Biology subjects

Lee, R. H.

Publications and source records attributed to Lee, R. H..

8 recordsLinked to original sources

bioRxiv2026

Lack of canonical PAR4 activation is associated with reduced arterial and venous thrombosis in mice.

BackgroundProtease-activated receptor 4 (PAR4) is the only functional thrombin receptor on mouse platelets. Expression and activation of platelet PAR4 was shown to influence hemostatic plug stability and contributes to thrombosis in different murine arterial and venous thrombosis models. PAR4 activation by thrombin and other serine proteases occurs at the canonical activation site at Arginine (Arg) 59 in mice and Arg47 in humans. If murine PAR4 has functional non-canonical activation sites as shown for other PARs is unknown. ObjectiveTo investigate canonical and potentially non-canonical PAR4 signaling in mice, we generated a mouse model expressing a functional, thrombin-cleavage resistant PAR4 by changing Arg59 to Alanine (Ala) 59 in murine PAR4 (PAR4R59A). PAR4R59A mice were used to assess the impact of impaired canonical (thrombin)-dependent PAR4 signaling on hemostasis and thrombosis in mice. MethodsWe analyzed platelet aggregation, platelet integrin activation and -granule release ex vivo. Hemostasis and thrombosis in PAR4R59A and their control mice was compared using the jugular vein needle puncture injury-induced hemostasis model, and the ferric chloride-induced carotid artery and electrolytic injury-induced femoral vein thrombosis models. ResultsPlatelets of PAR4R59A mice did not response to thrombin but responded normally to PAR4 agonist peptide (PAR4AP) stimulation in aggregation assays. Platelets of PAR4R59A and their control mice exhibited comparable responses to ADP, convulxin or PAR4AP regarding integrin activation and -granular release. PAR4R59A mice exhibited impaired hemostasis in the jugular vein needle puncture model, and were protected from ferric chloride-induced arterial thrombosis and from electrolytic injury induced thrombosis in the femoral vein. ConclusionThe novel PAR4R59A mouse expresses a thrombin-insensitive but still functional PAR4. We propose that the new mouse line will increase the in vivo investigation of canonical PAR4 signaling pathways and may reveal unknown non-canonical PAR4 signaling in different pathologies.

pathology↗

bioRxiv2025

RAP1-RHO small GTPase cross-talk mediates integrin-dependent and -independent platelet procoagulant response

Platelet adhesion and procoagulant activity are critical for primary and secondary hemostasis, respectively. The small GTPase RAP1 is a central regulator of platelet aggregation as it controls IIb{beta}3 integrin activation through direct interaction with the integrin adapter protein, TALIN-1 (Tln-1). In addition to their aggregation defect, activated platelets lacking RAP1 (Rap1mKO) exhibited a marked impairment in surface exposure of phosphatidylserine (PtdSer), a negatively charged phospholipid with procoagulant activity. However, the mechanisms by which RAP1 regulates PtdSer exposure are unclear. Here we investigated the hypothesis that RAP1 regulates platelet PtdSer exposure through cross-talk with small GTPases of the Rho family. Consistent with their defect in PtdSer exposure, Rap1mKO platelets showed reduced procoagulant activity in vitro and in vivo when compared to controls. Stimulated Rap1mKO platelets exhibited elevated RHOA-GTP levels, and inhibition of the RHOA effector, Rho associated coiled-coil kinase (ROCK), partially restored PtdSer exposure in these cells. A milder defect in PtdSer exposure was observed for platelets from Tln-1mR35/118E mice, i.e. mice with impaired RAP1-Tln-1 interaction but otherwise intact RAP1 signaling. ROCK inhibition fully restored PtdSer exposure in Tln-1mR35/118E platelets. Opening of the mitochondrial permeability transition pore, a cellular response critical to PtdSer exposure, was impaired in Rap1mKO platelets and restored by pretreatment of cells with the ROCK inhibitor. Our study provides first evidence that platelet RAP1 signaling affects hemostatic plug formation independent of its key role in platelet adhesion. Additionally, our studies strongly suggest that RAP1 regulates PtdSer exposure and procoagulant activity in a RHOA/integrin-dependent and -independent manner.

cell biology↗

bioRxiv2025

Mouse Single-Cell Long-Read Splicing Atlas by Ouro-Seq

Alternative splicing creates cellular and phenotypic diversity; thus, characterizing splicing patterns across diverse cell types is essential for a comprehensive understanding of these cell types. However, existing full-length single-cell RNA-sequencing methods suffer from multiple technical limitations, such as the pervasive presence of reverse transcription artifacts across genes. Here, we introduce Ouro-Seq, a novel single-cell long-read sequencing framework with a versatile artifact removal mechanism for characterizing full-length mRNA species expressed by individual cells. We also developed a computational pipeline that identifies genuine full-length cDNAs and normalizes full-length mRNA size distributions for integration. Using Ouro-Seq, we constructed the Mouse Single-Cell Long-Read Splicing Atlas by collecting full-length transcriptomes of 103,304 nuclei or cells from 12 major organs and tissues of adult mice. Our atlas comprehensively characterizes alternative splicing, alternative promoter usage, and alternative polyadenylation events across cell types, revealing previously unappreciated functional heterogeneity among individual cells. Different cell types exhibit largely distinct expression patterns of various isoforms, corresponding to their specific morphologies and biological functions. Our splicing atlas and generalized long-read frameworks will empower researchers to fully interpret biological variation across full-length transcriptomes of single cells.

genomics↗

bioRxiv2024

Utility of thromboelastography with platelet mapping (TEG-PM) for monitoring platelet transfusion in qualitative platelet disorders

Patients with pathogenic variants in RASGRP2 (inherited platelet disorder (IPD)-18) have normal platelet counts but show impaired platelet aggregation due to diminished activation of IIb{beta}3 integrin. This defect results in moderate to severe bleeding episodes, especially following surgical procedures, which require patients to be transfused with platelets and/or pro-hemostatic agents. We recently demonstrated that the hemostatic efficacy of transfused platelets is limited by dysfunctional endogenous platelets in a mouse model of IPD-18 (Rasgrp2-/- mice), as dysfunctional platelets were recruited to the forming hemostatic plug but did not participate in clot contraction. Consequently, higher amounts of transfused platelets were required to outcompete these dysfunctional cells and to reverse bleeding. We here studied the usefulness of thromboelastography with platelet mapping (TEG-PM), a method to evaluate platelet-dependent clot contraction, for ex vivo monitoring of the hemostatic potential in Rasgrp2-/- mice transfused with various amounts of wild-type (WT) platelets. Rasgrp2-/- whole blood samples did not contract in TEG-PM, consistent with a critical role of this protein in IIb{beta}3 activation. Addition of WT platelets improved TEG parameters (K time, -angle, MA) in a ratio dependent manner, consistent with our recent in vivo studies showing impaired hemostasis at a 5:1, but not at a 2:1 ratio of mutant to WT platelets. Interestingly, K and values were identified as better predictors of transfusion efficacy than MA, the most platelet-dependent TEG parameter. In conclusion, this proof-of-concept study supports the use of TEG-PM to monitor platelet transfusion ratios and hemostatic potential in IPD-18 and potentially other platelet disorders.

cell biology↗

bioRxiv2023

4D intravital imaging studies iden1fy platelets as the predominant cellular procoagulant surface in a mouse model of hemostasis

Interplay between platelets, coagulation/fibrinolytic factors, and endothelial cells (ECs) is necessary for effective hemostatic plug formation. This study describes a novel four-dimensional (4D) imaging platform to visualize and quantify hemostatic plug components with high spatiotemporal resolution. Fibrin accumulation following laser-induced endothelial ablation was observed at the EC-platelet plug interface, controlled by the antagonistic balance between fibrin generation and breakdown. Phosphatidylserine (PS) was first detected in close physical proximity to the fibrin ring, followed by exposure across the endothelium. Impaired PS exposure in cyclophilinD-/- mice resulted in a significant reduction in fibrin accumulation. Adoptive transfer and inhibitor studies demonstrated a key role for platelets, but not ECs, in fibrin generation during hemostatic plug formation. Inhibition of fibrinolysis with tranexamic acid (TXA) led to increased fibrin accumulation in WT mice, but not in cyclophilinD-/- mice or WT mice treated with antiplatelet drugs. These studies implicate platelets as the functionally dominant procoagulant surface during hemostatic plug formation. In addition, they suggest that impaired fibrin formation due to reduced platelet procoagulant activity is not reversed by TXA treatment.

cell biology↗

bioRxiv2021

O-GlcNAc transferase regulates glioblastoma acetate metabolism via regulation of CDK5-dependent ACSS2 phosphorylation

Glioblastomas (GBMs) preferentially generate acetyl-CoA from acetate as a fuel source to promote tumor growth. O-GlcNAcylation has been shown to be elevated by increasing O-GlcNAc transferase (OGT) in many cancers and reduced O-GlcNAcylation can block cancer growth. Here, we identify a novel mechanism whereby OGT regulates acetate-dependent acetyl-CoA production by regulating phosphorylation of acetyl-CoA synthetase 2 (ACSS2) by cyclin-dependent kinase 5 (CDK5). OGT is required and sufficient for GBM cell growth and regulates acetate conversion to acetyl-CoA. Elevating O-GlcNAcylation in GBM cells increases phosphorylation of ACSS2 on Ser-267 in a CDK5-dependent manner. Importantly, we show that ACSS2 Ser-267 phosphorylation regulates its stability by reducing polyubiquitination and degradation. ACSS2 Ser-267 is critical for OGT-mediated GBM growth as overexpression of ACSS2 Ser-267 phospho-mimetic rescues growth in vitro and in vivo. Importantly, we show that pharmacologically targeting OGT and CDK5 reduces GBM growth ex vivo. Thus, the OGT/CDK5/ACSS2 pathway may be a way to target altered metabolic dependencies in brain tumors.

cancer biology↗

bioRxiv2021

Opposing effects of competitive exclusion determine invasion outcomes and structure ant assemblages

Understanding how species phenotypic differences affect competition is key to explaining community assembly and predicting biodiversity responses. Many studies overlook the variable effects that species trait differences can have on the direction of competitive exclusion, which reverses depending on the specific mechanism at play. We performed a comprehensive trait-based study of an ant invasion integrating morphological, dietary, physiological and behavioral analyses. We found that trait differences between invasive and resident species were not only associated with niche differences which promoted the coexistence of dissimilar species, but also competitive ability differences which acted in the opposite fashion. Furthermore, competition along separate trait axes led to complex and contrasting patterns in the invaded assemblages, where species were at once similar (clustered) in some traits but also dissimilar (overdispersed) in others. Our results reveal that different aspects of phenotype may distinctly modulate the effect of competition in structuring ecological communities and functional diversity.

ecology↗

bioRxiv2020

Estrogen withdrawal alters oxytocin signaling in the paraventricular hypothalamus and dorsal raphe nucleus to increase postpartum anxiety

BackgroundEstrogen increases dramatically during pregnancy, but quickly drops below pre-pregnancy levels at birth and remains suppressed during the postpartum period. Clinical and rodent work suggests that this postpartum drop in estrogen results in an "estrogen withdrawal" state that is related to changes in affect, mood, and behavior. Most studies examining the effect of estrogen withdrawal on the brain have focused solely on the hippocampus. MethodsWe used a hormone-simulated pseudopregnancy model in Syrian hamsters, a first for this species. Ovariectomized females were given daily injections to approximate hormone levels during gestation and then withdrawn from estrogen to simulate postpartum estrogen withdrawal. Subjects were tested for behavioral assays of anxiety and anhedonia during estrogen withdrawal. Following sacrifice, neuroplasticity in oxytocin-producing neurons in the paraventricular nucleus of the hypothalamus (PVH) and its efferent targets was measured. ResultsEstrogen-withdrawn females had increased anxiety-like behaviors in the elevated plus and open field, but did not differ from controls in sucrose preference. Furthermore, estrogen-withdrawn females had more oxytocin-immunoreactive cells and oxytocin mRNA in the PVH, as well as an increase in oxytocin receptor density in the dorsal raphe nucleus (DRN). Finally, blocking oxytocin receptors in the DRN during estrogen withdrawal prevented the high-anxiety behavioral phenotype in estrogen-withdrawn females. ConclusionsEstrogen withdrawal alters oxytocin signaling in the PVH and DRN to increase anxiety-like behavior during the postpartum period. More broadly, these experiments suggest Syrian hamsters as a novel organism in which to model the effects of postpartum estrogen withdrawal on the brain and anxiety-like behavior.

neuroscience↗