Welcome and General Information
Welcome to The Platelet Society Online Meeting,
When we started planning this meeting we had envisioned hosting you in the stunning Staffordshire countryside that surrounds Keele. Little did we know that instead you would be hosting us in your houses, offices and labs around the world.
Whilst the COVID-19 pandemic has made it impossible to meet physically, it has allowed the global platelet research community to come together in new ways online. Through this challenging time for everyone, we have found comfort and inspiration from all those people conducting amazing research in the most challenging of circumstances. It is perhaps fitting then that this online meeting will allow us to gather delegates from across the globe and share the results of all these efforts.
We hope that we will be able to return to in-person meetings in the future, including the forthcoming Platelet Summer School.
The event has taken many twists and turns during its organisation, and we would not have got here without a team of people who have supported us along the way. The organising Committee would like to thank those people who have kindly volunteered their time to help us make this meeting possible. Thank you to Gayle Halford, Steve Thomas, Jon Gibbins, Sam Montague, Gagan Flora, Matthew Harper, Khalid Nasseem and Ingeborg Hers – we could not have done this without each of your contributions.
We hope you enjoy the meeting!
Alan, Amanda, Carly, David, Kirk & Paul
The Keele Platelet Society Organising Committee
Alan Harper, School of Medicine, Keele University
Amanda Unsworth, Centre for Bioscience, Manchester Metropolitan University
Carly Kempster, Institute of Metabolic and Cardiovascular Research, University of Reading
David Cabrera, School of Pharmacy and Bioengineering, Keele University
Kirk Taylor, National Heart & Lung Institute, Imperial College London
Paul Armstrong, Blizard Institute, Queen Mary University of London
At this meeting we will be using the ReAttendance platform for the oral communications and discussion panels and GatherTown for the Poster session.
Full details on how to use these platforms can be foud in the PDF document below.
The Platelet Society and Organising Committee are committed to making this event productive and enjoyable for everyone, regardless of sex, sexual orientation, disability, age, physical appearance, body size, ethnicity, nationality or religion. We will not tolerate harassment of participants in any form. By accessing the online conference platform, all attendees are agreeing to abide by this Code of Conduct.
Code of Conduct
Behave professionally. Harassment and sexist, racist, or exclusionary comments or jokes are not appropriate. Harassment includes sustained disruption of talks or other events, inappropriate physical contact, sexual attention or innuendo, deliberate intimidation, stalking, and photography or recording of an individual without consent. It also includes offensive or belittling comments related to gender, sexual orientation, disability, age, physical appearance, body size, ethnicity or religion.
All communication should be appropriate for a professional audience including people of many different backgrounds. Sexual language and imagery are not appropriate. Be kind to others. Do not insult or put down other attendees.
Incident reporting and resolution
If you observe someone making you or anyone else feel unsafe or unwelcome, please tell them so, and remind them of the Code of Conduct.
If you are hesitant about addressing the person yourself, report it as soon as possible to a member of the Organising Committee – see contact information below. The Committee is committed to addressing and resolving the matter to the best of their abilities.
Please use the following contact information and explain what happened and who was involved so that we can investigate.
Dr Alan Harper (he/him): A.G.S.firstname.lastname@example.org
Dr Amanda Unsworth (her/hers): A.Unsworth@MMU.ac.uk
Carly Kempster (her/hers): C.R.Kempster@pgr.reading.ac.uk
Dr David Cabrera (he/him): D.Cabrera@keele.ac.uk
Dr Kirk Taylor (he/him): K.A.Taylor@reading.ac.uk
Dr Paul Armstrong (he/him): P.C.Armstrong@QMUL.ac.uk
Thank you for your participation in the Platelet Society Conference and your efforts to keep our event welcoming, respectful, and friendly for all participants!
When someone is asked to stop any behaviour that makes others uncomfortable, they are expected to comply immediately. In response to inappropriate behaviour (e.g. sexual content, rudeness, unprofessional) organisers may take any action they deem appropriate, including warning the person in question, asking them to leave the event, or removing them from a mailing list.
Specific actions may include but are not limited to:
- asking the person to cease the inappropriate behaviour, and warning them that any further reports will result in other sanctions.
- requiring that the person avoid any interaction with another person for the remainder of the event.
- early termination of a talk that violates the policy.
- not publishing the video or slides of a talk that violates the policy.
- not allowing a speaker who violated the policy to give (further) talks at the event.
- immediately ending any event responsibilities or privileges held.
- requiring that the person immediately leave the event and not return.
- blocking the person on social media platforms (for one year).
- banning the person from future events (for one year).
- publishing an anonymous account of the harassment.
- reporting the incident to the person’s employer or educational institution.
The Platelet Society is delighted to announce the return of the Platelet Summer school in 2021. Hosted by the University of Reading, this will introduce new researchers to all aspects of platelet biology with sessions taught by leaders in the field from across Europe. 1st – 3rd September 2021
We have an exciting programme for you, spread over the three days of the meeting. Select the day below to explore the sessions.
Time (BST) Activity Speaker 12:30 Welcome Communications 1: Main Stage 12:40 Sex-related variations in the response of anti-platelet drug therapies targeting purinergic signaling pathways in sepsis Elisabetta Liverani 12:55 Proteolysis targeting chimeras: proteasomal degradation of BTK and TEC as a novel approach in thrombosis prevention Attila Munkacsi 13:10 Assessment of anti-platelet drug efficacy using a novel endothelialised thrombosis model. Ryan Riley 13:25 Break Career Workshop Main Stage 13:30 CV Masterclass (Theme 1)
Attaining a Research Fellowship or Academic Career
Patricia Munroe 13:45 CV Masterclass (Theme 2)
Getting Industry Ready
Louise Armstrong-Denby 14:00 Career Panels
Theme 1: Academic careers & research fellowships
Networking Rooms Career Panels
Theme 2: Industry & non-academic careers
Networking Rooms 14:45 Break Communications 2: Main Stage 15:00 Tissue-engineered human arteries replicate primary and secondary haemostatic functions seen in vivo: An alternative to mouse thrombosis models? Jacob Ranjbar 15:15 Platelet transcriptome yields progressive and predictive markers for subtype-specific risk in myeloproliferative neoplasms Anandi Krishnan 15:30 Elevated platelet-derived sGPVI is a biomarker of venous in-stent stenosis in patients with post-thrombotic syndrome Adam Gwozdz 15:45 Break Communications 3: Main Stage 15:50 Investigating the role of the novel, monoallelic GPIba G138V variant in patients with inherited bleeding Jack Yule 16:05 The importance of the GPIba intracellular tail in VWF-mediated platelet signalling events Adela Constantinescu-Bercu 16:20 Platelet Society Initiatives 16:30 Break 17:00 Poster Browsing & Social Networking GatherTown 19:00 Day 1 close
We are delighted to announce the following people who will be joining our career workshop panel.
Career Panel Members Industry & non-academic careers Dr Louise Armstrong-Denby Chief Commercial Officer, Visiopharm Dr Suze Kundu Head of Public Engagement, Digital Science Dr Melissa Hayman Research Adviser, British Heart Foundation Academic careers & research fellowships Prof. Patricia Munroe Professor of Molecular Medicine, QMUL Dr Nicholas Kirkby BHF Intermediate Fellow, Imperial College London Dr Mark Thomas Clinical Lecturer, University of Birmingham Dr Amanda Unsworth Lecturer, Manchester Metropolitan University Time (BST) Activity Speaker 12:30 Welcome Alan Harper Communications 1: Thromboinflammation Main Stage 12:40 Public Plenary Lecture: Clotting complexities of COVID-19 Nicola Mutch 13:10 Contribution of the TF-driven coagulation in the thrombotic complications of COVID-19 patients: insights into platelet and endothelial activation Marta Brambilla 13:25 Protecting the coronary microcirculation from thromboinflammatory events post-reperfusion injury – an intravital microscopy study of the beating mouse heart Neena Kalia 13:40 Altered platelet proteome, enhanced basal platelet procoagulant activity, and increased platelet-leukocyte interactions in patients with Covid-19 Lucy Goudswaard 13:55 Break Society Networking Main Stage 14:10 Platelet Summer School Jon Gibbins 14:15 Discussion Group Introduction Alan Harper 14:20 Discussion Group Break-out Rooms Networking Rooms 14:50 Break 15:05 Gold Sponsor Talk: Izon Science Main Stage Communications 2: Megkaryocyte and Platelet signalling Main Stage 15:15 Plenary Lecture: A Role for Platelet-Derived Vesicles in Megakaryopoiesis During Inflammation Kellie Machlus 15:45 Stimulation of the ssRNA virus immune receptor, Toll-like receptor 7, on megakaryocytes increases thrombopoesis Amie Waller 16:00 Tunability of platelet-derived extracellular vesicles Mari Palviainen 16:15 The intracellular tail of GPIba augments GPVI-mediated signaling in mice Isabelle Salles-Crawley 16:30 GPVI clustering investigation by Agent-Based Modelling (ABM) approach Chukiat Tantiwong 16:45 Break 17:00 Poster Session 18:00 Online Social 19:00 Day 2 Close Time (BST) Activity Speaker Equity, Diversity and Inclusion Spotlight:
Mentoring within The Platelet Society
Main Stage 12:30 Introduction Amanda Unsworth 12:40 Mentoring discussion Groups Networking Rooms 13:20 Break 13:30 Gold Sponsor Talk - Stago UK Main Stage Communications 3: New Horizons in Platelet Research Main Stage 13:40 Anti-GPVI nanobodies as a novel tool to inhibit thrombus formation on atherosclerotic plaque under flow Natalie Jooss 13:55 Mechanisms of fibronectin fibrillogenesis by platelets Ingmar Schoen 14:10 High-throughput, Functional Mechanophenotyping of Platelet Activation Induced by Bacterial Proteins Raghavendra Palankar 14:25 Generating large numbers of in vitro platelets from Human induced stem cell derived Megakaryocytes via a platelet extruding bioreactor. Daniel Howard 14:40 Plenary Lecture: Platelet-inspired Nanotechnologies for Applications in
Hemostasis, Thrombosis and Thromboinflammation
Anirban Sen Gupta 15:10 Break Gustav Born Lectures Main Stage 15:30 Introduction Alan Harper 15:35 Developing iPSC derived and bioprinted organoids for the study of platelet production and biology Abdullah Khan 15:55 Investigations into mitochondrial dynamics during platelet ageing and reactivity Harriet Allan 16:15 Magnetic nanoparticles as a therapeutic and diagnostics platform for cardiovascular disease David Cabrera 16:35 Prize Giving and Meeting Close
Abstracts – Monday
Sex-related variations in the response of anti-platelet drug therapies targeting purinergic signaling pathways in sepsis
1Department of Pharmaceutical Sciences North Dakota State University, Fargo ND, USA. 2Sol Sherry Thrombosis Research Centre, Philadelphia PA, USA.
E-mail of Presenting Author: elisabetta.liverani@ndUS.edu
Background: Sepsis is a major healthcare problem. There are sex-differences in the immune response to sepsis. Platelets isolated from male and females show a differential response to ADP, indicating that activation of ADP-receptors P2Y12 or P2Y1 are sex-specific. Anti-platelet therapy targeting P2Y12 , but not P2Y1, improved sepsis outcomes in male mice.
We aim to study sex differences in modulating P2Y12 or P2Y1 during sepsis.
Methods: Male and female wild type (WT), P2Y12 knock-out (KO) and P2Y1 KO mice underwent sham surgery or cecal ligation and puncture (CLP) to induce sepsis. Sepsis-induced changes in circulating white blood cells (WBC), neutrophils and platelets were measured 24 hrs post-surgery. Neutrophil infiltration in the lung was determined by myeloperoxidase (MPO) activity. P-selectin surface expression in platelets and platelet-leukocyte aggregates was determined by flow cytometry.
Results: Sepsis induced increased circulating WBC, platelet activation, platelet-leukocyte aggregates, and neutrophil trafficking to the lungs in male and female WT mice as compared to Sham. Platelet activation, aggregates and neutrophil infiltration were less severe in CLP WT female compared to WT male. Circulating WBC, neutrophils and platelets, platelet activation and neutrophil infiltration did not differ between WT CLP and CLP P2Y12 KO in female mice. CLP P2Y12 KO male mice significantly improved as compared to male WT. In female mice, platelet activation and aggregates were significantly reduced in CLP P2Y1 KO compared with CLP WT. Sepsis-induced MPO activity was reduced in CLP P2Y1 KO female.
Conclusions: Identifying sex-specific purinergic signalling targets will lead to a sex-related therapy in sepsis.
Proteolysis targeting chimeras: proteasomal degradation of BTK and TEC as a novel approach in thrombosis prevention
Attila Munkacsi1, Kamila Sledz1 and Ingeborg Hers1
1School of Physiology, Pharmacology and Neuroscience, Biomedical Sciences Building, University of Bristol, Bristol, BS8 1TD, UK.
E-mail of Presenting Author: email@example.com
Background: Bruton’s tyrosine kinase (BTK) plays an important role in platelet signalling downstream of GPVI receptor and has been proposed as a novel target to prevent thrombosis in high-risk patients. Current, clinically used BTK inhibitors (e.g. acalabrutinib, ibrutinib), however, have off target effects and are associated with an increased risk of bleeding.
Aims: Our study is focussed on the targeted chemical degradation of BTK in human platelets by using recently developed heterobifunctional small molecule degraders (PROteolysis TArgeting Chimeras, PROTACs) that utilise the proteasomal system to degrade BTK. We aim to explore the repurposing potential of PROTACs as novel anti-platelet therapy and to see whether these compounds can be used as pharmacological research tools to study platelet function.
Methods: Human platelet rich plasma (PRP) was treated with various concentrations of the PROTACs and incubated overnight at 30°C. Samples were then either lysed and subjected to immunoblotting, or were assessed for integrin aIIbb3 activation, P-selectin expression and Annexin V binding in response to the GPVI and PAR agonists CRP or a-thrombin, respectively.
Results: The multi-kinase degrader TL 12-186 and the BTK degraders DD 04-15 and DD 03-171 show potent BTK degradation, which can be overcome by addition of proteasomal inhibitors. GPVI-mediated integrin activation, P-selectin expression and PS exposure were significantly impaired, whereas PAR-mediated responses were unaffected. Tandem mass tagging confirmed the high specificity of BTK degradation.
Conclusions: Our data confirm the high susceptibility of human platelets to BTK degraders, therefore PROTACs can be successfully utilised in modulating and studying human platelet function.
Assessment of anti-platelet drug efficacy using a novel endothelialised thrombosis model.
Ryan Riley1, Amanda Unsworth1, Stephen White1, and Sarah Jones1
1Department of Life Science, Manchester Metropolitan University, Manchester, UK.
E-mail of Presenting Author: Ryan.firstname.lastname@example.org
Currently there is not a gold standard in vitro thrombosis model. Although current models have been beneficial in furthering our understanding of thrombosis, they have several limitations. One key limitation of current in vitro models is the absence of endothelial cells, which play a key role in regulating platelet activity in vivo. The aim of this study is to develop an endothelialized in vitro model of atherothrombosis, which enables anti-platelet drug efficacy to be assessed.
Direct endothelial cell contributions were measured by perfusing whole blood through Human umbilical vein endothelial cell (HUVECs) lined chambers, prior to perfusion over immobilized collagen. The ability of the model to assess anti-platelet drug efficacy was analysed using aspirin (30μM) and Clopidogrel (10μM).. The effects of endothelial dysfunction on thrombosis were investigated by exposing the HUVECs to tumour necrosis factor alpha (10ng/ml) for 24 hours . Endothelial activation was confirmed using western blotting.
The addition of healthy endothelial cells into the flow model significantly reduced thrombus size (20.61%; P<0.05) and thrombus surface coverage (32.77%; P<0.001). Aspirin and Clopidogrel both significantly decreased average thrombus size (P<0.05).The antithrombotic effects of the endothelial cells was diminished by stimulation with TNF-alpha leading to an increase in cell adhesion molecules VCAM-1 (P<0.05) and ICAM-1 (P<0.05). Endothelial activation significantly increased the size of the thrombus formed on the immobilized collagen (P<0.05).
The novel endothelialised thrombosis model presented in this study demonstrates the key role the endothelium plays in regulating platelet activity and the importance of its inclusion in in vitro thrombosis models.
As a research society our aim is to support our members regardless of their career stage. This is all the more important with Early Career Researchers! Surveys have shown that the majority of people taking on doctoral studies intend to pursue an academic career. However the route to that academic career is increasingly hard and uncertain and most will go on to pursue careers in non-university research, research support roles or even outside of science.
A PhD or postdoctoral role therefore is not just about doing experiments and publishing papers but also about gaining the transferable skills and experience that will help you to be successful in whatever career path you choose.
To start the workshop we have a CV masterclass to help you:
- understand what potential employers look for when recruiting
- sell your best self on paper
- identify skillsets and areas to improve
Our academic CV Masterclass is delivered by Prof. Patricia Munroe.
Prof. Munroe is Professor of Molecular Medicine at QMUL who leads research programmes identifying genetic variants for hypertension, cardiac arrhythmias and heart failure. In addition to her work being funded by the British Heart Foundation (BHF), Medical Research Council, NIHR and The Wellcome Trust, Prof Munroe is a Thomson Reuters Highly Cited Researcher and a past member of the BHF fellowship committee.
Our industry CV Masterclass is delivered by Dr. Louise Armstrong-Denby.
Dr Armstrong-Denby is currently Chief Commercial Officer for Visiopharm and has 20 years international business experience in commercialising technology particularly for cellular imaging and analysis in disease research and drug discovery. Her resume includes roles at PerkinElmer, Andor Technologies and Random 42. In addition to her current role, Dr. Armstrong-Denby is an industrial advisor and steering committee member of the Centre for Doctoral Training at Birmingham University, and a Trustee of the Royal Society of Chemistry.
Career Panel Q&A
After our masterclass presentations we have two concurrent career panel Q&A sessions in dedicated networking rooms to allow you to ask your questions. These sessions will run very much like a zoom call where we can have the maximum amount of interaction and discussion between panel members and attendees. You are welcome and encouraged to switch between the two sessions, so you get to hear the valuable advice being offered in both. (This will be done via a technician managed holding room so please be patient should you experience any delays.)
On our academic theme panel Prof. Munroe will be joined by Dr Mark Thomas (NIHR clinical lecturer), Dr Nicholas Kirkby (BHF intermediate fellow) and Dr Amanda Unsworth (Lecturer) to answer your academia and research fellowship related questions.
On our industry theme panel, Dr Armstrong-Denby is joined by Dr Suze Kundu (Head of Public Engagement, Digital Science) and Dr Melissa Hayman (Research Adviser, British Heart Foundation) to help answer your questions exploring non-academia career roles.
If you have any feedback for the Platelet Society following your these sessions please email this to email@example.com or include in the post-meeting survey.
Communications 2 & 3
Tissue-engineered human arteries replicate primary and secondary haemostatic functions seen in vivo: An alternative to mouse thrombosis models?
Jacob Ranjbar1, Ying Yang2, Alan G.S. Harper1
1 School of Medicine, University of Keele, UK.
2 School of Pharmacy and Bioengineering, University of Keele, UK.
E-mail of Presenting Author: firstname.lastname@example.org
Background: Intravital microscopy in mice is widely used to study in vivo thrombus formation. Previously we have developed a 3D, tissue-engineered human arterial construct (TEAC) that replicates the primary haemostatic properties of the native artery .
Aims: To assess whether our TEAC can trigger the activation of the extrinsic coagulation cascade, and whether this can be used to generate a human arterial thrombosis model to replace current in vivo studies.
Methods: Medial and adventitial arterial layers were replicated by seeding human smooth muscle and adventitial cells into a collagen hydrogel. Prothrombin times were measured by exposing these constructs to Platelet-poor plasma (PPP) from healthy human volunteers. A novel fluorogenic tissue factor assay was performed to assess tissue factor activity by incubation with factor VII and SN17a. Platelet activation was assessed using physiological flow conditions in a 3D printed flow chamber.
Results: Both the medial and adventitial layer constructs are able to trigger rapid coagulation when exposed to PPP. Both constructs have measurable tissue factor activity. Tissue factor activity in the medial layer construct was enhanced by culturing these in the presence of ascorbic acid. Ascorbic acid supplementation of the medial construct also significantly increased platelet adhesion and aggregation on this construct under flow conditions.
Conclusions: Our data demonstrates that our TEACs can replicate all of the major haemostatic processes of the native blood vessel and this was enhanced by supplementation of ascorbic acid into the culture media. The TEAC is a viable replacement to current murine thrombosis models.
This work is supported by an NC3R- and British Heart Foundation co-funded PhD studentship
Refs:  Musa et al. (2016) Tissue Eng Part C Methods 22, 691-699.
Platelet transcriptome yields progressive and predictive markers for subtype-specific risk in myeloproliferative neoplasms
Zhu Shen2, Wenfei Du2, Cecelia Perkins4, Lenn Fechter4, Vanita Natu3, Holden Maecker5, Jesse Rowley6, Jason Gotlib4, James Zehnder1,4, and Anandi Krishnan1.
1 Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA.
2 Department of Statistics, Stanford University, Stanford, CA, USA.
3 Stanford Functional Genomics Facility, Stanford University, Stanford, CA, USA.
4 Division of Hematology, Department of Medicine, Stanford University School of Medicine and
Stanford Cancer Institute, Stanford, CA, USA.
5 Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA.
6 Department of Internal Medicine, University of Utah School of Medicine, Salt Lake City, UT, USA.
E-mail of Presenting Author: email@example.com
Background: Genotype-phenotype relationships in the pathogenesis of classic, Philadelphia chromosome (Ph-)-negative myeloproliferative neoplasms (MPNs) remain elusive beyond their key driver mutations (JAK2,CALR or MPL in over 90% of patients). Platelets hold vast amounts of biologic information reflecting the diversity in the genes that encode their canonical functions, but also disease- and treatment-related factors.
Aims: Here, we probe the platelet transcriptome in chronic progressive MPNs with the primary aim of defining subtype-specific molecular differences and predicting the advanced phenotype, myelofibrosis.
Methods: Using RNA sequencing (RNA-seq), we profile disease-relevant gene expression and alternative splicing in purified platelets from 120 peripheral blood samples that constitute two independently collected (mutually validating) cohorts of the three chronic clinical phenotypes of MPN patients – essential thrombocythemia, ET (n=24), polycythemia vera, PV (n=33), and primary or secondary myelofibrosis, MF (n=42) – and healthy donors (n=21).
Results: The MPN platelet transcriptome discriminates each clinical phenotype and reveals an incremental molecular reprogramming that is independent of driver mutation status or therapy. Leveraging this dataset, in particular the progressive expression gradient noted across MPN, and a novel application of established Lasso-penalized regression modeling with 10-fold cross-validation, we predict the advanced subtype MF at high accuracy (AUC-ROC of 0.95) and offer a robust core set of < 10 MPN progression markers.
Conclusion: Our platelet transcriptome snapshot of chronic MPNs establishes a methodological foundation for deciphering disease risk stratification and progression beyond genetic data alone, thus presenting an important avenue toward personalized medicine in a wide range of age-related disorders.
Elevated platelet-derived sGPVI is a biomarker of venous in-stent stenosis in patients with post-thrombotic syndrome
Adam M. Gwozdz1, Prakash Saha1, Stephen A. Black1,Alexander P. Bye2, Neline Kriek2, Jonathan M. Gibbins2, and Alberto Smith1
1 Academic Department of Vascular Surgery, Section of Vascular Risk and Surgery, School of Cardiovascular Medicine and Science, St Thomas’ Hospital, King’s College London, UK
2 Institute for Cardiovascular and Metabolic Research, School of Biological Sciences, University of Reading, UK
E-mail of Presenting Author: firstname.lastname@example.org
Background: In-stent stenosis following intervention for post-thrombotic syndrome (PTS) occurs in ~30% of cases, despite therapeutic anticoagulation.
Aims: The aim of this study was to investigate whether platelets have a role in this process.
Methods: Blood samples were taken from case-matched patients before and after venous stent placement. Platelet activation markers (P-selectin and phosphatidylserine) and reactivity were determined by flow cytometry and plate-based aggregation, respectively. Soluble glycoprotein VI (sGPVI), shed during activation, was measured in plasma. Patients with in-stent stenosis requiring reintervention (>50% diameter reduction) were compared with those who did not during follow-up.
Results: Forty-five patients were recruited (median age: 43yrs, range: 33-55yrs; 65% female). Re-intervention was required in 19 patients (42%; median time: 3wks, range: 1day-3mths). There was no significant difference in platelet activation or reactivity after stenting, but P-selectin exposure pre-stent was significantly higher in patients who developed in-stent stenosis (2.7%±0.4 vs 1.7%±0.2; P<0.05). sGPVI levels before stent insertion were increased in patients who developed in-stent stenosis (18.9±3.6ng/mL vs. 7.4±0.9ng/mL; P<0.01). Platelet reactivity to collagen-related peptide, a GPVI-specific platelet agonist, was reduced in patients who developed in-stent stenosis (logEC50 = -6.5M±0.3 vs -7.2M±0.2; P<0.05; n=33).
Conclusions: Venous stenting does not activate platelets or alter platelet function, but patients who developed in-stent stenosis exhibited greater levels of pre-stent platelet activation, greater loss of platelet surface GPVI in the form of sGPVI and consequent reduction in reactivity to GPVI activation. sGPVI may have potential to risk-stratify patients undergoing deep venous reconstruction and predict who requires closer surveillance.
Investigating the role of the novel, monoallelic GPIba G138V variant in patients with inherited bleeding
J.I. Yule1,2, I. Almazny3, C.W. Smith3, R.J. Stapley3, N.J. Jooss3, J. Emsley1,2, N.V. Morgan3 and N.S. Poulter1,3
1Centre of Membrane Proteins and Receptors, University of Birmingham, Edgbaston, Birmingham B15 2TT. The University of Nottingham, University Park, Nottingham, NG7 2RD
2Centre for Biomolecular Sciences, School of Pharmacy, University of Nottingham, Nottingham, NG7 2JE
1,3Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK.
E-mail of Presenting Author: Jack.Yule@nottingham.ac.uk
Background: GPIbα-VWF binding is essential in tethering of platelets to a vascular injury site. The majority of GPIbα variants are linked with Bernard-Soulier syndrome and are homozygous. These typically present as macrothrombocytopenia with ablated GPIb-IX-V complex expression on the platelet surface.
Aims: Through the Genotyping and Phenotyping of Platelets Project (GAPP), an atypical monoallelic GPIbα variant (G138V) was discovered in two siblings who exhibit a mild thrombocytopenia and bleeding symptoms. This work aims to characterise this variant and uncover the mechanism by which it contributes to bleeding.
Methods: Standard platelet function assays (e.g. FACS, aggregometry and spreading) were carried out using blood samples from one patient to assess the effect of the G138V variant on receptor levels and platelet activation. A whole blood flow adhesion assay over different substrates was also used to quantify the effect of G138V on thrombus formation and platelet activation. Microscopy was used to look at GPIbα localisation.
Results: The patient has normal platelet surface GPIbα, but exhibits reduced platelet activation and aggregation responses to standard agonists. Flow adhesion showed impaired platelet deposition and activation on VWF, collagen and fibrinogen coated surfaces. Significantly more spread patient platelets contained a ‘centralised’ GPIbα structure compared to controls.
Conclusions: We observe a platelet functional defect in a patient with a heterozygous G138V variant of GPIbα. Patient platelets show reduced activation, adhesion and thrombus formation under flow conditions, which is likely to contribute to the observed bleeding. Further work is now required to evaluate the extent to which this variant affects GPIbα-VWF binding capacity.
The importance of the GPIba intracellular tail in VWF-mediated platelet signalling events
Adela Constantinescu-Bercu1, Yuxiao Alice Wang1, Kevin J Woollard2, Pierre Mangin3, Karen Vanhoorelbeke4, James TB Crawley1 and Isabelle I. Salles-Crawley1
1Centre for Haematology, Department of Immunology and Inflammation, Imperial College London, London, UK
2Centre for Inflammatory Disease, Department of Immunology and Inflammation, Imperial College London, London, UK
3Université de Strasbourg, INSERM, EFS Grand-Est, BPPS UMR-S 1255, FMTS, Strasbourg, France
4Laboratory for Thrombosis Research, KU Leuven, Kortrijk, Belgium.
E-mail of Presenting Author: email@example.com
Background: The role of the VWF A1 domain interaction with platelet GPIba has classically been limited to platelet capture at sites of vessel injury. We recently demonstrated that human VWF-bound platelets are ‘primed’ under flow, recruiting neutrophils and inducing NETosis via activated aIIbβ3.
Aims: To evaluate the (patho)physiological importance of VWF/flow-dependent signalling through GPIba.
Methods: GPIbaDsig/Dsig mice were generated via the CRISPR-Cas9 technology, by deleting the last 24 amino acids from the intracellular tail of GPIba. Platelet function was assessed ex vivo and in vivo.
Results: GpIbaDsig/Dsig mice exhibited normal tail-bleeding responses, and unchanged platelet/fibrin accumulation in the laser-induced thrombosis model. GpIbaDsig/Dsig platelets bound normally to VWF under flow, but had a reduced ability to form filopodia on VWF, in the presence of Botrocetin and GR144053, confirming that VWF-dependent signalling is impaired in these mice. GPIba+/+ primed platelets recruited neutrophils under low shear, in a manner that could be inhibited by aIIbβ3 blockade, but not by β2-integrin inhibition. Neutrophil recruitment was significantly increased in the absence of plasma. GPIbaDsig/Dsig platelets exhibited a significantly reduced ability to recruit neutrophils compared to GPIba+/+ platelets, confirming the importance of the intracellular tail of GPIba in mediating interactions between VWF-‘primed’ platelets and neutrophils under flow.
Summary/Conclusions: Our data suggest an important role for the GPIba intracellular tail to transduce signals downstream of the VWF A1-GPIbα interaction and mediate subsequent platelet-neutrophil interactions. Additional thrombosis models and platelet signalling assays are underway to fully appreciate the pathophysiological role of the GPIba-A1 signalling.
Abstracts – Tuesday 30th March
Communications 1: Thromboinflammation
Clotting Complexities of Covid-19
Dr Nicola J. Mutch
Aberdeen Cardiovascular & Diabetes Centre, School of Medicine, Medical Sciences and Nutrition, University of Aberdeen, UK.
Coronavirus disease 2019 (COVID-19) results from infection with coronavirus, SARS-CoV2, which emerged Wuhan, China in late 2019. To date COVID-19 has resulted in almost 2.5 million deaths worldwide and more than 100,000 in the UK alone. The severity of the disease varies with some people being asymptomatic, whilst for others it can lead to severe breathing difficulties that may require ventilation. It is now clear that the disease not only results in respiratory complications but also affects other organs, such as the heart and kidneys. Many patients experience abnormal blood clot formation which can lead to further complications such as heart attacks, strokes, deep vein thrombosis and clots in the lungs. Patients are given standard anticlotting drugs but surprising many (30-50%) still develop clots. Onset of severe COVID-19 results in a hyperinflammatory response which in turn disrupts the normal function of blood vessels and the composition of the blood. These factors promote ongoing blood clot formation. Blood clots, which are composed of blood cells including platelets and a scaffold protein called fibrin, can form within the lung but also in other areas of the body. One thing that has become evident over the past few months is that certain groups of individuals with additional risk factors, such as hypertension, diabetes, increased BMI and previous cardiovascular events are more of risk of developing severe COVID-19 disease. Understanding the key players promoting inflammatory dysregulation of haemostasis and their correlation with disease severity will permit early identification of severe COVID-19 disease and help predict those at risk of immunothrombotic complications. This may reshape the conventional approach to antithrombotic therapy in severely ill patients with COVID-19 and will indefinitely provide greater understanding of the complex aetiology of this multifaceted disease.
Contribution of the TF-driven coagulation in the thrombotic complications of COVID-19 patients: insights into platelet and endothelial activation
Marta Brambilla1, Paola Canzano1, Elena Tortorici2, Nicola Cosentino1, Benedetta Porro1, Martino Pengo2, Alice Bonomi1, Fabrizio Veglia1, GianFranco Parati2, Viviana Cavalca1, Emanuela Omodeo Salè1, Elena Tremoli1, Marina Camera1,3
1Centro Cardiologico Monzino IRCCS, Milan,
2Istituto Auxologico Italiano, Milan
3Department of Pharmaceutical Sciences, Università degli Studi di Milano, Milan, Italy
E-mail of Presenting Author: firstname.lastname@example.org
Background: The cytokine storm described in severe COVID-19 pneumonia may lead to consistent cell-based Tissue Factor (TF)-mediated activation of blood coagulation, procoagulant microvesicles (MVs) release and massive platelet activation.
Aims: To assess in 46 COVID-19 patients: 1) levels of TF+ circulating cells and MVs; 2) residual plasma thrombin generation (TG) capacity; 3) platelet and endothelial activation. Finally, through an in vitro approach, we verified whether: 4) plasma from COVID-19 patients could reproduce the platelet activation observed in vivo when added to blood cells from healthy subjects (HS); 5) treatment with antiplatelet drugs was effective in reverting platelet activation.
Methods: TF+-platelets, -monocytes, -granulocytes, and -platelet-leukocyte aggregates (PLA), P-selectin, percentage of PLA and MVs were evaluated by whole-blood-flow cytometry. Thrombin generation (TG) and nitric oxide (NO) and PGI2 synthesis were analyzed by CAT and LC-MS/MS, respectively. HS and coronary artery disease (CAD) patients were used for comparison.
Results: COVID-19 patients had higher levels of TF+-platelets, -granulocytes and -MVs (2-to-3-fold) than HS and CAD (p<0.0001). A residual MV-associated-TG was measured in plasma of patients treated with prophylactic anticoagulation. Ten-fold increase of P-selectin expression and PLA formation vs HS and CAD was also observed. Also, NO and PGI2 synthesis was deeply affected (p<0.0001). COVID-19 plasma, added to blood of HS, induced platelet activation similar to that observed in vivo. This effect was blunted by preincubation with aspirin and AR-C69931MX.
Conclusion: These results provide insights into the IL-6 mediated platelet activation that triggers the hypercoagulable state in COVID-19 suggesting the potential effectiveness of antiplatelet drugs.
Protecting the coronary microcirculation from thromboinflammatory events post-reperfusion injury – an intravital microscopy study of the beating mouse heart
Kobkaew Bumroongthai1, Juma El-Awaisi1, Dean P.J. Kavanagh1, Paul Genever2, Neena Kalia1
1Microcirculation Research Group, Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, B15 2TT, UK,
2Department of Biology, University of York, York, YO10 5DD.
E-mail of Presenting Author: email@example.com
Background: We have previously shown that myocardial ischaemia-reperfusion (IR) injury induces thromboinflammatory disturbances in the beating mouse heart microcirculation in vivo (Kavanagh et al.Cardiovascular Research 2019: 115:1918-32). The ability of bone marrow-derived mesenchymal stem cells (BM-MSCs), known to possess anti-inflammatory properties, and the anti-coagulant heparin, to confer coronary vasculoprotection was investigated intravitally.
Methods: BM-MSCs tested were: (i) tri-lineage differentiating CD317– Y201 cells or (ii) poorly differentiating CD317+ Y202 cells. Conventional 2D or 3D hanging drop methods were used to culture MSCs. The kinetics of MSC, neutrophil and platelet recruitment within coronary microvessels was imaged intravitally in the IR injured beating heart of anaesthetised mice (Ketamine/Medetomidine; i.p.). Myocardial infarction was assessed by TTC staining.
Results: Homing of 3D MSCs was significantly (p<0.05) higher than 2D MSCs. Only 3D Y201 MSCs significantly (p<0.05) reduced neutrophil recruitment within coronary microvessels, with no effect on platelets observed. Unfractionated heparin when administered alone reduced occlusive platelet microthrombi presence but concomitantly increased (p<0.001) neutrophil infiltration. Combination therapy enhanced 3D Y201 MSC homing, decreased microthrombus formation but did not modify neutrophil recruitment when compared to MSCs alone. Infarct size was decreased (p<0.05) in mice receiving 3D Y201 MSCs.
Conclusion: 3D culture of an MSC sub-population with high differentiating capacity showed greater immunomodulatory and cardioprotective capacity. Although heparin alone was an effective anti-platelet strategy, removal of obstructive microthrombi led to a remarkable infiltration of neutrophils within the beating coronary microcirculation. However, combination therapy, involving MSCs + heparin could be considered for treating myocardial reperfusion injury.
Altered platelet proteome, enhanced basal platelet procoagulant activity, and increased platelet-leukocyte interactions in patients with Covid-19
Lucy J. Goudswaard1, Chris M Williams1, Fergus Hamilton2, David Arnold3, Andrew Davidson4, Alastair W. Poole1, Ingeborg Hers1
1School of Physiology, Pharmacology and Neuroscience, Biomedical Sciences Building, University Walk, Bristol, BS8 1TD, United Kingdom.
2Department of Infection Sciences, North Bristol NHS Trust, BS10 5NB, Bristol, United Kingdom.
3 Academic Respiratory Unit, North Bristol NHS Trust, BS10 5NB, Bristol, UK.
4School of Cellular and Molecular Medicine, Biomedical Sciences Building, University Walk, Bristol, BS8 1TD, United Kingdom.
E-mail of Presenting Author: firstname.lastname@example.org
Background: Patients with severe coronavirus disease 19 (COVID-19) are at increased risk of thrombosis, which is associated with enhanced coagulopathy and increased mortality. Previous studies showed enhanced platelet aggregation in patients with Covid-19, however the underlying mechanism and how this contributes to thrombosis is still largely unknown.
Aim: To evaluate the platelet proteome, platelet functional responses and platelet/leukocytes aggregate formation in patients with Covid-19.
Methods: Platelets were isolated from patients with Covid-19 and healthy controls. Platelet lysates were subjected to TMT proteomic analysis. Platelet functional responses such as integrin aIIbb3 activation, P-selectin expression and PS exposure were analysed by FACS analysis.
Results: The platelet proteome was significantly altered with enrichment of ribosomal and mitochondrial proteins, and reduced levels of signalling proteins. Circulating platelets from Covid-19 patients furthermore showed enhanced PS exposure, whereas integrin aIIbb3 activation and P-selectin expression were unaltered. In contrast, agonist stimulated integrin aIIbb3 activation and PS exposure were significantly decreased in Covid-19 patients. Furthermore, we found high levels of platelet/leukocyte aggregate formation in Covid-19 patients, which was reduced by a blocking anti-P-selectin antibody, suggesting immuno-thrombosis is part of the Covid-19 phenotype. Interestingly, however platelets in these platelet/leukocyte aggregates did not show enhanced integrin aIIbb3 activation, suggesting they are generally in a low activation state, or having undergone activation followed by desensitisation.
Conclusions: Platelets from Covid-19 patients show significant changes in their proteome and enhanced basal PS exposure but impaired activation-dependent responses including integrin activation and PS exposure. High levels of platelet/leukocyte aggregates are present and given the increased thrombotic tendency in severely ill patients, these findings point to an immunothrombotic pathogenesis where enhanced basal procoagulant activity may also be important. It will be important now to determine whether these changes are the result of direct activation of platelets or leukocytes by viral contact or cellular infection.
Society Networking Session
One of the highlights of any meeting of The Platelet Society is having time at lunch and coffee breaks to get to meet one another, make new connections, and find out about each others current and future research plans. Whilst we cannot bring refreshments to your offices, we wanted to create an opportunity for you to meet and talk with people. In this networking session, we will allocate attendees to breakout rooms based off the preferences that everyone provided at registration.
3Rs approaches to platelet research(Rooms 1 and 2)
Treating platelets at the nanoscale (Rooms 3 and 4)
How do we artificially replace blood platelets? (Rooms 5 and 6)
Current and future challenges in platelet signalling (Rooms 7 and 8)
What are the next-generation anti-thrombotics? (Rooms 9 and 10)
Non-thrombotic roles for platelets in human disease (Rooms 11 and 12)
Big data and platelets: What are the future applications? (Rooms 13 and 14)
Clinical studies – how can we support translation of our research? (Rooms 15 and 16)
At the start of the session you will be moved into a networking room with other people who have expressed an interest in discussing these topics. We have tried to mix up groups as far as possible to . So you will be with people from different institutes, and at a variety of career stages. We hope that by putting together these randomized groups, that you will be able to network and make connections with people that can help you develop your research.
Please start by introducing yourself to the other people in your room, briefly describe your current research project(s) and what your interest was in this topic (whether this is from your current research project, or future interests). This will hopefully allow the group to find common interests that can be discussed further. In case you get stuck, here are some other questions that you might wish to discuss:
- What the key gaps in our knowledge in this research area?
- What are the current challenges and opportunities?
- What techniques and methodologies are you interested in working with, or sharing with others?
- Are there ways that the Platelet Society can help support research in this area?
If you have any feedback for the Platelet Society following your conversations please email this to email@example.com or in the post-meeting survey.
Communications 2: Megakaryocyte and Platelet Signalling
Synergistic use of qEV Size-exclusion Chromatography columns and Tunable Resistive Pulse Sensing technique in EV extraction and characterisation
Izon Science Europe SAS, Lyon, France. E-mail: firstname.lastname@example.org
The number of scientific publications that have described physiological functions of extracellular vesicles (EVs) has sharply increased in the last decade. EVs collectively cover various types of cell-derived membranous vesicles, such as exosomes, microvesicles, apoptotic bodies, and other membranous structures. These vesicles are usually extracted from different biological fluids, organs and corpuscles, such as blood plasma, conditioned cultured media and platelets. Hence, the demand for increased rigour in EVs extraction and characterisation is required, in which EVs isolation, purification, measurement and downstream analysis have become a real challenge in the research and clinical fields.
Izon Science has synergistically utilised Size-exclusion Chromatography (SEC) and Tunable Resistive Pulse Sensing (TRPS) techniques in terms of EV extraction and characterisation (size, number and surface charge/zeta potential). The qEV smart SEC columns have demonstrated an efficient method to isolate EVs from most biological fluids based on EV size. A combination of qEV columns with Izon’s Automatic Fraction Collector (AFC) can decrease the inaccuracies of EV sample collection volumes and the experimental time significantly.
Izon’s new generation TRPS instrument Exoid (a semi-automatic version of qNano) can measure down to 40 nm up to 10 um. Exoid incorporates the unparalleled accuracy of TRPS into a semi-automated machine which improves sensitivity and usability, representing a reliable method of measuring individual particles.
Data of EVs derived from plasma by qEV column, AFC and Exoid will be presented in the main talk.
A Role for Platelet-Derived Vesicles in Megakaryopoiesis During Inflammation
Dr Kellie Machlus
Harvard Medical School. Vascular Biology Program, Boston Children’s Hospital, USA. E-mail:email@example.com
During inflammation, steady-state hematopoiesis switches to emergency hematopoiesis to repopulate myeloid cells, with a bias toward the megakaryocytic lineage. Soluble inflammatory cues are thought to be largely responsible for these alterations. However, how these plasma factors rapidly alter the bone marrow (BM) is not understood. Inflammation also drives platelet activation, causing the release of platelet-derived extracellular vesicles (PEVs), which package diverse cargo and reprogram target cells. We hypothesized that PEVs infiltrate the BM, providing a direct mode of communication between the plasma and BM environments. To test this, we transfused fluorescent, wild-type platelets into recipient cMpl knockout mice before triggering systemic inflammation. Twenty hours post-infusion, we observed significant infiltration of donor platelet-derived particles in the BM. To determine if this phenomenon relates to humans, we extensively characterized both megakaryocyte-derived and PEVs generated in vitro and in vivo, and found enrichment of extracellular vesicles in bone marrow compared with autologous peripheral blood. Last, BM from cMpl knockout mice was cultured in the presence or absence of wild-type PEVs. After 72 hours, flow cytometry revealed increased megakaryocytes only in cultures with added PEVs, suggesting a PEV-mediated rescue of megakaryopoiesis. In conclusion, we report for the first time that plasma-residing PEVs infiltrate the BM. Further, PEVs interact with BM cells in vivo and in vitro, causing functional reprogramming that may represent a novel model of inflammation-induced hematopoiesis.
Stimulation of the ssRNA virus immune receptor, Toll-like receptor 7, on megakaryocytes increases thrombopoesis
Background: Toll-like receptors 7/8 (TLR7/8) are immune receptors expressed in megakaryocytes which detects single-stranded RNA viruses such as SARS-CoV-2. There is increasing evidence that in addition to raised platelet counts, severe infection with SARS-CoV-2 increases the risk of venous, arterial and microvascular coagulation.
Aims: To determine if ssRNA viruses are capable of increasing thrombopoeisis through direct interaction with megakaryocytes.
Methods: Single or double TLR7/8-/- iPSC derived megakaryocytes (iPSC-MKs) were produced using CRISPR Cas9 editing of iPSCs and subsequent forward programming using an doxycyclin inducible cassette overexpressing GATA1, FLI1 and TAL1. RT-qPCR and ELISA assays were used to confirm loss of functional TLR7/8 receptor. Mature cells were incubated with and without Gardiquimod (GDQ), a specific agonist of TLR7/8, that we have previously shown to increase platelet production in cord blood derived and mouse bone marrow derived megakaryocytes. GFP labelled SARS-CoV-2 virus was incubated with the TLR7/8-/- iPSC-MKs and wild-type iPSC-MKs
Results: Incubation with GDQ induced increased expression of IL1β in the parental iPSC-MKs, however in the TLR7-/- and TLR7/8-/- MKs, no increased expression was observed. This finding was further confirmed using IL1β ELISA assays. There was a significant increase in platelet production from the parental iPSC-MKs in response to incubation with GDQ, which was not seen in the TLR7-/- and TLR7/8-/- MKs. Incubation of the GFP-labelled SARS-CoV-2 virus with wild-type MKs did not lead to a significant increase in fluorescence. Only very low level viral sequences were found in the cells post-incubation demonstrating that penetration within the MKs and subsequent replication of SARS-CoV-2 is unlikely to be of significance. Studies are ongoing to ascertain whether SARS-CoV-2 induces outside in signalling leading to changes in transcription within the MKs (such as elevation of IL1β).
Conclusions: SARS-CoV-2 does not appear to penetrate and significantly replicate within MKs. It may be that the virus induces changes due to downstream signalling from surface receptors such as TLR7/8 leading to changes in platelet count and reactivity.
Tunability of platelet-derived extracellular vesicles
Background: The proteome of anuclear platelets comprises >5000 proteins and is impacted e.g. by age and disease. The platelet secretome of over 300 proteins contains e.g. growth factors and therefore, platelet products, such as platelet rich plasma are used in regenerative medicine. Platelets also release extracellular vesicles (EVs), both constitutively and upon activation.
Aims: To investigate 1) the tunability of platelet EVs by agonists engaging different critical platelet signaling pathways, 2) the characteristics of these EVs, and 3) macrophage responses to these EVs.
Methods: Isolated human platelets were activated by CRP (engaging GPVI), rhodocytin (CLEC-2), and by thrombin and collagen co-stimulus (all thrombin and collagen receptors). Agonist concentrations and the time of activation were optimized for maximal EV yield in the shortest possible time. EVs were isolated by ultracentrifugation using cushioned density-gradient, and then characterized by particle concentration, size distribution (NTA) and marker protein expression (Exoview R100). The inflammation-linked proteome of EVs was analyzed in a targeted array using Olink technology. Macrophages differentiated from THP-1 cells were treated with equal numbers of EVs for 6 and 24 hours, and the secretome of macrophages was analyzed with Luminex technology targeted for cytokines and chemokines.
Results: Although more CD63+/CD9+ EVs were generated from activated platelets when compared to non-activated platelets, activation by rhodocytin resulted in a markedly lower EV yield compared to CRP or TC co-stimulus. The signaling pathways engaged during activation significantly impacted on the inflammation-related molecular cargo. These findings were further supported by the variability of the agonist-, but also time-dependent changes in the secretome of the EV-treated macrophages reflecting the tunability of platelet-derived EVs.
Conclusions: Upon activating platelets, agonists have distinct impacts on the characteristics of secreted EVs, their proteome and functionality. These results imply that by differential activation it is possible to create tunable EVs e.g. for immunomodulatory purposes.
The intracellular tail of GPIba augments GPVI-mediated signaling in mice
Background: The platelet GPIbα-VWF A1 domain interaction is essential for platelet tethering under high shear conditions. GPIbα and GPVI signaling machineries have previously been suggested to be linked, but the molecular basis of their signalling overlap remains unclear.
Aims: To evaluate the importance of the GPIbα intracellular tail in platelet function and thrombus formation.
Methods: GpIbαDsig/Dsig mice, in which the last 24 amino acids of the intracellular tail were deleted, were generated via CRISPR-Cas9 technology. Platelet function and thrombus formation was assessed ex vivo and in vivo.
Results:GpIbαDsig/Dsig mice exhibited mildly reduced platelet count and slightly enlarged platelets compared to GpIbα+/+ littermates. Activation of GpIbαDsig/Dsig platelets with ADP, thrombin and rhodocytin was normal, but GpIbαDsig/Dsig platelets stimulated with collagen-related-peptide (CRP) exhibited markedly decreased P-selectin exposure and αIIbβ3 activation, suggesting a role for the GpIbα intracellular tail in GPVI-mediated signaling. Consistent with this, while haemostasis was normal in GpIbαDsig/Dsig mice, diminished tyrosine-phosphorylation, particularly pSYK was detected in CRP-stimulated GpIbαDsig/Dsig platelets as well as reduced platelet spreading on CRP. Although GpIbαDsig/Dsig platelets bound VWF normally under flow, they formed smaller aggregates than wild-type platelets on collagen-coated microchannels at low, medium and high shears. In response to both VWF and collagen binding, flow assays performed with plasma-free blood or in the presence of αIIbβ3– or GPVI-blockers suggested reduced αIIbβ3 activation contributes to the phenotype of the GpIbαDsig/Dsig platelets.
Conclusions: Together, our data reveal the importance of the intracellular tail of GPIbα in transducing VWF-GPIbα signals and also a previously uncharacterised role for this region in collagen-GPVI signaling events in platelets.
GPVI clustering investigation by Agent-Based Modelling (ABM) approach
Background: A key receptor that plays an important role in platelet activation is the collagen receptor glycoprotein VI (GPVI). GPVI dimerization, and higher-order clustering (i.e. oligomerization) is thought to be required for collagen binding and the downstream signalling. Development of a computational model of GPVI clustering and the signalling implications of this will help to understanding the regulation of inter-GPVI interactions.
Aim: To construct a computational model of a platelet membrane that generates spatiotemporal simulations of the dimerization and clustering of GPVI molecules.
Method: An artificial membrane was built using an Agent-Based Model (ABM), as a computational modelling approach that maps moving bodies within a simulation box as interacting agents. Visualisation occurs by implementing micro-autonomous steps within the modelled system, based on predefined rules. Modelling of laterally diffusing GPVI-GPVI interactions was in an environment with biochemically different regions of the plasma membrane, i.e. lipid rafts and non-lipid rafts. Parameters such as protein diffusivity, binding and dissociation constants, and size or fraction of raft area were varied within simulations to explore the effects on simulation outcomes.
Results: The developed model indicated that both monomeric and dimeric forms of GPVI preferentially localise in (nanosecond?) lipid raft areas. An increase in rafts leads to higher GPVI dimerization and higher-order clustering, but only until an optimal ratio of raft and non-raft area was reached Importantly, it appeared that the merging of nanosize rafts increased the overall level of dimerization and clustering.
Conclusion: Modelling of GPVI clustering by a newly developed Agent-Based approach revealed the importance of spatiotemporal intra-membrane heterogeneity in GPVI-clustering dependent platelet activation steps.
Abstracts – Wednesday
Equity, Diversity and Inclusion Spotlight
Equity, Diversity and Inclusion Spotlight: Mentoring within The Platelet Society.
Following discussions with our members, and in collaboration with the Society’s ECR working group, The Platelet Society would like to establish a mentoring programme to help develop and support our members at all levels.
We hope that our mentoring programme will build relationships between our members that will build confidence and support our members in their own development and career journey. As a mentor you will have the opportunity to use your experience and knowledge in a facilitative manner to support the development of our mentees, helping mentees progress, share concerns, make career decisions, and navigate the world both inside and outside of academia.
Welcome and Introduction (10 minutes) – main room.
A quick overview of the mentoring scheme, and objectives of the session.
Group discussions (30 minutes) – randomly allocated breakout rooms.
In this networking session, we will allocate attendees to breakout rooms, and would like you in your groups to discuss your experiences of mentorship and the purpose of the mentoring relationship. These discussions will enable us to develop a mentorship programme that meets the needs of our members.
We ask that you focus your discussions on the following topics:
- The purpose of the mentee-mentor relationship
- What are you expectations of mentorship?
- Mentorship experiences
- Group rather than individual mentoring
- Peer mentorship
- Would you like mentorship to be formal or informal?
We ask that one member of your group posts key discussion points on our Mentorship Padlet using the QR code or link below
Session Summary (10 minutes) – main room
After your group discussions we ask that you return to the main room, to share your thoughts and suggestions for the Society’s mentoring programme.
Thank you in advance for taking part in this networking session. If you are interested in taking part in the Society’s mentoring programme, either as a mentee, mentor or both, please complete the Mentorship questionnaire online at:
Communications 3: New Horizons in Platelet Research
Introduction to Stago UK, specialists in haemostasis assays and equipment
Stago is a pharmaceutical laboratory, founded in 1945, which now operates in the In Vitro Diagnosis (IVD) industry, wholly dedicated to the exploration of haemostasis and thrombosis.
Specialised in the fields of Haemostasis and Thrombosis, Stage invests in research and innovation to develop new and better performing reagents, systems and solutions. With 60 years of experience, Stago has acquirred a charismatic image in Haemostasis and is well recognised in the international scientific world.
With over 350 marketed products, Stago is a worldwide reference in haemostasis and a first class partner for biomedical laboratories. Stago also has a licensed training centre, offering theoretical and practical training courses at different levels.
Join us for a brief introduction to Stago and a few items we think will be of interest!
Anti-GPVI nanobodies as a novel tool to inhibit thrombus formation on atherosclerotic plaque under flow
Background: 17.9 million people die of cardiovascular diseases, which is often initiated by atherosclerosis and subsequent induction of thrombosis via atherosclerotic plaque rupture. It is postulated that this is mediated through the main platelet collagen receptor, GPVI. Due its platelet specificity and only minor bleeding tendencies observed in knockout mouse models, GPVI is an attractive target for the development of novel antiplatelet therapies.
Aims: We have raised a number of novel anti-GPVI nanobodies. Here, we aim to investigate the effect of 4 nanobodies on in vitro thrombus formation triggered by different collagens and atherosclerotic plaque material via whole blood microfluidics.
Methods: The Maastricht flow chamber was utilized to assess the effect of 3 GPVI inhibiting nanobodies and 1 non-inhibiting nanobody on thrombus formation. After pre-incubation with the nanobodies, whole blood was flown over pooled atherosclerotic plaque homogenate, Horm (Type I) collagen as well as human collagen Type III and aggregate formation and platelet activation investigated.
Results: Qualitative and quantitative analysis demonstrated that the GPVI inhibiting nanobodies decreased thrombus formation and reduced P-selectin surface expression and integrin IIb3 activation on all 3 surfaces. Further, the highly GPVI dependent phosphatidylserine exposure was almost completely abolished. However, platelet adhesion was not affected. None of the investigated parameters were affected by the control nanobody.
Conclusions: We show that novel anti-GPVI nanobodies are capable of inhibiting thrombus formation, in a GPVI dependent manner, in whole blood flown over pooled atherosclerotic plaque homogenate, Horm collagen and human collagen III. Therefore, these nanobodies warrant further investigation as potential anti-thrombotic agents.
Mechanisms of fibronectin fibrillogenesis by platelets
Background: Platelets are the first cells to arrive at wound sites. Later invading fibroblasts are thought to initiate tissue repair by laying down a provisional fibronectin matrix. This view is challenged by reports that platelets themselves can assemble fibronectin matrix, yet the underlying mechanisms and the physiological imporance remain largely unclear.
Aims: To elucidate the mechanisms governing the assembly of fibronectin fibrils by platelets.
Methods: Blood was obtained from healthy donors under Ethics approval REC1405. Washed platelets were seeded onto adhesion protein-coated substrates in the presence of fluorescently labeled fibronectin. Platelet traction forces were measured on micropost arrays. Fibronectin fibrils and adhesion sites were characterised by 3D super-resolution microscopy (dSTORM). The role of adhesion receptors and platelet contractility was assessed using specific inhibitors for integrins and myosin IIa.
Results: Platelets assembled fibronectin nanofibrils along fibrillar-like adhesions using αIIbβ3 (CD41/CD61, GPIIb-IIIa) rather than α5β1 integrins, in contrast to fibroblasts. Highly contractile platelets in contact with thrombus proteins (fibronectin, fibrin) pulled fibronectin fibrils along their apical membrane, whereas less contractile platelets on basement membrane proteins (collagen IV, laminin) generated less stretched planar meshworks beneath themselves. As probed by vinculin localisation within apical fibrillar adhesions, platelets on fibronectin generated similar fibre pulling forces as fibroblasts.
Conclusions: These are novel mechanobiology mechanisms by which platelets spearhead the fibrillogenesis, as well as the 2D versus 3D network architecture, of the first de novo ECM. Whether this matrix helps to pave the way for cell infiltration in vivo remains to be investigated.
High-throughput, Functional Mechanophenotyping of Platelet Activation Induced by Bacterial Proteins
Background: Pathogenic Gram-negative and Gram-positive bacteria and their secreted proteins interact directly and indirectly with human platelets and induce platelet activation. Whether this affects the viscoelastic properties of platelets that are relevant for hemodynamic platelet margination in vivo is not well understood.
Aims: To quantify changes in viscoelastic properties (i.e. biomechanical deformation) of single platelets induced by proteins from Staphylococcus aureus using high-throughput functional mechanophenotyping.
Methods: Functional mechanophenotyping of single platelets was performed by high-throughput, real-time 1D-imaging fluorescence, and deformability cytometry (RT-FDC). Washed platelets (n=9 donors) labeled with anti-CD41-FITC were incubated for 10 minutes with S. aureus proteins: phospholipase C (Plc), major autolysin (AtlA-1), extracellular adherence protein (EapD3D4 domain), chemotaxis inhibitory protein (CHIPS), and formyl peptide receptor-like 1 inhibitory protein (FLIPr). Platelet deformation and activation were detected by anti-CD62P-AlexaFluor647 in RT-FDC, simultaneously. Assessment of platelet cytoskeletal alterations was performed by fluorescence flow cytometry and microscopy.
Results: EapD3D4 and CHIPS lowered platelet deformation (i.e. increased stiffness) and induced platelet activation (CD62P+) simultaneously, whereas AtlA-1 induced lower deformation in the absence of platelet activation, with a concomitant increase in F-actin content. While both Plc and FLIPr failed to activate platelets, we observed Plc induced viscoelastic softening of platelets without a change in F-actin content.
Conclusions: Single platelet mechanophentyping reveals S. aureus proteins alter the intrinsic biomechanical properties as well as platelet activation in vitro. Intriguingly, some donors also exhibit a heterogeneous mechano-functional response to identical proteins. The in vivo relevance of this on hemodynamic during systemic infection by S.aureus warrants further investigation.
Generating large numbers of in vitro platelets from Human induced stem cell derived Megakaryocytes via a platelet extruding bioreactor
Background: Megakaryocytes (MK) derived from hiPSCs could be used as a scalable source of platelets for transfusion. However, the current average rate of platelet release per in vitro MK is below that of bone marrow by several orders of magnitude. To encourage the MK cellular structures and pseudopodia to release platelets we applied shear flow via a specialised bioreactor.
Aims: We hoped to significantly increase platelet release from hiPSC-derived MKs using a bespoke bioreactor to provide large numbers of platelets for functional testing.
Methods: A prototype system based on a microfluidic chamber (Thon et al 2014) developed by PlateletBioGenesis was used with our iPSC-derived MKs (Moreau et al 2016). For these experiments multiple bioreactors were loaded with approximately 12 million hiPSC MKs and operated with a recirculating reservoir. Collected platelets were analysed by flow cytometry and in vitro thrombus participation. Potential clinical effects were investigated by transfusion of platelets into a thrombocytopenic mouse model.
Results: Tens of millions of CalceinAM+ CD42/41++ in vitro platelets were produced per bioreactor. Labelled platelets were able to integrate into thrombi in whole blood flowing through collagen coated-chambers. In mice transfused with either control donor platelets or in vitro derived platelets, flow cytometry showed the presence of Cal/CD42/41+++ and reduced bleeding following resection of a 2mm section of tail at higher counts.
Conclusions:These small scale laboratory runs could produce 10s of millions of Cal+CD41+CD42+ in vitro platelets with the possibility to multiplex bioreactors. Transfused platelets could be found in the circulation and influence clotting.
Platelet-inspired Nanotechnologies for Applications in Hemostasis, Thrombosis and Thromboinflammation
Professor Anirban Sen Gupta
Department of Biomedical Engineering Western Reserve University, Cleveland, Ohio, USA. E-mail: firstname.lastname@example.org
Platelet transfusions are used in prophylactic management of bleeding risks, as well as emergency management of acute surgical and traumatic bleeding. However, natural platelet products suffer from challenges of (i) limited availability and portability, (ii) pathogenic contamination risks resulting in very short shelf-life (~5 days), and (iii) various biological side-effects. Current pathogen reduction technologies and recent research with temperature reduced (chilled, freeze-dried etc.) platelets have extended the shelf-life to a few weeks, but have not fully resolved the challenges of widespread availability both within and outside of hospitals (e.g. at point-of-injury), and the issues of variable hemostatic performance. In this framework, our research is focused on developing nanoparticle-based technologies that mimic, leverage or amplify platelet-mediated mechanisms of hemostasis, by modular mimicry of injury-site targeted adhesion and aggregation, coagulation amplification and secretion of hemostasis-augmenting agents. These technologies can potentially allow large scale manufacture, reproducible quality control, storage over months-to-years, and efficient hemostatic management of patients in both prophylactic and emergency settings, when natural platelet products are of limited availability. We have evaluated these technologies in vitro and in vivo with promising results, and are currently advancing them through academic research as well as through a biotech start-up I co-founded (Haima Therapeutics), towards clinical translation. Furthermore, these bio-inspired systems can be modularly refined or engineered to act as ‘targeted drug delivery’ vehicles for site-specific therapy in pathologies where platelets have critical mechanistic involvement, e.g. in thrombosis and thromboinflammation. To this end, we have engineered platelet-inspired systems that enable targeted fibrinolysis, for potential application in heart attack and stroke. We have also engineered drug delivery systems that target ‘platelet-neutrophil complexes’ for potential application in thrombo-inflammatory diseases, and have carried out pilot studies with them in vitro and in vivo. We continue to develop these technologies with a vision for potential applications in treating deep vein thrombosis, chronic wounds and cancer where platelet- and neutrophil-mediated processes become unique mechanistic drivers of disease pathology. Thus our research is at the interface of biology, chemistry, engineering and medicine, to create bioinspired technologies directed at efficiently addressing and resolving clinical challenges in hemostasis, thrombosis and thromboinflammation.
Gustav Born Lectures
Developing iPSC derived and bioprinted organoids for the study of platelet production and biology
Dr Abdullah O. Khan
Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, UK. E-mail: email@example.com
Organoids are self-organising, stem cell derived 3D structures which allow us to study complex cellular architecture and cell-cell interactions in vitro. Advances in organoid development have led to increasingly physiological models derived from human induced pluripotent stem cells (hiPSCs) for a number of different tissues and organs, most significantly the brain and gastrointestinal systems. My work aims to develop vascular organoids to better model the bone marrow and heart in dynamic, 3D models. These approaches employ directed differentiation to generate vascular networks in specialised tissues for the study of complex, environment driven diseases like fibrosis. We show a matrix specific effect on lineage commitment in developing vascular organoids which allows for the production of bone marrow cells, including haematopoeitic progenitors, specialist mesenchymal cells and megakaryocytes. Further specialisation of these differentiation protocols also allow for the generation of vascularised cardiac organoids, which we have begun to employ to assess the effects of SARS-CoV-2 antigens on vascular integrity and cardiomyocytes in vitro. Developing these approaches involves both differentiating self-organising cultures, and introducing bioprinting techniques to better recapitulate adult tissues. As we validate these models and employ them to address biological questions, future efforts are focussed on using 3D bioprinting and microfluidics to better recapitulate tissue specific physiology, and develop perfusable systems for the study of platelet biology.
Investigations into mitochondrial dynamics during platelet ageing and reactivity
Dr Harriet Allan
Blizard Institute, Queen Mary University London, London, UK. E-mail: firstname.lastname@example.org
Platelets are small, anucleate cell fragments fundamental for maintaining haemostasis. These unique cells are packaged with a range of organelles including α-granules, dense granules, the dense tubular system and mitochondria. Whilst the first three of these have been widely explored during platelet activation, mitochondria function is less well characterised. Here we have investigated mitochondrial dynamics in the context of platelet activation and ageing.
Platelets exist for approximately 10 days in a healthy individual, during which time their structural, transcriptomic and proteomic composition changes. We have shown that during platelet lifespan there is a decline in key mitochondrial proteins including citrate synthase and ATP synthase, which is accompanied by a reduction in the overall number of mitochondria per platelet from 11±1 mitochondria in young platelets to 5±1 mitochondria in old platelets. Similarly, platelet activation by TRAP-6 amide causes a decrease in the number of mitochondria from 8±1 in resting platelets to 4±1 in stimulated samples, without any changes in mitochondrial area. This reduction is accompanied by an increase in the number of platelet microvesicles. Using imaging flow cytometry, we were able to demonstrate the presence of mitochondria encapsulated within approximately 20% of these microvesicles, suggesting they are being packaged and released during platelet activation.
This work demonstrates marked changes in the content of mitochondria as platelets age. Furthermore, platelet mitochondria may have a role beyond the platelet, being important in intracellular communicator with other cells within the circulation and vasculature.
Magnetic nanoparticles as a therapeutic and diagnostics platform for cardiovascular disease
Dr David Cabrera
School of Pharmacy and Bioengineering, Guy Hilton Research Centre, Keele University, UK. Email: email@example.com
Magnetic nanoparticles (MNPs) are currently used in a wide range of biomedical applications. Thanks to their ability to generate targeted hyperthermic stimuli in tumour cells when exposed to alternating magnetic fields, MNPs have enabled us to improve the treatment of some of the most aggressive forms of cancer. However, this significant effort to conceive less invasive therapies to treat cancer contrast with the still scarce utilisation of this nanotechnology in cardiovascular diseases therapeutics. Current treatment of venous thromboembolism (VTE) involves the systemic administration of either anticoagulants to prevent further thrombus growth, or thrombolytic enzymes to try to lyse the clot. Yet systemic circulation of both pharmacological agents often leads to major unwanted bleeding in patients. In this talk I will discuss how magnetic nanoparticles can be utilised to improve current treatments of VTE. By functionalising MNPs with anti-fibrin antibodies and tissue plasminogen activator (tPA), MNPs can target offending clots to deliver tPA locally and potentially reduce secondary effects associated with systemic circulation of tPa. Additionally, the MNPs can be magnetically stimulated to deliver a localised heating response to the clot that can increase tPa activity, as well as breakdown the cellular barriers on the thrombus surface to all fibrinolytic enzymes to permeate deeper into the clot. By combining future in vitro studies in a humanised vein model and in vivo studies to confirm the biocompatibility of MNPs, my project aims to demonstrate the feasibility of using MNPs as a more effective and safer treatment for VTE.
Platelet Society Twitter Feed
Congratulations Giordano on completing the #londonmarathon2021 in 3:34:12
You can sponsor this heroic effort here 👇
Come on Giordano!
Not far to go now, you got this!!
Good luck @GiordanoPula!!
#RunGioRun #GioRuns #RunningBlood #BloodyRun #research #Platelets #BloodClots #bleeding #thrombosis
@plateletsociety @LondonMarathon https://twitter.com/plateletsociety/status/1444409942341980162
Giordano is putting in a stonking effort!
He’s crossed the 21k mark in 1:42!!
@SysmexUK @wearecellix @3i_inc @LabmedicsUK
No turning back now…
We’re so proud of our #MarathonMan Giordano. Please do sponsor him to help him get over the finish line with a PB.
It’s been a miserable day in London so every little helps!
Gus Born Lecture 2021
Dr Harriet Allen
Blizzard Institute, Queen Mary University London, London, UKAbstract
Gus born Lecture 2021
Dr David Cabrera
School of Pharmacy and Bioengineering, Keele University, UKAbstract