Targeting The PI3K/AKT Pathway To Inhibit Platelet Activation and Thrombus Formation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3513-3513
Author(s):  
Wenxiu Yi ◽  
Wei Li ◽  
Lijie Ren ◽  
Xinliang Mao ◽  
Li Zhu
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Platelet Function ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Venous Blood ◽  
Pi3k Pathway ◽  
Dependent Manner ◽  
Clot Retraction ◽  
Occlusion Time

Abstract The phosphatidylinositol 3' –kinase (PI3K)-Akt signaling pathway has been shown to be critical in modulating platelet function and increasing number of studies have been focusing on the development of PI3K inhibitors to modulate platelet function. We recently identified a novel small molecule compound S14161, namely 8-ethoxy-2-(4-fluorophenyl)-3-nitro-2H-chromene, displaying potent antileukemia and antimyeloma activity via inhibition of the PI3K pathway (Mao et al, Blood, 2011, 117:1986). In the present study, we evaluated the effect of S14161 on platelet activation and the underlying mechanisms. Gel-filtered human platelets were isolated from venous blood of healthy adults and the effect of S14161 on platelet aggregation in response to agonists was determined. Results showed that S14161 inhibited platelet aggregation induced by collagen, convulxin, thrombin, PAR1 agonist peptide SFLLRN, and U46619 in a dose dependent manner (2.5-10μM) with the most striking inhibition for collagen by 89.8% (P<0.001, n=3) and for U46619 by 94.3% (P<0.001, n=3), respectively compared to vehicle-treated samples when 10μM S14161 was used. Flow cytometry studies showed that S14161 inhibits convulxin- or thrombin-induced P-selectin expression and fibrinogen binding of single platelet. S14161 also inhibited platelet spreading on fibrinogen and clot retraction, processes mediated by outside-in signaling. Using a microfluidic chamber we demonstrated that incubation of S14161 decreases platelet adhesion on collagen-coated surface by about 80% at various time points of blood flow in the chambers. Western blot showed that similar to LY294002, the classic PI3K inhibitor, S14161 inhibited phosphorylation of Akt Ser473 and Akt Thr308 in response to collagen, thrombin, or U46619, implying the involvement of PI3K pathway. Additionally, S14161 inhibited MAPK/ERK1/2 phosphorylation. Finally, the effects of S14161 on thrombus formation in vivo were measured using a ferric chloride-induced carotid artery injury model in mice. The intraperitoneal injection of S14161 (2mg/kg) to male C57BL6/J mice significantly extended the first occlusion time (5.05±0.99 min, N=9) compared to the vehicle controls (3.72±0.95 min, N=8) (P<0.05), but did not increase the bleeding time (P>0.05). Taken together, our data showed that S14161 inhibits platelet activation and thrombus formation, and may be developed as a novel therapeutic agent for the prevention of thrombotic disorders. (This study was supported by National Natural Science Foundation of China 81170132 to Li Zhu) Disclosures: No relevant conflicts of interest to declare.

FUNDC2 regulates platelet activation through AKT/GSK-3β/cGMP axis

2018 ◽  
Vol 115 (11) ◽  
pp. 1672-1679 ◽  
Author(s):  
Qi Ma ◽  
Weilin Zhang ◽  
Chongzhuo Zhu ◽  
Junling Liu ◽  
Quan Chen
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Platelet Rich Plasma ◽  
Thrombus Formation ◽  
Mitochondrial Protein ◽  
Dependent Manner ◽  
Clot Retraction ◽  
Akt Phosphorylation ◽  
Gsk 3Β

Abstract Aims AKT kinase is vital for regulating signal transduction in platelet aggregation. We previously found that mitochondrial protein FUNDC2 mediates phosphoinositide 3-kinase (PI3K)/phosphatidylinositol-3,4,5-trisphosphate (PIP3)-dependent AKT phosphorylation and regulates platelet apoptosis. The aim of this study was to evaluate the role of FUNDC2 in platelet activation and aggregation. Methods and results We demonstrated that FUNDC2 deficiency diminished platelet aggregation in response to a variety of agonists, including adenosine 5′-diphosphate (ADP), collagen, ristocetin/VWF, and thrombin. Consistently, in vivo assays of tail bleeding and thrombus formation showed that FUNDC2-knockout mice displayed deficiency in haemostasis and thrombosis. Mechanistically, FUNDC2 deficiency impairs the phosphorylation of AKT and downstream GSK-3β in a PI3K-dependent manner. Moreover, cGMP also plays an important role in FUNDC2/AKT-mediated platelet activation. This FUNDC2/AKT/GSK-3β/cGMP axis also regulates clot retraction of platelet-rich plasma. Conclusion FUNDC2 positively regulates platelet functions via AKT/GSK-3β/cGMP signalling pathways, which provides new insight for platelet-related diseases.

scholarly journals Effect of alteplase on platelet function and receptor expression

2019 ◽  
Vol 47 (4) ◽  
pp. 1731-1739 ◽  
Author(s):  
Jun Lu ◽  
Peng Hu ◽  
Guangyu Wei ◽  
Qi Luo ◽  
Jianlin Qiao ◽  
...  
Keyword(s):  
Arachidonic Acid ◽  
Platelet Aggregation ◽  
Platelet Activation ◽  
Platelet Function ◽  
Adenosine Diphosphate ◽  
Receptor Expression ◽  
Light Transmittance ◽  
Dependent Manner ◽  
Clot Retraction ◽  
Platelet Receptors

Objective To investigate the role of alteplase, a widely-used thrombolytic drug, in platelet function. Methods Human 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. Results This 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. Conclusions Alteplase inhibits platelet aggregation and clot retraction without affecting platelet activation and surface receptor levels.

scholarly journals Phosphatidylinositol-3,4-Bisphosphate-Akt Signaling Pathway Promotes Platelet Activation

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1131-1131
Author(s):  
Jasna Marjanovic ◽  
Brad Rumancik ◽  
Luke Weber ◽  
Felix Wangmang ◽  
Dane Fickes ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Conflicts Of Interest ◽  
Glycogen Synthase ◽  
Thrombus Formation ◽  
Type I ◽  
Dependent Manner ◽  
Wild Type ◽  
Phosphorylated Akt

Abstract Phosphatidylinositol-3,4-bisphosphate (PtdIns(3,4)P2) is a messenger that accumulates in platelets in a phosphoinositide 3-kinase and platelet aggregation-dependent manner. PtdIns(3,4)P2 is broken down by inositol polyphosphate 4-phosphatases, type I (INPP4A) and type II (INPP4B). These enzymes do not catalyze hydrolysis of phosphoinositides other than PtdIns(3,4)P2, and therefore provide unique means for studying the role of this lipid in platelet activation. We have found that the dominant isoform of 4-phosphatases expressed in platelets is INPP4A and we have generated radiation chimera mice with the deficiency in INPP4A restricted to hematopoietic cell lineage. Compared to wild type platelets, agonist-stimulated INPP4A-deficient platelets accumulated higher levels of PtdIns(3,4)P2. An increase in platelet aggregation in INPP4A-deficient platelets was observed with all tested agonists. To study platelet function in vivo, we performed carotid artery injury mouse thrombosis model experiments. Time to occlusion was dramatically reduced in mice with INPP4A deficiency. These data support the hypothesis that by regulating PtdIns(3,4)P2 levels, INPP4A downregulates platelet aggregation and thrombus formation. To investigate mechanisms mediating INPP4A-dependent signals, we compared levels of phosphorylated Akt and phosphorylated glycogen synthase kinase (GSK) in wild type and INPP4A-deficient platelets in response to agonist stimulation. An increase in phospho-Akt levels was observed in INPP4A-deficient platelets, suggesting that in addition to its well-characterized regulator, PtdIns(3,4,5)P3, PtdIns(3,4)P2 can promote Akt activation. Interestingly, this was not accompanied by a significant increase in phospho-GSK levels, suggesting a possible novel mechanism involved in platelet aggregation. Disclosures No relevant conflicts of interest to declare.

The Endocannabinoid 2-Arachidonoylglycerol Regulates Platelet Function.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3904-3904
Author(s):  
Samantha Baldassarri ◽  
Alessandra Bertoni ◽  
Paolo Lova ◽  
Stefania Reineri ◽  
Chiara Sarasso ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Platelet Function ◽  
Cannabinoid Receptors ◽  
Thromboxane A2 ◽  
Human Platelets ◽  
Dependent Manner ◽  
Cb2 Receptor ◽  
Thromboxane A2 Receptor

Abstract 2-Arachidonoylglycerol (2-AG) is a naturally occurring monoglyceride that activates cannabinoid receptors and meets several key requisites of an endogenous cannabinoid substance. It is present in the brain and hematopoietic cells, including macrophages, lymphocytes and platelets. 2-AG is released from cells in a stimulus-dependent manner and is rapidly eliminated by uptake into cells and enzymatic hydrolysis in arachidonic acid and glycerol. 2-AG might exert a very fine control on platelet function either through mechanisms intertwining with the signal transduction pathways used by platelet agonists or through mechanisms modulating specific receptors. The aim of this study was to define the role of 2-AG in human platelets and characterize the mechanisms by which it performs its action. Platelets from healthy donors were isolated from plasma by differential centrifugations and gel-filtration on Sepharose 2B. The samples were incubated with 2-AG (10–100 μM) under constant stirring in the presence or absence of various inhibitors. Platelet aggregation was measured by Born technique. We have found that stimulation of human platelets with 2-AG induced irreversible aggregation, which was significantly enhanced by co-stimulation with ADP (1–10 μM). Furthermore, 2-AG-dependent platelet aggregation was completely inhibited by ADP scavengers, aspirin, and Rho kinase inhibitor, as well as by antagonists of the 2-AG receptor (CB2), of the ADP P2Y12 receptor, and of the thromboxane A2 receptor. We further investigated the role of endocannabinoids on calcium mobilization. Intracellular [Ca2+] was measured using FURA-2-loaded platelets prewarmed at 37°C under gentle stirring in a spectrofluorimeter. 2-AG induced rapid increase of cytosolic [Ca2+] in a dose-dependent manner. This effect was partially blocked by ADP scavengers and CB2 receptor antagonists. Furthermore, 2-AG-induced [Ca2+] mobilization was totally suppressed by aspirin or the thromboxane A2 receptor antagonist. These results suggest that 2-AG is able to trigger platelet activation, and that this action is partially mediated by CB2 receptor and ADP. Furthmore, 2-AG-dependent platelet activation is totally dependent on thromboxane A2 generation.

Anthocyanins Inhibit Platelet Activation and Attenuate Thrombus Growth In Both Human and Murine Thrombosis Models

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3197-3197 ◽  
Author(s):  
Yan Yang ◽  
Zhenyin Shi ◽  
Adili Reheman ◽  
Wuxun Jin ◽  
Conglei Li ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Platelet Function ◽  
Intravital Microscopy ◽  
Platelet Adhesion ◽  
Thrombus Formation ◽  
Vessel Occlusion ◽  
Shear Rates ◽  
Perfusion Chamber ◽  
Platelet Adhesion And Aggregation

Abstract Abstract 3197 Background: Thrombosis and cardiovascular diseases (CVDs) result from blood vessel occlusion by inappropriately activated platelets. They are the leading causes of morbidity and mortality worldwide. Anthocyanins are major phytochemicals abundant in plant food and have been shown to play a protective role against CVDs. Our previous studies have demonstrated that anthocyanins are antioxidative and prevent inflammation (J Biol Chem. 2005; 280:36792-01; Arterioscler Thromb Vasc Biol. 2007; 27:519-24), which may indirectly affect platelet function. It has also been reported that anthocyanins affect platelet activities in whole blood and platelet rich plasma (PRP). However, the direct effects of anthocyanins on platelet function and thrombus formation have not been studied. Methods: Here we investigated the effects of anthocyanins on thrombosis using purified platelets as well as several thrombosis models in vitro and in vivo. Cyaniding-3-gulucoside (Cy-3-g) and delphinidin-3-glucoside (Dp-3-g), the two predominantly bioactive compounds of anthocyanin preparations, were prepared from Polyphenol AS Company in Norway. Purified gel-filtered platelets and PRP from healthy human volunteers and C57BL/6J mice were incubated at 37°C for 10 minutes with different concentrations (0.5μM, 5μM and 50μM) of Cy-3-g, Dp-3-g or PBS buffer as a control. Platelet aggregation was assessed by aggregometry using 5μM ADP, 10μg/ml collagen, or 100μM thrombin receptor activating peptide (TRAP; AYPGKF) as agonists. Platelet adhesion and aggregation were assessed in response to an immobilized collagen matrix in an ex vivo perfusion chamber at both high (1800 s-1) and low (600 s-1) shear rates. The expression of activated GPIIbIIIa was determined via PAC-1 monoclonal antibody in flow cytometry. Lastly, the effects of anthocyanins on thrombus formation in C57BL/6J mice were assessed using a FeCl3-induced intravital microscopy thrombosis model. Results: Both Cy-3-g and Dp-3-g significantly inhibited platelet aggregation induced by collagen and TRAP in gel-filtered platelets, and inhibited aggregation induced by ADP, TRAP and collagen in human and mouse PRP. These inhibitory functions were observed at Cy-3-g and Dp-3-g doses as low as 0.5μM. Cy-3-g and Dp-3-g also reduced the surface expression of activated GPIIbIIIa on resting human platelets in a dose-dependent manner. These compounds also markedly reduced platelet adhesion and aggregation in perfusion chamber assays at both low and high shear rates. Using intravital microscopy, we further demonstrated that Cy-3-g and Dp-3-g decreased platelet deposition, destabilized thrombi, and prolonged the time required for thrombus formation and vessel occlusion. Conclusions: our data clearly demonstrated for the first time that anthocyanin compounds directly inhibited platelet activation, adhesion and aggregation, as well as attenuated thrombus growth at both arterial and veinous shear stresses. These effects on platelets likely contribute to the protective effects of anthocyanins against thrombosis and CVDs. Disclosures: No relevant conflicts of interest to declare.

Platelet ERp57 Is Required for Hemostasis and Thrombosis.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2168-2168
Author(s):  
Lu Wang ◽  
Yi Wu ◽  
Junsong Zhou ◽  
Syed S. Ahmad ◽  
Bulent Mutus ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Function ◽  
Protein Disulfide Isomerase ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Platelet Glycoprotein ◽  
Platelet Surface ◽  
Disulfide Isomerase ◽  
Protein Disulfide

Abstract Abstract 2168 Several members of the protein disulfide isomerase family of enzymes are important in platelet function and in thrombosis. Platelet protein disulfide isomerase (PDI) has been shown to have an important role in platelet function but is reported to not be required for thrombus formation in vivo. A novel platelet PDI called ERp57 mediates platelet aggregation but its role in thrombus formation is unknown. To determine the specific role of platelet-derived ERp57 in hemostasis and thrombosis we generated a megakaryocyte/platelet specific knockout. Despite normal platelet counts and platelet glycoprotein expression, mice with ERp57-deficient platelets had prolonged tail-bleeding times and thrombus occlusion times, and defective activation of the αIIbβ3 integrin and platelet aggregation. The aggregation defect was corrected by addition of exogenous ERp57 implicating surface ERp57 in platelet aggregation. Platelet surface ERp57 protein and activity increased substantially with platelet activation. We conclude that platelet-derived ERp57 is required for hemostasis and thrombosis and platelet function. Disclosures: No relevant conflicts of interest to declare.

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

2021 ◽  
Vol 12 ◽  
Author(s):  
Manting Huang ◽  
Minzhen Deng ◽  
Wenqiang Nie ◽  
Dezhi Zou ◽  
Huanlin Wu ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Cyclic Nucleotide ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Antiplatelet Agent ◽  
Protective Effects ◽  
Clot Retraction ◽  
Vasp Phosphorylation ◽  
Nucleotide Signaling

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

All-Trans Retinoic Acid Impairs Platelet Function and Thrombus Formation and Inhibits Protein Kinase CßI/δ Phosphorylation

2019 ◽  
Vol 119 (10) ◽  
pp. 1655-1664 ◽  
Author(s):  
Qi Luo ◽  
Guangyu Wei ◽  
Xiamin Wang ◽  
Xiaoqi Xu ◽  
Wen Ju ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Protein Kinase ◽  
Platelet Function ◽  
Thrombus Formation ◽  
Receptor Expression ◽  
Dependent Manner ◽  
Clot Retraction ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid ◽  
Arterial Thrombus

AbstractAll-trans retinoic acid (ATRA) is widely used for induction of complete remission in patients with acute promyelocytic leukemia (APL). ATRA also regulates protein kinase C (PKC) activity. Therapeutic use of ATRA reportedly interferes with hemostatic function in APL patients, including effects on coagulation or other vascular cells, although effects of ATRA on platelets remain unclear. This study aims to investigate the effect of therapeutic-relevant doses of ATRA on platelet function. Human platelets were preincubated with ATRA (0–20 μM) for 1 hour at 37°C, followed by analysis of aggregation, granule secretion, receptor expression by flow cytometry, platelet spreading, or clot retraction. Additionally, ATRA (10 mg/kg) was injected intraperitoneally into mice and tail bleeding time and arterial thrombus formation were evaluated. ATRA inhibited platelet aggregation and adenosine triphosphate release induced by collagen (5 μg/mL) or thrombin (0.05 U/mL) in a dose-dependent manner without affecting P-selectin expression or surface levels of glycoprotein (GP) Ibα, GPVI, or αIIbβ3. ATRA-treated platelets demonstrated reduced spreading on immobilized fibrinogen or collagen and reduced thrombin-induced clot retraction together with reduced phosphorylation of Syk and PLCγ2. In addition, ATRA-treated mice displayed significantly impaired hemostasis and arterial thrombus formation in vivo. Further, in platelets stimulated with either collagen-related peptide or thrombin, ATRA selectively inhibited phosphorylation of PKCßI (Ser661) and PKCδ (Thr505), but not PKCα or PKCßII phosphorylation (Thr638/641). In conclusion, ATRA inhibits platelet function and thrombus formation, possibly involving direct or indirect inhibition of PKCßI/δ, indicating that ATRA might be beneficial for the treatment of thrombotic or cardiovascular diseases.

Effect of an anti-sulfatide single-chain antibody probe on platelet function

2008 ◽  
Vol 99 (03) ◽  
pp. 552-557 ◽  
Author(s):  
Corie Shrimpton ◽  
Koichi Honke ◽  
Rolando Rumbaut ◽  
Jose Lopez ◽  
Perumal Thiagarajan ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Function ◽  
Thrombus Formation ◽  
Lag Phase ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Platelet Surface ◽  
Occlusion Time ◽  
Single Chain Fragment Variable

SummarySulfatide (galactocylceramide-3'-sulfate), a cell surface glycosphingolipid interacts with several cell adhesion molecules including fibrinogen, von Willebrand factor (VWF), P-selectin, thrombospondin (TSP) and laminin, which are involved in haemostasis.We have used a sulfatide-specific single-chain fragment variable (scFv) antibody probe PA38 and sulfatide-deficient mice to investigate the role of membrane sulfatide in platelet function. PA38 bound to platelets and binding increased following platelet activation. Sulfatide was localized as a large cluster towards the center of the platelet surface when examined in a confocal microscope. PA38 (20 μg/ml) inhibited the adhesion of activated platelets to fibrinogen,VWF, P-selectin,TSP1 and laminin by 30%, 30%,75%,20% and 35%,respectively,compared to a control scFv (p<0.05). Furthermore, PA38 inhibited collagen, ADP, thrombin and ristocetin-induced platelet aggregation in PRP by 25%, 30%, 18% and 20%, respectively, compared to the control scFv (p<0.05). In a PFA-100 platelet function assay, PA38 prolonged the occlusion time by 25% (p<0.05).Under flow PA38 decreased the thrombus formation on collagen by 31%, (p<0.01). Sulfatidedeficient mice displayed an extended lag-phase in collagen-induced platelet aggregation compared to wild type (p<0.05), though in-vivo haemostasis did not differ significantly.Thus, this study provides new evidence for a role for membrane sulfatide in platelet function.

Inhibition of PAR4 Signaling in Ethanol-Attenuation of Platelet Function.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3892-3892
Author(s):  
Shogo Kasuda ◽  
Yoshihiko Sakurai ◽  
Midori Shima ◽  
Masahiro Takeyama ◽  
Katsuhiko Hatake ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Phospholipase A2 ◽  
Platelet Activation ◽  
Platelet Function ◽  
Phase Curve ◽  
Peak Time ◽  
Dependent Manner ◽  
Low Concentrations ◽  
High Concentrations ◽  
Induced Aggregation

Abstract Background: Moderate consumption of alcohol beverages reduces the morbidity from coronary heart disease. Previous studies describing of inhibitory activity of ethanol (EtOH) on platelet function have substantiated this observation. However, the effects of EtOH on thrombin-related platelet activation remains to be fully elucidated, though platelet activation by thrombin is essential for normal hemostasis as well as relevant to pathophysiological conditions of thrombosis. Objectives: The aim of this study is to elucidate the effect of EtOH on α-thrombin-related platelet function by measuring platelet aggregation and intracellular calcium ([Ca2+]i). Materials and Methods: A dual-wavelength spectrofluorometer was used for measurement. α-thrombin, PAR1-activating peptide (AP) (10 μM) or PAR4-AP (25 μM) was added to fura2-AM loaded washed platelet preincubated with or without EtOH (40, 80, 160 and 320 mM). Results and Interpretations: First, the effects of EtOH on 0.5 nM of thrombin-induced platelet activation was assessed. The concentration 0.5 nM used is conceived to activate platelets only via PAR-1. EtOH did not affect platelet aggregation. EtOH inhibited rise of [Ca2+]i dose-dependently. [Ca2+]i peak time at which maximal rise of [Ca2+]i delayed in a dose-dependent manner. Secondly, 10 nM of thrombin was used as an agonist. Stimulation by high concentrations of thrombin (〉 5nM) results in cleavage of both PAR1 and PAR4. The changes in [Ca2+]i showed double-phase curve composed of transient spike and prolonged peak in the absence of EtOH. Although EtOH inhibited neither platelet aggregation nor the first phase of [Ca2+]i increasing, it reduced the second prolonged elevation of [Ca2+]i dose-dependently. To elucidate the inhibiting mechanism of EtOH more precisely, the effects of EtOH on PAR1-AP-induced platelet function were examined. Rise of [Ca2+]i gave a spike form and was almost unchanged even in the presence of high concentrations of EtOH, whereas platelet aggregation was reduced and dissociated in the presence of EtOH. Lastly, the effects of EtOH on PAR4-AP-induced platelet function was examined. Aggregation of PRP was quenched by high concentrations of EtOH but dissociation was not observed contrary to that observed in PAR1-AP-induced aggregation. Further, EtOH inhibited [Ca2+]i rise and delayed [Ca2+]i peak time dose-dependently. Our results provided a possible mechanism by which EtOH inhibits platelet activation. Reduction of the prolonged elevation of [Ca2+]i by high concentrations of thrombin suggested that EtOH inhibits PAR4 signaling not PAR1 since the second prolonged phase of [Ca2+]i is mediated by PAR4. Inhibition of PAR4-induced aggregation and [Ca2+]i elevation by EtOH supported the findings and EtOH might reduce Ca2+ influx through inhibition of PAR4. Furethermore, the difference between the platelet activation mechanisms of low concentrations of thrombin and PAR1-AP was suggested. PAR1-AP can aggregate platelets at least but might fail to activate phospholipase A2 required for sustaining stable aggregation since EtOH abolishes phospholipase A2 and thereby reduces thromboxane A2 generation. On the other, thrombin at low concentrations might have another pathway for activating platelet differently than PAR1-AP. Further characterization of the mechanisms involved in inhibition of platelet activation by EtOH may help develop new strategies to control thrombin-mediated platelet activation.

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