ADP-induced Aggregation Research Articles

A Heterozygous Mutation of G-Protein-Coupled Receptor 25 in a Family with Inherited Thrombocytopenia and Thrombosis

BackgroundInherited thrombocytopenia is associated with bleeding tendency and no association with thrombotic tendency has been reported. We found a Japanese family with autosomal dominant form of thrombocytopenia associated with arterial and venous thrombosis.AimsTo identify a causative gene for hereditary thrombocytopenia with thrombosis.MethodsA propositus was a 42-year-old Japanese woman with thrombocytopenia and deep vein thrombosis (DVT). She had normal activities of antithrombin, protein C, and protein S, and negative results for lupus anticoagulant and anti-cardiolipin antibodies. She has suffered from thrombocytopenia (20 - 50 x 109/L) for at least 20 years. Six members among her family pedigree had thrombocytopenia, and also suffered from thrombosis including mesenteric artery thrombosis, young-onset myocardial infarction, femoral artery thrombosis, repeated cerebral infarction, DVT, and pulmonary embolism. Importantly, the family members with these thrombotic disorders had exclusively thrombocytopenia. Whole exome sequencing was performed with venous blood from the 6 affected and 6 non-affected family members to identify a causative gene for thrombocytopenia.ResultsWe identified a heterozygous change (c.764G>T:p.G255V) in GPR25 gene, an uncharacterized G protein-coupled receptor. The mutation was completely linked with thrombocytopenia. The mutated residue was located at the transmembrane domain of GPR25. We generated anti-GPR25 monoclonal antibodies and confirmed that GPR25 was expressed on the platelet surface. The patient's platelets showed the same level of PAC1 (a monoclonal antibody recognizing the active conformation of integrin αIIbβ3) binding and P-selectin expression as platelets from healthy subjects. However, ADP-induced aggregation of patient's washed platelets was enhanced as compared with normal washed platelets. Moreover, the patient's platelets had an increase in thrombin + collagen-induced and A23187-induced Annexin V binding compared with platelets from healthy subjects.ConclusionWe identified a novel gene mutation responsible for inherited thrombocytopenia and thrombosis that may induce platelet hyper-reactivity. DisclosuresNo relevant conflicts of interest to declare.

Platelet activity is negatively modulated by tumor necrosis factor alpha through reductions of cytosolic calcium levels and integrin alphaIIbbeta3 phosphorylation

IntroductionTumor necrosis factor-alpha (TNF-α) exerts a critical role in inflammatory events through two distinct receptors, TNFR1 and TNFR2. Platelets have been recognized as important inflammatory cells, but little is known about the effects of TNF-α on the platelet activity. ObjectivesIn the present study we have studied the role of TNF-α on ADP-induced platelet aggregation and its downstream signaling (c-Src and fibrinogen receptor phosphorylation, cytosolic Ca2+ mobilization, cAMP and cGMP levels and cell viability). Methods and resultsWashed rat platelets were incubated with TNF-α (1–3000 pg/ml) for different time-periods (5–60 min) before the addition of ADP (5 μM) to induce platelet aggregation. TNF-α concentration- and time-dependently inhibits ADP-induced aggregation, which was significantly prevented by incubation with the non-selective TNF-α receptor antagonist R7050. TNF-α (300 pg/ml, 30 min) decreases thrombin-induced elevation of cytosolic Ca++ levels by 2.2- fold compared to untreated platelets. TNF-α decreases the cAMP levels, while significantly increases the intracellular cyclic cGMP levels. However, the pre-incubation of platelets with the guanylyl cyclase inhibitor ODQ, despite decreasing the cGMP levels, does not modify the inhibitory effect of TNF-α on ADP-induced platelet aggregation. Additionally, western blotting analysis showed that TNF-α significantly reduced (Tyr 416)-c-Src and (Tyr773)-β3 subunit of αIIbβ3 integrin phosphorylation. TNF-α does not affect the platelet viability in any condition tested. ConclusionTherefore, our results show that TNF-α negatively modulates ADP-induced aggregation via TNFR1/TNFR2 receptors by reducing cytosolic Ca++ levels and by inhibiting c-Src and fibrinogen receptor activation, which take place through cAMP- and cGMP-independent mechanisms.

PROTHROMBOTIC STATES IN WOMEN WITH INFERTILITY AND PSYCHOSOMATIC DISORDERS

Failures of in vitro fertilization (IVF) may be associated with prothrombotic states, the circulation of antiphospholipid antibodies (APA).The aim of the study was to establish the value of antiphospholipid antibodies in the effectiveness of infertility treatment by assisted reproductive technologies in women with psychosomatic disorders.Material and methods. 93 women with infertility were screened: 1 group - 32 women without severe psychosomatic disorders; group 2 - 61 women with psychosomatic disorders. The control group consisted of 30 fertile women.Results. Definition in the blood serum of the level of AFA to membrane phospholipids (phosphatidylethanolamine, phosphatidylserine, cardiolipinum), hemostasis (platelet count, ADP-induced platelet aggregation index, fibrinogen concentration, prothrombin index, activated partial thromboplastin time - APTT, D-dimer), homocysteine, molecular genetic study of polymorphous variants for β-fibrinogen (C148T, -455GA) genes. The frequency of significant AFA titres in the group of women without psychosomatic disorders was 18.9%, and if they were present - 44.3%, respectively. Only in 11.5% of women in 2 groups, elevated levels of APA were associated with one or more clinical criteria for antiphospholipid syndrome (APS). In patients with infertility and psychosomatic disorders, increased platelet aggregation in the context of relative thrombocytopenia, higher fibrinogen levels, soluble fibrin-monomeric complexes, and prolonged APTT with elevated D-dimer levels. Some patients have hyperhomocysteinemia. In patients with psychosomatic disorders, the frequency of the minor alleles of the locus C148T and -455GA of the β-fibrinogen gene was greater than 40% (25-30% in group 1).Conclusions. Allocated factors that adversely affect the efficiency of IVF at the patients with psychosomatic disorders: elevation of APA; reduction in the number of platelets; growth of the ADP-induced aggregation index; extension of APTT; increase of fibrinogen, D-dimer; homocysteine; the presence of the minor allele of the T polymorphic locus C148T of the β-fibrinogen gene. The presence of prothrombotic states associated with APS should be taken into account when preparing for IVF and make the appropriate correction for them.

Comparison of platelet function tests for the in vitro quality assessment of platelet concentrates produced under real-life conditions

Platelet quality in different platelet concentrates (PCs) has been the subject of several studies. Nonetheless, there is a lack of robust data on the correlation and agreement among platelet function tests as a prerequisite for the association of PC functionality in vitro with platelet function in vivo post PC transfusion. The purpose of our study was to correlate a larger panel of platelet function assays in PCs and to assess whether the methods agree sufficiently and can be used interchangeably. Twelve apheresis platelet concentrates in plasma (APC), 16 pooled platelet concentrates in plasma (PPC), and 12 PPC in T-sol (PPCA) were examined on days 1 and 4 after production. PCs were tested for platelet count, light transmission aggregation (LTA) induced by ADP, collagen, or TRAP; platelet ATP release induced by collagen; and spontaneous and ADP and TRAP-induced increase in CD62P and PAC1 expression measured by flow cytometry. All tests were performed in undiluted platelet-rich plasma, recalcified and mixed with an inhibitor of factor Xa and thrombin. Most platelet function parameters correlated significantly with each other, but agreement among methods was insufficient. A proper inverse correlation was observed between ADP-induced LTA and spontaneous platelet activation assessed by CD62P expression (r = −0.61, p < 0.0001). Spontaneous CD62P correlated also significantly with spontaneous PAC1 (r = 0.69, p < 0.0001) and inversely with TRAP-induced CD62P expression (r = −0.86, p < 0.0001). We found significant correlations among all flow cytometric assays measuring platelet CD62P and PAC1 expression induced by ADP or TRAP. Subsequent Bland Altman analysis revealed insufficient agreement between methods. With one exception (collagen-induced LTA compared with TRAP-induced LTA, percentage error = 16%) the limits of agreement expressed as percentage error exceeded the chosen acceptable difference of 30%.In APC, platelet count was 41% and 44% higher, respectively, than in PPC and PPCA (p < 0.0001). Spontaneous CD62P and PAC1 expression were significantly greater, and ADP-induced aggregation and agonist-induced increase in CD62P and PAC1 were significantly lower in PPCA compared to APC and PPC on day 4 of storage. ADP and TRAP-induced CD62P and PAC1 activatability fell significantly during storage between day 1 and day 4 in APC and PPCA, but not in PPC. In conclusion, different platelet function tests capture different aspects of platelet function and do not correlate and agree sufficiently to be used interchangeably.

Synergistic inhibitory effect of capsaicin and dihydrocapsaicin on in-vitro platelet aggregation and thromboxane formation.

: Capsaicinoids, including capsaicin (CAP) and dihydrocapsaicin (DHC), the pungent principles of pepper fruits, individually inhibit in-vitro platelet aggregation. However, their effects, when present together, are not known. The aims of this study were to compare the effects of CAP and DHC alone, and in combination in the ratio that they are found in chilies (∼60% CAP : 40% DHC), on in-vitro platelet aggregation, platelet count and thromboxane B2 (TXB2) formation. The effects of 12.5 and 6.25 μmol/l CAP and DHC individually, and in combination (CAP : DHC, 60 : 40) on arachidonic acid-induced, ADP-induced and collagen-induced aggregation, were investigated. Platelet count was determined preincubation and postincubation with CAP and DHC and in combination. TXB2 formation from platelets treated with arachidonic acid in the absence and presence of CAP and DHC individually, and in combination, was measured. Compared with control, CAP and DHC (12.5 μmol/l) inhibited arachidonic acid-induced aggregation by 23.2 and 25.3%, respectively (both P < 0.01). In combination, CAP and DHC exhibited further inhibition in arachidonic acid-induced aggregation (CAP : DHC, 3.75 : 2.5 μmol/l, 36.5%, P = 0.01; 7.5 : 5 μmol/l, 57.5%, P < 0.001), compared with control. Incubation of platelets with caspaicinoids did not significantly affect the platelet count. In addition, the CAP : DHC (7.5 : 5 μmol/l) combination significantly inhibited (P < 0.001) TXB2 formation, compared with the individual capsaicinoids. Capsaicinoids had no effect ADP-induced or collagen-induced aggregation. The combination of CAP and DHC produces a significantly greater inhibitory effect on arachidonic acid-induced platelet aggregation and subsequent TXB2 formation, compared with the individual capsaicinoids.

Application of Feedback System Control Optimization Technique in Combined Use of Dual Antiplatelet Therapy and Herbal Medicines.

Aim: Combined use of herbal medicines in patients underwent dual antiplatelet therapy (DAPT) might cause bleeding or thrombosis because herbal medicines with anti-platelet activities may exhibit interactions with DAPT. In this study, we tried to use a feedback system control (FSC) optimization technique to optimize dose strategy and clarify possible interactions in combined use of DAPT and herbal medicines.Methods: Herbal medicines with reported anti-platelet activities were selected by searching related references in Pubmed. Experimental anti-platelet activities of representative compounds originated from these herbal medicines were investigated using in vitro assay, namely ADP-induced aggregation of rat platelet-rich-plasma. FSC scheme hybridized artificial intelligence calculation and bench experiments to iteratively optimize 4-drug combination and 2-drug combination from these drug candidates.Results: Totally 68 herbal medicines were reported to have anti-platelet activities. In the present study, 7 representative compounds from these herbal medicines were selected to study combinatorial drug optimization together with DAPT, i.e., aspirin and ticagrelor. FSC technique first down-selected 9 drug candidates to the most significant 5 drugs. Then, FSC further secured 4 drugs in the optimal combination, including aspirin, ticagrelor, ferulic acid from DangGui, and forskolin from MaoHouQiaoRuiHua. Finally, FSC quantitatively estimated the possible interactions between aspirin:ticagrelor, aspirin:ferulic acid, ticagrelor:forskolin, and ferulic acid:forskolin. The estimation was further verified by experimentally determined Combination Index (CI) values.Conclusion: Results of the present study suggested that FSC optimization technique could be used in optimization of anti-platelet drug combinations and might be helpful in designing personal anti-platelet therapy strategy. Furthermore, FSC analysis could also identify interactions between different drugs which might provide useful information for research of signal cascades in platelet.

Effect of Nanocrystalline Particles of Chitin on Blood Components in Humans and Experimental Animals.

Nanocrystalline particles of chitin in the form of hydrosol in a concentration of 0.63 mg/ml have no effect on aggregation of human platelets and clotting time of platelet-poor plasma in coagulation tests. ADP-induced aggregation of human platelets was inhibited by these nanoparticles in concentrations of 0.63 and 1.00 mg/ml in comparison with the control. Intravenous administration of nanoparticles in a dose of 1 mg/kg to guinea pigs produced no anticoagulant effect. The nanocrystalline particles of chitin can be of interest as potential drug delivery systems.

Functional cyclophilin D moderates platelet adhesion, but enhances the lytic resistance of fibrin

In the course of thrombosis, platelets are exposed to a variety of activating stimuli classified as ‘strong’ (e.g. thrombin and collagen) or ‘mild’ (e.g. ADP). In response, activated platelets adhere to injured vasculature, aggregate, and stabilise the three-dimensional fibrin scaffold of the expanding thrombus. Since ‘strong’ stimuli also induce opening of the mitochondrial permeability transition pore (MPTP) in platelets, the MPTP-enhancer Cyclophilin D (CypD) has been suggested as a critical pharmacological target to influence thrombosis. However, it is poorly understood what role CypD plays in the platelet response to ‘mild’ stimuli which act independently of MPTP. Furthermore, it is unknown how CypD influences platelet-driven clot stabilisation against enzymatic breakdown (fibrinolysis). Here we show that treatment of human platelets with Cyclosporine A (a cyclophilin-inhibitor) boosts ADP-induced adhesion and aggregation, while genetic ablation of CypD in murine platelets enhances adhesion but not aggregation. We also report that platelets lacking CypD preserve their integrity in a fibrin environment, and lose their ability to render clots resistant against fibrinolysis. Our results indicate that CypD has opposing haemostatic roles depending on the stimulus and stage of platelet activation, warranting a careful design of any antithrombotic strategy targeting CypD.

Mechanistic insights into functional characteristics of native crotamine

The chemical composition of snake venoms is a complex mixture of proteins and peptides that can be pharmacologically active. Crotamine, a cell-penetrating peptide, has been described to have antimicrobial properties and it exerts its effects by interacting selectively with different structures, inducing changes in the ion flow pattern and cellular responses. However, its real therapeutic potential is not yet fully known. Bearing in mind that crotamine is a promising molecule in therapeutics, this study investigated the action of purified molecule in three aspects: I) antibacterial action on different species of clinical interest, II) the effect of two different concentrations of the molecule on platelet aggregation, and III) its effects on isolated mitochondria. Crotamine was purified to homogeneity in a single step procedure using Heparin Sepharose. The molecular mass of the purified enzyme was 4881.4 Da, as determined by mass spectrometry. To assess antibacterial action, changes in the parameters of bacterial oxidative stress were determined. The peptide showed antibacterial activity on Escherichia coli (MIC: 2.0 μg/μL), Staphylococcus aureus (MIC: 8–16 μg/μL) and methicillin-resistant Staphylococcus aureus (MIC: 4.0–8.0 μg/μL), inducing bacterial death by lipid peroxidation and oxidation of target proteins, determined by thiobarbituric acid reactive substances and sulfhydryl groups, respectively. Crotamine induced increased platelet aggregation (IPA) at the two concentrations analyzed (0.1 and 1.4 μg/μL) compared to ADP-induced aggregation of PRP. Mitochondrial respiratory parameters and organelle structure assays were used to elucidate the action of the compound in this organelle. The exposure of mitochondria to crotamine caused a decrease in oxidative phosphorylation and changes in mitochondrial permeability, without causing damage in the mitochondrial redox state. Together, these results support the hypothesis that, besides the antimicrobial potential, crotamine acts on different molecular targets, inducing platelet aggregation and mitochondrial dysfunction.

Inhibitory Effect of Flavonolignans on the P2Y12 Pathway in Blood Platelets.

Adenosine diphosphate (ADP) is the major platelet agonist, which is important in the shape changes, stability, and growth of the thrombus. Platelet activation by ADP is associated with the G protein-coupled receptors P2Y1 and P2Y12. The pharmacologic blockade of the P2Y12 receptor significantly reduces the risk of peripheral artery disease, myocardial infarction, ischemic stroke, and vascular death. Recent studies demonstrated the inhibition of ADP-induced blood platelet activation by three major compounds of the flavonolignans group: silybin, silychristin, and silydianin. For this reason, the aim of the current work was to verify the effects of silybin, silychristin, and silydianin on ADP-induced physiological platelets responses, as well as mechanisms of P2Y12-dependent intracellular signal transduction. We evaluated the effect of tested flavonolignans on ADP-induced blood platelets’ aggregation in platelet-rich plasma (PRP) (using light transmission aggregometry), adhesion to fibrinogen (using the static method), and the secretion of PF-4 (using the ELISA method). Additionally, using the double labeled flow cytometry method, we estimated platelet vasodilator-stimulated phosphoprotein (VASP) phosphorylation. We demonstrated a dose-dependent reduction of blood platelets’ ability to perform ADP-induced aggregation, adhere to fibrinogen, and secrete PF-4 in samples treated with flavonolignans. Additionally, we observed that all of the tested flavonolignans were able to increase VASP phosphorylation in blood platelets samples, which is correlated with P2Y12 receptor inhibition. All of these analyses show that silychristin and silybin have the strongest inhibitory effect on blood platelet activation by ADP, while silydianin also inhibits the ADP pathway, but to a lesser extent. The results obtained in this study clearly demonstrate that silybin, silychristin, and silydianin have inhibitory properties against the P2Y12 receptor and block ADP-induced blood platelet activation.

Vitamin D diminishes the high platelet aggregation of type 2 diabetes mellitus patients

Platelet activation is found in inflammatory conditions and implicated in the pathogenesis of chronic medical conditions, such as atherosclerosis, coronary vascular disease, cerebrovascular disease, and diabetes mellitus (DM). HbA1c is inversely related to vitamin D25 levels in individuals with and without DM. This study aimed to determine the relation between platelet aggregation, vitamin D and HbA1c among healthy individuals and those with Type 2 DM (T2DM). The direct effect of vitamin D1, 25 (calcitriol) on platelet aggregation was also investigated. The study included four groups: A. normoglycemic Control group: HbA1c<5.7%; B. Pre-diabetes (DM): 5.7% ≥ HbA1c ≤ 6.4%; C. DM on aspirin therapy: HbA1c>6.4%(+)Asp.; and D. DM not on aspirin therapy: HbA1c > 6.4%(−)Asp. Platelet aggregation was tested with and without calcitriol or saline pre-treatment, using collagen or adenosine diphosphate (ADP) as agonists. Platelet aggregation was higher in DM(−)Asp group compared to normoglycemic and DM(+)Asp, and higher, but not significant compared to pre-DM. The entire study population exhibited negative correlation between HbA1c and serum concentration of vitamin D25. Excluding DM(+)Asp, aggregation induced by collagen was significantly higher in patients with insufficient (<76 nmol/L) vitamin D25 compared to sufficient (≥76 nmol/L) vitamin D25. In this cohort, a negative correlation was found between serum concentrations of vitamin D25 and collagen-induced percent maximum (%max) aggregation and area under curve (AUC) aggregation. In the DM(−)Asp group, collagen-induced aggregation was reduced by approximately 25% after calcitriol treatment. Calcitriol decreased ADP-induced aggregation of control and DM(+)Asp groups to approximately 85% of saline treatment. We conclude that glycemic control is inversely associated with high platelet aggregation and low vitamin D25 levels. This elevated aggregation could be regulated by a novel, direct effect of calcitriol, indicating a beneficial effect of vitamin D on vascular complications related to diabetes. We offer a possible non-genomic mechanism for the vitamin D/Vitamin D receptor (VDR) pathway.

Effects of Juglans regia Root Bark Extract on Platelet Aggregation, Bleeding Time, and Plasmatic Coagulation: In Vitro and Ex Vivo Experiments.

Platelets have an important role in thrombosis and haemostasis. Hyperactivity of the platelets has been associated with thromboembolic diseases and represents the main cause of complications of cardiovascular diseases. Crude aqueous extract (CAE) of Juglans regia root bark was evaluated for bleeding time, antiaggregant activity by using agonists, thrombin, ADP, collagen, or arachidonic acid (in vitro and ex vivo), and anticoagulant activity by measuring the clotting parameters: activated partial thromboplastin time, prothrombin time, thrombin time, and fibrinogen dosage (in vitro and ex vivo). The result of this study reported that the strongest antiaggregant effect of CAE in vitro was observed on the ADP-induced aggregation with inhibitions up to 90 %, while, in ex vivo experiments, the inhibition (more than 80 %) was observed with all agonists. Anticoagulant effect of CAE significantly prolonged the TT and decreased the fibrinogen level in vitro and ex vivo without interfering with APTT and PT. The bleeding time in mice and rats was significantly increased by CAE. The antiplatelet and anticoagulant effect observed in this study suggest that Juglans regia could have antithrombotic and/or thrombolytic activities and provide an alternative therapy against thrombotic complications related to cardiovascular diseases.

Perioperative changes in platelet count and function in patients undergoing cardiac surgery

Background: Patients undergoing cardiac surgery are at increased risk of bleeding due to multifactorial coagulopathies. In the present study, we aimed at investigating the changes in platelet count and function during and after surgery as well as determining the association of the platelet dysfunction with bleeding and transfusion requirements in these patients. Methods: A total of 40 adult patients scheduled for elective valve coronary cardiac surgery were included in this prospective observational study. Changes in platelet count and function with ADP, acid arachidonic, and collagen (light transmission aggregometry) were analyzed at three time points: before CPB, after CPB, and 24 hours after end of surgery. Postoperative bleeding and intraoperative transfusion requirements were recorded. Results: There were a significant reverse correlation between CPB time and ADP-induced aggregation, particularly after CPB and postoperative AA-induced aggregation. There was not any significant correlation between platelet count and function at all-time points. Both platelet count and platelet aggregation significantly reduced during CPB. While platelet aggregation increased on postoperative Day 1, platelet count reduced by about 40% after CPB, and remained at this level postoperatively. Patients with abnormal ADP-induced aggregation had significant increased postoperative bleeding and transfusion requirements. Conclusion: The results of this study demonstrate that platelet count and platelet aggregation are reduced during CPB. Our results emphasized the effect of platelet dysfunction on increased postoperative bleeding and transfusion requirements. Perioperative monitoring of platelet function can be considered as a bleeding management strategy for implantation of PBM programs.

The role of adenosine diphosphate mediated platelet responsiveness for the stability of platelet integrity in citrated whole blood under ex vivo conditions.

BackgroundPlatelets are important for effective hemostasis and considered to be involved in pathophysiological processes, e.g. in cardiovascular diseases. Platelets provided for research or for therapeutic use are frequently separated from citrated whole blood (WB) stored for different periods of time. Although functionally intact platelets are required, the stability of platelet integrity, e.g. adenosine diphosphate (ADP) mediated responsiveness, has never been thoroughly investigated in citrated WB under ex vivo conditions.ObjectivesPlatelet integrity was evaluated at different time points in citrated WB units, collected from healthy donors and stored for 5 days at ambient temperature. The analysis included the measurement of activation markers, of induced light transmission aggregometry and of purinergic receptor expression or function. Inhibitory pathways were explored by determination of basal vasodilator-stimulated phosphoprotein (VASP)-phosphorylation, intracellular cyclic nucleotide levels and the content of phosphodiesterase 5A. Fresh peripheral blood (PB) samples served as controls.ResultsOn day 5 of storage, thrombin receptor activating peptide-6 (TRAP-6) stimulated CD62P expression and fibrinogen binding were comparable to PB samples. ADP induced aggregation continuously decreased during storage. Purinergic receptor expression remained unchanged, whereas the P2Y1 activity progressively declined in contrast to preserved P2Y12 and P2X1 function. Inhibitory pathways were unaffected except for a slight elevation of VASP phosphorylation at Ser239 on day 5.ConclusionAfter 5 days of storage in citrated WB, platelet responsiveness to TRAP-6 is sufficiently maintained. However, ADP-mediated platelet integrity is more sensitive to deterioration, especially after storage for more than 2 days. Decreasing ADP-induced aggregation is particularly caused by the impairment of the purinergic receptor P2Y1 activity. These characteristics should be considered in the use of platelets from stored citrated WB for experimental or therapeutic issues.

Neuromedin U potentiates ADP- and epinephrine-induced human platelet activation

Neuromedin U (NmU) is a pleiotropic hypothalamic neuropeptide involved in the gut–brain axis. It acts via both a Gαq/11-coupled receptor (NMUR1) and a Gαi-coupled receptor (NMUR2) in different cell types. Expression of both receptors was reported in platelets, but their significance for NmU signaling remains elusive. We studied the potential effects of NmU on human platelet activation. In platelet-rich plasma (PRP), NmU alone (up to 10μM) did not induce any measurable aggregation, but at nanomolar concentrations, it potentiated platelet aggregation by low (mean 0.47μM) ADP concentrations (from 25.9±3.6% to 74.8±2.7% maximal aggregation for ADP vs. ADP+NmU, 100nM, mean±SEM, n=13), accompanied by platelet P-selectin expression and intracellular calcium mobilization. Accordingly, platelet preincubation with NmU for 2min sensitized platelets for subsequent activation by ADP. When P2Y1 was inactivated by 50μM MRS2179, NmU comparably potentiated ADP-induced PRP aggregation, suggestive of cooperative activation with Gαi-coupled P2Y12. Likewise, NmU potentiated platelet aggregation by Gαi-operated epinephrine at subthreshold concentrations (99ng/ml, mean), but not that by Gαq-dependent serotonin (20μM). Platelet aggregation by NmU/epinephrine combination was fully inhibited by the Gαq inhibitor YM-254890 (1μM). qPCR detection and western blot analysis substantiated platelet expression of NMUR1 in different donors, a finding collectively complying with functionally relevant Gαq/11-mediated activation of platelet NMUR1 by NmU. Our findings advocate further studies on platelet sensitization by NmU, released during vascular activation and injury, to define its role as a modifier of platelet responsiveness to the physiological activation signals, operational in cardiovascular health and disease.

A Selective Biligand Inhibitor of CK2 Increases Caspase-3 Activity in Cancer Cells and Inhibits Platelet Aggregation.

Cancer cells express high levels of CK2, and its inhibition leads to apoptosis. CK2 has therefore emerged as a new drug target for cancer therapy. A biligand inhibitor ARC-772 was constructed by conjugating 4-(2-amino-1,3-thiazol-5-yl)benzoic acid and a carboxylate-rich peptoid. ARC-772 was found to bind CK2 with a Kd value of 0.3 nm and showed remarkable CK2 inhibitory selectivity in a panel of 140 protein kinases (Gini coefficient: 0.75 at c=100 nm). ARC-775, the acetoxymethyl ester prodrug of ARC-772, was efficiently taken up by cells. Once internalized, the inhibitor is activated by cellular esterase activity. In HeLa cancer cells ARC-775 was found to activate caspase-3 (an apoptosis marker) at sub-micromolar concentrations (EC50 =0.3 μm), a 20-fold lower extracellular concentration than CX-4945, the only CK2 inhibitor under clinical trials. At micromolar concentrations, ARC-775 was also found to inhibit ADP-induced aggregation of human platelets. The overall results of this study demonstrate that oligo-anionic biligand inhibitors have good potential for drug development.

Platelet aggregation under the action of drugs of glucosamine hydrochloride and acetylsalicylic acid in acute cold injury

General and local cooling is a common factor of the effect of the environment on the human body. Exposure to cold can act as a damaging factor causing development of the destructive processes with the various degree of severity in tissues. It causes a complex reaction in the organism, which functioning in conditions of hypothermia greatly depends on the state of the blood system, including its rheological properties.Aim. To determine the effect of the drugs studied on hemostasis, including platelet aggregation under conditions of acute cold injury.Materials and methods. The study was performed in summer on white rats male weighing 280-320 g (24 rats); they were kept in standard vivarium conditions in accordance with the GLP rules.Results. The state of platelet aggregation and the rectal temperature after acute cold injury (general cooling) in rats has been studied. When using “Glucosamine-C BCPP” and glucosamine hydrochloride the rectal temperature significantly increased in relation to the control pathology group, it indicated the improved condition of the animals. Acetylsalicylic acid (ASA) caused death of 50 % of the rats and did not affect the temperature decrease. Cold injury was accompanied by a significant (1.7 times) increase in the extent of ADP- and epinephrine-induced platelet aggregation, and the speed increased by 1.6-1.9 times. “Glucosamine-C BCPP” and glucosamine hydrochloride significantly reduced the intensity of ADP-induced aggregation, not being inferior ASA, and the rate of this process was at the trend level.Conclusions. In contrast to the moderate antiplatelet effect of “Glucosamine-С BCPP” and glucosamine hydrochloride on the model of adrenaline-induced aggregation ASA caused an excessive effect with 1.5 times reduction in the intensity of aggregation compared to the intact control. This fact indicates the danger of bleedings and confirms the benefits of drugs of glucosamine as frigoprotective agents.

ANTIPLATELET TREATMENT IN ST ELEVATION ACUTE CORONARY SYNDROME UNDER REAL CLINICAL CIRCUMSTANCES: THE DYNAMICS OF THROMBOCYTE FUNCTION

Aim. To evaluate the dynamics of thrombocyte functional activity (TFA) under antiplatelet treatment conditions which include the branded, and generic compounds as well, in ST elevation acute coronary syndrome patients (STEACS) in routine clinical practice. Material and methods. The open-label prospective study was done, including STEACS patients stratified according to the kind of antiplatelet in-patient treatment (original and/or generics). As an endpoint, we used the surrogate — functional activity of thrombocytes (TFA), measured by impedance and luminescent aggregatometry at 1 and 7 day from STEACS onset. Results. By the inclusion, baseline point all patients were comparable by TFA. On double antiplatelet therapy (DAT) by the 7th day of STEACS there was statistically significant difference of all ADP-induced thrombocyte aggregation. There was difference in ADP-induced platelet aggregation depending on the DAT variant, which included the original drug and generic. Conclusion. In STEACS patients the level of aggregation activity of platelets does significantly differ from the kind of antiplatelet treatment. Usage of the branded and generic compounds of the came antiplatelet agent by the same regimen does differ by different grade and dynamics of platelets activeness suppression.

Whole exome sequencing in the Framingham Heart Study identifies rare variation in HYAL2 that influences platelet aggregation.

Inhibition of platelet reactivity is a common therapeutic strategy in secondary prevention of cardiovascular disease. Genetic and environmental factors influence inter-individual variation in platelet reactivity. Identifying genes that contribute to platelet reactivity can reveal new biological mechanisms and possible therapeutic targets. Here, we examined rare coding variation to identify genes associated with platelet reactivity in a population-based cohort. To do so, we performed whole exome sequencing in the Framingham Heart Study and conducted single variant and gene-based association tests against platelet reactivity to collagen, adenosine diphosphate (ADP), and epinephrine agonists in up to 1,211 individuals. Single variant tests revealed no significant associations (p<1.44×10-7), though we observed a suggestive association with previously implicated MRVI1 (rs11042902, p = 1.95×10-7). Using gene-based association tests of rare and low-frequency variants, we found significant associations of HYAL2 with increased ADP-induced aggregation (p = 1.07×10-7) and GSTZ1 with increased epinephrine-induced aggregation (p = 1.62×10-6). HYAL2 also showed suggestive associations with epinephrine-induced aggregation (p = 2.64×10-5). The rare variants in the HYAL2 gene-based association included a missense variant (N357S) at a known N-glycosylation site and a nonsense variant (Q406*) that removes a glycophosphatidylinositol (GPI) anchor from the resulting protein. These variants suggest that improper membrane trafficking of HYAL2 influences platelet reactivity. We also observed suggestive associations of AR (p = 7.39×10-6) and MAPRE1 (p = 7.26×10-6) with ADP-induced reactivity. Our study demonstrates that gene-based tests and other grouping strategies of rare variants are powerful approaches to detect associations in population-based analyses of complex traits not detected by single variant tests and possible new genetic influences on platelet reactivity.

Assessment of platelet function in patients receiving tirofiban early after primary coronary intervention.

BackgroundFollowing percutaneous coronary intervention, combined antiplatelet therapy is necessary. Platelet function testing (PFT) has prognostic value and may be applied in the risk assessment of acute coronary syndrome. In case of combined antiplatelet therapy, PFT may require special laboratory methods, as different antiplatelet agents may influence test results.Materials and methodsPlatelet functions were measured in stent thrombosis-segment elevation myocardial infarction patients receiving aspirin, clopidogrel, and tirofiban. The first sampling was obtained immediately after the termination of administration of tirofiban. The second sample was drawn at a randomly assigned time between 1 and 6 h. The third sampling was done after a minimum of 24 h of tirofiban cessation. Adenosine diphosphate (ADP)- and thrombin receptor-activating peptide (TRAP)-induced aggregations were measured.ResultsThirty-seven patients were included. Both TRAP- and ADP-induced aggregation values were significantly lower immediately after tirofiban termination, than after 24 h [TRAP: 26.41 ± 25.00 units (U) vs. 109.86 ± 23.69 U, p < 0.0001; ADP: 17.43 ± 10.10 U vs. 43.92 ± 23.35 U, p ≤ 0.0001]. Elimination half-life of tirofiban and clopidogrel were 1.34 ± 0.49 and 1.269 ± 0.78, respectively.ConclusionADP-induced residual platelet reactivity is significantly influenced by the presence of concurrent glycoprotein IIb/IIIa inhibitor. In patients receiving combined antiplatelet treatment, ADP-receptor-specific efficiency measurements are valid only after total elimination of GPIIb/IIIa inhibitors.