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The clustering of platelet glycoprotein receptors with cytosolic YxxL and YxxM motifs, including GPVI, CLEC-2 and PEAR1, triggers activation via phosphorylation of the conserved tyrosine residues and recruitment of the tandem SH2 (Src homology 2) domain effector proteins, Syk and PI 3-kinase. We have modelled the clustering of these receptors with monovalent, divalent and tetravalent soluble ligands and with transmembrane ligands based on the law of mass action using ordinary differential equations and agent-based modelling. The models were experimentally evaluated in platelets and transfected cell lines using monovalent and multivalent ligands, including novel nanobody-based divalent and tetravalent ligands, by fluorescence correlation spectroscopy. Ligand valency, receptor number, receptor dimerisation, receptor phosphorylation and a cytosolic tandem SH2 domain protein act in synergy to drive receptor clustering. Threshold concentrations of a CLEC-2-blocking antibody and Syk inhibitor act in synergy to block platelet aggregation. This offers a strategy for countering the effect of avidity of multivalent ligands and in limiting off-target effects.

The contact activation system (CAS) or contact pathway is central to the crosstalk between coagulation and inflammation and contributes to diverse disorders affecting the cardiovascular system. CAS initiation contributes to thrombosis but is not required for hemostasis and can trigger plasma coagulation the intrinsic pathway [through factor XI (FXI)] and inflammation bradykinin release. Activation of factor XII (FXII) is the principal starting point for the cascade of proteolytic cleavages involving FXI, prekallikrein (PK), and cofactor high molecular weight kininogen (HK) but the precise location and cell receptor interactions controlling these reactions remains unclear. FXII, PK, FXI, and HK utilize key protein domains to mediate binding interactions to cognate cell receptors and diverse ligands, which regulates protease activation. The assembly of contact factors has been demonstrated on the cell membranes of a variety of cell types and microorganisms. The cooperation between the contact factors and endothelial cells, platelets, and leukocytes contributes to pathways driving thrombosis yet the basis of these interactions and the relationship with activation of the contact factors remains undefined. This review focuses on cell receptor interactions of contact proteins and FXI to develop a cell-based model for the regulation of contact activation.

Glycoprotein (GP) VI (GPVI) plays a major role in thrombosis but not haemostasis, making it a promising antithrombotic target. The primary role of GPVI on the surface of platelets is a signalling receptor for collagen, which is one of the most potent thrombotic sub-endothelial components that is exposed by atherosclerotic plaque rupture. Inhibition of GPVI has therefore been investigated as a strategy for treatment and prevention of atherothrombosis, such as during stroke and acute coronary syndromes. A range of specific GPVI inhibitors have been characterized, and two of these inhibitors, glenzocimab and revacept, have completed Phase II clinical trials in ischaemic stroke. In this review, we summarize mechanisms of GPVI activation and the latest progress of clinically tested GPVI inhibitors, including their mechanisms of action. By focusing on what is known about GPVI activation, we also discuss whether alternate strategies could be used to target GPVI.

Hypertrophic cardiomyopathy (HCM) is a genetic disease characterized by diastolic dysfunction and is frequently associated with sudden cardiac death. HCM is associated with pathogenic variants in Z-disk proteins, a key structural component of the sarcomere. Alpha-actinin 2 (ACTN2), is a crucial Z-disk protein important for cross-linking actin filaments. Few ACTN2 variants have been identified, with a limited focus on the mechanisms by which these variants alter protein structure and function. Thereby, this study investigated the biophysical characterisation of multiple ACTN2 variants, as well as assessing the functional impact of the ACTN2-M228T variant in cellular models.ACTN2 missense variants were identified using the Human Mutation Gene Database and characterized with established in silico tools. Structural modelling approaches were then used to predict the impact of HCM-associated variants on ACTN2. To further assess their structural consequences, ACTN2 variants were recombinantly expressed, purified, and analysed using mass photometry, X-ray crystallography, small-angle X-ray scattering, actin-binding and thermal denaturation assays. Additionally, functional and molecular assessments of the ACTN2-M228T variant were performed using induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM).Characterization of the identified ACTN2 variants revealed 18 variants distributed among the different domains of the protein. Structural modeling predictions showed that these variants impact the protein structure through distinct mechanisms, including impaired actin-binding, disrupted dimerization, and reduced protein stability. Actin-binding assays of six variants identified that two variants exhibited enhanced binding, two demonstrated reduced binding, and two showed no change. Additionally, the R327C and R457C variants, predicted to disrupt dimerization, were crystalized, with structures revealing no effect on dimerization. Other variants showed reduced protein stability as evidenced by decreased solubility, impaired thermal stability, alterations in structural conformation, and increased aggregation.The functional impact of the ACTN2-M228T variant was evaluated using iPSC-CMs. Cardiomyocytes harbouring the mutant gene displayed developmental delays, decreased contractility, and upregulation of fibrosis and hypertrophy markers. Protein aggregation and destabilization were confirmed by immunofluorescence and biochemical fractionation techniques. Furthermore, protein degradation mechanisms were examined using inhibitors targeting the ubiquitin-proteosome system and the autophagy-lysosomal pathway, revealing that mutant cells demonstrated autophagy upregulation.Taken together, this study provides valuable insights into how ACTN2 variants impact protein structure and function, contributing to HCM pathogenesis. These findings enhance our understanding of disease mechanisms and may inform the development of targeted therapeutic strategies.Introduction Hypertrophic cardiomyopathy (HCM) is a genetic cardiac disease marked by diastolic dysfunction and left ventricular hypertrophy, often linked with sudden cardiac death (Harris et al., 2006). Genetic studies have associated genetic variants in Z-disk proteins, including Alpha-actinin-2 (ACTN2), with HCM. ACTN2 stabilises the contractile muscle apparatus by anchoring actin filaments (Sjöblom et al., 2008). While a few ACTN2 variants linked to HCM have been identified, research has primarily focused on the actin-binding domain, with no studies examining variants in the full-length protein. Notably, the ACTN2 M228T variant was identified in 11 HCM-affected family members (Girolami et al., 2014), and recent findings from our group indicate that homozygous mice harbouring this mutation are embryonically lethal, possibly due to ACTN2 destabilization (Broadway-Stringer et al., 2023).The precise mechanisms by which ACTN2 variants contribute to HCM remain poorly understood. Herein, we investigated the biophysical implications of ACTN2 variants on protein structure and the functional consequences of the ACTN2-M228T variant to better elucidate potential disease mechanisms.MethodsMissense variants in ACTN2 were identified using the Human Mutation Gene Database (HGMD), followed by in silico characterization. Structural modelling was employed to investigate disease mechanisms. Recombinant mutant proteins were generated via mutagenesis, expressed in, and purified from BL21 bacteria. Structural alterations in ACTN2 variants were examined using biophysical techniques, including mass photometry, X-ray crystallography, small-angle X-ray scattering, actin-binding, and thermal assays. Functional and molecular assessments of the ACTN2-M228T homozygous (Hom) variant in induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM), described in (Cumberland et al., 2023), included contractility analysis, immunofluorescence staining, biochemical fractionation and proteosome and protease inhibitor treatments.Results1. R327C and R457C ACTN2 variants did not disrupt dimerisationUsing the HGMD, 76 ACTN2 variants were identified and further characterized using in silico tools. Of 45 variants predicted to be pathogenic, 20 were associated with HCM. Two of these variants (R327C and R457C) were found to stabilise the ACTN2 dimer structure (figure 1A ). However, neither variant disrupted dimer formation after 48 hours of incubation with high-salt concentration (2.5M NaCl) and rigorous shaking (figure 1B ). Thermal assays showed that the R327C variant had a lower melting temperature than wild-type (figure 1C ) and exhibited increased aggregate formation (figure 1D ).Abstract B Figure 1(A) R327 and R457 contribute to stabilizing the ACTN2 dimer interface ( red). (B) Both variants in solution do not disrupt dimer formation after high salt incubation. (C) R327C variant demonstrates decreased thermal stability. (D) R327C variant is prone to aggregate formation based on molecular weight calculations using mass photometry[Figure omitted. See PDF]2. G111V, M228T, and T247M variants display impaired thermal stability and actin-binding, with M228T showing aggregate formation at 60°CThree ACTN2 variants, G111V, M228T and T247M, showed decreased thermal stability indicating impaired function (figure 2A ). Actin binding assays revealed increased binding affinity to actin for the M228T and T247M variants, and decreased affinity for G111V (figure 2B ). In addition, small angle X-ray scattering (SAXS) analysis of the M228T variant highlighted aggregate formation at 53–60°C (figure 2C-D ).Abstract B Figure 2(A) Thermal assays show a 10°C decrease in melting temperature (Tm) in three ACTN2 variants. (B) Actin-binding assay revealing increased actin binding affinity for M228T and T247M variants and decreased affinity in G111V. (C) SAXS log 10 profile of M228T variant demonstrating aggregate formation at 60°C. (D) M228T variant protein profile displaying aggregation[Figure omitted. See PDF]3. M228T mutant cardiomyocytes display decreased contractility and developmental delaysM228T Hom iPSC-CMs displayed a significant decrease in contraction amplitude and increase in relaxation time relative to wild-type (figure 3A ). Mutant cells also indicated developmental delays evident by decreased MYH7 protein and increased MYH6 mRNA (figure 3B ). In addition, the cells demonstrated a significant upregulation in fetal-gene programme markers including ANKRD1 and FHL1, collagen markers such as COL1A and COL3A, and cardiac stress markers NPPA and NPPB.Abstract B Figure 3(A) M228T Hom iPSC-CMs display decrease contractility analysed using MUSCLE MOTION software. (B) The Hom cells indicate significant decreased in MYH7 protein and increase in MYH6 mRNA. (C) The Hom cells reveal upregulation of fetal-gene markers, fibrosis, and cardiac stress markers. Unpaired t-test was used. Values are the means ± S.D. * p < 0.05; Total n = 5 (Protein), and n=8 (mRNA)[Figure omitted. See PDF]4. M228T cells display protein aggregation, destabilisation and autophagy activationImmunofluorescence staining of mutant cardiomyocytes showed aggregates in ACTN2 and sarcomeric proteins including myomesin, titin, cardiac troponin T, and actin (figure 4A ). Mutant cardiomyocytes showed destabilised ACTN2 using biochemical fractionation techniques (figure 4B ). In addition, M228T Hom cells showed upregulation in autophagy markers (LC3I and LC3II) (figure 4C ).Abstract B Figure 4(A) M228T Hom iPSC-CMs show protein aggregation of ACTN2 (in green), and cardiac proteins (TNNT2: Cardiac-troponin-T, Phalloidin: stain for F-actin, Myomesin; TTN-T12: stain for titin Z-disk portion, in red). (B) Mutant cells highlight significant decrease in soluble ACTN2 upon biochemical fractionation and decrease in myofilament fraction. (C) The cells also show upregulation of autophagy markers. Unpaired t-test was used. Values are the means ± S.D. * p < 0.05; Total n = 5 biological replicates[Figure omitted. See PDF]DiscussionCollectively, this study enhances our understanding of the impact of ACTN2 variants on protein structure and function. Structural modelling elucidated the mechanisms through which these variants contribute to HCM. The biophysical characterisation showed that two variants do not disrupt dimerisation, while three variants affected actin binding. Other variants compromised protein stability, as demonstrated through various assays. Therefore, employing structural analysis before functional studies is an important step in evaluating the pathogenicity of these variants.Moreover, the iPSC-CM model incorporating the M228T v

Hypertrophic cardiomyopathy (HCM) is a genetic disease characterized by diastolic dysfunction and is frequently associated with sudden cardiac death. HCM is associated with pathogenic variants in Z-disk proteins, a key structural component of the sarcomere. Alpha-actinin 2 (ACTN2), is a crucial Z-disk protein important for cross-linking actin filaments. Few ACTN2 variants have been identified, with a limited focus on the mechanisms by which these variants alter protein structure and function. Thereby, this study investigated the biophysical characterisation of multiple ACTN2 variants, as well as assessing the functional impact of the ACTN2-M228T variant in cellular models. ACTN2 missense variants were identified using the Human Mutation Gene Database and characterized with established in silico tools. Structural modelling approaches were then used to predict the impact of HCM-associated variants on ACTN2. To further assess their structural consequences, ACTN2 variants were recombinantly expressed, purified, and analysed using mass photometry, X-ray crystallography, small-angle X-ray scattering, actin-binding and thermal denaturation assays. Additionally, functional and molecular assessments of the ACTN2-M228T variant were performed using induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM).Characterization of the identified ACTN2 variants revealed 18 variants distributed among the different domains of the protein. Structural modeling predictions showed that these variants impact the protein structure through distinct mechanisms, including impaired actin-binding, disrupted dimerization, and reduced protein stability. Actin-binding assays of six variants identified that two variants exhibited enhanced binding, two demonstrated reduced binding, and two showed no change. Additionally, the R327C and R457C variants, predicted to disrupt dimerization, were crystalized, with structures revealing no effect on dimerization. Other variants showed reduced protein stability as evidenced by decreased solubility, impaired thermal stability, alterations in structural conformation, and increased aggregation.The functional impact of the ACTN2-M228T variant was evaluated using iPSC-CMs. Cardiomyocytes harbouring the mutant gene displayed developmental delays, decreased contractility, and upregulation of fibrosis and hypertrophy markers. Protein aggregation and destabilization were confirmed by immunofluorescence and biochemical fractionation techniques. Furthermore, protein degradation mechanisms were examined using inhibitors targeting the ubiquitin-proteosome system and the autophagy-lysosomal pathway, revealing that mutant cells demonstrated autophagy upregulation.Taken together, this study provides valuable insights into how ACTN2 variants impact protein structure and function, contributing to HCM pathogenesis. These findings enhance our understanding of disease mechanisms and may inform the development of targeted therapeutic strategies.

Hypertrophic cardiomyopathy (HCM) is a genetic disease often associated with sudden cardiac death and linked to Z-disc genetic variants. Alpha-actinin 2 (ACTN2) is a key Z-disc protein critical for stabilising the contractile muscle apparatus. A novel missense ACTN2 variant, M228T, was identified in 2014 in a family of 11 HCM patients. A previous in vivo study by our group showed embryonic lethality in mice with the M228T homozygous copy.1–3 However, the mechanism by which this missense variant impacts ACTN2 protein and leads to disease has not been widely investigated. In this study, we examine the functional implications of the ACTN2 M228T variant using biochemical and cellular models.The ACTN2 M228T variant was recombinantly expressed using E.coli, and the structural and thermal stability of the mutant protein was assessed. Functional implications of this variant were further assessed using induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs).Structural modelling predictions revealed that the ACTN2 M228T variant adversely impacts the binding of ACTN2 to actin. Actin-binding assays showed increased binding affinity. Thermal assays showed decreased thermal stability, and enzymatic digestion using thermolysin showed reduced structural stability. Additional assays also showed increased aggregate formation.Moreover, the functional implications of M228T variant were assessed using an iPSC-CM model with mutant cardiomyocytes showing protein degradation, destabilisation, upregulation of hypertrophy and fibrosis. Mutant cells demonstrated impairment cellular quality control and autophagy, suggesting involvement of additional mechanisms.In summary, this study provides valuable insights into the impact of M228T variant on the protein structure and function. These approaches help facilitate our understanding of disease pathways in cardiomyopathy-linked ACTN2 variants.References Noureddine M, et al. Atrial electrical alterations with intact cardiac structure and contractile function in a mouse model of an HCM-linked ACTN2 variant. J Mol Cell Cardiol Plus. 2025 May 17;12:100455. doi: 10.1016/j.jmccpl.2025.100455. PMID: 40503000; PMCID: PMC12153375. Broadway-Stringer S, et al. Insights into the role of a cardiomyopathy-causing genetic variant in ACTN2. Cells. 2023 Feb 24;12(5):721. doi: 10.3390/cells12050721. PMID: 36899856; PMCID: PMC10001372. Girolami F, et al. Novel α-actinin 2 variant associated with familial hypertrophic cardiomyopathy and juvenile atrial arrhythmias: a massively parallel sequencing study. Circ Cardiovasc Genet. 2014 Dec;7(6):741–50. doi: 10.1161/CIRCGENETICS.113.000486. Epub 2014 Aug 30. PMID: 25173926.

Understanding the pathways involved in the formation and stability of the core and shell regions of a platelet-rich arterial thrombus may result in new ways to treat arterial thrombosis. The distinguishing feature between these two regions is the absence of fibrin in the shell which indicates that in vitro flow-based assays over thrombogenic surfaces, in the absence of coagulation, can be used to resemble this region. In this study, we have investigated the contribution of Syk tyrosine kinase in the stability of platelet aggregates (or thrombi) formed on collagen or atherosclerotic plaque homogenate at arterial shear (1000 s−1). We show that post-perfusion of the Syk inhibitor PRT-060318 over preformed thrombi on both surfaces enhances thrombus breakdown and platelet detachment. The resulting loss of thrombus stability led to a reduction in thrombus contractile score which could be detected as early as 3 min after perfusion of the Syk inhibitor. A similar loss of thrombus stability was observed with ticagrelor and indomethacin, inhibitors of platelet adenosine diphosphate (ADP) receptor and thromboxane A2 (TxA2), respectively, and in the presence of the Src inhibitor, dasatinib. In contrast, the Btk inhibitor, ibrutinib, causes only a minor decrease in thrombus contractile score. Weak thrombus breakdown is also seen with the blocking GPVI nanobody, Nb21, which indicates, at best, a minor contribution of collagen to the stability of the platelet aggregate. These results show that Syk regulates thrombus stability in the absence of fibrin in human platelets under flow and provide evidence that this involves pathways additional to activation of GPVI by collagen.

The use of CRISPR-Cas9 genome editing to introduce endogenously expressed tags has the potential to address a number of the classical limitations of single molecule localisation microscopy. In this work we present the first systematic comparison of inserts introduced through CRISPR-knock in, with the aim of optimising this approach for single molecule imaging. We show that more highly monomeric and codon optimised variants of mEos result in improved expression at the TubA1B locus, despite the use of identical guides, homology templates, and selection strategies. We apply this approach to target the G protein-coupled receptor (GPCR) CXCR4 and show a further insert dependent effect on expression and protein function. Finally, we show that compared to over-expressed CXCR4, endogenously labelled samples allow for accurate single molecule quantification on ligand treatment. This suggests that despite the complications evident in CRISPR mediated labelling, the development of CRISPR-PALM has substantial quantitative benefits.

In specialised cells, the expression of specific isoforms of tubulin and their subsequent post-translational modifications are thought to drive and co-ordinate unique morphologies and behaviours. The mechanisms by which β1 tubulin (encoded by TUBB1), the platelet and megakaryocyte lineage restricted β tubulin isoform, drives these processes remains poorly understood. We investigate the effects of two key tubulin post-translational polymodifications (polyglutamylation and polyglycylation) on the glutamate rich C-terminus of β1 tubulin using a cohort of thrombocytopenic patients, human induced pluripotent stem cell (iPSC) derived megakaryocytes, and healthy human donor platelets. We find that while megakaryocytes (MKs) are positive for both polymodifications, polyglycylation is substantially reduced on platelets. On platelet activation, the marginal band becomes heavily polyglutamylated, which drives the mobilisation of motor proteins, including axonemal dynein, to achieve the shape change required for the haemostatic role of platelets. We show that a number of modifying enzymes (Tubulin Tyrosine Like Ligases (TTLLs) and Cytosolic Carboxypeptidases (CCPs)) are up-regulated through MK maturation. In platelets, a single polyglutamylase (TTLL7) is expressed to mediate the polyglutamylation of the marginal band required for shape change on activation. Finally, we report a novel disease causing gene in multiple families (TTLL10) resulting in bleeding despite normal platelet production and function. This work highlights the importance of a complex regulatory mechanism driven by both cell specific tubulin isoform expression and differential post-translational modification to drive specialist function, the loss of which results in disease states. Competing Interest Statement The authors have declared no competing interest. Footnotes * This version of the manuscript has been updated to include data from TUBB1 CRISPR knock-out iPSC line. This data supplements work performed in iPSC derived megakaryocytes which shows differential patterns of polymodification. V3 of this preprint includes minor changes to the language to clarify details of some results. V4 of this manuscript includes improved images and data from a series of constructs used to show the effect of a C-terminal truncation on the protein.

The use of CRISPR-Cas9 genome editing to introduce endogenously expressed tags has the potential to address a number of the classical limitations of single molecule localisation microscopy. In this work we present the first systematic comparison of inserts introduced through CRISPR knock-in, with the aim of optimising this approach for single molecule imaging. We show that more highly monomeric and codon optimised variants of mEos result in improved expression at the TubA1B locus, despite the use of identical guides, homology templates, and selection strategies. We apply this approach to target the G protein-coupled receptor (GPCR) CXCR4, and show a further insert dependent effect on expression and protein function. Finally, we show that compared to over-expressed CXCR4, endogenously labelled samples allow for accurate single molecule quantification on ligand treatment. This suggests that despite the complications evident in CRISPR mediated labelling, the development of CRISPR-PALM has substantial quantitative benefits.