Traditional Chinese medicine practices used in COVID-19 (Sars-cov 2/Coronavirus-19) treatment in clinic and their effects on the cardiovascular system.

OBJECTIVE: The aim of this study was to evaluate the effectiveness of plants used in the formulations of traditional Chinese medicine (TCM), which were also used in clinical trials to treat patients with the novel coronavirus COVID-19, and to assess their effects on the cardiovascular system. METHODS: A literature review of PubMed, ResearchGate, ScienceDirect, the Cochrane Library, and TCM monographs was conducted and the effects of the plants on the cardiovascular system and the mechanisms of action in COVID-19 treatment were evaluated. RESULTS: The mechanism of action, cardiovascular effects, and possible toxicity of 10 plants frequently found in TCM formulations that were used in the clinical treatment of COVID-19 were examined. CONCLUSION: TCM formulations that had been originally developed for earlier viral diseases have been used in COVID-19 treatment. Despite the effectiveness seen in laboratory and animal studies with the most commonly used plants in these formulations, the clinical studies are currently insufficient according to standard operating procedures. More clinical studies are needed to understand the safe clinical use of traditional plants.

A Pharmacodynamic Study of CN-218, a Novel Antiplatelet and Antithrombotic Agent Primarily Targeting the P2Y12 Receptor.

PURPOSE: Drugs inhibiting the platelet P2Y12 receptor, such as clopidogrel and prasugrel, are potent antithrombotic agents and are widely used in cardiovascular disease. However, the adverse effects of these drugs have limited their clinical use. For example, clopidogrel resistance occurs in approximately one third of patients, while prasugrel increases the risk of major bleeding. Therefore, new generations of such drugs are of clinical interest. METHODS: In this study, the pharmacodynamics of a new P2Y12 antagonist, CN-218, was compared with that of clopidogrel and prasugrel in rats and mice. The differences between CN-218 and clopidogrel include deuteration of the 7-position methyl carboxylate and the introduction of cinnamate in the 2-position of thiophene. RESULTS: CN-218 had an antiaggregatory efficacy that was at least five times more potent than that of clopidogrel but not as potent as that of prasugrel. It had a significant impact on activated partial thromboplastin time (APTT), whereby the APTT of CN-218-treated rats was approximately 9 s longer than that of the vehicle- or clopidogrel-treated group, while it had no impact on prothrombin time (PT) in rats. CN-218 had a similar potent antithrombotic effect to that of prasugrel and clopidogrel and also reduced the risk of bleeding compared to prasugrel. CONCLUSION: CN-218 may be a promising antithrombotic agent, with potent antiplatelet and significant anticoagulant activity, as well as a lower risk of bleeding compared to clopidogrel and prasugrel.

The activation of high mobility group Box1 and toll-like receptor 4 is involved in clopidogrel-induced gastric injury through p38 MAPK.

Clopidogrel-induced gastric injury is an important clinical problem. However, the exact mechanism was still unclarified. This study aimed to investigate the role of high mobility group box protein 1 (HMGB1) and the toll-like receptor 4 (TLR4) pathway in normal human gastric epithelial (GES-1) cells under clopidogrel exposure. Morphological alterations of the gastric epithelial cells were observed under a microscope. A laser scanning confocal microscope was used to determine the distribution of HMGB1 and TLR4 in clopidogrel-induced injury. MTT was used to compare the viability of GES-1 cell among the pretreated Cli-095 (TLR4 inhibitor) group, pretreated ethyl pyruvate (HMGB1 inhibitor) group, clopidogrel group, and control group. Moreover, expression of the HMGB1, TLR4, tight junction (TJ) proteins occludin and the phosphorylation of the mitogen-activated protein kinases (MAPK) p38 were examined by western blot. We found the expression of HMGB1 and TLR4 in the cytoplasm increased after clopidogrel administration. Besides, inhibited TLR4, which is a receptor of HMGB1, prevented the injury and occludin reducing caused by clopidogrel challenge. Furthermore, blocking HMGB1 or TLR4 activity by ethyl pyruvate (HMGB1 inhibitor) or cli-095(TLR4 inhibitor) can partially diminish the activation of p38MAPK. Gastric mucosal damages observed by clopidogrel challenge are associated with HMGB1, TLR4, through p38MAPK signal pathway.

Pioglitazone prevents sevoflurane­induced neuroinflammation and cognitive decline in a rat model of chronic intermittent hypoxia by upregulating hippocampal PPAR­Î³.

Post­operative cognitive dysfunction is a common complication after anesthesia and surgery. Sevoflurane (SEV), a widely used inhalational anesthetic, can exaggerate neuroinflammation and cause cognitive dysfunction under chronic intermittent hypoxia (CIH) conditions by downregulating hippocampal peroxisome proliferator­activated receptor­Î³ (PPAR­Î³). In the present study, it was examined whether treatment with PPAR­Î³ agonist pioglitazone (PIO) is beneficial in counteracting SEV­induced neuroinflammation and cognitive decline in a rat model of CIH. Rats were exposed to CIH for 4 weeks. After 2 weeks of CIH, these animals underwent either 2.6% SEV or control (CON) exposure for 4 h. PIO (60 mg/kg) or vehicle (VEH) was administered orally twice daily for 2 weeks, starting one day prior to SEV or CON exposure. Compared with CIH­CON+VEH rats, CIH­SEV+VEH rats exhibited significant cognitive decline as indicated by increased latency to locate the hidden platform and shorter dwell­time in the goal quadrant in the Morris Water Maze task. Molecular studies revealed that CIH­SEV+VEH rats had increased proinflammatory cytokine expression and microglial activation in the hippocampus, which were associated with decreased PPAR­Î³ activity. Notably, SEV­induced cognitive decline and increases in proinflammatory cytokine expression and microglial activation were prevented by PIO, which increased hippocampal PPAR­Î³ activity. PIO also increased hippocampal PPAR­Î³ activity in CIH­CON rats but did not alter proinflammatory cytokine expression and microglial activation as well as cognitive function. Additionally, expression of hippocampal PPAR­α and PPAR­ß, two other PPAR isotypes, were comparable among the groups. These data suggest that PIO prevents SEV­induced exaggeration of neuroinflammation and cognitive decline under CIH conditions by upregulating hippocampal PPAR­Î³. PIO may have the potential to prevent anesthetic SEV­induced cognitive decline in surgical patients with obstructive sleep apnea.

Optimizing Platelet GPVI Inhibition versus Haemostatic Impairment by the Btk Inhibitors Ibrutinib, Acalabrutinib, ONO/GS-4059, BGB-3111 and Evobrutinib.

Ibrutinib and acalabrutinib are approved for B cell malignancies and novel Bruton's tyrosine kinase (Btk) inhibitors undergo clinical testing also in B cell-driven autoimmune disorders. Btk in platelets mediates platelet activation via glycoprotein (GP) VI, which is crucial for atherosclerotic plaque-induced platelet thrombus formation. This can be selectively inhibited by Btk inhibitors. Since patients on second-generation Btk inhibitors apparently show less bleeding than patients on ibrutinib, we compared the effects of ibrutinib and four novel irreversible Btk inhibitors on GPVI-dependent platelet aggregation in blood and in vitro bleeding time. Low concentrations of collagen which induced the same low degree of GPVI-mediated platelet aggregation as atherosclerotic plaque material were applied. IC50 values for collagen (0.2-0.5 µg/mL)-induced platelet aggregation after 15-minute pre-incubation were: ibrutinib 0.12 µM, BGB-3111 0.51 µM, acalabrutinib 1.21 µM, ONO/GS-4059 1.20 µM and evobrutinib 5.84 µM. Peak venous plasma concentrations of ibrutinib (0.5 µM), acalabrutinib (2 µM) and ONO/GS-4059 (2 µM) measured after anti-proliferative dosage inhibited collagen-induced platelet aggregation, but did not increase PFA-200 closure time on collagen/epinephrine. Closure times were moderately increased by 2- to 2.5-fold higher concentrations of these inhibitors, but not by BGB-3111 (1 µM) and evobrutinib (10 µM). Prolonging platelet drug exposure to 60 minutes lowered IC50 values of any Btk inhibitor for GPVI-mediated aggregation by several fold, and 5- to 10-fold below anti-proliferative therapeutic drug plasma levels. In conclusion, low blood concentrations of ibrutinib and the novel Btk inhibitors suffice for GPVI selective platelet inhibition relevant for atherothrombosis but do not impair primary haemostasis.

Sevoflurane impairs learning and memory of the developing brain through post-transcriptional inhibition of CCNA2 via microRNA-19-3p.

The molecular mechanisms underlying sevoflurane (SEVO)-induced impairment of learning and memory remain unclear. Specifically, a role of microRNAs (miRNAs) in the control of the neuron proliferation in the developing brain exposed to SEVO has not been reported previously. Here, we studied the effects of SEVO exposure on the neural cell proliferation, and on the learning and memory of neonatal rats. We found that SEVO exposure significantly decreased neuron cell proliferation, reduced BDNF levels in brain, and impaired learning and memory of neonatal rats in Morris water maze test and Plus-Maze discriminative avoidance task (PM-DAT), likely through downregulation of CCNA2 protein. Next, we used bioinformatic tools to predict CCNA2-binding microRNAs (miRNAs), and found that miR-19-3p was upregulated in neurons exposed to SEVO. Moreover, miR-19-3p functionally inhibited the protein translation of CCNA2 in a human neural cell line, HCN-2. Furthermore, intracranial injection of adeno-associated virus carrying antisense of miR-19-3p under a CMV promoter into the neonatal rats significantly alleviated SEVO exposure-induced impairment of neuron cell proliferation, as well as the learning and memory of the rats. Together, our data suggest that SEVO-induced upregulation of miR-19-3p post-transcriptionally inhibits CCNA2, which contributes to the SEVO-associated impairment of learning and memory of the neonatal rats.

An αIIb ß3 antagonist prevents thrombosis without causing Fc receptor γ-chain IIa-mediated thrombocytopenia.

Essentials FcγRIIa-mediated thrombocytopenia is associated with drug-dependent antibodies (DDAbs). We investigated the correlation between αIIb ß3 binding epitopes and induction of DDAbs. An FcγRIIa-transgenic mouse model was used to evaluate thrombocytopenia among anti-thrombotics. An antithrombotic with binding motif toward αIIb ß-propeller domain has less bleeding tendency. SUMMARY: Background Thrombocytopenia, a common side effect of Arg-Gly-Asp-mimetic antiplatelet drugs, is associated with drug-dependent antibodies (DDAbs) that recognize conformation-altered integrin αIIb ß3 . Objective To explore the correlation between αIIb ß3 binding epitopes and induction of DDAb binding to conformation-altered αIIb ß3 , we examined whether two purified disintegrins, TMV-2 and TMV-7, with distinct binding motifs have different effects on induction of αIIb ß3 conformational change and platelet aggregation in the presence of AP2, an IgG1 inhibitory mAb raised against αIIb ß3 . Methods We investigated the possible mechanisms of intrinsic platelet activation of TMV-2 and TMV-7 in the presence of AP2 by examining the signal cascade, tail bleeding time and immune thrombocytopenia in Fc receptor γ-chain IIa (FcγRIIa) transgenic mice. Results TMV-7 has a binding motif that recognizes the αIIb ß-propeller domain of αIIb ß3 , unlike that of TMV-2. TMV-7 neither primed the platelets to bind ligand, nor caused a conformational change of αIIb ß3 as identified with the ligand-induced binding site mAb AP5. In contrast to eptifibatide and TMV-2, cotreatment of TMV-7 with AP2 did not induce FcγRIIa-mediated platelet aggregation and the downstream activation cascade. Both TMV-2 and TMV-7 efficaciously prevented occlusive thrombosis in vivo. Notably, both eptifibatide and TMV-2 caused severe thrombocytopenia mediated by FcγRIIa, prolonged tail bleeding time in vivo, and repressed human whole blood coagulation indexes, whereas TMV-7 did not impair hemostatic capacity. Conclusions TMV-7 shows antiplatelet and antithrombotic activities resulting from a mechanism different from that of all other tested αIIb ß3 antagonists, and may offer advantages as a therapeutic agent with a better safety profile.

ADPase CD39 Fused to Glycoprotein VI-Fc Boosts Local Antithrombotic Effects at Vascular Lesions.

BACKGROUND: GPVI (Glycoprotein VI) is the essential platelet collagen receptor in atherothrombosis. Dimeric GPVI-Fc (Revacept) binds to GPVI binding sites on plaque collagen. As expected, it did not increase bleeding in clinical studies. GPVI-Fc is a potent inhibitor of atherosclerotic plaque-induced platelet aggregation at high shear flow, but its inhibition at low shear flow is limited. We sought to increase the platelet inhibitory potential by fusing GPVI-Fc to the ectonucleotidase CD39 (fusion protein GPVI-CD39), which inhibits local ADP accumulation at vascular plaques, and thus to create a lesion-directed dual antiplatelet therapy that is expected to lack systemic bleeding risks. METHODS AND RESULTS: GPVI-CD39 effectively stimulated local ADP degradation and, compared with GPVI-Fc alone, led to significantly increased inhibition of ADP-, collagen-, and human plaque-induced platelet aggregation in Multiplate aggregometry and plaque-induced platelet thrombus formation under arterial flow conditions. GPVI-CD39 did not increase bleeding time in an in vitro assay simulating primary hemostasis. In a mouse model of ferric chloride-induced arterial thrombosis, GPVI-CD39 effectively delayed vascular thrombosis but did not increase tail bleeding time in vivo. CONCLUSIONS: GPVI-CD39 is a novel approach to increase local antithrombotic activity at sites of atherosclerotic plaque rupture or injury. It enhances GPVI-Fc-mediated platelet inhibition and presents a potentially effective and safe molecule for the treatment of acute atherothrombotic events, with a favorable risk-benefit ratio.

Trowaglerix Venom Polypeptides As a Novel Antithrombotic Agent by Targeting Immunoglobulin-Like Domains of Glycoprotein VI in Platelet.

OBJECTIVE: Currently prescribed antiplatelet drugs have 1 common side effect-an increased risk of hemorrhage and thrombocytopenia. On the contrary, bleeding defects associated with glycoprotein VI (GPVI) expression deficiency are usually slightly prolonged bleeding times. However, GPVI antagonists are lacking in clinic. APPROACH AND RESULTS: Using reverse-phase high-performance liquid chromatography and sequencing, we revealed the partial sequence of trowaglerix α subunit, a potent specific GPVI-targeting snaclec (snake venom C-type lectin protein). Hexapeptide (Troα6 [trowaglerix a chain hexapeptide, CKWMNV]) and decapeptide (Troα10) derived from trowaglerix specifically inhibited collagen-induced platelet aggregation through blocking platelet GPVI receptor. Computational peptide design helped to design a series of Troα6/Troα10 peptides. Protein docking studies on these decapeptides and GPVI suggest that Troα10 was bound at the lower surface of D1 domain and outer surface of D2 domain, which was at the different place of the collagen-binding site and the scFv (single-chain variable fragment) D2-binding site. The newly discovered site was confirmed by inhibitory effects of polyclonal antibodies on collagen-induced platelet aggregation. This indicates that D2 domain of GPVI is a novel and important binding epitope on GPVI-mediated platelet aggregation. Troα6/Troα10 displayed prominent inhibitory effect of thrombus formation in fluorescein sodium-induced platelet thrombus formation of mesenteric venules and ferric chloride-induced carotid artery injury thrombosis model without prolonging the in vivo bleeding time. CONCLUSIONS: We develop a novel antithrombotic peptides derived from trowaglerix that acts through GPVI antagonism with greater safety-no severe bleeding. The binding epitope of polypeptides on GPVI is novel and important. These hexa/decapeptides have therapeutic potential for developing ideal small-mass GPVI antagonists for arterial thrombogenic diseases.

Recombinant GPVI-Fc added to single or dual antiplatelet therapy in vitro prevents plaque-induced platelet thrombus formation.

The efficiency of current dual antiplatelet therapy might be further improved by its combination with a glycoprotein (GP) VI-targeting strategy without increasing bleeding. GPVI-Fc, a recombinant dimeric fusion protein binding to plaque collagen and concealing binding sites for platelet GPVI, acts as a lesion-focused antiplatelet drug, and does not increase bleeding in vivo. We investigated, whether GPVI-Fc added in vitro on top of acetylsalicylic acid (ASA), the P2Y12 antagonist ticagrelor, and the fibrinogen receptor antagonist abciximab alone or in combination would increase inhibition of platelet activation by atherosclerotic plaque. Under static conditions, GPVI-Fc inhibited plaque-induced platelet aggregation by 53 %, and increased platelet inhibition by ASA (51 %) and ticagrelor (64 %) to 66 % and 80 %, respectively. Under arterial flow, GPVI-Fc inhibited plaque-induced platelet aggregation by 57 %, and significantly increased platelet inhibition by ASA (28 %) and ticagrelor (47 %) to about 81 % each. The triple combination of GPVI-Fc, ASA and ticagrelor achieved almost complete inhibition of plaque-induced platelet aggregation (93 %). GPVI-Fc alone or in combination with ASA or ticagrelor did not increase closure time measured by the platelet function analyzer (PFA)-200. GPVI-Fc added on top of abciximab, a clinically used anti-fibrinogen receptor antibody which blocks platelet aggregation, strongly inhibited total (81 %) and stable (89 %) platelet adhesion. We conclude that GPVI-Fc added on top of single or dual antiplatelet therapy with ASA and/or a P2Y12 antagonist is likely to improve anti-atherothrombotic protection without increasing bleeding risk. In contrast, the strong inhibition of platelet adhesion by GPVI-Fc in combination with GPIIb/IIIa inhibitors could be harmful.

Toxin Fused with SUMO Tag: A New Expression Vector Strategy to Obtain Recombinant Venom Toxins with Easy Tag Removal inside the Bacteria.

Many animal toxins may target the same molecules that need to be controlled in certain pathologies; therefore, some toxins have led to the formulation of drugs that are presently used, and many other drugs are still under development. Nevertheless, collecting sufficient toxins from the original source might be a limiting factor in studying their biological activities. Thus, molecular biology techniques have been applied in order to obtain large amounts of recombinant toxins into Escherichia coli. However, most animal toxins are difficult to express in this system, which results in insoluble, misfolded, or unstable proteins. To solve these issues, toxins have been fused with tags that may improve protein expression, solubility, and stability. Among these tags, the SUMO (small ubiquitin-related modifier) has been shown to be very efficient and can be removed by the Ulp1 protease. However, removing SUMO is a labor- and time-consuming process. To enhance this system, here we show the construction of a bicistronic vector that allows the expression of any protein fused to both the SUMO and Ulp1 protease. In this way, after expression, Ulp1 is able to cleave SUMO and leave the protein interest-free and ready for purification. This strategy was validated through the expression of a new phospholipase D from the spider Loxosceles gaucho and a disintegrin from the Bothrops insularis snake. Both recombinant toxins showed good yield and preserved biological activities, indicating that the bicistronic vector may be a viable method to produce proteins that are difficult to express.

Shear-sensitive nanocapsule drug release for site-specific inhibition of occlusive thrombus formation.

Essentials Vessel stenosis due to large thrombus formation increases local shear 1-2 orders of magnitude. High shear at stenotic sites was exploited to trigger eptifibatide release from nanocapsules. Local delivery of eptifibatide prevented vessel occlusion without increased tail bleeding times. Local nanocapsule delivery of eptifibatide may be safer than systemic antiplatelet therapies. SUMMARY: Background Myocardial infarction and stroke remain the leading causes of mortality and morbidity. The major limitation of current antiplatelet therapy is that the effective concentrations are limited because of bleeding complications. Targeted delivery of antiplatelet drug to sites of thrombosis would overcome these limitations. Objectives Here, we have exploited a key biomechanical feature specific to thrombosis, i.e. significantly increased blood shear stress resulting from a reduction in the lumen of the vessel, to achieve site-directed delivery of the clinically used antiplatelet agent eptifibatide by using shear-sensitive phosphatidylcholine (PC)-based nanocapsules. Methods PC-based nanocapsules (2.8 × 1012 ) with high-dose encapsulated eptifibatide were introduced into microfluidic blood perfusion assays and into in vivo models of thrombosis and tail bleeding. Results Shear-triggered nanocapsule delivery of eptifibatide inhibited in vitro thrombus formation selectively under stenotic and high shear flow conditions above a shear rate of 1000 s-1 while leaving thrombus formation under physiologic shear rates unaffected. Thrombosis was effectively prevented in in vivo models of vessel wall damage. Importantly, mice infused with shear-sensitive antiplatelet nanocapsules did not show prolonged bleeding times. Conclusions Targeted delivery of eptifibatide by shear-sensitive nanocapsules offers site-specific antiplatelet potential, and may form a basis for developing more potent and safer antiplatelet drugs.

Chemical composition, vasorelaxant, antioxidant and antiplatelet effects of essential oil of Artemisia campestris L. from Oriental Morocco.

BACKGROUND: Artemisia campestris L. (Asteraceae) is a medicinal herb traditionally used to treat hypertension and many other diseases. Hence, this study is aimed to analyze the essential oil of A. campestris L (AcEO) and to investigate the antiplatelet, antioxidant effects and the mechanisms of its vasorelaxant effect. METHODS: The chemical composition of AcEO was elucidated using GC/MS analysis. Then, the antioxidant effect was tested on DPPH radical scavenging and on the prevention of ß-carotene bleaching. The antiplatelet effect was performed on the presence of the platelet agonists: thrombin and ADP. The mechanism of action of the vasorelaxant effect was studied by using the cellular blockers specified to explore the involvement of NO/GC pathway and in the presence of calcium channels blockers and potassium channels blockers. RESULTS: AcEO is predominated by the volatiles: spathulenol, ß-eudesmol and p-cymene. The maximal antioxidant effect was obtained with the dose 2 mg/ml of AcEO. The dose 1 mg/ml of AcEO showed a maximum antiplatelet effect of, respectively 49.73% ±9.54 and 48.20% ±8.49 on thrombin and ADP. The vasorelaxation seems not to be mediated via NOS/GC pathway neither via the potassium channels. However, pretreatment with calcium channels blockers attenuated this effect, suggesting that the vasorelaxation is mediated via inhibition of L-type Ca2+ channels and the activation of SERCA pumps of reticulum plasma. CONCLUSION: This study confirms the antioxidant, antiplatelet and vasorelaxant effects of A.campestris L essential oil. However, the antihypertensive use of this oil should be further confirmed by the chemical fractionation and subsequent bio-guided assays.

Ticagrelor reversal: in vitro assessment of four haemostatic agents.

AIM: Management of ticagrelor-induced bleeding is challenging as platelet transfusion is ineffective. An effective strategy is needed. This study aimed to investigate in vitro the efficacy of four haemostatic drugs (HDs), namely recombinant activated factor VII (rFVIIa), fibrinogen concentrate (Fib), tranexamic acid (TXA) and factor XIII concentrate (FXIII) to improve the haemostatic capacity in the presence of ticagrelor. METHODS: Blood was spiked with ticagrelor then supplemented by either HD or control. Several assays were performed: ADP-induced platelet aggregation measured by impedance aggregometry, light transmission and two global assays, thrombolastography with the platelet mapping device (TEG-PM) and a platelet-dependent thrombin generation assay (TGA). RESULTS: Ticagrelor inhibited ADP-induced platelet aggregation and decreased the clot strength maximum amplitude (MA) in TEG-PMADP. None of the HDs corrected these parameters. However, rFVIIa shortened the coagulation time R using TEG-PMthrombin and the time to peak prolonged by ticagrelor in TGA. Fib increased MAthrombin and FXIII decreased LY30. TXA had no effects. CONCLUSIONS: Whereas none of the HDs corrected ticagrelor-induced platelet inhibition, rFVIIa shortened coagulation times, Fib increased clot firmness and FXIII decreased fibrinolysis. Consequently, they may bypass ticagrelor effects by acting on fibrin formation or fibrinolysis. Further studies are needed to confirm these data in vivo.

Aspirin and Clopidogrel Inhibit Aneurysm Healing after HydroCoil Implantation in External Carotid Artery Aneurysm Model.

PURPOSE: To understand whether the use of antiplatelet agents leads to less intra-aneurismal tissue formation following coil implantation in a rat end-pouch external carotid artery (ECA) aneurysm model. METHODS: End-pouch ECA aneurysms were created in adult rats and were then embedded with either platinum or HydroCoils. Rats were treated either with aspirin, clopidogrel, aspirin + clopidogrel, or saline for 2 weeks after coil implantation. At 2 weeks after coil implantation, rats were sacrificed and the aneurysm pouch was removed for histological and immunohistochemical analysis. A blinded single observer calculated the percentage of the organized area and the residual length of elastic lamina within the aneurysm. Student's t-test was used to compare data from image analysis between the different groups. RESULTS: Within the platinum group, the organized tissue area was not affected by antiplatelet administration (aspirin versus saline, P = .83; clopidogrel versus saline, P = .46; aspirin + clopidogrel versus saline, P = .54). For the HydroCoil group, the organized tissue area was significantly reduced (aspirin versus saline, P = .02; clopidogrel versus saline, P = .04; aspirin + clopidogrel versus saline, P = .02) in rats treated with antiplatelet agents; however, no difference (aspirin versus clopidogrel, P = .8; aspirin versus aspirin + clopidogrel, P = .3; clopidogrel versus aspirin + clopidogrel, P = .5) was found among type or combination of antiplatelets administered. HydroCoil-treated aneurysms had a similar number of macrophages compared to the platinum group (P = .3819); however, the HydroCoil group had significant suppression of macrophages in the groups treated with combined antiplatelets (P = .02). CONCLUSION: Following HydroCoil implantation, the area of organized tissue is diminished significantly in a rat end-pouch ECA aneurysm model treated with antiplatelets.

The effectiveness of platelet supplementation for the reversal of ticagrelor-induced inhibition of platelet aggregation: An in-vitro study.

BACKGROUND: Management of ticagrelor-induced bleeding is challenging, as no antidote is currently available. Platelet transfusion, usually proposed to reverse antiplatelet drugs, has been suggested to be ineffective but few data are available. OBJECTIVE: To assess the efficacy of platelet supplementation to restore platelet aggregation inhibited by ticagrelor. DESIGN: In vitro study. SETTING: Blood samples were obtained from the French Blood Bank Institute. PARTICIPANTS: Healthy blood donors. INTERVENTIONS: Whole blood from healthy donors was spiked with ticagrelor or aspirin (used as a positive control). MAIN OUTCOME MEASURES: Platelet aggregation was investigated with impedance aggregometry on whole blood [expressed in ohms (V)] and light transmission aggregometry (expressed in %) on platelet-rich plasma using ADP or arachidonic acid as agonists for ticagrelor or aspirin, respectively. Platelet supplementation was defined as the addition of washed platelet suspension increasing at least 60% of whole blood platelet count. RESULTS: Ticagrelor (3.25 mM) inhibited ADP-induced platelet aggregation compared with control either in whole blood (2 vs. 13 V, P < 0.05) or in platelet-rich plasma (15 vs. 75% P < 0.05). Aspirin (25 mM) inhibited arachidonic acid-induced aggregation (1 vs. 7.5 V, P < 0.05 in whole blood and 5 vs. 77.5%, P = 0.01 in platelet-rich plasma). Platelet supplementation completely restored arachidonic acid-induced platelet aggregation in whole blood (10 vs. 1 V, P = 0.008) and platelet-rich plasma (73 vs. 5%, P < 0.01) in aspirin-treated samples, whereas it failed to correct ADP-induced aggregation (2 vs. 2 V in whole blood and 13.5 vs. 15% in platelet-rich plasma, P > 0.05) in ticagrelor-treated samples. We also report a case of a ticagrelor-treated patient in whom platelet transfusion failed to restore ADP-induced platelet aggregation. CONCLUSION: Platelet supplementation restored platelet aggregation in aspirin-spiked but not in ticagrelor-spiked samples. These results do not support the use of platelet transfusion to reverse the effects of ticagrelor.

The mechanism and risk factors of clopidogrel-induced liver injury.

CONTEXT AND OBJECTIVE: Clopidogrel (CLP) is a prodrug which is widely used as a platelet aggregation inhibitor. Hepatotoxicity is rare but a potentially serious adverse reaction that is associated with CLP. Thiophene in CLP (the thienopyridine derivative) is a group that is easily oxidated by cytochrome P450 enzymes (CYP450s) to generate reactive metabolites (RMs), it may be implicated in the mechanism of CLP-induced hepatotoxicity. CYP2C19 and CYP2B6 are important CYP450s involved in the metabolism and activation of CLP, and the aim of this study is to investigate whether the metabolites of CYP2C19 and CYP2B6 are associated with the CLP-induced liver injury. METHOD: Primary rat hepatocytes are applied to evaluate the hepatotoxicity of CLP. Glutathione-depleted mouse model is used to evaluate whether this toxicity of CLP is metabolized by CYP450s. We also used HepG2 cells co-incubated with recombinant CYP2B6 and CYP2C19 enzymes to further assess whether the metabolites of CYP2C19 and CYP2B6 are associated with the CLP-induced hepatocellular toxicity. RESULT: CLP in high dose (100 µM and 300 µM) showed cytotoxicity in primary rat hepatocytes assay. Administration of CLP with l-buthionine-S, R-sulfoxinine (BSO) for seven days enhanced the liver injury of CLP. The level of ALT, AST and TBIL in plasma increased significantly, and the histopathological results showed the obvious liver injury; Pretreatment of 1-aminobenzotriazole, a nonspecific inhibitor of CYP450s, suppressed CLP-induced hepatotoxicity; CLP showed a dose-dependent toxicity in HepG2/CYP2C19 enzyme and HepG2/CYP2B6 enzyme models. CONCLUSION: High activities of CYP2C19 and CYP2B6 are the risk factors for hepatocellular toxicity of CLP.

Characterization of a novel function-blocking antibody targeted against the platelet P2Y1 receptor.

OBJECTIVE: Platelet hyperactivity is associated with vascular disease and contributes to the genesis of thrombotic disorders. ADP plays an important role in platelet activation and activates platelets through 2 G-protein-coupled receptors, the Gq-coupled P2Y1 receptor (P2Y1R), and the Gi-coupled P2Y12 receptor. Although the involvement of the P2Y1R in thrombogenesis is well established, there are no antagonists that are currently available for clinical use. APPROACH AND RESULTS: Our goal is to determine whether a novel antibody targeting the ligand-binding domain, ie, second extracellular loop (EL2) of the P2Y1R (EL2Ab) could inhibit platelet function and protect against thrombogenesis. Our results revealed that the EL2Ab does indeed inhibit ADP-induced platelet aggregation, in a dose-dependent manner. Furthermore, EL2Ab was found to inhibit integrin GPIIb-IIIa activation, dense and α granule secretion, and phosphatidylserine exposure. These inhibitory effects translated into protection against thrombus formation, as evident by a prolonged time for occlusion in a FeCl3-induced thrombosis model, but this was accompanied by a prolonged tail bleeding time. We also observed a dose-dependent displacement of the radiolabeled P2Y1R antagonist [(3)H]MRS2500 from its ligand-binding site by EL2Ab. CONCLUSIONS: Collectively, our findings demonstrate that EL2Ab binds to and exhibits P2Y1R-dependent function-blocking activity in the context of platelets. These results add further evidence for a role of the P2Y1R in thrombosis and validate the concept that targeting it is a relevant alternative or complement to current antiplatelet strategies.

Comparison of cytotoxic and anti-platelet activities of polyphenolic extracts from Arnica montana flowers and Juglans regia husks.

Polyphenolic compounds of plant origin are well known to be beneficial to human health: they exert protective effects on haemostasis and have a particular influence on blood platelets. However, the anti-platelet properties of polyphenolic compounds observed so far have not been weighed against their potential cytotoxic action against platelets. The aim of this study was to demonstrate that anti-platelet and cytotoxic effects on blood platelets may interfere and therefore, may often lead to confusion when evaluating the properties of plant extracts or other agents towards blood platelets. The anti-platelet and cytotoxic in vitro effects of plant extracts obtained from the husks of walnuts (J. regia) and flowers of arnica (A. montana) on platelet reactivity and viability were examined. Platelet function was assessed using standard methods (flow cytometry: P-selectin expression, activation of GPIIbIIIa complex, vasodilator-stimulated phosphoprotein, VASP index; turbidimetric and impedance aggregometry) and newly set assays (flow cytometric monitoring of platelet cytotoxicity). The results reveal that none of the studied plant extracts demonstrated cytotoxicity towards blood platelets. The phenolic acid-rich extract of A. montana (7.5 and 15 µg/ml) significantly reduced the ADP-induced aggregation in both whole blood and PRP, and decreased the platelet reactivity index (PRI; VASP phosphorylation) in whole blood, while showing excellent antioxidant capacity. The extract of J. regia husks significantly reduced ADP-induced platelet aggregation in whole blood when applied at 7.5 µg/ml, and only slightly decreased the PRI at 15 µg/ml. Both examined extracts suppressed platelet hyper-reactivity, and such influence did not interfere with cytotoxic effects of the extracts. Thus, its high polyphenol content, excellent antioxidant capacity and distinct anti-platelet properties, in combination with its lack of toxicity, make the extract of A. montana flowers a possible candidate as an anti-platelet agent or a compounding diet supplement.

Gastric-sparing nitric oxide-releasable 'true' prodrugs of aspirin and naproxen.

Nitric oxide-releasing non-steroidal anti-inflammatory drugs (NO-NSAIDs) are gaining attention as potentially gastric-sparing NSAIDs. Herein, we report a novel class of '1-(nitrooxy)ethyl ester' group-containing NSAIDS as efficient NO releasing 'true' prodrugs of aspirin and naproxen. While an aspirin prodrug exhibited comparable oral bioavailability and antiplatelet activity (i.e., TXB2 inhibition) to those of aspirin, a naproxen prodrug exhibited better bioavailability than naproxen. These promising NO-NSAIDs protected experimental rats from gastric damage. We therefore believe that these promising NO-NSAIDs could represent a new class of potentially 'Safe NSAIDs' for the treatment of arthritic pain, inflammation and cardiovascular disorders in the case of NO-aspirin.