Collagen-stimulated Platelet Aggregation Research Articles

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.

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.  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.  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.

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.

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.

Oleocanthal-rich extra virgin olive oil demonstrates acute anti-platelet effects in healthy men in a randomized trial

The phenolic profiles of extra virgin olive oils (EVOOs) may influence their cardiovascular benefits. In a randomized crossover of acute EVOO intake on platelet function, participants (n=9) consumed 40mL of EVOO weekly. EVOOs were matched for total phenolic content and were either tyrosol-poor with 1:2 oleacein/oleocanthal (D2i0.5), or 2:1 oleacein/oleocanthal (D2i2), or predominantly tyrosol (D2i0). Ibuprofen provided a platelet inhibition control. Blood was collected pre- and 2h post-EVOO intake. D2i0.5 and D2i2 reduced 1µg/mL collagen-stimulated maximum platelet aggregation (Pmax), with effects best correlated to oleocanthal intake (R=0.56, P=0.002). Total phenolic intake was independently correlated to eicosanoid production inhibition, suggesting that cyclooxygenase blockade was not responsible for the Pmax inhibition. Five participants exhibited >25% ΔPmax declines with D2i0.5 and D2i2 intake and plasma metabolomic profiles discriminated subjects by oil responsivity. Platelet responses to acute EVOO intake are associated with oil phenolic composition and may be influenced by diet.

Sphingosine 1-phosphate (S1P) suppresses the collagen-induced activation of human platelets via S1P4 receptor

Sphingosine 1-phosphate (S1P) is as an extracellular factor that acts as a potent lipid mediator by binding to specific receptors, S1P receptors (S1PRs). However, the precise role of S1P in human platelets that express S1PRs has not yet been fully clarified. We previously reported that heat shock protein 27 (HSP27) is released from human platelets accompanied by its phosphorylation stimulated by collagen. In the present study, we investigated the effect of S1P on the collagen-induced platelet activation. S1P pretreatment markedly attenuated the collagen-induced aggregation. Co-stimulation with S1P and collagen suppressed collagen-induced platelet activation, but the effect was weaker than that of S1P-pretreatment. The collagen-stimulated secretion of platelet-derived growth factor (PDGF)-AB and the soluble CD40 ligand (sCD40L) release were significantly reduced by S1P. In addition, S1P suppressed the collagen-induced release of HSP27 as well as the phosphorylation of HSP27. S1P significantly suppressed the collagen-induced phosphorylation of p38 mitogen-activated protein kinase. S1P increased the levels of GTP-bound Gαi and GTP-bound Gα13 coupled to S1PPR1 and/or S1PR4. CYM50260, a selective S1PR4 agonist, but not SEW2871, a selective S1PR1 agonist, suppressed the collagen-stimulated platelet aggregation, PDGF-AB secretion and sCD40L release. In addition, CYM50260 reduced the release of phosphorylated-HSP27 by collagen as well as the phosphorylation of HSP27. The selective S1PR4 antagonist CYM50358, which failed to affect collagen-induced HSP27 phosphorylation, reversed the S1P-induced attenuation of HSP27 phosphorylation by collagen. These results strongly suggest that S1P inhibits the collagen-induced human platelet activation through S1PR4 but not S1PR1.

Honokiol as a specific collagen receptor glycoprotein VI antagonist on human platelets: Functional ex vivo and in vivo studies

Honokiol, derived from Magnolia officinalis, has various pharmacological properties. Platelet activation plays a critical role in cardiovascular diseases. Honokiol has been reported to inhibit collagen-stimulated rabbit platelet aggregation. However, detailed further studies on the characteristics and functional activity of honokiol in platelet activation are relatively lacking. In the present study, honokiol specifically inhibited platelet aggregation and Ca+2 ion mobilization stimulated with collagen or convulxin, an agonist of glycoprotein (GP) VI, but not with aggretin, an agonist of integrin α2β1. Honokiol also attenuated the phosphorylation of Lyn, PLCγ2, PKC, MAPKs, and Akt after convulxin stimulation. Honokiol have no cytotoxicity in zebrafish embryos. Honokiol diminished the binding of anti-GP VI (FITC-JAQ1) mAb to human platelets, and it also reduced the coimmunoprecipitation of GP VI-bound Lyn after convulxin stimulation. The surface plasmon resonance results revealed that honokiol binds directly to GP VI, with a KD of 289 μM. Platelet function analysis revealed that honokiol substantially prolonged the closure time in human whole blood and increased the occlusion time of thrombotic platelet plug formation in mice. In conclusion, honokiol acts as a potent antagonist of collagen GP VI in human platelets, and it has therapeutic potential in the prevention of the pathological thrombosis.

27- Hydroxycholesterol reverses estradiol induced inhibition of platelet aggregation in postmenopausal women

The decline of estrogen levels increases cardiovascular risk in women. Platelets express estrogen receptors and 17β-estradiol- (E2) can produce a protective effect on thrombus formation. The hydroxylation of cholesterol generates several sterols and 27-hydroxycholesterol (27HC) predominates in circulation. To evaluate the effect of 27HC as an endogenous antagonist of the anti-aggregating properties of E2 in platelets of postmenopausal women. Platelet function of postmenopausal women was evaluated ex-vivo. Platelets pre-incubated with 27HC in the presence or absence of E2, were stimulated with collagen. Aggregation was evaluated using turbidimetry using a Chrono-log aggregometer. Collagen-stimulated platelet aggregation was significantly inhibited by E2. The inhibitory effect of E2 on collagen-stimulated platelet aggregation was significantly reversed in the presence of 27HC. The suppressive effect of E2 on platelet aggregation is inhibited by 27HC, which could contribute to increase cardiovascular risk in postmenopausal women.

Dose Responses of Ibuprofen In Vitro on Platelet Aggregation and Coagulation in Human and Pig Blood Samples

Ibuprofen is commonly used by warfighters in the deployed environment. This study investigated its dose effects on in vitro coagulation in human and pig blood. Blood samples were collected from 6 normal volunteers and 6 healthy pigs and processed to make platelet-adjusted samples (100 × 10(3)/μL, common transfusion trigger in trauma). Ibuprofen was added to the samples at concentrations of 0 μg/mL (control), the concentration from the highest recommended oral dose (163 μg/mL, 1×), and 2×, 4×, 8×, 10×, 12×, 16×, and 20×. Platelet aggregation by Chrono-Log aggregometer and coagulation by rotational thrombelastogram (Rotem) were assessed at 15 minutes after the addition of ibuprofen. A robust inhibition of ibuprofen on arachidonic acid-induced platelet aggregation was observed at all doses tested in human or pig blood. Collagen-stimulated platelet aggregation was inhibited starting at 1× in human blood and 4× in pig blood. Rotem measurements were similarly compromised in pig and human blood starting at 16×, except clot formation time was prolonged at 1× in human blood (all p < 0.05). Ibuprofen inhibited platelet aggregation at recommended doses, and compromised coagulation at higher doses. Human blood was more sensitive to ibuprofen inhibition. Further effort is needed to investigate ibuprofen dose responses on coagulation in vivo.

Effect of Ibuprofen dose on platelet aggregation and coagulation in blood samples from pigs.

Ibuprofen is commonly used by Soldiers in the deployed environment. This study investigated its dose-effects on in vitro coagulation. Blood samples were collected from 4 normal healthy pigs and were processed to make platelet-adjusted (100×10(3)/μL) blood samples. Ibuprofen was added to the samples at doses of 0 μg/mL (control), recommended oral dose (163 μg/mL, 1×), 2×, 4×, 8×, 10×, 12×, 16×, and 20×. Arachidonic acid or collagen-stimulated platelet aggregation was assessed at 15 minutes after the addition of ibuprofen. Coagulation was assessed with measurements of prothrombin time (PT) and activated partial thromboplastin time (aPTT), and thrombelastography by Rotem. A robust inhibition of ibuprofen on arachidonic acid-induced platelet aggregation was observed at all doses tested. Collagen-stimulated platelet aggregation was inhibited to 71%±5% and 10%±5% of the control values at ibuprofen doses of 4× and 20×, respectively (both p<0.05). No changes were observed in PT at any dose, but aPTT was prolonged at dose of 16× and 20×. Rotem measurements of coagulation time, clot formation time, maximum clot firmness, and A10 were compromised at dose 16× and 20× (all p<0.05). Ibuprofen inhibited platelet aggregation at recommended doses, but did not compromise aPTT or coagulation profile until at 16 times the recommended doses and higher. Further effort is needed to clarify whether there are different dose-responses between human and pig blood samples in trauma situations.

Onion (Allium cepa L.) peel extract has anti-platelet effects in rat platelets.

The effects of onion peel extract (OPE) in collagen (5 μg/mL)-stimulated washed rat platelet aggregation were investigated. OPE inhibited platelet aggregation via inhibition of aggregation-inducing molecules, intracellular Ca2+ and thromboxane A2 (TXA2) by blocking cyclooxygenase-1 (COX-1) and TXA2 synthase (TXAS) activities in a dose-dependent manner. In addition, OPE elevated the formation of cyclic adenosine monophosphate (cAMP), aggregation-inhibiting molecule, but not cyclic guanosine monophosphate (cGMP). High performance liquid chromatography (HPLC) analysis of OPE revealed that OPE contains quercetin, one of the major flavonoids, which has anti-platelet effect. In conclusion, we suggest that OPE is an effective inhibitor of collagen-stimulated platelet aggregation in vitro. Therefore, it can be a promising and safe strategy for anti-cardiovascular diseases.

Effect of Cordycepin-Enriched WIB801C from Cordyceps militaris Suppressing Fibrinogen Binding to Glycoprotein IIb/IIIa.

In this study, we investigated the effects of cordycepin-enriched (CE)-WIB801C, a n-butanol extract of Cordyceps militaris-hypha on collagen-stimulated platelet aggregation. CE-WIB801C dose dependently inhibited collagen-induced platelet aggregation, and had a synergistic effect together with cordycepin (W-cordycepin) from CE-WIB801C on the inhibition of collagen-induced platelet aggregation. CE-WIB801C and cordycepin stimulated the phosphorylation of VASP (Ser157) and the dephosphorylation of PI3K and Akt, and inhibited the binding of fibrinogen to glycoprotein IIb/IIIa (αIIb/β3) and the release of ATP and serotonin in collagen-induced platelet aggregation. A-kinase inhibitor Rp-8-Br-cAMPS reduced CE-WIB801C-, and cordycepin-increased VASP (Ser157) phosphorylation, and increased CE-WIB801C-, and cordycepin-inhibited the fibrinogen binding to αIIb/β3. Therefore, we demonstrate that CE-WIB801C-, and cordycepin-inhibited fibrinogen binding to αIIb/β3 are due to stimulation of cAMP-dependent phosphorylation of VASP (Ser157), and inhibition of PI3K/Akt phosphorylation. These results strongly indicate that CE-WIB801C and cordycepin may have preventive or therapeutic potential for platelet aggregation-mediated diseases, such as thrombosis, myocardial infarction, atherosclerosis, and ischemic cerebrovascular disease.

Acetaminophen and meloxicam inhibit platelet aggregation and coagulation in blood samples from humans.

Acetaminophen (Ace) and meloxicam (Mel) are the two types of analgesic and antipyretic medications. This study investigated the dose responses of acetaminophen and meloxicam on platelet aggregation and coagulation function in human blood samples. Blood samples were collected from six healthy humans and processed to make platelet-adjusted (100 × 10 cells/μl) blood samples. Acetaminophen (Tylenol, Q-PAP, 100 mg/ml) was added at the doses of 0 μg/ml (control), 214 μg/ml (the standard dose, 1 ×), 4 ×, 8 ×, 10 ×, 12 ×, 16 ×, and 20 ×. Similarly, meloxicam (Metacam, 5 mg/ml) was added at doses of 0 μg/ml (control), 2.85 μg/ml (the standard dose, 1 ×), 4 ×, 8 ×, 10 ×, 12 ×, 16 ×, and 20 ×. Fifteen minutes after the addition of acetaminophen and/or meloxicam, platelet aggregation was stimulated with collagen (2 μg/ml) or arachidonic acid (0.5 mmol/l) and assessed using a Chrono-Log 700 aggregometer. Coagulation function was assessed by prothrombin time (PT), activated partial thromboplastin time (aPTT), and using Rotem thrombelastogram. A robust inhibition by acetaminophen and/or meloxicam was observed in arachidonic acid-stimulated platelet aggregation starting at 1 × dose. Collagen-stimulated platelet aggregation was inhibited by ACE starting at 1 × (78 ± 10% of control), and by meloxicam starting at 4 × (72 ± 5% of control, both P < 0.05). The inhibitions by acetaminophen and meloxicam combined were similar to those by acetaminophen or meloxicam. aPTT was prolonged by meloxicam starting at 4 ×. No changes were observed in PT or any of Rotem measurements by acetaminophen and/or meloxicam. Acetaminophen and meloxicam compromised platelet aggregation and aPTT. Further effort is warranted to characterize the effects of acetaminophen and meloxicam on bleeding in vivo.

THE RELATIONSHIP BETWEEN THE METABOLITES OF THE DIETARY FLAVONOID QUERCETIN AND HAEMOSTASIS, THROMBOSIS AND PLATELET FUNCTION

Flavonoids are plant secondary metabolites with beneficial effects on cardiovascular health. One notable flavonoid due to its anti-platelet properties is quercetin (3,3′,4′,5,7-pentahydroxyflavone). Upon consumption, quercetin is metabolized in the small intestine and liver, yielding methylated, sulphated and glucuronidated metabolites; their anti-platelet effects (function and signalling) and relevance at concentrations achieved in-vivo is not understood; a long term goal of my PhD is to investigate and model these effects. Methods Platelet aggregation was performed by light-transmission aggregometry. Fibrinogen-binding and P-Selectin exposure were measured by flow cytometry. Results Consistent with previous studies, quercetin inhibited collagen-stimulated (5 µg/ml) platelet aggregation in a dose-dependent manner; significant inhibition was observed as low as 3 µM (lower than previously reported). Above 6 µM, aggregation was inhibited >90%. In-vivo, numerous agonists are present at lower concentrations than typically used in in-vitro assays. Agonist concentrations that individually are subthreshold for aggregation, when added together, may synergise and stimulate substantial functional responses. The ability and potency of quercetin to inhibit fibrinogen-binding to platelets stimulated by this synergistic response between thrombin and CRP (Collagen-Related-Peptide) was therefore explored. Quercetin inhibited significantly fibrinogen-binding at concentrations as low as 500 nM. This was associated with substantial inhibition of alpha-granule secretion. Fibrinogen-binding and P-Selectin exposure stimulated by 0.1 µg/ml and 1 µg/ml CR P alone was also significantly inhibited by quercetin, at concentrations as low as 500 nM. Conclusions Quercetin inhibits platelet function at concentrations lower than previously recognised and within potential physiological concentrations. Development of this work will allow elucidation and modelling of the effects of quercetin9s main metabolites.

Sex differences in platelet reactivity and cardiovascular and psychological response to mental stress in patients with stable ischemic heart disease: insights from the REMIT study.

Although emotional stress is associated with ischemic heart disease (IHD) and related clinical events, sex-specific differences in the psychobiological response to mental stress have not been clearly identified. We aimed to study the differential psychological and cardiovascular responses to mental stress between male and female patients with stable IHD. Patients with stable IHD enrolled in the REMIT (Responses of Mental Stress-Induced Myocardial Ischemia to Escitalopram) study underwent psychometric assessments, transthoracic echocardiography, and platelet aggregation studies at baseline and after 3 mental stress tasks. Mental stress-induced myocardial ischemia (MSIMI) was defined as the development or worsening of regional wall motion abnormality, reduction of left ventricular ejection fraction (LVEF) ≥8% by transthoracic echocardiography, and/or ischemic ST-segment change on electrocardiogram during 1 or more of the 3 mental stress tasks. In the 310 participants with known IHD (18% women, 82% men), most baseline characteristics were similar between women and men (including heart rate, blood pressure, and LVEF), although women were more likely to be nonwhite, living alone (p < 0.001), and unmarried (p < 0.001); they also had higher baseline depression and anxiety (p < 0.05). At rest, women had heightened platelet aggregation responses to serotonin (p = 0.007) and epinephrine (p = 0.004) compared with men. Following mental stress, women had more MSIMI (57% vs. 41%; p < 0.04), expressed more negative (p = 0.02) and less positive emotion (p < 0.001), and demonstrated higher collagen-stimulated platelet aggregation responses (p = 0.04) than men. Men were more likely than women to show changes in traditional physiological measures, such as blood pressure (p < 0.05) and double product. In this exploratory analysis, we identified clear, measurable, and differential responses to mental stress in women and men. Further studies should test the association of sex differences in cardiovascular and platelet reactivity in response to mental stress and long-term outcomes. (Responses of Myocardial Ischemia to Escitalopram Treatment [REMIT]; NCT00574847).

Compartmentalisation of cAMP-dependent signalling in blood platelets: The role of lipid rafts and actin polymerisation

Prostacyclin (PGI2) inhibits blood platelets through the activation of membrane adenylyl cyclases (ACs) and cyclic adenosine 3',5'-monophosphate (cAMP)-mediated signalling. However, the molecular mechanism controlling cAMP signalling in blood platelet remains unclear, and in particular how individual isoforms of AC and protein kinase A (PKA) are coordinated to target distinct substrates in order to modulate platelet activation. In this study, we demonstrate that lipid rafts and the actin cytoskeleton may play a key role in regulating platelet responses to cAMP downstream of PGI2. Disruption of lipid rafts with methyl-beta-cyclodextrin (MβCD) increased platelet sensitivity to PGI2 and forskolin, a direct AC cyclase activator, resulting in greater inhibition of collagen-stimulated platelet aggregation. In contrast, platelet inhibition by the direct activator of PKA, 8-CPT-6-Phe-cAMP was unaffected by MβCD treatment. Consistent with the functional data, lipid raft disruption increased PGI2-stimulated cAMP formation and proximal PKA-mediated signalling events. Platelet inhibition, cAMP formation and phosphorylation of PKA substrates in response to PGI2 were also increased in the presence of cytochalasin D, indicating a role for actin cytoskeleton in signalling in response to PGI2. A potential role for lipid rafts in cAMP signalling is strengthened by our finding that a pool of ACV/VI and PKA was partitioned into lipid rafts. Our data demonstrate partial compartmentalisation of cAMP signalling machinery in platelets, where lipid rafts and the actin cytoskeleton regulate the inhibitory effects induced by PGI2. The increased platelet sensitivity to cAMP-elevating agents signalling upon raft and cytoskeleton disruption suggests that these compartments act to restrain basal cAMP signalling.