The Endocannabinoid 2-Arachidonoylglycerol Regulates Platelet Function.

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.

Download Full-text

Role of phosphoinositide 3-kinase β in platelet aggregation and thromboxane A2 generation mediated by Gi signalling pathways

Biochemical Journal ◽

10.1042/bj20100166 ◽

2010 ◽

Vol 429 (2) ◽

pp. 369-377 ◽

Cited By ~ 64

Author(s):

Analia Garcia ◽

Soochong Kim ◽

Kamala Bhavaraju ◽

Simone M. Schoenwaelder ◽

Satya P. Kunapuli

Keyword(s):

Platelet Aggregation ◽

Critical Role ◽

Thromboxane A2 ◽

Inhibitory Effect ◽

Human Platelets ◽

P2y12 Receptor ◽

Functional Responses ◽

Induce Platelet Aggregation ◽

Erk Phosphorylation

PI3Ks (phosphoinositide 3-kinases) play a critical role in platelet functional responses. PI3Ks are activated upon P2Y12 receptor stimulation and generate pro-aggregatory signals. P2Y12 receptor has been shown to play a key role in the platelet aggregation and thromboxane A2 generation caused by co-stimulation with Gq or Gz, or super-stimulation of Gi pathways. In the present study, we evaluated the role of specific PI3K isoforms α, β, γ and δ in platelet aggregation, thromboxane A2 generation and ERK (extracellular-signal-regulated kinase) activation. Our results show that loss of the PI3K signal impaired the ability of ADP to induce platelet aggregation, ERK phosphorylation and thromboxane A2 generation. We also show that Gq plus Gi- or Gi plus Gz-mediated platelet aggregation, ERK phosphorylation and thromboxane A2 generation in human platelets was inhibited by TGX-221, a PI3Kβ-selective inhibitor, but not by PIK75 (a PI3Kα inhibitor), AS252424 (a PI3Kγ inhibitor) or IC87114 (a PI3Kδ inhibitor). TGX-221 also showed a similar inhibitory effect on the Gi plus Gz-mediated platelet responses in platelets from P2Y1−/− mice. Finally, 2MeSADP (2-methyl-thio-ADP)-induced Akt phosphorylation was significantly inhibited in the presence of TGX-221, suggesting a critical role for PI3Kβ in Gi-mediated signalling. Taken together, our results demonstrate that PI3Kβ plays an important role in ADP-induced platelet aggregation. Moreover, PI3Kβ mediates ADP-induced thromboxane A2 generation by regulating ERK phosphorylation.

Download Full-text

Effect of alteplase on platelet function and receptor expression

Journal of International Medical Research ◽

10.1177/0300060519829991 ◽

2019 ◽

Vol 47 (4) ◽

pp. 1731-1739 ◽

Cited By ~ 1

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.

Download Full-text

ADRENALINE (ADR) INTERACTIONS WITH THROMBIN AND COLLAGEN -EFFECTS ON PLATELET ARACHIDONATE RELEASE AND 5HT SECRETION AND ROLE OF ENDOGENOUSLY FORMED THROMBOXANE A2 (TxA2)

10.1055/s-0038-1643384 ◽

1987 ◽

Author(s):

Y Patel ◽

S Krishnamurthi ◽

V V Kakkar

Keyword(s):

Arachidonic Acid ◽

Platelet Aggregation ◽

Adenylate Cyclase ◽

Synergistic Effects ◽

Thromboxane A2 ◽

Human Platelets ◽

Presence And Absence ◽

Pre Treatment ◽

Arachidonate Release

We have examined the effect of combinations of ADR + thrombin (T) and ADR + collagen (C) on platelet arachidonate release and 5HT secretion, and assessed the role of endogenously formed TxA2 on these responses using indomethacin (I). Washed, human platelets prelabelled with [3H]-arachidonic acid (AA) or [14C]-5HT were used, ADR was added 10 sec before T or C and the reaction was terminated 3 min later. In the range 1-100μM, ADR induced no detectable aggregation or 5HT secretion but potentiated platelet aggregation when added with sub-threshold concentrations of T or C, which on their own induced no aggregation. At 2-4 fold higher concentrations of T and C (threshold for 5HT secretion), 5HT secretion and AA/TXB2 release were also potentiated by ADR (1-10μM) by 30-50%. Pre-treatment of platelets with I (10μM) abolished threshold T and C-induced 5HT secretion, as well as its potentiation by ADR. However, approximately 2-fold and 5-fold higher concentrations of T and C respectively were able to induce 'I-insensitive'secretion, which was further potentiated by ADR. In I-treated platelets, C-induced AA release and its potentiation by ADR were also abolished suggesting a role for endogenously formed TxA2 This was confirmed by addition of the TxA2 mimetic, U46619 (0.3μM), which potentiated C-induced AA release in the presence and absence of ADR, even though it induced no AA release on its own or, in combination with ADR alone in the absence of collagen. The latter suggests agonist specificity regarding the ability of TxA2 to synergistically stimulate AA release. Finally, unstirred platelets in PRP pre-incubated with ADR (10μM) for 120 min lost their responsiveness to ADR, when eventually stirred; however, these 'ADR-desensitised' platelets when washed and resuspended, were able to demonstrate synergistic effects on secretion when stimulated with ADR+T or ADR+C. This is analogous to the previously demonstrated ability of ADR to inhibit adenylate cyclase even in 'ADR-desensitised' platelets and re-inforces the separation regarding the mechanisms underlying the various effects of ADR on platelets.

Download Full-text

Anandamide inhibits transport-related oxygen consumption in the loop of Henle by activating CB1 receptors

AJP Renal Physiology ◽

10.1152/ajprenal.00239.2012 ◽

2013 ◽

Vol 304 (4) ◽

pp. F376-F381 ◽

Cited By ~ 12

Author(s):

Guillermo B. Silva ◽

Douglas K. Atchison ◽

Luis I. Juncos ◽

Néstor H. García

Keyword(s):

Oxygen Consumption ◽

Receptor Antagonist ◽

Cannabinoid Receptors ◽

Cb1 Receptors ◽

Dependent Manner ◽

Cb2 Receptor ◽

Na Transport ◽

Concentration Dependent Manner ◽

Selective Agonist

The energy required for active Na chloride reabsorption in the thick ascending limb (TAL) depends on oxygen consumption and oxidative phosphorylation (OXP). In other cells, Na transport is inhibited by the endogenous cannabinoid anandamide through the activation of the cannabinoid receptors (CB) type 1 and 2. However, it is unclear whether anandamide alters TAL transport and the mechanisms that could be involved. We hypothesized that anandamide inhibits TAL transport via activation of CB1 receptors and NO. For this, we measured oxygen consumption (QO2) in TAL suspensions to monitor the anandamide effects on transport and OXP. Anandamide reduced QO2 in a concentration-dependent manner. During Na-K-2Cl cotransport and Na/H exchange inhibition, anandamide did not inhibit TAL QO2. To test the role of the cannabinoid receptors, we used specific agonists and antagonists of CB1 and CB2 receptors. The CB1-selective agonist WIN55212–2 reduced QO2 in a concentration-dependent manner. Also, the CB1 receptor antagonist rimonabant blocked the effect of anandamide on QO2. In contrast, the CB2-selective agonist JHW-133 had no effect on QO2, while the CB2 receptor antagonist AM-630 failed to block the anandamide effects on QO2. To confirm these results, we measured CB1 and CB2 receptor expression and only CB1 expression was detected. Because CB1 receptors are strong nitric oxide synthase (NOS) stimulators and NO inhibits transport in TALs, we evaluated the role of NO. Anandamide stimulated NO production and the NOS inhibitor NG-nitro-l-arginine methyl ester blocked the anandamide effects on QO2. We conclude that anandamide inhibits TAL Na transport-related QO2 via activation of CB1 receptor and NOS.

Download Full-text

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

Blood ◽

10.1182/blood.v104.11.3892.3892 ◽

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.

Download Full-text

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

Blood ◽

10.1182/blood.v122.21.3513.3513 ◽

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.

Download Full-text

Intracellular Ca2+ rise in human platelets induced by polymorphonuclear-leucocyte-derived cathepsin G

Biochemical Journal ◽

10.1042/bj2880741 ◽

1992 ◽

Vol 288 (3) ◽

pp. 741-745 ◽

Cited By ~ 18

Author(s):

M Molino ◽

M Di Lallo ◽

G de Gaetano ◽

C Cerletti

Keyword(s):

Plasma Membrane ◽

Platelet Aggregation ◽

Platelet Activation ◽

Human Platelets ◽

Polymorphonuclear Leucocyte ◽

Cation Channel ◽

Cathepsin G ◽

Dependent Manner ◽

Bivalent Cation ◽

Platelet Suspension

Cathepsin G, a serine protease released by polymorphonuclear-leucocyte azurophilic granules upon stimulation, activates human platelets, inducing an increase in intra-platelet Ca2+ concentration ([Ca2+]i) in a concentration-dependent manner (50-200 nM). The [Ca2+]i rises elicited by low (50-80 nM) cathepsin G concentrations in fura-2-loaded platelets showed a biphasic mode, with a first small peak followed by a greater and more prolonged Ca2+ transient. Higher (100-200 nM) cathepsin G concentrations induced a monophasic increase in intracellular Ca2+. Acetylsalicylic acid, nordihydroguaiaretic acid and ketanserin did not affect platelet activation by cathepsin G, whereas the ADP-scavenger system phosphocreatine/creatine kinase significantly decreased Ca2+ mobilization, platelet aggregation and 5-hydroxytryptamine secretion by cathepsin G. Preventing cathepsin G-induced platelet aggregation with the synthetic peptide RGDSP (Arg-Gly-Asp-Ser-Pro) did not significantly affect cathepsin G-induced Ca2+ transients. Ni2+ (4 mM), a bivalent-cation-channel inhibitor, decreased the cathepsin G-induced fluorescence rise by more than 90%. This effect was reversed by either decreasing Ni2+ or increasing cathepsin G concentration. Preventing Ca2+ influx across the plasma membrane with 4 mM-EGTA totally abolished Ca2+ transients. However, EGTA also strongly decreased catalytic activity of cathepsin G, which is essential for platelet activation. Evidence of a rapid and sustained bivalent-cation channel opening in the platelet membrane was obtained by adding Mn2+ to the platelet suspension 30 s or 3 min after cathepsin G. No accumulation of InsP3 could be detected when platelets were stimulated with cathepsin G. All these data indicate that cathepsin G induces a [Ca2+]i increase mainly through an influx across the plasma membrane. This massive Ca2+ entry is probably due to opening of receptor-operated channels and is amplified by endogenous ADP release.

Download Full-text

Src family kinase–mediated and Erk-mediated thromboxane A2 generation are essential for VWF/GPIb-induced fibrinogen receptor activation in human platelets

Blood ◽

10.1182/blood-2005-05-1933 ◽

2005 ◽

Vol 106 (10) ◽

pp. 3410-3414 ◽

Cited By ~ 80

Author(s):

Analia Garcia ◽

Todd M. Quinton ◽

Robert T. Dorsam ◽

Satya P. Kunapuli

Keyword(s):

Platelet Aggregation ◽

Phospholipase C ◽

Platelet Activation ◽

Kinase Inhibitor ◽

Thromboxane A2 ◽

Receptor Activation ◽

Src Family Kinases ◽

Mitogen Activated Protein Kinase ◽

Dependent Manner ◽

Src Family Kinase

AbstractThe binding of von Willebrand factor (VWF) to the platelet membrane glycoprotein Ib-IX (GPIb-IX) results in platelet activation. In this study, we sought to clarify previous conflicting reports and to elucidate the mechanism of activation and the precise role of extracellular signal-regulated kinase (Erk) in VWF-induced platelet activation. Erk2 is activated in platelets on stimulation with VWF/ristocetin in a time-dependent manner. VWF-induced Erk2 phosphorylation and thromboxane A2 (TXA2) release were completely blocked by PP2, an Src family kinase inhibitor, suggesting that Erk is downstream of Src family kinases. U73122, a phospholipase C inhibitor, also abolished TXA2 generation and Erk phosphorylation. Although VWF fostered the agglutination of platelets regardless of any additional treatment, the inhibition of mitogen-activated protein kinase kinase (MEK) with U0126 abolished VWF-induced platelet aggregation and thromboxane production in non–aspirin-treated washed platelets. However, in platelets treated with aspirin, VWF failed to cause any aggregation. Thus, we conclude that VWF stimulation of platelets results in phospholipase A2 activation through Erk stimulation and that Src family kinases and phospholipase C play essential roles in this event. We further conclude that VWF-induced platelet aggregation does not directly depend on Erk activation but has an absolute requirement for Src/Erk-mediated TXA2 generation.

Download Full-text

The Gas6 Receptor Axl Enhances Platelet Activation Responses through Stimulation of PI3kinase- and PLC-Dependent Signaling Pathways.

Blood ◽

10.1182/blood.v104.11.630.630 ◽

2004 ◽

Vol 104 (11) ◽

pp. 630-630

Author(s):

Weston R. Gould ◽

Sangita Baxi ◽

Lisa A. Perrin ◽

Robert J. Leadley

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Intracellular Signaling ◽

Human Platelets ◽

Dense Granule ◽

Calcium Mobilization ◽

Dependent Manner ◽

Induced Aggregation ◽

Granule Release ◽

Stimulation Of

Abstract At the site of vascular injury, platelet activation is paramount in supporting formation of a platelet plug and generating a functional surface for the protein elements of coagulation. Recently, we demonstrated that the receptors for the α-granule constituent Gas6, support and enhance platelet aggregation and dense-granule release. The current study examined additional affects of Gas6 signaling in human platelets and sought to decipher intracellular signaling mechanisms initiated by stimulation of Axl, a Gas6 platelet receptor. Flow cytometry analyses indicated that all three Gas6 receptors, Axl, Sky, and Mer were present on the platelet surface. Blockade of Gas6, Sky, or Mer by specific antibodies not only inhibited TRAP- and ADP-induced platelet aggregation and dense granule release, but also prevented thrombin mediated clot retraction by as much as 55%. Furthermore, intracellular calcium mobilization in response to TRAP activation was greater than 80% inhibited in the presence of each of these blocking antibodies. A highly specific antibody directed toward Axl (< 2% cross reactivity with Sky and Mer) activated Axl leading to an enhancement of TRAP and ADP induced aggregation and degranulation. Stimulation of human platelets by this Axl agonist led to a modest, but sustained increase in calcium mobilization suggesting that Axl signaling incorporated activation of PLC. The increase in calcium mobilization was sensitive to wortmannin, demonstrating that PLC activation occurred concurrent with or downstream of PI3K. Indeed, additional experiments to ascertain the intracellular mediators of Axl activity identified a two-fold increase in specific phosphorylation of Akt downstream of PI3K as well as a similar increase in phosphorylation of PLCγ. TRAP stimulation of human platelets also increased the phosphorylation levels of Akt and PLCγ in a Gas6 dependent manner as a Gas6 blocking antibody reduced the levels of Akt and PLCγ phosphorylation by 50%. Overall, these studies suggest that Gas6 enhancement of human platelet activation occurs through the low-level stimulation of the intracellular signaling molecules Akt and PLCγ, serving at the juncture of several mediators of platelet activation. These events also increase levels of cytoplasmic calcium, further supporting an enhancement of activation observed in response to low levels of known platelet agonists. Thus, platelet Gas6 functions to support platelet activation at the very early stages of the hemostatic response to injury.

Download Full-text

In vitro Effects of Aprosulate sodium, a Novel Anticoagulant, On Platelet Activation: Possible Mechanism for Antiplatelet Action

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650661 ◽

1996 ◽

Vol 76 (05) ◽

pp. 786-790 ◽

Cited By ~ 1

Author(s):

Atsuhiro Sugidachi ◽

Norbert Breiter ◽

Taketoshi Ogawa ◽

Fumitoshi Asai ◽

Hiroyuki Koike

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Anticoagulant Activity ◽

Acid Amide ◽

Human Platelets ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Free System ◽

Induced Aggregation

SummaryAprosulate sodium, a bis-lactobionic acid amide derivative, is a novel synthetic polyanion with potent anticoagulant activities. In the present study, the effects of aprosulate on platelet aggregation were investigated in a plasma-free system. Aprosulate inhibited thrombin (0.03-0.3 U/ml)-induced aggregation in rat washed platelets in a concentration-dependent manner, with an IC50 value of 0.38 Μg/ml. In contrast, aprosulate, at up to 10 Μg/ml, did not affect collagen (1 Μg/ml) - or ADP (3 ΜM)-induced aggregation. In fura 2-loaded platelets, aprosulate (1-10 Μg/ml) inhibited intracellular Ca2+ mobilization induced by thrombin, but not that by ADP. Protamine, a highly basic protein, abolished aprosulate-mediated inhibition of thrombin-induced platelet aggregation, suggesting that the observed inhibition is primarily due to the negative charge contained on the aprosulate molecule. In human platelets, aprosulate inhibited thrombin-induced aggregation, but failed to inhibit platelet aggregation induced by SFLLRN, a synthetic tethered ligand of a thrombin receptor. Antiplatelet profiles of aprosulate were largely similar to those of heparin, although heparin inhibited both thrombin- and collagen-induced aggregation. These in vitro studies indicate that aprosulate is capable of inhibiting thrombin-induced platelet activation and that this effect is independent of its anticoagulant activity. These results suggest that the polyanionic feature of aprosulate plays an essential role in promoting its antiplatelet activities, and that a plausible mechanism to explain the observed inhibition conferred by this agent, would be one which involves blocking the platelet-thrombin interaction.

Download Full-text