Non-canonical non-genomic morphogen signaling in anucleate platelets: a critical determinant of prothrombotic function in circulation.

Circulating platelets derived from bone marrow megakaryocytes play a central role in thrombosis and hemostasis. Despite being anucleate, platelets express several proteins known to have nuclear niche. These include transcription factors and steroid receptors whose non-genomic functions are being elucidated in platelets. Quite remarkably, components of some of the best-studied morphogen pathways, namely Notch, Sonic Hedgehog (Shh), and Wnt have also been described in recent years in platelets, which regulate platelet function in the context of thrombosis as well as influence their survival. Shh and Notch pathways in stimulated platelets establish feed-forward loops of autocrine/juxtacrine/paracrine non-canonical signaling that helps perpetuate thrombosis. On the other hand, non-canonical Wnt signaling is part of a negative feedback loop for restricting platelet activation and possibly limiting thrombus growth. The present review will provide an overview of these signaling pathways in general. We will then briefly discuss the non-genomic roles of transcription factors and steroid receptors in platelet activation. This will be followed by an elaborate description of morphogen signaling in platelets with a focus on their bearing on platelet activation leading to hemostasis and thrombosis as well as their potential for therapeutic targeting in thrombotic disorders.

Ayurvedic preparations of Raudra Rasa inhibit agonist-mediated platelet activation and restrict thrombogenicity without affecting cell viability.

Ayurveda is considered to be one of the most ancient forms of medicine still practiced. The Ayurvedic preparation Raudra Rasa and its derivatives have been widely employed against cancer since the 12th century, but the effect of these traditional formulations on platelet function and signaling has not previously been examined. Here we demonstrate that Raudra Rasa and its derivatives significantly reduce thrombin-induced integrin activation and granule secretion in platelets, as observed by reduced PAC-1 binding and P-selectin externalization, respectively. These formulations also inhibited thrombin-stimulated phosphatidylserine exposure, mitochondrial reactive oxygen species generation, and mitochondrial transmembrane potential in platelets. Consistent with the above, Raudra Rasa significantly reduced thrombin-induced tyrosine phosphorylation of the platelet proteins, as well as phosphorylation of the enzymes AKT and GSK-3ß. In summary, Raudra Rasa inhibits agonist-mediated platelet activation without affecting cell viability, suggesting it may have therapeutic potential as an anti-platelet/anti-thrombotic agent.

Necroptosis executioner MLKL plays pivotal roles in agonist-induced platelet prothrombotic responses and lytic cell death in a temporal order.

Necroptosis is a form of programmed cell death executed by receptor-interacting serine/threonine protein kinase 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like (MLKL). Platelets are circulating cells that play central roles in haemostasis and pathological thrombosis. In this study we demonstrate seminal contribution of MLKL in transformation of agonist-stimulated platelets to active haemostatic units progressing eventually to necrotic death on a temporal scale, thus attributing a yet unrecognized fundamental role to MLKL in platelet biology. Physiological agonists like thrombin instigated phosphorylation and subsequent oligomerization of MLKL in platelets in a RIPK3-independent but phosphoinositide 3-kinase (PI3K)/AKT-dependent manner. Inhibition of MLKL significantly curbed agonist-induced haemostatic responses in platelets that included platelet aggregation, integrin activation, granule secretion, procoagulant surface generation, rise in intracellular calcium, shedding of extracellular vesicles, platelet-leukocyte interactions and thrombus formation under arterial shear. MLKL inhibition, too, prompted impairment in mitochondrial oxidative phosphorylation and aerobic glycolysis in stimulated platelets, accompanied with disruption in mitochondrial transmembrane potential, augmented proton leak and drop in both mitochondrial calcium as well as ROS. These findings underscore the key role of MLKL in sustaining OXPHOS and aerobic glycolysis that underlie energy-intensive platelet activation responses. Prolonged exposure to thrombin provoked oligomerization and translocation of MLKL to plasma membranes forming focal clusters that led to progressive membrane permeabilization and decline in platelet viability, which was prevented by inhibitors of PI3K/MLKL. In summary, MLKL plays vital role in transitioning of stimulated platelets from relatively quiescent cells to functionally/metabolically active prothrombotic units and their ensuing progression to necroptotic death.

Platelet function suggests cardioembolic aetiology in cryptogenic stroke.

Platelet-monocyte (PMA) and platelet-neutrophil aggregations (PNA) play critical roles in the evolution of acute ischemic stroke (AIS). The present study investigates the mechanistic basis of platelet responsiveness in cryptogenic stroke compared with cardioembolic stroke. Platelet from 16 subjects, each from cryptogenic and cardioembolic stroke groups and 18 age-matched healthy controls were subjected to different investigations. Compared to healthy controls, platelet-monocyte and platelet-neutrophil interactions were significantly elevated in cryptogenic (2.7 and 2.1 times) and cardioembolic stroke (3.9 and 2.4 times). P-selectin expression on platelet surface was 1.89 and 2.59 times higher in cryptogenic and cardioembolic strokes, respectively, compared to healthy control. Cell population with [Ca2+i] in either stroke group was significantly outnumbered (by 83% and 72%, respectively, in cryptogenic and cardioembolic stroke) in comparison to healthy controls. Noteworthy, TEG experiment revealed that the cryptogenic stroke exhibited significant decline in Reaction Time (R) and amplitude of 20 mm (K) (by 32% and 33%, respectively) while thrombin burst (α-angle) was augmented by 12%, which reflected substantial boost in thrombus formation in cryptogenic stroke. Although TEG analysis reveals a state of hypercoagulability in patients with cryptogenic stroke. However, platelets from both stroke subtypes switch to a 'hyperactive' phenotype.

Energy metabolism in platelets fuels thrombus formation: Halting the thrombosis engine with small-molecule modulators of platelet metabolism.

Platelets are circulating cells central to haemostasis that follows vessel injury, as well as thrombosis that ensues as a consequence of pathological stasis or plaque rupture. Platelet responses to various stimuli that mediate these processes are all energy-intensive. Hence, platelets need to adapt their energy metabolism to fulfil the requirements of clot formation while overcoming the adversities of the thrombus niche such as restricted access to oxygen and nutrient. In the present review, we describe the changes in energy metabolism of platelets upon agonist challenge and their underlying molecular mechanisms. We briefly discuss the metabolic flexibility and dependency of stimulated platelets in terms of choice of energy substrates. Finally, we discuss how targeting the metabolic vulnerabilities of stimulated platelets such as aerobic glycolysis and/or beta oxidation of fatty acids could forestall platelet activation and thrombus formation. Thus, we present a case for modulating platelet energy metabolism using small-molecules as a novel anti-platelet strategy in the management of vaso-occlusive disorders like acute myocardial infarction, ischemic stroke, deep vein thrombosis and pulmonary embolism.

Fatty acid oxidation fuels agonist-induced platelet activation and thrombus formation: Targeting ß-oxidation of fatty acids as an effective anti-platelet strategy.

Platelet mitochondria possess remarkable plasticity for oxidation of energy substrates, where metabolic dependency on glucose or fatty acids is higher than glutamine. Since platelets metabolize nearly the entire pool of glucose to lactate rather than fluxing through mitochondrial tricarboxylic acid cycle, we posit that majority of mitochondrial ATP, which is essential for platelet granule secretion and thrombus formation, is sourced from oxidation of fatty acids. We performed a comprehensive analysis of bioenergetics and function of stimulated platelets in the presence of etomoxir, trimetazidine and oxfenicine, three pharmacologically distinct inhibitors of ß-oxidation. Each of them significantly impaired oxidative phosphorylation in unstimulated as well as thrombin-stimulated platelets leading to a small but consistent drop in ATP level in activated cells due to a lack of compensation from glycolytic ATP. Trimetazidine and oxfenicine attenuated platelet aggregation, P-selectin externalization and integrin αIIb ß3 activation. Both etomoxir and trimetazidine impeded agonist-induced dense granule release and platelet thrombus formation on collagen under arterial shear. The effect of inhibitors on platelet aggregation and dense granule release was dose- and incubation time- dependent with significant inhibition at higher doses and prolonged incubation times. Neither of the inhibitors could protect mice from collagen-epinephrine-induced pulmonary embolism or prolong mouse tail bleeding times. However, mice pre-administered with etomoxir, trimetazidine and oxfenicine were protected from ferric chloride-induced mesenteric thrombosis. In conclusion, ß-oxidation of fatty acids sustains ATP level in stimulated platelets and is therefore essential for energy-intensive agonist-induced platelet responses. Thus, fatty acid oxidation may constitute an attractive therapeutic target for novel antiplatelet agents.

Notch signaling functions in noncanonical juxtacrine manner in platelets to amplify thrombogenicity.

Background: Notch signaling dictates cell fate decisions in mammalian cells including megakaryocytes. Existence of functional Notch signaling in enucleate platelets remains elusive. Methods: Transcripts/peptides of Notch1 and Delta-like ligand (DLL)-4 were detected in platelets isolated from human blood by RT-qPCR, Western analysis and flow cytometry. Platelet aggregation, granule secretion and platelet-leukocyte interaction were analyzed by lumi-aggregometry and flow cytometry. Platelet-derived extracellular vesicles were documented with Nanoparticle Tracking Analyzer. Platelet thrombus on immobilized collagen was quantified using microfluidics platform. Intracellular calcium was monitored by fluorescence spectrophotometry. Whole blood coagulation was studied by thromboelastography. Ferric chloride-induced mouse mesenteric arteriolar thrombosis was imaged by intravital microscopy. Results: We demonstrate expression of Notch1, its ligand DLL-4 and their respective transcripts in human platelets. Synthesis and surface translocation of Notch1 and DLL-4 were upregulated by thrombin. DLL-4, in turn, instigated neighbouring platelets to switch to 'activated' phenotype through cleavage of Notch receptor and release of its intracellular domain (NICD), which was averted by inhibition of γ-secretase and phosphatidylinositol-3-kinase (PI3K). Inhibition of Notch signaling, too, restrained agonist-induced platelet activation, and significantly impaired arterial thrombosis in mice. Strikingly, prevention of DLL-4-Notch1 interaction by a blocking antibody abolished platelet aggregation and extracellular vesicle shedding induced by thrombin. Conclusions: Our study presents compelling evidence in support of non-canonical juxtacrine Notch signaling within platelet aggregates that synergizes with physiological agonists to generate occlusive intramural thrombi. Thus, Notch pathway can be a potential anti-platelet/anti-thrombotic therapeutic target. Funding: Research was supported by grants received by DD from JC Bose Fellowship (JCB/2017/000029), ICMR (71/4/2018-BMS/CAR), DBT (BT/PR-20645/BRB/10/1541/2016) and SERB (EMR/2015/000583). SNC, ME and VS are recipients of ICMR-Scientist-C, CSIR-SRF and UGC-SRF support, respectively. Funders had no role in design, analysis and reporting of study.

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.