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

ETHNOPHARMACOLOGICAL RELEVANCE: Qilong 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 STUDY: This study aimed to investigate the protective effect and possible mechanism of QC on platelet function in MIRI rats. MATERIALS AND METHODS: The 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. RESULTS: We 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 ß-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. CONCLUSIONS: QC improves MIRI-induced hemorheological disorders, which might be partly attributed to the inhibition of platelet activation via CD36-mediated platelet signaling pathways.

Therapeutic effects and molecular mechanisms of natural products in thrombosis.

Thrombotic disorders, such as myocardial infarction and stroke, are the leading cause of death in the global population and have become a health problem worldwide. Drug therapy is one of the main antithrombotic strategies, but antithrombotic drugs are not completely safe, especially the risk of bleeding at therapeutic doses. Recently, natural products have received widespread interest due to their significant efficacy and high safety, and an increasing number of studies have demonstrated their antithrombotic activity. In this review, articles from databases, such as Web of Science, PubMed, and China National Knowledge Infrastructure, were filtered and the relevant information was extracted according to predefined criteria. As a result, more than 100 natural products with significant antithrombotic activity were identified, including flavonoids, phenylpropanoids, quinones, terpenoids, steroids, and alkaloids. These compounds exert antithrombotic effects by inhibiting platelet activation, suppressing the coagulation cascade, and promoting fibrinolysis. In addition, several natural products also inhibit thrombosis by regulating miRNA expression, anti-inflammatory, and other pathways. This review systematically summarizes the natural products with antithrombotic activity, including their therapeutic effects, mechanisms, and clinical applications, aiming to provide a reference for the development of new antithrombotic drugs.

Identification of linoleic acid as an antithrombotic component of Wenxin Keli via selective inhibition of p-selectin-mediated platelet activation.

Atrial fibrillation significantly increases the risk of thromboembolism and stroke. Wenxin Keli (WXKL) is a widely used Chinese patent medicine against arrhythmia but if it has antithrombotic activity is unknown. Since platelet activation is a critical factor in thrombosis and the key target for many antithrombotic drugs, this study aims to demonstrate the antithrombotic efficacy of WXKL. In vitro platelet activation experiments showed that WXKL significantly inhibited platelet adhesion and aggregation. The potential active monomers in WXKL were screened by in silico prediction and in vitro platelet aggregation/adhesion assays. From WXKL chemical fractions and more than 40 monomers, linoleic acid (LA) was identified as the strongest antiplatelet compound. Oral administration of WXKL (1.2 g/kg/day) and LA (50 mg/kg/day) for 7 days significantly improved FeCl3-induced carotid thrombus formation in ICR mice without prolonging bleeding time. Flow cytometry showed that both WXKL and LA inhibited the release of p-selectin after platelet activation. ELISA showed that WXKL and LA also inhibited the expression of 6-Keto-PGF1α in plasma of mice with thrombus, but had no obvious effect on the expression of TXB2. WXKL inhibited platelet activation by broadly inhibiting the phosphorylation of protein kinase B (Akt), mitogen-activated protein kinases (MAPKs) and phospholipase C (PLC) ß3. In contrast, LA only inhibited the phosphorylation of PLCß3. In conclusion, WXKL and its active component LA showed good antiplatelet and antithrombotic efficacy in vivo and in vitro. Mechanistically, the multicomponent Chinese medicine WXKL acts on multiple targets in the platelet activation pathway whereas its active monomer linoleic acid acts specifically on phospholipase C ß3.

Curcumin by activation of adenosine A2A receptor stimulates protein kinase a and potentiates inhibitory effect of cangrelor on platelets.

Curcumin is a natural polyphenol derived from the turmeric plant (Curcuma longa) which exhibits numerous beneficial effects on different cell types. Inhibition of platelet activation by curcumin is well known, however molecular mechanisms of its action on platelets are not fully defined. In this study, we used laser diffraction method for analysis of platelet aggregation and Western blot for analysis of intracellular signaling mechanisms of curcumin effects on platelets. We identified two new molecular mechanisms involved in the inhibitory effects of curcumin on platelet activation. Firstly, curcumin by activation of adenosine A2A receptor stimulated protein kinase A activation and phosphorylation of Vasodilator-stimulated phosphoprotein. Secondly, we demonstrated that curcumin even at low doses, which did not inhibit platelet aggregation, potentiated inhibitory effect of ADP receptor P2Y12 antagonist cangrelor which partly could be explained by activation of adenosine A2A receptor.

Effects of Nutrients on Platelet Function: A Modifiable Link between Metabolic Syndrome and Neurodegeneration?

Metabolic syndrome increases the risk of vascular dementia and other neurodegenerative disorders. Recent studies underline that platelets play an important role in linking peripheral with central metabolic and inflammatory mechanisms. In this narrative review, we address the activation of platelets in metabolic syndrome, their effects on neuronal processes and the role of the mediators (e.g., serotonin, platelet-derived growth factor). Emerging evidence shows that nutritional compounds and their metabolites modulate these interactions-specifically, long chain fatty acids, endocannabinoids and phenolic compounds. We reviewed the role of activated platelets in neurovascular processes and nutritional compounds in platelet activation.

Rutaecarpine, an Alkaloid from Evodia rutaecarpa, Can Prevent Platelet Activation in Humans and Reduce Microvascular Thrombosis in Mice: Crucial Role of the PI3K/Akt/GSK3ß Signal Axis through a Cyclic Nucleotides/VASP-Independent Mechanism.

The role of activated platelets in acute and chronic cardiovascular diseases (CVDs) is well established. Therefore, antiplatelet drugs significantly reduce the risk of severe CVDs. Evodia rutaecarpa (Wu-Chu-Yu) is a well-known Chinese medicine, and rutaecarpine (Rut) is a main bioactive component with substantial beneficial properties including vasodilation. To address a research gap, we investigated the inhibitory mechanisms of Rut in washed human platelets and experimental mice. At low concentrations (1-5 µM), Rut strongly inhibited collagen-induced platelet aggregation, whereas it exerted only a slight or no effect on platelets stimulated with other agonists (e.g., thrombin). Rut markedly inhibited P-selectin expression; adenosine triphosphate release; [Ca2+]i mobilization; hydroxyl radical formation; and phospholipase C (PLC)γ2/protein kinase C (PKC), mitogen-activated protein kinase, and phosphoinositide 3-kinase (PI3K)/Akt/glycogen synthase kinase-3ß (GSK3ß) phosphorylation stimulated by collagen. SQ22536 (an adenylate cyclase inhibitor) or ODQ (a guanylate cyclase inhibitor) did not reverse Rut-mediated antiplatelet aggregation. Rut was not directly responding to vasodilator-stimulated phosphoprotein phosphorylation. Rut significantly increased the occlusion time of fluorescence irradiated thrombotic platelet plug formation. The findings demonstrated that Rut exerts a strong effect against platelet activation through the PLCγ2/PKC and PI3K/Akt/GSK3ß pathways. Thus, Rut can be a potential therapeutic agent for thromboembolic disorders.

Dietary Antiplatelets: A New Perspective on the Health Benefits of the Water-Soluble Tomato Concentrate Fruitflow®.

Our understanding of platelet functionality has undergone a sea change in the last decade. No longer are platelets viewed simply as regulators of haemostasis; they are now acknowledged to be pivotal in coordinating the inflammatory and immune responses. This expanded role for platelets brings new opportunities for controlling a range of health conditions, targeting platelet activation and their interactions with other vascular cells. Antiplatelet drugs may be of wider utility than ever expected but often cause platelet suppression too strong to be used out of clinical settings. Dietary antiplatelets represent a nutritional approach that can be efficacious while safe for general use. In this review, we discuss potential new uses for dietary antiplatelets outside the field of cardiovascular health, with specific reference to the water-soluble tomato extract Fruitflow®. Its uses in different aspects of inflammation and immune function are discussed, highlighting exercise-induced inflammation, mediating the effects of air pollution, and controlling thrombotic aspects of the immune response. Potential future developments in women's health, erectile dysfunction, and the allergic response indicate how broad the utility of dietary antiplatelets can be.

Pharmacologic targeting of Cdc42 GTPase by a small molecule Cdc42 activity-specific inhibitor prevents platelet activation and thrombosis.

Gene targeting of Cdc42 GTPase has been shown to inhibit platelet activation. In this study, we investigated a hypothesis that inhibition of Cdc42 activity by CASIN, a small molecule Cdc42 Activity-Specific INhibitor, may down regulate platelet activation and thrombus formation. We investigated the effects of CASIN on platelet activation in vitro and thrombosis in vivo. In human platelets, CASIN, but not its inactive analog Pirl7, blocked collagen induced activation of Cdc42 and inhibited phosphorylation of its downstream effector, PAK1/2. Moreover, addition of CASIN to washed human platelets inhibited platelet spreading on immobilized fibrinogen. Treatment of human platelets with CASIN inhibited collagen or thrombin induced: (a) ATP secretion and platelet aggregation; and (b) phosphorylation of Akt, ERK and p38-MAPK. Pre-incubation of platelets with Pirl7, an inactive analog of CASIN, failed to inhibit collagen induced aggregation. Washing of human platelets after incubation with CASIN eliminated its inhibitory effect on collagen induced aggregation. Intraperitoneal administration of CASIN to wild type mice inhibited ex vivo aggregation induced by collagen but did not affect the murine tail bleeding times. CASIN administration, prior to laser-induced injury in murine cremaster muscle arterioles, resulted in formation of smaller and unstable thrombi compared to control mice without CASIN treatment. These data suggest that pharmacologic targeting of Cdc42 by specific and reversible inhibitors may lead to the discovery of novel antithrombotic agents.

Vaccine-Induced Immune Thrombotic Thrombocytopenia (VITT): Targeting Pathomechanisms with Bruton Tyrosine Kinase Inhibitors.

A series of cases with rare thromboembolic incidents including cerebral sinus vein thrombosis (some of them fatal) and concomitant thrombocytopenia occurring shortly after vaccination with the coronavirus disease 2019 (COVID-19) vaccine AZD1222 (Vaxzevria) have caused significant concern and led to its temporary suspension in many countries. Immediate laboratory efforts in four of these patients have identified a tentative pathomechanism underlying this syndrome termed initially vaccine-induced prothrombotic immune thrombocytopenia (VIPIT) and renamed recently vaccine-induced immune thrombotic thrombocytopenia (VITT). It encompasses the presence of platelet-activating antibodies to platelet factor-4/heparin complexes, possibly emulated by polyanionic constituents of AZD1222, and thus resembles heparin-induced thrombocytopenia (HIT). Because these immune complexes bind and activate platelets via Fcγ receptor IIA (FcγRIIA), high-dose intravenous immunoglobulin G has been suggested for treatment of VITT in addition to non-heparin anticoagulants. Here we propose inhibitors of Bruton tyrosine kinase (Btk) approved for B cell malignancies (e.g., ibrutinib) as another therapeutic option in VITT, as they are expected to pleiotropically target multiple pathways downstream of FcγRIIA-mediated Btk activation, for example, as demonstrated for the effective inhibition of platelet aggregation, dense granule secretion, P-selectin expression and platelet-neutrophil aggregate formation stimulated by FcγRIIA cross-linking. Moreover, C-type lectin-like receptor CLEC-2- and GPIb-mediated platelet activation, the interactions and activation of monocytes and the release of neutrophil extracellular traps, as encountered in HIT, could be attenuated by Btk inhibitors. As a paradigm for emergency repurposing of approved drugs in COVID-19, off-label use of Btk inhibitors in a low-dose range not affecting haemostatic functions could thus be considered a sufficiently safe option to treat VITT.

Anti-Platelet Properties of Phenolic and Nonpolar Fractions Isolated from Various Organs of Elaeagnus rhamnoides (L.) A. Nelson in Whole Blood.

Sea buckthorn (Elaeagnus rhamnoides (L.) A. Nelson) is a shrub growing in coastal areas. Its organs contain a range of bioactive substances including vitamins, fatty acids, various micro and macro elements, as well as phenolic compounds. Numerous studies of sea buckthorn have found it to have anticancer, anti-ulcer, hepatoprotective, antibacterial, and antiviral properties. Some studies suggest that it also affects the hemostasis system. The aim of the study was to determine the effect of six polyphenols rich and triterpenic acids rich fractions (A-F), taken from various organs of sea buckthorn, on the activation of blood platelets using whole blood, and to assess the effect of the tested fractions on platelet proteins: fraction A (polyphenols rich fraction from fruits), fraction B (triterpenic acids rich fraction from fruits), fraction C (polyphenols rich fraction from leaves), fraction D (triterpenic acids rich fraction from leaves), fraction E (polyphenols rich fraction from twigs), and fraction F (triterpenic acids rich fraction from twigs). Hemostasis parameters were determined using flow cytometry and T-TAS (Total Thrombus-formation Analysis System). Additionally, electrophoresis was performed under reducing and non-reducing conditions. Although all tested fractions inhibit platelet activation, the greatest anti-platelet activity was demonstrated by fraction A, which was rich in flavonol glycosides. In addition, none of the tested fractions (A-F) caused any changes in the platelet proteome, and their anti-platelet potential is not dependent on the P2Y12 receptor.

In vitro antiplatelet activity of extract and its fractions of Paulownia Clone in Vitro 112 leaves.

BACKGROUND: Paulownia Clone in Vitro 112, also known as Oxytree is a hybrid of Paulownia elongata and Paulownia fortunei, developed under laboratory conditions. Its seeds are sterile, making it a noninvasive variety that can only be propagated in the laboratory. In China, species from the Paulownia genus (Paulowniaceae) are widely used in traditional medicine for the treatment of infectious diseases, such as gonorrhea and erysipelas. It has a broad spectrum of bioactivity, including neuroprotective, antioxidant, antibacterial, antiphlogistic, antiviral, and cytotoxic actions. However, the antiplatelet potential of Paulownia Clone in Vitro 112 has not yet been described. STUDY DESIGN: The aim of our study was thus to examine the effect of an extract and four fractions from leaves of Paulownia Clone in Vitro 112 on various parameters of platelet activation in an in vitro model. METHODS: Composition of the investigated extract and fractions was determined by UHPLC-UV-MS. The following parameters of platelet activation were investigated: nonenzymatic lipid peroxidation in resting platelets; enzymatic lipid peroxidation (AA metabolism) in platelets activated by thrombin; superoxide anion (O2-.) generation in the resting and activated platelets; platelet adhesion to collagen type I and fibrinogen; platelet aggregation stimulated by various physiological agonists, such as ADP, collagen, and thrombin. The effect of the extract and fractions on extracellular LDH activity, a marker of cell damage, was also determined. RESULTS: Verbascoside a phenylethnanoid glycoside, was the main secondary metabolite of the extract from leaves of oxytree (constituting approximately 45 % of all compounds). There were also iridoids, such as catalpol, aucubin, and 7-hydroxytomentoside, as well as flavonoids, such as luteolin and apigenin glycosides. Moreover, the extract had stronger antiplatelet properties than the fractions. For example, the extract at 10 µg/mL inhibited five parameters of platelet activation. CONCLUSIONS: Our results show that Paulownia Clone in Vitro 112 leaves are a new valuable source of compounds with antiplatelet potential.

Juglone prevents human platelet aggregation through inhibiting Akt and protein disulfide isomerase.

BACKGROUND/PURPOSE: Juglone, a natural compound widely found in Juglandaceae plants, has been suggested as a potential drug candidate for treating cancer, inflammation, and diabetic vascular complications. In the present study, the antiplatelet effect and underlying mechanisms of juglone were investigated for the first time. STUDY DESIGN/METHODS: Human platelet aggregation and activation were measured by turbidimetric aggregometry, flow cytometry, and Western blotting. In vitro antithrombotic activity of juglone was assessed using collagen-coated flow chambers under whole-blood flow conditions. The effect of juglone on protein disulfide isomerase (PDI) activity was determined by the dieosin glutathione disulfide assay. RESULTS: Juglone (1 - 5 µM) inhibited platelet aggregation and glycoprotein (GP) IIb/IIIa activation caused by various agonists. In a whole blood flow chamber system, juglone reduced thrombus formation on collagen-coated surfaces under arterial shear rates. Juglone abolished intracellular Ca2+ elevation and protein kinase C activation caused by collagen, but had no significant effect on that induced by G protein-coupled receptor agonists. In contrast, Akt activation caused by various agonists were inhibited in juglone-treated platelets. Additionally, juglone showed inhibitory effects on both recombinant human PDI and platelet surface PDI at concentrations similar to those needed to prevent platelet aggregation. CONCLUSION: Juglone exhibits potent in vitro antiplatelet and antithrombotic effects that are associated with inhibition of Akt activation and platelet surface PDI activity.

Combination of Panax notoginseng saponins and aspirin potentiates platelet inhibition with alleviated gastric injury via modulating arachidonic acid metabolism.

High platelet reactivity and gastric mucosal injury after aspirin (ASA) treatment are associated with poor compliance and an increased risk of cardiovascular events. Panax notoginseng saponins (PNS) have been widely used for the treatment of coronary heart disease (CHD) in addition to antiplatelet drugs in China; however, the joint effect and possible mechanism of PNS in addition to ASA on platelet activation and gastric injury remain unclear. This study was designed to investigate the combinational effects of PNS with ASA, and to explore the underlying mechanism via arachidonic acid (AA) metabolism pathway using lipidomic analysis. In a randomized, assessor-blinded trial, 42 patients with stable coronary heart disease (SCHD) and chronic gastritis were randomly assigned to receive ASA (n = 21) or PNS + ASA (n = 21) for 2 months. Compared with ASA alone, PNS + ASA further inhibited CD62p expression, GPIIb-IIIa activation and platelet aggregation and led to increased platelet inhibition rate. PNS + ASA suppressed the activity of platelet cyclooxygenase (COX)-1, and decreased the production of TXB2, PGD2, PGE2, 11-HETE, the downstream oxylipids of AA/COX-1 pathway in platelets, compared with ASA alone. The severity of dyspepsia assessment (SODA) results showed that patients in PNS + ASA group exhibited relieved dyspeptic symptoms as compared with those in ASA group, which might be associated with enhanced secretion of gastrin and motilin. In vivo study of myocardial infarction rats demonstrated that PNS attenuated ASA-induced gastric mucosal injury, which was related to markedly boosted gastric level of 6,15-diketo-13,14-dihydro-prostaglandin (PG)F1α, 13,14-dihydro-15-keto-PGE2 and PGE2 from AA/PG pathway in response to PNS + ASA compared with ASA alone. In summary, our study demonstrated that the combination of PNS and ASA potentiated the antiplatelet effect of ASA via AA/COX-1/TXB2 pathway in platelets, and mitigated ASA-related gastric injury via AA/PG pathway in gastric mucosa.

Understanding the Activation of Platelets in Diabetes and Its Modulation by Allyl Methyl Sulfide, an Active Metabolite of Garlic.

BACKGROUND: Diabetes mellitus (DM) is a chronic metabolic disorder associated with higher risk of having cardiovascular disease. Platelets play a promising role in the pathogenesis of cardiovascular complications in diabetes. Since last several decades, garlic and its bioactive components are extensively studied in diabetes and its complications. Our aim was to explore the antiplatelet property of allyl methyl sulfide (AMS) focusing on ameliorating platelet activation in diabetes. METHOD: We used streptozotocin- (STZ-) induced diabetic rats as model for type 1 diabetes. We have evaluated the effect of allyl methyl sulfide on platelet activation by administrating AMS to diabetic rats for 10 weeks. Flow cytometry-based analysis was used to evaluate the platelet activation, platelet aggregation, platelet macrophage interaction, and endogenous ROS generation in the platelets obtained from control, diabetes, and AMS- and aspirin-treated diabetic rats. RESULTS: AMS treatment for 10 weeks effectively reduced the blood glucose levels in diabetic rats. Three weeks of AMS (50 mg/kg/day) treatment did not reduce the activation of platelets but a significant (p < 0.05) decrease was observed after 10 weeks of treatment. Oral administration of AMS significantly (p < 0.05) reduced the baseline and also reduced ADP-induced aggregation of platelets after 3 and 10 weeks of treatment. Furthermore, 10 weeks of AMS treatment in diabetic rats attenuated the endogenous ROS content (p < 0.05) of platelets and platelet macrophage interactions. The inhibition of platelet activation in diabetic rats after AMS treatment was comparable with aspirin treatment (30 mg/kg/day). CONCLUSION: We observed an inhibitory effect of allyl methyl sulfide on platelet aggregation, platelet activation, platelet macrophage interaction, and increased ROS levels in type 1 diabetes. Our data suggests that AMS can be useful to control cardiovascular complication in diabetes via inhibition of platelet activation.

Quercitrin inhibits platelet activation in arterial thrombosis.

BACKGROUND: The ingestion of flavonoids has been reported to be associated with reduced cardiovascular disease risk. Quercitrin is a common flavonoid in nature, and it exhibits antioxidant properties. Although the process of thrombogenesis is intimately related to cardiovascular disease risk, it is unclear whether quercitrin plays a role in thrombogenesis. PURPOSE: The aim of this study was to examine the antiplatelet effect of quercitrin in platelet activation. METHODS: Platelet aggregation, granule secretion, calcium mobilization, and integrin activation were used to assess the antiplatelet activity of quercitrin. Antithrombotic effect was determined in mouse using ferric chloride (FeCl3)-induced arterial thrombus formation in vivo and thrombus formation on collagen-coated surfaces under arteriolar shear in vitro. Transection tail bleeding time was used to evaluate whether quercitrin inhibited primary hemostasis. RESULTS: Quercitrin significantly impaired collagen-related peptide-induced platelet aggregation, granule secretion, reactive oxygen species generation, and intracellular calcium mobilization. Outside-in signaling of αIIbß3 integrin was significantly inhibited by quercitrin in a concentration-dependent manner. The inhibitory effect of quercitrin resulted from inhibition of the glycoprotein VI-mediated platelet signal transduction during cell activation. Further, the antioxidant effect is derived from decreased phosphorylation of components of the TNF receptor-associated factor 4/p47phox/Hic5 axis signalosome. Oral administration of quercitrin efficiently blocked FeCl3-induced arterial thrombus formation in vivo and thrombus formation on collagen-coated surfaces under arteriolar shear in vitro, without prolonging bleeding time. Studies using a mouse model of ischemia/reperfusion-induced stroke indicated that treatment with quercitrin reduced the infarct volume in stroke. CONCLUSIONS: Our results demonstrated that quercitrin could be an effective therapeutic agent for the treatment of thrombotic diseases.

Effect of leech-centipede medicine on improving erectile function in DIED rats via PKC signalling pathway-related molecules.

ETHNOPHARMACOLOGICAL RELEVANCE: Leeches (pinyin name Shui Zhi; Latin scientific name Hirudo; Hirudinea; Hirudinidae) and centipedes (pinyin name Wu Gong; Latin scientific name Scolopendridae; Chilopoda; Scolopendridae) are traditional Chinese medicines, and they belong to the family entomology. A combination of leech and centipede is used as an effective medicine to promote blood circulation and remove blood stasis in traditional Chinese medicine, and "leech-centipede" medicine has been used in many prescriptions to treat diabetic vascular disease, including diabetic erectile dysfunction (DIED). However, its specific mechanism remains unclear and requires in-depth study. AIM OF THE STUDY: This study aimed to investigate the mechanism of "leech-centipede" medicine to improve erectile dysfunction-associated diabetes by detecting PKC pathway-related molecules. MATERIALS AND METHODS: The active ingredients of "leech-centipede" medicine were identified using high performance liquid chromatography (HPLC). Fifty male SPF rats were injected with streptozotocin to induce the DM model. Eight weeks later, the DMED model was validated with apomorphine. The DIED rats were divided into five groups-T,P,DD,DZ, and DG-and were separately treated with tadalafil, pathway inhibitor LY333531 and low-, medium-, and high-dose "leech-centipede" medicine for 8 weeks. After treatment, the blood glucose level was measured, erectile function with apomorphine was assessed, the LOX-1, sE-selectin, sICAM-1, SOD, and MDA in serum was evaluated by enzyme-linked immunosorbent assay, and flow cytometry was performed. After the collection of penile tissue, the related protein and mRNA expression was assessed by Western blotting and PCR, and the tissue and ultrastructure were analysed by HE staining, immunohistochemistry and scanning electron microscopy. RESULTS: After treatment, the erectile function of rats was significantly improved in the T,P,DD,DZ, and DG groups compared with that in the model group. Thus, "leech-centipede" medicine can significantly reduce the levels of LOX-1, sE-selectin, sICAM-1, EMPs and CD62P to protect vascular endothelial function and anti-platelet activation, improving DIED rat erectile function. Additionally, "leech-centipede" medicine can increase SOD expression and decrease MDA expression, reducing the possibility of oxidative stress injury in DIED rats and improving the antioxidant capacity. Moreover, "leech-centipede" therapy can dramatically reduce the protein and mRNA expression of DAG, PKCß, NF-κB, and ICAM-1, improve vascular endothelial injury in DIED rats and inhibit abnormal platelet activation. CONCLUSION: "leech-centipede" medicine can improve erectile dysfunction by inhibiting the expression of PKC pathway-related molecules in DIED rats and protects endothelial function and anti-platelet activation.

Effects of nitrite and far-red light on coagulation.

Nitric oxide, NO, has been explored as a therapeutic agent to treat thrombosis. In particular, NO has potential in treating mechanical device-associated thrombosis due to its ability to reduce platelet activation and due to the central role of platelet activation and adhesion in device thrombosis. Nitrite is a unique NO donor that reduces platelet activation in that it's activity requires the presence of red blood cells whereas NO activity of other NO donors is blunted by red blood cells. Interestingly, we have previously shown that red blood cell mediated inhibition of platelet activation by adenosine diphosophate (ADP) is dramatically enhanced by illumination with far-red light that is likely due to photolysis of red cell surface bound NO congeners. We now report the effects of nitrite, far-red light, and their combination on several measure of blood coagulation using a variety of agonists. We employed turbidity assays in platelet rich plasma, platelet activation using flow cytometry analysis of a fluorescently labeled antibody to the activated platelet fibrinogen binding site, multiplate impedance-based platelet aggregometry, and assessment of platelet adhesion to collagen coated flow-through microslides. In all cases, the combination of far-red light and nitrite treatment decreased measures of coagulation, but in some cases mono-treatment with nitrite or light alone had no effect. Perhaps most relevant to device thrombosis, we observed that platelet adhesions was inhibited by the combination of nitrite and light treatment while nitrite alone and far-red light alone trended to decrease adhesion, but the results were mixed. These results support the potential of combined far-red light and nitrite treatment for preventing thrombosis in extra-corporeal or shallow-tissue depth devices where the far-red light can penetrate. Such a combined treatment could be advantageous due to the localized treatment afforded by far-red light illumination with minimal systemic effects. Given the role of thrombosis in COVID 19, application to treatment of patients infected with SARS Cov-2 might also be considered.

Noncanonical Effects of Oral Thrombin and Factor Xa Inhibitors in Platelet Activation and Arterial Thrombosis.

Nonvitamin K oral anticoagulants (NOACs) or direct oral anticoagulants comprise inhibitors of factor Xa (rivaroxaban, apixaban, edoxaban) or factor IIa (dabigatran). Both classes efficiently interfere with the final or penultimate step of the coagulation cascade and showed superior net clinical benefit compared with vitamin K antagonists for prevention of thromboembolic events in patients with AF and for prevention and therapy of deep vein thrombosis and pulmonary embolism. None the less, accumulating data suggested, that there may be differences regarding the frequency of atherothrombotic cardiovascular events between NOACs. Thus, the optimal individualized NOAC for each patient remains a matter of debate. Against this background, some basic and translational analyses emphasized NOAC effects that impact on platelet activity and arterial thrombus formation beyond inhibition of plasmatic coagulation. In this review, we will provide an overview of the available clinical and translational evidence for so-called noncanonical NOAC effects on platelet activation and arterial thrombosis.

Mumefural Improves Blood Flow in a Rat Model of FeCl3-Induced Arterial Thrombosis.

Mumefural (MF), a bioactive component of the processed fruit of Prunus mume Sieb. et Zucc, is known to inhibit platelet aggregation induced by agonists in vitro. In this study, we investigated the anti-thrombotic effects of MF using a rat model of FeCl3-induced arterial thrombosis. Sprague-Dawley rats were intraperitoneally injected with MF (0.1, 1, or 10 mg/kg) 30 min before 35% FeCl3 treatment to measure the time to occlusion using a laser Doppler flowmeter and to assess the weight of the blood vessels containing thrombus. MF treatment significantly improved blood flow by inhibiting occlusion and thrombus formation. MF also prevented collagen fiber damage in injured vessels and inhibited the expression of the platelet activation-related proteins P-selectin and E-selectin. Moreover, MF significantly reduced the increased inflammatory signal of nuclear factor (NF)-κB, toll-like receptor 4 (TLR4), tumor necrosis factor (TNF)-α, and interleukin (IL)-6 in blood vessels. After administration, MF was detected in the plasma samples of rats with a bioavailability of 36.95%. Therefore, we suggest that MF may improve blood flow as a candidate component in dietary supplements for improving blood flow and preventing blood circulation disorders.

Lagopsis supina extract and its fractions exert prophylactic effects against blood stasis in rats via anti-coagulation, anti-platelet activation and anti-fibrinolysis and chemical characterization by UHPLC-qTOF-MS/MS.

Lagopsis supina (Steph.) IK. -Gal. ex Knorr. has been used for centuries as an empiric treatment for blood stasis syndrome in China without scientific validation. The aim of this study was to evaluate for the first time the chemical profiling, efficacy and mechanism of L. supina ethanol extract (LS) and its four fractions (LSA∼D) in Dextran 500-induced acute blood stasis model rats. Oral administration of LS (229.0∼916.0 mg/kg) and LSC (17.6∼70.4 mg/kg) once daily for seven consecutive days significantly improved microcirculation hemodynamics function (blood flow, blood concentration and blood flow velocity), hemorheological parameters (whole blood viscosity, whole blood reduced viscosity, plasma viscosity, platelet aggregation rate, hematokrit, erythrocyte assembling index and erythrocyte deformation index), and coagulation parameters (thrombin time, prothrombin time, activated partial thromboplastin time, fibrinogen and antithrombin III). Furthermore, their markedly down-regulated thromboxane B2 and 6-keto-prostaglandin F1α levels. In addition, it significantly decreased tissue-type plasminogen activator (t-PA), urokinasetype plasminogen activator (u-PA) and plasminogen activator inhibitor-1 (PAI-1) levels, as well as PAI-1/t-PA and PAI-1/u-PA rations. In parallel, 51 chemical constituents were identified from LS based on ultra-high-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UHPLC-qTOF-MS/MS), and quantitative analysis showed that the two major constituents of stachysoside A and acteoside were present in 0.90 ± 0.01 and 1.36 ± 0.01 mg/g of the L. supina whole plant, respectively. These findings suggest that LS and LSC possess prominent anti-blood stasis effect on rats by modulating the anti-coagulation, anti-platelet activation and anti-fibrinolysis, and supports the traditional folk use of this plant.