Erythrocyte interaction with neutrophil extracellular traps in coronary artery thrombosis following myocardial infarction.

Cardiovascular disease, including myocardial infarction (MI), is the leading cause of death globally. Current antithrombotic medications used during MI treatment are predominantly directed towards platelet inhibition and, to a lesser extent, anticoagulation. Bleeding is a major risk of such treatment and could be circumvented by targeting other causative factors essential for arterial thrombus formation. We sought to re-evaluate the cellular composition of arterial thrombus in order to better understand mechanisms that lead to coronary artery thrombosis in acute MI. We performed detailed histological and immunohistochemical analysis of coronary artery thrombi aspirated from 26 patients undergoing emergency percutaneous coronary intervention for acute ST elevated myocardial infarction (STEMI). Coronary arterial thrombi had an unanticipated cellular heterogeneity. Neutrophil extracellular traps (NETs) were observed in thrombi as identified by anti-citrullinated histone 3 and anti-myeloperoxidase staining. Increased abundance of NETs was seen directly surrounding erythrocytes. Extracellular iron and erythrocyte fragments were also associated with areas of NETs suggesting a possible link. Our results shed light on potential involvement of erythrocytes in coronary arterial thrombosis through activation of platelets and induction of NETs. If supported by further in vitro and in vivo studies, novel therapies to inhibit NET formation or coagulation activation by erythrocyte release products, could bolster current myocardial infarction treatment.

Fragment-Based Discovery of Inhibitors of the Bacterial DnaG-SSB Interaction.

In bacteria, the DnaG primase is responsible for synthesis of short RNA primers used to initiate chain extension by replicative DNA polymerase(s) during chromosomal replication. Among the proteins with which Escherichia coli DnaG interacts is the single-stranded DNA-binding protein, SSB. The C-terminal hexapeptide motif of SSB (DDDIPF; SSB-Ct) is highly conserved and is known to engage in essential interactions with many proteins in nucleic acid metabolism, including primase. Here, fragment-based screening by saturation-transfer difference nuclear magnetic resonance (STD-NMR) and surface plasmon resonance assays identified inhibitors of the primase/SSB-Ct interaction. Hits were shown to bind to the SSB-Ct-binding site using 15N-¹H HSQC spectra. STD-NMR was used to demonstrate binding of one hit to other SSB-Ct binding partners, confirming the possibility of simultaneous inhibition of multiple protein/SSB interactions. The fragment molecules represent promising scaffolds on which to build to discover new antibacterial compounds.

Fragment-based screening by protein crystallography: successes and pitfalls.

Fragment-based drug discovery (FBDD) concerns the screening of low-molecular weight compounds against macromolecular targets of clinical relevance. These compounds act as starting points for the development of drugs. FBDD has evolved and grown in popularity over the past 15 years. In this paper, the rationale and technology behind the use of X-ray crystallography in fragment based screening (FBS) will be described, including fragment library design and use of synchrotron radiation and robotics for high-throughput X-ray data collection. Some recent uses of crystallography in FBS will be described in detail, including interrogation of the drug targets ß-secretase, phenylethanolamine N-methyltransferase, phosphodiesterase 4A and Hsp90. These examples provide illustrations of projects where crystallography is straightforward or difficult, and where other screening methods can help overcome the limitations of crystallography necessitated by diffraction quality.