Membrane Ballooning in Aggregated Platelets is Synchronised and Mediates a Surge in Microvesiculation:Synchronised ballooning and microvesiculation

AbstractHuman platelet transformation into balloons is part of the haemostatic response and thrombus architecture. Here we reveal that in aggregates of platelets in plasma, ballooning in multiple platelets occurs in a synchronised manner. This suggests a mechanism of coordination between cells, previously unrecognised. We aimed to understand this mechanism, and how it may contribute to thrombus development. Using spinning-disc confocal microscopy we visualised membrane ballooning in human platelet aggregates adherent to collagen-coated surfaces. Within an aggregate, multiple platelets undergo ballooning in a synchronised fashion, dependent upon extracellular calcium, in a manner that followed peak cytosolic calcium levels in the aggregate. Synchrony was observed in platelets within but not between aggregates, suggesting a level of intra-thrombus communication. Blocking phosphatidylserine, inhibiting thrombin or blocking PAR1 receptor, largely prevented synchrony without blocking ballooning itself. In contrast, inhibition of connexins, P2Y12, P2Y1 or thromboxane formation had no effect on synchrony or ballooning. Importantly, synchronised ballooning was closely followed by a surge in microvesicle formation, which was absent when synchrony was blocked. Our data demonstrate that the mechanism underlying synchronised membrane ballooning requires thrombin generation acting effectively in a positive feedback loop, mediating a subsequent surge in procoagulant activity and microvesicle release.</jats:p

Glycoprotein Ib-V-IX, a Receptor for von Willebrand Factor, Couples Physically and Functionally to the Fc Receptor y-Chain, Fyn, and Lyn to Activate Human Platelets

The adhesion molecule von Willebrand factor (vWF) activates platelets upon binding 2 surface receptors, glycoprotein (GP) Ib-V-IX and integrin aIIbb3. We have used 2 approaches to selectively activate GP Ib using either the snake venom lectin alboaggregin-A or mutant recombinant forms of vWF (DA1-vWF and RGGS-vWF) with selective binding properties to its 2 receptors. We show that activation of GP Ib induces platelet aggregation, secretion of 5-hydroxy tryptamine (5-HT), and an increase in cytosolic calcium. Syk becomes tyrosine phosphorylated and activated downstream of GP Ib, and associates with several tyrosinephosphorylated proteins including the Fc receptor g-chain through interaction with Syk SH2 domains. GP Ib physically associates with the g-chain in GST-Syk-SH2 precipitates from platelets stimulated through GP Ib, and 2 Src family kinases, Lyn and Fyn, also associate with this signaling complex. In addition, GP Ib stimulation couples to tyrosine phosphorylation of phospholipase Cg2. The Src familyspecific inhibitor PP1 dose-dependently inhibits phosphorylation of Syk, its association with tyrosine-phosphorylated g-chain, phosphorylation of PLCg2, platelet aggregation, and 5-HT release. The results indicate that, upon activation, GP Ib is physically associated with FcR g-chain and members of the Src family kinases, leading to phosphorylation of the g-chain, recruitment, and activation of Syk. Phosphorylation of PLCg2 also lies downstream of Src kinase activation and may critically couple early signaling events to functional platelet responses

Platelets protect cardiomyocytes from ischaemic damage

AbstractPlatelets are classically known for their roles in bleeding control and occlusive thrombus formation causing ischemic tissue damage. Recently, nonclassical roles for platelets have been described, many of which may be mediated by the heterogeneous cargo that platelets secrete from granular stores upon activation. Using an in vitro model of ischemic injury to ventricular cardiomyocytes, we observed that platelets, through secreted factors, delayed the rate of cardiomyocyte death during ischemia. This protective effect appeared independent of platelet dense granule cargo, but required α-granule components stromal cell-derived factor-1α and transforming growth factor-β1. Protein kinase C activity within cardiomyocytes was responsible for mediating the protective signals initiated by the released platelet cargo. Importantly, pretreating platelets with a P2Y12 antagonist, but not the cyclooxygenase inhibitor aspirin, substantially attenuated this protective effect. These findings therefore reveal a paradoxically protective role for platelet activation during cardiac ischemia and could have important implications for the use of antiplatelet therapeutics in the management of myocardial infarction.</jats:p

Do platelets promote cardiac recovery after myocardial infarction:Roles beyond occlusive ischemic damage

Our understanding of platelet function has traditionally focused on their roles in physiological hemostasis and pathological thrombosis, with the latter being causative of vessel occlusion and subsequent ischemic damage to various tissues. In particular, numerous in vivo studies have implicated causative roles for platelets in the pathogenesis of ischemia-reperfusion (I/R) injury to the myocardium. However, platelets clearly have more complex pathophysiological roles, particularly as a result of the heterogeneous nature of biologically active cargo secreted from their granules or contained within released microparticles or exosomes. While some of these released mediators amplify platelet activation and thrombosis through autocrine or paracrine amplification pathways, they can also regulate diverse cellular functions within the localized microenvironment and recruit progenitor cells to the damage site to facilitate repair processes. Notably, there is evidence to support cardioprotective roles for platelet mediators during I/R injury. As such, it is becoming more widely appreciated that platelets fulfill a host of physiological and pathological roles beyond our basic understanding. Therefore, the purpose of this perspective is to consider whether platelets, through their released mediators, can assume a paradoxically beneficial role to promote cardiac recovery after I/R injury. </jats:p

Absence of platelet phenotype in mice lacking the motor protein myosin Va.

BACKGROUND: The motor protein myosin Va plays an important role in the trafficking of intracellular vesicles. Mutation of the Myo5a gene causes Griscelli syndrome type 1 in humans and the dilute phenotype in mice, which are both characterised by pigment dilution and neurological defects as a result of impaired vesicle transport in melanocytes and neuroendocrine cells. The role of myosin Va in platelets is currently unknown. Rab27 has been shown to be associated with myosin Va cargo vesicles and is known to be important in platelet dense granule biogenesis and secretion, a crucial event in thrombus formation. Therefore, we hypothesised that myosin Va may regulate granule secretion or formation in platelets. METHODOLOGY/PRINCIPAL FINDINGS: Platelet function was studied in vitro using a novel Myo5a gene deletion mouse model. Myo5a(-/-) platelets were devoid of myosin Va, as determined by immunoblotting, and exhibited normal expression of surface markers. We assessed dense granule, α-granule and lysosomal secretion, integrin α(IIb)β(3) activation, Ca(2+) signalling, and spreading on fibrinogen in response to collagen-related peptide or the PAR4 agonist, AYPGKF in washed mouse platelets lacking myosin Va or wild-type platelets. Surprisingly, Myo5a(-/-) platelets showed no significant functional defects in these responses, or in the numbers of dense and α-granules expressed. CONCLUSION: Despite the importance of myosin Va in vesicle transport in other cells, our data demonstrate this motor protein has no non-redundant role in the secretion of dense and α-granules or other functional responses in platelets

A novel complete-surface-finding-algorithm for online surface scanning with limited view sensors

Robotised Non-Destructive Testing (NDT) has revolutionised the field, increasing the speed of repetitive scanning procedures and ability to reach hazardous environments. Application of robot-assisted NDT within specific industries such as remanufacturing and Aersopace, in which parts are regularly moulded and susceptible to non-critical deformation has however presented drawbacks. In these cases, digital models for robotic path planning are not always available or accurate. Cutting edge methods to counter the limited flexibility of robots require an initial pre-scan using camera-based systems in order to build a CAD model for path planning. This paper has sought to create a novel algorithm that enables robot-assisted ultrasonic testing of unknown surfaces within a single pass. Key to the impact of this article is the enabled autonomous profiling with sensors whose aperture is several orders of magnitude smaller than the target surface, for surfaces of any scale. Potential applications of the algorithm presented include autonomous drone and crawler inspections of large, complex, unknown environments in addition to situations where traditional metrological profiling equipment is not practical, such as in confined spaces. In simulation, the proposed algorithm has completely mapped significantly curved and complex shapes by utilising only local information, outputting a traditional raster pattern when curvature is present only in a single direction. In practical demonstrations, both curved and non-simple surfaces were fully mapped with no required operator intervention. The core limitations of the algorithm in practical cases is the effective range of the applied sensor, and as a stand-alone method it lacks the required knowledge of the environment to prevent collisions. However, since the approach has met success in fully scanning non-obstructive but still significantly complex surfaces, the objectives of this paper have been met. Future work will focus on low-accuracy environmental sensing capabilities to tackle the challenges faced. The method has been designed to allow single-pass scans for Conformable Wedge Probe UT scanning, but may be applied to any surface scans in the case the sensor aperture is significantly smaller than the part