The Asp272-Glu282 Region of Platelet Glycoprotein Ib Interacts with the Heparin-binding Site of -Thrombin and Protects the Enzyme from the Heparin-catalyzed Inhibition by Antithrombin III

Platelet glycoprotein Ib (GpIb) mediates interaction with both von Willebrand factor and thrombin. Thrombin binds to GpIb via its heparin-binding site (HBS) (De Candia, E., De Cristofaro, R., De Marco, L., Mazzucato, M., Picozzi, M., and Landolfi, R. (1997) Thromb. Haemostasis 77, 735–740; De Cristofaro, R., De Candia, E., Croce, G., Morosetti, R., and Landolfi, R. (1998) Biochem. J. 332, 643–650). To identify the thrombin-binding domain on GpIbα, we examined the effect of GpIbα1–282, a GpIbα fragment released by the cobra venom mocarhagin on the heparin-catalyzed rate of thrombin inhibition by antithrombin III (AT). GpIbα1–282 inhibited the reaction in a dose-dependent and competitive fashion. In contrast, the GpIbα1–271 fragment, produced by exposing GpIbα1–282 to carboxypeptidase Y, had no effect on thrombin inhibition by the heparin-AT complex. Measurements of the apparent equilibrium constant of the GpIbα1–282 binding to thrombin as a function of different salts (NaCl and tetramethyl-ammonium chloride) concentration (0.1–0.2 M) indicated a large salt dependence (Γ± = −4.5), similar to that pertaining to the heparin binding to thrombin. The importance of thrombin HBS in its interaction with GpIbα was confirmed using DNA aptamers, which specifically bind to either HBS (HD22) or the fibrinogen recognition site of thrombin (HD1). HD22, but not HD1, inhibited thrombin binding to GpIbα1–282. Furthermore, the proteolytic derivative γT-thrombin, which lacks the fibrinogen recognition site, binds to GpIbα via its intact HBS in a reaction that is inhibited by HD22. Neither α- nor γT-thrombin bound to GpIbα1–271, suggesting that the Asp272–Glu282 region of GpIbα may act as a “heparin-like” ligand for the thrombin HBS, thereby inhibiting heparin binding to thrombin. It was also demonstrated that intact platelets may dose-dependently inhibit the heparin-catalyzed thrombin inhibition by AT at enzyme concentrations <5 nM. Altogether, these findings show that thrombin HBS binds to the region of GpIbα involving the Asp272–Glu282 segment, protecting the enzyme from the inactivation by the heparin-AT system

Binding of thrombin to glycoprotein Ib accelerates the hydrolysis of Par-1 on intact platelets

The activation of human platelets by α-thrombin is mediated at least in part by cleavage of protease-activated G-protein-coupled receptors, PAR-1 and PAR-4. Platelet glycoprotein Ibα also has a high affinity binding site for α-thrombin, and this interaction contributes to platelet activation through a still unknown mechanism. In the present study the hypothesis that GpIbα may contribute to platelet activation by modulating the hydrolysis of PAR-1 on the platelet membrane was investigated. Gel-filtered platelets from normal individuals were stimulated by α-thrombin, and the kinetics of PAR-1 hydrolysis by enzyme was followed with flow cytometry using an anti-PAR-1 monoclonal antibody (SPAN 12) that recognizes only intact PAR-1 molecules. This strategy allowed measurement of the apparentk cat/K m value for thrombin hydrolysis of PAR-1 on intact platelets, which was equal to 1.5 ± 0.1 × 107 m −1sec−1. The hydrolysis rate of PAR-1 by thrombin was measured under conditions in which thrombin binding to GpIb was inhibited by different strategies, with the following results. 1) Elimination of GpIbα on platelet membranes by mocarhagin treatment reduced the k cat/K m value by about 6-fold. 2) A monoclonal anti-GpIb antibody reduced the apparent k cat/K m value by about 5-fold. 3) An oligonucleotide DNA aptamer, HD22, which binds to the thrombin heparin-binding site (HBS) and inhibits thrombin interaction with GpIbα, reduced the apparentk cat/K m value by about 5-fold. 4) Displacement of α-thrombin from the binding site on GpIb using PPACK-thrombin reduced the apparentk cat/K m value by about 5-fold, and 5) mutation at the HBS of thrombin (R98A) caused a 5-fold reduction of the apparentk cat/K m value of PAR-1 hydrolysis. Altogether these results show that thrombin interaction with GpIb enhances the specificity of thrombin cleavage of PAR-1 on intact platelets, suggesting that GpIb may function as a “cofactor” for PAR-1 activation by thrombin

Fibrinogen-elongated Chain Inhibits Thrombin-induced Platelet Response, Hindering the Interaction with Different Receptors

The expression of the elongated fibrinogen γ chain, termed γ′, derives from alternative splicing of mRNA and causes an insertion sequence of 20 amino acids. This insertion domain interacts with the anion-binding exosite (ABE)-II of thrombin. This study investigated whether and how γ′ chain binding to ABE-II affects thrombin interaction with its platelet receptors, i.e. glycoprotein Ibα (GpIbα), protease-activated receptor (PAR) 1, and PAR4. Both synthetic γ′ peptide and fibrinogen fragment D*, containing the elongated γ′ chain, inhibited thrombin-induced platelet aggregation up to 70%, with IC50 values of 42 ± 3.5 and 0.47 ± 0.03 μm, respectively. Solid-phase binding and spectrofluorimetric assays showed that both fragment D* and the synthetic γ′ peptide specifically bind to thrombin ABE-II and competitively inhibit the thrombin binding to GpIbα with a mean Ki ≈ 0.5 and ≈35 μm, respectively. Both these γ′ chain-containing ligands allosterically inhibited thrombin cleavage of a synthetic PAR1 peptide, of native PAR1 molecules on intact platelets, and of the synthetic chromogenic peptide d-Phe-pipecolyl-Arg-p-nitroanilide. PAR4 cleavage was unaffected. In summary, fibrinogen γ′ chain binds with high affinity to thrombin and inhibits with combined mechanisms the platelet response to thrombin. Thus, its variations in vivo may affect the hemostatic balance in arterial circulation

Disruption of Phthorimaea operculella (Lepidoptera: Gelechiidae) oviposition by the application of host plant volatiles

Phthorimaea operculella is a key pest of potato. The authors characterised the P. operculella olfactory system, selected the most bioactive host plant volatiles and evaluated their potential application in pest management. The electrophysiological responses of olfactory receptor neurons (ORNs) housed in long sensilla trichodea of P. operculella to plant volatiles and the two main sex pheromone components were evaluated by the single-cell recording (SCR) technique. The four most SCR-active volatiles were tested in a laboratory oviposition bioassay and under storage warehouse conditions. The sensitivity of sensilla trichodea to short-chained aldehydes and alcohols and the existence of ORNs tuned to pheromones in females were characterised. Male recordings revealed at least two types of ORN, each of which typically responded to one of the two pheromone components. Hexanal, octanal, nonanal and 1-octen-3-ol significantly disrupted the egg-laying behaviour in a dose-dependent manner. Octanal reduced the P. operculella infestation rate when used under storage conditions. This work provides new information on the perception of plant volatiles and sex pheromones by P. operculella. Laboratory and warehouse experiments show that the use of hexanal, octanal, nonanal and 1-octen-3-ol as host recognition disruptants and/or oviposition deterrents for P. operculella control appears to be a promising strategy

Sensory Adaptation of Antennae and Sex Pheromone-Mediated Flight Behavior in Male Oriental Fruit Moths (Leptidoptera: Tortricidae) After Prolonged Exposure to Single and Tertiary Blends of Synthetic Sex Pheromone

Sensory adaptation has been measured in the antennae of male Grapholita molesta (Busck) after 15 min of exposure to its main pheromone compound (Z)-8-dodecen-1-yl acetate (Z8-12:OAc) at the aerial concentration of 1 ng/m(3) measured in orchards treated with pheromone for mating disruption. Exposing males to this aerial concentration of Z8-12:OAc for 15 min, however, had only a small effect on their ability to orientate by flight to virgin calling females in a flight tunnel. Experiments were undertaken to determine if exposure to the main pheromone compound in combination with the two biologically active minor compounds of this species, (E)-8-dodecen-1-yl acetate (E8-12:OAc) and (Z)-8-dodecen-1-ol (Z8-12:OH) would induce greater levels of sensory adaptation and have a greater effect on male sexual behavior. The exposure of male antennae to 0.5 g/m(3) air of one of the three pheromone compounds induced sensory adaptation to this compound and to the other two pheromone compounds demonstrating cross adaptation. Average percentage sensory adaptation to a pheromone compound was similar after 15 min of exposure to 1 ng/m(3) air of Z8-12:OAc, or to 1 ng/m(3) air of a 1:1:1 or 93:6:1 blend of Z8-12:OAc, E8-12:OAc, and Z8-12:OH. The exposure of males to 1 ng/m(3) air of Z8-12:OAc or the two ratios of Z8-12:OAc, E8-12:OAc, and Z8-12:OH for 15 min had no effect on their ability to orientate to a virgin calling female. The implications of these results for the operative mechanisms of sex pheromone-mediated mating disruption of this species are discussed

MaMaDroid: Detecting Android malware by building markov chains of behavioral models (extended version)

As Android has become increasingly popular, so has malware targeting it, thus motivating the research community to propose different detection techniques. However, the constant evolution of the Android ecosystem, and of malware itself, makes it hard to design robust tools that can operate for long periods of time without the need for modifications or costly re-training. Aiming to address this issue, we set to detect malware from a behavioral point of view, modeled as the sequence of abstracted API calls. We introduce MaMaDroid, a static-analysis based system that abstracts app’s API calls to their class, package, or family, and builds a model from their sequences obtained from the call graph of an app as Markov chains. This ensures that the model is more resilient to API changes and the features set is of manageable size. We evaluate MaMaDroid using a dataset of 8.5K benign and 35.5K malicious apps collected over a period of six years, showing that it effectively detects malware (with up to 0.99 F-measure) and keeps its detection capabilities for long periods of time (up to 0.87 F-measure two years after training). We also show that MaMaDroid remarkably overperforms DroidAPIMiner, a state-of-the-art detection system that relies on the frequency of (raw) API calls. Aiming to assess whether MaMaDroid’s effectiveness mainly stems from the API abstraction or from the sequencing modeling, we also evaluate a variant of it that uses frequency (instead of sequences), of abstracted API calls. We find that it is not as accurate, failing to capture maliciousness when trained on malware samples that include API calls that are equally or more frequently used by benign apps

Does Platform Migration Compromise Content Moderation? {Evidence} from {r/The\_Donald} and {r/Incels}

When toxic online communities on mainstream platforms face moderation measures, such as bans, they may migrate to other platforms with laxer policies or set up their own dedicated website. Previous work suggests that, within mainstream platforms, community-level moderation is effective in mitigating the harm caused by the moderated communities. It is, however, unclear whether these results also hold when considering the broader Web ecosystem. Do toxic communities continue to grow in terms of user base and activity on their new platforms? Do their members become more toxic and ideologically radicalized? In this paper, we report the results of a large-scale observational study of how problematic online communities progress following community-level moderation measures. We analyze data from r/The_Donald} and r/Incels, two communities that were banned from Reddit and subsequently migrated to their own standalone websites. Our results suggest that, in both cases, moderation measures significantly decreased posting activity on the new platform, reducing the number of posts, active users, and newcomers. In spite of that, users in one of the studied communities (r/The_Donald) showed increases in signals associated with toxicity and radicalization, which justifies concerns that the reduction in activity may come at the expense of a more toxic and radical community. Overall, our results paint a nuanced portrait of the consequences of community-level moderation and can inform their design and deployment

Do Platform Migrations Compromise Content Moderation? Evidence from r/The_Donald and r/Incels

When toxic online communities on mainstream platforms face moderation measures, such as bans, they may migrate to other platforms with laxer policies or set up their own dedicated websites. Previous work suggests that within mainstream platforms, community-level moderation is effective in mitigating the harm caused by the moderated communities. It is, however, unclear whether these results also hold when considering the broader Web ecosystem. Do toxic communities continue to grow in terms of their user base and activity on the new platforms? Do their members become more toxic and ideologically radicalized? In this paper, we report the results of a large-scale observational study of how problematic online communities progress following community-level moderation measures. We analyze data from r/The_Donald and r/Incels, two communities that were banned from Reddit and subsequently migrated to their own standalone websites. Our results suggest that, in both cases, moderation measures significantly decreased posting activity on the new platform, reducing the number of posts, active users, and newcomers. In spite of that, users in one of the studied communities (r/The_Donald) showed increases in signals associated with toxicity and radicalization, which justifies concerns that the reduction in activity may come at the expense of a more toxic and radical community. Overall, our results paint a nuanced portrait of the consequences of community-level moderation and can inform their design and deployment

Kek, Cucks, and God Emperor Trump: A Measurement Study of 4chan's Politically Incorrect Forum and its Effects on the Web

The discussion-board site 4chan has been part of the Internet's dark underbelly since its inception, and recent political events have put it increasingly in the spotlight. In particular, /pol/, the “Politically Incorrect'” board, has been a central figure in the outlandish 2016 US election season, as it has often been linked to the alt-right movement and its rhetoric of hate and racism. However, 4chan remains relatively unstudied by the scientific community: little is known about its user base, the content it generates, and how it affects other parts of the Web. In this paper, we start addressing this gap by analyzing /pol/ along several axes, using a dataset of over 8M posts we collected over two and a half months. First, we perform a general characterization, showing that /pol/ users are well distributed around the world and that 4chan's unique features encourage fresh discussions. We also analyze content, finding, for instance, that YouTube links and hate speech are predominant on /pol/. Overall, our analysis not only provides the first measurement study of /pol/, but also insight into online harassment and hate speech trends in social media