RGS10 shapes the hemostatic response to injury through its differential effects on intracellular signaling by platelet agonists.

Platelets express ≥2 members of the regulators of G protein signaling (RGS) family. Here, we have focused on the most abundant, RGS10, examining its impact on the hemostatic response in vivo and the mechanisms involved. We have previously shown that the hemostatic thrombi formed in response to penetrating injuries consist of a core of fully activated densely packed platelets overlaid by a shell of less-activated platelets responding to adenosine 5\u27-diphosphate (ADP) and thromboxane A2 (TxA2). Hemostatic thrombi formed in RGS10-/- mice were larger than in controls, with the increase due to expansion of the shell but not the core. Clot retraction was slower, and average packing density was reduced. Deleting RGS10 had agonist-specific effects on signaling. There was a leftward shift in the dose/response curve for the thrombin receptor (PAR4) agonist peptide AYPGKF but no increase in the maximum response. This contrasted with ADP and TxA2, both of which evoked considerably greater maximum responses in RGS10-/- platelets with enhanced Gq- and Gi-mediated signaling. Shape change, which is G13-mediated, was unaffected. Finally, we found that free RGS10 levels in platelets are actively regulated. In resting platelets, RGS10 was bound to 2 scaffold proteins: spinophilin and 14-3-3γ. Platelet activation caused an increase in free RGS10, as did the endothelium-derived platelet antagonist prostacyclin. Collectively, these observations show that RGS10 serves as an actively regulated node on the platelet signaling network, helping to produce smaller and more densely packed hemostatic thrombi with a greater proportion of fully activated platelets

The influence of bovine serum albumin on β-lactoglobulin denaturation, aggregation and gelation

peer-reviewedThe effect of bovine serum albumin (BSA) on the heat-induced denaturation, aggregation and subsequent acid-induced gelation of β-lactoglobulin (β-lg) was investigated in this work. Changes in the denaturation kinetics of β-lg during heating at 78 °C were determined by monitoring the disappearance of the native protein by reverse-phase chromatography. Replacing β-lg with increasing amounts of BSA, while keeping the total protein concentration constant at 5% (w/w), significantly increased the denaturation rate of β-lg from 2.57±0.30×10−3(g L−1)(1−n)s−1 to 5.07±0.72×10−3(g L−1)(1−n)s−1 (β-lg: BSA ratio of 3:1 w/w). The reaction order for β-lg was 1.40±0.09. Partial replacement of β-lg with BSA (β-lg: BSA ratio of 3:1 w/w) significantly increased the reaction order to 1.67±0.13. Heat-induced aggregates between β-lg and BSA were studied by dynamic light scattering, two-dimensional electrophoresis and size exclusion chromatography. The partial replacement of β-lg with BSA significantly changed the gelling properties of the acid-induced gels. A rapid rate of acidification resulted in a significant decrease, while a slow acidification rate resulted in a significant increase in gel strength. Size exclusion chromatography demonstrated that intermolecular disulphide bond formation occurred during both heat-induced denaturation/aggregation and subsequent acid-induced gelation. Results clearly indicate that BSA contributed to the formation of these disulphide bonds.This work was funded under the Food Institutional Research Measure (FIRM) of the National Development Plan 2000-2006. J. Kehoe is funded by the Teagasc Walsh Fellowship schem

The generalized identification of truly interfacial molecules (ITIM) algorithm for nonplanar interfaces

We present a generalized version of the ITIM algorithm for the identification of interfacial molecules, which is able to treat arbitrarily shaped interfaces. The algorithm exploits the similarities between the concept of probe sphere used in ITIM and the circumsphere criterion used in the α-shapes approach, and can be regarded either as a reference-frame independent version of the former, or as an extended version of the latter that includes the atomic excluded volume. The new algorithm is applied to compute the intrinsic orientational order parameters of water around a dodecylphosphocholine and a cholic acid micelle in aqueous environment, and to the identification of solvent-reachable sites in four model structures for soot. The additional algorithm introduced for the calculation of intrinsic density profiles in arbitrary geometries proved to be extremely useful also for planar interfaces, as it allows to solve the paradox of smeared intrinsic profiles far from the interface. © 2013 American Institute of Physics