[GDF-15 and the risk of bleeding in patients with stable CAD receiving multicomponent antithrombotic therapy: the results of the prospective REGATA register].

AIM: To evaluate the prognostic value of GDF-15 in relation the development of bleeding and events in stable CAD patients, receiving combined antithrombotic therapy. MATERIALS AND METHODS: The data was obtained from the prospective registry REGATA, 343 CAD patients (249 males), median age 68 [IQR 62; 75] years) were enrolled. Patients with sinus rhythm and concomitant PAD received acetylsalicylic acid in combination with rivaroxaban 2.5 mg bid (31.8%) or clopidogrel (24.8%). Other 43.4% with concomitant atrial fibrillation (AF) received direct oral anticoagulants in combination with antiplatelet therapy after elective percutaneous coronary interventions. Median follow-up was 12 months [IQR 9.0; 18.0]. The safety end point was major and clinically relevant bleedings (type 2-5) according to the BARC classification. Plasma samples for GDF-15 identification were taken at the inclusion and analyzed using ELISA assay. RESULTS: Frequency of BARC 2-5 bleedings was 16% (BARC 2 - 46; BARC 3 - 9; BARC 4-5 - 0), median GDF-15 level was 1185.0 pg/ml [850.0; 1680.0]. In patients with AF and concomitant MFA, the level of GDF-15 was significantly higher than in the subgroups of patients with only AF or MFA (p=0.0022). According to the quintile analysis, GDF-15 values in the top three quintiles of distribution (cut-off value >943 pg/ml) were associated with higher frequency of bleeding events: 23.2% versus 5.1%; p=0.0001. The multivariable logistic regression model demonstrated that bleeding events were independently associated with GDF-15 level>943 pg/ml (OR 2.65, 95% CI 1.11-6.30; p=0.0275), AF (OR 2.61, 95% CI 1.41-4.83; p=0.0023) and chronic kidney disease (OR 1.92, 95% CI 1.03-3.60; p=0.0401). Clinical factors determining the risk of bleeding events also determined a GDF-15 elevation. CONCLUSION: Assessment of GDF-15 level may improve bleeding risk stratification in CAD patients with concomitant AF and/or PAD receiving combined antithrombotic therapy.

[Albuminuria as a marker of atherosclerosis burden and a possible predictor of adverse events in patients with polyvascular disease].

BACKGROUND: The role of albuminuria as a marker of the atherosclerosis burden and a predictor of prognosis in patients with polyvascular disease (PD) has been little studied. AIM: To evaluate the prevalence, association with atherosclerosis burden, and prognostic value of albuminuria in relation to cardiovascular and bleeding complications in patients with PD. MATERIALS AND METHODS: The data was obtained from the prospective registry REGATA-1 (NCT04347200). Seventy four patients (75.7% males, median age 67 [61-69] years) with PD (CAD and peripheral arterial disease) were enrolled. All patients received aspirin and rivaroxaban 2.5 mg. The albumin-creatinine ratio in a single morning urine sample, estimated glomerular filtration rate (eGFR), and von Willebrand factor levels were determined. RESULTS: Mild albuminuria (10-29 mg/g) was detected in 45.9% of patients, moderate and severe (≥30 mg/g) - in 29.7%; eGFR<60 ml/min - in 21.7%, chronic kidney disease (CKD) according to the full KDIGO criteria (eGFR and/or albuminuria ≥30 mg/g) - twice as often (39.2%). The frequency of nephroprotective therapy prescription was insufficient. The level of albuminuria did not correlate with von Willebrand factor (endothelial dysfunction marker), but was associated with affecting of 4-5 vascular beds (ROC AUC 0.775; p=0.011). During the follow-up (12 [8-18] months) 3 patients developed MACE, 11 - BARC 2-3 bleedings. Neither albuminuria nor eGFR were predictors of MACE, bleeding, or net clinical benefit. CKD (KDIGO) was also not associated with bleedings. CKD (KDIGO) was independent predictor of MACE (in significant multiple regression model beta - coefficient for CKD was 0.097; p=0.042), however, the small number of end points allows us to speak only of a hypothesis-generating trend. The implementation of CKD (KDIGO) has increased the predictive value of the REACH score. CONCLUSION: Albuminuria is highly prevalent in patients with PD. It is a marker of atherosclerosis burden. CKD, diagnosed taking into account the level of albuminuria, can be used in a comprehensive assessment of cardiovascular risk in this category of patients.

Prediction-Determining Outcomes and Their Predictors in Atrial Fibrillation Patients Receiving Multicomponent Antithrombotic Therapy in Real Clinical Practice.

Aim      Searching for clinical, angiographic, and biochemical predictors of cardiovascular complications (CVC) and hemorrhagic complications in patients with atrial fibrillation (AF) receiving a multicomponent antithrombotic therapy (MAT) for an elective percutaneous coronary intervention (PCI). Patients with ischemic heart disease (IHD) and AF who require MAT for PCI are at a high risk of thrombotic complications (stroke, systemic embolism, coronary events) and hemorrhage. This warrants searching for new risk factors determining prediction of the outcome.Materials and methods This study included 207 patients (146 males aged 70.1±8.3 years) with IHD and AF who received direct oral anticoagulants (DOAC) as a part of their MAT therapy. Median duration of the follow-up was 12 [8.0; 12.0] months. The efficacy endpoint was a sum of CVCs combining cardiovascular death, ischemic stroke, venous thromboembolic complications, acute coronary syndrome (ACS), and requirement for an unscheduled PCI. "Coronary events", including ACS and requirement for an unscheduled PCI were analyzed separately. The safety endpoint was BARC type 2-5 bleeding. Upon admission, biomarkers (growth-differentiation factor 15 (GDF-15), D-dimer, thrombin-activated fibrinolysis inhibitor (TAFI), and plasminogen activator inhibitor-1 (PAI-1)) were measured for all patients. Searching for prognostically significant indexes was performed with the Cox proportional hazards regression.Results Incidence of all CVCs was 16.4 %. Independent predictors of CVC included the DOAC treatment at a reduced dose (odds ratio (OR) 2.5 at 95 % confidence interval (CI) 1.02-6.15; p=0.0454), GDF-15 >1191 pg /ml (OR 3.76 at 95 % CI, 1.26-11.18; p=0.0172), PAI-1 >13.2 U/ml (OR 2.67 at 95 % CI, 1.13-6,26; p=0.0245). Incidence of coronary complications was 9.2 %. Independent predictors of coronary complications included a SYNTAX index >26.5 (OR 4.5 at 95 % CI, 1.45-13.60; p=0.0090), PCI for chronic coronary occlusion (OR 3.21 at 95 % CI, 1.10-9.33; p=0.0326), a GDF-15 >1191 pg/ml (ОR 4.70 at 95 % CI, 1.32-16.81; p=0.0172). Incidence of BARC type 2-5 bleeding was 26.1 %. The only independent predictor for hemorrhage complications was the total PRECISE-DAPT score >30 (ОR 3.22; 95 % CI, 1.89-5.51; р<0.0001).Conclusion      Three independent predictors of CVC were identified for patients with IHD and AF treated with MAT following an elective PCI: treatment with a reduced dose of DOAC, GDF-15 >1191 pg /ml, and PAI-1>13.2 U/ml. Independent predictors of coronary complications included a SYNTAX index >26.5, PCI for chronic coronary occlusion, and GDF-15 >1191 pg/ml. The factor associated with a risk of bleeding was the total PRECISE-DAPT score >30.

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.

[Renal function in patients receiving long-term warfarin therapy: A five-year prospective follow-up]. / Funktsiia pochek u bol'nykh, dlitel'no prinimaiushchikh varfarin (5-letnee prospektivnoe nabliudenie).

AIM: To investigate the prognostic value of renal function and to estimate glomerular filtration rate (GFR) changes during a 5-year follow-up of patients receiving warfarin therapy. SUBJECTS AND METHODS: 200 patients (124 men, 76 women) mainly from a group at high risk for thromboembolic events (mean CHA2DS2-VASc scores, 3.25±1.89) were examined. The patients' mean age was 62.3±9.4 years; the follow-up period was 5 years. 74% of the patients received warfarin monotherapy (international normalized ratio (INR) 2.0 to 3.0); 36% took vitamin K antagonists in combination with one or two antiplatelet agents. The CKD-EPI formula was used to estimate GFR in all the patients at baseline and throughout the investigation once a year. RESULTS: GFR less than 70.9 ml/min/1.73 m2 was found to be a predictor of fatal and nonfatal thrombotic events. The decreased GFR was unassociated with the development of major and clinically relevant hemorrhagic complications within 5 years of warfarin therapy. The initial decline in renal function (GFR <70.9 ml/min/1.73 m2) was associated only with an increased rate of recurrent minor hemorrhagic complications. During 5-year warfarin therapy, there was a significant decrease in GFR from 97.1±24.85 to 91.9±28.9 ml/min/1.73 m2; at the same time, a rapidly progressive loss of renal function (GFR ≥3 ml/min/1.73 m2/year) was recorded in 25.9% of the patients. Discriminant analysis showed that a baseline left ventricular ejection fraction of <40% was a predictor for the rapidly progressive loss of kidney function. CONCLUSION: Long-term warfarin therapy achieved the therapeutic range for INR is safe in the environment of the created patronage system. The initial decrease in GFR is a predictor of thrombotic events and is unassociated with an increased risk of bleeding.

Effect of Fibrinogen on Platelet Reactivity Measured by the VerifyNow P2Y12 Assay.

The VerifyNow assay is based upon the ability of activated platelets to cross-link beads coated with fibrinogen. However, fibrinogen is an abundant protein of blood, and therefore it may affect test results by competing with fibrinogen of beads for binding to platelets. To test this assumption, we assessed the influence of artificial alteration of fibrinogen level in blood samples obtained from donors (n = 9) and patients on clopidogrel therapy (n = 8) on the results of the VerifyNow P2Y12 assay. Fibrinogen level was altered by adding to blood samples 1/10 volume of fibrinogen solution (10.56 g/liter) or corresponding buffer. Relative to baseline, addition of buffer significantly increased platelet reactivity, whereas addition of fibrinogen decreased it. Analysis of the relationship between change in platelet reactivity values (dBase and dPRU) and change in fibrinogen concentration (dFg) revealed strong negative correlations: dBase = -63.3 × dFg - 27.1 (r = -0.924, p < 0.0005) and dPRU = -54.4 × dFg - 21.8 (r = -0.764, p < 0.0005). Thus, the results of our experiments suggest that: (i) blood fibrinogen strongly influences results of the VerifyNow P2Y12 assay, and (ii) correcting for fibrinogen effect may be needed to improve the accuracy of the test in the measuring of antiplatelet effect of clopidogrel therapy.

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.

[Risk Factors of Recurrent Bleedings at Therapeutic International Normalized Ratio in Patients on Long-Term Warfarin Therapy].

AIM: to investigate parameters of fibrinolysis in patients on long-term warfarin (W) therapy, and assess their relation to the risk of recurrent bleeding occurring at therapeutic international normalized ratio (INR). MATERIALS AND METHODS: Our prospective study involved 78 W-naive patients (40 men, age 64.3+/-12.2 years). Follow up period was 5.6+/-3.4 months. INR was measured monthly; determination of coagulation parameters (D-dimer, fibrinogen, complex plasmin-2-antiplasmin [PAP] and thrombin-activatable fibrinolysis inhibitor [TAFI] was performed before and after at least 3 months of W therapy. RESULTS: During follow-up bleedings occurred in 47 (60.3%) patients, 26 patients (33.3%) had recurrent bleedings at therapeutic INR and 21 patients (26.9%) had single bleeding. Mean time in therapeutic range (TTR) was >70.

[HAS-BLED and HEMORR2HAGES Scales in Assessment of Bleeding Risk in Patients on Long-Term Warfarin Therapy].

UNLABELLED: Aim of the study was to elucidate value of HAS-BLED and HEMORR2HAGES scales for prediction bleedings in patients receiving long-term warfarin (W) therapy. MATERIAL AND METHODS: The study involved 119 patients (72 men) aged 60.9 ± 9.6 years with atrial fibrillation or venous thromboembolic complications. Follow up period was 5.6 ± 3.4 years. All bleedings were categorized as 1) single bleeding with INR > 4.0 during the 1st month of W therapy; 2) any single bleeding after 1st month of W therapy; 3) recurrent bleedings. CYP2C9 and VKORC1 (G3673A) genotypic variants were determined by PCR. Patients were divided into low (< 3 points of HAS-BLED scale, n = 58; < 4 points of HEMORR2HAGES scale, n = 109) and high (3 points of HAS-BLED scale, n = 61, ≥ 4 points of HEMORR2HAGES scale, n = 10) bleeding risk groups. RESULTS: There was no relationship between total HAS-BLED, HEMORR2HAGES scores and numbers of all as well as category 1 and 2 bleedings. The difference in bleeding frequency between high and low risk groups was significant only for recurrent bleedings. There were 22 (36.1%) and 5 (8.6%) recurrent bleedings among 61 and 58 patients with high and low-risk HAS-BLED score, respectively (p = 0.0048). Recurrent bleedings also occurred more frequently among patients with high risk (7/10, 70%) compared with low risk (20/109, 18.35%) HEMORR2HAGES score (p = 0.018). Subgroups of high and low bleeding risk according to HAS-BLED and HEMORR2HAGES scores differed only by proportion of patients with recurrent bleedings. High W sensitivity represented by 2*/2*, 2*/3*, 3*/3* CYP2C9 and/or AA VKORC1 homozygosis was detected in 25 of 119 patients. Six of 8 patients (75%) with category 1 bleedings were carriers of any polymorpism. CONCLUSION: HAS-BLED and HEMORR2HAGES scales performed best in predicting recurrent bleedings in patients on long term W therapy. Single bleedings with INR > 4.0 during 1st month of W therapy were associated with reduced W metabolism (AA VKORC1 or/and CYP2C9 allelic variants 2*/2*, 2*/3*, 3*/3*).

[HAS-BLED and HEMORR2HAGES Scales in Assessment of Bleeding Risk in Patients on Long-Term Warfarin Therapy].

Aim of the study was to elucidate value of HAS-BLED and HEMORR2HAGES scales for prediction bleedings in patients receiving long-term warfarin (W) therapy. MATERIAL AND METHODS: The study involved 119 patients (72 men) aged 60.9+/-9.6 years with atrial fibrillation or venous thromboembolic complications. Follow up period was 5.6 +/-3.4 years. All bleedings were categorized as 1) single bleeding with INR>4.0 during the 1st month of W therapy; 2) any single bleeding after 1st month of W therapy; 3) recurrent bleedings. CYP29 and VKORC1 (G3673A) genotypic variants were determined by PCR. Patients were divided into low (<3 points of HAS-BLED scale, n=58; <4 points.

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.

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.

The fibrinolysis system: regulation of activity and physiologic functions of its main components.

This review summarizes basic properties and mechanisms of activation and inhibition of main components of the fibrinolytic system that, acting in accord with the system of blood coagulation, provides temporal formation of fibrin clots at sites of vascular injury for the time required for the regeneration of vascular wall. Impairments in the fibrinolytic system may cause bleedings or thrombotic complications in patients. The predictive value of some components of the fibrinolytic system with respect to the development of complications of atherothrombosis is considered.

Urokinase plasminogen activator system in humans with stable coronary artery disease.

1. The present study compares plasma urokinase plasminogen activator (uPA) peptide levels, plasma plasminogen inhibitor (PAI-1) activity and urokinase receptors (uPAR) on peripheral blood monocytes of patients with stable coronary artery disease (SCAD) and healthy volunteers. 2. Urokinase plasminogen activator levels were analysed by ELISA and PAI-1 activity was determined by a plasmin generation method using the chromogenic substrate S2390. Relative uPAR numbers and the adhesion molecules CD11b/CD18 on peripheral blood monocytes were estimated using specific antibodies and flow cytometry. 3. Patients with SCAD were found to have higher plasma uPA peptide levels than age-matched healthy subjects (10.40 +/- 0.99 vs 8.25 +/- 0.53 pmol/L, respectively; P < 0.05). 4. Plasma PAI-1 activity was also higher in patients with SCAD than in healthy subjects (13.6 +/- 2.5 vs 5.2 +/- 1.0 IU/mL, respectively; P < 0.05). 5. Relative uPAR and CD11b/CD18 adhesion molecules were similar on peripheral blood monocytes of patients with SCAD and in healthy subjects. 6. The data indicate a pattern of expression/activity of uPA and PAI-1 in patients with SCAD suggestive of an impaired fibrinolytic ability.