Noncanonical Sonic Hedgehog signaling amplifies platelet reactivity and thrombogenicity.

Sonic Hedgehog (Shh) is a morphogen in vertebrate embryos that is also associated with organ homeostasis in adults. We report here that human platelets, though enucleate, synthesize Shh from preexisting mRNAs upon agonist stimulation, and mobilize it for surface expression and release on extracellular vesicles, thus alluding to its putative role in platelet activation. Shh, in turn, induced a wave of noncanonical signaling in platelets leading to activation of small GTPase Ras homolog family member A and phosphorylation of myosin light chain in activated protein kinase-dependent manner. Remarkably, agonist-induced thrombogenic responses in platelets, which include platelet aggregation, granule secretion, and spreading on immobilized fibrinogen, were significantly attenuated by inhibition of Hedgehog signaling, thus, implicating inputs from Shh in potentiation of agonist-mediated platelet activation. In consistence, inhibition of the Shh pathway significantly impaired arterial thrombosis in mice. Taken together, the above observations strongly support a feed-forward loop of platelet stimulation triggered locally by Shh, similar to ADP and thromboxane A2, that contributes significantly to the stability of occlusive arterial thrombus and that can be investigated as a potential therapeutic target in thrombotic disorders.

Fibrinogen Mitigates Prion-Mediated Platelet Activation and Neuronal Cell Toxicity.

Prion peptide (PrP) misfolds to infectious scrapie isoform, the ß pleat-rich insoluble fibrils responsible for neurodegeneration and fatal conformational diseases in humans. The amino acid sequence 106-126 from prion proteins, PrP(106-126), is highly amyloidogenic and implicated in prion-induced pathologies. Here, we report a novel interaction between PrP(106-126) and the thrombogenic plasma protein fibrinogen that can lead to mitigation of prion-mediated pro-thrombotic responses in human platelets as well as significant decline in neuronal toxicity. Thus, prior exposure to fibrinogen-restrained PrP-induced rise in cytosolic calcium, calpain activation, and shedding of extracellular vesicles in platelets while it, too, averted cytotoxicity of neuronal cells triggered by prion peptide. Interestingly, PrP was found to accelerate fibrin-rich clot formation, which was resistant to plasmin-mediated fibrinolysis, consistent with enhanced thrombus stability provoked by PrP. We propose that PrP-fibrinogen interaction can be clinically exploited further for prevention and management of infectious prion related disorders. Small molecules or peptides mimicking PrP-binding sites on fibrinogen can potentially mitigate PrP-induced cellular toxicity while also preventing the negative impact of PrP on fibrin clot formation and lysis.

Volk eye check ocular measurement device for objective periorbital measurements: A prospective study.

BACKGROUND: Volk Eye Check (VEC) is a portable digital ocular measurement device that automatically captures clinical photographs with facial measurements in real time (Volk Optical Inc), claiming to reduce measurement errors. The accuracy and repeatability of this ocular measurement device in the periorbital region has not been reported. AIM: To report the reproducibility and repeatability of periocular biometric measurements using Volk Eye Check Ocular measurement device. METHODS: Prospective, single blind, comparative study. Two experts performed digital photography of 100 volunteers using the standard photography technique using Oculoplasty module of the Volk Eye Check ocular measurement device. Each expert photographed the volunteer twice, to obtain two sets of automated printouts of 13 periorbital biometrics that were measured automatically by the ocular measurement device. Bland Altman plot and multiple comparisons of means from linear mixed-effects model fit by REML using simultaneous contrasts with p values reported by Bonferroni method were used as statistical tests to analyze following parameters: MRD1 (margin reflex distance-1), MRD2 (margin reflex distance-2), PFH (palpebral fissure height), HVID (horizontal visible iris diameter), ALL (aperture length at lateral limbus), and ALM (aperture length at medial limbus). RESULTS: The mean inter-observer difference in measurement (mm) was as follows: MRD1 (0.04), MRD2 (0.02), HVID (0.01), PFH (0.03), ALL (0.05), ALM (0.08). The mean intra-observer difference in measurement (mm) was as follows: MRD1 (0.02), MRD2 (0.09), HVID (0.0), PFH (0.09), ALL (0.06), ALM (0.05). CONCLUSION: Periorbital biometric measurements using Volk Eye Check ocular measurement devices are highly reproducible and repeatable. The oculoplasty module of Volk Eye Check ocular measurement device can provide reliable periorbital measurements for routine clinical use and for objective clinical studies.

Lacrimal drainage system anomalies in microphthalmia anophthalmia coloboma complex.

Purpose: To study the prevalence and types of lacrimal drainage system (LDS) anomalies inchildren with congenital microphthalmia anophthalmia coloboma (MAC) complex.Methods: This is a prospective, consecutive, non-comparative, case series of LDS anomalies in 31 children presenting with MAC complex. Previously untreated children with MAC complex, enrolled for socket surface and volume expansion during the study period from January 2017 to April 2018 were included.Results: Thirty-one patients with MAC complex were evaluated for LDS anomalies. Incidence of LDS anomalies in children presenting with MAC complex was 68% (42 of 62 lacrimal systems evaluated). Mean age of the patients at the time of examination under anaesthesia was 27 ± 25 (median 15 months, range 3-108 months). Proximal bicanalicular block (BCB) was the commonest LDS anomaly, noted in 15 (48%) cases. In patients with unilateral ocular disease (n = 20), LDS was affected in 14 (70%) patients and in 9 (45%) patients ipsilateral side LDS was affected. In patients with bilateral ocular disease (n = 11), LDS was affected in 7 (64%) patients. Three patients with unilateral anophthalmos (25%) had an ipsilateral upper canalicular block and contralateral nasolacrimal duct obstruction, of which one patient had a single canalicular wall hypoplasia along with CNLDO. No patient had punctal anomalies.Conclusions: LDS anomalies are seen in nearly two-thirds (68%) of children with MAC complex. The lacrimal drainage system anomaly is not limited to the side of the MAC complex. Commonest LDS anomaly is proximal bicanalicular blocks while punctal anomalies are unusual.

Glutamate induces synthesis of thrombogenic peptides and extracellular vesicle release from human platelets.

Platelets are highly sensitive blood cells, which play central role in hemostasis and thrombosis. Platelet dense granules carry considerable amount of neurotransmitter glutamate that is exocytosed upon cell activation. As platelets also express glutamate receptors on their surface, it is pertinent to ask whether exposure to glutamate would affect their signalling within a growing thrombus. In this study we demonstrate that, glutamate per se induced synthesis of thrombogenic peptides, plasminogen activator inhibitor-1 and hypoxia-inducible factor-2α, from pre-existing mRNAs in enucleate platelets, stimulated cytosolic calcium entry, upregulated RhoA-ROCK-myosin light chain/myosin light chain phosphatase axis, and elicited extensive shedding of extracellular vesicles from platelets. Glutamate, too, incited platelet spreading and adhesion on to immobilized matrix under arterial shear, raised mitochondrial transmembrane potential associated with generation of reactive oxygen species and induced activation of AMP-activated protein kinase in platelets. Taken together, glutamate switches human platelets to pro-activation phenotype mediated mostly through AMPA receptors and thus targeting glutamate receptors may be a promising anti-platelet strategy.

Aerobic glycolysis fuels platelet activation: small-molecule modulators of platelet metabolism as anti-thrombotic agents.

Platelets are critical to arterial thrombosis, which underlies myocardial infarction and stroke. Activated platelets, regardless of the nature of their stimulus, initiate energy-intensive processes that sustain thrombus, while adapting to potential adversities of hypoxia and nutrient deprivation within the densely packed thrombotic milieu. We report here that stimulated platelets switch their energy metabolism to aerobic glycolysis by modulating enzymes at key checkpoints in glucose metabolism. We found that aerobic glycolysis, in turn, accelerates flux through the pentose phosphate pathway and supports platelet activation. Hence, reversing metabolic adaptations of platelets could be an effective alternative to conventional anti-platelet approaches, which are crippled by remarkable redundancy in platelet agonists and ensuing signaling pathways. In support of this hypothesis, small-molecule modulators of pyruvate dehydrogenase, pyruvate kinase M2 and glucose-6-phosphate dehydrogenase, all of which impede aerobic glycolysis and/or the pentose phosphate pathway, restrained the agonist-induced platelet responses ex vivo These drugs, which include the anti-neoplastic candidate, dichloroacetate, and the Food and Drug Administration-approved dehydroepiandrosterone, profoundly impaired thrombosis in mice, thereby exhibiting potential as anti-thrombotic agents.