Biology subjects

Nieman, M. T.

Publications and source records attributed to Nieman, M. T..

9 recordsLinked to original sources

bioRxiv2026

Nonviral, ultrasound-triggered gene delivery platform via gas-core cationic nanobubbles

Despite their promise, lipid nanoparticle gene delivery systems have repeatedly failed clinical trials and struggle to achieve efficient, localized transfection in target tissues. The majority of endocytosed nanoparticles are degraded before nucleic acid release, and an inability to track particle distribution in vivo prevents validation of successful delivery. Alternatively, nanobubbles (NBs) are lipid-shelled, gas-core preclinical ultrasound contrast agents and stimuli-responsive drug delivery vehicles. Under varying acoustic pressures, NBs expand, contract, and burst, releasing cargo in an externally controlled, site-specific manner while scattering unique echoes for simultaneous ultrasound visualization. Here, we introduce a cationic nanobubble (CNB) formulation with a +42.3 mV zeta potential, 265 nm diameter, and 2.43x1011 NBs/mL concentration. CNBs produce stable ultrasound contrast, electrostatically load plasmid DNA onto their surface, and internalize into >99% of human prostate cancer cells within 15 minutes in vitro. CNBs remain brightly echogenic intracellularly and induce sonication-dependent expression of green fluorescent protein (GFP). In vivo, CNBs generate contrast in mouse livers for 50 minutes after intravenous administration. Therapeutic ultrasound stimulation over the liver causes a sharp reduction in ultrasound contrast, visualizing localized cavitation in the target organ and inducing a 2.5-fold increase in anti-GFP mean fluorescence intensity relative to the untransfected control. Importantly, no GFP expression is observed without ultrasound stimulation, supporting a mechanism for selective and site-specific gene delivery. This study presents a highly stable CNB capable of efficient DNA loading and ultrasound-dependent gene expression. These results provide a foundation for the future development of CNB platforms to advance image-guided, ultrasound-triggered gene therapy.

bioengineering↗

bioRxiv2025

Platelets drive immune suppression and glioblastoma growth in a sex-dependent manner via PAR4 signaling

Sex differences in cancer outcome, including that of glioblastoma (GBM), are shaped by biological, hormonal, and immunological factors and impact disease progression, treatment responses, survival, and tumor microenvironment (TME) interactions. Platelets regulate the immune responses and tumor progression in many cancers, but it is not clear how they contribute to these sex-based differences by affecting the dynamics of the TME. Here, we show that GBM patients exhibit heightened platelet reactivity driven by PAR4 signaling. In murine GBM models, both pharmacological inhibition of PAR4 using BMS986120 and genetic deletion of PAR4 significantly prolong survival in females but not males. This survival advantage is estrogen dependent: it is preserved in chromosomal male-hormonal female mice within the four-core genotype model and is rescued in ovariectomized mice treated with estrogen. The survival benefit is TME specific and is mediated by platelet-driven enhancement of CD8 T cell infiltration into the tumor. Inhibition of platelet PAR4 signaling increases calcium signaling through an estrogen-dependent interaction between PAR4 and estrogen receptor {beta} (ER{beta})--a receptor interaction not previously described. PAR4-activated platelets within the TME suppress CD8 T cell function, and depletion of CD8 T cells abolishes both the tumor-induced platelet reactivity and the survival benefit conferred by PAR4 inhibition. These findings establish platelet-mediated PAR4 signaling as a critical driver of tumor progression and identify sex-specific immune responses as key to therapeutic efficacy.

cancer biology↗

bioRxiv2024

Elucidating the dynamics of Integrin αIIb3 from native platelet membranes by cryo-EM with build and retrieve method

Platelets fulfill their essential physiological roles sensing the extracellular environment through their membrane proteins. The native membrane environment provides essential regulatory cues that impact the protein structure and mechanism of action. Single-particle cryogenic electron microscopy (cryo-EM) has transformed structural biology by allowing high-resolution structures of membrane proteins to be solved from homogeneous samples. Our recent breakthroughs in data processing now make it feasible to obtain atomic-level-resolution protein structures from crude preparations in their native environments by integrating cryo-EM with the "Build-and-Retrieve" (BaR) data processing methodology. We applied this iterative bottom-up methodology on resting human platelet membranes for an in-depth systems biology approach to uncover how lipids, metal binding, post-translational modifications, and co-factor associations in the native environment regulate platelet function at the molecular level. Here, we report using cryo-EM followed by the BaR method to solve the unmodified integrin IIb{beta}3 structure directly from resting human platelet membranes in its inactivated and intermediate states at 2.75[A] and 2.67[A], respectively. Further, we also solved a novel dimer conformation of IIb{beta}3 at 2.85[A] formed by two intermediate-states of IIb{beta}3. This may indicate a previously unknown self-regulatory mechanism of IIb{beta}3 in its native environment. In conclusion, our data show the power of using cryo-EM with the BaR method to determine three distinct structures including a novel dimer directly from natural sources. This approach allows us to identify unrecognized regulation mechanisms for proteins without artifacts due to purification processes. These data have the potential to enrich our understanding of platelet signaling circuitry. Key pointsO_LIWe report the first structural analysis of platelet membrane proteins extracted directly from human platelet membranes. C_LIO_LIOur novel structural-omics approach allowed us to solve integrin IIb{beta}3 structures in two distinct states from resting human platelets. C_LIO_LIRegulatory cues of integrin IIb{beta}3 were preserved from the native resource and revealed on the models with atomic resolutions. C_LIO_LIThis study opens the potential to build the platelet membrane protein atlas to understand platelet physiology. C_LI

biochemistry↗

bioRxiv2024

Mice with Reduced PAR4 Reactivity show Decreased Venous Thrombosis and Platelet Procoagulant Activity

BackgroundHypercoagulation and thrombin generation are major risk factors for venous thrombosis. Sustained thrombin signaling through PAR4 promotes platelet activation, phosphatidylserine exposure, and subsequent thrombin generation. A single-nucleotide polymorphism in PAR4 (rs2227376) changes proline to leucine extracellular loop 3 (P310L), which decreases PAR4 reactivity and is associated with a lower risk for venous thromboembolism (VTE) in a GWAS meta-analysis. ObjectiveThe goal of this study is to determine the mechanism for the association of rs2227376 with reduced risk for VTE in using mice with a homologous mutation (PAR4-P322L). MethodsVenous thrombosis was examined using our recently generated PAR4-P322L mice in the inferior vena cava stasis and stenosis models. Coagulation and clot stability was measured using rotational thromboelastometry (ROTEM). Thrombin generating potential was measured in platelet-rich plasma. Phosphatidylserine surface expression and platelet-neutrophil aggregates were analyzed using flow cytometry. ResultsPAR4P/L and PAR4L/L had reduced incidence and size of venous clots at 48 hours. PAR4P/L and PAR4L/L platelets had progressively decreased phosphatidylserine in response to thrombin and convulxin, which led to decreased thrombin generation and decreased PAR4-mediated platelet-neutrophil aggregation. ConclusionsThe leucine allele in extracellular loop 3, PAR4-322L leads to fewer procoagulant platelets and decreased endogenous thrombin potential. This decreased ability to generate thrombin offers a mechanism for PAR4s role in VTE highlighting a key role for PAR4 signaling.

physiology↗

bioRxiv2023

A Mouse Model of the Protease Activated Receptor 4 (PAR4) Pro310Leu Variant has Reduced Platelet Reactivity

BackgroundProtease activated receptor 4 (PAR4) mediates thrombin signaling on platelets and other cells. Our recent structural studies demonstrated a single nucleotide polymorphism in extracellular loop 3 (ECL3), PAR4-P310L (rs2227376) leads to a hypo-reactive receptor. ObjectivesThe goal of this study was to determine how the hypo-reactive PAR4 variant in ECL3 impacts platelet function in vivo using a novel knock-in mouse model (PAR4-322L). MethodsA point mutation was introduced into the PAR4 gene, F2rl3, via CRISPR/Cas9 to create PAR4-P322L, the mouse homolog to human PAR4-P310L. Platelet response to PAR4 activation peptide (AYPGKF), thrombin, ADP, and convulxin was monitored by IIb{beta}3 integrin activation and P-selectin translocation using flow cytometry or platelet aggregation. In vivo responses were determined by the tail bleeding assay and the ferric chloride-induced carotid artery injury model. ResultsPAR4-P/L and PAR4-L/L platelets had a reduced response to AYPGKF and thrombin measured by P-selectin translocation or IIb{beta}3 activation. The response to ADP and convulxin was unchanged among genotypes. In addition, both PAR4-P/L and PAR4-L/L platelets showed a reduced response to thrombin in aggregation studies. There was an increase in the tail bleeding time for PAR4-L/L mice. The PAR4-P/L and PAR4-L/L mice both showed an extended time to arterial thrombosis. ConclusionsPAR4-322L significantly reduced platelet responsiveness to AYPGKF and thrombin, which is in agreement with our previous structural and cell signaling studies. In addition, PAR4-322L had prolonged arterial thrombosis time. Our mouse model provides a foundation to further evaluate the role of PAR4 in other pathophysiological contexts. EssentialsO_LIA mouse model was created to represent the PAR4-P310L sequence variant. C_LIO_LIPAR4-P322L leads to reduced platelet reactivity in response to PAR4-activation peptide and thrombin, while the ADP and GPVI signaling pathways were unaffected. C_LIO_LIThe PAR4-P322L mutation decreases time to occlusion in a mouse model of arterial thrombosis. C_LIO_LIThe PAR4-P322L mouse model provides a foundation to further explore the role of PAR4 in hemostasis and thrombosis. C_LI

physiology↗

bioRxiv2023

Prothrombin Knockdown Protects Podocytes and Reduces Proteinuria in Glomerular Disease

Chronic kidney disease (CKD) is a leading cause of death, and its progression is driven by glomerular podocyte injury and loss, manifesting as proteinuria. Proteinuria includes urinary loss of coagulation zymogens, cofactors, and inhibitors. Importantly, both CKD and proteinuria significantly increase the risk of thromboembolic disease. Prior studies demonstrated that anticoagulants reduced proteinuria in rats and that thrombin injured cultured podocytes. Herein we aimed to directly determine the influence of circulating prothrombin on glomerular pathobiology. We hypothesized that (pro)thrombin drives podocytopathy, podocytopenia, and proteinuria. Glomerular proteinuria was induced with puromycin aminonucleoside (PAN) in Wistar rats. Circulating prothrombin was either knocked down using a rat-specific antisense oligonucleotide or elevated by serial intravenous infusions of prothrombin protein, which are previously established methods to model hypo- (LoPT) and hyper-prothrombinemia (HiPT), respectively. After 10 days (peak proteinuria in this model) plasma prothrombin levels were determined, kidneys were examined for (pro)thrombin co-localization to podocytes, histology, and electron microscopy. Podocytopathy and podocytopenia were determined and proteinuria, and plasma albumin were measured. LoPT significantly reduced prothrombin colocalization to podocytes, podocytopathy, and proteinuria with improved plasma albumin. In contrast, HiPT significantly increased podocytopathy and proteinuria. Podocytopenia was significantly reduced in LoPT vs. HiPT rats. In summary, prothrombin knockdown ameliorated PAN-induced glomerular disease whereas hyper-prothrombinemia exacerbated disease. Thus, (pro)thrombin antagonism may be a viable strategy to simultaneously provide thromboprophylaxis and prevent podocytopathy-mediated CKD progression.

physiology↗

bioRxiv2020

Complement factor C4a does not activate protease activated receptor 1 (PAR1) or PAR4 on human platelets.

BackgroundProtease activated receptor 1 (PAR1) and PAR4 are key thrombin signal mediators for human platelet activation and aggregation in response to vascular injury. They are primarily activated by thrombin cleavage of the N-terminus to expose a tethered ligand. In addition to the canonical activation by thrombin, a growing panel of proteases can also elicit PAR1- or PAR4-mediate signal transduction. Recently, complement factor C4a was reported as the first endogenous agonist for both PAR1 and PAR4. Further, it is the first endogenous non-tethered ligand that activates PAR1 and PAR4. These studies were conducted with human microvascular cells; the impact of C4a on platelet PARs is unknown. ObjectivesThe goal of this study was to interrogate PAR1 and PAR4 activation by C4a on human platelets. MethodsPlatelet rich plasma (PRP) were isolated from healthy donors. PRP was stimulated with C4a and the platelet aggregation was measured. HEK293 Flp-In T-rex cells were used to further test if C4a stimulation can initiate PAR1- or PAR4-mediated Gq signaling, which was measured by intracellular calcium mobilization. ResultsC4a failed to elicit platelet aggregation via PAR1- or PAR4-mediated manner. In addition, no PAR1- or PAR4-mediated calcium mobilization was observed upon C4a stimulation on HEK293 cells. ConclusionsComplement factor C4a does not activate PAR1 or PAR4 on human platelets. These data show that PAR1 and PAR4 activation by C4a on microvascular cells likely requires a cofactor, which re-enforces the concept of cell-type specific regulation of protease signaling. EssentialsO_LIC4a is an agonist for both PAR1 and PAR4 on human microvascular cells. C_LIO_LIWe sought to determine if C4a activates human platelets through PAR1 or PAR4. C_LIO_LIC4a does not activate PAR1 and PAR4 on human platelets. C_LIO_LIThis re-enforces the concept of cell-type specific regulation of PAR signaling. C_LI

cell biology↗

bioRxiv2020

Cadherin-6 mediates thrombosis in vivo

Background Platelet adhesion is the critical process mediating stable thrombus formation. Previous reports of cadherin-6 on human platelets have demonstrated its role in platelet aggregation and thrombus formation.Objectives We aimed to further characterize the importance of cadherin-6 in thrombosis in vivo.Methods Cadherin-6 platelet expression was evaluated by western blotting, flow cytometry and immunoprecipitation. Thrombosis was evaluated using the FeCl3 and Rose Bengal carotid artery models in C57Bl6 mice treated with anti-cadherin-6 or IgG and wild-type or Cdh6-/- mice. Platelet function was compared in wild type and Cdh6-/- mice using tail-clip assays and aggregometry.Results Human platelet expression of cadherin-6 was confirmed at ~3,000 copies per platelet. Cdh6-/- mice or those treated with anti-Cadherin-6 antibody showed an increased time to occlusion in both thrombosis models. Cadherin-6 was not expressed on mouse platelets, and there were no differences in tail bleeding times or platelet aggregation in wild-type versus Cdh6-/- mice.Conclusions Cadherin-6 plays an essential role in thrombosis in vivo. However, cadherin-6 is not expressed on murine platelets. These data are in contrast to human platelets, which express a functional cadherin-6/catenin complex. The essential, platelet-independent role for cadherin-6 in hemostasis may allow it to be an effective and safe therapeutic target.EssentialsCadherin-6 function in thrombus formation was investigated in vivo using two murine models of thrombosis.Blocking or deleting cadherin-6 significantly delayed time to occlusionHuman platelets express cadherin-6, but murine platelets do not.Competing Interest StatementThe authors have declared no competing interest.View Full Text

pathology↗

bioRxiv2019

Expression and purification of Protease Activated Receptor 4 (PAR4) and analysis with histidine hydrogen deuterium exchange.

Protease activated receptors (PARs) are G-protein coupled receptors (GPCRs) that are activated by proteolyis of the N-terminus, which exposes a tethered ligand that interacts with the receptor. Numerous studies have focused on the signaling pathways mediated by PARs. However, the structural basis for initiation of these pathways is unknown. Here, we describe a strategy for the expression and purification of PAR4. This is the first PAR family member to be isolated without stabilizing modifications for biophysical studies. We monitored PAR4 activation with histidine-hydrogen deuterium exchange (His-HDX). PAR4 has 9 histidines that are spaced throughout the protein allowing a global view of solvent accessible and non-accessible regions. Peptides containing each of the 9 His residues were used to determine the t1/2 for each His residue in apo or thrombin activated PAR4. The thrombin cleaved PAR4 had a 2-fold increase (p > 0.01) in t1/2 values observed for four histidine residues (His180, His229, His240, and His380) demonstrating that these regions have decreased solvent accessibility upon thrombin treatment. In agreement, thrombin cleaved PAR4 also was resistant to thermolysin digestion. In contrast, activation with the PAR4 agonist peptide was digested at the same rate as apo PAR4. Further analysis showed the C-terminus is protected in thrombin activated PAR4 compared to uncleaved or agonist peptide treated PAR4. The studies described here are the first to examine the tethered ligand activation mechanism for a PAR family member using biophysically and shed light on the overall conformational changes that follow activation of PARs by a protease.

biochemistry↗