Assessment of platelet functional activity in healthy individuals and patients receiving antiplatelet therapy. Possible inconsistencies between aggregation and flow cytometry tests.

Platelet functional activity was assessed in healthy volunteers (HV, n=92), patients with stable angina pectoris (SA, n=42) and acute coronary syndrome (ACS, n=73), treated with acetylsalicylic acid (ASA) + clopidogrel and ASA + ticagrelor, respectively. In all HV and patients we have compared parameters of platelet aggregation (maximum light transmission and velocity, Tmax and Vmax) and parameters, characterizing exposure of platelet activation markers, evaluated by flow cytometry. HV platelets were activated by 10 µM, 1 µM TRAP, and 20 µM, 5 µM, 2.5 µM ADP; patient platelets were activated by 10 µM TRAP and by 20 µM and 5 µM ADP. Strong and significant correlations between the aggregation and flow cytometry parameters (the r correlation coefficient from 0.4 up to >0.6) most frequently were registered in HV platelet during activation by 1 µM TRAP and in SA patients during platelet activation by 20 µM and 5 µM ADP. However, in many other cases these correlations were rather weak (r < 0.3) and sometimes statistically insignificant. In HV the differences in PAC-1 binding parameters between platelets activated by 10 µM TRAP (the strongest agonist) and all ADP concentrations were negligible (≤ 10%), while CD62P binding (at all ADP concentrations) and LTA parameters for (5 µM and 2.5 µM ADP) were significantly lower (by 40-60%). Antiplatelet therapy in patients decreased all parameters as compared to HV, but to varying extents. For 10 µM TRAP the MFI index for PAC-1 binding (40-50% decrease) and for both ADP concentrations the Tmax values (60-85% decrease) appeared to be the most sensitive in comparison with the other parameters that decreased to a lesser extent. The data obtained indicate a possibility of inconsistency between different LTA and flow cytometry parameters in assessing platelet activity and efficacy of antiplatelet drugs.

[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.

[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.

Maternal incompatibilities with fetal human platelet alloantigens -1a, -1b and -15 are the main causes of neonatal alloimmune thrombocytopenia in Russia.

AIM: Mechanisms underlying the development of neonatal alloimmune thrombocytopenia (NAIT) in in Russia have been studied. MATERIALS AND METHODS: Genetic polymorphisms of human platelet alloantigens (HPA) -1, -2, -3, -4, -5, and -15 were evaluated in 27 families having the newborns with NAIT. NAIT was diagnosed according to the following criteria: (1) newborn with thrombocytopenia; (2) mother with no thrombocytopenia and no increase of platelet associated IgG, (3) presence of antibodies reacting with paternal platelets in maternal plasma / serum. HPA genotyping revealed incompatibilities in 23 out of 27 tested families. In these 23 families HPA-1 conflicts were detected in 16 ones (70%). In 8 cases mothers were homozygous carriers of rare HPA-1b allele and in another 8 cases - of HPA-1a allele which cased incompatibilities with fetal HPA-1a and HPA-1b respectively. In 5 out of 23 families (22%) there were incompatibilities with fetal HPA-15 (HPA-15a, n=2 and HPA-15b, n=3), in 1 family - with HPA-5b (4%), and in 1 family - with HPA-3b (4%) alloantigens. CONCLUSION: In conclusion the main causes of NAIT in Russia were HPA-1a and -1b conflicts and HPA-15 conflicts were the second frequent ones.

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.

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.

[Platelet aggregation upon acetylsalicylic acid and clopidogrel treatment and glycoprotein IIb/IIIa content in patients with acute coronary syndrome].

Interaction between aggregating activity of platelets and glycoprotein (GP) IIb/IIIa (fibrinogen receptor) content on their surface was investigated in patients with acute coronary syndrome (ACS). Eighty nine ACS patients were included into the study - 69 with and 20 without elevation of ST segment. Blood was collected within the first hour of admission to the clinic (1 day), and then at 3-5 and 8-12 days. All patients received standard antiaggregant therapy - acetylsalicylic acid - ASA (thromboxane A2 synthesis inhibitor) and clopidogrel (ADP receptor antagonist). Platelet aggregation was analyzed at the first time point when patients had already taken ASA but not clopidogrel, and then (3-5 and 8- 12 days) upon combined therapy with both preparations. Aggregation was induced by 5 and 20 uM ADP and measured by turbidimetric method. In comparison with the initial level (1 day, ASA) at days 3-5, i.e. after development of clopidogrel effect, platelet aggregation was decreased by 54 and 40% upon its stimulation with 5 and 20 uM ADP, and was not further changed at days 8-12. GP IIb/IIIa content on platelet surface was determined by binding of 125I-labelled monoclonal antibody CRC64. GP IIb/IIIa number varied from 31100 to 73000 per platelet with the mean level of 48500 +/- 8400 (mean +/- standard deviation). No differences were detected between mean GP IIb/IIIa number at 1, 3-5 and 8-12 days after ACS onset. Upon repeat GP IIb/IIIa measurement coefficient of variation was 6.1% demonstrating the stability of this parameter in each patient. Positive correlation between platelet aggregation and GP IIb/IIIa content was detected at the first day - correlation coefficients (r) 0.425 and 0.470 for 5 and 20 uM ADP (n=57, p<0.001). However positive association between these parameters was not revealed at 3-5 and 8-12 days, when patients received not only ASA but clopidogrel as well (r from -0.054 to -0.237, p>0.05). These results indicates that variations of GP IIb/IIIa content affect platelet aggregating activity within first hours of ACS upon ASA treatment. However after saturation with clopidogrel this factor has no significant influence on platelet aggregation, at least on aggregation induced by ADP which receptor is the target of this antiaggregant. Under such conditions aggregation parameters are presumably influenced first of all by individual characteristics of clopidogrel pharmacokinetics.

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.

[Von Willebrand factor and soluble P-selectin in patients with non ST-Elevation acute coronary syndrome during treatment with glycoprotein IIb/IIIa antagonist eptifibatide].

Soluble P-selectin (marker of activation of platelets) and von Willebrand factor (marker of activation of endothelium) were measured in 44 patients with non ST-Elevation acute coronary syndrome (NSTEACS) 21 of whom received glycoprotein IIb/IIIa antagonist eptifibatide (two 180 mg/kg boluses with 10 min interval followed by infusion of 2 mcg/kg/min during first 24 hours and 1.3 mcg/kg/min during subsequent 48 hours). Measurements were made within first 2 hours, in 2--3 and 10--14 days after development of NSTEACS. During first 2 hours P-selectin content was 1.6 times higher than in healthy donors, but in 2--3 and 10--14 days after onset of NSTEACS it did not differ from normal values. Contrary to P-selectin value of von Willebrand factor was elevated both during first 2 hours (1.3 times) and in 2--3 days (2 times) compared with healthy donors. Some elevation of von Willebrand factor (1.2 times) persisted after 10--14 days after development of NSTEACS. The results obtained have shownd that elevated activity of endothelium persists at least for 2--3 days after onset of NSTEACS, while activity of platelets during this period decreases. In none of temporal points there have been revealed differences in contents of P-selectin and von Willebrand factor between groups of patients receiving and not receiving glycoprotein IIb/IIIa antagonists. No differences were also found between these parameters in groups of patients favorable and unfavorable outcomes (myocardial infarction, angina recurrence) of the disease during 30 days of follow-up.

[Immune state of arterial hypertension and hypercholesterolemia patients engaged into construction materials production].

The authors studied immune state in individuals engaged in construction materials production, in accordance with presence or absence of arterial hypertension and hypercholesterolemia. Findings are depressed nonspecific resistance parameters and T-cell reactions in immune state.

[Aminopeptidases in human leukemia cells]. / Aminopeptidazy v leikoznykh kletkakh cheloveka.

Aminopeptidase activity in lymphoid cells of patients with various lymphoproliferative diseases was studied. The enzyme activity was detected in lysates of all leukemic B- and T-cells. The lymphoid cells contained aminopeptidases of at least two classes: metallo- and SH-dependent enzymes. The SH-dependent aminopeptidase with pH optimum 8.5-9.0 was revealed in lymphoid cells for the first time, and it seems to belong to a poorly studied aminopeptidase family.

Proteolytic enzymes in human leukemic lymphoid cells. III. Aminopeptidases, angiotensin-converting enzyme, and its inhibitor in cells of different immunological phenotype.

Activities of plasma membrane proteinases such as angiotensin-converting enzyme (ACE), aminopeptidases, and dipeptidyl peptidase IV (DPP-IV) were determined in lymphoid cells of various immunological phenotype which were obtained from 30 patients with lymphoproliferative diseases. The enzyme activities significantly varied depending on the immunological phenotype and stage of cell differentiation, but no correlation was found between activities of ACE, DPP-IV, and aminopeptidases in the cells of different type. The cell lysates studied contained at least two classes of aminopeptidases: metal- and sulfhydryl-dependent enzymes. A sulfhydryl-dependent aminopeptidase with activity optimum at pH 8. 5-9.0 was found for the first time and is suggested to be from a poorly studied aminopeptidase family. In addition to ACE, lysates of leukemic T- and B-cells were found to contain an inhibitor of ACE which was not previously described for these cells.

[A comparative study of aspartyl and cysteine proteinases and their inhibitors in human B- and T-cell leukemias]. / Sravnitel'noe izuchenie aspartil'nykh, tsisteinovykh porteinaz i ikh ingibitorov v leikoznykh B- i T-limfotsitakh cheloveka.

A procedure was developed for simultaneous isolation of aspartyl and cysteine proteinases as well as of the cysteine-proteinase inhibitors. Affinity chromatography using pepstatin-Sepharose enabled one to isolate aspartyl proteinases, while inhibitors of cysteine-proteinases were isolated by affinity chromatography on CM-papain-Sepharose; further purification of the enzymes was carried out using ion exchange chromatography and gel filtration. Partially purified preparations of cathepsin D as well as of cysteine-proteinases and their inhibitors were obtained. Some physicochemical and enzymatic properties of the enzymes and inhibitors obtained were studied.

[Proteolytic enzymes in human lymphocytic leukemia cells. I. Activity of dipeptidylaminopeptidase IV, plasminogen activator and cathepsins B and L in cells with different immunologic phenotype]. / Proteoliticheskie fermenty v leikoznykh limfoidnykh kletkakh cheloveka. I. Aktivnost' dipeptidilaminopeptidazy IV, aktivatora plazminogena i katepsinov B i L v kletkakh s raznym immunologicheskim fenotipom.

Immunological and biochemical study of lymphoid cells obtained from 20 patients with various forms of lymphoproliferative disorders has been carried out. It was found that different phenotypes of lymphoid cells at various stages of differentiation have different activity levels of dipeptidyl aminopeptidase IV (DAP IV), plasminogen activator (urokinase type) and cathepsins B + L. The highest proteinase activity values were found in the lysates of just those leukemic T-cells whose phenotypes corresponded to the initial stages of thymic differentiation or to activated T-cells. The 10 times lowered activity was found in the cell phenotypes of mature T-helpers and T-suppressors, and the activity of the both was at virtually the same level. In lymphoid cells of the B-lineage (from pre-B to mature B-lymphocytes) the proteinase activities did not differ essentially: they were 2 to 3 times lower than in the lymphoid progenitors. It was suggested that the regulated activity changes in some proteinases occur during differentiation along the T- or B-pathways. It is likely that the increases in DAP IV and cathepsins B + L activities are associated with the activation of mature lymphoid T- and B-cells. No direct correlation was found between the activity of either proteinase and the expression of any of the surface markers under study.

[Proteolytic enzymes in human lymphocytic leukemia cells. II. Comparative characteristics of proteolytic enzymes and their inhibitors in three differing types of lymphoid cells]. / Proteoliticheskie fermenty v leikoznykh limfoidnykh kletakh cheloveka. II. Sravnitel'naia kharakteristika proteoliticheskikh fermentov i ikh ingibitorov v trekh razlichaiushchikhsia tipakh limfoidnykh kletok.

A comparative study of proteolytic enzymes and their inhibitors in three human leukemic lymphoid cell populations has been carried out. The lysates of all lymphoid cells contained cathepsins D, B, L and H as well as serine trypsin-like proteinases, several aminopeptidases, dipeptidyl aminopeptidase IV and plasminogen activator (urokinase type). The activities of individual proteinases and their ratios in all cell types under study varied essentially, suggesting that lymphoid cells with different functions have different sets of proteolytic enzymes. FPLC chromatography of the lysates revealed the presence of inhibitors of cysteine and serine trypsin-like proteinases. The procedure for isolation of cathepsins D, B, L and H and of their inhibitors has been proposed and partially purified protein preparations obtained. Some properties of cathepsins B and L and those of their inhibitor have been examined.

Dipeptidylpeptidase IV in human leukemic B- and T-cells.

Dipeptidylpeptidase IV (DPP-IV) activity in lymphoid cells of patients with various lymphoproliferative diseases has been assayed. The enzyme activity was detected in lysates of all leukemic T- and B-cells studied. High DPP-IV activity levels (5-7 fold higher than those in mature Th and Ts) were found in T-cells with phenotypes corresponding either to the early thymic stages of maturation, or to activated T-lymphocytes, or to some atypic T-cells. In most leukemic B-cells with phenotypes of mature and late B-lymphocytes, the DPP-IV activity was lower than in mature T-cells; however, in some B-cells carrying the activation markers CD 25 and CD 30 the activity was rather high. High DPP-IV activity was seen in the majority of leukemic B-cells expressing CD 25 (87%) and CD 11c (82%) antigens. Experiments with intact cells have shown that DPP-IV is present in the plasma membrane of leukemic B- and T-cells studied. Thus DPP-IV cannot be considered as a marker of activated T-cells alone because a significant DPP-IV activity was also detected in a number of activated leukemic B-cells and in early T-cells.

[Protein kinase activity in lymphoid cells in various forms of lymphoproliferative disorders]. / Aktivnost' proteinaz v limfoidnykh kletkakh pri raznykh formakh limfoproliferativnykh zabolevanii.

Activities of dipeptidyl-aminopeptidase IV, urokinase-like plasminogen activator, cathepsins B and L were studied in lymphoid cells of patients with various forms of lymphoproliferative disorders. Activity of the enzymes studied was found in all the T- and B-cell, although rate and ratio of the enzymatic activity were dissimilar in various cell types. The highest rate of activity exhibited cells at early stages of maturation obtained from patients with acute lymphoblastic leukemia, while low level of the proteinase activity was detected in cells of patients with chronic lymphoid leukemia, non-Hodgkin lymphoma, hairy cell leukemia and Sezary disease, corresponding to mature T- and B-subpopulations. As shown by analysis of the cells immunological phenotype and their proteolytic activity, the rate of lymphoid cells differentiation correlated with level of proteinases activity. Series of proteinases were firstly studied in human malignant lymphoid cells with known phenotype. The enzyme assay may be used in diagnosis and treatment of patients with lymphoproliferative disorders.

Activity of some proteinases and glycosidases in human leukemic lymphoid cells at various stages of differentiation.

Activities of some glycosidases and proteinases in human leukemic lymphoid cells at various stages of differentiation have been compared. It was found that cells with different immunological phenotypes gave different enzymic spectra. Glycosidases and proteinases in lymphoid cell precursors had higher activity level than the enzymes in mature T- and B- cells. In cells of B- lineage, all activities were lower than in common precursor of lymphoid cells. In T-cells at the earlier stages of thymic differentiation, activities of all proteinases and most of glycosidases were higher than in common precursor cells whereas in mature T-helpers and T-suppressors the activities were markedly lower. Most of hydrolases in mature T-cells were twice more active than the enzymes in mature B-cells. The opposite-directional changes in activities of some hydrolases at the earlier stages of differentiation of lymphoid cells along B- or T- cells pathways are suggested.

[Contribution of lysosomal enzymes into degradation of short- and long-lived proteins in human embryonal fibroblasts with abnormal chromosome complement]. / Issledovanie vklada lizosomal'nykh fermentov v degradatsiiu korotko- i dolgozhivushchikh belkov émbrional'nykh fibroblastov cheloveka s anomal'nym naborom khromosom.

Degradation of short- and long-life proteins was studied in human embryonal fibroblasts with normal karyotype, trisomy and triploidy. Degradation of both short- and long-life proteins by means of lysosomal enzymes was elevated in fibroblasts with anomalous karyotype at the logarithmic phase of growth as compared with normal state, when lysosomotropic agent NH4Cl was used. In transition to stationary phase of growth participation of the lysosomal system in degradation of long-life proteins was increased in fibroblasts with normal karyotype, whereas degradation of these proteins in aneuploid fibroblasts maintained at the level found during the logarithmic phase of growth. Lysosomal enzymes did not participate apparently in degradation of short-life proteins in fibroblasts with normal and anomalous karyotype at both phases of growth. The data obtained suggest that chromosomal disbalance was related to impaired regulation of protein degradation as well as to possible alteration in the ratio of cytosol and lysosomal proteolysis.