The Critical Roles of SIN1 in Platelet Activation and Myocardial Infarction

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3715-3715
Author(s):  
Yanyan Xu ◽  
Xue Chen ◽  
Junling Liu
Keyword(s):  
Myocardial Infarction ◽  
Platelet Activation ◽  
Complex Analysis ◽  
Conflicts Of Interest ◽  
Heart Function ◽  
Platelet Rich Plasma ◽  
Clot Retraction ◽  
Interacting Protein ◽  
St Segment Elevation ◽  
Mtorc2 Complex

Abstract Mammalian stress-activated protein kinase interacting protein 1 (SIN1) is an essential subunit of the mTORC2 complex, which regulates Akt activation by phosphorylation of Akt at Ser473 residue. Despite the function of Akt in platelet activation and thrombosis was well studied, the role of SIN1 in platelet activation and thrombosis remains unknown. In this study, we observed that megakaryocyte/platelet specific SIN1 deficiency caused 30% reduction of platelet counts in peripheral blood probably by blockage of megakaryocyte differentiation and enhancing platelet apoptosis, suggesting that SIN1 had an important role in thrombopoiesis. More importantly, SIN1 deficiency caused a defect in platelet aggregation in response to low level of thrombin, U46619, ADP and collagen. SIN1 deficiency also exhibited diminished ability of platelet to spread on immobilized fibrinogen and the decreased rate of clot retraction in platelet-rich plasma containing SIN1 deficient platelets. mTORC2 complex analysis revealed that the expression levels of Rictor, another mTORC2 component, were significantly diminished in SIN1 deficient platelet. And SIN1 deficiency attenuated agonist-induced phosphorylation of Akt at Ser473, Thr308 and Thr450, and Gsk3β at Ser9 in platelet. SIN1 could be phosphorylated at Thr86, which correlated with the phosphorylation of Akt at Ser473 in activated platelets. Further study demonstrated that the phosphorylation levels of SIN1 at Thr86 and Akt at Ser473 and Thr450, but not at Thr308 were enhanced in the platelets collected from ST-segment elevation myocardial infarction (STEMI) patients, indicating that SIN1 activation correlated with myocardial infarction process. A mouse model of chronic myocardial infarction (MI) was performed and the results demonstrated that platelet-specific SIN1 deficient mice had less platelet activation, reduced MI size, and improved post-MI heart function. In conclusion, SIN1 plays critical roles in platelet activation, MI and post-MI heart failure, therefore serves as a target for therapeutic intervention in the thrombosis and myocardial infarction. Disclosures No relevant conflicts of interest to declare.

Circulating Human Platelets Express LRP-1 and uPAR mRNAs and Synthesize the Proteins: The Complex LRP-1, uPAR, PAI-1 and uPA Play a Role in Modulating Fibrinolysis in Platelet-Rich Thrombi

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1116-1116
Author(s):  
Olga Panes ◽  
Valeria Matus ◽  
César González ◽  
Claudia G Sáez ◽  
Jaime Pereira ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Conflicts Of Interest ◽  
Platelet Rich Plasma ◽  
Human Platelets ◽  
Platelet Membrane ◽  
Fibrinolytic System ◽  
Clot Lysis ◽  
Clot Retraction ◽  
Tetrameric Complex ◽  
Pai 1

Abstract Abstract 1116 Platelets are intrinsic components of hemostatic and pathological clots, and are essential for clot retraction. However, their role and sequential involvement in clot stabilization and lysis are still poorly understood. Human platelets contain several components of the fibrinolytic system, including functional PAI-1, TAFI, uPA and α 2-antiplasmin. Moreover, platelets possess a rich transcriptome and synthesize several proteins, among them, PAI-1. Using a global, modified clot lysis time assay in platelet-rich plasma (CLT-PRP; Panes et al., Platelets 2012) we found that the CLT-PRP was significantly longer than that of CLT in platelet-free plasma (PFP), reflecting a down-regulation of the fibrinolytic process. However, the prolonged CLT in subjects receiving tranexamic acid was normalized earlier in PRP than in PPP, denoting some pro-fibrinolytic activity in clots formed in a platelet milieu. Aim: to study the presence, origin, association and functional role of components of the fibrinolytic system in human platelets. Also, we aim to getting insight into the dynamic balance and modulation of the fibrinolytic process by the interplay of pro- and anti-fibrinolytic platelet factors. Methods and Results: in washed, leukocyte-free human platelets we detected expression of LRP-1, uPAR, PAI-1 mRNAs, and synthesis of these proteins (metabolic radiolabeling). Neither uPA mRNA nor synthesis of uPA was evidenced. All of these proteins, including uPA were detected in membrane or cytosol fractions by western blotting (WB). LRP-1 and uPAR were present in the outer leaflet of platelet membranes, with increased uPAR labeling after platelet activation (confocal microscopy-immunofluorescence). Non-stimulated whole platelets exhibit a low basal uPA activity (specific chromogenic substrate) selectively inhibited by amiloride. uPA activity falls slightly immediately after VWF-Ristocetin (VWF-R) and TRAP stimulation, but recovers to basal levels after 15min. Biotinylated washed platelets were immunoprecipitated (IP) with α -uPAR MoAb at different times before and after activation with either TRAP or VWF-Ristocetin. Co-precipitations with LRP-1, PAI-1 and uPA were detected in WB only after platelet activation with TRAP for 5 min, denoting the formation of a tetrameric complex, likely involved in endocytosis and receptor recycling. Interestingly, 5min after TRAP stimulation, uPA was sharply reduced, disappearing at 15 min, either in membrane or cytosol fractions, suggesting degradation of the protein. Similar pattern of co-precipitations were observed when IP was done with α -LRP-1 MoAb. Co-precipitations were more prominent in purified platelet membrane than in cytosolic fractions. Conclusions: human platelets express LRP-1, uPAR and PAI-1 mRNAs, and synthesize these proteins. uPA activity is present in whole, purified, washed platelets, and the protein is likely bound to the external platelet membrane. Co-precipitation of all these fibrinolytic components presumably denotes the formation of a tetrameric complex with endocytic and recycling capacities, as demonstrated in other cell lineages. Sequential IP′s after platelet activation disclose the disappearance of uPA, but not of PAI-1, from the complex, probably explained by a degradation process. Taken together, these results suggest that platelets play a predominantly antifibrinolytic role during early stages of formation of platelet-rich clots. 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.

Abstract 39: Platelet Reactivity to Prostaglandin E2 is a Novel Modifiable Risk Factor for St-Elevation Myocardial Infarction

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Eitan A Friedman ◽  
Elias V Haddad ◽  
Valentinas Joksas ◽  
Shi Huang ◽  
Meng Xu ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Cardiovascular Risk ◽  
Platelet Activation ◽  
Platelet Reactivity ◽  
Platelet Rich Plasma ◽  
Coronary Intervention ◽  
Prostaglandin E ◽  
Platelet Response ◽  
Increased Risk ◽  
St Elevation

Background: Patients with lower thresholds for platelet activation are at increased risk for primary and recurrent myocardial infarction (MI) and overall cardiovascular (CV) mortality. We have demonstrated that there are two phenotypes of platelet response to Prostaglandin E 2 (PGE 2 ), such that it increases threshold for aggregation in 45% of individuals (inhibitory) and lowers threshold for aggregation in 55% (potentiating). As PGE 2 is present in atherosclerotic plaques, and its receptors are present on platelets, biologic variability in PGE 2 responses may have clinical implications. We hypothesized that patients with higher thresholds for platelet activation would have a lower risk of thrombotic CV events, specifically ST-Elevation MI (STEMI). Methods: 85 patients undergoing percutaneous coronary intervention for stable or unstable coronary disease were phenotyped for PGE 2 response. Platelet rich plasma was treated with various concentrations of U46,619 (thromboxane agonist) with or without PGE 2 100 nM, and phenotype determined by light aggregometry. Analysis of the maximum PGE 2 effect (maximum aggregation with PGE 2 minus maximum aggregation without it) was performed using linear and non-linear statistical methods. Results: Traditional cardiovascular risk factors were similar between groups. A higher percentage of patients with the potentiating phenotype had a history of STEMI than those with the inhibitory phenotype. Logistic regression using restricted cubic spline showed that the predicted probabilities of STEMI increased from 0.04 (at the strongest inhibitory phenotype) to 0.43 (at the median phenotype). The OR of phenotype at the median relative to that at the 10th quantile was 7.4 (95 % CI=1.6, 34.8). Conclusions: PGE 2 inhibitory phenotype confers a decreased lifetime risk of STEMI in individuals at high risk for CV events. We have previously shown that an EP3 receptor antagonist converts the potentiating to the inhibitory phenotype. Thus, the PGE2 phenotype may be a novel marker of cardiovascular risk that may also identify patients who would benefit from an EP3 antagonist.

Fucosyltransferase VI Induces Platelet Activation: A Novel Property of a Plasma Glycosyltransferase.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4016-4016
Author(s):  
José-Tomás Navarro ◽  
Shwan Tawfiq ◽  
Roland Wohlgemuth ◽  
Karin M. Hoffmeister ◽  
Robert Sackstein
Keyword(s):  
Flow Cytometry ◽  
Platelet Aggregation ◽  
Platelet Activation ◽  
Light Transmission ◽  
Conflicts Of Interest ◽  
Platelet Rich Plasma ◽  
Surface Protein ◽  
Surface Flow ◽  
Platelet Surface ◽  
Biochemical Studies

Abstract Abstract 4016 Poster Board III-952 A number of glycosyltransferases are present in human plasma with the α(1→3) fucosyltransferase, Fucosyltransferase VI (FTVI), having the highest plasma concentration. Notably, elevated plasma levels of FTVI are associated with a variety of cancers and correlate with tumor load/progression. The well-known association of neoplasia with thromboembolic complications prompted us to examine whether FTVI has direct effect(s) on platelet function. We obtained human platelets from blood of healthy donors and separated from platelet-rich plasma by differential centrifugation. Freshly isolated platelets (x108/ml) were stirred and exposed at 37°C to varying concentrations (20, 40, 60 and 80 mU/mL) of glycosyltransferases FTVI, β-1-4-galactosyltransferase-I (βGalT-I), or α,2-3-N-sialyltransferase (α2,3-N-ST), or to 1 U/mL thrombin. Platelet aggregation and activation was assessed by aggregometry (light transmission) or by flow cytometry of FSC/SSC characteristics and of surface expression of P-Selectin, respectively. FT-VI reproducibly induced platelet aggregation and activation, whereas other glycosyltransferases (β4GalT-I and α2,3-N-ST) had no effect on platelets. FTVI activation of platelets was concentration-dependent, and the aggregation curve for FTVI was one wave, similar to that for thrombin. FTVI-induced platelet activation was independent of catalytic conversion of surface glycans, but was inhibited by FTVI denaturation, indicating that FTVI-induced platelet activation is a lectin-mediated process. To determine the membrane target(s) mediating FTVI-induced platelet activation, biochemical studies were performed after catalytic exofucosylation of the platelet surface. Flow cytometry after platelet exofucosylation showed formation of the carbohydrate structure sLex, detected by the mAb Heca452, but no formation of Lex (CD15). Western blot showed that enforced fucosylation induced sLex on a single platelet surface protein, and further biochemical studies revealed that this protein is GPIbα. These findings unveil a previously unrecognized property of FTVI as an activator of platelets, mediated via a specific lectin/carbohydrate interaction on GP1ba, and offer novel perspectives on the pathobiology of tumor-associated thrombogenesis. Disclosures: No relevant conflicts of interest to declare.

Suppression of Murine Platelet Activation by the β Isoform of the Catalytic Subunit of Protein Phosphatase 2B

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 190-190
Author(s):  
Francisca C. Gushiken ◽  
Nawaf Alrehani ◽  
Subhashree Pradhan ◽  
Lavanya Kailasam ◽  
Rolando Rumbaut ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Protein Phosphatase ◽  
Conflicts Of Interest ◽  
Fibrin Clot ◽  
Catalytic Subunit ◽  
Low Doses ◽  
Clot Retraction ◽  
Protein Phosphatase 2B ◽  
Platelet Functions

Abstract Abstract 190 Signal transduction mediated by the kinases and phosphatases are critical for platelet activation at the site of vascular injury. Compared to the kinases, a role for phosphatases in platelet activation is less understood. Our previous studies have focused on the roles of serine/threonine protein phosphatase 1 (PP1) and 2A (PP2A) in regulating integrin αIIbβ3functions. However, platelets also express protein phosphatase 2B (PP2B) and its role in platelet function is unexplored. PP2B-Aα and PP2B-Aβ constitute two ubiquitous isoforms of the PP2B catalytic subunit. Due to the general concerns regarding the specificity of the PP2B inhibitors, we have utilized mice deficient in the β isoform of the catalytic subunit of PP2B (PP2B-Aβ) to explore the role of PP2B in platelet functions. Mice lacking PP2B-Aα are short lived and are not considered in this study. Loss of PP2B-Aβ did not cause any compensatory increase in the PP2B-Aα levels in platelets. Compared to the wild type (WT) platelets, PP2B-Aβ−/− platelets displayed increased aggregation in response to low doses of protease-activated receptor 4-activating peptide (PAR4-AP), ADP, collagen and collagen related peptide (CRP). Enhanced α granule secretion in response to the low doses of PAR4-AP and CRP was noticed in PP2B-Aβ−/− platelets, relative to the WT platelets. Functions regulated by the outside-in αIIbβ3 integrin signaling like adhesion to immobilized fibrinogen and fibrin clot retraction were enhanced in the PP2B-Aβ−/− platelets. These studies indicate that PP2B-Aβ negatively regulate platelet functions in vitro. Consistent with these observations, PP2B-Aβ−/− mice exhibited a shorter tail bleeding time compared to the WT mice. In a FeCl3 induced endothelial denudation injury model, PP2B-Aβ−/− mice showed decreased time to occlusion in the carotid artery, and reduced number of emboli compared to the WT mice. These studies indicate that PP2B-Aβ suppress multiple murine platelet functions that contribute to an occlusive thrombi. Unlike a positive thrombus promoting role for the PP1cγ that was noticed in our previous study, PP2B-Aβ suppressed murine platelet activation, suggesting that different subtypes of Ser/Thr phosphatases have distinct roles in murine platelet activation. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Receptor-interacting protein kinase 3 promotes platelet activation and thrombosis

2017 ◽  
Vol 114 (11) ◽  
pp. 2964-2969 ◽  
Author(s):  
Yiwen Zhang ◽  
Jian Zhang ◽  
Rong Yan ◽  
Jingluan Tian ◽  
Yang Zhang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Protein Kinase ◽  
Platelet Activation ◽  
Thrombus Formation ◽  
Critical Role ◽  
In Vivo Model ◽  
Clot Retraction ◽  
Interacting Protein ◽  
Bone Marrow Derived Cells

Previous studies have shown that receptor-interacting protein kinase 3 (RIP3) is involved in many important biological processes, including necroptosis, apoptosis, and inflammation. Here we show that RIP3 plays a critical role in regulating platelet functions and in vivo thrombosis and hemostasis. Tail bleeding times were significantly longer in RIP3-knockout (RIP3−/−) mice compared with their wild-type (WT) littermates. In an in vivo model of arteriole thrombosis, mice lacking RIP3 exhibited prolonged occlusion times. WT mice repopulated with RIP3−/− bone marrow-derived cells had longer occlusion times than RIP3−/− mice repopulated with WT bone marrow-derived cells, suggesting a role for RIP3-deficient platelets in arterial thrombosis. Consistent with these findings, we observed that RIP3 was expressed in both human and mice platelets. Deletion of RIP3 in mouse platelets caused a marked defect in aggregation and attenuated dense granule secretion in response to low doses of thrombin or a thromboxane A2 analog, U46619. Phosphorylation of Akt induced by U46619 or thrombin was diminished in RIP3−/− platelets. Moreover, RIP3 interacted with Gα13. Platelet spreading on fibrinogen and clot retraction were impaired in the absence of RIP3. RIP3 inhibitor dose-dependently inhibited platelet aggregation in vitro and prevented arterial thrombus formation in vivo. These data demonstrate a role for RIP3 in promoting in vivo thrombosis and hemostasis by amplifying platelet activation. RIP3 may represent a novel promising therapeutic target for thrombotic diseases.

scholarly journals Prothrombotic markers and early spontaneous recanalization in ST-segment elevation myocardial infarction

2007 ◽  
Vol 98 (08) ◽  
pp. 420-426 ◽  
Author(s):  
Emilie Lanoy ◽  
Didier Tcheche ◽  
Laurent Feldman ◽  
Annie Bezeaud ◽  
Eduardo Anglès-Cano ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Plasminogen Activator ◽  
Platelet Activation ◽  
Plasminogen Activator Inhibitor ◽  
Platelet Glycoprotein ◽  
Activation Markers ◽  
St Segment Elevation ◽  
St Segment ◽  
Circulating Microparticles ◽  
The Difference

SummaryWe tested the hypothesis that selected prothrombotic biomarkers might be associated with early spontaneous coronary recanalization in patients with ST-segment elevation acute myocardial infarction (STEMI). We prospectively enrolled 123 patients with STEMI including 53 patients with spontaneous coronary recanalization (cases) and 70 patients with persistent occlusion (controls) at the time of emergent coronary angiography and before angioplasty. All had received aspirin and heparin. Blood samples were collected immediately before angioplasty to measure soluble P-selectin, circulating microparticles originating from platelets (PMPs), granulocytes (GMPs), endothelial cells (EMPs); tissue factor-associated MP (TF-MP); soluble platelet glycoprotein V (sGPV) and prothrombin F1+2; tissue plasminogen activator (tPA), plasminogen activator inhibitor (PAI-1) and plasmin-antiplasmin (PAP). A sub-group of 70 patients (35 cases, 35 controls) was available for flow cytometry analysis of platelet P-selectin and activated GPIIb-IIIa. Baseline clinical characteristics did not differ between groups except for more frequent hypertension and dyslipidemia in controls. Platelet activation markers and PMP did not differ between the two groups. Controls had higher numbers of EMPs and GMPs compared to cases, but the difference was no longer significant when corrected for risk factors. Controls differed from cases by higher plasma levels of sGPV [64 (47–84) ng/ml vs. 53 (44–63) ng/ml] and PAP [114(65–225) ng/ml vs. 88 (51–147) ng/ml].The difference persisted after adjustment for risks factors (p=0.031 and 0.037, respectively). Persistent occlusion of the infarct related artery is associated with some markers related to higher thrombin (sGPV) and plasmin (PAP) production but is not associated with markers of platelet activation.

LPS Mediates the Activation of Platelet by Toll-Like Receptor 4.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3001-3001
Author(s):  
Liping Ma ◽  
Jing Wei ◽  
Jian-Xing Chang ◽  
Cheng Zhang ◽  
Zhi-Xin Pei ◽  
...  
Keyword(s):  
Western Blot ◽  
Platelet Activation ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Conflicts Of Interest ◽  
Human Platelet ◽  
Platelet Rich Plasma ◽  
Toll Like Receptor ◽  
Toll Like Receptor 4 ◽  
Platelet Suspension

Abstract Abstract 3001 Poster Board II-978 Lipopolysaccsharide (LPS) is a principal outer membrane component of gram-negative bacteria. It initiates an inflammatory response to infection by activating Toll-like receptor-4 (TLR4) on various tissues or cells in host. Platelet contributes to the inflammation process through respond to invading pathogens, membrane adhesion molecule (CD62P, P-selectin) and CD40L on platelet are the indexes to determine platelet activation. The present experiment was designed to investigate the expression of Toll-like receptor 4 (TLR4) on platelet and to determine whether platelet TLR4 involves in platelet activation induced by Lipopolysaccsharide (LPS). Human platelet-rich plasma (PRP) and platelet suspension obtained from 15 healthy people were pretreated with a concentration of 0.2μg/ml of LPS in the presence or absence of thrombin (1 U/ml) for 1 hour. The expressions of TLR4,CD62P and CD40L on platelets were detected through flow cytometry, and platelet TLR4 was further determined by performing western blot analysis. The results show that the percentage of TLR4-positive platelet induced by thrombin was increased by 32.34% compared with the resting platelets (25.44%, P<0.01). TLR4 expression on platelets treated with LPS was remarkedly elevated in the presence or absent of thrombin. However, the expression level was much higher in presence of both than thrombin alone( 39.16%,P<0.01). Moreover, the similar results were found in Western blot analysis. Synchronously, the expressions of CD62P and CD40L on resting platelets were 6.39% and 2.45%, they were also markedly increased when treated with thrombin(42.68% and 14.80%) and LPS respectively, and the increase of CD62P was more significant when stimulated with both of LPS and thrombin(63.03%). Although anti-TLR4 antibody inhibited significantly the increases of TLR4, CD62P and CD40L on platelets induced by LPS, it didn't effect their increases induced by thrombin. The experiment results support the evidence that functional TLR4 can be expressed on human platelet. It may involve in platelet activation as an important mediator of LPS- induced CD62P and CD40L expressions on platelets. Disclosures: No relevant conflicts of interest to declare.

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.

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