Suppression Of Platelet Aggregation Research Articles

Qilong capsule prevents myocardial ischemia/reperfusion injury by inhibiting platelet activation via the platelet CD36 signaling pathway

Ethnopharmacological relevanceQilong capsule (QC) is developed from the traditional Chinese medicine formula Buyang Huanwu Decoction, which has been clinically used to invigorate Qi and promote blood circulation to eliminate blood stasis. Myocardial ischemia‒reperfusion injury (MIRI) can be attributed to Qi deficiency and blood stasis. However, the effects of QC on MIRI remain unclear. Aim of the studyThis study aimed to investigate the protective effect and possible mechanism of QC on platelet function in MIRI rats. Materials and methodsThe left anterior descending artery of adult Sprague‒Dawley rats was ligated for 30 min and then reperfused for 120 min with or without QC treatment. Then, the whole blood viscosity, plasma viscosity, coagulation, platelet adhesion rate, platelet aggregation, and platelet release factors were evaluated. Platelet CD36 and its downstream signaling pathway-related proteins were detected by western blotting. Furthermore, the active components of QC and the molecular mechanism by which QC regulates platelet function were assessed via molecular docking, platelet aggregation tests in vitro and BLI analysis. ResultsWe found that QC significantly reduced the whole blood viscosity, plasma viscosity, platelet adhesion rate, and platelet aggregation induced by ADP or AA in rats with MIRI. The inhibition of platelet activation by QC was associated with reduced levels of β-TG, PF-4, P-selectin and PAF. Mechanistically, QC effectively attenuated the expression of platelet CD36 and thus inhibited the activation of Src, ERK5, and p38. The active components of QC apparently suppressed platelet aggregation in vitro and regulated the CD36 signaling pathway. ConclusionsQC improves MIRI-induced hemorheological disorders, which might be partly attributed to the inhibition of platelet activation via CD36-mediated platelet signaling pathways.

Management of multiple cerebral embolisms due to polycythemia vera with acquired von Willebrand syndrome

AbstractPolycythemia vera (PV) is a rare blood disorder associated with thrombosis or acquired von Willebrand syndrome (AvWS); however, their coexistence is unusual. Here, we report about a 59‐year‐old man with PV who developed both ischemic stroke (IS) and AvWS. Although the patient had no history of thrombosis, he had refractory epistaxis 2 years prior. Brain magnetic resonance imaging and angiography (MRI and MRA) revealed new IS and mural thrombi at the left distal carotid artery. Laboratory investigations showed elevated hemoglobin and hematocrit levels with mild leukocytosis and thrombocytosis, corresponding to PV. Additionally, suppressed platelet aggregation and activity despite normal antigen of von Willebrand factor indicated AvWS. Appropriate repeated phlebotomies successfully resolved the MRA abnormalities and prevented IS recurrence, although the patient refused the recommended treatment with hydroxyurea. This case highlights the need for careful management to prevent recurrent IS and major bleeding in patients with PV who develop IS accompanying AvWS.

Eugenol Suppresses Platelet Activation and Mitigates Pulmonary Thromboembolism in Humans and Murine Models.

Platelets assume a pivotal role in the pathogenesis of cardiovascular diseases (CVDs), emphasizing their significance in disease progression. Consequently, addressing CVDs necessitates a targeted approach focused on mitigating platelet activation. Eugenol, predominantly derived from clove oil, is recognized for its antibacterial, anticancer, and anti-inflammatory properties, rendering it a valuable medicinal agent. This investigation delves into the intricate mechanisms through which eugenol influences human platelets. At a low concentration of 2 μM, eugenol demonstrates inhibition of collagen and arachidonic acid (AA)-induced platelet aggregation. Notably, thrombin and U46619 remain unaffected by eugenol. Its modulatory effects extend to ATP release, P-selectin expression, and intracellular calcium levels ([Ca2+]i). Eugenol significantly inhibits various signaling cascades, including phospholipase Cγ2 (PLCγ2)/protein kinase C (PKC), phosphoinositide 3-kinase/Akt/glycogen synthase kinase-3β, mitogen-activated protein kinases, and cytosolic phospholipase A2 (cPLA2)/thromboxane A2 (TxA2) formation induced by collagen. Eugenol selectively inhibited cPLA2/TxA2 phosphorylation induced by AA, not affecting p38 MAPK. In ADP-treated mice, eugenol reduced occluded lung vessels by platelet thrombi without extending bleeding time. In conclusion, eugenol exerts a potent inhibitory effect on platelet activation, achieved through the inhibition of the PLCγ2-PKC and cPLA2-TxA2 cascade, consequently suppressing platelet aggregation. These findings underscore the potential therapeutic applications of eugenol in CVDs.

Synergy by Ristocetin and CXCL12 in Human Platelet Activation: Divergent Regulation by Rho/Rho-Kinase and Rac.

CXCL12, belonging to the CXC chemokine family, is a weak agonist of platelet aggregation. We previously reported that the combination of CXCL12 and collagen at low doses synergistically activates platelets via not CXCR7 but CXCR4, a specific receptor for CXCL12 on the plasma membrane. Recently, we reported that not Rho/Rho kinase, but Rac is involved in the platelet aggregation induced by this combination. Ristocetin is an activator of the von Willebrand factor that interacts with glycoprotein (GP) Ib/IX/V, which generates thromboxane A2 via phospholipase A2 activation, resulting in the release of the soluble CD40 ligand (sCD40L) from human platelets. In the present study, we investigated the effects of a combination of ristocetin and CXCL12 at low doses on human platelet activation and its underlying mechanisms. Simultaneous stimulation with ristocetin and CXCL12 at subthreshold doses synergistically induce platelet aggregation. A monoclonal antibody against not CXCR7 but CXCR4 suppressed platelet aggregation induced by the combination of ristocetin and CXCL12 at low doses. This combination induces a transient increase in the levels of both GTP-binding Rho and Rac, followed by an increase in phosphorylated cofilin. The ristocetin and CXCL12-induced platelet aggregation as well as the sCD40L release were remarkably enhanced by Y27362, an inhibitor of Rho-kinase, but reduced by NSC23766, an inhibitor of the Rac-guanine nucleotide exchange factor interaction. These results strongly suggest that the combination of ristocetin and CXCL12 at low doses synergistically induces human platelet activation via Rac and that this activation is negatively regulated by the simultaneous activation of Rho/Rho-kinase.

Controlled release nanomedicine (CRNM) of aspirin using “biomimetic niosomal nanoparticles (BNNs)”for Covid-19 and cardiovascular treatment: DOE based optimization

Biomimetic strategies can be adopted to improve biopharmaceutical aspects. Subsequently, Biomimetic reconstitutable pegylated amphiphilic lipid nanocarriers have high translational potential for systemic controlled drug delivery; however, such an improvised system for systemic aspirin delivery exploring nanotechnology is not available. Systemic administration of aspirin and its controlled delivery can significantly control blood clotting events, leading to stroke, which has immediate applications in cardiovascular diseases and Covid-19. In this work, we are developing aspirin sustained release pegylated amphiphilic self-assembling nanoparticles to develop reconstitutable aspirin injections by solvent-based co-precipitation method with phase inversion technique that leads to novel “biomimetic niosomal nanoparticles (BNNs).” DOE led optimization is done to develop Design of space for optimized particles. Upon reconstitution of solid powder, the particle size was 144.8 ± 12.90 nm with a surface charge of -29.2 ± 2.24 mV. The entrapment efficiency was found to be 49 ± 0.15%, wherein 96.99 ± 1.57% of the drug was released in 24hr showing super case II transport-based drug release mechanism. The formulation has the least hemolysis while showing significant suppression of platelet aggregation. MTT assay does not show any significant cytotoxicity. This is a potential nanoparticle that can be explored for developing aspirin injection, which is not available.

Ticagrelor versus Clopidogrel in the Dual Antiplatelet Regimen for Unruptured Intracranial Aneurysm Treated with Stent-Assisted Coil Embolization: A Single-Center Cohort Study

Dual antiplatelet therapy (DAPT) of aspirin plus clopidogrel is commonly utilized in patients with unruptured intracranial aneurysms treated with stent-assisted coil (SAC) embolization. However, the unpredictable clopidogrel efficacy of the 5% to 55% non-responders limits its use. Ticagrelor, as a potential alternative of clopidogrel, is an antiplatelet agent with low resistance rates but uncertain efficacy and safety in these patients. A single-center, cohort study was performed to compare the efficacy and safety of ticagrelor with clopidogrel in the DAPT regimen in patients with unruptured intracranial aneurysms and treated with SAC. The patients with clopidogrel resistance identified as inadequate adenosine diphosphate (ADP) inhibition rate determined by thromboelastography were treated with ticagrelor instead, and both drugs achieved adequate suppression of platelet aggregation when stents were implanted. The occurrence of major adverse cardiovascular and cerebrovascular events (MACCE) and bleeding events was recorded through 6-month follow-up. Data from 86 patients with 99 unruptured intracranial aneurysms and treated by SAC with clopidogrel were compared with those from 108 patients with 111 aneurysms and treated with ticagrelor. Neither the baseline characteristics nor the incidence of the MACCE or bleeding events differ between the groups. Ticagrelor exerted significantly higher ADP inhibition rate than that of the clopidogrel. Multivariable logistic regression analysis demonstrated that the incidence of MACCE was relate to hematocrit and fibrinogen levels. Ticagrelor appeared to be as effective and safe as clopidogrel for SAC in unruptured intracranial aneurysms. Hematocrit and fibrinogen levels were independent risk factors for the incidence of MACCE.

Quinonoid dihydropteridine reductase, a tetrahydrobiopterin-recycling enzyme, contributes to 5-hydroxytryptamine-associated platelet aggregation in mice

Quinonoid dihydropteridine reductase (QDPR) regenerates tetrahydrobiopterin (BH4), which is an essential cofactor for catecholamine and serotonin (5-hydroxytryptamine, 5-HT) biosynthesis. Serotonin is known as an important platelet agonist, but its role under BH4-synthesizing or recycling enzymes deficiency is unknown. In the present study, we evaluated the effect of Qdpr gene disruption on platelet aggregation using knockout (Qdpr−/−) mice. Platelet aggregation was monitored by light transmission aggregometry using adenosine diphosphate (ADP) and collagen as agonists. We also assessed how platelet aggregation was modified by 5-HT recovery through supplementation with 5-hydroxytryptophan (5-HTP), a 5-HT precursor, or by blocking the serotonin 5-HT2A receptor. Platelet aggregation in the Qdpr−/− mice was significantly suppressed in comparison with that in wild-type (Qdpr+/+) mice, particularly at the maintenance phase of aggregation. 5-HT storage was decreased in Qdpr−/− platelets, and 5-HTP supplementation recovered not only the intraplatelet 5-HT levels but also platelet aggregation. In addition, 5-HT signal blockade using sarpogrelate suppressed platelet aggregation in Qdpr+/+ mice, and platelets in Qdpr−/− mice were hardly affected. Our results indicate that QDPR deficiency suppresses platelet aggregation by impairing 5-HT biosynthesis in mice.

Orally delivered rutin in lipid-based nano-formulation exerts strong antithrombotic effects by protein disulfide isomerase inhibition

Thrombosis occurs in both macrovasculature and microvasculature, causing various cardio-cerebral vascular diseases. The lack of effective and safe antithrombotic drugs leads to a public health crisis. Mounting evidence suggests that protein disulfide isomerase (PDI) plays a critical role in the initial stage of thrombus formation, motivating the research of the feasibility of PDI inhibitors as novel anti-thrombotics. Rutin, one of the most potent PDI inhibitors, was reported to suppress platelet aggregation and thrombosis in animal models, but further studies and clinical translation were restricted due to its low aqueous solubility and oral bioavailability. In this work, we fabricated rutin-loaded lipid-based nano-formulation (NanoR) and characterized their physical–chemical properties, release profiles, pharmacokinetic process, and pharmacodynamic function against thrombosis in macrovessels and microvessels. NanoR provided increased solubility and dissolution of rutin to achieve earlier T max and higher C max than the sodium salt of rutin (NaR) after oral gavage. Ex vivo studies demonstrated that NanoR significantly inhibited thrombin generation and clot formation in the plasma of mice. Importantly, such effect was reversed by exogenous recombinant PDI, demonstrating the specificity of the NanoR. In direct current-induced arterial thrombosis model and ferric chloride-induced microvascular thrombosis model, NanoR exhibited greatly enhanced antithrombotic activity compared with NaR. NanoR also showed good safety performance according to tail bleeding assay, global coagulation tests, and histological analysis. Overall, our current results indicated that NanoR offers a promising antithrombotic treatment with potential for clinical translation.

DK-MGAR101, an extract of adventitious roots of mountain ginseng, improves blood circulation by inhibiting endothelial cell injury, platelet aggregation, and thrombus formation

BackgroundSince ginsenosides exert an anti-thrombotic activity, blood flow-improving effects of DK-MGAR101, an extract of mountain ginseng adventitious roots (MGAR) containing various ginsenosides, were investigated in comparison with an extract of Korean Red Ginseng (ERG). MethodsIn Sprague-Dawley rats orally administered with DK-MGAR101 or ERG, oxidative carotid arterial thrombosis was induced with FeCl3 (35%), and their blood flow and occlusion time were measured. To elucidate underlying mechanisms, the cytoprotective activities on rat aortic endothelial cells (RAOECs) exposed to hydrogen peroxide (H2O2) were confirmed. In addition, the inhibitory activities of DK-MGAR101 and ERG on agonist-induced platelet aggregation, thromboxane B2 production, and ATP granule release from stimulated platelets as well as blood coagulation were analyzed. ResultsDK-MGAR101 containing high concentrations of Rb1, Rg1, Rg3, Rg5, and Rk1 ginsenosides (55.07 mg/g) was more effective than ERG (ginsenosides 8.45 mg/g) in protecting RAOECs against H2O2 cytotoxicity. DK-MGAR101 was superior to ERG not only in suppressing platelet aggregation, thromboxane B2 production, and granule release, but also in delaying blood coagulation, FeCl3-induced arterial occlusion, and thrombus formation. ConclusionsThe results indicate that DK-MGAR101 prevents blood vessel occlusion by suppressing platelet aggregation, thrombosis, and blood coagulation, in addition to endothelial cell injury.

Ligustrazine suppresses platelet aggregation through inhibiting the activities of calcium sensors

Ligustrazine is widely used for the treatment of cardiovascular diseases in traditional Chinese medication. It has been reported that Ligustrazine decreases the concentration of intracellular calcium ions (Ca2+); however, the underlying mechanism remains unknown. In the present study, the effect of Ligustrazine on adenosine diphosphate (ADP)-induced platelet aggregation was evaluated using a turbidimetric approach. The changes in concentration of intracellular Ca2+ stimulated by ADP was measured using fluo-4, a fluorescent Ca2+ indicator dye. The mRNA expression of stromal interaction molecule l (STIM1) and Orai1, calcium sensor, was determined using real-time PCR. In addition, the protein expression of STIM1, Orai1, and serum/glucocorticoid-regulated protein kinase 1 (SGK1) was determined using Western blot analysis. The data demonstrated that Ligustrazine significantly suppressed platelet aggregation in a dose-dependent manner and reduced the concentration of intracellular Ca2+ triggered by ADP. Our data showed that Ligustrazine treatment inhibited the expression of STIM1 and Orai1 induced by ADP at both mRNA and protein levels, and suppressed the protein expression of SGK1. Taken together, our data indicated that Ligustrazine suppressed platelet aggregation by partly inhibiting the activities of calcium sensors, thereby suggesting that Ligustrazine may be a promising candidate for the treatment of platelet aggregation.

LIPOIC ACID NANODISPERSIONS: PREPARATION, ANTIPLATELET AND ANTIOXIDANT PROPERTIES

Nanoemulsions (NEs) with lipoic acid (LA) with a particle size from 50 to 350 nm were obtained, characterized by 85±10% efficiency of inclusion (EF) of LA into nanoparticles (NPs), its slow release from nanoforms, and also having high stability during long-term storage at room temperature. Nanoemulsions containing LA have been shown to suppress platelet aggregation (Pt) caused by arachidonic acid (AA).

STUDY THE FUNCTIONAL ACTIVITY OF PLATELETS AGAINST THE BACKGROUND OF ANTIPLATELET THERAPY IN PATIENTS OF THE CARDIOLOGICAL HOSPITAL

Antiplatelet drugs are widely used in clinical practice to reduce the risk of adverse cardiovascular events in patients with cardiovascular diseases. It is noted that resistance to antiplatelet agents is determined in a number of patients, which may increase the risk of cardiovascular catastrophes. Given the importance of the antiplatelet component in preventive and therapeutic strategies, the inability to achieve its potential benefit in patients with atherothrombosis is an important clinical problem. Aim of the study. Studying the features of platelet aggregation in vitro in patients with coronary heart disease, acute disorders of cerebral circulation and acute coronary syndrome receiving acetylsalicylic acid or acetylsalicylic acid in combination with clopidogrel during hospital treatment. Materials and methods. The subjects were divided into 3 groups: 1 - patients of the Department of Cardiology - received acetylsalicylic acid - Cardiomagnil ® a dose of 75 mg / day; group 2 - Departments of acute coronary syndrome - took acetylsalicylic acid - Acecardol ® - 100 mg once a day in combination with clopidogrel – 75 mg per day; group 3 - patients of the Department of patients with acute disorders of cerebral circulation took Acecardol ® 100 mg once a day. Platelet aggregation activity was studied in all patients after 5 days of regular intake of antiplatelet agents. Platelet aggregation was studied by optical aggregatometry using a laser aggregometer «Biola». As agonists of aggregation were used: ADP (up to 5 mmol/L), epinephrine (10 mg/ml), collagen (1 mg/ml) and ristocetin (ristomycin) (15 mg/ml). Results and discussion. Insufficient effect on the background of taking antiplatelet agents was observed in 27.2% of cases in patients of group 1 and 8.2% in patients of group 2 (p=0.0034). In patients with acute disorders of cerebral circulation (group 3), the insufficient effect of acetylsalicylic acid was noted in 16.3% of cases, which was significantly less than in group 1 (p=0.026). Patients with acute coronary syndrome receiving double antiplatelet therapy achieved pronounced suppression of aggregation in 54% of cases, which was statistically significantly more than in group 1 - 23.3% (p=0.0004). In patients of group 3, a pronounced antiplatelet effect was observed in 41.2% of cases, which was significantly more than in patients of group 1 (p=0.0054). Thus, the use of acetylsalicylic acid in combination with clopidogrel in patients with acute coronary syndrome provided a more pronounced suppression of platelet aggregation. This is consistent with the data of other researchers who found that in 6% of patients with the use of two antiplatelet agents, the desired result was not obtained [14, 15]. It was noted that in patients with acute cerebrovascular accident who received only acetylsalicylic acid, platelet aggregation decreased to a greater extent than in patients with coronary heart disease (group 1). Perhaps this is due to the fact that patients of these groups received different medications of acetylsalicylic acid. Findings. The use of two antiplatelet agents does not always lead to optimal effect. In this regard, the expediency of aggregatometry to assess the effect of acetylsalicylic acid is obvious. Particular attention should be paid to patients receiving acetylsalicylic acid an outpatient basis, since the low effectiveness of antiplatelet therapy increases the risk of myocardial infarction, stroke.

Amyloid β protein negatively regulates human platelet activation induced by thrombin receptor-activating protein.

Amyloid β protein deposition in cerebral vessels, a characteristic of Alzheimer's disease, is a risk factor for intracerebral hemorrhage. Amyloid β protein directly modulates human platelet function; however, the exact mechanism of action is unclear. Therefore, we investigated the effects of amyloid β protein on human platelet activation using an aggregometer with laser scattering. Amyloid β protein decreased platelet aggregation induced by thrombin receptor-activating protein, but not by collagen and ADP. Amyloid β protein also suppressed platelet aggregation induced by SCP0237 and A3227. Platelet-derived growth factor-AB secretion and phosphorylated-heat shock protein 27 release by thrombin receptor-activating protein were inhibited by amyloid β protein. Additionally, thrombin receptor-activating protein-induced phosphorylation of JNK and p38 MAP kinase was reduced by amyloid β protein. Collectively, our results strongly suggest that amyloid β protein negatively regulates protease-activated receptor-elicited human platelet activation. These findings may indicate a cause of intracerebral hemorrhage due to amyloid β protein.

The antiplatelet effect of mirtazapine is mediated by co-blocking 5-HT2A and α2-adrenergic receptors on platelets: An in vitro human plasma-based study

Mirtazapine (MTZ) is a noradrenergic and specific serotonergic antidepressant that has been associated with an increased risk of bleeding. However, there is insufficient evidence confirming this association. We hypothesised that 5-HT2A and α2 receptor-mediated inhibitory effects of MTZ on platelets suppress platelet aggregation and increase the risk of bleeding. In this study, we examined the antiplatelet effect of MTZ on human platelets to test our hypothesis. Blood samples for platelet aggregation tests were obtained from 14 healthy volunteers. The antiplatelet effect of MTZ was evaluated using light transmission aggregometry. MTZ significantly suppressed platelet aggregation mediated both by the synergistic interaction of serotonin (5-HT) and adrenaline and the synergistic interaction of ADP and 5-HT or adrenaline. In conclusion, MTZ exerts its antiplatelet effects by co-blocking the 5-HT2A and α2-adrenergic receptors on platelets and also suppresses platelet aggregation induced by ADP and 5-HT or adrenaline. Therefore, when MTZ is used, especially for patients with a high risk of bleeding, the significance of its use must be considered carefully. In addition, the platelet aggregation pattern by adrenaline + 5-HT, ADP + adrenaline, and ADP + 5-HT was similar between humans and mice; however, this study did not directly compare the effects of MTZ on human and murine platelets. Therefore, under the conditions for inducing platelet aggregation using adrenaline + 5-HT, ADP + adrenaline, and ADP + 5-HT, mouse platelets can be used in the evaluation of the efficacy of antiplatelet drugs in humans.

Naringenin Inhibits Platelet Activation and Arterial Thrombosis Through Inhibition of Phosphoinositide 3-Kinase and Cyclic Nucleotide Signaling.

Citrus flavanoids intake can reduce the risk of cardiovascular diseases. Naringenin, a natural predominant flavonoid abundant in citrus fruits, possesses protective effects against atherothrombotic diseases. As platelet activation plays central roles in atherothrombogenesis, we studied the effects of naringenin on platelet activation, signaling, thrombosis and hemostasis. Naringenin dose-dependently inhibited agonist-induced platelet aggregation in vitro, and exhibited more-potent efficacy on ADP-induced platelet aggregation. It also suppressed platelet aggregation stimulated by ADP ex vivo. Naringenin inhibited ADP-induced platelet α-granule secretion, fibrinogen binding, intracellular calcium mobilization and platelet adhesion on collagen-coated surface. Naringenin also inhibited platelet spreading on fibrinogen and clot retraction, processes mediated by outside-in integrin signaling. Mechanism studies indicated that naringenin suppressed PI3K-mediated signaling and phosphodiesterase activity in platelets, in addition to increasing cGMP levels and VASP phosphorylation at Ser239. Furthermore, naringenin-induced VASP phosphorylation and inhibition of platelet aggregation were reversed by a PKA inhibitor treatment. Interestingly, naringenin inhibited thrombus formation in the (FeCl3)-induced rat carotid arterial thrombus model, but not cause a prolonged bleeding time in mice. This study suggests that naringenin may represent a potential antiplatelet agent targeting PI3K and cyclic nucleotide signaling, with a low bleeding risk.

12-Lipoxygenase Regulates Platelet Reactivity and Thrombosis Through Formation Of Unique Oxylipins

Platelet activation plays a central role in hemostasis and thrombosis and is regulated through a complex milieu of intracellular signaling events vital for prevention of excessive clot formation and vessel occlusion. There is strong evidence that oxylipins (oxygenated fatty acids) derived from omega-3 (ω-3) and w-6 fatty acids offer cardio-protection; however the underlying mechanism for this protection is not well understood. Previously, we showed that ex vivo fatty acid supplementation of human platelets with the ω-6 fatty acid (dihomo-γ-linolenic acid (DGLA)) significantly inhibited platelet-induced aggregation. The anti-platelet effect exerted by DGLA depended on oxidation by 12-lipoxygenase (12-LOX) to form unique oxylipins with inhibitory activity toward platelet function. Here, for the first time, we show the underlying signaling mechanisms by which 12-LOX functions as an essential regulator of endogenous fatty acid metabolism protecting against excessive platelet activation and thrombosis. To delineate the mechanisms by which 12-LOX regulates platelet reactivity, we investigated upstream signaling correlates leading to platelet aggregation, including calcium mobilization, Rap1 activation, and GP IIb/IIIa activation. Using mice deficient in platelet 12-LOX (12-LOX-/-), we were able to show functional 12-LOX is required for the regulation of DGLA inhibition of PAR4- and collagen-mediated platelet aggregation. Platelets from wild-type mice treated with DGLA or 12-HETrE (oxidized form of DGLA) showed significant suppression of platelet-mediated aggregation, whereas DGLA was unable to suppress platelet aggregation in platelets from 12-LOX-/- mice. Importantly, treatment with 12-HETrE significantly attenuated platelet aggregation in 12-LOX-/- mice. Further, DGLA was unable inhibit calcium mobilization and Rap1 activation in mice deficient for 12-LOX, while calcium mobilization and Rap1 activation were significantly inhibited by 12-HETrE. Overall, our data support 12-LOX playing an important role in both hemostasis and thrombosis. The loss of 12-LOX activity results in an inability of fatty acids to be oxidized to their respective bioactive lipid products. The formation of these oxylipins (12-HETrE in particular) is essential for prevention of platelet activation leading to atherothrombus formation and stroke. Hence, this study is the first to show that the anti-platelet effect mediated by fatty acids is due in large part to their oxylipins production by 12-LOX and the formation of potently anti-platelet metabolites. Disclosures:No relevant conflicts of interest to declare.

Novel serotonin 5-HT2A receptor antagonists derived from 4-phenylcyclohexane-5-spiro-and 5-methyl-5-phenyl-hydantoin, for use as potential antiplatelet agents

BackgroundAntiplatelet drugs have been used in the treatment of acute coronary syndromes and for the prevention of recurrent events. Unfortunately, many patients remain resistant to the available antiplatelet treatment. Therefore, there is a clinical need to synthesize novel antiplatelet agents, which would be associated with different pathways of platelet aggregation, to develop an alternative or additional treatment for resistant patients. Recent studies have revealed that 5-HT2A receptor antagonists could constitute alternative antiplatelet therapy.MethodsBased on the structures of the conventional 5-HT2A receptor ligands, two series of compounds with 4-phenylcyclohexane-5-spiro- or 5-methyl-5-phenyl-hydantoin core linked to various arylpiperazine moieties were synthesized and their affinity for 5-HT2A receptor was assessed. Further, we evaluated their antagonistic potency at 5-HT2A receptors using isolated rat aorta and cells expressing human 5-HT2A receptors. Finally, we studied their anti-aggregation effect and compared it with ketanserin and sarpogrelate, the reference 5-HT2A receptor antagonists. Moreover, the structure–activity relationships were studied following molecular docking to the 5-HT2A receptor model.ResultsFunctional bioassays revealed some of the synthesized compounds to be moderate antagonists of 5-HT2A receptors. Among them, 13, 8-phenyl-3-(3-(4-phenylpiperazin-1-yl)propyl)-1,3-diazaspiro[4.5]decane-2,4-dione, inhibited collagen stimulated aggregation (IC50 = 27.3 μM) being more active than sarpogrelate (IC50 = 66.8 μM) and comparable with ketanserin (IC50 = 32.1 μM). Moreover, compounds 2–5, 9–11, 13, 14 inhibited 5-HT amplified, ADP- or collagen-induced aggregation.ConclusionsOur study confirmed that the 5-HT2A antagonists effectively suppress platelet aggregation and remain an interesting option for the development of novel antiplatelet agents with an alternative mechanism of action.

Antiplatelet Effect of Mirtazapine via Co-blocking of the 5-HT2A and α2-Adrenergic Receptors on Platelets.

Mirtazapine (MTZ) is a noradrenergic and specific serotonergic antidepressant. MTZ is reportedly associated with an increased risk of bleeding. However, the underlying mechanism remains unclear. In this study, we investigated the antiplatelet effect of MTZ in mice via light transmission aggregometry to elucidate the mechanism of MTZ-induced bleeding. The results of the ex vivo study showed that the oral administration of MTZ (20 or 100 mg/kg) significantly suppressed platelet aggregation mediated by the synergic interaction of 5-hydroxytryptamine (5-HT) and adrenaline. Additionally, MTZ significantly suppressed platelet aggregation, mediated by the synergic interaction of ADP and 5-HT or adrenaline. Similar results were obtained in vitro, under the condition of 5-HT- and adrenaline-induced platelet aggregation. Overall, the results suggest that MTZ exerts antiplatelet effect by co-blocking 5-HT2A and α2-adrenergic receptors on platelets and suppresses platelet aggregation mediated by ADP, increased by either 5-HT or adrenaline. Thus, a detailed monitoring of bleeding is recommended for patients taking MTZ.

Platelets and Regulatory T Cells May Induce a Type 2 Immunity That Is Conducive to the Progression and Fibrogenesis of Endometriosis.

Endometriosis is a hormonal disease, as well as a chronic inflammatory disease. While various immune cells are documented to be involved in endometriosis, there is a wanton lack of a bigger picture on how these cells are coordinated to work concertedly. Since endometriotic lesions experience cyclical bleeding, they are fundamentally wounds that undergo repeated tissue injury and repair (ReTIAR). In this study, we attempted to characterize the role of platelets and regulatory T cells (Tregs) in modulating the lesional immune microenvironment and its subsequent effects on lesional progression and fibrogenesis. Through two mouse experiments, we show that, by disrupting predominantly a type 2 immune response in lesional microenvironment, both platelets and Tregs depletion decelerated lesional progression and fibrogenesis, likely through the suppression of the TGF-β1/Smad3 and PDGFR-β/PI3K/Akt signaling pathways. In particular, platelet depletion resulted in significantly reduced lesional expression of thymic stromal lymphopoietin (TSLP), leading to reduced aggregation of macrophages and alternatively activated (M2) macrophages, and of Tregs, T helper 2 (Th2) and Th17 cells but increased aggregation of Th1 cells, in lesions, which, in turn, yields retarded fibrogenesis. Similarly, Tregs depletion resulted in suppression of platelet aggregation, and reduced aggregation of M2 macrophages, Th2 and Th17 cells but increased aggregation of Th1 cells, in lesions. Thus, both platelet and Tregs depletion decelerated lesional progression and fibrogenesis by disrupting predominantly a type 2 immunity in lesional microenvironment. Taken together, this suggests that both platelets and Tregs may induce a type 2 immunity in lesional microenvironment that is conducive to lesional progression and fibrogenesis.

PEAR1 rs12041331 polymorphisms and the risk of adverse cardiovascular outcomes in patients with acute coronary syndrome and/or percutaneous coronary intervention: a systematic review and meta-analysis

Abstract Background Platelet endothelial aggregation receptor-1 (PEAR1), expressed in endothelium, platelets, and other tissues, is a platelet transmembrane tyrosine kinase receptor involved in platelet aggregation and platelet-platelet contact. The minor allele (A) in intron 1 of the PEAR1 gene (rs12041331, G>A) is associated with an reduced PEAR1 protein expression and suppressed platelet aggregation response toward multiple agonists. But current evidences on the association between PEAR1 rs12041331 polymorphisms and cardiovascular outcomes in patients with acute coronary syndrome (ACS) and/or percutaneous coronary intervention (PCI) are discordant. Purpose To characterize and quantify the association between PEAR1 rs12041331 polymorphisms and cardiovascular outcomes in patients with ACS and/or PCI. Methods We conducted a systematic review and meta-analysis by searching MEDLINE via PubMed, EMBASE, Cochrane Central Register of Controlled Trials, SinoMed, CNKI, and Wanfang Data before Dec 7, 2019 to identify studies evaluating the association between PEAR1 rs12041331 polymorphisms and cardiovascular outcomes in patients with ACS and/or PCI. The primary outcome was the major adverse cardiovascular outcomes (MACEs) defined by each study. We adopted the Mantel-Haenszel method to calculate the relative risks (RRs) with 95% confidence intervals (CIs) and the corresponding P values using the random effect model. To assess the effect of ethnicity, we performed the subgroup analyses per ethnic population using the fix effect model. Results Among 542 citations identified along with 1 from additional sources, we included 4 studies, which documented 218 MACEs in 8180 patients. The A allele frequency in each study was comparable with that reported in the 1000 Genome Project, but varied among ethnic populations, that is 45.8% in East Asians, 46.5% in American Africans, and 9.2% in Caucasians, adopted from the 1000 Genome Project. A-allele carriers are associated with a 54% increase in MACEs than non-A-carriers (RR, 1.54; 95% CI, 1.10–2.16; P=0.01) with a low but non-significant heterogeneity. Subgroup analyses showed that A-allele carriers tend to have more MACEs than non-A-allele carriers despite of ethnicity, that is 1.32-fold increase in East Asians (RR, 1.32; 95% CI, 0.99–1.77; P=0.06), 2.43-fold in Caucasians (RR, 2.43; 95% CI, 0.99–5.98; P=0.05), and 3.56-fold (RR, 3.56; 95% CI, 1.08–11.70; P=0.04) in American Africans. Conclusions Our study indicates that the PEAR1 rs12041331 is prognostic in patients with ACS and/or PCI and treated with DAPT, which might be not caused by the associations with pharmacological response to antiplatelet agents. Further investigations are thus required to address the unrevealing mechanisms of PEAR1, especially on the development of atherosclerosis and the occurrence of acute adverse ischemic events. Figure 1 Funding Acknowledgement Type of funding source: None