2-Aminoethoxydiphenylborate (2-APB) inhibits release of phosphatidylserine-exposing extracellular vesicles from platelets.

Activated, procoagulant platelets shed phosphatidylserine (PS)-exposing extracellular vesicles (EVs) from their surface in a Ca2+- and calpain-dependent manner. These PS-exposing EVs are prothrombotic and proinflammatory and are found at elevated levels in many cardiovascular and metabolic diseases. How PS-exposing EVs are shed is not fully understood. A clearer understanding of this process may aid the development of drugs to selectively block their release. In this study we report that 2-aminoethoxydiphenylborate (2-APB) significantly inhibits the release of PS-exposing EVs from platelets stimulated with the Ca2+ ionophore, A23187, or the pore-forming toxin, streptolysin-O. Two analogues of 2-APB, diphenylboronic anhydride (DPBA) and 3-(diphenylphosphino)-1-propylamine (DP3A), inhibited PS-exposing EV release with similar potency. Although 2-APB and DPBA weakly inhibited platelet PS exposure and calpain activity, this was not seen with DP3A despite inhibiting PS-exposing EV release. These data suggest that there is a further target of 2-APB, independent of cytosolic Ca2+ signalling, PS exposure and calpain activity, that is required for PS-exposing EV release. DP3A is likely to inhibit the same target, without these other effects. Identifying the target of 2-APB, DPBA and DP3A may provide a new way to inhibit PS-exposing EV release from activated platelets and inhibit their contribution to thrombosis and inflammation.

Using Yoda-1 to mimic laminar flow in vitro: A tool to simplify drug testing.

The endothelium is an attractive drug target and an important site of adverse drug reactions. Endothelial dysfunction is strongly associated with inflammation and contributes to drug-induced cardiovascular toxicity. Endothelial cells in the circulation are exposed to haemodynamic forces including shear stress. Including shear stress may improve future endothelial cell drug discovery or toxicity screening. Piezo-1 is required for endothelial cells to respond to shear stress. In this study, we investigated whether a small molecule activator of Piezo-1, Yoda-1, can mimic the effect of laminar flow-induced shear stress on endothelial cell inflammation, and endothelial cytotoxicity in response to the chemotherapy agent, doxorubicin. First, we tested whether Yoda-1 could mimic the effects of shear stress of expression of the endothelial adhesion molecules, ICAM-1 and VCAM-1. Human umbilical vein endothelial cells (HUVEC) were cultured in static conditions (with or without Yoda-1) or under laminar flow-induced shear stress (5 dyn/cm2). Yoda-1 and laminar flow had similar anti-inflammatory effects, reducing the ability of TNF-α to induce ICAM-1 and VCAM-1 expression. We then tested whether Yoda-1 could mimic the effect of shear stress on doxorubicin-induced cytotoxicity. Both laminar flow and Yoda-1 treatment of static cultures increased the cytotoxicity of doxorubicin. These findings show that Piezo-1 activation with Yoda-1 in static culture leads to an endothelial cell phenotype that mimics endothelial cells under laminar flow. Pharmacological activation of Piezo-1 may be a useful approach to mimic constant shear stress in static cultures, which may improve endothelial drug discovery and toxicity testing.

Cross-reactivity of anti-HMGB1 antibodies for HMGB2.

HMGB1 and HMGB2 are DNA-interacting proteins but can also have extracellular actions during inflammation. Despite their relatively high homology, they may have distinct roles, making it essential to be able to differentiate between the two. Here we examine the specificity of five commercially-available anti-HMGB1 antibodies. By Western blotting of recombinant proteins and HMGB1-/- mouse embryonic fibroblasts, we identified only one HMGB1 antibody that, under our experimental conditions, did not also detect HMGB2. Selecting specific antibodies for HMGB1 and HMGB2 allowed identification of distinct HMGB1 and HMGB2 subcellular pools in primary neutrophils.