European Research Council–funded grant: maintenance of platelet homeostasis

Platelets, or thrombocytes, are small anucleate cell fragments of megakaryocytes (MKs) that are vital for thrombosis and haemostasis. By their very nature, they are primary causes of cardiovascular disease-associated morbidity and mortality, obstructing damaged and inflamed blood vessels. However, all conventional anti-platelet therapeutics have bleeding complications, including relatively mild antagonists, such as aspirin, prasugrel, and ticagrelor used extensively in cardiovascular medicine. Further, reversing the inhibitory effects of anti-platelet agents in instances of trauma and surgical intervention is lacking. There is also an urgent need for an unlimited supply of transfusable platelets to circumvent issues related to donor-derived platelets. Addressing these unmet clinical needs requires filling major gaps in our knowledge of the homeostatic mechanisms regulating the number and reactivity of platelets in the circulation in health and disease. The primary outstanding question in the field remains what the triggering mechanism of platelet production or thrombopoiesis is and why MKs do not release activated platelets in a haphazard manner in the bone marrow and other sites of thrombopoiesis (Figure 1A). The primary driver of MK development, proliferation, and maturation (megakaryopoiesis) is the cytokine thrombopoietin, but it does not explain when and where platelets are made and their biological activity. The focus to date has been on putative stimulatory factors, including cytokines, chemokines, extracellular matrix, and mechanical forces thought to drive thrombopoiesis, but none induces physiological platelet counts. Additionally, we still do not fully comprehend how platelets are inhibited in the circulation. The search must go beyond the usual suspects. Maintenance of platelet homeostasis by tyrosine phosphatases and vascular heparan sulfates. (A) Thrombopoietin is the primary driver of megakaryopoiesis, but it does not explain when and where megakaryocytes produce platelets (thrombopoiesis) and platelet inhibition in the circulation. (B) Defining and harnessing the hypothetical megakaryocyte/platelet checkpoint governed by the G6b-B-Shp1/2 inhibitory complex may provide new and improved way of inhibiting thrombosis in cardiovascular disease and beyond What is conspicuously overlooked are intrinsic inhibitory mechanisms that maintain MKs and platelets in a resting state and extrinsic cues that release this brake system, allowing asymmetric proplatelet production to proceed towards the vascular lumen, whilst platelets remain inactive. Drawing on the concept of the immune checkpoint that regulates the adaptive immune cell response to self and has been effectively exploited to augment the immune response to cancer cells, we hypothesize that a MK/platelet checkpoint regulates proplatelet formation and platelet reactivity to their environment. Defining and harnessing this checkpoint underpins the European Research Council (ERC)-funded grant Maintenance of Platelet Homeostasis by Tyrosine Phosphatases and Vascular Glycans (MENTOR) (Figure 1B).1 We postulate that the MK/platelet-specific co-inhibitory receptor G6b-B (MPIG6B) forms a complex with the non-transmembrane protein-tyrosine phosphatases (PTPs) Shp1 and Shp2 that acts as a gatekeeper of this checkpoint, loss of which severely impairs platelet production and function.2–5 The strength, duration, and compartmentalization of this inhibitory signal in the plasma membrane are regulated by intrinsic PTPs and kinases that lie upstream of G6b-B and extrinsic vascular heparan sulfate ligands that mediate homo- and hetero-clustering with itself and activation receptors, respectively.6 Our working model is that binding of MK G6b-B with heparan sulfates in the vessel wall inhibits inhibitory signalling from G6b-B, thereby initiating MK polarization and asymmetric platelet production, whereas kiss-and-go interactions of platelet G6b-B with heparan sulfates in the endothelial glycocalyx lining the luminal surface of blood vessels maintain platelets in a resting state by augmenting its inhibitory signal. Disseminated and persistent changes in the endothelial glycocalyx have been associated with endothelial dysfunction, vascular inflammation, and disease.7,8 Studies on the effects of heparan sulfates and structurally distinct sulfated glycosaminoglycans, including chondroitin sulfate on MKs and platelets have been largely exploratory and empirical to date, but demonstrate clear effects on MK and platelet function,6,9,10 providing a potential link between vascular heparan sulfates and platelet homeostasis in the context of cardiovascular disease. However, heparan sulfates vary enormously biochemically, biophysically, and spatiotemporally throughout the vasculature under pathophysiological conditions, and it remains unknown which specific forms of heparan sulfates G6b-B binds and how they regulate downstream signalling. Moreover, platelet-derived heparanase may be involved in this regulator mechanism through the release of soluble heparan sulfate degradation products and unmasking cryptic ligands. By advancing this alternative model of thrombopoiesis and defining how the MK/platelet checkpoint is regulated, we can proceed to harnessing G6b-B and other key components of this system through the use of novel antibody-based homo- and hetero-bifunctional compounds that modulate the threshold of MK/platelet activation. In this way, the MK/platelet homeostatic equilibrium can be shifted towards increased or decreased platelet production and reactivity, providing a more refined and versatile means of controlling thrombosis in cardiovascular disease compared with conventional competitive antagonists of platelet activation pathways. Discoveries may also yield lead compounds for augmenting platelet production in vitro for transfusion medicine. This innovative ERC-funded grant will be led by Dr. Yotis Senis, who will be joining the Cardiovascular and Nutrition Research Centre (C2VN) in the Faculty of Medicine, Aix-Marseille University in the summer of 2025. The C2VN is under the joint auspices of Aix-Marseille University, Institut National de la Santé et de la Recherche Médicale (Inserm), and Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE). Primary areas of research at the C2VN include vascular biology, extracellular vesicles, and platelet-based thrombotic and haemostatic disorders, with state-of-the-art flow cytometry, imaging, and animal facilities. A multidisciplinary approach will be taken to address key objectives of this project in collaboration with an international network of leading experts in glycobiology, biophysics and mathematical modelling, antibody engineering, and heparan sulfate medicinal chemistry. Megakaryocyte, platelet and endothelial cell biology, signal transduction, and pathophysiology involving genetically modified mouse models will be conducted in the Senis laboratory in the C2VN, setting the stage for future non-human primate studies and clinical trials in cardiovascular disease. Y.A.S. is funded by Inserm, the Agence Nationale pour la Recherche (ANR-23-TARGITT-CE18-0020-01), and the European Research Council (ERC-MENTOR-101141783). All authors declare no disclosure of interest for this contribution.