Natasha E. Barrett

Future innovations in anti-platelet therapies

Platelets have long been recognized to be of central importance in haemostasis, but their participation in pathological conditions such as thrombosis, atherosclerosis and inflammation is now also well established. The platelet has therefore become a key target in therapies to combat cardiovascular disease. Anti‐platelet therapies are used widely, but current approaches lack efficacy in a proportion of patients, and are associated with side effects including problem bleeding. In the last decade, substantial progress has been made in understanding the regulation of platelet function, including the characterization of new ligands, platelet‐specific receptors and cell signalling pathways. It is anticipated this progress will impact positively on the future innovations towards more effective and safer anti‐platelet agents. In this review, the mechanisms of platelet regulation and current anti‐platelet therapies are introduced, and strong, and some more speculative, potential candidate target molecules for future anti‐platelet drug development are discussed.

A dual role for integrin-linked kinase in platelets: regulating integrin function and alpha-granule secretion.

Integrin-linked kinase (ILK) has been implicated in the regulation of a range of fundamental biological processes such as cell survival, growth, differentiation, and adhesion. In platelets ILK associates with β1- and β3-containing integrins, which are of paramount importance for the function of platelets. Upon stimulation of platelets this association with the integrins is increased and ILK kinase activity is up-regulated, suggesting that ILK may be important for the coordination of platelet responses. In this study a conditional knockout mouse model was developed to examine the role of ILK in platelets. The ILK-deficient mice showed an increased bleeding time and volume, and despite normal ultrastructure the function of ILK-deficient platelets was decreased significantly. This included reduced aggregation, fibrinogen binding, and thrombus formation under arterial flow conditions. Furthermore, although early collagen stimulated signaling such as PLCγ2 phosphorylation and calcium mobilization were unaffected in ILK-deficient platelets, a selective defect in α-granule, but not dense-granule, secretion was observed. These results indicate that as well as involvement in the control of integrin affinity, ILK is required for α-granule secretion and therefore may play a central role in the regulation of platelet function.

Platelet 12-Lipoxygenase Activation via Glycoprotein VI. Involvement of Multiple Signaling Pathways in Agonist Control of H(P)ETE Synthesis

Lipoxygenases (LOX) contribute to vascular disease and inflammation through generation of bioactive lipids, including 12-hydro(pero)xyeicosatetraenoic acid (12-H(P)ETE). The physiological mechanisms that acutely control LOX product generation in mammalian cells are uncharacterized. Human platelets that contain a 12-LOX isoform (p12-LOX) were used to define pathways that activate H(P)ETE synthesis in the vasculature. Collagen and collagen-related peptide (CRP) (1 to 10 μg/mL) acutely induced platelet 12-H(P)ETE synthesis. This implicated the collagen receptor glycoprotein VI (GPVI), which signals via the immunoreceptor-based activatory motif (ITAM)-containing FcRμ chain. Conversely, thrombin only activated at high concentrations (> 0.2 U/mL), whereas U46619 and ADP alone were ineffective. Collagen or CRP-stimulated 12-H(P)ETE generation was inhibited by staurosporine, PP2, wortmannin, BAPTA/AM, EGTA, and L-655238, implicating src-tyrosine kinases, PI3-kinase, Ca2+ mobilization, and p12-LOX translocation. In contrast, protein kinase C (PKC) inhibition potentiated 12-H(P)ETE generation. Finally, activation of the immunoreceptor tyrosine-based inhibitory motif (ITIM)–containing platelet endothelial cell adhesion molecule (PECAM-1) inhibited p12-LOX product generation. This study characterizes a receptor-dependent pathway for 12-H(P)ETE synthesis via the collagen receptor GPVI, which is negatively regulated by PECAM-1 and PKC, and demonstrates a novel link between immune receptor signaling and lipid mediator generation in the vasculature.

Platelet endothelial cell adhesion molecule-1 regulates collagen-stimulated platelet function by modulating the association of phosphatidylinositol 3-kinase with Grb-2-associated binding protein-1 and linker for activation of T cells

Summary.  Background: Platelet activation by collagen depends on signals transduced by the glycoprotein (GP)VI–Fc receptor (FcR)γ‐chain collagen receptor complex, which involves recruitment of phosphatidylinositol 3‐kinase (PI3K) to phosphorylated tyrosines in the linker for activation of T cells (LAT). An interaction between the p85 regulatory subunit of PI3K and the scaffolding molecule Grb‐2‐associated binding protein‐1 (Gab1), which is regulated by binding of the Src homology 2 domain‐containing protein tyrosine phosphatase‐2 (SHP‐2) to Gab1, has been shown in other cell types to sustain PI3K activity to elicit cellular responses. Platelet endothelial cell adhesion molecule‐1 (PECAM‐1) functions as a negative regulator of platelet reactivity and thrombosis, at least in part by inhibiting GPVI–FcRγ‐chain signaling via recruitment of SHP‐2 to phosphorylated immunoreceptor tyrosine‐based inhibitory motifs in PECAM‐1. Objective: To investigate the possibility that PECAM‐1 regulates the formation of the Gab1–p85 signaling complexes, and the potential effect of such interactions on GPVI‐mediated platelet activation in platelets. Methods: The ability of PECAM‐1 signaling to modulate the LAT signalosome was investigated with immunoblotting assays on human platelets and knockout mouse platelets. Results: PECAM‐1‐associated SHP‐2 in collagen‐stimulated platelets binds to p85, which results in diminished levels of association with both Gab1 and LAT and reduced collagen‐stimulated PI3K signaling. We therefore propose that PECAM‐1‐mediated inhibition of GPVI‐dependent platelet responses result, at least in part, from recruitment of SHP‐2–p85 complexes to tyrosine‐phosphorylated PECAM‐1, which diminishes the association of PI3K with activatory signaling molecules, such as Gab1 and LAT.

Platelet Endothelial Cell Adhesion Molecule-1 Inhibits Platelet Response to Thrombin and von Willebrand Factor by Regulating the Internalization of Glycoprotein Ib via AKT/Glycogen Synthase Kinase-3/Dynamin and Integrin αIIbβ3

Objective— Platelet endothelial cell adhesion molecule-1 (PECAM-1) regulates platelet response to multiple agonists. How this immunoreceptor tyrosine-based inhibitory motif–containing receptor inhibits G protein-coupled receptor–mediated thrombin-induced activation of platelets is unknown. Approach and Results— Here, we show that the activation of PECAM-1 inhibits fibrinogen binding to integrin &agr;IIb&bgr;3 and P-selectin surface expression in response to thrombin (0.1–3 U/mL) but not thrombin receptor–activating peptides SFLLRN (3×10−7–1×10−5 mol/L) and GYPGQV (3×10−6–1×10−4 mol/L). We hypothesized a role for PECAM-1 in reducing the tethering of thrombin to glycoprotein Ib&agr; (GPIb&agr;) on the platelet surface. We show that PECAM-1 signaling regulates the binding of fluorescein isothiocyanate–labeled thrombin to the platelet surface and reduces the levels of cell surface GPIb&agr; by promoting its internalization, while concomitantly reducing the binding of platelets to von Willebrand factor under flow in vitro. PECAM-1–mediated internalization of GPIb&agr; was reduced in the presence of both EGTA and cytochalasin D or latrunculin, but not either individually, and was reduced in mice in which tyrosines 747 and 759 of the cytoplasmic tail of &bgr;3 integrin were mutated to phenylalanine. Furthermore, PECAM-1 cross-linking led to a significant reduction in the phosphorylation of glycogen synthase kinase-3&bgr; Ser9, but interestingly an increase in glycogen synthase kinase-3&agr; pSer21. PECAM-1–mediated internalization of GPIb&agr; was reduced by inhibitors of dynamin (Dynasore) and glycogen synthase kinase-3 (CHIR99021), an effect that was enhanced in the presence of EGTA. Conclusions— PECAM-1 mediates internalization of GPIb&agr; in platelets through dual AKT/protein kinase B/glycogen synthase kinase-3/dynamin-dependent and &agr;IIb&bgr;3-dependent mechanisms. These findings expand our understanding of how PECAM-1 regulates nonimmunoreceptor signaling pathways and helps to explains how PECAM-1 regulates thrombosis.

A Classroom Deployment of a Haptic System for Learning Cell Biology

The use of haptic systems in the classroom for enhancing science education is an underexplored area. In the education literature, it has been reported that certain concepts in science education are difficult for students to grasp and, as a result, misconceptions can be formed in the students’ knowledge. We conducted a study with 62 Year 8 (typically 12–13 years old) students who used a haptic application to study cell biology, specifically the concept of diffusion across a cell membrane. The preliminary analysis of the feedback from the students suggests opportunities for haptic applications to enhance their learning, and also highlights a number of points to consider in the design of the application, including the choice of haptic interface and the design of the virtual environment.