Biology subjects

Poole, A. W.

Publications and source records attributed to Poole, A. W..

7 recordsLinked to original sources

bioRxiv2026

Disintegrin-like and Cysteine-rich Domains Govern Enzymatic Activity and Substrate Recognition in Echis Snake Venom Metalloproteinases

Snake venom metalloproteinases (SVMPs) are major drivers of pathology following viper envenomation and represent important targets for the development of next-generation recombinant antivenoms. PIII SVMPs are among the most potent haemorrhagic toxins and contain disintegrin-like (Dis) and cysteine-rich (C-rich) accessory domains. Despite their biomedical importance, the mechanistic roles of these accessory domains in substrate recognition and catalysis remain poorly understood. We produced recombinant full-length and domain-deletion variants of two functionally distinct PIII SVMPs: the broadly proteolytic, cytotoxic cPIII and the highly specific prothrombin activator Ecarin. Proteins were expressed as latent zymogens in insect cells, auto-activated by Zn2+, and analysed using enzymatic, blood clotting, and cell-based assays. Progressive removal of the C-rich and Dis domains reduced zymogen auto-activation and markedly diminished catalytic activity in both toxins. In cPIII, domain deletion caused a stepwise loss of proteolytic and cytotoxic activity without altering substrate preference. In Ecarin, removal of the accessory domains strongly impaired prothrombin activation, and thus plasma clotting, demonstrating a critical role in substrate recognition. Conversely, deletion of the C-rich domain increased fibrinogenolytic activity, revealing a substrate-dependent gatekeeping function. Deglycosylation showed that N-linked glycans modulate SVMP activity in a construct-dependent manner. Recombinant Ecarin closely recapitulated the biochemical properties of the native venom-derived toxin. Our data support a model in which PIII SVMP accessory domains enhance substrate positioning and catalytic efficiency while selectively restricting access to non-cognate substrates. These findings establish accessory-domain-mediated substrate recognition as a key determinant of SVMP function, informing rational antivenom design.

biochemistry↗

bioRxiv2025

Selective degradation of platelet BTK by PROTAC NX-5948 provides antithrombotic benefits without affecting haemostasis

Current antithrombotic therapies are effective in reducing thrombotic events but are limited by their associated risk of bleeding. BTK acts as a key signalling switch that drives platelet activation during thrombosis but is largely dispensable for routine haemostasis. It is an important non-redundant signalling mediator downstream of the GPVI and CLEC-2 receptors, plays a key role in thrombosis with minimal involvement in haemostasis, making it an attractive antithrombotic target. While BTK inhibitors effectively reduce thrombosis, their clinical use has been limited due to off-target effects. Protein degraders may overcome this limitation by enabling the ubiquitin proteasomal system to selectively target and degrade BTK. We here assessed the ability of the BTK degraders NX-2127 and NX-5948, currently in clinical trials for B cell pathologies, to target platelet BTK for degradation. NX-2127 and NX-5948 induced concentration-dependent degradation of BTK in washed platelets, platelet-rich plasma and whole blood. NX-5948 showed no hook effect and outperformed NX-2127 in potency, efficacy, and degradation kinetics. TMT proteomic analysis confirmed selective BTK degradation by NX-5948 with no evidence of major off-target effects. BTK degradation impaired CRP-mediated integrin IIb{beta}3 activation, P-selectin expression, platelet aggregation and in vitro thrombosis, with PAR-1 mediated platelet function being left intact. Dosing mice with NX-5948 led to efficacious degradation of platelet BTK and impaired CRP-, but not thrombin-, mediated ex vivo platelet function. In vivo, arterial thrombosis was markedly reduced, without an increase in bleeding time. Together, these results highlight NX-5948 as a potent, selective BTK degrader with antithrombotic potential and minimal haemostatic impact. Key PointsO_LIThe BTK degrader NX-5948 potently and selectively degrades platelet BTK and suppresses thrombus formation without affecting bleeding. C_LIO_LITargeting BTK degradation offers a new antithrombotic strategy that spares haemostasis and may aid patients intolerant to DAPT. C_LI

cell biology↗

bioRxiv2023

CryoET reveals actin filaments within platelet microtubules

Crosstalk between the actin and microtubule cytoskeletons is essential for many cellular processes. Recent studies have shown that microtubules and F-actin can assemble to form a composite structure where F-actin occupies the microtubule lumen. Whether these cytoskeletal hybrids exist in physiological settings and how they are formed is unclear. Here, we show that the short-crossover Class I actin filament previously identified inside microtubules in human HAP1 cells is cofilin-bound F-actin. Lumenal F-actin can be reconstituted in vitro, but cofilin is not essential. Moreover, actin filaments with both cofilin-bound and canonical morphologies reside within human platelet microtubules under physiological conditions. We propose that stress placed upon the microtubule network during motor-driven microtubule looping and sliding may facilitate the incorporation of actin into microtubules.

cell biology↗

bioRxiv2022

Alterations in platelet proteome signature and impaired platelet integrin αIIbβ3 activation in patients with COVID-19

BackgroundPatients with coronavirus disease-19 (COVID-19) are at increased risk of thrombosis, which is associated with altered platelet function and coagulopathy, contributing to excess mortality. ObjectivesWe aimed to characterise the mechanism of altered platelet function in COVID-19 patients. MethodsThe platelet proteome, platelet functional responses and platelet-neutrophil aggregates were compared between patients hospitalised with COVID-19 and healthy control subjects using Tandem Mass Tag (TMT) proteomic analysis, Western blotting and flow cytometry. ResultsCOVID-19 patients showed a different profile of platelet protein expression (858 altered out of 5773 quantified). Levels of COVID-19 plasma markers were enhanced in COVID-19 platelets. Gene ontology (GO) pathway analysis demonstrated that levels of granule secretory proteins were raised, whereas some platelet activation proteins, such as the thrombopoietin receptor and PKC, were lowered. Basally, COVID-19 platelets showed enhanced phosphatidylserine (PS) exposure, with unaltered integrin IIb{beta}3 activation and P-selectin expression. Agonist-stimulated integrin IIb{beta}3 activation and PS exposure, but not P-selectin expression, were significantly decreased in COVID-19 patients. COVID-19 patients had high levels of platelet-neutrophil aggregates, even under basal conditions, compared to controls. This interaction was disrupted by blocking P-selectin, demonstrating that platelet P-selectin is critical for the interaction. ConclusionsOverall, our data suggests the presence of two platelet populations in patients with COVID-19: one with circulating platelets with an altered proteome and reduced functional responses and another with P-selectin expressing neutrophil-associated platelets. Platelet driven thromboinflammation may therefore be one of the key factors enhancing the risk of thrombosis in COVID-19 patients. Essentials- COVID-19 patient platelet function and platelet proteins were compared with healthy controls - Proteomic analysis of platelets indicated that COVID-19 decreased platelet activation proteins - Agonist induced PS exposure and integrin IIb{beta}3 activation were impaired in COVID-19 - COVID-19 led to maximal levels of P-selectin dependent platelet-neutrophil aggregates

cell biology↗

bioRxiv2021

Platelet generation from circulating megakaryocytes is triggered in the lung vasculature

Platelets, small hemostatic blood cells, are derived from megakaryocytes, although the generation process is not clear. Only small numbers of platelets have been produced in systems outside the body, where bone marrow and lung are proposed as sites of platelet generation. Here we show that perfusion of megakaryocytes ex vivo through the mouse lung vasculature generates very large numbers of platelets, up to 3,000 per megakaryocyte. Despite their large size, megakaryocytes were able repeatedly to passage through the lung vasculature, leading to enucleation and fragmentation to generate platelets intravascularly. Using the ex vivo lung and a novel in vitro microfluidic chamber we determined the contributions of oxygenation, ventilation and endothelial cell health to platelet generation, and showed a critical role for the actin regulator TPM4. One-Sentence SummaryMegakaryocytes form platelets intravascularly in the lung, dependent upon oxygenation, endothelium and megakaryocyte TPM4

physiology↗

bioRxiv2021

A role for SHARPIN in platelet linear protein ubiquitination and function

SHARPIN (Src homology 3 and multiple ankyrin repeat domains protein (SHANK)- associated RH domain-interacting protein) as part of the linear ubiquitin chain assembly complex (LUBAC) catalyses the addition of linear (Met1-linked) ubiquitin chains to substrates. As part of this complex SHARPIN acts as a multi-functional modulator of immune/inflammatory responses through regulation of NfkB activation. In addition, SHARPIN can act as a negative regulator of integrin function. Despite platelets being anucleate cells several studies have determined potential roles for both ubiquitination and NfkB in regulating platelet function. However, little is known about either linear ubiquitination and/or SHARPIN in mouse platelets. In this study, we evaluated platelet function in mice with impaired SHARPIN expression. We confirmed that SHARPIN was expressed in platelets from wild-type mice but not in mice homozygous for SHARPINcpdm allele (cpdm/cpdm) and that this correlated with a reduction in linear ubiquitination. Platelet function in response to thrombin was unaffected. In contrast, CRP-XL-and U46619-mediated platelet responses and thrombus formation under flow on a collagen-coated surface were significantly reduced in the cpdm/cpdm mice. This was associated with impaired U46619-mediated intracellular signalling as well as a reduction in CRP-mediated ERK phosphorylation. Despite the reported role for Nf{kappa}B in regulating platelet function, inhibiting I{kappa}B phosphorylation did not recapitulate the cpdm/cpdm phenotype. Together, these data indicate that the lack of SHARPIN and linear ubiquitination results in impaired thrombosis and platelet functional responses to CRP and U46619. This phenotype is independent of Nf{kappa}B pathway inhibition but may involve alternative signalling pathways regulated by linear ubiquitination. Key PointsO_LISHARPIN plays an essential role in platelet linear protein ubiquitination and CRP and U46619-mediated platelet function C_LIO_LIIn vitro thrombosis is significantly impaired in SHARPIN deficient mice C_LI

cell biology↗

bioRxiv2018

Epigenetic regulation of PAR4-related platelet activation: mechanistic links between environmental exposure and cardiovascular disease

Protease-activated receptor 4 (PAR4) is a potent thrombin receptor. Epigenetic control of the F2RL3 locus (which encodes for PAR4) via DNA methylation is associated with both smoking and cardiovascular disease. We examined the association between DNA hypomethylation at F2RL3 and risk of cardiovascular disease, focusing on acute myocardial infarction (AMI) (n=853 cases / 2,352 controls). We used in vitro cell models to dissect the role of DNA methylation in regulating expression of F2RL3. We investigated the interplay between F2RL3 DNA methylation and platelet function in human (n=41). Lastly, we used Mendelian randomization to unify observational and functional work by assessing evidence for causal relationships using data from UK Biobank (n=407,141) and CARDIoGRAMplusC4D (n=184,305). Observationally, one standard deviation (SD) decrease in DNA methylation at F2RL3 was associated with a 25% increase in the odds of AMI. In vitro, short-term exposure of cells to cigarette smoke reduced F2RL3 DNA methylation and increased gene expression. Transcriptional assays flagged a role for a CEBP recognition sequence in modulating the enhancer activity of F2RL3 exon 2. Lower DNA methylation at F2RL3 was associated with increased platelet reactivity in human. The estimated casual odds ratio of ischaemic heart disease was 1.03 (95% CI: 1.00, 1.07) per 1 SD decrease in F2RL3 DNA. In conclusion, we show that DNA methylation-dependent platelet activation is part of a complex system of features contributing to cardiovascular health. Tailoring therapeutic intervention to new knowledge of F2RL3/PAR4 function should be explored to ameliorate the detrimental effects of this risk factor on cardiovascular health.\n\nOne sentence summaryDNA methylation-dependent platelet activation is a likely causal contributor to cardiovascular health.

epidemiology↗