h. Reoxygenation was carried out at different pH-6.4 (preservation of acidosis after hypoxia), 7.0, and 7.4 (partial and complete re-alkalization, respectively). Hypoxia only slightly (by approximately 30%) increased the cell adhesion molecule ICAM-1 content on the cell surface, whereas reoxygenation more than doubled its expression. The reoxygenation effect depended on the medium acidity, and ICAM-1 increase was more pronounced at pH 7.0 compared to that at pH 6.4 and 7.4. Neither hypoxia nor reoxygenation induced expression of two other cell adhesion molecules, VCAM and E-selectin. Incubation of cells under hypoxic conditions but not reoxygenation stimulated secretion of von Willebrand factor and increased its concentration in the culture medium by more than 4 times. The percentage of cells containing apoptosis marker, activated caspase-3, was increased by approximately 1.5 times upon hypoxia as well as hypoxia/reoxygenation. Maximal values were achieved when reoxygenation was performed at pH 7.0. These data show that hypoxia/reoxygenation stimulate pro-inflammatory activation (ICAM-1 expression) and apoptosis (caspase-3 activation) of endothelial cells, and the extracellular pH influences both processes.

Damage and activation of endothelial cells during in vitro hypoxia.

We studied the effect of hypoxia on activation and stimulation of apoptosis in cultured endothelial cells. The effect of hypoxia was compared to that of apoptosis-inducing agents (tumor necrosis factor and bacterial lipopolysaccharide). Incubation of endothelial cells for 24 h under hypoxic conditions (2% O2, 5% CO2, and 93% N2) increased secretion of von Willebrand factor, but had no effect on the expression of cell adhesion molecule ICAM-1. Tumor necrosis factor and lipopolysaccharide did not stimulate secretion of von Willebrand factor, but significantly increased the expression of ICAM-1. These data attest to significant differences in the mechanisms of endothelium activation under hypoxic conditions and during treatment with tumor necrosis factor or lipopolysaccharide. Hypoxia stimulated apoptosis in endothelial cells, which was seen from the increase in the number of annexin V-binding cells and activation of caspase-3. Similar changes were revealed in the presence of tumor necrosis factor and lipopolysaccharide. Hence, damage to endothelial cells caused by hypoxia and these compounds is mediated by similar mechanisms.

Optimum conditions for culturing of human bone marrow and adipose tissue mesenchymal precursor cells.

We present a technology of culturing of human mesenchymal stem cells under conditions excluding the presence of animal sera or additional growth factors, but preserving high proliferative potential and the capacity of these cells to multilineage differentiation. Human umbilical serum was used as the alternative material. We found that in the presence of human umbilical serum mesenchymal stem cells more effectively proliferate and retain their differentiation capacity. The proposed technology yields 109-1010 morphologically and functionally identical cells.

[Results of clinical trials of a novel glycoprotein IIb-IIIa antagonist framon in high-risk coronary angioplasty].

New glycoprotein (GP) IIb-IIIa antagonist preparation framon (Monafram), is the F(ab')(2) fragment of a monoclonal antibody FRaMon directed against GP IIb-IIIa. This preparation blocks GP IIb-IIIa binding with fibrinogen and inhibits platelet aggregation both in vitro and upon intravenous administration. Safety and ability of framon to prevent thrombotic complications in high risk coronary angioplasty (CA) was evaluated in the present study. FRAMON was injected intravenously into 153 patients just before the start of procedure as a single bolus at the dose of 0.25 mg/kg. Control group was formed of 126 patients who underwent angioplasty without GP IIb-IIIa blockers. After framon administration there were no allergic reactions or major bleedings, deep thrombocytopenia (< 50000/microl) developed in 1 patient (< 1%), and antibodies against framon were detected in less than 5% of patients. Number of unfavorable outcomes (cardiovascular death, myocardial infarction, angina recurrence) within 1 month after CA was 3 times higher in control group than in the group of patients treated with framon (11.4% and 3.3%, respectively, p = 0.018). The effect of framon was most strongly pronounced within the first day after procedure -- administration of the drug reduced number of acute thromboses from 6.5% to 0.7% (p = 0.013). Significant differences between numbers of end points was still preserved at 6 months after procedure (25.7 and 14.2% in control and framon groups, respectively, p = 0.023). The data obtained proved safety and clinical efficacy of framon administration in coronary angioplasty with high risk of thrombotic complications.

[Characteristics of immune system in patients with metabolic syndrome]. / Osobennosti sostoianiia immunnoi sistemy u bol'nykh s metabolicheskim sindromom.

AIM: To characterize features of immune status in patients with metabolic syndrome (MS). MATERIAL AND METHODS: 41 MS patients entered the study (mean age 55.9 +/- 9.7 years). Blood free triiodthyronine (T3f), thyroxine (T4f), TTH, antibodies to thyroglobulin (abTG), thyroid peroxidase (AbTP) were studied with enzyme immunoassay; levels of CD3+, CD4+, CD8+, CD16+, CD72+ were studied with monoclonal antibodies; IgG, IgA, IgM were measured by radial immunodiffusion in gel by Manchini. The size and the structure of the thyroid were investigated with ultrasound. RESULTS: Thyroid pathology was in 48.8% patients with MS, chronic infectious diseases (CID)--in 51.2%. MS patients free of thyroid pathology and CID had elevated blood levels of IgG, IgA, dysimmunoglobulinemia and low relative number of CD3+ lymphocytes, close correlations of T3free with the levels of CD3+, CD4+, CD8+, T4free with CD72+ (r = -0.97 to +0.92), T4free with IgA, IgG (r = -0.96, r = -0.90), TTH and IgA (r = -0.89), weak negative correlations of uric acid with the levels of CD3+, CD4+, CD72+ and positive with CD8+, CD16+, immunoglobulins. Combination of MS with thyroid pathology and/or CID was characterized with aggravated defects in T- and B-cell immunity, fall in IgG, IgA and changed direction of correlations of thyroid and immune statuses. CONCLUSION: Immune status in MS patients was characterized by stimulation of humoral immunity, dysimmunoglobulinemia, T-cell immunity deficiency. This may be related to chronic hyperinsulinemia, dyslipidemia, antigenic stimulation with modified lipoproteins. Thyroid hormones levels positively correlate with concentrations of immunocytes and negatively--with immunoglobulins.

[Effect of the mitogen-activated kinase inhibitors on monocyte protein-1-stimulated chemotaxis of cells]. / Deistvie ingibitorov mitogen-aktiviruemykh kinaz na khemotaksis kletok, stimulirovannyi monotsitarnym belkom-1.

Monocyte chemoattractant protein (MCP-1) is a major chemoattractant for monocytes and T-lymphocytes although it can cause migration of the HUVECs. We used monocytic cell line THP-1, monocytes of human peripheral blood, and HUVECs to study MCP-1 receptor-mediated cell migration. We showed that THP-1 and the monocytes chemotaxis was decreased in presence of specific inhibitors of p 38 MAP-kinase. Furthermore, it was almost completely diminished by inhibitor of tyrosine kinases. In contrast, MCP-1-stimulated migration of HUVECs was abrogated by specific inhibitor of ERK1/2 MAP-kinases and, to a lesser extent, by blocking tyrosine kinases. These results suggest that intracellular signal pathways activated by MCP-1 in monocytes and HUVECs, are distinct.

Safety, inhibition of platelet aggregation and pharmacokinetics of Fab'2 fragments of the anti-glycoprotein IIb-IIIa monoclonal antibody FRaMon in high-risk coronary angioplasty.

The purpose of the study was to evaluate safety, effects on platelet aggregation and pharmacokinetics of F(ab')(2) fragments of anti-glycoprotein (GP) IIb-IIIa murine monoclonal antibody FRaMon (F(ab')(2) FRaMon) upon its intravenous administration in patients undergoing high-risk coronary angioplasty. Patients were treated before angioplasty with F(ab')(2) FRaMon at 0.2 mg/kg (n = 17) and 0.25 mg/kg (n = 12) bolus or with abciximab at 0.25 mg/kg bolus + 12 h infusion at 0.125 microg/kg per min (n = 29). F(ab')(2) FRaMon at both doses decreased platelet aggregation induced by 20 microM ADP to <10, <20, <40 and <70% of the predrug level at 1, 12, 24 and 72 h after injection, respectively. No significant differences were observed between F(ab')(2) FRaMon and abciximab antiaggregatory effects. In none of the patients did F(ab')(2) FRaMon cause allergic reactions, major bleedings or deep thrombocytopenia. Antibodies against F(ab')(2) FRaMon were detected in one patient. Free F(ab')(2) FRaMon was cleared from plasma within 12 h, while platelet-bound preparation occupied >95, 70-80 and 40-50% of GP IIb-IIIa at 1 and 12-24 h and 3 days after injection, respectively. Thrombotic complications within the first month after angioplasty in groups treated with F(ab')(2) FRaMon and abciximab were observed in one and two patients, respectively. The data obtained have shown that F(ab')(2) FRaMon at bolus administration to patients undergoing coronary angioplasty caused no serious side effects and at comparative dosage inhibited platelet aggregation with the same efficacy as abciximab at bolus + infusion administration.

[Pharmacodynamics, safety, and clinical effects of a novel glycoprotein IIb/IIIa antagonist framon during high risk coronary angioplasty]. / Farmakodinamika, bezopasnost' i klinicheskie éffekty novogo antagonista glikoproteidov IIb/IIIa framona pri koronarnoi angioplastike vysokogo riska.

Preparation framon [F(ab')2 fragments of the anti-glycoprotein (GP) IIb/IIIa monoclonal antibody (FRaMon)] blocks fibrinogen binding to GP IIb/IIIa and platelet aggregation. Dynamics of platelet aggregation inhibition, safety, and clinical effects of framon were studied in high-risk coronary angioplasty. Twenty seven patients underwent angioplasty with framon, 29 - with abciximab and 28 - with no GP IIb/IIIa antagonists. Framon at 0.2 mg/kg (n=16) and 0.25 mg/kg (n=11) bolus administration inhibited platelet aggregation induced by 20 mcM ADP by more than 90%, 80%, 60% and 30% in comparison with the predrug level 1, 12, 24 and 72 h after injection, respectively. Almost the same dynamics of aggregation inhibition was observed upon abciximab administration at 0.25 mg/kg bolus + 0.125 mcg/kg/min infusion for 12 h. No signs of individual intolerance and side effects including allergic reactions and bleedings were detected in patients treated with framon. Slight decrease of platelet count (15-20%) was observed on the first day after framon administration. Antibodies against framon were detected in 1 out of 22 tested patients. Free (nonbound to platelets) framon was completely removed from the circulation 12 h after injection. The number of endpoints (death, myocardial infarction and indications for repeat revascularization) within 1 year after angioplasty was approximately the same in the groups with framon and abciximab - 7 of 25 (28%) and 7 of 28 (25%), respectively, and more than 1.5 fold higher in the group without GP IIb/IIIa blockers - 12 of 27 (44,4%).

Studies of bivalent copper ion binding to poly C.

Ultraviolet differential spectra of single-stranded poly C, taken in the presence of Cu2+ ions, are studied at various ionic strengths and temperatures. Coordinational and conformational components of these spectra are obtained. The Cu2+ ion coordination site on the polynucleotide bases is found to be N(3) and possibly O(2). The direction of the poly C absorption band shift due to ion binding and conformational transitions is established. At low ionic strengths of the solution Cu2+ ions cause the helical parts of poly C to melt. At high ones the formation of double-stranded parts was observed in addition to the above effect. The calculated concentration dependences of ion-poly C bases association constants show that binding is cooperative at any ionic strength.

Binding of divalent copper and calcium ions to poly I.

The effect of Cu2+ ions on the ultraviolet differential ( UVD ) spectra of single-stranded poly I was studied and the coordination (delta epsilon b) and conformation (delta epsilon c) components of the spectra calculated. The comparison of delta epsilon b and the UVD spectrum of protonated IMP leads to the conclusion that N(7) of inosine-5'-monophosphate (IMP) is a coordinating site for Ca2+ and Cu2+ ions on the polymer bases. The binding of Ca2+ and Cu2+ ions causes differently directed displacements of the four absorption bands of poly I, which are observed in the wavenumber range (50-34) X 10(3) cm-1. The calculation of concentration dependencies for the association constants (K") of Ca2+ and Cu2+ ions binding to poly I bases shows that the binding is cooperative. The K" values for the poly I + Ca2+ complex are two orders of magnitude lower than those for the poly I + Cu2+ complex. At low ion concentrations, binding to the poly I phosphates predominates and increases the degree of the polynucleotide helicity. At higher concentrations the spectra are mainly affected by the ion binding to bases, which results in melting of the helical parts of poly I. At Ca2+ concentrations exceeding 10(-3) M light-scattering aggregates are formed. The degree of monomer order in them is close to that observed in multistranded helices of poly I.