Abstract
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 GSK3β 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 (5.05±0.99 min, n = 9) compared to the vehicle controls (3.72±0.95 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.
Highlights
Platelets play a critical role in atherothrombosis that leads to myocardial infarction and ischemic stroke [1,2]
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]
In this study we showed that S14161 inhibits platelet aggregation in the presence of a variety of platelet agonists including collagen, thrombin, ADP and U46619
Summary
Platelets play a critical role in atherothrombosis that leads to myocardial infarction and ischemic stroke [1,2]. 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 aIIbb 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]. Tremendous effort has been made in the past years on the identification of novel pharmacological reagents with both effective and safe antiplatelet effect
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