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.

The role of platelet glycoprotein IIb / IIIa inhibitors in current treatment of acute coronary syndrome.

Current management of patients with acute coronary syndrome (ACS) includes a dual antiplatelet therapy with acetylsalicylic acid and a platelet P2Y12 receptor inhibitor. For patients without a high risk of bleeding, prasugrel and ticagrelor are preferred, since their effect is more pronounced, less dependent on metabolism of a specific patient, and occurs faster that the effect of clopidogrel. The prescription rate of platelet glycoprotein IIb/IIIa (GP IIb / IIIa) receptor inhibitors has considerably decreased. However, these drugs remain relevant in percutaneous coronary interventions in patients with a high risk of coronary thrombosis or a massive coronary thrombus, in thrombotic complications of the procedure, and in the "no-reflow" phenomenon. The intravenous route of GP IIb / IIIa inhibitor administration provides their effectiveness in patients with difficulties of drug intake or with impaired absorption of oral medications. This review presents clinical and pharmacological characteristics of various GP IIb / IIIa inhibitors and data of randomized clinical studies and registries of recent years that evaluated results of their use in patients with ACS.

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.

DNA Aptamers to Thrombin Exosite I. Structure-Function Relationships and Antithrombotic Effects.

DNA aptamers (oligonucleotides) interacting with thrombin exosite I contain G-quadruplex, two T-T, and one T-G-T loops in their structure. They prevent exosite I binding with fibrinogen and thrombin receptors on platelet surface, thereby suppressing thrombin-stimulated formation of fibrin from fibrinogen and platelet aggregation. Earlier, we synthesized original antithrombin aptamer RE31 (5'-GTGACGTAGGTTGGTGTGGTTGGGGCGTCAC-3') that contained (in addition to G-quadruplex) a hinge region connected to six pairs of complementary bases (duplex region). In this study, we compared properties of RE31 aptamer and its analogues containing varying number of bases in the duplex region and nucleotide insertions in the hinge region. Reduction in the number of nucleotides in the duplex region by 1 to 4 pairs (in comparison with RE31 aptamer) resulted in the decrease of the structural stability of aptamers (manifested as lower melting temperatures) and their ability to inhibit thrombin-stimulated fibrin formation in human blood plasma in tests of thrombin, prothrombin, and activated partial thromboplastin times. However, an increase in the number of bases by 1 to 2 pairs did not cause significant changes in the stability and antithrombin activity of the aptamers. Insertions into the hinge region of RE31 aptamer decreased its antithrombin activity. Investigation of RE31 antithrombotic properties demonstrated that RE31 (i) slowed down thrombin formation in human blood plasma (thrombin generation test), (ii) accelerated lysis of fibrin clot by tissue plasminogen activator in in vitro model, and (iii) suppressed arterial thrombosis in in vivo model. Based on the obtained data, RE31 aptamer can be considered as a potentially effective antithrombotic compound.

[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.

Diagnostics of thrombocytopenias.

Laboratory methods used for the diagnostics of thrombocytopenias are reviewed. Differential diagnosis is usually carried out between immune and hypoproductive forms of thrombocytopenia. Immune thrombocytopenias are caused by appearance in blood of antiplatelet abtibodies and accelerated destruction of platelets sensibilized by those antibodies, and hypoproductive thrombocytopenias - by impaired platelet production in the bone marrow. Main directions of the laboratory diagnostics of thrombocytopenias - analysis of auto- and alloautoantibodies and evaluation of platelet production and turnover in the blood stream. The following methods are used for the investigation of antiplatelet antibodies: 1) measurement of platelet associated immunoglobulins; 2) determination of circulating antibodies reacting with platelets; 3) determination of antibodies using antigen specific methods - by their reactivity with isolated platelet antigens (glycoproteins). Efficacy of platelet production could be assessed by measuring in blood the amount of "young" (reticulated) platelets. One more method for the evaluation of platelet production as well as the rate of platelet turnover - measurement of plasma soluble glycocalicin, glycoprotein Ib fragment shed from the surface of platelets upon their destruction in spleen and liver. In patients with immune thrombocytopenia autoantibodies are evaluated in all cases, the percentage of reticulated platelets is significantly increased and the amount of plasma glycocalicin is within the normal range or increased. In patients with hypoproductive thrombocytopenia autoantibodies are not detected or detected at low level, the percentage of reticulated platelets is within the normal range or slightly increased and the amount of plasma glycocalicin is lowered. Diagnostics of hapten forms of immune thromocytopenias (heparin-induced thrombocytopenia and others) and of alloimmune thrombocytopenias (neonatal alloimmune thrombocytopenia in particular) are considered in the separate sections of this review.

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.

[Factors influencing platelet aggregation in patients with acute coronary syndrome].

AIM: To study factors influencing platelet aggregation in patients with acute coronary syndrome (ACS). SUBJECTS AND METHODS: The investigation enrolled 147 patients with ACS. Their blood was sampled on days 1, 3-5, and 8-12 days after the onset of ACS. All the patients received acetylsalicylic acid (ASA) 300 mg on day 1, then 100 mg/day and clopidogrel 300-600 mg on day 1, then 75-150 mg/day. Platelet aggregation was analyzed in 65 patients on day 1 after ASA intake, but prior to clopidogrel therapy. The aggregation was induced by 5 and 20 pmol of ADP. RESULTS: With the use of clopidogrel 75 mg/day on day 3-5, platelet aggregation was reduced by 2.1 and 1.7 times for 5 and 20 µmol of ADP, respectively, as compared to day 1 (ASA without clopidogrel) and remained unchanged on days 8-12. Increasing the dose of clopidogrel up to 150 mg/day potentiated its antiaggregatory effect. On day 1 (ASA without clopidogrel), there was a direct correlation between platelet aggregation levels and mean platelet volume (MPV) (correlation coefficients (r), 0.526 (p < 0.001) and 0.368 (p = 0.015) for 5 and 20 µmol of ADP, and between platelet aggregation levels and glycoprotein (GP) IIb-IIIa (r = 0.387; p = 0.002 and r = 0.411 (p < 0.001) for 5 and 20 µmol of ADP. No similar correlations were found on days 3-5 and 8-12 of administration of ASA and clopidogrel. The genetic polymorphism of GP lIb-Illa (GP Ila Leu33Pro) was not noted to affect platelet aggregation. Examining the effects of genetic variations in cytochrome P450 isoform CYP2C19 (a clopidogrel metabolizer) revealed the enhanced aggregation stimulated with 20 µmol of ADP in the carriers of slowly clopidogrel-metabolizing haplotype of CYP2C19 (differences were found on days 3-5 as compared to rapidly and routinely metabolizing haplotypes). CONCLUSION: In the patients with ACS, platelet aggregation is influenced by MPV, GP IIb-IIIa levels, and CYP2C19 polymorphism and is not by GP IIb-IIIa polymorphism.

[Mean platelet volume: interactions with platelet aggregation activity and glycoprotein IIb-IIIa and Ib expression levels].

Increased mean platelet volume (MPV) is an independent risk factor of thrombotic events in patients with cardiovascular diseases. Interactions of MPV with platelet aggregation activity and contents of glycoprotein (GP) IIb-IIIa (alphaIIb/beta3 integrin, fibrinogen receptor) and GP Ib (von Willebrand factor receptor) were investigated in this study. Investigation was performed in a group of healthy volunteers (n = 38) and in a group of patients with acute coronary syndrome (ACS). In patients blood was collected at days 1, 3-5 and 8-12 after ACS development. As an antiaggregant therapy all patients received acetylsalicylic acid (ASA, inhibitor of thromboxane A2 synthesis) and most of them--clopidogrel (ADP receptor antagonist) with the exception of part of the patients (n = 44) at day 1 who had not taken clopidogrel before first blood collection. In volunteers platelet aggregation was stimulated by 1.25, 2.5, 5 and 20 M ADP, and in patients--by 5 and 20 M ADP. GP IIb-IIIa and GP Ib content on platelet surface was measured using 125I-labelled monoclonal antibodies. GP IIb-IIIa and GP Ib genetic polymorphisms were determined in ACS patients. In healthy donors significant correlations between MPV and aggregation levels were revealed at 1.25 and 2.5 M ADP (coefficients of correlation (r)--0.396 and 0.373, p < 0.05) and at 5 and 20 those interactions did not reach significant level (r--0.279 and 0.205, p > 0.05). Correlations between MPV and aggregation levels were observed at day 1 of ACS in a subgroup of patients who received ASA but had not started clopidogrel treatment (r--0.526, p < 0.01 and 0.368, p < 0.05 for 5 and 20 M ADP respectively). Interactions between these parameters were not registered upon combined treatment with ASA and clopidogrel. Strong direct correlations between MPV and GP IIb-IIIa and GP Ib contents were detected in healthy donors and ACS patients (at all time points) -r from 0.439 to 0.647 (p < or = 0.001 for all correlations). Genetic polymorphisms of GP IIb-IIIa (GP IIIa Leu33Pro) and GP Ib ((-5)T/C (Kozak) and Thr145Met) identified in ACS patients did not affect expression levels of corresponding glycoproteins. The data obtained indicated that increased MPV values correlate with increased platelet aggregation activity and enhanced GP IIb-IIIa and GP Ib expression.

Spontaneous platelet aggregation in patients with acute coronary syndrome.

Spontaneous platelet aggregation was evaluated in patients with acute coronary syndrome on days 1, 3-5, and 8-12 of the disease. On day 1, aggregation was analyzed after aspirin, but before clopidogrel administration; during other periods after both antiaggregants. The mean levels of spontaneous aggregation after antithrombotic therapy did not change during different periods after the onset of acute coronary syndrome, in contrast to ADP-induced aggregation that decreased after the development of clopidogrel effects (days 3-5 and 8-12). Spontaneous aggregation during different periods directly correlated (r>0.4, p<0.01) with spontaneous and ADP-induced aggregation during different periods (r=0.372, r=0.447, and r=0.543 on days 1, 3-5, and 8-12, respectively; p<0.01). No relationship between spontaneous aggregation and plasma concentration of von Willebrand's factor was detected. Spontaneous aggregation was completely suppressed after in vitro addition of prostaglandin E1 (platelet activation inhibitor), slightly (by ≈20%) decreased in the presence of antibodies to glycoprotein Ib, blocking its reactions with von Willebrand's factor, and did not change in the presence of aptamer inhibiting thrombin activity.

[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.

Glycoprotein IIb-IIIa content and platelet aggregation in healthy volunteers and patients with acute coronary syndrome.

Glycoprotein (GP) IIb-IIIa (αIIbß3-integrin) is the central receptor of platelet aggregation. Activated GP IIb-IIIa binds fibrinogen or von Willebrand factor, which forms molecular bridges between aggregating platelets. This review summarizes data on the relationship between GP IIb-IIIa expression on the platelet surface and platelet aggregating activity. GP IIb-IIIa number, measured as maximal binding of complex-specific monoclonal antibody, varied by approximately two fold in both healthy volunteers (n = 35) and patients with acute coronary syndrome (ACS) (n = 65). In healthy volunteers positive associations were observed between GP IIb-IIIa number and the level of ADP-induced aggregation when this relationship was analysed in untreated platelet-rich plasma (PRP) as well as upon in vitro addition of aspirin or non-saturating concentrations of GP IIb-IIIa blockers. In the same group of volunteers almost no differences in aggregating activity were detected between donors carrying the GP IIIa Pro33 allele (n = 15) and those with the GP IIIa Leu33Leu33 genotype (n = 20). No significant relationships were revealed between platelet aggregability and variations of plasma fibrinogen concentration. Positive correlation of the level of ADP-induced aggregation and GP IIb-IIIa content was detected in patients with ACS within the first hour upon admission to the hospital when they had already received aspirin, but not clopidogrel. However, there were no correlations between these parameters at days 3-5 and days 8-12 (before discharge). At these time points patients were treated not only with aspirin but were saturated with clopidogrel as well. In ACS patients we also evaluated the expression of another platelet adhesive receptor, GP Ib, and found a significant positive correlation between GP IIb-IIIa and GP Ib content. A strong association was also revealed between the number of both receptors and mean platelet volume. The latter observation indicated that individual variations of the number of glycoprotein molecules are mainly affected by platelet size but not the density of their expression on the platelet membrane. Possible usefulness of measuring GP IIb-IIIa content as a marker of increased platelet reactivity is discussed.

Characteristics of a new DNA aptamer, direct inhibitor of thrombin.

Characteristics of a new antithrombin DNA-aptamer RE31 were studied. This aptamer inhibited thrombin formation in human plasma catalyzed by exogenous (lengthening of thrombin time) and endogenous thrombin (lengthening of partial prothrombin time and activated partial thromboplastin time). In addition, the aptamer completely suppressed thrombin-induced aggregation of human platelets. On the other hand, RE31 did not reduce amidolytic activity of thrombin towards the short peptide substrate, in other words, did not modify the state of enzyme active center. By the capacity to inhibit clotting reactions, RE31 was superior to the previously described highly effective 31-component antithrombin aptamer 31TBA (thrombin binding aptamer, TBA). The effect of RE31 was species-specific: it inhibited human thrombin activity more effectively than activities of rat and rabbit thrombins.

[Glycoprotein IIb-IIIa antagonist Monafram in primary angioplasty of patients with acute coronary syndrome without st segment elevation].

Glycoprotein (GP) llb-llla anagonist monafram is the F(ab)2 fragments of anti GP llb llla monoclonal antibody FraMon (CRC64). Efficacy and safety of monafram in primary coronary angioplasty of patients with acute coronary syndrome without ST segment elevation (non ST ACS) was evaluated in this study. Monafram was introduced intravenously to 284 patients just before angioplasty at standard dosage - 0.25 mg/kg as single i.v. bolus. Control group included 203 patients. All patients received aspirin (loading dose 300 mg and then 75 mg daily) and more than 90% - clopidogrel (loading dose 300-600 mg and then 75 mg daily). Within 30 days of follow up period monafram decreased by more than 2.5 fold the total amount of unfavorable outcomes (cardiovascular death, myocardial infarction and indications for repeat revascularization due to angina recurrence) - from 19.2% to 7.4% (p<0.001). The rate of indications for revascularization was most strongly decreased - by more than 7 times - from 7.9% to 1.1% (p<0.001). The number of myocardial infarctions was reduced by more than 2 times - from 8.4% to 3.9% (p=0.057). The amount of lethal outcomes did not differ between two groups (2.9% and 2.4% in the control and monafram groups, respectively). In the control group 8.9% patients received monafram during primary angioplasty due to urgent indications. Monafram did not cause any allergic reaction in all tested patients. Major bleeding was registered in one (less than 0.5%) and deep thrombocytopenia (<20000 platelets per 1 ul) - in 3 (1.1%) out of 284 patients. The data obtained indicated that monafram decreased the number of thrombotic complications in non ST ACS patients undergoing angioplasty upon the dual antiplatelet therapy (aspirin+clopidogrel) and without significant increase of dangerous side effects.

Inhibition of thrombin activity with DNA-aptamers.

The effects of two DNA aptamers (oligonucleotides) 15TBA and 31TBA (15- and 31-mer thrombin-binding aptamers, respectively) on thrombin activity were studied. Both aptamers added to human plasma dose-dependently increased thrombin time (fibrin formation upon exposure to exogenous thrombin), prothrombin time (clotting activation by the extrinsic pathway), and activated partial thromboplastin time (clotting activation by the intrinsic pathway). At the same time, these aptamers did not modify amidolytic activity of thrombin evaluated by cleavage of synthetic chromogenic substrate. Aptamers also inhibited thrombin-induced human platelet aggregation. The inhibitory effects of 31TBA manifested at lower concentrations than those of 15TBA in all tests. These data indicate that the studied antithrombin DNA aptamers effectively suppress its two key reactions, fibrin formation and stimulation of platelet aggregation, without modifying active center of the thrombin molecule.