Biology subjects

Roytenberg, R.

Publications and source records attributed to Roytenberg, R..

4 recordsLinked to original sources

bioRxiv2026

Regulator of G Protein Signaling 6 Negatively Regulates Platelet Activation and Arterial Thrombosis in Mice

BackgroundPlatelet activation via G protein-coupled receptors (GPCRs) is central to arterial thrombosis. P2Y12 is a canonical Gi-coupled receptor mediating ADP-dependent platelet activation, yet the role of Regulator of G protein Signaling 6 (RGS6), a modulator of Gi signaling, in platelet function and thrombosis remains unclear. ObjectivesTo determine the role of RGS6 in platelet activation and arterial thrombosis and to define its impact on P2Y12/Gi signaling. MethodsArterial thrombosis was assessed using a FeCl{square}-induced carotid artery injury model in wild-type (WT) and Rgs6-/- mice. Platelet aggregation was measured ex vivo. Signaling pathways were analyzed by Western blot in ADP-stimulated platelets. P2Y12/Gi signaling was further evaluated using a cAMP-responsive luciferase reporter assay in HEK293 cells. ResultsMale Rgs6-/- mice exhibited significantly accelerated thrombosis compared with WT controls. Rgs6-/- platelets showed enhanced ADP-induced aggregation, whereas collagen-induced aggregation was unchanged. In ADP-stimulated platelets, RGS6 deficiency altered signaling kinetics, characterized by delayed Akt phosphorylation and reduced PKA and VASP phosphorylation. In a heterologous cAMP-luciferase assay, RGS6 attenuated P2Y12/Gi-mediated suppression of cAMP. Two-way ANOVA demonstrated significant effects of ADP and RGS6 expression on luciferase activity, with no interaction, indicating that RGS6 modulates signaling magnitude rather than agonist sensitivity. Pharmacologic inhibition of P2Y12 with clopidogrel abolished the genotype-dependent difference in thrombosis in vivo. ConclusionsRGS6 acts as a negative regulator of platelet P2Y12/Gi signaling and thrombus formation. Loss of RGS6 enhances ADP-dependent platelet activation and accelerates arterial thrombosis, establishing RGS6 as an endogenous brake on platelet activation.

cell biology↗

bioRxiv2024

Sodium/Potassium ATPase Alpha 1 Subunit Fine-tunes Platelet GPCR Signaling Function and is Essential for Thrombosis

BackgroundThrombosis is a major cause of myocardial infarction and ischemic stroke. The sodium/potassium ATPase (NKA), comprising and {beta} subunits, is crucial in maintaining intracellular sodium and potassium gradients. However, the role of NKA in platelet function and thrombosis remains unclear. MethodsWe utilized wild-type (WT, 1+/+) and NKA 1 heterozygous (1+/-) mice, aged 8 to 16 weeks, of both sexes. An intravital microscopy-based, FeCl3-induced carotid artery injury thrombosis model was employed for in vivo thrombosis assessment. Platelet transfusion assays were used to evaluate platelet NKA 1 function on thrombosis. Human platelets isolated from healthy donors and heart failure patients treated with/without digoxin were used for platelet function and signaling assay. Complementary molecular approaches were used for mechanistic studies. ResultsNKA 1 haplodeficiency significantly reduced its expression on platelets without affecting sodium homeostasis. It significantly inhibited 7.5% FeCl3-induced thrombosis in male but not female mice without disturbing hemostasis. Transfusion of 1+/-, but not 1+/+, platelets to thrombocytopenic WT mice substantially prolonged thrombosis. Treating WT mice with low-dose ouabain or marinobufagenin, both binding NKA 1 and inhibiting its ion-transporting function, markedly inhibited thrombosis in vivo. NKA 1 formed complexes with leucine-glycine-leucine (LGL)-containing platelet receptors, including P2Y12, PAR4, and thromboxane A2 receptor. This binding was significantly attenuated by LGL>SFT mutation or LGL peptide. Haplodeficiency of NKA 1 in mice or ouabain treatment of human platelets notably inhibited ADP-induced platelet aggregation. While not affecting 10% FeCl3-induced thrombosis, NKA 1 haplodeficiency significantly prolonged thrombosis time in mice treated with an ineffective dose of clopidogrel. ConclusionNKA 1 plays an essential role in enhancing platelet activation through binding to LGL-containing platelet GPCRs. NKA 1 haplodeficiency or inhibition with low-dose ouabain and marinobufagenin significantly inhibited thrombosis and sensitized clopidogrels anti-thrombotic effect. Targeting NKA 1 emerges as a promising antiplatelet and antithrombotic therapeutic strategy.

cell biology↗

bioRxiv2024

Thymidine Phosphorylase Mediates SARS-CoV-2 Spike Protein Enhanced Thrombosis in K18-hACE2TG Mice

COVID-19, caused by SARS-CoV-2, is associated with arterial and venous thrombosis, thereby increasing mortality. SARS-CoV-2 spike protein (SP), a viral envelope structural protein, is implicated in COVID-19-associated thrombosis. However, the underlying mechanisms remain unknown. Thymidine phosphorylase (TYMP), a newly identified prothrombotic protein, is upregulated in the plasma, platelets, and lungs of patients with COVID-19 but its role in COVID-19-associated thrombosis is not defined. In this study, we found that wild-type SARS-CoV-2 SP significantly promoted arterial thrombosis in K18-hACE2TG mice. SP-accelerated thrombosis was attenuated by inhibition or genetic ablation of TYMP. SP increased the expression of TYMP, resulting in the activation of signal transducer and activator of transcription 3 (STAT3) in BEAS-2B cells, a human bronchial epithelial cell line. A siRNA-mediated knockdown of TYMP inhibited SP-enhanced activation of STAT3. Platelets derived from SP-treated K18-hACE2TG mice also showed increased STAT3 activation, which was reduced by TYMP deficiency. Activated STAT3 is known to potentiate glycoprotein VI signaling in platelets. While SP did not influence ADP- or collagen-induced platelet aggregation, it significantly shortened activated partial thromboplastin time and this change was reversed by TYMP knockout. Additionally, platelet factor 4 (PF4) interacts with SP, which also complexes with TYMP. TYMP enhanced the formation of the SP/PF4 complex, which may potentially augment the prothrombotic and procoagulant effects of PF4. We conclude that SP upregulates TYMP expression, and TYMP inhibition or knockout mitigates SP-enhanced thrombosis. These findings indicate that inhibition of TYMP may be a novel therapeutic strategy for COVID-19-associated thrombosis. Key PointsO_LISARS-CoV-2 spike protein, thymidine phosphorylase, and platelet factor 4 form a complex that may promote clot formation. C_LIO_LIInhibiting thymidine phosphorylase attenuates SARS-CoV-2 spike protein-enhanced thrombosis, platelet activation, and coagulation. C_LI

cell biology↗

bioRxiv2017

Ancient antagonism between CELF and RBFOX families tunes mRNA splicing outcomes

Over 95% of human multi-exon genes undergo alternative splicing, a process important in normal development and often dysregulated in disease. We sought to analyze the global splicing regulatory network of CELF2 in human T cells, a well-studied splicing regulator critical to T cell development and function. By integrating high-throughput sequencing data for binding and splicing quantification with sequence features and probabilistic splicing code models, we find evidence of splicing antagonism between CELF2 and the RBFOX family of splicing factors. We validate this functional antagonism through knockdown and overexpression experiments in human cells and find CELF2 represses RBFOX2 mRNA and protein levels. Because both families of proteins have been implicated in the development and maintenance of neuronal, muscle, and heart tissues, we analyzed publicly available data in these systems. Our analysis suggests global, antagonistic co-regulation of splicing by the CELF and RBFOX proteins in mouse muscle and heart in several physiologically relevant targets including proteins involved in calcium signaling and members of the MEF2 family of transcription factors. Importantly, a number of these co-regulated events are aberrantly spliced in mouse models and human patients with diseases that affect these tissues including heart failure, diabetes, or myotonic dystrophy. Finally, analysis of exons regulated by ancient CELF family homologs in chicken, and Drosophila suggests this antagonism is conserved through evolution.

genomics↗