Illustrated capsules from the Advanced Course in Platelet Research

Abstract

This series of illustrated capsules summarizes the presentations made by the speakers at the first International Advanced Course in Platelet Research held in Murcia (Spain) from 27 to 28 September, 2024. This is the first course to receive a Fundamental Research Workshop Grant from the International Society on Thrombosis and Haemostasis (ISTH) and was also supported administratively and scientifically by the Spanish Society of Thrombosis and Haemostasis (SETH). This unique course focused on new methodologies applied in platelet research and how these are increasing our understanding of platelet formation, their multifunctionality in different physiological and pathological contexts, and contributing to the development of new platelet-targeted therapies to improve the management of hemostatic/thrombotic pathologies. It aligns with the objectives of several Scientific and Standardization Committees of the ISTH, including Platelet Physiology and Genomics in Thrombosis and Haemostasis, as well as with the academic objectives of the ISTH and SETH. The program was designed by the coordinator (J. Rivera), and the scientific advisory board (SAB: S.P. Watson, K. Freson, A. Balduini, and J. Di Paola) and comprised 9 scientific sessions with 25 presentations, each with time for extensive open discussion. Additionally, 33 abstract posters were presented, with the 3 highest scoring selected as oral presentations. The course was held in a single location and with an informal atmosphere to facilitate networking among participants. The course received very positive feedback from the 140 attendees. The course was supported by the ISTH, SETH, University of Murcia, CIBERER-ISCIII, Fundación Séneca (22426/OC/24), the United Kingdom Platelet Society and various pharmaceutical companies. We believe that the extraordinary scientific and human experience of this course may act as a stimulus for future courses.

New Concepts in Megakaryopoiesis and Thrombopoiesis

How should we study megakaryopoiesis nowadays?

Christian Andrea Di Buduo, Vittorio Abbonante, Alessandro Malara, Alessandra Balduini

Department of Molecular Medicine, University of Pavia, Pavia, Italy

Department of Biomedical Engineering, Tufts University, Medford, MA, USA

Reproducing the biophysical and biological characteristics of the human bone marrow niche is essential for advancing human blood cell production ex vivo. This has broad implications for studying normal and malignant hematopoiesis, validating novel therapies, and applications in transfusion medicine. The bone marrow, composed of hematopoietic stem and progenitor cells (HSPCs) within an extracellular matrix framework, plays a key role in this process. Recent bioengineering advances, such as three-dimensional scaffolds and organoids, recreate essential features of the native tissue to support HSPCs function ex vivo. These models are poised to transform hematologic research by enabling dynamic studies of human hematopoiesis. However, controlling the interaction of diverse cell types remains challenging.

In the International Society on Thrombosis and Haemostasis (ISTH) Advanced Course on Platelet Research in Murcia, we presented innovative silk-based bone marrow models developed by our group. These models support human HSPC functions on-demand and enable detailed studies of human platelet production, focusing on biophysical cues and cell-to-cell interactions in both health and disease. Figure made with BioRender.com

• [1]Di Buduo CA, Lunghi M, Kuzmenko V, Laurent PA, Della Rosa G, Del Fante C, et al. Bioprinting soft 3D models of hematopoiesis using natural silk fibroin-based bioink efficiently supports platelet differentiation. Adv Sci (Weinh) 2024;11:e2308276. https://doi.org/10.1002/advs.202308276
• [2]Di Buduo CA, Laurent PA, Zaninetti C, Lordier L, Soprano PM, Ntai A, et al. Miniaturized 3D bone marrow tissue model to assess response to thrombopoietin-receptor agonists in patients. Elife 2021;10:e58775. https://doi.org/10.7554/eLife.58775
• [3]Di Buduo CA, Wray LS, Tozzi L, Malara A, Chen Y, Ghezzi CE, et al. Programmable 3D silk bone marrow niche for platelet generation ex vivo and modeling of megakaryopoiesis pathologies. Blood 2015;125:2254–64.

Can we really produce platelets in vitro for clinical use?

Amie K. Waller

Department of Haematology, University of Cambridge, Cambridge, UK

(1) Inducible pluripotent stem cell lines for the generation of megakaryocytes (MKs) must be both efficient [1] and good manufacturing process level quality. (2) Forward programming of human inducible pluripotent stem cell lines using a 3-transcription factor system [2] results in an output of mature MKs. Improvements in efficiency have been achieved by inserting a doxycycline inducible all-in-one vector into a single safe harbor. (3) Advances in bioreactor design, including recapitulating the bone marrow environment, have improved the numbers of platelets that can be produced and studying the supernatants of human platelet apheresis donors has provided small molecule candidates for enhancing the production of platelets from these MKs. (4) The opportunity to genetically engineer MKs, and therefore the platelets that are produced from them, allows added benefits, like “supercharging” with tissue factors or producing human leukocyte antigen null (universal) platelets. (5) Using gravity-based concentration device, outputs from bioreactors can be volume reduced while maintaining platelet function.

• [1]Evans AL, Dalby A, Foster HR, Howard D, Waller AK, Taimoor M, et al. Transfer to the clinic: refining forward programming of hPSCs to megakaryocytes for platelet production in bioreactors. Blood Adv 2021;5:1977–90.
• [2]Moreau T, Evans AL, Vasquez L, Tijssen MR, Yan Y, Trotter MW, et al. Large-scale production of megakaryocytes from human pluripotent stem cells by chemically defined forward programming. Nat Commun 2016;7:11208. Erratum in: Nat Commun 2017;8:15076.

Emerging Technologies for Understanding Platelet Function (I)

Clustering of single transmembrane receptors driven by the law of mass action

Steve P. Watson¹, Eleyna M. Martin¹, Lloyd Bridge²

¹Department of Cardiovascular Sciences, College of Medicine and Health, University of Birmingham, UK

²Department of Mathematics, University of the West of England, Frenchay Campus, Bristol, UK

Platelets are activated by single transmembrane proteins that signal through tyrosine-based motifs in their cytosolic tails, namely glycoprotein VI (GPVI), C-type lectin-like receptor 2 (CLEC-2), Fc receptor γ RIIA (FcγRIIA) and platelet and endothelial aggregation receptor-1 (PEAR1) (upper left). Three of these receptors signal through an immunoreceptor tyrosine-based activation motif, defined by 1 or 2 YxxL sequences, while PEAR1 signals via a single YxxM sequence. The receptors signal through 3 families of tyrosine kinases (TKs) belong to the Sarcoma (Src), spleen TK (Syk), and Bruton’s TK (BTK) families leading to activation of phospholipase Cγ and phosphatidylinositol 3-kinase (PI3-kinase)/protein kinase B (Akt). These receptors support the nonhemostatic roles of platelets including the regulation of inflammation and host defense and are targets for a new antiplatelet agent in thromboinflammatory disorders. This class of receptor is activated by multivalent ligands which induce clustering, which is also supported by receptor dimerization and potentially crosslinking of their C-terminals by tandem SH2 domain-containing proteins Syk and PI3-kinase (lower left). Modeling of the binding of a ligand (L) to a receptor (R) using ordinary differential equations illustrates the bell-shaped relationship between ligand valency and occupancy, as shown for a tetravalent ligand (lower right). A bell-shaped relationship however is not seen in functional studies in platelets suggesting that other factors regulate ligand binding such as receptor homo-dimerization and release of secondary mediators [1]. We have raised nanobodies in llamas to recombinant domains of the 4 receptors and used these to generate agonists, antagonists, and probes for them. We have generated di-, tri-, and tetravalent ligands using a linker sequence and mouse Fc domain to study the relationship between ligand valency and receptor activation (upper right). With reagents, we have reported that a valency of 3 is sufficient to activate CLEC-2, GPVI, and PEAR1 in human platelets but that a valency of 4 is required to activate FcγRIIA [2].

• [1]Maqsood Z, Clark JC, Martin EM, Cheung YFH, Morán LA, Watson SET, et al. Experimental validation of computerised models of clustering of platelet glycoprotein receptors that signal via tandem SH2 domain proteins. PLoS Comp Biol 2002:18:e1010708. https://doi.org/10.1371/journal.pcbi.1010708
• [2]Martin EM, Clark JC, Montague SJ, Morán LA, Di Y, Bull LJ, et al. Trivalent nanobody-based ligands mediate powerful activation of GPVI, CLEC-2 and PEAR1 in human platelets whereas FcγRIIA requires a tetravalent ligand. J Thromb Haemost 2024:22:271–85.

Multi-parameter platelet function analysis for donor or patient phenotyping and stratification

Jonathan Gibbins

University of Reading, UK

Antiplatelet medication has the potential to substantially reduce the risk of thrombotic disease. Levels of platelet reactivity and responses vary substantially in the population, which is a stable characteristic. Some patients have highly reactive platelets, while the platelets of others may be relatively sluggish. This means that antiplatelet drugs may benefit some patients but put others at risk of serious bleeding. Indeed, the potential benefits of such treatments do not outweigh the risk of bleeding for primary prevention, even in patients with multiple cardiovascular disease risk factors, and are, therefore, no longer recommended for such use. If we could reliably measure platelet function in a clinical setting, more personalized approaches to the administration of drugs such as aspirin or the family of ADP-receptor antagonists may be possible. Platelet phenomics analysis allows detailed platelet function analysis in a simplified format incorporating unsupervised clustering approaches to define patient phenotype groups. Into these phenotype groups, recognized cardiovascular disease risk factors cluster, indicating that this approach may be used for patient stratification. Recent advances in understanding pharmacogenomic influences on the metabolism (and therefore bioavailability) of ADP-receptor antagonists, particularly clopidogrel, point toward the future combination of platelet function analysis and pharmacogenomic analysis for the identification of patients that would most benefit and choice of the most appropriate medication. The potential ability to identify patients who may experience on-therapy bleeding may allow antiplatelet drugs to be used more effectively for primary prevention.

• [1]Dunster JL, Bye AP, Kriek N, Sage T, Mitchell JL, Kempster C, et al. Multiparameter phenotyping of platelet reactivity for stratification of human cohorts. Blood Adv 2021;5:4017–30.
• [2]NICE Guideline DG59. CYP2C19 genotype testing to guide clopidogrel use after ischaemic stroke or transient ischaemic attack. 2024; https://www.nice.org.uk/guidance/dg59.

Generating human platelet protein “knockouts” using proteolysis targeted chimeras

Ingeborg Hers

School of Physiology, Pharmacology and Neuroscience, University of Bristol, Biomedical Sciences Building, University Walk, Bristol, United Kingdom

Platelets lack a nucleus, prohibiting traditional molecular approaches to protein knockdown. Our current understanding of hemostasis and thrombosis therefore derives largely from genetic animal models and pharmacologic approaches. Proteolysis-targeting chimeras (PROTACs) are an innovative class of small molecules designed to selectively degrade proteins. They work by bringing an E3 ubiquitin ligase (eg, Cereblon) in proximity to the target protein, leading to polyubiquitination and subsequent proteasomal degradation. PROTACs targeting BTK, a key signaling molecule downstream of the collagen receptor GPVI, rapidly degrade BTK and impair CRP-mediated platelet function and in vitro thrombosis on collagen [1]. The successful application of PROTACs in human platelets highlights the feasibility and promise of this approach to advance our understanding of platelet function and thrombosis. This may pave the way for future novel, more precise antithrombotic therapies with reduced bleeding risks. Parts of the graphics have been created using BioRender.com.

• [1]Trory JS, Munkacsi A, Sledz KM, Vautrinot J, Goudswaard LJ, Jackson ML, et al. Chemical degradation of BTK/TEC as a novel approach to inhibit platelet function. Blood Adv 2023;7:1692–6.

Advances in Flow Cytometry and Microscopy

How can high-resolution 3D imaging help understand platelet formation?

Claire Masson, Anita Eckly

Université de Strasbourg, INSERM, EFS Grand Est, BPPS UMR_S1255, FMTS, France

Electron microscopy (EM) has a long history in MK cellular biology. It encompasses standard transmission and scanning EM, innovative 3D methods, and correlative microscopy, providing valuable insights into the rare substructures of MKs in their native bone marrow microenvironment [1]. Through these methods, we have discovered that MKs form interconnected podosomes, which generate the force required to merge the apical and basal endothelial membranes, forming transendothelial pores through which MKs extend their initial proplatelets. This process ensures the proper delivery of the large protrusions, structurally different from the in vitro proplatelets, which anchor to the luminal face of the sinusoid through an actin-rich shoulder [2]. These innovative approaches can also aid in understanding the complex role of MK membrane buds and microvesicles in platelet formation and functions by revealing detailed structural granular and microtubule arrangements [3]. Questions remain, and future directions for improving our understanding of thrombopoiesis will likely require novel approaches that can evaluate dynamic MK behavior at a high-resolution level.

• [1]Scandola C, Erhardt M, Rinckel J-Y, Proamer F, Gachet C, Eckly A. Use of electron microscopy to study megakaryocytes. Platelets 2020;31:589–98.
• [2]Eckly A, Scandola C, Oprescu A, Michel D, Rinckel J-Y, Proamer F, et al. Megakaryocytes use in vivo podosome-like structures working collectively to penetrate the endothelial barrier of bone marrow sinusoids. J Thromb Haemost 2020;18:2987–3001.
• [3]Carminita E, Becker IC, Italiano JE. What it takes to be a platelet: evolving concepts in platelet production. Circ Res 2024;135:540–9.

How should we use advanced microscopy to improve our understanding of platelet function?

Natalie S. Poulter

Department of Cardiovascular Sciences, College of Medicine and Health, University of Birmingham, UK

Centre of Membrane Proteins and Receptors (COMPARE), Universities of Birmingham and Nottingham, Midlands, UK

Microscopy has played a significant role in understanding platelet biology to date. Over recent years, there has been great advancement in fluorescent microscopy techniques which are able to resolve structures below the resolution limit of light (∼200 nm). These so-called superresolution microscopy techniques have been successfully applied to various aspects of platelet biology, including receptor distribution [1] and cytoskeletal arrangements [2]. Some techniques have also been proposed as methods to replace EM in the diagnosis of platelet-related disorders such as Hermansky-Pudlak Syndrome [3]. However, the choice of which microscopy technique to use should be driven by the biological question you want to answer. The availability of suitable probes for your protein of interest and the analysis methods you have available to you will also influence this choice. Just because a technique is new and exciting, does not mean that it is the most appropriate technique to use.

• [1]Pallini C, Pike JA, O’Shea C, Andrews RK, Gardiner EE, Watson SP, et al. Immobilized collagen prevents shedding and induces sustained GPVI clustering and signaling in platelets. Platelets 2021;32:59–73.
• [2]Poulter NS, Pollitt AY, Davies A, Malinova D, Nash GB, Hannon MJ, et al. Platelet actin nodules are podosome-like structures dependent on Wiskott-Aldrich syndrome protein and Arp2/3 complex. Nat Commun 2015;6:7254.
• [3]Clauser S, Cramer-Bordé E. Role of platelet electron microscopy in the diagnosis of platelet disorders. Semin Thromb Hemost 2009;35:213–23.

The Critical Role of Metabolism in Platelet Biology

miRNA deficiency modulates platelet metabolism promoting hyperactivation and thrombosis: from mechanism to therapy

Beatriz Martínez-García, Sonia Aguila

Centro Regional de Hemdonación, IMIB-Pascual Parrilla, Servicio de Hematología Hospital Universitario Morales Meseguer, Universidad de Murcia

miRNAs are small noncoding RNA that regulate gene expression. Platelets have a great miRNA cargo that modulates their activation as it has been described in several studies. The role of miRNAs in platelet metabolism remains unknown. The presence of miR-SNPs in miRNA genes impacts on mature miRNA levels or their target interaction. Thus, the miR-SNP that reduced miRNA levels was associated with higher thrombotic and cardiovascular events and platelet hyperreactivity. Our study showed that a mouse miRNA deficiency model drove metabolic reprogramming causing platelet hyperactivation with different agonists in static and flow conditions and promoting a prothrombotic phenotype. The knockout mice presented lower survival rate and larger aggregates in the lungs following pulmonary embolism challenge. Accordingly, the treatment with miRNA mimic modulated platelet response protecting mice against thrombosis and the subsequent death. Therefore, miRNA therapy could be a promising approach to prevent fatal thrombotic and cardiovascular events in individuals with reduced miRNA levels in thromboinflammatory situations.

How is nitric oxide metabolism regulating platelet function?

Paolo Gresele, Stefania Momi

Department of Medicine and Surgery, Section of Internal and Cardiovascular Medicine, University of Perugia, Perugia, Italy

Nitric oxide (NO), a gaseous radical molecule, plays a crucial antithrombotic and antiatherosclerotic role in the cardiovascular system.

While the generation from and function of endothelium-derived NO is undisputed, the very existence of NO-synthase in platelets and the role of platelet-derived NO (PDNO) in the regulation of platelet function have been a matter of controversy.

Evidence that human platelets generate NO in vitro has been given using a NO-selective electrode during aggregation, immunofluorescence of DAF-FM Diacetate (4-Amino-5-Methylamino-2',7'-Difluorofluorescein Diacetate)-loaded platelets under flow conditions, flow cytometry [1,2].

Evidence that PDNO inhibits platelet activation in vitro has been given by light transmission aggregometry preincubating with eNOS inhibitors (L-MeArg) or substrate (L-Arg), by assessing platelet recruitment, and platelet adhesion on a collagen substrate.

Evidence that PDNO prevents thrombosis in vivo has been given in arterial damage-induced thrombosis in transgenic mice [3], in coronary heart disease patients, from genetic studies in a kindred with familial early myocardial infarction.

• [1]Freedman JE, Loscalzo J, Barnard MR, Alpert C, Keaney JF, Michelson AD. Nitric oxide released from activated platelets inhibits platelet recruitment. J Clin Invest 1997;100:350–6.
• [2]Cozzi MR, Guglielmini G, Battiston M, Momi S, Lombardi E, Miller EC, et al. Visualization of nitric oxide production by individual platelets during adhesion in flowing blood. Blood 2015;125:697–705.
• [3]Wen L, Feil S, Wolters M, Thunemann M, Regler F, Schmidt K, et al. A shear-dependent NO-cGMP-cGKI cascade in platelets acts as an auto-regulatory brake of thrombosis. Nat Commun 2018;9:4301.

Platelet heterogeneity in health and disease

Paul Amstrong

Blizard Institute, Faculty of Medicine and Dentistry, Queen Mary University of London, London, UK

Platelets play a key role in thrombosis and hemostasis, and their heterogeneity is often categorized by function and molecular properties. Platelet dysfunction can manifest as hypo-reactivity, seen in bleeding disorders [1], or hyperreactivity, which is harder to detect but linked to increased thrombotic risk. Studies have associated heightened platelet reactivity with factors such as age, sex, and race, though the genetic and molecular mechanisms are still under investigation. Intraindividual platelet variation, including differences in size, density, and lifespan, has long been recognized, but the functional consequences remain unclear. Advances in flow cytometry have provided valuable insights, revealing that older platelets exhibit loss of cytoskeletal proteins and mitochondria, which influences their thrombotic capacity [2]. New findings suggest distinct subpopulations of platelets with varying phenotypes may be clinically relevant [3], but it is unclear whether these arise from genetic programming or in response to environmental factors. Improved understanding of platelet heterogeneity will allow us to tailor and target more effective interventions in the future.

• [1]Palma-Barqueros V, Revilla N, Sánchez A, Zamora Cánovas A, Rodriguez-Alén A, Marín-Quílez A, et al. Inherited platelet disorders: an updated overview. Int J Mol Sci 2021;22:4521.
• [2]Allan HE, Hayman MA, Marcone S, Chan MV, Edin ML, Maffucci T, et al. Proteome and functional decline as platelets age in the circulation. J Thromb Haemost 2021;19:3095–112.
• [3]Josefsson EC, Ramström S, Thaler J, Lordkipanidzé M, COAGAPO study group. Consensus report on markers to distinguish procoagulant platelets from apoptotic platelets: communication from the Scientific and Standardization Committee of the ISTH. J Thromb Haemost 2023;21:2291–9.

Emerging Technologies for Understanding Platelet Function (II)

Megakaryocyte and platelet Transcriptomics yield biological discoveries

Matthew Rondina

University of Utah Health, Huntsman Cancer Institute & Molecular Medicine Program

Anucleate platelets are now recognized to have a complex and dynamic transcriptome that may be significantly altered during inherited and acquired diseases [1]. Although still incompletely understood, many changes in the platelet transcriptome are thought to be driven by signal-dependent packaging of RNAs from the platelet precursor, the MK. In addition, exogenous RNAs may also be taken up by platelets from other cells, tissues, and tumors as they circulate through the vasculature [2]. As platelets are abundant in the circulation, only relatively small amounts of peripheral blood isolated from patients (or from experimental model systems) are necessary to profile the platelet RNA signature [3]. Changes in platelet RNA can be leveraged for previously unrecognized insights into human health and disease.

• [1]Davizon-Castillo P, Rowley JW, Rondina MT. Megakaryocyte and platelet transcriptomics for discoveries in human health and disease. Arterioscler Thromb Vasc Biol 2020;40:1432–40.
• [2]Sun S, Jin C, Si J, Lei Y, Chen K, Cui Y, et al. Single-cell analysis of ploidy and the transcriptome reveals functional and spatial divergency in murine megakaryopoiesis. Blood 2021;138:1211–24.
• [3]Thibord F, Johnson AD. Sources of variability in the human platelet transcriptome. Thromb Res 2023;231:255–63.

What is the future of proteomics in platelet research?

Sara Troitiño and Ángel García

Center for Research in Molecular Medicine and Chronic Diseases (CiMUS) – Universidade de Santiago de Compostela, and Instituto de Investigación Sanitaria de Santiago (IDIS), Santiago de Compostela, Spain

It was back in 2012 when the first comprehensive and quantitative study of the platelet proteome was published, reporting the identification of almost 4000 unique proteins [1]. Later studies completed the picture and today the platelet proteome comprises almost 6000 unique proteins [2]. All the above, together with recent advances in mass spectrometry and quantitative analyses of post-translational modifications, such as phosphorylation, paved the way for more clinically orientated studies. Thus, during the last 15 years proteomics has been applied to the study of diseases where platelet function is altered, such as acute coronary syndromes, arterial thrombosis (AT), diabetes, sepsis, or obesity, among others [2,3]. The goal of Platelet Clinical Proteomics studies is to find novel platelet-related biomarkers and drug targets that allow a better diagnosis and treatment of disease. The latter can be done through a drug discovery strategy to pharmacologically modulate the identified targets. The main challenge for the future is to standardize the methodology for sample preparation and proteomic analysis so it can be implemented in the clinic minimizing technical variations and increasing reproducibility between labs.

• [1]Burkhart JM, Vaudel M, Gambaryan S, Radau S, Walter U, Martens L, et al. The first comprehensive and quantitative analysis of human platelet protein composition allows the comparative analysis of structural and functional pathways. Blood 2012;120:e73–82.
• [2]Huang J, Zhang P, Solari FA, Sickmann A, Garcia A, Jurk K, et al. Molecular proteomics and signaling of human platelets in health and disease. Int J Mol Sci 2021;22:9860.
• [3]Barrachina MN, Hermida-Nogueira L, Moran LA, Casas V, Hicks SM, Sueiro AM, et al. Phosphoproteomics analysis of platelets in severe obesity uncovers platelet reactivity and signalling pathways alterations. Arterioscler Thromb Vasc Biol 2021;41:478–90.

Inherited Platelet Disorders. Educational Approach of Diagnosis and Management

How will you approach the diagnosis and clinical management of inherited platelet disorders (IPD)?

José M. Bastida

Department of Hematology, Complejo Asistencial Universitario de Salamanca (CAUSA), Instituto de Investigación Biomédica de Salamanca (IBSAL), Universidad de Salamanca (USAL), Salamanca, Spain

Diagnosing and treating IPDs is challenging. Clinical suspicion is crucial, based on factors such as bleeding tendency, evaluated by ISTH-BAT, family history, and specific systemic symptoms. Initial laboratory tests should rule out common acquired causes of thrombocytopenia and/or bleeding and, ideally, provide specific findings in the blood film [1]. Useful platelet phenotyping includes light transmission aggregometry, flow cytometry, and EM. Genetic diagnosis now relies on high-throughput sequencing methods [1]. Documenting informed consent, classifying the variants with consensus criteria, and data sharing to facilitate curation are encouraged [1,2]. Patients should be referred to specialized centers able to perform more complex tests and integrate the information [1,2]. Clinical management of IPD patients integrates general recommendations and the use of classical prohemostatic treatments depending on the severity of the bleeding episode, type of disorder, or the invasive procedure. TPO mimetics could be beneficial for some IPDs, particularly as bridging therapies while Hematopoietic stem cell transplant can be an appropriate option for severe disorders [3]. However, it is important to evaluate the risk-benefit before making any decisions. Novel drugs based on bispecific or monoclonal antibodies are being investigated, mainly for Glanzmann Thrombasthenia, with promising data in vitro and clinical trials [4].

• [1]Gomez K, Anderson J, Baker P, Biss T, Jennings I, Lowe G, et al. Clinical and laboratory diagnosis of heritable platelet disorders in adults and children: a British Society for Haematology Guideline. Br J Haematol 2021;195:46–72.
• [2]Megy K, Downes K, Morel-Kopp M-C, Bastida JM, Brooks S, Bury L, et al. GoldVariants, a resource for sharing rare genetic variants detected in bleeding, thrombotic, and platelet disorders: communication from the ISTH SSC Subcommittee on Genomics in Thrombosis and Hemostasis. J Thromb Haemost 2021;19:2612–7.
• [3]Bastida JM, Gonzalez-Porras JR, Rivera J, Lozano ML. Role of thrombopoietin receptor agonists in inherited thrombocytopenia. Int J Mol Sci 2021;22:4330.
• [4]Gandhi PS, Zivkovic M, Østergaard H, Bonde AC, Elm T, Løvgreen MN, et al. A bispecific antibody approach for the potential prophylactic treatment of inherited bleeding disorders. Nat CardioVasc Res 2024;3:166–85.

The Iberian contribution to the knowledge of IPD

Ana Marín Quilez, Ana Sánchez Fuentes, José Rivera

Servicio de Hematología, Hospital Universitario Morales Meseguer, Centro Regional de Hemodonación, Universidad de Murcia, IMIB-Pascual Parrilla, CIBERER-U765, Murcia, Spain. Grupo Español de Alteraciones Plaquetarias Congénitas (GEAPC), Spanish Society of Thrombosis and Haemostasis (SETH)

The Spanish Group of Inherited Platelet Disorders has been running, since 2008, a multicenter project aiming to facilitate the diagnosis and characterization of IPD in Spain, and potentially other countries such as Portugal. To date, about 450 patients, from more than 300 unrelated pedigrees, have been diagnosed, corresponding to 44 different types of IPD types. We have pioneering description of very rare IPD families. This image summarized some of our more relevant contributions to the knowledge of IPD: 1) the use of platelet transcriptomics as a tool to assess the pathogenicity of new RUNX1 variants and to identify new biomarkers of the disease; 2) SRC-RT is associated not only with impaired platelet production but also, potentially, with immune deregulation and increased platelet consumption; 3) genetic variants in TPM4 delocalizes tropomyosin-4 in the spreading structures, altering the platelet cytoskeleton and remodeling; 4) characterization of 5 novel patients with the rare GALE-RT, showing that mutant and dysfunctional GALE reduces platelet formation by impairing glycosylation of GPIbα and β1 integrin and reducing externalization to MKs and platelet membranes; and 5) a new variant affecting a N-glycosylation sequence in COX-1 with exert a dominant negative effect causing platelet dysfunction.

• [1]Palma-Barqueros V, Bastida JM, López Andreo MJ, Zámora-Cánovas A, Zaninetti C, Ruiz-Pividal JF, et al. Platelet transcriptome analysis in patients with germline RUNX1 mutations. J Thromb Haemost 2023;21:1352–65.
• [2]Palma-Barqueros V, Revilla N, Zaninetti C, Galera AM, Sánchez-Fuentes A, Zámora-Cánovas A, et al. Src-related thrombocytopenia: a fine line between a megakaryocyte dysfunction and an immune-mediated disease. Blood Adv 2022;6:5244–55.
• [3]Marín-Quílez A, Vuelta E, Díaz-Ajenjo L, Fernández-Infante C, García-Tuñón I, Benito R, et al. A novel nonsense variant in TPM4 caused dominant macrothrombocytopenia, mild bleeding tendency and disrupted cytoskeleton remodeling. J Thromb Haemost 2022;20:1248–55.
• [4]Marín-Quílez A, Di Buduo CA, Díaz-Ajenjo L, Abbonante V, Vuelta E, Soprano PM, et al. Novel variants in GALE cause syndromic macrothrombocytopenia by disrupting glycosylation and thrombopoiesis. Blood 2023;141:406–21.
• [5]Palma-Barqueros V, Crescente M, de la Morena ME, Chan MV, Almarza E, Revilla N, et al. A novel genetic variant in PTGS1 affects N-glycosylation of cyclooxygenase-1 causing a dominant-negative effect on platelet function and bleeding diathesis. Am J Hematol 2021;96:E83–E88.

Gene Therapy in severe IPD: Dream or reality?

Raul Torres-Ruiz^1,2,3,4, Paula Ojeda-Walczuk²

¹Molecular Cytogenetics and Genome Editing Unit, Human Cancer Genetics Program, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid, Spain

²Division of Hematopoietic Innovative Therapies, Biomedical Innovation Unit, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT), Madrid, Spain

³Advanced Therapies Unit, Instituto de Investigación Sanitaria Fundación Jiménez Díaz, Madrid Spain

⁴Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Madrid Spain

Glanzmann Thrombasthenia (GT) is a rare inherited platelet disorder characterized by severe bleeding due to mutations in the ITGA2B or ITGB3 genes, which impair platelet function, and by hence clot formation [1]. Current treatments, such as hemostatic drugs and allogeneic transplants, are limited, highlighting the need for more targeted therapies. Gene therapy using autologous HSPCs is emerging as a promising alternative [2]. However, precise transgene regulation is critical to avoid adverse effects. We propose the employment of CRISPRi (dCas9-KRAB) screening [3] to identify MK-specific promoters/enhancers, facilitating safer, lineage-specific lentiviral vectors for GT and related inherited platelet disorders with improved safety and therapeutic accuracy.

• [1]Palma-Barqueros V, Revilla N, Sánchez A, Zamora Cánovas A, Rodriguez-Alén A, Marín-Quílez A, et al. Inherited platelet disorders: an updated overview. Int J Mol Sci 2021;22:4521.
• [2]Wilcox DA. Gene therapy for platelet disorders. Platelets 2019:1191–205.
• [3]Fulco CP, Munschauer M, Anyoha R, Munson G, Grossman SR, Perez EM, et al. Systematic mapping of functional enhancer-promoter connections with CRISPR interference. Science 2016;354:769–73.

Advanced Exploration of Genome Variation in Platelets, Bleeding and Thrombotic Disorders

Why is the genetic basis of many platelet disorders still unknown?

Neil V. Morgan

Department of Cardiovascular Sciences, School of Medical Sciences, College of Medicine and Health, University of Birmingham, Birmingham, UK

Platelet disorders are extremely heterogeneous and can be caused by both reduced platelet number and platelet dysfunction and often associated with clinical bleeding. Although many genetic causes of platelet disorders are known, it is well recognized that >50% still have no obvious genetic cause despite ongoing large sequencing studies worldwide [1,2]. Studies have focused around developing platelet-specific gene panels and whole exome and genome sequencing to identify the candidate genetic variants. By utilizing in silico prediction tools and genome databases it is possible to model and narrow down the culprit gene variant(s). Many genetic variants may be missed and could be present in noncoding regions of the genome or be due to complex large genomic rearrangements, increasingly becoming detectable by current technologies [3]. It is also thought that platelet disorders could be multifactorial with genetic predisposition along with environmental factors. Candidate gene variants can be explored further via functional investigations to define their pathophysiology in platelet disease, but importantly link back to the well-defined patient platelet phenotype.

• [1]Daly ME, Leo VC, Lowe GC, Watson SP, Morgan NV. What is the role of genetic testing in the investigation of patients with suspected platelet function disorders? Br J Haematol 2014;165:193–203.
• [2]Johnson B, Lowe GC, Futterer J, Lordkipanidzé M, MacDonald D, Simpson MA, et al. UK GAPP Study Group. Whole exome sequencing identifies genetic variants in inherited thrombocytopenia with secondary qualitative function defects. Haematologica 2016;101:1170–9.
• [3]Turro E, Astle WJ, Megy K, Gräf S, Greene D, Shamardina O, et al. Whole-genome sequencing of patients with rare diseases in a national health system. Nature 2020;583:96–102.

How are novel long sequencing technologies increasing our knowledge of bleeding and thrombotic disorders

Belen de la Morena

Servicio de Hematología, Hospital Universitario Morales Meseguer, Centro Regional de Hemodonación, Universidad de Murcia, IMIB-Pascual Parrilla, CIBERER-U765, Murcia, Spain

Genetic analysis with next generation sequencing methods based on short-reads yields a positive diagnosis in up to 80% of cases, particularly those with a confirmed intermediate phenotype. Third generation sequencing methods like nanopore sequencing has emerged as a powerful approach for filling this gap in genetic diagnosis [1]. This technology enables real-time reading of long DNA/RNA molecules without fragmentation or manipulation, producing long-reads that allow (1) the detection and characterization at a nucleotide resolution of structural variants (SVs); (2) the identification of complex genetic mechanisms; and (3) detecting epigenetic or epitranscriptomics modifications. We showed examples of how this technology has allowed the dissection of SVs in antithrombin deficiency including the resolution of complex SVs [2] and resolved cases with unknown molecular defect, such as detecting complex mechanisms in Glanzmann thrombastenia [3] and insertions of retrotransposons in antithrombin deficiency [4].

• [1]Oehler JB, Wright H, Stark Z, Mallett AJ, Schmitz U. The application of long-read sequencing in clinical settings. Hum Genomics 2023;17:73.
• [2]de la Morena-Barrio B, Orlando C, Sanchis-Juan A, García JL, Padilla J, de la Morena-Barrio ME, et al. Molecular dissection of structural variations involved in antithrombin deficiency. J Mol Diagn 2022;24:462–475.
• [3]Zamora-Cánovas A, de la Morena-Barrio B, Marín-Quilez A, Sierra-Aisa C, Male C, Fernández-Mosteirin N, et al. Targeted long-read sequencing identifies and characterizes structural variants in cases of inherited platelet disorders. J Thromb Haemost 2024;22:851–859.
• [4]de la Morena-Barrio B, Stephens J, de la Morena-Barrio ME, Stefanucci L, Padilla J, Miñano A, et al. Long-read sequencing identifies the first retrotransposon insertion and resolves structural variants causing antithrombin deficiency. Thromb Haemost 2022;122:1369–1378.

New concepts in genetic diversity of inherited platelet disorders

Kato Ramaekers, My Tran, Koen De Wispelaere, Kathleen Freson

Center for Molecular and Vascular Biology, Department of Cardiovascular sciences, KULeuven, Belgium

IPDs are a heterogeneous group of mucocutaneous bleeding disorders of variable severity that relate to defects in platelet formation and function. They can be caused by 69 known genetic defects (gene list via www.isth.org/general/custom.asp?page=GinTh_GeneLists) but still many genetic causes remain unknown even after a decade of using next generation sequencing approaches. Even if whole-genome sequencing is used, nearly 50% of all IPD patients remain undiagnosed [1]. Platelet RNA sequencing (RNA-seq) studies are expected to provide information on gene expression and splicing in platelets from IPD patients [1]. We have initiated platelet RNA-seq to explore disease mechanisms (eg, for SLFN14 [2]) and assist in the gene discovery path of unexplained IPD. Platelets are enucleated cell fragments that contain RNA delivered their progenitor cell, the MK [3]. It is currently unknown how good the platelet transcriptome mimics that of the MK. We have started to compare platelet and MK transcriptomes under healthy conditions and for some IPD know to affect proplatelet formation. Multiomics approaches to study IPD are still an unexplored field and it can be expected that some more IPD will be discovered over the coming years.

• [1]Ver Donck F, Labarque V, Freson K. Hemostatic phenotypes and genetic disorders. Res Pract Thromb Haemost 2021;5:e12637.
• [2]Ver Donck F, Ramaekers K, Thys C, Van Laer C, Peerlinck K, Van Geet C, et al. Ribosome dysfunction underlies SLFN14-related thrombocytopenia. Blood 2023;141:2261–74.
• [3]De Wispelaere K, Freson K. The analysis of the human megakaryocyte and platelet coding transcriptome in healthy and diseased subjects. Int J Mol Sci 2022;23:7647.

Platelets Beyond Hemostasis

How do platelets contribute to venous thrombosis

Wolfgang Bergmeier

Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, USA

Blood Research Center, University of North Carolina, Chapel Hill, USA

Platelets are well-known for their detrimental role in AT. However, studies in humans and animal models also indicate a key role for platelets in venous thrombosis (VT). Compared with AT, where platelet-rich thrombi form under high shear stress conditions at sites of plaque rupture and extracellular matrix and smooth muscle cell exposure, VT is thought to be initiated by reduced blood flow and endothelial cell activation, leading to local inflammation and the formation of a thrombus enriched in fibrin and erythrocytes [1]. Studies in mouse models, especially the inferior vena cava stenosis model, have provided important mechanistic insights on how platelets affect the different stages of VT pathogenesis: initiation, propagation, and consolidation [2,3]. Major conclusions from these studies include:

• •Platelets contribute significantly to all stages of VT pathogenesis
• •Platelets are critical for leukocyte adhesion and activation during VT initiation
• •Leukocyte activation during VT initiation is impaired by antiplatelet drugs (aspirin, P2Y12 inhibitors) and inhibitors of immunoreceptor tyrosine-based activation motif signaling
• •Neutrophil extracellular traps and leukocyte-expressed tissue factor are critical for thrombin generation and fibrin formation in VT; during the propagation and consolidation phases, platelets sense and respond to thrombin and fibrin through protease-activated receptor 4 (PAR4) and glycoprotein VI (GPVI)
• •
  Mechanistic similarities exist for how platelets contribute to inflammatory hemostasis and VT initiation; however, low-flow-environment at sites of flow restriction also allows for platelet-leukocyte aggregate formation and the generation of a procoagulant environment needed for VT pathogenesis

  • [1]
    Wolberg AS, Rosendaal FR, Weitz JI, Jaffer IH, Agnelli G, Baglin T, et al. Venous thrombosis. Nat Rev Dis Primers 2015;1:15006.
  • [2]
    von Brühl M-L, Stark K, Steinhart A, Chandraratne S, Konrad I, Lorenz M, et al. Monocytes, neutrophils, and platelets cooperate to initiate and propagate venous thrombosis in mice in vivo. J Exp Med 2012;209:819–35.
  • [3]
    Mwiza JMN, Lee RH, Paul DS, Holle LA, Cooley BC, Nieswandt B, et al. Both G protein-coupled and immunoreceptor tyrosine-based activation motif receptors mediate venous thrombosis in mice. Blood 2022;139:3194–203.

Anti-PF4–antibody disorders

Andreas Greinacher

Institut für Transfusionsmedizin Universitätsmedizin Greifswald, Germany

Antiplatelet factor 4 (PF4) antibodies can cause several antibody mediated prothrombotic disorders [1]. They cause thromboinflammation and one of the most prothrombotic diseases in clinical medicine. Heparin-induced thrombocytopenia (HIT) was the first of these disorders, which is triggered by treatment with the widely used anticoagulant heparin. Heparin forms multimolecular complexes with PF4. This induces a conformational change in PF4 and exposure of neoepitopes to which some individuals develop IgG antibodies. The resulting immune complexes activate platelets, monocytes, granulocytes, and endothelial cells, inducing a cascade of thrombin generation [2]. HIT can persist, even when heparin treatment is stopped, becoming autoimmune HIT. Here, the anti-PF4 antibodies bind to PF4 as autoantibodies, which causes a conformational change in PF4 recruiting the heparin dependent antibodies. Rarely these antibodies occur spontaneously without pre-exposure with heparin (= spontaneous HIT). During the COVID-19 pandemic another group of anti-PF4 antibodies was observed, triggered by adenovirus vector based COVID-19 vaccines and named Vaccine Induced Thrombosis and Thrombocytopenia syndrome (VITT). These antibodies are superaggressive, bind to PF4 at another site than HIT antibodies and form immunecomplexes with PF4 without the need of an additional exogenous cofactor [3]. Since 2022 an increasing number of patients is observed in whom the same VITT-like antibodies occur after virus infections, or in individuals with monoclonal gammopathy. As the antibodies and the clinical picture resemble VITT, this is called VITT-like disorders. Rapid diagnosis and treatment with anticoagulants and additional inhibition of platelet and monocyte Fc-receptors strongly improve patient outcome.

• [1]Greinacher A, Warkentin TE. Thrombotic anti-PF4 immune disorders: HIT, VITT, and beyond. Hematology Am Soc Hematol Educ Program 2023;2023:1–10.
• [2]Greinacher A, Warkentin TE. Platelet factor 4 triggers thrombo-inflammation by bridging innate and adaptive immunity. Int J Lab Hematol 2023;45(S2):11–22.
• [3]Warkentin TE, Greinacher A. Laboratory testing for VITT antibodies. Semin Hematol 2022;59:80–8.

Aberrant platelet metabolism and activity in myeloproliferative neoplasms

Fan He, Stephen Oh, Jorge Di Paola

Department of Medicine and Pediatrics, Washington University School of Medicine in Saint Louis, Saint Louis, MO, USA

Platelets from patients with myeloproliferative neoplasms (MPNs) exhibit a hyperreactive phenotype [1]. Here, we found elevated P-selectin exposure and platelet-leukocyte aggregates in MPN platelets. Single-cell RNA-seq analysis of peripheral blood cells from primary samples revealed significant enrichment of transcripts related to platelet activation, mTOR, and oxidative phosphorylation in MPN platelets. Elevated PI3K/AKT/mTOR signaling and mitochondrial activity in MPN platelets were validated via platelet metabolomics, proteomic profiling, and seahorse mito stress assay. α-Ketoglutarate (α-KG) suppresses PI3K/AKT/mTOR signaling, ATP generation, and platelet activation in MPN platelets. Oral α-KG supplementation of JAK2 V617F mice decreases splenomegaly and reduces hematocrit, monocyte, and platelet counts. Our results reveal a previously unrecognized metabolic disorder in conjunction with aberrant PI3K/AKT/mTOR signaling that contributes to platelet hyperreactivity in MPNs [2].

• [1]Davizon-Castillo P, McMahon B, Aguila S, Bark D, Ashworth K, Allawzi A, et al. TNF-α-driven inflammation and mitochondrial dysfunction define the platelet hyperreactivity of aging. Blood 2019;134:727–40.
• [2]He F, Laranjeira AB, Kong T, Lin S, Ashworth KJ, Liu A, et al. Multiomic profiling reveals metabolic alterations mediating aberrant platelet activity and inflammation in myeloproliferative neoplasms. J Clin Invest 2024;134:e172256. https://doi.org/10.1172/jci172256

Immune Thrombocytopenia—Diagnosis and Treatment

Complex immunopathogenesis of immune thrombocytopenia

John W. Semple

Division of Hematology and Transfusion Medicine, Lund University, Lund, Sweden

Department of Pharmacology, University of Toronto, Toronto, Canada

Immune thrombocytopenia is the most common hematologic autoimmune disorder where immune-mediated destruction of platelets and MKs can lead to bleeding, fatigue, and sometimes thrombosis [1]. Platelets are normally removed by macrophages (M) during senescence and destroyed in lysosomal compartments within the spleen. An inflammatory insult (eg, infection) can activate antigen-presenting cell (macrophages [M] and dendritic cell) and alter their processing and presentation pathways. Excessive polarization of M toward the M1 phenotype and proinflammatory dendritic cells leads to the overproduction of proinflammatory cytokines (eg, IL-1β, TNF-α, and IL-6), which induce the expression of inflammatory cytokines (such as IL-2) in T helper (Th) 0 (naïve T cells) that support the autoimmune response [2]. The Th0 differentiate into several proinflammatory Th subsets such as T-follicular–helper cells, Th1 and Th17 cells. This unbalanced Th-cell differentiation excessively activates downstream effector cells such as autoreactive B cells differentiating into antibody-secreting plasma cells, natural killer cells and CD⁸⁺ cytotoxic T cells (CTLs) and CD⁸⁺ terminally differentiated effector memory CD⁸⁺ T cells. These effector cell responses can destroy platelets and MKs through a variety of processes such as Fc-receptor–mediated phagocytosis, complement activation and perforin/granzyme target cell destruction [3]. Superimposed on these above scenarios, platelets can be rapidly desialylated and recognized by the Ashwell Morrell receptor causing their further destruction by liver hepatocytes. Taken together, these effector cell events are finally completed to drive the thrombocytopenia observed in immune thrombocytopenia.

• [1]Provan D, Semple JW. Recent advances in the mechanisms and treatment of immune thrombocytopenia. EBioMedicine 2022;76:103820.
• [2]Zufferey A, Kapur R, Semple JW. Pathogenesis and therapeutic mechanism in immune thrombocytopenia (ITP). J Clin Med 2017;6:16.
• [3]Semple JW, Rebetz J, Maouia A, Kapur R. An update on the pathophysiology of immune thrombocytopenia. Curr Opin Hematol 2020;27:423–9.

How should we treat ITP in 2024?

María L. Lozano

Servicio de Hematología, Hospital Universitario Morales Meseguer, Centro Regional de Hemodonación, Universidad de Murcia, Murcia, Spain

In 2024, management decisions are still largely based on guidelines and patient preferences, leading to uniform therapeutic choices. However, these choices often result in a trial-and-error approach, with varied outcomes. While some patients achieve platelet responses on treatment or sustained responses off treatment, others may face complications like thrombosis.

In the near future, treatment will become more personalized through the integration of artificial intelligence. Artificial intelligence will analyze clinical characteristics and specific biomarkers, allowing for tailored therapeutic choices. This approach aims to increase the likelihood of platelet responses to treatment, reduce the risk of thrombosis, and enhance the probability of achieving sustained responses off treatment. The shift toward personalized medicine promises more targeted and effective treatments for patients with immune thrombocytopenia, moving away from the current trial-and-error methodology.