Transoral Robotic Surgery for a Rare Case of Venolymphatic Malformation in Parapharyngeal Space.

Venous malformations are low flow endothelial malformations with aberrant and ectatic venous channels. They are defects in vascular growth which causes functional and cosmetic impairment. Gradual growth in size of the lesion occurs due to venous congestion or thrombosis. Venous malformations in parapharyngeal space are a rare entity and are difficult to diagnose. Case Report. 13 year old boy presented with a history of hyposmia and progressive difficulty in breathing for a duration of 2.5 years. MRI face and neck with contrast showed a 4.5 × 4.3x3.6 cm lesion in the left parapharyngeal space. CT angiogram of brain and neck demonstrated a heterogeneously enhancing mass in the left parapharyngeal region. PET scan illustrated an ill-defined mass in the left pre styloid parapharyngeal space. Biopsy from the lesion showed features consistent with venolymphatic malformation. Flexible laryngoscopy showed a bulge over the left soft palate region with narrowing of nasopharyngeal lumen. Patient underwent transoral robotic surgery for complete excision of the mass. Post-operative period was uneventful. He has been on follow up for the past 1 year with no evidence of any residual or recurrent disease. Venolymphatic malformation is a rare lesion in the parapharyngeal space which is difficult to diagnose pre operatively. Surgical excision is the preferred modality of treatment for deep seated lesions in the parapharyngeal space. The advent of transoral robotic surgery have reduced the morbidity and improved clearance for such cases.

Lipid remodeling in megakaryocyte differentiation and platelet biogenesis.

Lipid remodeling, from fatty acid transport and de novo lipid synthesis, is necessary for megakaryocyte differentiation and platelet production. Dietary saturated fatty acids, impaired fatty acid transport and/or dysfunction in lipid biogenesis can contribute to low platelet counts.

Platelet-derived TLT-1 promotes tumor progression by suppressing CD8+ T cells.

Current understanding of tumor immunosuppressive mechanisms forms the basis for modern day immunotherapies. Immunoregulatory role of platelets in cancer remains largely elusive. Platelets from non-small cell lung cancer (NSCLC) patients revealed a distinct activation phenotype. TREM-like transcript 1 (TLT-1), a platelet protein, was increased along with enhanced extracellular release from NSCLC platelets. The increased platelet TLT-1 was also evident in humanized mice with patient-derived tumors. In immunocompetent mice with syngeneic tumors, TLT-1 binding to T cells, in vivo, led to suppression of CD8 T cells, promoting tumor growth. We identified direct interaction between TLT-1 and CD3ε on T cells, implicating the NF-κB pathway in CD8 T cell suppression. Anti-TLT-1 antibody rescued patients' T cells from platelet-induced suppression ex vivo and reduced tumors in mice in vivo. Clinically, higher TLT-1 correlated with reduced survival of NSCLC patients. Our findings thus identify TLT-1 as a platelet-derived immunosuppressor that suppresses CD8 T cells and demonstrate its therapeutic and prognostic significance in cancer.

Platelet in thrombo-inflammation: Unraveling new therapeutic targets.

In the broad range of human diseases, thrombo-inflammation appears as a clinical manifestation. Clinically, it is well characterized in context of superficial thrombophlebitis that is recognized as thrombosis and inflammation of superficial veins. However, it is more hazardous when developed in the microvasculature of injured/inflamed/infected tissues and organs. Several diseases like sepsis and ischemia-reperfusion can cause formation of microvascular thrombosis subsequently leading to thrombo-inflammation. Thrombo-inflammation can also occur in cases of antiphospholipid syndrome, preeclampsia, sickle cell disease, bacterial and viral infection. One of the major contributors to thrombo-inflammation is the loss of normal anti-thrombotic and anti-inflammatory potential of the endothelial cells of vasculature. This manifest itself in the form of dysregulation of the coagulation pathway and complement system, pathologic platelet activation, and increased recruitment of leukocyte within the microvasculature. The role of platelets in hemostasis and formation of thrombi under pathologic and non-pathologic conditions is well established. Platelets are anucleate cells known for their essential role in primary hemostasis and the coagulation pathway. In recent years, studies provide strong evidence for the critical involvement of platelets in inflammatory processes like acute ischemic stroke, and viral infections like Coronavirus disease 2019 (COVID-19). This has encouraged the researchers to investigate the contribution of platelets in the pathology of various thrombo-inflammatory diseases. The inhibition of platelet surface receptors or their intracellular signaling which mediate initial platelet activation and adhesion might prove to be suitable targets in thrombo-inflammatory disorders. Thus, the present review summarizes the concept and mechanism of platelet signaling and briefly discuss their role in sterile and non-sterile thrombo-inflammation, with the emphasis on role of platelets in COVID-19 induced thrombo-inflammation. The aim of this review is to summarize the recent developments in deciphering the role of the platelets in thrombo-inflammation and discuss their potential as pharmaceutical targets.

Unfolded Protein Response Differentially Modulates the Platelet Phenotype.

BACKGROUND: Unfolded protein response (UPR) is a multifaceted signaling cascade that alleviates protein misfolding. Although well studied in nucleated cells, UPR in absence of transcriptional regulation has not been described. Intricately associated with cardiovascular diseases, platelets, despite being anucleate, respond rapidly to stressors in blood. We investigate the UPR in anucleate platelets and explore its role, if any, on platelet physiology and function. METHODS: Human and mouse platelets were studied using a combination of ex vivo and in vivo experiments. Platelet lineage-specific knockout mice were generated independently for each of the 3 UPR pathways, PERK (protein kinase RNA [PKR]-like endoplasmic reticulum kinase), XBP1 (X-binding protein), and ATF6 (activating transcription factor 6). Diabetes patients were prospectively recruited, and platelets were evaluated for activation of UPR under chronic pathophysiological disease conditions. RESULTS: Tunicamycin induced the IRE1α (inositol-requiring enzyme-1alpha)-XBP1 pathway in human and mouse platelets, while oxidative stress predominantly activated the PERK pathway. PERK deletion significantly increased platelet aggregation and apoptosis and phosphorylation of PLCγ2, PLCß3, and p38 MAPK. Deficiency of XBP1 increased platelet aggregation, with higher PLCß3 and PKCδ activation. ATF6 deletion mediated a relatively modest effect on platelet phenotype with increased PKA (protein kinase A). Platelets from diabetes patients exhibited a positive correlation between disease severity, platelet activation, and protein aggregation, with only IRE1α-XBP1 activation. Moreover, IRE1α inhibition increased platelet aggregation, while clinically approved chemical chaperone, sodium 4-phenylbutyrate reduced the platelet hyperactivation. CONCLUSIONS: We show for the first time, that UPR activation occurs in platelets and can be independent of genomic regulation, with selective induction being specific to the source and severity of stress. Each UPR pathway plays a key role and can differentially modulate the platelet activation pathways and phenotype. Targeting the specific arms of UPR may provide a new antiplatelet strategy to mitigate thrombotic risk in diabetes and other cardiovascular diseases.

A guide to molecular and functional investigations of platelets to bridge basic and clinical sciences.

Platelets have been shown to be associated with pathophysiological process beyond thrombosis, demonstrating critical additional roles in homeostatic processes, such as immune regulation, and vascular remodeling. Platelets themselves can have multiple functional states and can communicate and regulate other cells including immune cells and vascular smooth muscle cells, to serve such diverse functions. Although traditional platelet functional assays are informative and reliable, they are limited in their ability to unravel platelet phenotypic heterogeneity and interactions. Developments in methods such as electron microscopy, flow cytometry, mass spectrometry, and 'omics' studies, have led to new insights. In this Review, we focus on advances in platelet biology and function, with an emphasis on current and promising methodologies. We also discuss technical and biological challenges in platelet investigations. Using coronavirus disease 2019 (COVID-19) as an example, we further describe the translational relevance of these approaches and the possible 'bench-to-bedside' utility in patient diagnosis and care.

Thrombocytopathy and endotheliopathy: crucial contributors to COVID-19 thromboinflammation.

The core pathology of coronavirus disease 2019 (COVID-19) is infection of airway cells by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that results in excessive inflammation and respiratory disease, with cytokine storm and acute respiratory distress syndrome implicated in the most severe cases. Thrombotic complications are a major cause of morbidity and mortality in patients with COVID-19. Patients with pre-existing cardiovascular disease and/or traditional cardiovascular risk factors, including obesity, diabetes mellitus, hypertension and advanced age, are at the highest risk of death from COVID-19. In this Review, we summarize new lines of evidence that point to both platelet and endothelial dysfunction as essential components of COVID-19 pathology and describe the mechanisms that might account for the contribution of cardiovascular risk factors to the most severe outcomes in COVID-19. We highlight the distinct contributions of coagulopathy, thrombocytopathy and endotheliopathy to the pathogenesis of COVID-19 and discuss potential therapeutic strategies in the management of patients with COVD-19. Harnessing the expertise of the biomedical and clinical communities is imperative to expand the available therapeutics beyond anticoagulants and to target both thrombocytopathy and endotheliopathy. Only with such collaborative efforts can we better prepare for further waves and for future coronavirus-related pandemics.

Role of Platelet Mitochondria: Life in a Nucleus-Free Zone.

Platelets are abundant, small, anucleate circulating cells, serving many emerging pathophysiological roles beyond hemostasis; including active critical roles in thrombosis, injury response, and immunoregulation. In the absence of genomic DNA transcriptional regulation (no nucleus), platelets require strategic prepackaging of all the needed RNA and organelles from megakaryocytes, to sense stress (e.g., hyperglycemia), to protect themselves from stress (e.g., mitophagy), and to communicate a stress response to other cells (e.g., granule and microparticle release). Distinct from avian thrombocytes that have a nucleus, the absence of a nucleus allows the mammalian platelet to maintain its small size, permits morphological flexibility, and may improve speed and efficiency of protein expression in response to stress. In the absence of a nucleus, platelet lifespan of 7-10 days, is largely determined by the mitochondria. The packaging of 5-8 mitochondria is critical in aerobic respiration and yielding metabolic substrates needed for function and survival. Mitochondria damage or dysfunction, as observed with several disease processes, results in greatly attenuated platelet survival and increased risk for thrombovascular events. Here we provide insights into the emerging roles of platelets despite the lack of a nucleus, and the key role played by mitochondria in platelet function and survival both in health and disease.

Venous thrombosis at altitude presents with distinct biochemical profiles: a comparative study from the Himalayas to the plains.

High-altitude (HA) hypoxia exposure is believed to induce venous thromboembolism (VTE) in otherwise healthy individuals, although this needs to be fully established. The present study aims to ascertain the role of HA exposure in aggravating any predisposition toward VTE and to explore whether the etiology of HA-induced VTE is different from that of VTE closer to sea level. We compared manifestation-matched male VTE patients from HA (HAPs) and VTE patients from the plains closer to sea level (SLPs) for 54 parameters, including coagulation-related, fibrinolytic, and thrombophilic variables, as well as markers for stress and inflammatory response and platelet and endothelial activation. Our results established an association between HA hypoxia and VTE in alterations of primarily hemostatic variables. Approximately 96% of HAPs presented with ≥10 altered parameters out of 54 studied compared with 7% of SLPs. Elevated platelet count, von Willebrand factor, and clotting factors and altered coagulation exhibited significant associations with VTE events and altitude exposure (all P < .05). Additionally, most VTEs at HA were associated with younger age groups, unlike those on the plains. A receiver operator characteristic curve analysis revealed differences between HAPs and SLPs for CD40 ligand (area under the curve [AUC], 0.90; 95% confidence interval [CI], 0.84-0.96]), P-selectin (0.79; 0.70-0.88), platelet factor-4 (0.90; 0.84-0.96), intracellular adhesion molecule-1 (0.86; 0.79-0.93), vascular cell adhesion molecule-1 (0.97; 0.95-0.99), vascular endothelial growth factor (0.87; 0.8-0.94), FLT4 (0.94; 0.89-0.99), and Toll-like receptor-2 (0.98; 0.96-1.0) (all P < .05). In conclusion, this study suggests that HA exposure perturbs the molecules associated with vascular integrity and contributes to the early onset of VTE.

Mitochondrial MsrB2 serves as a switch and transducer for mitophagy.

Mitophagy can selectively remove damaged toxic mitochondria, protecting a cell from apoptosis. The molecular spatial-temporal mechanisms governing autophagosomal selection of reactive oxygen species (ROS)-damaged mitochondria, particularly in a platelet (no genomic DNA for transcriptional regulation), remain unclear. We now report that the mitochondrial matrix protein MsrB2 plays an important role in switching on mitophagy by reducing Parkin methionine oxidation (MetO), and transducing mitophagy through ubiquitination by Parkin and interacting with LC3. This biochemical signaling only occurs at damaged mitochondria where MsrB2 is released from the mitochondrial matrix. MsrB2 platelet-specific knockout and in vivo peptide inhibition of the MsrB2/LC3 interaction lead to reduced mitophagy and increased platelet apoptosis. Pathophysiological importance is highlighted in human subjects, where increased MsrB2 expression in diabetes mellitus leads to increased platelet mitophagy, and in platelets from Parkinson's disease patients, where reduced MsrB2 expression is associated with reduced mitophagy. Moreover, Parkin mutations at Met192 are associated with Parkinson's disease, highlighting the structural sensitivity at the Met192 position. Release of the enzyme MsrB2 from damaged mitochondria, initiating autophagosome formation, represents a novel regulatory mechanism for oxidative stress-induced mitophagy.

CELA2A mutations predispose to early-onset atherosclerosis and metabolic syndrome and affect plasma insulin and platelet activation.

Factors that underlie the clustering of metabolic syndrome traits are not fully known. We performed whole-exome sequence analysis in kindreds with extreme phenotypes of early-onset atherosclerosis and metabolic syndrome, and identified novel loss-of-function mutations in the gene encoding the pancreatic elastase chymotrypsin-like elastase family member 2A (CELA2A). We further show that CELA2A is a circulating enzyme that reduces platelet hyperactivation, triggers both insulin secretion and degradation, and increases insulin sensitivity. CELA2A plasma levels rise postprandially and parallel insulin levels in humans. Loss of these functions by the mutant proteins provides insight into disease mechanisms and suggests that CELA2A could be an attractive therapeutic target.

Age associated non-linear regulation of redox homeostasis in the anucleate platelet: Implications for CVD risk patients.

BACKGROUND: Aging is a complex physiological phenomenon, intricately associated with cardiovascular pathologies, where platelets play a central pathophysiological role. Although antiplatelets are commonly employed to prevent and treat major adverse cardiovascular events, aging associated intraplatelet changes remain largely unexplored. METHODS: Platelets were studied in high cardiovascular risk patients (aged 40-100 years) comparing them to younger healthy subjects. This was followed by cross sectional and longitudinal mice studies. Flow cytometry, biochemical and molecular assays were used to study platelets comprehensively. FINDINGS: CVD Patients were categorized in the age groups 40-59, 60-79, and 80-100 years. Progressive decline in platelet health was observed in the 40-79 years age cohort, marked by increase in oxidative stress, hyperactivation and apoptotic markers. Paradoxically, this was reversed in patients aged above 79 years and the improved platelet phenotype was associated with lower oxidative damage. The platelets from the very old (80-100 year) group were found to be preloaded with increased antioxidants, which also contributed to higher resistance against induced redox insults. Cross sectional mouse studies excluded the effect of comorbidities and medications. Longitudinal mouse studies implicate an adaptive increase in antioxidant levels as the mechanism. INTERPRETATION: We report a novel age associated, non-linear redox regulation in platelets in both humans and mice. In advanced age, there occurs an adaptive increase in platelet antioxidants, reducing the intracellular ROS and leading to a healthier platelet phenotype. Clinically, our results advocate the use of less aggressive antiplatelet therapies for CVD in the elderly population. FUND: Study funded by NIH-NHLBI, RO1-HL122815 and RO1-HL115247.

Epithelial (E)-Cadherin is a Novel Mediator of Platelet Aggregation and Clot Stability.

Cadherins play a major role in mediating cell-cell adhesion, which shares many parallels with platelet-platelet interactions during aggregate formation and clot stabilization. Platelets express epithelial (E)-cadherin, but its contribution to platelet function and/or platelet production is currently unknown. To assess the role of E-cadherin in platelet production and function in vitro and in vivo, we utilized a megakaryocyte-specific E-cadherin knockout mouse model. Loss of E-cadherin in megakaryocytes does not affect megakaryocyte maturation, platelet number or size. However, platelet dysfunction in the absence of E-cadherin is revealed when conditional knockout mice are challenged with acute antibody-mediated platelet depletion. Unlike wild-type mice that recover fully, knockout mice die within 72 hours post-antibody administration, likely from haemorrhage. Furthermore, conditional knockout mice have prolonged tail bleeding times, unstable clot formation, reduced clot retraction and reduced fibrin deposition in in vivo injury models. Murine platelet aggregation in vitro in response to thrombin and thrombin receptor activating peptide is compromised in E-cadherin null platelets, while aggregation in response to adenosine diphosphate (ADP) is not significantly different. Consistent with this, in vitro aggregation of primary human platelets in response to thrombin is decreased by an inhibitory E-cadherin antibody. Integrin activation and granule secretion in response to ADP and thrombin are not affected in E-cadherin null platelets, but Akt and glycogen synthase kinase 3ß (GSK3ß) activation are attenuated, suggesting a that E-cadherin contributes to aggregation, clot stabilization and retraction that is mediated by phosphoinositide 3-kinase/Akt/GSK3ß signalling. In summary, E-cadherin plays a salient role in platelet aggregation and clot stability.

Genome-Wide Expression Analysis Suggests Hypoxia-Triggered Hyper-Coagulation Leading to Venous Thrombosis at High Altitude.

Venous thromboembolism (VTE), a multi-factorial disease, is the third most common cardiovascular disease. Established genetic and acquired risk factors are responsible for the onset of VTE. High altitude (HA) also poses as an additional risk factor, predisposing individuals to VTE; however, its molecular mechanism remains elusive. This study aimed to identify genes/pathways associated with the pathophysiology of deep vein thrombosis (DVT) at HA. Gene expression profiling of DVT patients, who developed the disease, either at sea level or at HA-DVT locations, resulted in differential expression of 378 and 875 genes, respectively. Gene expression profiles were subjected to bioinformatic analysis, followed by technical and biological validation of selected genes using quantitative reverse transcription-polymerase chain reaction. Both gene ontology and pathway analysis showed enrichment of genes involved in haemostasis and platelet activation in HA-DVT patients with the most relevant pathway being 'response to hypoxia'. Thus, given the environmental condition the differential expression of hypoxia-responsive genes (angiogenin, ribonuclease, RNase A family, 5; early growth response 1; lamin A; matrix metallopeptidase 14 [membrane-inserted]; neurofibromin 1; PDZ and LIM domain 1; procollagen-lysine 1, 2-oxoglutarate 5-dioxygenase 1; solute carrier family 6 [neurotransmitter transporter, serotonin], member 4; solute carrier family 9 [sodium/hydrogen exchanger], member 1; and TEK tyrosine kinase, endothelial) in HA-DVT could be a determining factor to understand the pathophysiology of DVT at HA.

MicroRNA-145 Impedes Thrombus Formation via Targeting Tissue Factor in Venous Thrombosis.

Venous thromboembolism (VTE), the third leading cardiovascular complication, requires more understanding at molecular levels. Here, we have identified miR-145 as a key molecule for regulating thrombus formation in venous thrombosis (VT) employing network based bioinformatics approach and in vivo experiments. Levels of miR-145 showed an inverse correlation with thrombus load determined by coagulation variables. MiRNA target prediction tools and in vitro study identified tissue factor (TF) as a target gene for miR-145. The restoration of miR-145 levels in thrombotic animals via in vivo miR-145 mimic delivery resulted in decreased TF level and activity, accompanied by reduced thrombogenesis. MiR-145 levels were also reduced in VT patients and correlated with increased TF levels in patients, thereby, confirming our preclinical findings. Our study identifies a previously undescribed role of miRNA in VT by regulating TF expression. Therefore, restoration of miR-145 levels may serve as a promising therapeutic strategy for management of VT.

Activation of NLRP3 inflammasome complex potentiates venous thrombosis in response to hypoxia.

Venous thromboembolism (VTE), caused by altered hemostasis, remains the third most common cause of mortality among all cardiovascular conditions. In addition to established genetic and acquired risk factors, low-oxygen environments also predispose otherwise healthy individuals to VTE. Although disease etiology appears to entail perturbation of hemostasis pathways, the key molecular determinants during immediate early response remain elusive. Using an established model of venous thrombosis, we here show that systemic hypoxia accelerates thromboembolic events, functionally stimulated by the activation of nucleotide binding domain, leucine-rich-containing family, pyrin domain containing 3 (NLRP3) inflammasome complex and increased IL-1ß secretion. Interestingly, we also show that the expression of NLRP3 is mediated by hypoxia-inducible factor 1-alpha (HIF-1α) during these conditions. The pharmacological inhibition of caspase-1, in vivo knockdown of NLRP3, or HIF-1α other than IL-1ß-neutralizing antibodies attenuated inflammasome activation and curtailed thrombosis under hypoxic conditions. We extend the significance of these preclinical findings by studying modulation of this pathway in patients with altitude-induced venous thrombosis. Our results demonstrate distinctive, increased expression of NLRP3, caspase-1, and IL-1ß in individuals with clinically established venous thrombosis. We therefore propose that an early proinflammatory state in the venous milieu, orchestrated by the HIF-induced NLRP3 inflammasome complex, is a key determinant of acute thrombotic events during hypoxic conditions.

Endothelin-1 Gene Polymorphism and Its Level Predict the Risk of Venous Thromboembolism in Male Indian Population.

OBJECTIVES: Genes related to endothelial function are responsible for the regulation of vascular functions. AIM: The aim of this study is to investigate whether endothelial gene-associated polymorphism and their plasma levels can be used to predict the risk for venous thromboembolism (VTE). METHODS: We studied 133 patients with VTE and 164 healthy controls. Endothelin (EDN) G8002A, EDN T1370G, EDN 3A/4A, eNOSG894T, angiotensin-converting enzyme I/D, vascular endothelial growth factor C936T, and endothelial cell protein C receptor A6936G polymorphism was genotyped by restriction fragment length polymorphism. Plasma levels of endothelin 1 (EDN1), endothelial nitric oxide synthase, and angiotensin-converting enzyme were measured by enzyme-linked immunoassay kit. RESULTS: The genotype and allele frequency between control and patients with VTE were significantly altered only for EDN T1370G polymorphism. The plasma EDN1 concentration was relatively higher in patients with VTE ( P = .0017) compared to healthy controls and showed an association with the EDN1 gene polymorphism in male Indian population. Logistic regression model analysis for EDN T1370G indicated a significant association between EDN G allele and occurrence of VTE. CONCLUSION: The EDN1 gene polymorphism may play a significant role in predicting individual's susceptibility toward VTE and its clinical progression.