In platelets stimulated by thrombin to secrete and aggregate, cofilin is rapidly dephosphorylated leading to its activation. Cofilin by severing existing actin filaments and stimulating F-actin polymerization on newly created barbed ends dynamizes the actin cytoskeleton. We previously found that cofilin dephosphorylation is Ca 2؉ -dependent and occurs upstream of degranulation in stimulated platelets. We report now in thrombinstimulated platelets that Rac1 and class II PAKs (PAK4/5/6) were rapidly (within 5 seconds) activated, whereas PAK1/2 (class I PAKs) phosphorylation was slower. The Rac1-specific inhibitor NSC23766 blocked phosphorylation of class II PAKs, but not PAK1/2. Moreover, inhibition of the Ca 2؉ /calmodulindependent phosphatase calcineurin inhibited Rac1 activation and class II PAKs phosphorylation. Prevention of Rac1 activation by calcineurin inhibition or NSC23766 also blocked cofilin dephosphorylation and platelet granule secretion indicating that a calcineurin/Rac1/ class II PAKs pathway regulates cofilin dephosphorylation leading to secretion.We further found that PI3-kinases were activated downstream of Rac1, but were not involved in regulating cofilin dephosphorylation and secretion in thrombinstimulated platelets. Our study unravels a Ca 2؉ -dependent pathway of secretion in stimulated platelets as a signaling pathway linking Rac1 activation to actin dynamics: calcineurin3Rac13class II PAKs3cofilin activation. We further demonstrate that this pathway is separate and independent of the protein kinase C (PKC) pathway mediating secretion. (Blood. 2009;114:415-424)

BackgroundPlatelet activation requires rapid remodeling of the actin cytoskeleton which is regulated by small GTP-binding proteins. By using the Rac1-specific inhibitor NSC23766, we have recently found that Rac1 is a central component of a signaling pathway that regulates dephosphorylation and activation of the actin-dynamising protein cofilin, dense and α-granule secretion, and subsequent aggregation of thrombin-stimulated washed platelets.ObjectivesTo study whether NSC23766 inhibits stimulus-induced platelet secretion and aggregation in blood.MethodsHuman platelet aggregation and ATP-secretion were measured in hirudin-anticoagulated blood and platelet-rich plasma (PRP) by using multiple electrode aggregometry and the Lumi-aggregometer. Platelet P-selectin expression was quantified by flow cytometry.ResultsNSC23766 (300 μM) inhibited TRAP-, collagen-, atherosclerotic plaque-, and ADP-induced platelet aggregation in blood by 95.1%, 93.4%, 92.6%, and 70%, respectively. The IC50 values for inhibition of TRAP-, collagen-, and atherosclerotic plaque-, were 50 ± 18 μM, 64 ± 35 μM, and 50 ± 30 μM NSC23766 (mean ± SD, n = 3-7), respectively. In blood containing RGDS to block integrin αIIbβ3-mediated platelet aggregation, NSC23766 (300 μM) completely inhibited P-selectin expression and reduced ATP-secretion after TRAP and collagen stimulation by 73% and 85%, respectively. In ADP-stimulated PRP, NSC23766 almost completely inhibited P-selectin expression, in contrast to aspirin, which was ineffective. Moreover, NSC23766 (300 μM) decreased plaque-stimulated platelet adhesion/aggregate formation under arterial flow conditions (1500s-1) by 72%.ConclusionsRac1-mediated signaling plays a central role in secretion-dependent platelet aggregation in blood stimulated by a wide array of platelet agonists including atherosclerotic plaque. By specifically inhibiting platelet secretion, the pharmacological targeting of Rac1 could be an interesting approach in the development of future antiplatelet drugs.

Atherosclerosis has an important inflammatory component. Macrophages accumulating in atherosclerotic arteries produce prostaglandin E(2) (PGE(2)), a main inflammatory mediator. Platelets express inhibitory receptors (EP(2), EP(4)) and a stimulatory receptor (EP(3)) for this prostanoid. Recently, it has been reported in ApoE(-/-) mice that PGE(2) accumulating in inflammatory atherosclerotic lesions might contribute to atherothrombosis after plaque rupture by activating platelet EP(3), and EP(3) blockade has been proposed to be a promising new approach in anti-thrombotic therapy. The aim of our investigation was to study the role of PGE(2) in human atherosclerotic plaques on human platelet function and thrombus formation. Plaque PGE(2) might either activate or inhibit platelets depending on stimulation of either EP(3) or EP(4), respectively. We found that the two EP(3)-antagonists AE5-599 (300 nM) and AE3-240 (300 nM) specifically and completely inhibited the synergistic effect of the EP(3)-agonist sulprostone on U46619-induced platelet aggregation in blood. However, these two EP(3)-antagonists neither inhibited atherosclerotic plaque-induced platelet aggregation, GPIIb/IIIa exposure, dense and alpha granule secretion in blood nor reduced plaque-induced platelet thrombus formation under arterial flow. The EP(4)-antagonist AE3-208 (1-3 μM) potentiated in combination with PGE(2) (1 μM) ADP-induced aggregation, demonstrating that PGE(2) enhances platelet aggregation when the inhibitory EP(4)-receptor is inactivated. However, plaque-induced platelet aggregation was not augmented after platelet pre-treatment with AE3-208, indicating that plaque PGE(2) does not stimulate the EP(4)-receptor. We found that PGE(2) was present in plaques only at very low levels (15 pg PGE(2)/mg plaque). We conclude that PGE(2) in human atherosclerotic lesions does not modulate (i.e. stimulate or inhibit) atherothrombosis in blood after plaque rupture.

To cite this article: Bampalis VG, Dwivedi S, Shai E, Brandl R, Varon D, Siess W. Effect of 5-HT 2A receptor antagonists on human platelet activation in blood exposed to physiologic stimuli and atherosclerotic plaque. J Thromb Haemost 2011; 9: 2112-5.New antiplatelet therapies are needed, because the two types of clinically used platelet inhibitors, i.e. the cyclo-oxygenase-1 inhibitor aspirin and P2Y 12 receptor antagonists (clopidogrel and prasugrel), even in combination, are not optimal in preventing major atherothrombotic events, and are associated with increased bleeding [1,2]. Concerning this challenge, a recent study, published in this journal, has reported encouraging results showing that 5-HT 2A receptor inhibition by a new antagonist (APD791) improves coronary patency in the in vivo canine model [3].5-HT 2A receptor antagonists have been long known to inhibit 5-hydroxytryptamine (5-HT)-dependent platelet aggregation [4][5][6][7][8]; however, they have not been successfully introduced as antiplatelet agents in the clinic. 5-HT stored in platelet-dense granules and released from them upon platelet stimulation might serve as a feedback mediator by binding to 5-HT 2A receptors on the platelet surface and support platelet aggregation [9]. Indeed, 5-HT added exogenously to plateletrich plasma (PRP) or blood synergizes with several platelet stimuli in inducing platelet aggregation [5,6,8,10]. A short survey of the literature shows that 5-HT 2A receptor antagonists have unequivocally been shown to inhibit 5-HT-potentiated platelet aggregation of PRP in humans and many other species in vitro and ex vivo [3,6,8,11,12] and 5-HT-stimulated thrombus formation in mice in vivo [13]. There are also animal studies reporting inhibition of aggregation induced by physiologic platelet stimuli (collagen, thrombin, ADP, and epinephrine) by 5-HT 2A receptor antagonists in vitro and ex vivo, and in thrombosis models in vivo, particularly in the Folts model of coronary occlusion in the dog [3,11,12,[14][15][16][17]. However, the results in humans are conflicting [5,[18][19][20].In light of the conflicting results reported in the literature and the recent study in dogs, we decided to perform, for the first time, a comprehensive study of the effects of 5-HT 2A antagonists on platelets in human blood in vitro under static and arterial flow conditions, after stimulation with physiologic agonists and a pathophysiologically relevant platelet agonist, i.e. human atherosclerotic plaque homogenate.We studied three different 5-HT 2A receptor antagonists: ketanserin [21], R-96544 [12], and sarpogrelate [22]. Sarpogrelate is a drug introduced clinically as a therapeutic agent for the treatment of peripheral artery occlusive disease [23]. It has been shown recently that sarpogrelate treatment inhibits 5-HTpotentiated platelet aggregation in patients with ischemic stroke [24].Hirudin-anticoagulated blood from healthy volunteers was used in all experiments. Multiple electrode aggregometry was used to measure platelet aggregation in blood (Dyn...

In Ultra Deep Sub Micron technology nodes, particularly 45nm and below, multiple power supplies are needed to achieve optimum performance. In such SoC's, level shifters play an important role in translating the signals from one voltage level to another. The conventional level shifters suffer from the contention between the pull up and pull down transistors which leads to the increase in delay and the power consumption, so the existing techniques are unable to address the requirement of wide range of voltage translation at lower core voltages below 1V. In this paper a new voltage up level shifter has been proposed and designed for up shifting the signal levels for a wide range of voltage levels. This circuit consists of level shifter block and reference voltage generation circuit, which generates a constant voltage depending up on the ratio between pre charge rate and discharge rate to avoid the contention between pull up and pull down transistors of level shifter block. It results in faster transition and less power compared to earlier level shifters. The proposed circuit is designed in 45nm CMOS technology using PO-WERSPICE, consumes only 89.4nw power and gives an average delay of 70ps at Vin =0.45V, VDDH=1.05V while occupying an area of 1.67um2.