Collagen-stimulated Platelets Research Articles

Response of human blood platelets to preparations from leaves of Paulownia Clon in Vitro 112

Paulownia Clon in Vitro 112, also called the Oxytree, is a fast-growing hybrid of two trees belonging to the Paulowniaceae family – P. elongata and P. fortunei. It demonstrates a wide range of biological effects (including antioxidant, anti-inflammatory, antibacterial, and neuroprotective) due to the high concentration of secondary metabolites. Our previous results showed an in vitro antioxidant and antiplatelet activity of the extract and four fractions (A–D) from the leaves of Paulownia Clon in Vitro 112 in human plasma and washed blood platelets. Here, we used a microchip flow chamber-based thrombus formation analysis system (T-TAS) and flow cytometry to assess the anticoagulant and antiplatelet activity of the extract and four fractions with different chemical content (A-D) from Paulownia Clon in Vitro 112 leaves in human whole blood. Two tested fractions: fraction C and D (at the concentrations of 5 and 50 μg/mL) inhibited the exposition of the active form of GPIIb/IIIa (integrin αIIbβ3) on the surface of blood platelets stimulated by ADP and collagen. The antiplatelet activity of fraction C is likely due to its high verbascoside content and the presence of apigenin’s derivatives. Fraction D contains triterpenoids, including ursolic, pomoleic, and maslinic acid, which could be responsible for decreased activation of ADP- and collagen-stimulated blood platelets. These results suggest that fractions C and D might be promising sources of phytochemicals with antiplatelet activity, which are important for prophylaxis and treatment of cardiovascular diseases associated with hyperactivation of blood platelets. However, further research is needed to ascertain which exact compounds and mechanisms are responsible for this phenomenon.

The Anti-Aggregative Potential of Resolvin E1 on Human Platelets.

Resolvin E1 is a metabolite of eicosapentaenoic acid (EPA) which is one of the omega-3 polyunsaturated fatty acids (omega-3 PUFAs). The antiplatelet properties of omega-3 PUFAs are well known, but the effect of resolvin E1 on platelets via the collagen receptors is extremely poorly reported. We investigated the effect of resolvin E1 on collagen-induced platelet aggregation, activation, and reactivity, and also platelet membrane fluidity. The ultimate and statistically significant results showed that resolvin E1 may inhibit platelet reactivity due to the reduction of collagen-induced platelet aggregation in platelet-rich plasma and isolated platelets, but not in whole blood. Also, resolvin E1 significantly reduced P-selectin exposure on collagen-stimulated platelets. Moreover, we demonstrated that resolvin E1 can maintain platelet membrane structure (without increasing membrane fluidity). The association between platelet reactivity and membrane fluidity, including resolvin E1 and collagen receptors requires further research. However, the goal of this study was to shed light on the molecular mechanisms behind the anti-aggregative effects of resolvin E1 on platelets, which are still not fully clarified. We also indicate an innovative research direction focused on further analysis and then use of omega-3 PUFAs metabolites as antiplatelet compounds for future applications in the treatment and prevention of cardiovascular diseases.

Effect of fruitflow on platelet function: A randomized placebo-controlled clinical trial in middle-aged and elderly subjects

• Fruitflow decreased the platelet aggregation rate ex vivo . • Fruitflow reduced the plasma TXB 2 , 6-keto-PGF 1α and PF4 level in subjects. • Fruitflow and aspirin didn’t exert synergistic effect on platelet aggregation. • Fruitflow supplementation for 7 days did not change coagulation. To investigate the effect of fruitflow, a water-soluble tomato extract, on platelet function in participants. 190 participants were completed this clinical trial. The participants were randomly divided into four groups: placebo, fruitflow, aspirin, and fruitflow + aspirin. These groups received the respective supplements for 7 days. Fruitflow intervention for 7 days decreased platelet aggregation ratio by 7.7% and 10.2% in adenosine diphosphate (ADP)- and collagen-stimulated platelets, respectively. Aspirin decreased platelet aggregation ratio by 9.4% and 39% in ADP- and collagen-stimulated platelets, respectively. The combination of fruitflow and aspirin did not exert synergistic inhibitory effect on platelet aggregation. Fruitflow also significantly reduced plasma thromboxane B 2 (TXB 2 ), 6-keto-prostaglandin F 1α (6-keto-PGF 1α ), and platelet factor 4 (PF4) levels. Fruitflow intervention decreased platelet aggregation rate, this suggests that fruitflow is a functional food for reducing platelet activity.

Glabridin, a Bioactive Flavonoid from Licorice, Effectively Inhibits Platelet Activation in Humans and Mice.

Platelets are crucial for hemostasis and arterial thrombosis, which may lead to severe cardiovascular diseases (CVDs). Thus, therapeutic agents must be developed to prevent pathological platelet activation. Glabridin, a major bioalkaloid extracted from licorice root, improves metabolic abnormalities (i.e., obesity and diabetes) and protects against CVDs and neuronal disorders. To the best of our knowledge, no studies have focused on glabridin’s effects on platelet activation. Therefore, we investigated these effects in humans and mice. Glabridin exhibited the highest inhibitory effects on collagen-stimulated platelet aggregation and moderate effects on arachidonic-acid-stimulated activation; however, no effects were observed for any other agonists (e.g., thrombin or U46619). Glabridin evidently reduced P-selectin expression, ATP release, and intracellular Ca2+ ([Ca2+]i) mobilization and thromboxane A2 formation; it further reduced the activation of phospholipase C (PLC)γ2/protein kinase C (PKC), phosphoinositide 3-kinase (PI3K)/Akt/glycogen synthase kinase-3β (GSK3β), mitogen-activated protein kinase (MAPK), and NF-κB. In mice, glabridin reduced the mortality rate caused by acute pulmonary thromboembolism without altering bleeding time. Thus, glabridin effectively inhibits the PLCγ2/PKC cascade and prevents the activation of the PI3K/Akt/GSK3β and MAPK pathways; this leads to a reduction in [Ca2+]i mobilization, which eventually inhibits platelet aggregation. Therefore, glabridin may be a promising therapeutic agent for thromboembolic disorders.

Fruitflow inhibits platelet function by suppressing Akt/GSK3\u03b2, Syk/PLC\u03b32 and p38 MAPK phosphorylation in collagen-stimulated platelets

BackgroundPlatelets play an important role in the progression of atherosclerosis and cardiovascular events. The inhibition of platelet function is a main strategy to reduce risk of cardiovascular events. Some studies have shown that tomato extracts inhibit platelet function, but the molecular mechanisms remain unclear. Fruitflow is a water-solute tomato extract and the main ingredients including flavonoids, adenosine, chlorogenic acid, phytosterols, naringenin, and carotenoids. The present study investigated the effects of fruitflow on adenosine diphosphate (ADP)- and collagen- stimulated platelet aggregation, platelet adhesion, and levels of thromboxane B2 (TXB2), 6-keto-prostaglandin F1α (PGF1α), and platelet factor 4 (PF4) and explored the underlying molecular mechanisms.MethodsPlatelet-rich plasma (PRP) was used for measurement of platelet aggregation, TXB2, 6-keto- PGF1α, and PF4 levels. Platelet aggregation was analyzed using a Chrono-Log aggregometer. TXB2, 6-keto- PGF1α, and PF4 levels were determined using enzyme-linked immunosorbent assay kits. Immunoblotting was used to detect protein expression and phosphorylation on washed platelets. Platelet adhesion and spreading were determined by immunofluorescence.ResultsFruitflow (1, 3, 10 and 100 μg/ml) dose-dependently inhibited platelet aggregation that was induced by ADP and collagen. Fruitflow (100 μg/ml) treatment completely suppressed ADP- and collagen-stimulated platelet aggregation. Fruitflow (100 μg/ml) significantly decreased TXB2 and 6-keto-PGF1α generation and PF4 release in ADP- and collagen-stimulated platelets. Treatment with fruitflow effectively blocked collagen-induced platelet spreading. To determine the potential molecule mechanism of action of fruitflow, we investigated the protein expression and phosphorylation of several signaling molecules in collagen-activated platelets. Fruitflow dose-dependently suppressed Akt, Glycogen synthase kinase-3β (GSK-3β), spleen tyrosine kinase (Syk) and phospholipase Cγ2 (PLCγ2) and p38 MAPK phosphorylation that was induced by collagen.ConclusionFruitflow inhibited platelet aggregation and reduced TXB2, 6-keto-PGF1α, and PF4 levels in ADP- and collagen-stimulated platelets. The mechanism of action of fruitflow may be associated with the suppression of Akt/GSK3β, Syk/PLCγ2, and p38 MAPK phosphorylation in collagen-activated platelets. Fruitflow is a natural product derived from tomato and can be used as a health food for decreasing platelet activity.

Thrombopoietin and collagen in low doses cooperatively induce human platelet activation.

AimIn acute medicine, we occasionally treat life‐threatening conditions such as sepsis and trauma, which cause severe thrombocytopenia. Serum thrombopoietin levels have been reported to increase under the condition of thrombocytopenia related to severity. Collagen is a crucial activator of platelets, and Rho family members, such as Rho/Rho‐kinase and Rac, play roles as active molecules involved in the intracellular signaling pathways in platelet activation. The present study aimed to elucidate the effects of thrombopoietin (TPO) on subthreshold low‐dose collagen‐stimulated human platelets in terms of Rho/Rho‐kinase and Rac.MethodsPlatelet‐rich plasma donated from healthy volunteers was stimulated by the subthreshold low‐dose of collagen after pretreatment with TPO and/or NSC23766, an inhibitor of the Rac‐guanine nucleotide exchange factor interaction, or Y27632, an inhibitor of Rho‐kinase. Platelet aggregation was measured using an aggregometer based on laser‐scattering methods. Proteins involved in intracellular signaling were analyzed using western blotting, and the secretion of platelet‐derived growth factor‐AB from activated platelets was determined using an enzyme‐linked immunosorbent assay.ResultsUnder the existence of TPO, the low dose of collagen remarkably elicited the aggregation and platelet‐derived growth factor‐AB secretion of platelets, which were suppressed by NSC23766 and Y27632. The combination of TPO and collagen considerably induced a transient increase of guanosine triphosphate (GTP)‐binding Rac and GTP‐binding Rho followed by an increase of phosphorylated cofilin, a Rho‐kinase substrate.ConclusionThese results strongly suggest that TPO and collagen in low doses cooperatively potentiate human platelet activation through both Rac and Rho/Rho‐kinase mediated pathways.

Toll-like receptor 4 activation in platelets from myocardial infarction patients

IntroductionPlatelet toll-like receptor 4 (TLR4) is overexpressed in patients with myocardial infarction (MI) but it remains to elucidate if it is activated and the potential trigger. MethodsSerum levels of lipopolysaccharides (LPS) and platelet aggregation (PA) by collagen alone or in combination with a TLR4 inhibitor (TLR4i) were studied ex vivo in platelets from 40 MI patients and 40 controls matched for age, sex and atherosclerotic risk factors; platelet TIR domain-containing adaptor protein (TIRAP) and TIRAP-MyD88 interaction were also investigated by western blot and co-immunoprecipitation, respectively. In vitro experiments were conducted to see if LPS triggers platelet TIRAP phosphorylation. ResultsSerum LPS was significantly higher in patients compared to controls (29.5±7.1 vs 16.2±3.8 pg/mL; p<0.001). Collagen-stimulated platelets from MI pre-treated with TLR4i showed a significant decrease of PA compared to platelets stimulated with collagen. Ex vivo study showed that TIRAP phosphorylation as well as TIRAP-MyD88 co-immunoprecipitation were higher in patients compared to controls. In vitro study showed that LPS, at concentrations like those found in MI, dose-dependently activated TIRAP and amplified the platelet response to the agonist, an effect blunted by TLR4i. ConclusionThe study provides evidence that in MI patients platelet TLR4 is activated and suggests circulating LPS as potential trigger.

Different forms of vitamin E and metabolite 13’-carboxychromanols inhibit cyclooxygenase-1 and its catalyzed thromboxane in platelets, and tocotrienols and 13’-carboxychromanols are competitive inhibitors of 5-lipoxygenase

Cyclooxygenase (COX-1 and COX-2)- and 5-lipoxygenase (5-LOX)-catalyzed biosynthesis of eicosanoids play important roles in inflammation and chronic diseases. The vitamin E family has four tocopherols and tocotrienols. We have shown that the metabolites of δ-tocopherol (δT) and δ-tocotrienol (δTE), i.e., δT-13’-carboxychromanol (COOH) and δTE-13’-COOH, respectively, inhibit COX-1/-2 and 5-LOX activity, but the nature of how they inhibit 5-LOX is not clear. Further, the impact of tocopherols and tocotrienols on COX-1/-2 or 5-LOX activity has not been fully delineated. In this study, we found that tocopherols and tocotrienols inhibited human recombinant COX-1 with IC50s of 1–12 µM, and suppressed COX-1-mediated formation of thromboxane in collagen-stimulated rat's platelets with IC50s of 8–50 µM. None of the vitamin E forms directly inhibited COX-2 activity. 13’-COOHs inhibited COX-1 and COX-2 enzyme activity with IC50s of 3–4 and 4–10 µM, respectively, blocked thromboxane formation in collagen- and ionophore-stimulated rats’ platelets with IC50s of 1.5-2.5 µM, and also inhibited COX-2-mediated prostaglandins in stimulated cells. Using enzyme kinetics, we observed that δT-13’-COOH, δTE-13’-COOH and δTE competitively inhibited 5-LOX activity with Ki of 1.6, 0.8 and 2.2 µM, respectively. These compounds decreased leukotriene B4 from stimulated neutrophil-like cells without affecting translocation of 5-LOX from cytosol to the nucleus. Our study reveals inhibitory effects of vitamin E forms and 13’-COOHs on COX-1 activity and thromboxane formation in platelets, and elucidates mechanisms underlying their inhibition of 5-LOX. These observations are useful for understanding the role of these compounds in disease prevention and therapy.

Water-Soluble Tomato Extract Fruitflow Alters the Phosphoproteomic Profile of Collagen-Stimulated Platelets.

Platelet hyperactivity is a risk factor for cardiovascular disease and thrombosis. Recent studies reported that the tomato extract Fruitflow inhibited platelet function, but the molecular mechanism is still unclear. The present study used proteomics to quantitatively analyze the effect of fruitflow on the inhibition of collagen-stimulated platelets and validated the involvement of several signaling molecules. Fruitflow significantly inhibited human platelet aggregation and P-selectin expression that were induced by collagen. Proteomics analysis revealed that compared fruitflow-treated collagen-stimulated platelets with only collagen-stimulated platelets, 60 proteins were upregulated and 10 proteins were downregulated. Additionally, 66 phosphorylated peptides were upregulated, whereas 37 phosphorylated peptides were downregulated. Gene Ontology analysis indicated that fruitflow treatment downregulated phosphoinositide 3-kinase (PI3K)/protein kinase B and guanosine triphosphatase-mediated signal transduction in collagen-activated platelets. Biological validation indicated that fruitflow decreased Akt, glycogen synthase kinase 3β, p38 mitogen-activated protein kinase (MAPK), and heat shock protein (Hsp27) phosphorylation in collagen-stimulated platelets. Fruitflow recovered cyclic adenosine monophosphate levels in collagen-activated platelets and reduced protein kinase A substrate phosphorylation that was induced by collagen. These findings suggest that fruitflow is a functional food that can inhibit platelet function, conferring beneficial effects for people who are at risk for platelet hyperactivity-associated thrombosis.

Ginsenoside Rk1 suppresses platelet mediated thrombus formation by downregulation of granule release and αIIbβ3 activation

Background and objectiveSynthetic ginsenoside compounds G-Rp (1,3, and 4) and natural ginsenosides in Panax ginseng 20(S)-Rg3, Rg6, F4 and Ro have inhibitory actions on human platelets. However, the inhibitory mechanism of ginsenoside Rk1 (G-Rk1) is still unclear thus, we initiated investigation of the anti-platelet mechanism by G-Rk1 from Panax ginseng. MethodologyOur study focused to investigate the action of G-Rk1 on agonist-stimulated human platelet aggregation, inhibition of platelet signaling molecules such as fibrinogen binding with integrin αIIbβ3 using flow cytometry, intracellular calcium mobilization, fibronectin adhesion, dense granule secretion, and thromboxane B2 secretion. Thrombin-induced clot retraction was also observed in human platelets. Key ResultsCollagen, thrombin, and U46619-stimulated human platelet aggregation were dose-dependently inhibited by G-Rk1, while it demonstrated a more effective suppression on collagen-stimulated platelet aggregation using human platelets. Moreover, G-Rk1 suppressed collagen-induced elevation of Ca2+ release from endoplasmic reticulum, granule release, and αIIbβ3 activity without any cytotoxicity. Conclusions and implicationsThese results indicate that G-Rk1 possess strong anti-platelet effect, proposing a new drug candidate for treatment and prevention of platelet-mediated thrombosis in cardiovascular disease.

Reduction of NF-κB Signals in Platelets and Prolongation of Platelet Plug Formation against High Shear Flow in Whole Blood on Human Subject by Columbianadin

Myocardial infarction and cerebral ischemic stroke during the process of arterial thrombosis are prominently causes of death worldwide. Platelets are anucleated cells and play a critical factor in these diseases. Columbianadin (CBN), a coumarin derivative from plants, inhibits effective platelet activation. In this study, platelet function analysis revealed that the closure time of the platelet plug in human whole blood significantly prolonged by CBN, whereas CBN did not pointedly prolong the bleeding time in mice. BAY11-7082 (an inhibitor of IκB kinase) and MG-132 (an inhibitor of proteasome) inhibited collagen-stimulated platelet aggregation and ATP-release in human platelets, BAY11-7082 exhibited a higher potency than MG-132. Moreover, CBN markedly reduced NF-κB activation (e.g., IκBα and p65 phosphorylation) and reversed IκBα degradation in activated platelets. We investigated intercellular signaling events between mitogen-activated protein kinases and NF-κB, and found that BAY11-7082 abolished JNK1/2 and ERK1/2 phosphorylation. Interestingly, SP600125 (an inhibitor of JNK) but not PD98059 (an inhibitor of ERK) had no effect in NF-κB activation in activated platelets. Moreover, CBN but not BAY11-7082 significantly reduced hydroxyl radical (HO●) formation in platelets. Therefore, we propose that CBN inhibits NF-κB activation in human platelets and could present a potent clinical treatment for thromboembolic diseases.

Carbon Dot Nanoparticles Exert Inhibitory Effects on Human Platelets and Reduce Mortality in Mice with Acute Pulmonary Thromboembolism.

The inhibition of platelet activation is considered a potential therapeutic strategy for the treatment of arterial thrombotic diseases; therefore, maintaining platelets in their inactive state has garnered much attention. In recent years, nanoparticles have emerged as important players in modern medicine, but potential interactions between them and platelets remain to be extensively investigated. Herein, we synthesized a new type of carbon dot (CDOT) nanoparticle and investigated its potential as a new antiplatelet agent. This nanoparticle exerted a potent inhibitory effect in collagen-stimulated human platelet aggregation. Further, it did not induce cytotoxic effects, as evidenced in a lactate dehydrogenase assay, and inhibited collagen-activated protein kinase C (PKC) activation and Akt (protein kinase B), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) phosphorylation. The bleeding time, a major side-effect of using antiplatelet agents, was unaffected in CDOT-treated mice. Moreover, our CDOT could reduce mortality in mice with ADP-induced acute pulmonary thromboembolism. Overall, CDOT is effective against platelet activation in vitro via reduction of the phospholipase C/PKC cascade, consequently suppressing the activation of MAPK. Accordingly, this study affords the validation that CDOT has the potential to serve as a therapeutic agent for the treatment of arterial thromboembolic disorders

CcMP-II Metalloproteinase from Cerastes Cerastes Snake Venom Acts both on Blood Vessel ECM and Platelets

Background: Snake venoms contain various metalloproteinases that are highly toxic, inducing in severe bleeding by interfering with the blood coagulation and degrading the basement membrane or extracellular matrix (ECM) components. It has been suggested that hemorrhagic metalloproteinases interact in a specific way with platelet surface proteins resulting in an alteration of platelet function.&nbsp; Hemorrhage and tissue necrosis are common manifestations of viperid envenomations in humans, largely due to the actions of prominent metalloproteinases. CcMP-II a weakly hemorrhagic metalloproteinase purified from Cerastes cerastes venom, as already described it belongs to the class P-II snake venom metalloproteinase. Methods: In this study, we characterized the biological effects of CcMP-II, including the ability of the enzyme to hydrolyze extracellular matrix components and plasma proteins, as well as its histopathological effects induced in capillaries. Then a study of the anti-aggregating activity was carried out and controlled for 10 min using a Chronolog aggregometer (ServiBIO) (USTHB, Faculty of Biological Sciences, BP 32, El-Alia Bab Ezzouar, 16111, Algiers, Algeria in 2019). The histopathological changes induced by CcMP-II were also analyzed and the 1 µm semi-thin sections are then stained with Toluidine Blue and the observation of these sections was realized using a photomicroscope. Toluidine Blue, magnification: 100x. Results: Obtained results showed that CcMP-II metalloproteinase hydrolyzed selectively the A α-chain of fibrinogen. This enzyme hydrolyzed also laminin and type IV collagen in time- and dose-dependent manner. CcMP-II inhibits collagen-induced platelet aggregation of human PRP with an IC50 value of 0.11 nM. &nbsp;Pathological changes induced by CcMP-II after intramuscular injection in mice gastrocnemius were also studied. Histological study showed that the main morphological alterations in capillary vessels are characterized by a separation of endothelial cells from the surrounding basement membrane and reduction in the width of these cells, together with loss of basement membrane in some areas, through which erythrocytes and plasma are extravasated. Conclusion: In conclusion, CcMP-II, as a member of the P-II class of SVMPs, presents a selective specificity toward fibrinogen, laminin and type IV collagen. It was an α- fibrinogenase metalloproteinase, which inhibits collagen-stimulated platelet aggregation. This study will contribute to understanding better the functional mechanisms of the metalloproteinases that may be useful in the development of therapeutic agents related to thrombotic disorders.

A novel role of MMP2 in regulating platelet NOX2 activation

NOX2 has a key role for cellular production of reactive oxidant species (ROS) and although the mechanism of its activation is well known, little is known about its regulation. Metallo-proteinases (MMPs) regulate numerous protein activities both in physiological and pathological conditions but their interplay with NOX2 and ROS formation is still unclear. We performed experimental studies in human platelets and polymorphonuclear leukocytes (PMNs) to investigate the interplay of MMP2 with NOX2 activity.In collagen-stimulated platelets and in PMA-stimulated PMNs from healthy subjects, an immediate burst of ROS was detected at 10 min to then decline at 20 min. Coincidentally, sNOX2-dp, a split-off product of NOX2, increased and peaked at 10 min. ROS production was persistent whereas sNOX2dp is not released in cells treated with MMP2 inhibitor compared to other MMPs inhibitors. Western blot analysis showed the highest MMP2 expression on the cell membrane 10 min after stimulation. Moreover, the co-immunoprecipitation assay confirms the interaction between MMP2 and NOX2 that formed an active immuno-complex. Treating cells with NOX2ds-tat, an inhibitor of NADPH oxidase, significantly reduced ROS formation, sNOX2-dp, MMP2 expression and MMP2-NOX2-complex, which were all restored if cells were added with H2O2.The study provides the first evidence that MMP2 has a key role in blunting platelet NOX2 activity and eventually ROS formation.

Olive polyphenol reduces the collagen-elicited release of phosphorylated HSP27 from human platelets.

Hydroxytyrosol (HT) and oleuropein (OLE) are natural polyphenols found in extra virgin olive oil. Accumulating evidence indicates that ingestion of olive oil contributes to reduce the risk of cardiovascular diseases and stroke. It has been reported that HT and OLE inhibit human platelet aggregation. We have shown that collagen induces the phosphorylation of heat shock protein 27 (HSP27) in human platelets, resulting in the release of HSP27, an extracellular pro-inflammatory agent. In this study, we investigated the effects of HT and OLE on the collagen-stimulated human platelet activation. The PDGF-AB secretion and the soluble CD40 ligand (sCD40L) release by collagen were reduced by HT or OLE. HT and OLE significantly suppressed the phosphorylation of HSP27 and the release of phosphorylated-HSP27. These findings suggest that olive polyphenol reduces the collagen-stimulated phosphorylation of HSP27 in human platelets and the release. Our results may provide a novel anti- inflammatory effect of olive polyphenol.

Antiplatelet Activity of Acylphloroglucinol Derivatives Isolated from Dryopteris crassirhizoma.

Platelets are an important component of the initial response to vascular endothelial injury; however, platelet dysfunction induces the acute clinical symptoms of thrombotic disorders, which trigger severe cardiovascular diseases such as myocardial infarction, ischemia, and stroke. In this study, we investigated the Dryopteris crassirhizoma’s antiplatelet activity. A water extract of D. crassirhizoma (WDC) was partitioned into dichloromethane (DCM), ethyl acetate, n-butyl alcohol, and water. Among these four fractions, the DCM fraction potently inhibited the collagen-stimulated platelet aggregation in a concentration-dependent manner. From this fraction, five different acylphloroglucinol compounds and one flavonoid were isolated by activity-guided column chromatography. They were identified by comparing their mass, 1H-, and 13C-NMR spectral data with those reported in the literature. Quantifying the six compounds in WDC and its DCM fraction by high-performance liquid chromatography (HPLC) revealed that butyryl-3-methylphloroglucinol (compound 4) was the most abundant in these samples. Additionally, butyryl-3-methylphloroglucinol showed the strongest inhibitory activity in the collagen- and arachidonic acid (AA)-induced platelet aggregation, with inhibition ratios of 92.36% and 89.51% in the collagen and AA-induced platelet aggregation, respectively, without cytotoxicity. On the active concentrations, butyryl-3-methylphloroglucinol significantly suppressed the convulxin-induced platelet activation. Regarding the structure–activity relationships for the five acylphloroglucinol compounds, our results demonstrated that the functional butanonyl, methoxy, and hydroxy groups in butyryl-3-methylphloroglucinol play important roles in antiplatelet activity. The findings indicate that acylphloroglucinols, including butyryl-3-methylphloroglucinol from D. crassirhizom, possess an antiplatelet activity, supporting the use of this species for antiplatelet remedies.

Effect of alteplase on platelet function and receptor expression.

ObjectiveTo investigate the role of alteplase, a widely-used thrombolytic drug, in platelet function.MethodsHuman platelets were incubated with different concentrations of alteplase followed by analysis of platelet aggregation in response to adenosine diphosphate (ADP), collagen, ristocetin, arachidonic acid or epinephrine using light transmittance aggregometry. Platelet activation and surface levels of platelet receptors GPIbα, GPVI and αIIbβ3 were analysed using flow cytometry. The effect of alteplase on clot retraction was also examined.ResultsThis study demonstrated that alteplase significantly inhibited platelet aggregation in response to ADP, collagen and epinephrine in a dose-dependent manner, but it did not affect ristocetin- or arachidonic acid-induced platelet aggregation. Alteplase did not affect platelet activation as demonstrated by no differences in P-selectin levels and PAC-1 binding being observed in collagen-stimulated platelets after alteplase treatment compared with vehicle. There were no changes in the surface levels of the platelet receptors GPIbα, GPVI and αIIbβ3 in alteplase-treated platelets. Alteplase treatment reduced thrombin-mediated clot retraction.ConclusionsAlteplase inhibits platelet aggregation and clot retraction without affecting platelet activation and surface receptor levels.

Aquaporins in human platelets: intracellular localization and possible role in granule and lysosome secretion.

This study was undertaken to establish the presence and the role of aquaporins (AQPs) in human platelets. Immunodetection with polyclonal antibodies and fluorescent microscopy suggest the presence of AQP isoforms - 0-7 and 9-12 - localized (in resting platelets) in the plasma membrane and in the dense and alpha granules. In thrombin- or monensin-treated platelets, the granules' AQPs become visible in the whole cell body, indicating the granules' swelling. In our studies on the role of AQPs in platelet responses we used tetrachloroauric acid (HAuCl4), a classical water channel blocker. We found that 10-100 µM of Au(III) inhibited the hypotonicity-, monensin (simulating the action of Na+/H+ exchanger)-, and collagen-evoked platelet swelling and reduced tritiated water uptake by platelets treated by collagen or monensin, indicating its ability to block water channels in these cells. HAuCl4, at the concentrations reducing water influx, did not induce cell lysis, alter the plasma membrane shape or the -SH group content. The inhibitor also failed to affect Na+ and Cl--related osmotic gradient formation and protein kinase D2 phosphorylation. In platelets activated by threshold concentrations of collagen, the thrombin receptor activating peptide, ADP, calcium ionophore A23187, phorbol ester and arachidonic acid, HAuCl4 (100 µM) completely inhibited secretion of ATP from dense granules but failed to reduce platelet aggregation. In collagen-stimulated platelets, HAuCl4 (10-100 µM) reduced secretion from dense and alpha granules, as well as lysosomes, in a dose-dependent manner. We conclude that human platelets possess numerous AQPs subtypes localized in the plasma and granule membranes. AQP-mediated water fluxes may be crucial for platelet volume regulation as well as secretion from dense and alpha granules and lysosomes.

Mechanisms of attenuation of clot formation and acute thromboembolism by syringic acid in mice

Several in vitro studies have proposed syringic acid as a benzoic acid derivative found in plant foods, which can reduces collagen-stimulated platelet aggregation. Nonetheless, to date, there are no reports elucidating the role of syringic acid in platelet activation and thrombosis. Syringic acid inhibited clot formation and procoagulant protease activity and induced blood clot degradation. Syringic acid decreased the expression of density-enhanced phosphatase-1 (DEP-1)/protein tyrosine phosphatase-1B (PTP1B)/αIIbβ3, as well as the phosphorylation of kinases in collagen/epinephrine-stimulated platelets both in vitro and in vivo. Moreover, syringic acid inhibited the secretion of granule constituents, clot retraction, and FeCl3-induced vascular occlusion of the carotid artery. Syringic acid (up to 100 μM) did not show any effect on the viability of RAW 264.7 cells. Our findings demonstrate that syringic acid attenuate the development of thrombosis and thromboembolism by inhibiting fibrin clot formation, coagulant factors, and platelet stimulation through DEP-1/PTP1B/αIIbβ3/kinases.

Novel iridium (III)‑derived organometallic compound for the inhibition of human platelet activation.

Since cisplatin achieved clinical success, transition metal platinum (Pt) drugs have been effectively used for the treatment of cancer. Iridium (Ir) compounds are considered to be potential alternatives to Pt compounds, as they possess promising anticancer effects with minor side effects. Platelet activation is associated with the metastasis and progression of cancer, and also with arterial thrombosis. Therefore, it is necessary to develop novel, effective antithrombotic agents. An Ir (III)-derived complex, [Ir (Cp*) 1-(2-pyridyl)-3-(3-me-thoxyphenyl)imidazo[1,5-a]pyridine Cl]BF4 (Ir-3), was developed as a novel antiplatelet drug. Ir-3 exerted more potent inhibitory activity on platelet aggregation stimulated by collagen compared with other agonists, including thrombin. In collagen-activated platelets, Ir-3 also inhibited adenosine trisphosphate release, intracellular Ca+2 mobilization and surface P-selectin expression, as well as the phosphorylation of phospholipase Cγ2 (PLCγ2), protein kinase C (PKC), protein kinase B (Akt) and c-Jun N-terminal kinase (JNK) 1, but not p38 mitogen-activated protein kinase or extracellular signal-regulated kinases. Ir-3 did not markedly affect phorbol 12, 13-dibutyrate-stimulated platelet aggregation. Neither the adenylate cyclase inhibitor SQ22536 nor the guanylate cyclase inhibitor 1H-[1, 2, 4] oxadiazolo [4,3-a]quinoxalin-1-one significantly reversed the Ir-3-mediated inhibition of platelet aggregation. Furthermore, Ir-3 had no considerable diminishing effects on OH radical signals in collagen-stimulated platelets or Fenton reaction solution. In conclusion, Ir-3 serves a novel function in the inhibition of platelet aggregation through inhibiting the PLCγ2-PKC cascade, and the subsequent suppression of Akt and JNK1 activation. Therefore, Ir-3 may be a potential novel therapeutic agent for the treatment of thromboembolic disorders, or the interplay between platelets and tumor cells which contributes to tumor cell proliferation and progression.