Ca2+ waves coordinate purinergic receptor–evoked integrin activation and polarization

The integrin GPIIb/IIIa is highly abundant on the surface of platelets and can be activated by intracellular Ca2+ signaling in an “inside-out” manner to bind to the adhesive ligand fibrinogen. Bye et al. imaged intracellular Ca2+ signaling and fibrinogen binding events in primary rat megakaryocytes activated through the ADP-stimulated receptors P2Y1 and P2Y12. The authors found that signaling by both receptors was required for full integrin activation, which depended on P2Y1-stimulated Ca2+ signaling and P2Y12-stimulated activation of the kinase PI3K. In addition, fibrinogen binding became polarized in these cells in a manner dependent on the direction of ADP-stimulated Ca2+ waves

Induction skull melt spinning of reactive metal alloys

An apparatus for melting and rapid solidification casting of metal alloys has a crucible for molding a metal charge. The crucible has side walls, a top and a bottom having an orifice therein. Collectively, the side walls, top and bottom define an interior of the crucible. A portion of the dimensions of the side walls of the side walls and bottom is divided by longitudinal slits into at least two segments. A nozzle is disposed partially within the crucible and extends through the orifice. The nozzle has a first end in communication with the interior of tune crucible. A second end of the nozzle has a nozzle orifice therein for defining a stream of molten metal alloy. A cooling mechanism cools the top, side walls and bottom of the crucible. The apparatus has mechanisms for inducing alternating electrical currents within the metal charge and within the nozzle, and for establishing and maintaining pressure within the interior of the crucible. A positioning mechanism positions the crucible and nozzle means relative to a quenching mechanisms that includes a rapidly moving chill substrate. The crucible, nozzle and quenching mechanism are housed within an enclosure that provides there within a controlled atmosphere having positive or negative pressure

Validation of Different Combination of Three Reversing Half-Hitches Alternating Posts (RHAPs) Effects on Arthroscopic Knot Integrity

Introduction. With arthroscopic techniques being used, the importanceof knot tying has been examined. Previous literaturehas examined the use of reversing half-hitches on alternatingposts (RHAPs) on knot security. Separately, there has been researchregarding different suture materials commonly used inthe operating room. The specific aim of this study was to validatethe effect of different stacked half-hitch configuration anddifferent braided suture materials on arthroscopic knot integrity. Methods. Three different suture materials tied withfive different RHAPs in arthroscopic knots were compared.A single load-to-failure test was performed andthe mean ultimate clinical failure load was obtained. Results. Significant knot holding strength improvement wasfound when one half-hitch was reversed as compared to baselineknot. When two of the half-hitches were reversed, therewas a greater improvement with all knots having a mean ultimateclinical failure load greater than 150 newtons (N). Comparisonof the suture materials demonstrated a higher meanultimate clinical failure load when Force Fiber® was used andat least one half-hitch was reversed. Knots tied with eitherForce Fiber® or Orthocord® showed 0% chance of knot slippagewhile knots tied with FiberWire® or braided fishing linehad about 10 and 30% knot slippage chances, respectively. Conclusion. A significant effect was observed in regards to bothstacked half-hitch configuration and suture materials used onknot loop and knot security. Caution should be used with tyingthree RHAPs in arthroscopic surgery, particularly witha standard knot pusher and arthroscopic cannulas. The findingsof this study indicated the importance of three RHAPsin performing arthroscopic knot tying and provided evidenceregarding discrepancies of maximum clinical failure loads observedbetween orthopaedic surgeons, thereby leading to bettersurgical outcomes in the future. KS J Med 2017;10(2):35-39

Ibrutinib inhibits platelet integrin αIIbβ3 outside-in signaling and thrombus stability but not adhesion to collagen

OBJECTIVE: Ibrutinib is an irreversible Bruton tyrosine kinase inhibitor approved for treatment of Waldenstrom macroglobulinemia, chronic lymphocytic leukemia, and mantle cell lymphoma that increases the risk of bleeding among patients. Platelets from ibrutinib-treated patients exhibit deficiencies in collagen-evoked signaling in suspension; however, the significance of this observation and how it relates to bleeding risk is unclear, as platelets encounter immobile collagen in vivo. We sought to clarify the effects of ibrutinib on platelet function to better understand the mechanism underlying bleeding risk. APPROACH AND RESULTS: By comparing signaling in suspension and during adhesion to immobilized ligands, we found that the collagen signaling deficiency caused by ibrutinib is milder during adhesion to immobilized collagen. We also found that platelets in whole blood treated with ibrutinib adhered to collagen under arterial shear but formed unstable thrombi, suggesting that the collagen signaling deficiency caused by ibrutinib may not be the predominant cause of bleeding in vivo. However, clot retraction and signaling evoked by platelet adhesion to immobilized fibrinogen were also inhibited by ibrutinib, indicating that integrin αIIbβ3 outside-in signaling is also effected in addition to GPVI signaling. When ibrutinib was combined with the P2Y12 inhibitor, cangrelor, thrombus formation under arterial shear was inhibited additively. CONCLUSIONS: These findings suggest that (1) ibrutinib causes GPVI and integrin αIIbβ3 platelet signaling deficiencies that result in formation of unstable thrombi and may contribute toward bleeding observed in vivo and (2) combining ibrutinib with P2Y12 antagonists, which also inhibit thrombus stability, may have a detrimental effect on hemostasis

A risk model for recreational craft accidents

The Recreational Craft Platform (RCP, Norwegian: Fritidsbåtplattformen) is being developed to collect and merge available data on recreational craft accidents and thereby enable stakeholders to actively take measures to achieve the vision. The Norwegian Maritime Authority (NMA) will be the owner of the platform, using it to analyse the causes and risks associated with recreational craft, and to identify and evaluate risk reducing measures to reduce the number of accidents significantly. This paper presents the risk model for recreational craft accidents, developed together with the NMA that will support NMA's and their partners work to achieve these goals. The risk model was developed following NMA's existing risk modelling approach, with focus on the accident frequency of motorboats, sailing boats and personal watercraft. The model will be employed to assess effectiveness of measure and visualizing the contributing factors within the RCP.publishedVersio

The metabolites of the dietary flavonoid quercetin possess potent antithrombotic activity, and interact with aspirin to enhance antiplatelet effects

Quercetin, a dietary flavonoid, has been reported to possess antiplatelet activity. However, its extensive metabolism following ingestion has resulted in difficulty elucidating precise mechanisms of action. In this study, we aimed to characterize the antiplatelet mechanisms of two methylated metabolites of quercetin—isorhamnetin and tamarixetin—and explore potential interactions with aspirin. Isorhamnetin and tamarixetin inhibited human platelet aggregation, and suppressed activatory processes including granule secretion, integrin αIIbβ3 function, calcium mobilization, and spleen tyrosine kinase (Syk)/linker for activation of T cells (LAT) phosphorylation downstream of glycoprotein VI with similar potency to quercetin. All three flavonoids attenuated thrombus formation in an in vitro microfluidic model, and isoquercetin, a 3-O-glucoside of quercetin, inhibited thrombosis in a murine laser injury model. Isorhamnetin, tamarixetin, and quercetin enhanced the antiplatelet effects of aspirin more-than-additively in a plate-based aggregometry assay, reducing aspirin IC50 values by an order of magnitude, with this synergy maintained in a whole blood test of platelet function. Our data provide mechanistic evidence for the antiplatelet activity of two quercetin metabolites, isorhamnetin and tamarixetin, and suggest a potential antithrombotic role for these flavonoids. In combination with their interactions with aspirin, this may represent a novel avenue of investigation for the development of new antithrombotic strategies and management of current therapies

RXR ligands negatively regulate thrombosis and hemostasis

OBJECTIVE: Platelets have been found to express intracellular nuclear receptors including the Retinoid X receptors (RXRα and RXRβ). Treatment of platelets with ligands of RXR has been shown to inhibit platelet responses to ADP and thromboxane A2, however the effects on responses to other platelet agonists as well as the underlying mechanism has not been fully characterised. APPROACH AND RESULTS: The effect of 9-cis-retinoic acid (9-cis-RA), docosahexaenoic acid and synthetic ligand for RXR, methoprene acid on collagen receptor (GPVI) agonists and Thrombin stimulated platelet function; including aggregation, granule secretion, integrin activation, calcium mobilisation, integrin αIIbβ3 outside-in signalling and thrombus formation in vitro and in vivo were determined. Treatment of platelets with RXR ligands resulted in attenuation of platelet functional responses following stimulation by GPVI agonists and thrombin and inhibition of integrin αIIbβ3 outside-in signalling. Treatment with 9-cis-RA caused inhibition of thrombus formation in vitro and an impairment of thrombosis and haemostasis in vivo. Both RXR ligands stimulated protein kinase A activation, measured by VASP S157 phosphorylation, that was found to be dependent on both cAMP and NFκB activity. CONCLUSIONS: This study identifies a widespread, negative regulatory role for RXR in the regulation of platelet functional responses and thrombus formation and describes novel events that lead to the upregulation of PKA, a known negative regulator of many aspects of platelet function. This mechanism may offer a possible explanation for the cardioprotective effects described in vivo following treatment with RXR ligands

Novel anti-platelet properties of dietary cucurbitacins

Cucurbitacins are naturally occurring tetracyclic terpenes, present in foods such as cucumber and pumpkin, which elicit a range of anti-tumour, anti-inflammatory and anti-atherosclerotic effects. These dietary compounds modulate cellular functions through a variety of mechanisms, including dysregulation of the actin cytoskeleton and disruption of integrin function. Integrin outside-in signalling and cytoskeletal rearrangements are critical for stable thrombus formation and clot retraction following platelet adhesion at the site of vessel damage. We investigated the effects of cucurbitacins on platelet function and thrombus formation using human washed platelets, platelet rich plasma and whole blood in in vitro platelet function assays. We identified potent anti-platelet and anti-thrombotic effects of cucurbitacins B,E and I in human platelets. Treatment of platelets with cucurbitacins resulted in attenuation of platelet aggregation and fibrinogen binding evoked by ADP, TRAP6, collagen and CRP-XL. However, treatment with cucurbitacins did not significantly alter signalling events such as alpha granule secretion or mobilisation of intracellular calcium. We found that cucurbitacins potently inhibit integrin-mediated events, including adhesion and spreading on fibrinogen, fibronectin, collagen and laminin surfaces and cause a significant attenuation of clot retraction. Further investigation of cytoskeletal dynamics found treatment with cucurbitacins increased F actin polymerisation in a manner similar Jasplakinolide which has previously been shown to impair integrin activation, platelet spreading and clot retraction. The inhibitory effects of cucurbitacins on platelet integrin function and cytoskeletal dynamics resulted in the formation of highly unstable thrombi with reduced density under conditions of arterial shear. Our research identifies, anti-platelet and anti-thrombotic effects of dietary cucurbitacins that are linked to dysregulation of platelet cytoskeletal dynamics and integrin activity

Interspecies differences in protein expression do not impact the spatiotemporal regulation of glycoprotein VI mediated activation

Background Accurate protein quantification is a vital prerequisite for generating meaningful predictions when using systems biology approaches, a method that is increasingly being used to unravel the complexities of sub cellular interactions and as part of the drug discovery process. Quantitative proteomics, flow cytometry and western blotting have been extensively used to define human platelet protein copy numbers, yet for mouse platelets, a model widely used for platelet research, evidence is largely limited to a single proteomic dataset in which the total amount of proteins were generally comparatively higher than those found in human platelets. Objectives To investigate the functional implications of discrepancies between levels of mouse and human proteins in the GPVI signalling pathway using a systems pharmacology model of GPVI Methods The protein copy number of mouse platelet receptors was determined using flow cytometry. The Virtual Platelet, a mathematical model of Glycoprotein VI (GPVI) signalling, was used to determine the consequences of protein copy number differences observed between human and mouse platelets. Results and conclusion Despite the small size of mouse platelets compared to human platelets they possessed a greater density of surface receptors alongside a higher concentration of intracellular signalling proteins. Surprisingly the predicted temporal profile of Syk activity was similar in both species with predictions supported experimentally. Super resolution microscopy demonstrates that the spatial distribution of Syk is similar between species, suggesting that the spatial distribution of receptors and signalling molecules in activated platelets, rather than their copy number, is important for signalling pathway regulation

Platelet factor XIII-A regulates platelet function and promotes clot retraction and stability.

Factor XIII (FXIII) is an important proenzyme in the hemostatic system. The plasma-derived enzyme activated FXIII cross-links fibrin fibers within thrombi to increase their mechanical strength and cross-links fibrin to fibrinolytic inhibitors, specifically α2-antiplasmin, to increase resistance to fibrinolysis. We have previously shown that cellular FXIII (factor XIII-A [FXIII-A]), which is abundant in the platelet cytoplasm, is externalized onto the activated membrane and cross-links extracellular substrates. The contribution of cellular FXIII-A to platelet activation and platelet function has not been extensively studied. This study aims to identify the role of platelet FXIII-A in platelet function. We used normal healthy platelets with a cell permeable FXIII inhibitor and platelets from FXIII-deficient patients as a FXIII-free platelet model in a range of platelet function and clotting tests. Our data demonstrate that platelet FXIII-A enhances fibrinogen binding to the platelet surface upon agonist stimulation and improves the binding of platelets to fibrinogen and aggregation under flow in a whole-blood thrombus formation assay. In the absence of FXIII-A, platelets show reduced sensitivity to agonist stimulation, including decreased P-selectin exposure and fibrinogen binding. We show that FXIII-A is involved in platelet spreading where a lack of FXIII-A reduces the ability of platelets to fully spread on fibrinogen and collagen. Our data demonstrate that platelet FXIII-A is important for clot retraction where clots formed in its absence retracted to a lesser extent. Overall, this study shows that platelet FXIII-A functions during thrombus formation by aiding platelet activation and thrombus retraction in addition to its antifibrinolytic roles