NAADP/SERCA3-Dependent Ca 2+ Stores Pathway Specifically Controls Early Autocrine ADP Secretion Potentiating Platelet Activation

Rationale: Ca 2+ signaling is a key and ubiquitous actor of cell organization and its modulation controls many cellular responses. SERCAs (sarco-endoplasmic reticulum Ca 2+ -ATPases) pump Ca 2+ into internal stores that play a major role in the cytosolic Ca 2+ concentration rise upon cell activation. Platelets exhibit 2 types of SERCAs, SERCA2b and SERCA3 (SERCA3 deficient mice), which may exert specific roles, yet ill-defined. We have recently shown that Ca 2+ mobilization from SERCA3-dependent stores was required for full platelet activation in weak stimulation conditions. Objective: To uncover the signaling mechanisms associated with Ca 2+ mobilization from SERCA3-dependent stores leading to ADP secretion. Methods and Results: Using platelets from wild-type or Serca3 -deficient mice, we demonstrated that an early (within 5–10 s following stimulation) secretion of ADP specifically dependent on SERCA3 stored Ca 2+ is exclusively mobilized by nicotinic acid adenosine dinucleotide-phosphate (NAADP): both Ca 2+ mobilization from SERCA3-dependent stores and primary ADP secretion are blocked by the NAADP receptor antagonist Ned-19, and reciprocally both are stimulated by permeant NAADP. In contrast, Ca 2+ mobilization from SERCA3-dependent stores and primary ADP secretion were unaffected by inhibition of the production of IP3 (inositol-1,4,5-trisphosphate) by phospholipase-C and accordingly were not stimulated by permeant IP3. Conclusions: Upon activation, an NAADP/SERCA3 Ca 2+ mobilization pathway initiates an early ADP secretion, potentiating platelet activation, and a secondary wave of ADP secretion driven by both an IP3/SERCA2b-dependent Ca 2+ stores pathway and the NAADP/SERCA3 pathway. This does not exclude that Ca 2+ mobilized from SERCA3 stores may also enhance platelet global reactivity to agonists. Because of its modulating effect on platelet activation, this NAADP-SERCA3 pathway may be a relevant target for anti-thrombotic therapy. Graphic Abstract: A graphic abstract is available for this article.

Data-Driven Modeling Identifies TIRAP-Independent MyD88 Activation Complex and Myddosome Assembly Strategy in LPS/TLR4 Signaling

TLR4 complexes are essential for the initiation of the LPS-induced innate immune response. The Myddosome, which mainly contains TLR4, TIRAP, MyD88, IRAK1/4 and TRAF6 proteins, is regarded as a major complex of TLR4. Although the Myddosome has been well studied, a quantitative description of the Myddosome assembly dynamics is still lacking. Furthermore, whether some unknown TLR4 complexes exist remains unclear. In this study, we constructed a SWATH-MS data-based mathematical model that describes the component assembly dynamics of TLR4 complexes. In addition to Myddosome, we suggest that a TIRAP-independent MyD88 activation complex is formed upon LPS stimulation, in which TRAF6 is not included. Furthermore, quantitative analysis reveals that the distribution of components in TIRAP-dependent and -independent MyD88 activation complexes are LPS stimulation-dependent. The two complexes compete for recruiting IRAK1/4 proteins. MyD88 forms higher-order assembly in the Myddosome and we show that the strategy to form higher-order assembly is also LPS stimulation-dependent. MyD88 forms a long chain upon weak stimulation, but forms a short chain upon strong stimulation. Higher-order assembly of MyD88 is directly determined by the level of TIRAP in the Myddosome, providing a formation mechanism for efficient signaling transduction. Taken together, our study provides an enhanced understanding of component assembly dynamics and strategies in TLR4 complexes.

Respective contributions of single and compound granule fusion to secretion by activated platelets

Key Points Weak stimulation favors the fusion of single granules with the platelet surface and stronger stimulation induces granule-granule fusion. VAMP8 participates in single and compound exocytosis.

Digital signaling decouples activation probability and population heterogeneity

Digital signaling enhances robustness of cellular decisions in noisy environments, but it is unclear how digital systems transmit temporal information about a stimulus. To understand how temporal input information is encoded and decoded by the NF-κB system, we studied transcription factor dynamics and gene regulation under dose- and duration-modulated inflammatory inputs. Mathematical modeling predicted and microfluidic single-cell experiments confirmed that integral of the stimulus (or area, concentration × duration) controls the fraction of cells that activate NF-κB in the population. However, stimulus temporal profile determined NF-κB dynamics, cell-to-cell variability, and gene expression phenotype. A sustained, weak stimulation lead to heterogeneous activation and delayed timing that is transmitted to gene expression. In contrast, a transient, strong stimulus with the same area caused rapid and uniform dynamics. These results show that digital NF-κB signaling enables multidimensional control of cellular phenotype via input profile, allowing parallel and independent control of single-cell activation probability and population heterogeneity.

Detecting Electronic Initiators Using Electromagnetic Emissions

The accurate and timely discovery of radio receivers can assist in the detection of radio-controlled explosives. By detecting radio receivers, it is possible to indirectly infer the presence of an explosive device. Radio receivers unintentionally emit low-power radio signals during normal operation. By using a weak stimulation signal, it is possible to inject a known signal into these unintended emissions. This process is known as stimulated emissions. Unlike chemical traces, these stimulated emissions can propagate through walls and air-tight containers. The following case study discusses methods for detecting and locating two different types of radio receivers. Functional stimulated emissions detectors are constructed, and their performance is analyzed. Stimulated emissions are capable of detecting super-regenerative receivers at distances of at least one hundred meters and accurately locating superheterodyne receivers at distances of at least fifty meters. These results demonstrate a novel technique for detecting potential explosive threats at stand-off detection distances.

Selective release of molecules from Weibel-Palade bodies during a lingering kiss

Exocytosis of specialized endothelial cell secretory organelles, Weibel-Palade bodies (WPBs), is thought to play an important role in regulating hemostasis and intravascular inflammation. The major WPB core proteins are Von Willebrand factor (VWF) and its propolypeptide (Proregion), constituting more than 95% of the content. Although the composition of the WPBs can be fine-tuned to include cytokines and chemokines (eg, interleukin-8 [IL-8] and eotaxin-3), it is generally assumed that WPB exocytosis is inextricably associated with secretion of VWF. Here we show that WPBs can undergo a form of exocytosis during which VWF and Proregion are retained while smaller molecules, such as IL-8, are released. Imaging individual WPBs containing fluorescent cargo molecules revealed that during weak stimulation approximately 25% of fusion events result in a failure to release VWF or Proregion. The WPB membrane protein P-selectin was also retained; however, the membrane tetraspannin CD63 was released. Accumulation or exclusion of extracellular fluorescent dextran molecules ranging from 3 kDa to 2 mDa show that these events arise due to the formation of a fusion pore approximately 12 nm in diameter. The pore behaves as a molecular filter, allowing selective release of WPB core and membrane proteins. WPB exocytosis is not inextricably associated with secretion of VWF.

A New Role for FcγRIIA in the Potentiation of Human Platelet Activation Induced by Weak Stimulation.

The low affinity receptor for immunoglobulin G, FcγRIIA, is expressed in human platelets, mediates heparin-associated thrombocytopenia, and participates in platelet activation induced by von Willebrand factor. Activation of FcγRIIA occurs upon clustering of the receptor induced by immunocomplexes, and consists in the phosphorylation of two tyrosine residues within the ITAM, typically promoted by an associated Src kinase. The phosphorylated receptor acts as a docking site for SH2 domain-containing signaling proteins, including the tyrosine kinase Syk. This event initiates an intracellular tyrosine kinase-based signaling cascade that eventually leads to phosphorylation and activation of phospholipase C (PLC) γ2, and elicits cellular responses. To date, very little is known on the possible involvement of FcγRIIA in platelet activation induced by soluble agonists. We have found that stimulation of platelets with agonists acting on G-protein-coupled receptors resulted in Src-kinase-mediated tyrosine phosphorylation of FcγRIIA. Treatment of platelets with the blocking monoclonal antibody IV.3 against FcγRIIA, but not with control IgG, inhibited platelet aggregation induced by TRAP1, TRAP4, the thromboxane A2 analogue U46619, and low concentrations of thrombin. By contrast, platelet aggregation induced by high doses of thrombin was unaffected by blockade of FcγRIIA. We also found that the anti-FcγRIIA monoclonal antibody IV.3 inhibited pleckstrin phosphorylation and calcium mobilization induced by low, but not high, concentrations of thrombin. Thrombin- and U46619-induced tyrosine phosphorylation of Syk and PLCγ2, which represent substrates typically involved in FcγRIIA-mediated signaling, was clearly reduced by incubation with anti-FcγRIIA antibody IV.3. Morever, we were able to demonstrated that platelet stimulation by thrombin induced the association of FcγRIIA with Syk. Signaling through immunoreceptor typically takes places in characteristic membrane microdomains called lipid rafts. Upon stimulation with thrombin, FcγRIIA relocated in lipid rafts, and thrombin-induced tyrosine phosphorylation of FcγRIIA occurred within these membrane domains. Controlled disruption of lipid rafts by depleting membrane cholesterol prevented tyrosine phosphorylation of FcγRIIA, and impaired platelet aggregation induced by U46619 or by low, but not high, concentrations of thrombin. These results indicate that FcγRIIA can be activated in human platelets downstream G-protein-coupled receptors, and initiates a tyrosine kinase-based signaling pathway that significantly contributes to platelet activation and aggregation in response to weak stimulation.