Current antithrombotic therapies are effective in reducing thrombotic events but are limited by their associated risk of bleeding. BTK acts as a key signalling switch that drives platelet activation during thrombosis but is largely dispensable for routine haemostasis. It is an important non-redundant signalling mediator downstream of the GPVI and CLEC-2 receptors, plays a key role in thrombosis with minimal involvement in haemostasis, making it an attractive antithrombotic target. While BTK inhibitors effectively reduce thrombosis, their clinical use has been limited due to off-target effects. Protein degraders may overcome this limitation by enabling the ubiquitin proteasomal system to selectively target and degrade BTK. We here assessed the ability of the BTK degraders NX-2127 and NX-5948, currently in clinical trials for B cell pathologies, to target platelet BTK for degradation. NX-2127 and NX-5948 induced concentration-dependent degradation of BTK in washed platelets, platelet-rich plasma and whole blood. NX-5948 showed no hook effect and outperformed NX-2127 in potency, efficacy, and degradation kinetics. Proteomic analysis confirmed selective BTK degradation by NX-5948 with no evidence of major off-target effects. BTK degradation impaired CRP-mediated integrin αIIbβ₃ activation, P-selectin expression, platelet aggregation and in vitro thrombosis, with PAR-1 mediated platelet function being left intact. Dosing mice with NX-5948 led to efficacious degradation of platelet BTK and impaired CRP-, but not thrombin-, mediated ex vivo platelet function. In vivo, arterial thrombosis was markedly reduced, without an increase in bleeding time. Together, these results highlight NX-5948 as a potent, selective BTK degrader with antithrombotic potential and minimal haemostatic impact.

Class I PI3Kβ is critical in controlling platelet activation and preserving thrombus stability under high shear. PI3Kβ is unique among class I phosphoinositide 3-kinases (PI3Ks) in being activated by Rho-family GTPases Rac and Cdc42, positioning it as a potential key link between Rho-family GTPases and platelet signaling. Here, we combined pharmacological inhibition with genetic approaches to define a direct Rho-PI3Kβ signaling axis in mouse platelets. Platelets from knockin mice carrying two point mutations within the Rho-binding domain (RBD) of the PI3Kβ catalytic subunit p110β exhibited impaired GPVI-mediated platelet signaling, functional responses, and spreading on fibrinogen. Pharmacological inhibition with the PI3Kβ-selective inhibitor AZD6482 demonstrated that these responses largely depend on the p110β RBD. Inhibition of Rac/Cdc42 in wild-type platelets phenocopied the knockin defects, supporting a direct role for Rho GTPases in PI3Kβ activation. Conversely, Rac activation was impaired in RBD-mutant and AZD6482-treated platelets, revealing a Rac-PI3Kβ feedback loop that amplifies GPVI-dependent platelet activation.

Prostacyclin (PGI2, epoprostenol) and its more stable analogues iloprost and cicaprost are used in the treatment of pulmonary arterial hypertension (PAH) and other related diseases. Currently, PGI2 therapy is the most effective treatment for PAH, but is administered intravenously due to its instability under physiological conditions. We considered creating more chemically stable hybrids of PGI2 by merging essential features of iloprost/cicaprost with a more stable C-7 fluorinated PGI2, which maintained the cyclic enol ether. The synthesis employed our key bicyclic enal and furnished the required PGI2 analogues in just 7-8 steps, providing the most expedient route to this class of molecules. This led to the discovery of compound 9, a picomolar-potent, IP receptor-selective, and chemically stable PGI2 analogue that combined the ω-side chain of cicaprost with the C-7 difluorinated enol ether of PGI2. This compound provides the most potent PGI2 analogue tested to date.

Ischaemic heart disease and myocardial infarction remain the leading causes of mortality worldwide. This occurs when a coronary artery becomes occluded, leading to ischaemic damage which can develop into heart failure. However, current treatments do not directly promote myocardial repair. Although platelets mediate the primary ischaemic damage, they also release numerous beneficial pro-reparative mediators including soluble factors and extracellular vesicles, sub-micron sized mediators of intercellular communication. This study aimed to assess the cardioprotective efficacy of these secretome elements. To this end, murine platelets were stimulated ex vivo to trigger secretion. The full releasate secretome was then segregated into its components: molecular releasate, small- and large extracellular vesicles. These were injected into the myocardium at the time of permanent coronary artery ligation in murine models of myocardial infarction. Injection of full releasate led to significantly better cardiac function compared with vehicle from 7d to 21d post infarct. This benefit was predominantly mediated by the small extracellular vesicle fraction that, when isolated and injected into the infarcting myocardium, successfully preserved cardiac function and enhanced tissue revascularisation. Thus, this study establishes platelet-derived small extracellular vesicles as promising therapeutic candidates for MI which could harness the endogenous regenerative potential of platelets in clinic.

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.

Interleukin-6 (IL-6) is a pleiotropic cytokine with critical roles in immune regulation, inflammation, and haematopoiesis. While its functions in host defence and tissue repair are well established, accumulating evidence suggests that IL-6 also can directly and indirectly modulate megakaryocyte and platelet biology. This review examines the mechanistic basis supporting IL-6-mediated platelet hyper-responsiveness, in addition to its effect on megakaryopoiesis and thrombopoiesis in thromboinflammatory disease states. We discuss how IL-6-mediated trans-signalling may sensitizes platelets to activation, and that this may be exclusive to glycoprotein VI (GPVI) stimulation due to Janus kinase (JAK)–signal transducer 2 crosstalk, in addition to other mechanisms that may contribute to priming platelets. We further highlight clinical evidence linking IL-6 to thrombotic complications in cardiovascular disease and infection (e.g., COVID-19 and sepsis). Given the emerging interest in IL-6-targeting therapies as anti-inflammatory and anti-thrombotic agents, a thorough understanding of how IL-6 can drive platelet responsiveness is crucial.

Purpose of review Proteolysis-targeted chimeras (PROTACs) are heterobifunctional compounds that selectively target proteins for degradation and are an emerging therapeutic modality to treat diseases such as cancer and neurodegenerative disorders. This review will widen the area of application by highlighting the ability of PROTACs to remove proteins from the anucleate platelets and evaluate their antithrombotic potential. Recent findings Proteomic and biochemical studies demonstrated that human platelets possess the Ubiquitin Proteasomal System as well as the E3 ligase cereblon (CRBN) and therefore may be susceptible to PROTAC-mediated protein degradation. Recent findings confirmed that CRBN ligand-based PROTACs targeting generic tyrosine kinases, Btk and/or Fak lead to efficacious and selective protein degradation in human platelets. Downregulation of Btk, a key player involved in signalling to thrombosis, but not haemostasis, resulted in impaired in-vitro thrombus formation. Summary Platelets are susceptible to targeted protein degradation by CRBN ligand-based PROTACs and have limited ability to resynthesise proteins, ensuring long-term downregulation of target proteins. Therefore, PROTACs serve as an additional research tool to study platelet function and offer new therapeutic potential to prevent thrombosis. Future studies should focus on enhancing cell specificity to avoid on-target side effects on other blood cells.

Background and Purpose Thromboxane A(2) (TXA(2)) is a prostanoid produced during platelet activaton, important in enhancing platelet reactivity by activation of TP receptors. However, due to the short half-life, studying TXA(2) signalling is challenging. To enhance our understanding of TP receptor-mediated platelet biology, we therefore synthesised mono and difluorinated TXA(2) analogues and explored their pharmacology on heterologous and endogenously expressed TP receptor function. Experimental Approach Platelet functional and signalling responses were studied using aggregometry, Ca2+ mobilisation experiments and immunoblotting and compared with an analogue of the TXA(2) precursor prostaglandin H-2, U46619. G alpha(q)/G alpha(s) receptor signalling was determined using a bioluminescence resonance energy transfer (BRET) assay in a cell line overexpression system. Key Results BRET studies revealed that F-TXA(2) and F-2-TXA(2) promoted receptor-stimulated TP receptor G-protein activation similarly to U46619. Unexpectedly, F-2-TXA(2) caused reversible aggregation in platelets, whereas F-TXA(2) and U46619 induced sustained aggregation. Blocking the IP receptor switched F-2-TXA(2)-mediated reversible aggregation into sustained aggregation. Further BRET studies confirmed F-2-TXA(2)-mediated IP receptor activation. F-2-TXA(2) rapidly and potently stimulated platelet TP receptor-mediated protein kinase C/P-pleckstrin, whereas IP-mediated protein kinase A/P-vasodilator-stimulated phosphoprotein was more delayed. Conclusion and Implications F-TXA(2) is a close analogue to TXA(2) used as a selective tool for TP receptor platelet activation. In contrast, F-2-TXA(2) acts on both TP and IP receptors differently over time, resulting in an initial wave of TP receptor-mediated platelet aggregation followed by IP receptor-induced reversibility of aggregation. This study reveals the potential difference in the temporal aspects of stimulatory and inhibitory pathways involved in platelet activation.

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

Thousands of proteins circulate in the bloodstream; identifying those which associate with weight and intervention-induced weight loss may help explain mechanisms of diseases associated with adiposity. We aimed to identify consistent protein signatures of weight loss across independent studies capturing changes in body mass index (BMI). We analysed proteomic data from studies implementing caloric restriction (Diabetes Remission Clinical trial) and bariatric surgery (By-Band-Sleeve), using SomaLogic and Olink Explore1536 technologies, respectively. Linear mixed models were used to estimate the effect of the interventions on circulating proteins. Twenty-three proteins were altered in a consistent direction after both bariatric surgery and caloric restriction, suggesting that these proteins are modulated by weight change, independent of intervention type. We also integrated Mendelian randomisation (MR) estimates of the effect of BMI on proteins measured by SomaLogic from a UK blood donor cohort as a third line of causal evidence. These MR estimates provided further corroborative evidence for a role of BMI in regulating the levels of six proteins including alcohol dehydrogenase-4, nogo receptor and interleukin-1 receptor antagonist protein. These results indicate the importance of triangulation in interrogating causal relationships; further study into the role of proteins modulated by weight in disease is now warranted.

Understanding the differences between physiological hemostasis and pathological thrombosis at molecular and cellular levels is a long-standing goal of platelet and coagulation biology. Such a distinction will pave the way for designing strategies to pharmacologically inhibit thrombosis without an increased concomitant tendency to bleed. Given that bleeding is the most common adverse effect associated with antithrombotics, with some cases being life-threatening, it is essential to distinguish between hemostasis and thrombosis. A principal difference is that hemostasis and thrombosis do not occur in the same local environment; while hemostasis occurs within tissue in the extravascular space, thrombosis is essentially an intra-vascular process (Figure 1). Additionally, although hemostasis and thrombosis share common pathways at a molecular level, increasing evidence has demonstrated that they are mechanistically distinct. For example, animal model gene knockout (KO) or inhibition of coagulation factor XI (FXI), 1,2 FXII, 3,4 glycoprotein VI (GPVI), 5-8 water channel aquaporin-1 (AQP1), 9 or protein kinase C alpha 10 show no signi fi cant bleeding defects but markedly affect arterial thrombosis. Additionally, pharmacologic inhibition of FXI 11,12 and FXII 13 in preclinical trials provides evidence that hemostasis and thrombosis can be uncoupled. In this commentary, we propose the hypothesis that platelet procoagulant membrane ballooning and microvesiculation, collectively termed procoagulant membrane dynamics (PMD), which amplify the procoagulant response in platelets, could provide a mechanistic distinction between hemostasis and thrombosis

Background: Patients with COVID-19 are at increased risk of thrombosis, which is associated with altered platelet function and coagulopathy, contributing to excess mortality.Objectives: To characterize the mechanism of altered platelet function in COVID-19 patients.Methods: The platelet proteome, platelet functional responses, and platelet-neutrophil aggregates were compared between patients hospitalized with COVID-19 and healthy control subjects using tandem mass tag proteomic analysis, Western blotting, and flow cytometry.Results: COVID-19 patients showed a different profile of platelet protein expression (858 altered of the 5773 quantified). Levels of COVID-19 plasma markers were enhanced in the platelets of COVID-19 patients. Gene ontology pathway analysis demonstrated that the levels of granule secretory proteins were raised, whereas those of platelet activation proteins, such as the thrombopoietin receptor and protein kinase C & alpha;, were lowered. Basally, platelets of COVID-19 patients showed enhanced phosphatidylserine exposure, with unaltered integrin & alpha;IIb & beta;3 activation and P-selectin expression. Agonist-stimulated integrin & alpha;IIb & beta;3 activation and phosphatidylserine exposure, but not P-selectin expression, were decreased in COVID-19 patients. COVID-19 patients had high levels of platelet-neutrophil aggregates, even under basal conditions, compared to controls. This association was disrupted by blocking P-selectin, demonstrating that platelet P-selectin is critical for the interaction.Conclusions: Overall, our data suggest the presence of 2 platelet populations in patients with COVID-19: one of circulating platelets with an altered proteome and reduced functional responses and another of P-selectin-expressing neutrophil-associated platelets. Platelet-driven thromboinflammation may therefore be one of the key factors enhancing the risk of thrombosis in COVID-19 patients.

Deep vein thrombosis (DVT) is the formation of a blood clot in a deep vein. DVT can lead to a venous thromboembolism (VTE), the combined term for DVT and pulmonary embolism, a leading cause of death and disability worldwide. Despite the prevalence and associated morbidity of DVT, the underlying causes are not well understood. Our aim was to leverage publicly available genetic summary association statistics to identify causal risk factors for DVT. We conducted a Mendelian randomization phenome-wide association study (MR-PheWAS) using genetic summary association statistics for 973 exposures and DVT (6,767 cases and 330,392 controls in UK Biobank). There was evidence for a causal effect of 57 exposures on DVT risk, including previously reported risk factors (e.g. body mass index-BMI and height) and novel risk factors (e.g. hyperthyroidism and varicose veins). As the majority of identified risk factors were adiposity-related, we explored the molecular link with DVT by undertaking a two-sample MR mediation analysis of BMI-associated circulating proteins on DVT risk. Our results indicate that circulating neurogenic locus notch homolog protein 1 (NOTCH1), inhibin beta C chain (INHBC) and plasminogen activator inhibitor 1 (PAI-1) influence DVT risk, with PAI-1 mediating the BMI-DVT relationship. Using a phenome-wide approach, we provide putative causal evidence that hyperthyroidism, varicose veins and BMI enhance the risk of DVT. Furthermore, the circulating protein PAI-1 has a causal role in DVT aetiology and is involved in mediating the BMI-DVT relationship.

Platelets, small hemostatic blood cells, are derived from megakaryocytes. Both bone marrow and lung are principal sites of thrombopoiesis although underlying mechanisms remain unclear. Outside the body, however, our ability to generate large number of functional platelets is poor. Here we show that perfusion of megakaryocytes ex vivo through the mouse lung vasculature generates substantial platelet numbers, up to 3000 per megakaryocyte. Despite their large size, megakaryocytes are able repeatedly to passage through the lung vasculature, leading to enucleation and subsequent platelet generation intravascularly. Using ex vivo lung and an in vitro microfluidic chamber we determine how oxygenation, ventilation, healthy pulmonary endothelium and the microvascular structure support thrombopoiesis. We also show a critical role for the actin regulator Tropomyosin 4 in the final steps of platelet formation in lung vasculature. This work reveals the mechanisms of thrombopoiesis in lung vasculature and informs approaches to large-scale generation of platelets.

The tyrosine kinase BTK plays an important role in platelet function downstream of GPVI and CLEC2 receptors and has been proposed as a novel target to prevent thrombosis in patients that are at increased risk. However, current clinically approved BTK inhibitors have off target effects and are associated with an increased bleeding risk. In this study, we therefore explored whether BTK can be targeted for degradation in human platelets by using recently developed heterobifunctional molecules that employ the proteasomal system to break down BTK. Here we confirm that human platelets are highly susceptible to BTK degraders with the generic tyrosine kinase degrader TL12-186, and the BTK degraders DD-04-15 and DD-03-171 leading to breakdown of BTK and its closely related kinase TEC, an effect that was prevented by proteasomal inhibitors. Tandem Mass Tag proteomic analysis confirmed high selectivity with TL12-186 degrading BTK/TEC, FAK/PYK2 and FER, whereas DD-04-15 and DD-03-171 degraded BTK/TEC only. GPVI-mediated platelet integrin α IIb β 3 activation, P-selectin expression, and phosphatidyl-serine exposure were largely impaired upon BTK/TEC degradation, with PAR-1-mediated responses left intact. This is the first study to demonstrate that chemical protein degraders can be successfully employed in anucleate human platelets to modulate their function.

Higher body mass index (BMI) is a risk factor for thrombosis. Platelets are essential for hemostasis but contribute to thrombosis when activated pathologically. We hypothesized that higher BMI leads to changes in platelet characteristics, thereby increasing thrombotic risk. The effect of BMI on platelet traits (measured by Sysmex) was explored in 33 388 UK blood donors (INTERVAL study). Linear regression showed that higher BMI was positively associated with greater plateletcrit (PCT), platelet count (PLT), immature platelet count (IPC), and side fluorescence (SFL, a measure of mRNA content used to derive IPC). Mendelian randomization (MR), applied to estimate a causal effect with BMI proxied by a genetic risk score, provided causal estimates for a positive effect of BMI on both SFL and IPC, but there was little evidence for a causal effect of BMI on PCT or PLT. Follow-up analyses explored the functional relevance of platelet characteristics in a pre-operative cardiac cohort (COPTIC). Linear regression provided observational evidence for a positive association between IPC and agonist-induced whole blood platelet aggregation. Results indicate that higher BMI raises the number of immature platelets, which is associated with greater whole blood platelet aggregation in a cardiac cohort. Higher IPC could therefore contribute to obesity-related thrombosis.

Sphingosine 1-phosphate (S1P) is a bioactive signalling sphingolipid that is increased in diseases such as obesity and diabetes. S1P can modulate platelet function, however the direction of effect and S1P receptors (S1PRs) involved are controversial. Here we describe the role of S1P in regulating human platelet function and identify the receptor subtypes responsible for S1P priming. Human platelets were treated with protease-activated receptor 1 (PAR-1)-activating peptide in the presence or absence of S1P, S1PR agonists or antagonists, and sphingosine kinases inhibitors. S1P alone did not induce platelet aggregation but at low concentrations S1P enhanced PAR1-mediated platelet responses, whereas PAR1 responses were inhibited by high concentrations of S1P. This biphasic effect was mimicked by pan-S1PR agonists. Specific agonists revealed that S1PR1 receptor activation has a positive priming effect, S1PR2 and S1PR3 have no effect on platelet function, whereas S1PR4 and S1PR5 receptor activation have an inhibitory effect on PAR-1 mediated platelet function. Although platelets express both sphingosine kinase 1/2, enzymes which phosphorylate sphingosine to produce S1P, only dual and SphK2 inhibition reduced platelet function. These results support a role for SphK2-mediated S1P generation in concentration-dependent positive and negative priming of platelet function, through S1PR1 and S1PR4/5 receptors, respectively.