Jon M. Gibbins

Platelet signaling: a complex interplay between inhibitory and activatory networks.

The role of platelets in hemostasis and thrombosis is dependent on a complex balance of activatory and inhibitory signaling pathways. Inhibitory signals released from the healthy vasculature suppress platelet activation in the absence of platelet receptor agonists. Activatory signals present at a site of injury initiate platelet activation and thrombus formation; subsequently, endogenous negative signaling regulators dampen activatory signals to control thrombus growth. Understanding the complex interplay between activatory and inhibitory signaling networks is an emerging challenge in the study of platelet biology, and necessitates a systematic approach to utilize experimental data effectively. In this review, we will explore the key points of platelet regulation and signaling that maintain platelets in a resting state, mediate activation to elicit thrombus formation, or provide negative feedback. Platelet signaling will be described in terms of key signaling molecules that are common to the pathways activated by platelet agonists and can be described as regulatory nodes for both positive and negative regulators.

Integrin-linked kinase regulates the rate of platelet activation and is essential for the formation of stable thrombi

Integrin‐linked kinase (ILK) and its associated complex of proteins are involved in many cellular activation processes, including cell adhesion and integrin signaling. We have previously demonstrated that mice with induced platelet ILK deficiency show reduced platelet activation and aggregation, but only a minor bleeding defect. Here, we explore this apparent disparity between the cellular and hemostatic phenotypes.

A high-density immunoblotting methodology for quantification of total protein levels and phosphorylation modifications

The components of many signaling pathways have been identified and there is now a need to conduct quantitative data-rich temporal experiments for systems biology and modeling approaches to better understand pathway dynamics and regulation. Here we present a modified Western blotting method that allows the rapid and reproducible quantification and analysis of hundreds of data points per day on proteins and their phosphorylation state at individual sites. The approach is of particular use where samples show a high degree of sample-to-sample variability such as primary cells from multiple donors. We present a case study on the analysis of >800 phosphorylation data points from three phosphorylation sites in three signaling proteins over multiple time points from platelets isolated from ten donors, demonstrating the technique’s potential to determine kinetic and regulatory information from limited cell numbers and to investigate signaling variation within a population. We envisage the approach being of use in the analysis of many cellular processes such as signaling pathway dynamics to identify regulatory feedback loops and the investigation of potential drug/inhibitor responses, using primary cells and tissues, to generate information about how a cell’s physiological state changes over time.

188 DRUG-ELUTING STENTS CONTAINING IBUPROFEN: A NOVEL STRATEGY TO REDUCE RESTENOSIS AND PREVENT LATE IN-STENT THROMBOSIS

Introduction Drug-eluting stents (DES) have reduced the rates of restenosis to less than 10%; however, increased incidence of late in-stent thrombosis, is now a major clinical problem of using DES for its consequences in terms of morbidity and mortality. Interference with the process of re-endothelialization appears to play a major role in this latent complication. Thus, the search for molecular compounds capable of preventing restenosis that also can reduce the risk of late in-stent thrombosis is of utmost importance. Previously, we have shown that certain non-steroidal anti-inflammatory drugs (NSAIDs) are capable of inhibiting rat vascular smooth muscle cell proliferation, a key element in the process of restenosis post stent deployment. Here, we evaluated whether proliferation and migration of human coronary artery smooth muscle cells (HCASMCs) is reduced by the NSAID, ibuprofen, whether this correlated with changes in the phenotype of HCASMCs and the potential molecular mechanisms involved. Also, we investigated whether human coronary artery endothelial cell (HCAEC) migration was affected by ibuprofen. Methods Cell proliferation was evaluated by trypan blue exclusion. Cell migration was assessed by wound healing assay and by time lapse videomicroscopy. Protein expression was assessed by immunoblotting and morphology by immunocytochemistry. The involvement of the PPARγ pathway was studied with the selective agonist, troglitazone, and with the PPARγ antagonists, PGF2α and GW9662. Results We show that ibuprofen inhibited proliferation as well as migration of HCASMCs in a dose-dependent manner (IC50 of 680 and 410 µM, respectively). Interestingly, we found that ibuprofen induced a switch in HCASMCs towards a differentiated phenotype, with a characteristic spindle shape, and a significant increase in the expression of contractile protein markers, e.g. SMα-actin, SM22α and F-actin. PPARγ antagonists, PGF2α and GW9662, almost completely abrogated the proliferative and migratory responses of HCASMCs, as well the changes in morphology. However, PPARγ antagonists did not affect the expression pattern of contractile proteins, suggesting that these effects are mediated by a PPARγ-independent pathway. Importantly, ibuprofen did not affect migration of HCAECs even at high doses (up to 1000 µM), suggesting that the effects of ibuprofen on HCASMCs are selective. Interestingly, the protein levels of PPARγ are higher in HCAECs compared to those in HCASMCs. Conclusions Taken together, our results suggest that ibuprofen could be an effective treatment for the development of novel DES. Its effects on migration and proliferation of HCASMCs, and induction of a differentiated phenotype, could translate into reduced rates of restenosis, whilst the lack of an effect on HCAEC migration could result in a reduced risk of late in-stent thrombosis. Further studies in animal models will be required to evaluate the performance of an ibuprofen DES in vivo.

Platelet-Derived Inhibitors of Platelet Activation

Negative regulators of platelet activation are a relatively unexplored aspect of platelet physiology yet have an important role in tempering thrombus development by contributing much needed negative regulation to a process that is amplified by several positive feedback mechanisms. Some negative regulators, such as RASA3 and JAM-A, act as gatekeepers that modulate key mediators of activation and provide barriers that must be deactivated to permit full activation and stable thrombus formation. Other negative regulators, such as PECAM-1 and other proteins that signal through ITIMs, come into play once platelets are activated and provide restraining, negative feedback for activatory pathways. Many platelet-derived inhibitors have been identified but not fully characterised and so questions remain regarding the mechanisms that underlie the effects on platelet activity following their activation, inhibition or genetic disruption. However, dysregulation of inhibitory signals is believed to contribute to enhanced risk of thrombosis in diseases such as diabetes and other pathological conditions. In this chapter we have described platelet-derived inhibitors of platelet function that are secreted by or expressed within platelets themselves to provide inhibition or negative regulation to the processes that underpin activation.