Difluorinated thromboxane A2 reveals crosstalk between platelet activatory and inhibitory pathways by targeting both the TP and IP receptors.

BACKGROUND AND PURPOSE: Thromboxane A2 (TXA2) 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 TXA2 signalling is challenging. To enhance our understanding of TP receptor-mediated platelet biology, we therefore synthesised mono and difluorinated TXA2 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 TXA2 precursor prostaglandin H2, U46619. Gαq/Gαs 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-TXA2 and F2-TXA2 promoted receptor-stimulated TP receptor G-protein activation similarly to U46619. Unexpectedly, F2-TXA2 caused reversible aggregation in platelets, whereas F-TXA2 and U46619 induced sustained aggregation. Blocking the IP receptor switched F2-TXA2-mediated reversible aggregation into sustained aggregation. Further BRET studies confirmed F2-TXA2-mediated IP receptor activation. F2-TXA2 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-TXA2 is a close analogue to TXA2 used as a selective tool for TP receptor platelet activation. In contrast, F2-TXA2 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.

A phenome-wide approach to identify causal risk factors for deep vein thrombosis.

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.

Using trials of caloric restriction and bariatric surgery to explore the effects of body mass index on the circulating proteome.

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.

Highly efficient platelet generation in lung vasculature reproduced by microfluidics.

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.

Alterations in platelet proteome signature and impaired platelet integrin αIIbß3 activation in patients with COVID-19.

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 Cα, were lowered. Basally, platelets of COVID-19 patients showed enhanced phosphatidylserine exposure, with unaltered integrin αIIbß3 activation and P-selectin expression. Agonist-stimulated integrin αIIbß3 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.

Higher body mass index raises immature platelet count: potential contribution to obesity-related thrombosis.

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.

Opposing Roles of GSK3α and GSK3ß Phosphorylation in Platelet Function and Thrombosis.

One of the mechanisms by which PI3 kinase can regulate platelet function is through phosphorylation of downstream substrates, including glycogen synthase kinase-3 (GSK3)α and GSK3ß. Platelet activation results in the phosphorylation of an N-terminal serine residue in GSK3α (Ser21) and GSK3ß (Ser9), which competitively inhibits substrate phosphorylation. However, the role of phosphorylation of these paralogs is still largely unknown. Here, we employed GSK3α/ß phosphorylation-resistant mouse models to explore the role of this inhibitory phosphorylation in regulating platelet activation. Expression of phosphorylation-resistant GSK3α/ß reduced thrombin-mediated platelet aggregation, integrin αIIbß3 activation, and α-granule secretion, whereas platelet responses to the GPVI agonist collagen-related peptide (CRP-XL) were significantly enhanced. GSK3 single knock-in lines revealed that this divergence is due to differential roles of GSK3α and GSK3ß phosphorylation in regulating platelet function. Expression of phosphorylation-resistant GSK3α resulted in enhanced GPVI-mediated platelet activation, whereas expression of phosphorylation-resistant GSK3ß resulted in a reduction in PAR-mediated platelet activation and impaired in vitro thrombus formation under flow. Interestingly, the latter was normalised in double GSK3α/ß KI mice, indicating that GSK3α KI can compensate for the impairment in thrombosis caused by GSK3ß KI. In conclusion, our data indicate that GSK3α and GSK3ß have differential roles in regulating platelet function.

Effects of adiposity on the human plasma proteome: observational and Mendelian randomisation estimates.

BACKGROUND: Variation in adiposity is associated with cardiometabolic disease outcomes, but mechanisms leading from this exposure to disease are unclear. This study aimed to estimate effects of body mass index (BMI) on an extensive set of circulating proteins. METHODS: We used SomaLogic proteomic data from up to 2737 healthy participants from the INTERVAL study. Associations between self-reported BMI and 3622 unique plasma proteins were explored using linear regression. These were complemented by Mendelian randomisation (MR) analyses using a genetic risk score (GRS) comprised of 654 BMI-associated polymorphisms from a recent genome-wide association study (GWAS) of adult BMI. A disease enrichment analysis was performed using DAVID Bioinformatics 6.8 for proteins which were altered by BMI. RESULTS: Observationally, BMI was associated with 1576 proteins (P < 1.4 × 10-5), with particularly strong evidence for a positive association with leptin and fatty acid-binding protein-4 (FABP4), and a negative association with sex hormone-binding globulin (SHBG). Observational estimates were likely confounded, but the GRS for BMI did not associate with measured confounders. MR analyses provided evidence for a causal relationship between BMI and eight proteins including leptin (0.63 standard deviation (SD) per SD BMI, 95% CI 0.48-0.79, P = 1.6 × 10-15), FABP4 (0.64 SD per SD BMI, 95% CI 0.46-0.83, P = 6.7 × 10-12) and SHBG (-0.45 SD per SD BMI, 95% CI -0.65 to -0.25, P = 1.4 × 10-5). There was agreement in the magnitude of observational and MR estimates (R2 = 0.33) and evidence that proteins most strongly altered by BMI were enriched for genes involved in cardiovascular disease. CONCLUSIONS: This study provides evidence for a broad impact of adiposity on the human proteome. Proteins strongly altered by BMI include those involved in regulating appetite, sex hormones and inflammation; such proteins are also enriched for cardiovascular disease-related genes. Altogether, results help focus attention onto new proteomic signatures of obesity-related disease.

Sphingosine-1-phosphate modulates PAR1-mediated human platelet activation in a concentration-dependent biphasic manner.

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.

Synthesis, Stability, and Biological Studies of Fluorinated Analogues of Thromboxane A2.

Platelet activation results in the generation of thromboxane A2 (TxA2), which promotes thrombus formation by further amplifying platelet function, as well as causing vasoconstriction. Due to its role in thrombus formation and cardiovascular disease, its production is the target of antiplatelet drugs such as aspirin. However, the study of TxA2-stimulated cellular function has been limited by its instability (t 1/2 = 32 s, pH = 7.4). Although more stable analogues such as U46619 and difluorinated 10,10-F2-TxA2 have been prepared, we targeted a closer mimic to TxA2 itself, monofluorinated 10-F-TxA2, since the number of fluorine atoms can affect function. Key steps in the synthesis of F-TxA2 included α-fluorination of a lactone bearing a ß-alkoxy group, and a novel synthesis of the strained acetal. F-TxA2 was found to be 105 more stable than TxA2, and surprisingly was only slightly less stable than F2-TxA2. Preliminary biological studies showed that F-TxA2 has similar potency as TxA2 toward inducing platelet aggregation but was superior to F2-TxA2 in activating integrin αIIbß3.

Rapamycin restrains platelet procoagulant responses via FKBP-mediated protection of mitochondrial integrity.

BACKGROUND AND PURPOSE: Rapamycin is a potent immunosuppressant and anti-proliferative agent used clinically to prevent organ transplant rejection and for coating coronary stents to counteract restenosis. Rapamycin complexes with the immunophilin FKBP12, which subsequently binds and inhibits mTORC1. Despite several reports demonstrating that rapamycin affects platelet-mediated responses, the underlying mechanism of how it alters platelet function is poorly characterised. This study aimed to elucidate the effect of rapamycin on platelet procoagulant responses. EXPERIMENTAL APPROACH: The effect of rapamycin on platelet activation and signalling was investigated alongside the catalytic mTOR inhibitors KU0063794 and WYE-687, and the FKBP12-binding macrolide FK506. KEY RESULTS: Rapamycin affects platelet procoagulant responses by reducing externalisation of the procoagulant phospholipid phosphatidylserine, formation of balloon-like structures and local generation of thrombin. Catalytic mTOR kinase inhibitors did not alter platelet procoagulant processes, despite having a similar effect as rapamycin on Ca2+ signalling, demonstrating that the effect of rapamycin on procoagulant responses is independent of mTORC1 inhibition and not linked to a reduction in Ca2+ signalling. FK506, which also forms a complex with FKBP12 but does not target mTOR, reduced platelet procoagulant responses to a similar extent as rapamycin. Both rapamycin and FK506 prevented the loss of mitochondria integrity induced by platelet activation, one of the central regulatory events leading to PS externalisation. CONCLUSIONS AND IMPLICATIONS: Rapamycin suppresses platelet procoagulant responses by protecting mitochondrial integrity in a manner independent of mTORC1 inhibition. Rapamycin and other drugs targeting FKBP immunophilins could aid the development of novel complementary anti-platelet therapies.

PI3K inhibitors in thrombosis and cardiovascular disease.

Phosphoinositide 3-kinases (PI3Ks) are lipid kinases that regulate important intracellular signalling and vesicle trafficking events via the generation of 3-phosphoinositides. Comprising eight core isoforms across three classes, the PI3K family displays broad expression and function throughout mammalian tissues, and the (patho)physiological roles of these enzymes in the cardiovascular system present the PI3Ks as potential therapeutic targets in settings such as thrombosis, atherosclerosis and heart failure. This review will discuss the PI3K enzymes and their roles in cardiovascular physiology and disease, with a particular focus on platelet function and thrombosis. The current progress and future potential of targeting the PI3K enzymes for therapeutic benefit in cardiovascular disease will be considered, while the challenges of developing drugs against these master cellular regulators will be discussed.

Redundant role of ASK1-mediated p38MAPK activation in human platelet function.

Apoptosis signal-regulating kinase 1 (ASK1) is a member of mitogen-activated protein kinase kinase kinase (MAP3K) family, which recently has been implicated in the regulation of p38 MAPK/PLA2/thromboxane (TxA2) generation, as well as P2Y12 signalling in murine platelets. ASK1 has therefore been proposed as a potential target for anti-thrombotic therapy. At present it is unknown whether ASK1 also contributes to TxA2 formation and platelet function in human. In this study we therefore examined the role of ASK1 using the ASK1 inhibitor selonsertib (GS-4997). We established that ASK1 is responsible for p38 phosphorylation and TxA2 formation in murine platelets, with both GS4997 and p38 inhibitors reducing TxA2 formation. Similar to murine platelets, activation of human platelets resulted in the rapid and transient phosphorylation of ASK1 and the MAP2Ks MMK3/4/6. In contrast, phosphorylation of p38 and its substrate; MAPKAP-kinase2 (MAPKAPK2) was much more sustained. In keeping with these findings, inhibition of ASK1 blocked early, but not later p38/MAPKAPK2 phosphorylation. The latter was dependent on non-canonical autophosphorylation as it was blocked by the p38 inhibitor; SB203580 and the SYK inhibitor; R406. Furthermore, ASK1 and p38 inhibitors had no effect on PLA2 phosphorylation, TxA2 formation and platelet aggregation, demonstrating that this pathway is redundant in human platelets. Together, these results demonstrate that ASK1 contributes to TxA2 formation in murine, but not human platelets and highlight the importance of confirming findings from genetic murine models in humans.

Carbonic Anhydrase Inhibitors suppress platelet procoagulant responses and in vivo thrombosis.

Carbonic anhydrase (CA) inhibitors have a long history of safe clinical use as mild diuretics, in the treatment of glaucoma and for altitude sickness prevention. In this study, we aimed to determine if CA inhibition may be an alternative approach to control thrombosis. We utilized a high-resolution dynamic imaging approach to provide mechanistic evidence that CA inhibitors may be potent anti-procoagulant agents in vitro and effective anti-thrombotics in vivo. Acetazolamide and methazolamide, while sparing platelet secretion, attenuated intracellular chloride ion entry and suppressed the procoagulant response of activated platelets in vitro and thrombosis in vivo. The chemically similar N-methyl acetazolamide, which lacks CA inhibitory activity, did not affect platelet procoagulant response in vitro. Outputs from rotational thromboelastometry did not reflect changes in procoagulant activity and reveal the need for a suitable clinical test for procoagulant activity. Drugs specifically targeting procoagulant remodeling of activated platelets, by blockade of carbonic anhydrases, may provide a new way to control platelet-driven thrombosis without blocking essential platelet secretion responses.

Identification of PtdIns(3,4)P2 effectors in human platelets using quantitative proteomics.

After decades in PtdIns(3,4,5)P3's shadow, PtdIns(3,4)P2 has now emerged as a bona fide regulator of important cellular events, including endocytosis and cell migration. New understanding of PtdIns(3,4)P2's cellular roles has been possible via novel approaches to observe and quantify cellular PtdIns(3,4)P2 dynamics, alongside methods to target the kinases and phosphatases governing phosphoinositide turnover. Despite this, the mechanisms by which PtdIns(3,4)P2 orchestrates its cellular roles remain more poorly understood, most notably because, to date, few PtdIns(3,4)P2 effectors have been identified. Here, we develop and apply an affinity-proteomics strategy to conduct a global screen for PtdIns(3,4)P2 interactors in human platelets; a primary cell type with striking PtdIns(3,4)P2 accumulation. Through an integrated approach, coupling affinity capture of PtdIns(3,4)P2-binding proteins to both label-free and isobaric tag-based quantitative proteomics, we identify a diverse PtdIns(3,4)P2 interactome. Included are long-established PtdIns(3,4)P2-binding proteins such as PLEKHA1, PLEKHA2, AKT and DAPP1, and a host of potentially novel effectors, including MTMR5, PNKD, RASA3 and GAB3. The PtdIns(3,4)P2 interactome shows an enrichment of pleckstrin homology (PH) domain-containing proteins, and through bioinformatics and array analyses we characterise the PH domain of MTMR5 and define its phosphoinositide selectivity. The interactome is also diverse in function, including several proteins known to support protein trafficking and cytoskeletal mobilisation. Such proteins have the ability to drive key platelet events, and to fulfil recently-defined roles for PtdIns(3,4)P2 in a wider range of cell types. Moreover, this study will serve as a valuable resource for the future characterisation of effector-driven PtdIns(3,4)P2 function.

A Negative Feedback Loop Regulates Integrin Inactivation and Promotes Neutrophil Recruitment to Inflammatory Sites.

Neutrophils are abundant circulating leukocytes that are rapidly recruited to sites of inflammation in an integrin-dependent fashion. Contrasting with the well-characterized regulation of integrin activation, mechanisms regulating integrin inactivation remain largely obscure. Using mouse neutrophils, we demonstrate in this study that the GTPase activating protein ARAP3 is a critical regulator of integrin inactivation; experiments with Chinese hamster ovary cells indicate that this is not restricted to neutrophils. Specifically, ARAP3 acts in a negative feedback loop downstream of PI3K to regulate integrin inactivation. Integrin ligand binding drives the activation of PI3K and of its effectors, including ARAP3, by outside-in signaling. ARAP3, in turn, promotes localized integrin inactivation by negative inside-out signaling. This negative feedback loop reduces integrin-mediated PI3K activity, with ARAP3 effectively switching off its own activator, while promoting turnover of substrate adhesions. In vitro, ARAP3-deficient neutrophils display defective PIP3 polarization, adhesion turnover, and transendothelial migration. In vivo, ARAP3-deficient neutrophils are characterized by a neutrophil-autonomous recruitment defect to sites of inflammation.