Modulation of Platelet Activation and Thrombus Formation Using a Pan-PI3K Inhibitor S14161

Citation: Yi W, Li Q, Shen J, Ren L, Liu X, et al. ( Modulation of Platelet Activation and Thrombus Formation Using a Pan-PI3K Inhibitor S14161 Wenxiu Yi 0 Qiang Li 0 Jian Shen 0 Lijie Ren 0 Xiaohui Liu 0 Qi Wang 0 Sudan He 0 Qingyu Wu 0 Hu Hu 0 Xinliang Mao 0 Li Zhu 0 Zhenyu Li, University of Kentucky, United States of America 0 1 Cyrus Tang Hematology Center, Collaborative Innovation Center of Hematology, MOH Key Lab of Thrombosis and Hemostasis, Jiangsu Institute of Hematology, the First Affiliated Hospital, Soochow University , Suzhou , China , 2 Department of Pathology and Pathophysiology, Zhejiang University , Hangzhou , China The phosphatidylinositol 3-kinase (PI3K) signaling pathway is critical in modulating platelet functions. In the present study, we evaluated the effect of S14161, a recently identified pan-class I PI3K inhibitor, on platelet activation and thrombus formation. Results showed that S14161 inhibited human platelet aggregation induced by collagen, thrombin, U46619, and ADP in a dose-dependent manner. Flow cytometric studies showed that S14161 inhibited 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 S14161 decreased platelet adhesion on collagen-coated surface by about 80%. Western blot showed that S14161 inhibited phosphorylation of Akt at both Ser473 and Thr308 sites, and GSK3b at Ser9 in response to collagen, thrombin, or U46619. Comparable studies showed that S14161 has a higher potential bioavailability than LY294002, a prototypical inhibitor of pan-class I PI3K. 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 (2 mg/kg) to male C57BL/6 mice significantly extended the first occlusion time (5.0560.99 min, n = 9) compared to the vehicle controls (3.7260.95 min, n = 8) (P,0.05), but did not prolong the bleeding time (P.0.05). Taken together, our data showed that S14161 inhibits platelet activation and thrombus formation without significant bleeding tendency and toxicity, and considering its potential higher bioavailability, it may be developed as a novel therapeutic agent for the prevention of thrombotic disorders. - Funding: This work was supported in part by grants from the National Science Foundation of China (81071410, 81170132), Jiangsu Provinces Key Discipline of Medicine (XK201118), the Priority Academic Program Development of Jiangsu Higher Education Institutions of China (L.Z.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Platelets play a critical role in atherothrombosis that leads to myocardial infarction and ischemic stroke [1,2]. Once vascular injury occurs, the binding of the platelet glycoprotein (GP)Ib complex to von Willebrand factor (VWF) on the injured vessel wall initiates platelet tethering and subsequent adhesion [3]. The exposed collagen in the vascular wall and locally generated thrombin activate platelets and initiate hemostasis. The binding of collagen to GPVI on platelets results in receptor clustering and thereby stimulates phosphorylation of specific tyrosine residues within an associated trans-membrane protein, the Fc receptor cchain (FcRc-chain). This leads to the recruitment of signaling proteins such as Src kinase, the tyrosine kinase Syk, PLCc2, phosphatidylinositol 3-kinase (PI3K) and mitogen activated protein kinases (MAPKs), resulting in the inside-out activation of the integrin aIIbb3 and the release of the secondary mediators, such as ADP and thromboxane A2 (TxA2), culminating in platelet aggregation mediated by fibrinogen [4,5], or other ligands binding to aIIbb3 [6,7]. The modulation of platelet activity using specific pharmacological agents has proven to be a successful strategy for the prevention of thrombosis. The successful introduction of antiplatelet drugs, such as antagonists of ADP and aIIbb3, and inhibitors of COX-1 and phosphodiesterase, has led to considerable improvements in the management of cardiovascular diseases [8]. However, the risk of uncontrolled bleeding due to their inherent antihemostatic effects limited their clinical use [9]. Therefore, tremendous effort has been made in the past years on the identification of novel pharmacological reagents with both effective and safe antiplatelet effect. The recent search for compounds to prevent platelet activation has been focusing on the ones that modulate PI3K pathway. PI3K is a critical transmitter of intracellular signaling during platelet activation [1012], capable of triggering a wide variety of responses like phosphorylation of pleckstrin, activation of PLCc [13], Rap1b and AKT [1417], and mediating several important platelet responses like platelet shape change and stabilization of platelet aggregation [18]. Platelets contain PI3K class IA (p110a, p110b and p110d), class IB (p110c), and class II (C2a) [19]. Knock-out mouse models showed that PI3Kc acts as an important effector of P2Y12 while PI3K-IA as a key effector of collagen receptors [10,12]. PI3K activation leads to the phosphorylation of AKT or protein kinase B, which is a critical player in platelet function [20,21]. Targeting the PI3K/AKT is thus becoming an emerging strategy in the control of platelet-associated diseases. So far, more than 50 inhibitors for the PI3K/AKT/mTOR pathway are under clinical evaluation at different phases [22]. However, none of these PI3K inhibitors has been approved for modulating platelet activation and thrombus formation clinically. We recently identified S14161, or 8-ethoxy-2-(4-fluorophenyl)-3 -nitro-2H-chromene, as a novel PI3K inhibitor, that displays promising effects against multiple myeloma and leukemia cells with minimal toxicity [23]. However, whether S14161 affects platelet activation and thrombus formation is unknown. Therefore, in this study, we investigated the effect of S14161 on platelet activation in response to a variety of agonists and thrombus formation. We showed that S14161 significantly inhibited agonistinduced platelet activation and thrombus formation via inhibiting PI3K/AKT pathway. Methods and Materials Animals and human samples All animal procedures were approved by the University Committee on Animal Care of Soochow University (20140431) and each researcher followed the approved procedures when caring for or sacrificing mice and when conducting protocols involving mice. C57BL/6 mice were housed at a constant room temperature, humidity, and light cycle (12:12 hr light-dark) with free access to water and were fed chow diet. At age 68 weeks, mice were (...truncated)