Upon blood vessel injury, platelets are exposed to adhesive proteins in the vascular wall and soluble agonists, which initiate platelet activation, leading to formation of hemostatic thrombi. Pathological activation of platelets can induce occlusive thrombosis, resulting in ischemic events such as heart attack and stroke, which are leading causes of death globally. Platelet activation requires intracellular signal transduction initiated by platelet receptors for adhesion proteins and soluble agonists. Whereas many platelet activation signaling pathways have been established for many years, significant recent progress reveals much more complex and sophisticated signaling and amplification networks. With the discovery of new receptor signaling pathways and regulatory networks, some of the long-standing concepts of platelet signaling have been challenged. This review provides an overview of the new developments and concepts in platelet activation signaling.

Objective Reactive oxygen species (ROS) are known to regulate platelet activation; however, the mechanisms of ROS production during platelet activation remain unclear. Platelets express different isoforms of nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidases (NOXs). Here we investigated the role of NOX1 and NOX2 in ROS generation and platelet activation using NOX1 and NOX2 knockout mice. Approach and Results NOX1−/Y platelets showed selective defects in G protein-coupled receptor (GPCR)-mediated platelet activation induced by thrombin and thromboxane A2 analog U46619, but were not affected in platelet activation induced by collagen-related peptide (CRP), a glycoprotein VI (GPVI) agonist. In contrast, NOX2−/− platelets showed potent inhibition of CRP-induced platelet activation, and also showed partial inhibition of thrombin-induced platelet activation. Consistently, production of ROS was inhibited in NOX1−/Y platelets stimulated with thrombin, but not CRP, whereas NOX2−/− platelets showed reduced ROS generation induced by CRP or thrombin. Reduced ROS generation in NOX1/2 deficient platelets is associated with impaired activation of Syk and phospholipase Cγ2 (PLCγ2), but minimally affected mitogen-activated protein kinase pathways. Interestingly, laser-induced arterial thrombosis was impaired but the bleeding time was not affected in NOX2−/− mice. WT thrombocytopenic mice injected with NOX2−/− platelets also showed defective arterial thrombosis, suggesting an important role for platelet NOX2 in thrombosis in vivo but not hemostasis. Conclusions NOX1 and NOX2 play differential roles in different platelet activation pathways and in thrombosis. ROS generated by these enzymes promotes platelet activation via the Syk/PLCγ/calcium signaling pathway.

The critical roles of integrins in thrombosis have enabled the successful development and clinical use of the first generation of integrin antagonists as represented by abciximab (Reopro), eptifibatide (Integrilin), and tirofiban (Aggrastat). These integrin αIIb β3 antagonists are potent anti-thrombotics, but also have significant side effects. In particular, their induction of ligand-induced integrin conformational changes is associated with thrombocytopenia. Increased bleeding risk prevents integrin antagonists from being used at higher doses and in patients at risk for bleeding. To address the ligand-induced conformational changes caused by current integrin antagonists, compounds that minimally induce conformational changes in integrin αIIb β3 have been developed. Recent studies on the mechanisms of integrin signaling suggest that selectively targeting integrin outside-in signaling mechanisms allows for potent inhibition of thrombosis while maintaining hemostasis in animal models.

Sexual dimorphisms are typically attributed to the hormonal differences arising once sex differentiation has occurred. However, in some sexually dimorphic diseases that differ in frequency but not severity, the differences cannot be logically connected to the sex hormones. Therefore, we asked whether any aspect of sexual dimorphism could be attributed to chromosomal rather than hormonal differences. Cells taken from mice at d 10.5 postconception (PC) before sexual differentiation, at d 17.5 PC after the first embryonic assertion of sexual hormones, and at postnatal day 17 (puberty) were cultured and exposed to 400 microM ethanol or 20 microM camptothecin or to infection with influenza A virus (multiplicity of infection of 5). The results showed that untreated male and female cells of the same age grew at similar rates and manifested similar morphology. However, they responded differently to the applied stressors, even before the production of fetal sex hormones. Furthermore, microarray and qPCR analyses of the whole 10.5 PC embryos also revealed differences in gene expression between male and female tissues. Likewise, the exposure of cells isolated from fetuses and adolescent mice to the stressors and/or sex hormones yielded expression patterns that reflected chromosomal sex, with ethanol feminizing male cells and masculinizing female cells. We conclude that cells differ innately according to sex irrespective of their history of exposure to sex hormones. These differences may have consequences in the course of sexually dimorphic diseases and their therapy.