[Platelet adhesion mediated by von Willebrand factor in patients with premature coronary artery disease].

Aim    To study platelet adhesion mediated by von Willebrand factor (VWF) in patients with premature ischemic heart disease (IHD).Material and methods    This study enrolled 58 patients with stable IHD, including 45 men younger than 55 years with the first manifestation of IHD at the age of <50 years and 13 women younger than 65 years with the first manifestation of IHD at the age of <60 years. The control group consisted of 33 patients, 13 men younger than 55 years and 20 women younger than 65 years without IHD. Platelet adhesion to the collagen surface at the shear rate of 1300 s-1 was studied by evaluating the intensity of scattered laser light from the collagen-coated optical substrate in a flow chamber of a microfluidic device after 15-min circulation of whole blood in the chamber. Decreases in platelet adhesion after addition to the blood of monoclonal antibodies (mAb) to platelet receptors glycoproteins Ib (GPIb) to inhibit the receptor interaction with VWF were compared for patients of both groups. Results    In patients with premature IHD, the decrease in platelet adhesion following the platelet GPIb receptor inhibition was significantly less than in patients of the control group (74.8 % (55.6; 82.7) vs. 28.9 % (-9.8; 50,5), p <0.001). For the entire sample, the median decrease in platelet adhesion following the GPIb receptor inhibition was 62.8 % (52.2; 71.2). With an adjustment for traditional risk factors of IHD, a decrease in platelet adhesion of >62.8% after blocking GPIb receptors increased the likelihood of premature IHD (OR=9.84, 95 % CI: 2.80-34.59; p <0.001).Conclusion    Blocking the interaction of GPIb receptors with VWF in patients with premature IHD and increased shear rate induced a greater decrease in platelet adhesion than in patients without this disease. This suggested that an excessive interaction of VWF with platelets might contribute to the pathogenesis of premature IHD.

Monomeric form of C-reactive protein in the assessment of the residual inflammatory cardiovascular risk in patients with subclinical carotid atherosclerosis.

Aim      To study the relationship between monomeric C-reactive protein (mCRP) and the progression of asymptomatic carotid atherosclerosis in patients with a moderate risk for cardiovascular diseases (CVD) as assessed with the SCORE model.Material and methods  The study included 80 men and women aged 53.1±5.8 years assigned to the category of a moderate risk for CVDs by the SCORE model with a low-density lipoprotein cholesterol (LDL-C) level of 2.7-4.8 mmol/l and asymptomatic, hemodynamically insignificant (<50% luminal narrowing) carotid atherosclerosis according to ultrasonic data. All patients were prescribed atorvastatin to achieve a LDL-C level <2.6 mmol/l. After 7 years of follow-up, ultrasonic examination of carotid arteries was performed, and concentrations of high-sensitivity C-reactive protein (hsCRP) and mCRP were measured.Results A concentration of LDL-C <2.6 mmol/l was achieved in all patients. The progression of atherosclerosis as determined by an increased number of atherosclerotic plaques (ASPs), was observed in 45 (56 %) patients. At 7 months of follow-up, concentrations of cCRP were higher in the group of patients with progressive carotid atherosclerosis, while the levels of hsCRP did not differ between the groups. Increased mCRP concentrations were associated with changes in variables of the "atherosclerotic load", including the number of ASPs, total ASP height, and the intima-media thickness (IMT). In patients with a median mCRP concentration of 5.2 [3.3; 7.1] µg/l and more, the increases in mean ACP number and total ASP height were considerably higher than in patients with mCRP concentrations lower than the median (3.9 and 2.7 times, respectively), whereas the odds ratio for the progression of asymptomatic carotid atherosclerosis was 5.5 (95 % confidence interval, CI: 2.1-14.6; p=0.001). ROC analysis showed that the concentration of hsCRP had no predictive value for prognosis of asymptomatic carotid atherosclerosis (p=0.16), while the area under the ROC curve (AUC) for mCRP was 0.75±0.056 (95 % CI: 0.64-0.86; p=0.001).Conclusion      According to the results of 7-year follow-up, the plasma concentration of mCRP was significantly higher in patients with an increased number of ASPs than in patients without this increase. An increased level of mCRP may indicate a higher inflammatory risk of CVD.

Complex Interaction of Platelets, von Willebrand Factor and Leukocytes, in Whole Blood at High Shear Rates Is Mediated by Platelet GPIIb/IIIa Receptor.

We studied the contribution of von Willebrand factor (vWF) into blood cell adhesion to collagen-coated surfaces in whole blood of healthy volunteers. Adhesion of blood cells to collagen I was measured at shear rate of 2300 sec-1. The interaction of platelet GPIIb/IIIa receptor with vWF was blocked with monoclonal anti-GPIIb/IIIa antibodies. The degree of cell adhesion was quantified by measuring the intensity of scattered light after 15-min perfusion: in samples with blocked GPIIb/IIIa it decreased to 0.39±0.13 V vs 0.06±0.03 V in control samples (p=0.002). Under a fluorescence microscope, intensively stained structures consisting of vWF, platelets, and leukocytes attached to the collagen surface were observed. After blockade of GPIIb/IIIa, these structures were absent. Leukocyte recruitment at high shear rates is a time-dependent process sensitive to complex interaction of vWF, leukocytes, and platelets, in which the platelet GPIIb/IIIa receptor is essential.

Kinetics of Platelet Adhesion to Protein-Coated Surface in Whole Blood Samples at High Flow Rates.

We studied platelet adhesion to fibrinogen-coated surface in whole blood samples under conditions of high flow rates. The degree of platelet adhesion was evaluated by the intensity of laser light scattered from protein-coated optical surface with adhered platelets. The intensity of adhesion in whole blood samples at high flow rates was by 2.7 (2.4; 4.1) times higher than in platelet-rich plasma samples. Among the factors intensifying platelet adhesion in the whole blood at high flow rates, von Willebrand factor is of utmost importance. At low flow rates, platelet adhesion almost totally depends on platelet-fibrinogen interaction. At high flow rates, the interactions of platelets with both fibrinogen and von Willebrand factor become equally important.

[Acute Coronary Syndrome Registry of High Risk Patients: 30-Day Outcome].

AIM: The aim of the study is to evaluate important additional cardiovascular (CV) risk factors of major adverse cardiac events (MACE) in patients with acute coronary syndrome (ACS) during the first 30 days after index event. MATERIALS AND METHODS: Overall 750 patients with ACS were enrolled in the single center prospective registry from 2012-2015yy. 569 patients received dual antiplatelet therapy and in 425 cases platelet function testing (PFT) were performed. Most of the patients characterized as high risk elderly patients with multiple CV risk factors and high comorbidity index. RESULTS: At 30-day follow-up the mortality rate was 10,1%. Singlevariate analysis showed strong association between MACE and age, atrial fibrillation, stroke, chronic kidney disease, low ejection fraction, type 2 myocardial infarction (T2MI). Multivariate analysis showed that high-on-treatment platelet reactivity (PFT> 45%) with odds ratio 4.418 (p=0.0001), chronic kidney disease (OR 6.538 p=0.001) and T2MI (OR 1.925 p=0.0001) were significantly associated with adverse outcome. CONCLUSION: ACS registry showed high mortality level in real-life practice compared with randomized clinical trials due to the high prevalence of elderly patients with high comorbidity index. Patients with T2MI have significantly more severe prognosis and chronic kidney disease associated with increased MACE. PFT in this category of patients is reasonable for more accurate risk stratification.

[Relation of Functional Activity of Platelets to Prognosis of Unfavorable Cardiovascular Events in Patients with Acute Coronary Syndrome. Results of a Registry Study].

AIM: to assess relation ofhigh functional activity ofplatelets to prognosis ofunfavorable cardiovascular events in patients with Acute Coronary Syndrome (ACS). MATERIALS: The study was based on the data of a single center ACS registry conducted in the Central Clinical Hospital of the Presidential Affairs Department of Russian Federation. Of 529 included patients in 425 without contraindications to double antiplatelet therapy we carried out analysis of dependence of 30 days level of unfavorable events on parameters of functional activity of platelets. RESULTS: High on-treatment platelet reactivity (HTPR) was found to be associated with 3.5 increase of mortality in the group of patients with high cardiovascular risk. Logistic model of prognosis of unfavorable events based on multifactorial analysis of data from patients with measured platelet aggregation included chronic kidney disease, type of myocardial infarction, and degree ofplatelet aggregation >45%. C -statistic was equal to 0.77. We also present in this paper discussion of problems related to studying approaches to individualization of anti-aggregation therapy in real clinical practice and problems of organization ofsimilar studies. CONCLUSION: The study showed that patients with ACS increased platelet aggregation, as well as chronic kidney disease and type 2 MI are associated with a 30 day prognosis of adverse events.

Real-Time Recording of Platelet Adhesion to Fibrinogen-Coated Surface under Flow Conditions.

We present a testing system allowing real-time recording of the kinetics of platelet adhesion to fibrinogen-coated surface under flow conditions. The system consists of an optical flow chamber, semiconductor laser, two photodetectors, analog-to-digital converter, computer, and peristaltic pump. Platelet adhesion to fibrinogen-coated surface is recorded with two photodetectors and analyzed by the intensity of total internal reflection and scattered laser radiation at the boundary of the blood sample and fibrinogen-coated optical surface. Kinetics of platelet adhesion was studied as a function of shear rate and platelet concentration. The specificity of platelet adhesion with proteins on the surface of the flow chamber was verified by blocking IIb/IIIa glycoprotein complex on platelets with Fab2 fragments of monoclonal antibodies.

[The CD45-positive thrombocytes and neutrophils/lymphocytes level ratio in evaluation of risk of development of re-stenosis шт patients with chronic ischemic heart disease and diabetes mellitus type II.]

The purpose of study is to investigate significance of detection of number of CD45-positive thrombocytes and ratio neutrophils/lymphocytes in evaluation of risk of development of re-stenosis after implantation of drug-eluting stents in patients with chronic ischemic heart disease and diabetes mellitus type II. The examination was applied to 126 patients with chronic forms of ischemic heart disease who passed through repeat coronary angiography during first year after implantation of drug-eluting stents. The patients were separated on two groups depending on availability of diabetes mellitus type II. In both groups, the patients passed through comparing of angiography and clinical characteristics. Also, a logistic model was developed prognosticating development of re-stenosis. The re-stenosis developed more often in patients with diabetes mellitus type II (54.5%) than in patients without diabetes mellitus type II (32.4%; p=0.01). The comparative analysis of more than 35 characteristics permitted to establish risk factors of development of re-stenosis with the highest prognosticating capacity among patients with diabetes mellitus type II. These risk factors were CD45-positive thrombocytes and ratio neutrophils/lymphocytes. To prognosticate development of re-stenosis a logistic regression model was developed comprising number of circulating CD45-positive thrombocytes, ratio neutrophils/lymphocytes and availability of diabetes mellitus type II. The model demonstrated high prognosticating value as regards development of re-stenosis: OR = 15.1 (95% CI 4.81-31, p < 0.001). The square under ROC-curve: AUC = 0.83 (95% CI 0,72-0,92; р < 0,001). The increasing of number of circulating CD45-positive thrombocytes and ratio neutrophils/lymphocytes can be considered as a valuable risk factors of development of re-stenosis in case of diabetes mellitus type II.

Acetylsalicylic Acid Produces Different Effects on the Production of Active Oxygen Species by Activated Platelets in Different Inflammatory Diseases.

We studied the effect of acetylsalicylic acid on ROS generation by platelets in patients after surgical interventions and in patients with bronchial asthma was studied. Platelets stimulated with platelet-activating factor are characterized by weak luminol-enhanced chemiluminescence in healthy people and patients after operations with laparoscopic incisions. Addition of platelet activation factor to platelet samples from patients after open abdominal surgery caused intensive chemiluminescence that was suppressed after platelet incubation with acetylsalicylic acid. At the same time, platelets of patients with aspirin-sensitive asthma did not respond to addition of platelet activating factor, but after incubation with acetylsalicylic acid, an intensive burst of chemiluminescence was detected with a maximum in 5-10 sec after the addition of a platelet-activating factor. In patients with bronchial asthma tolerant to aspirin, platelet activation factor did not induce chemiluminescence irrespective of incubation with acetylsalicylic acid.

Relationship between the Level of Circulating CD45+ Platelets and Development of Restenosis after Implantation of Drug-Eluting Stents to Patients with Coronary Heart Disease.

The study was carried out in 126 patients with stable angina pectoris, who underwent elective coronary artery stenting with drug-eluting stents and follow-up angiography within 6-12 months thereafter. Five significant risk factors of restenosis were identified by binary comparisons of different variables. The logistic regression equation that included the level of CD45-positive platelets, diabetes, small vessel stenting, number of simultaneously implanted stents in one patient, and lesion length demonstrates the highest level of prediction of in-stent restenosis (OR=22.8; p<0.001). ROC-analysis demonstrated high prognostic value of the logit model (area under ROC curve 0.87, p<0.001). The data suggest that a close relationship exists between the development of restenosis and the level of circulating CD45+ 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.

[Level of Soluble Receptor for Glycation End-Products in Patients With Restenosis After Coronary Artery Stenting].

The purpose of research - the study of the level of soluble receptor for advanced glycation end products (CNG) in patients with coronary heart disease (CHD) with angina pectoris who have emerged restenosis after implantation of drug-eluting stents.

[Role of eosinophil granulocytes in the occurrence of restenosis in patients after coronary stenting with drug-eluting stents].

AIM: To study the role of eosinophil granulocytes in the development of restenosis after drug-eluting stent (DES) implantation. MATERIALS AND METHODS: The blood levels of eosinophils, eosinophil cationic protein (ECP), immunoglobulin E (IgE), and C-reactive protein (CRP) were compared in 170 patients with coronary heart disease (CHD) and stable angina who had undergone recoronarography within the first year after endovascular myocardial revascularization using DES. The blood level of eosinophils was determined by the results obtained employing the FACS Calibur flow cytofluorometer (Becton Dickinson, USA). That of ECP, IgE, and CRP was estimated by enzyme immunoassay by means of an Immulite-100 analyzer (Siemens, Germany). The data were presented as median (25th percentile and 75th percentile). RESULTS: The blood eosinophil level was 272 (234; 292) cells/µl in patients with restenosis while that was 134 (85; 156) cells/µl in those without restenosis (p = 0.002). The plasma ECP level proved to be greater in the patients who had developed restenosis after DES implantation than in those who had not [17.7 (11.2; 24) and 9 (6.4; 12.9) ng/ml, respectively (p = 0.017). At 6 months after DES implantation, the level of ECP was significantly higher (8.6 (7.3; 9.7) ng/ml) than the baseline level (p = 0.04). Later on, there was an even greater increase in ECP levels. No significant changes were noted in blood ECP levels after bare stent implantation. CONCLUSION: The findings suggest that there is a relationship between the occurrence of restenosis and the enhanced activity of eosinophil granulocytes in CHD patents after DES implantation.

[Eosinophilic cation protein and development of restenosis of drug covered stents].

Aim of the study was to assess activation of eosinophils as well as allergic and inflammatory reactions of the body in development of restenosis after implantation of stents with drug covering. We included into the study 32 patients with ischemic heart disease (IHD) and stable angina subjected to repeat coronary angiography during first year after endovascular myocardial revascularization with the help of stents with drug covering, and 11 healthy persons. Levels of eosinophilic cation protein (ECP), immunoglobulin E (IgE), and C-reactive protein (CRP) in blood plasma of patients and healthy persons was determined by immunoenzyme assay. According to results of angiographic study patients were divided into 2 groups: the first comprised 19 patients in whom no instent restenosis was found, the second comprised 13 patients in whom formation of restenosis at least in one stent was noted. Patients in these groups did not differ by age, sex, smokers and nonsmokers ratio, presence of hyperlipidemia, and angiographical characteristics of involved arteries. In patients with restenosis of stents blood plasma ECP level was 17.7 (11.2-24.0) g/ml and significantly higher than in patients without restenosis [9.0 (6.4-12.9) g/ml; p=0.017]. Blood level of IgE in these groups of patients did not differ [58.8 (42.1-164.0) and 52.9 (12.8-76.1) mg/ml, respectively; p=0.40] and did not differ from IgE level in blood of healthy volunteers [32.0 (21.2-80.8) mg/ml; p=0.91]. CRP level in patients with IHD was higher than in healthy volunteers [0.36 (0.1-0.75) mg/ml; p=0.0008)], but did not differ significantly in groups of patients with and without restenosis [2.38 (0.30-4.08) and 1.63 (0.61-2.47) mg/ml, respectively; p=0.52]. It was found that in the group of patients with low blood level of ECP (<11 g/ml) restenoses were revealed in 19% while in the group with higher blood level of ECP (>11 g/ml) - in 62% of cases (p=0,019). In patients subjected to coronary stenting with higher level of ECP in blood we noted more frequent development of restenoses than in patients with low level of this protein. The data obtained allow to suggest presence of relationship between development of restenosis and elevated activity of eosinophilic granulocytes in patients with IHD after revascularization.

[Stromal progenitor cells and blood leukocytes after implantation of drug-eluting stents].

Aim of the study was to assess participation in development of restenosis of circulating in blood progenitor cells of stromal line of differentiation and polymorphonuclear granulocytes. We compared levels of osteonectin positive progenitor cells, neutrophils, eosinophils, and basophils in blood of patients with ischemic heart disease (IHD) in whom according to data of angiographic study after endovascular myocardial revascularization with the help of stents with drug coating (Cypher, Cordis Corp, USA) restenosis was detected (n=15), in patients without restenosis (n=23), and in healthy persons (n=17). Levels of stromal progenitor cells and polymorphonuclear granulocytes in blood were measured with the help of methods of flow cytometry. In groups of patients with IHD with and without restenosis number of osteonectin positive cells in blood was higher than in healthy subjects (2.4+/-0.7 and 2.5+/-0.9 vs 1.5+/-0.5 cells/ microL, respectively, p=0.004) without significant differences between groups (p=0.59). These 2 groups of patients did not differ by numbers of leukocytes, neutrophils, and basophils in blood. At the same time we found that in patients with restenosis number of eosinophils in blood was significantly greater than in the group of patients without restenosis (262+/-68 vs 124+/-67 cells/ microL, respectively p<0.001). Moreover in patients with level of eosinophils exceeding 170 cells/ microL rate of development of restenosis was 74% against 5% in patients with number of eosinophils less than 170 cells/ microL (p<0.001). Thus level of stromal progenitor cells in blood of patients with IHD was higher than in healthy persons and remained equally high in groups with and without restenosis. Number of blood eosinophilic leukocytes in patients who had been subjected to coronary stenting in whom later restenosis developed was significantly higher than in patients without restenosis. The data obtained indicate at the presence of link between development of in - stent restenosis and elevated content of eosinophilic granulocytes in blood of patients with IHD.

Deglycosylation of apo B-containing lipoproteins increase their ability to aggregate and to promote intracellular cholesterol accumulation in vitro.

Sub-fractions of all apo B-100 containing lipoproteins (low density lipoproteins, very low density lipoproteins and intermediate density lipoproteins) with reduced contents of sialic acid were found in vivo in human blood. These lipoproteins were inclined to spontaneously form aggregates and were able to stimulate accumulation of cholesterol in cells cultured from human aortic intima. In vitro treatment of apo B-containing lipoproteins with 2,6- and 2,3-specific sialidases, alpha-mannosidase, endoglycosidases F1 or F2 or peptide-N-glycanase F also stimulated aggregation and increased the ability of these particles to potentiate cholesterol accumulation in cells of the intact human aortic intima. So, deglycosylation of various apo B-containing lipoproteins possibly occurs in the blood, decreases their resistance to aggregation and increases the ability of these particles to stimulate accumulation of cholesterol in human aortic intima cells, thereby increasing their atherogenic potential.

Detection of circulating stromal stem cells with osteogenic potential in the blood of coronary patients by laser flow cytometry.

The number of cells expressing antigen to osteonectin was appreciably increased in the blood of coronary patients, but no cells of this kind were detected in donors. The number of CD34+ stem cells in these patients virtually did not differ from that in normal subjects. A close relationship between atherosclerosis development and presence of stromal stem cells with osteogenic potential in the blood is hypothesized.

[Platelet aggregation in patients with hypertrophic cardiomyopathy]. / Sostoianie agregatsii trombotsitov u bol'nykh s gipertroficheskoi kardiomiopatiei.

AIM: To examine correlation between platelet aggregation and the degree of left ventricular hypertrophy (LVH) in patients with hypertrophic cardiomyopathy (HC). MATERIAL AND METHODS: Routine clinical examination, ECG, echo-CG, measurement of platelet aggregation, routine biochemical blood tests were made in 45 HC males and 15 healthy controls. RESULTS: HC patients were characterized with high induced and spontaneous platelet aggregation which increased with left ventricular myocardial mass. In LVH, platelets were especially sensitive to the aggregation inductor U-46619, thromboxane A2 analogue. CONCLUSION: It is demonstrated that there is a positive correlation between platelet aggregation and the degree of LVH in HC patients.

[Platelet aggregation in patients with various forms of left ventricular hypertrophy and its changes at long-term follow-up and treatment]. / Aggregatsiia trombotsitov u bol'nykh s razlichnymi formami gipertrofii levogo zheludochka i ee dinamika pri dlitel'nom nabliudenii i lechenii.

AIM: To study platelet aggregation (PA) in patients with left ventricular hypertrophy (LVH) in essential hypertension (EH) and hypertrophic cardiomyopathy (HTCM), and to assess aspirin treatment effects. MATERIAL AND METHODS: A general clinical examination, echocardiography, 24-h monitoring of ECG and arterial pressure, bicycle exercise, test for platelet aggregation, routine blood biochemical tests were performed in 30 males with EH and LVH, 30 males with HTCM and 10 healthy controls. RESULTS: It was found that the patients had high platelet sensitivity to aggregation inductors as well as high spontaneous aggregation. LVH severity correlated positively with activation of spontaneous and induced platelet aggregation. This dependence is unrelated to arterial pressure or the disease duration. Patients with episodes of silent myocardial ischemia had a more pronounced rise in spontaneous and induced platelet aggregation than those without silent ischemia. A long-term administration of aspirin significantly lowered spontaneous and induced platelet aggregation, depression of ST segment. The above changes occurred both in HTCM and in hypertensive patients with LVH. CONCLUSION: Platelet aggregation correlates with severity of LVH and episodes of silent myocardial ischemia both in patients with hypertension, LVH and HTCM.

[Determination of the activity of Willebrand's factor by laser analyzer of platelet aggregation]. / Opredelenie aktivnosti faktora Villebranda pri pomoshchi lazernogo analizatora agregatsii trombotsitov.

A modification of von Willebrand' factor/ristocetin cofactor activity test is proposed. Agglutination of formalin-treated platelets is monitored in the aggregation analyzer from changes in the light transmission in the specimen. The test is sensitive and reproducible. It is intended for clinical laboratories to be used in patients with hematological diseases and the endotheliopathy syndrome of different etiology.